Recent Advances in Bioenergy Research

Sardar Swaran Singh National Institute of Renewable Energy
Kapurthala, India

2014
Recent Advances in Bioenergy Research
Volume III
Editors
SACHIN KUMAR
A. K. SARMA
S. K. TYAGI
Y. K. YADAV
ii

ISBN 978-81-927097-2-7
Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala-2014
Electronic version published by SSS-NIRE
ALL RIGHTS RESERVED

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CONTENTS
Preface xv
Contributors xvii
Part-I: Biomass and Energy Management 1
1 Thermogravimetric characterization of wood stalks as gasification 2
and pyrolysis feedstock
Rakesh Punia, Sachin Kumar
Abstract 2
1.1 Introduction 2
1.2 Materials and methodology 4
1.3 Results and discussion 6
1.4 Conclusion 10
References 11
2 Assessment of solid waste management and energy recovery 13
from waste materials in Lucknow Zoo: a case study
Vinayak V. Pathak, Richa Kothari, A.K. Chopra, Lhaihoichong Singson
Abstract 13
2.1 Introduction 13
2.2 Materials and methods 15
2.4 Conclusion 21
References 21
3 A bioprospection of euphorbia cotinifolia for biofuel: 23
chromatography study
Punam Puri, Amita Mahajan, Anjana Bhatia, Navjot Kaur
Abstract 23
3.1 Introduction 23
3.2 Objectives 24
3.3 Methodology 25
3.5 Discussion 29
3.6 Conclusion 29
References 29
4 Cost effective electrical power generation in punjab using 31
agricutural biomass
Suman
Abstract 31
4.1 Introduction 31
4.2 Status of bio-energy resources in Punjab 32

iv

4.3 Power consumption in Punjab 34
4.4 Existing technologies for biomass conversion 36
4.5 Biomass as a coal substitute 37
4.6 Environmental criteria 37
4.7 Conclusion 38
References 39
5 Development of quality testing methodologies of fuel briquettes 40
Madhurjya Saikia, Bichitra Bikash
Abstract 40
5.1 Introduction 40
5.2 Parameters of quality assessment 41
5.3 Parameters of combustion characteristics of briquettes 43
5.4 Conclusion 45
References 45
Part-II: Thermochemical Conversion 48
6 Thermal and catalytic cracking of non-edible oil seeds to liquid fuel 49
Krushna Prasad Shadangi, Kaustubha Mohanty
Abstract 49
6.1 Introduction 49
6.2 Pyrolysis and its types 51
6.3 Process parameters that affect the yield 51
6.4 Fuel properties of seed pyrolytic oil 53
6.5 Catalytic pyrolysis of non-edible seeds 55
6.6 Conclusion 56
References 56
7 Evaluation of micro gasifier cookstove performance with 58
handmade biomass pellets using region-specific fuels and
assessment of deployment potential
Debkumar Mandal, Vikas Dohare, Vijay H. Honkalaskar, Anurag Garg,
Upendra V. Bhandarkar, Virendra Sethi
Abstract 58
7.1 Introduction 59
7.4 Conclusions 67
References 67
8 Production of hydrocarbon liquid by pyrolysis of Camellia sinensis (tea) 68
seed deoiled cake and characterization of products
Nabajit Dev Choudhury, Priyanko Protim Gohai,

Bichitra Bikash,
v

Sashi Dhar Baruah, Rupam Kataki
Abstract 68
8.1 Introduction 68
8.4 Conclusions 77
References 77
9 Comparative study of different biomass cookstove model: 79
An experimental study
K. Pal, A.K. Pandey, P Gera, S.K. Tyagi
Abstract 79
9.1 Introduction 79
9.2 Mathematical modeling 81
9.4 Analysis of cookstove 91
9.6 Conclusions 95
References 96
Part-III: Biochemical Conversion 98
10 Bioprospecting halotolerant cellulase from saline environment of 99
Bhitarkanika National Park, Odisha
Dash Indira, Sahoo Moumita, Dethose Ajay, C.S. Kar, R. Jayabalan
Abstract 99
10.1 Introduction 100
10.5 Conclusions 105
References 107
11 Isolation and molecular characterization of cellulolytic fungi used 110
for conversion of sugarcane biomass for bioethanol production
A.M. Chetan, K.M. Harinikumar, P. Bhavani, H.B. Manoj Kumar,
T. Madhu, Ningaraj Dalawai
Abstract 110
11.2 Material and methods 111
11.3 Result and discussion 114
References 120
12 Application of thermostable cellulase in bioethanol production from 121
lignocellulosic waste
Neha Srivatsava, Rekha Rawat, Harinder Singh Oberoi
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Abstract 121
12.2 Bioethanol production: current production status and challenges 123
12.3 Microorganism for thermostable cellulase production 124
12.4 Thermostable enzymes 125
12.5 Application of thermostable cellulases 128
12.6 Concluding remarks 129
References 129
13 Endoglucanases: characterization and its role in bioconversion of 135
cellulosic biomass
Rekha Rawat, Neha Srivastava, Harinder Singh Oberoi
Abstract 135
13.2 Mechanism of cellulolysis 136
13.3 Classification of endoglucanases 137
13.4 Structure of endoglucanases 137
13.5 Mechanism of cellulose hydrolysis by endoglucanases 139
13.6 Microbial sources of endoglucanase enzyme 139
13.7 Application of endoglucanases 140
13.8 Significance of thermostable endoglucanases 142
13.9 Factors responsible for thermal stability 142
13.10Conclusion 144
References 144
14 Comparative study of fermentation efficiency for bioethanol 149
production by isolates
Richa Arora, Shuvashish Behera, Sachin Kumar
Abstract 149
14.4 Conclusion 154
References 154
15 Sweet Sorghum - An ideal feedstock for bioethanol production 156
Reetika Sharma, Gurvinder Singh Kocher, Harinder Singh Oberoi
Abstract 156
15.2 Origin and biology of sweet sorghum 158
15.3 Cultivation and harvesting of sweet sorghum 159
15.4 Inherent advantages of sweet sorghum 160
15.5 Technical hurdles 163
15.6 Bioethanol production from sweet sorghum 163
15.7 Energy ratio and environmental sustainability 165
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15.8 Small-medium scale bioethanol production plant from sweet sorghum 166
References 168
16 Fermentation of glucose and xylose sugar for the production of 175
ethanol and xylitol by the newly isolated NIRE-GX1 yeast
Shuvashish Behera, Richa Arora, Nilesh Kumar Sharma, Sachin Kumar
Abstract 175
16.2 Material and method 177
16.4 Conclusion 180
References 181
17 Comparative bioethanol production by S. cerevisiae and Z. mobilis 183
from saccharified Sweet Potato Root Flour (Ipomoea batata L)
using immobilized - amylases and glucoamylase
Preeti Krishna Dash, Sonali Mohapatra, Manas Ranjan Swain,
Hrudaya Nath Thatoi
Abstract 183
17.4 Importance of enzyme immobilization 189
17.5 Conclusion 191
References 191
18 Genetic modifications in yeast for simultaneous utilization of 194
glucose and xylose
Nilesh Kumar Sharma, Shuvashish Behera, Sachin Kumar
Abstract 194
18.2 Necessity of pentose (C5) sugar fermenting organisms 196
18.3 Problems with pentose (C5) sugar fermenting yeast 197
18.4 Need of genetic engineering for xylose fermentation 198
18.5 Conclusion and future prospects 199
References 202
19 To optimize the process of alcohol production from banana peel 208
Mohit Jain, Anand Kumar Gupta, Sayan Chatterjee
Abstract 208
19.2 Materials used 210
19.3 Methodology 211
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References 216
20 Commercial production of bio-CNG & organic manure from 218
pressmud biomethanation
Preetam Holkar, A.V. Mohan Rao, K.K. Meher
Abstract 218
20.2 Feed preparation 220
20.3 CSTR digesters 220
20.4 Biogas cleaning process 222
20.5 BIO-CNG production & composition 223
20.6 Biogas utilization / storage devices cascades 224
20.7 Biogas based power 224
20.8 Digestate (Organic Manure) 224
References 226
21 Feasibility of filling biogas in cylinders 228
S.S. Sooch, Jasdeep Singh Saini
Abstract 228
21.3 Discussion 231
References 231
22 Effect of pretreatment on bioconversion of wheat straw for 232
the production of biogas
Nishshesh Singh, Vivek Saini, Pranshu Gupta, Rajan Sharma,
G Sanjay Kumar, Avanish K. Tiwari
Abstract 232
References 240
23 Potential of wheat straw for biogas production using thermophiles 242
Richa Singh, Shuvashish Behera, Yogendra Kumar Yadav, Sachin Kumar
Abstract 242
23.4 Conclusion 247
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References 248
24 Ultrasonic pretreatment to enhance biohydrogen production 250
from food waste
Abhijit Gadhe, Shriram Sonawane, Mahesh Varma
Abstract 250
24.4 Conclusion 261
References 261
25 Biological hydrogen production by facultative anaerobic bacteria 263
Enterobacter aerogens (MTCC 8100)
Virendra Kumar, Richa Kothari, S.K.Tyagi
Abstract 263
References 271
26 Enhanced biohydrogen production from glycerol using pretreated 273
mixed culture
Anbalagan Krishnasamy, Mohanraj Sundaresan, Kodhaiyolii Shanmugam,
Pugalenthi Velan
Abstract 273
References 279
Part-IV: Chemical Conversion 282
27 Isolation and characterization of freshwater microalgae Scenedesmus 283
from contaminated field samples for bioenergy generation
Mayur M. Phukan, B.K. Konwar
Abstract 283
27.3 Discussion 286
References 294
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28 Prospects of biodiesel production from non-edible oil seeds of 296
North East India: a review
Debashis Sut, Rupam Kataki
Abstract 296
28.2 Non-edible vegetable oils resources 299
28.3 Fatty acid profiles of the biodiesel 302
28.4 Properties of the biodiesel 303
28.5 Conclusion 304
References 304
29 A critical review of enzymatic transesterification: 308
a sustainable technology for biodiesel production
Neetu Singh, M.K. Jha, A.K. Sarma
Abstract 308
29.2 Biodiesel 309
29.3 Lipases as biocatalysts in biodiesel synthesis 313
29.4 Lipase immobilization 313
29.5 Variables affecting the enzymatic transesterification 315
29.6 Conclusion 317
References 317
30 Single step reaction for biodiesel production of Jatropha curcus seeds 323
Sanjaykumar N. Dalvi, Swati R. Sonawane
Abstract 323
References 328
31 Production of biodiesel from edible and non-edible oils: 329
a comparative study
A.D. Singh, R. Rao, L.B. Reddy, H.K. Raghuvanshi, A.I. Kankia, H. Sharma,
S. Srivastava, Debjani Mukherjee
Abstract 329
31.2 Scope for the study 331
31.3 Research methodology 331
References 335
32 Production of biodiesel from neem oil using synthesized iron nanocatalyst 337
Mookan Rengasamy, Sundaresan Mohanraj, Krishnasamy Anbalagan,
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Shanmugam Kodhaiyolii, Velan Pugalenthi
Abstract 337
References 345
33 Influence of free fatty acids content in catalytic activity of [BSMIM] Cl 349
ionic liquid for biodiesel production from non edible acidic oils
Subrata Das, Ashim Jyoti Thakur, Dhanapati Deka
Abstract 349
33.4 Conclusion 354
References 355
34 Analysis of physical properties and biodiesel production from 357
different accessions of Jatropha curcas
Dheeraj Singh, Chiranjib Banerjee, Animesh Sinha, Diwaker Prasad Nirala,
Santosh Prasad, Rajib Bandopadhyay
Abstract 357
34.2 Source: Jatropha Curcus 359
34.3 Different criteria which effect the biodiesel production 361
34.5 Production of biodiesel 363
34.7 Conclusion 366
References 367
35 Analysis of exhaust emission from a diesel engine fueled with 369
transesertified vegetable oils
Hemanandh J., Narayanan K.V.
Abstract 369
35.2 Background 370
35.3 Methodology 371
35.4 Results and discussions 374
35.5 Conclusion 377
References 378
36 Genetic enhancement of Pongamia pinnata for bio-energy 380
M.V.R. Prasad
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Abstract 380
36.2 Carbon sequestration by Pongamia 382
36.3 Sybiotic nitrogen fixation and soil amelioration by Pongamia 383
36.4 Selection of elite trees 383
36.5 Vegetative propagation 384
36.6 Vayusap plantations 384
References 388
Part-V: Electrochemical Processes 390
37 Evaluation of electrical properties under different operating conditions 391
of bio-electrochemical system treating thin stillage
S. Ghosh Ray, M.M. Ghangrekar
Abstract 391
37.3 Experimental results and discussion 397
38.4 Conclusion 400
References 401
38 Effect of salinity, acetate addition and alteration of sediment on 404
performance of benthic microbial fuel cells
D.A. Jadhav, M.M. Ghangrekar
Abstract 404
38.5 Future perspectives 415
References 416
39 Biohydrogen production using single-chamber membrane-free 419
microbial electrolysis cell with a stainless steel cathode
Sundaresan Mohanraj, Krishnasamy Anbalagan, Kodhaiyolii Shanmugam,
Velan Pugalenthi
Abstract 419
References 426
40 Feasibility of interlinking two technologies for simultaneously 428
two bioenergies generation
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Prashant Pandey, Vikas Shinde, S.P. Kale, R.L. Deopurkar
Abstract 428
40.3 Results 433
40.4 Discussion 438
References 439
41 Inhibitory effects of fluoride on bacterial metabolism present 441
in microbial fuel cells
Iti Sharma, M.M. Ghangrekar
Abstract 441
41.2 Materials and method 444
41.3 Results 446
41.4 Discussion 449
References 451
Part-VI: Hybrid Systems 454
42 Conversion of plastic wastes into liquid fuels a review 455
Arun Joshi, Rambir, Rakesh Punia
Abstract 455
42.2 Target of waste plastics into liquid fuel 458
42.3 Plastics recycling technologies 459
42.4 Process technology 460
42.5 Advantages of process of fuel production 461
42.4 Conclusion and recommendation 463
References 464
43 Kinetics of NOx reduction in BioDeNOx process water: effect of 466
temperature and iron chelate
B. Chandrashekhar, Heena Tabassum, Nidhi Sahu, Padmaraj Pai, R.A. Pandey
Abstract 466
43.3 Analytical methods 470
43.4 Conclusion 476
References 476

xiv

44 Status of waste treatment, utilization and management in agro processing 478
Yogender Singh, Y.K. Yadav
Abstract 478
44.2 Agro processing industrial wastes treatment/utilization 481
44.3 Waste water in agriculture and food processing 483
44.4 Importance of waste management 485
44.5 Challenges in waste management 485
References 485
45 Development of nano based thermic fluid: rheological aspects of 487
new energy system
Vijay Juwar, Shriram Sonawane
Abstract 487
45.2 Materials and method 488
References 496

xv

Preface

Due to increasing prices of petroleum products, shortage of electricity supply, degradation of
environment and availability of millions of tons of surplus biomass, R&D activities in the
area of bio-energy including biodiesel, bioethanol, biomethanation, biomass gasification,
biomass cookstove, etc. have received the tremendous attention all over the world. Keeping
this trend in mind, the Govt. of India has already initiated the blending of 5% ethanol in the
gasoline, which is likely to increase up to 10% in the coming years. While, the Jatropha
Mission has been initiated for the promotion of Biodiesel in transportation and agriculture
sectors and it is expected that by 2020 at least 10% of the liquid fuel used in transportation
sector can be replaced by biodiesel. Similarly, MNRE has already initiated the dissemination
of 10 millions of improved biomass cookstoves in the 12
th
five year plan through carbon
revenue. The renewable energy currently has made remarkable share (12.5%) of total primary
commercial energy supply of 228 GW, while the major share of around 70% of the total
generation capacity is from thermal (coal, gas, oil).
Since, energy security and diversification of the energy mix is a major policy driver for
renewables. Growth of renewables generally contributes to energy diversification, in terms of
the technology portfolio and geographical sources. Use of renewables can also reduce fuel
imports and insulate the economy to some extent from fossil fuel price rises and swings. This
not only increases the certainty for energy security but also symbolize the steady economic
growth of a country. However, the concentrated growth of variable renewables can make it
harder to balance power systems, which must be duly addressed. The electricity sector in
India had an installed capacity of around 228 GW, the world's fifth largest. Non-renewable
Power Plants constitute 87.55% of the installed capacity, and Renewable Power Plants
constitute of around 12.45% of total installed capacity while the major share is of biomass
based energy generation.
After receiving the great response of first two volumes on Recent Advances in Bioenergy
Research, we are introducing the third volume in the form of a book. The book is divided in
six parts viz. Part-I: Biomass and Energy Management; Part-II: Thermo-chemical Conversion;
Part-III: Chemical Conversion; Part-IV: Biochemical Conversion; Part-V: Electrochemical
Processes; and Part-VI: Hybrid Systems. Each section includes respective chapters from
xvi

Eminent Academician, Scientists and Researchers in the field. We are grateful for their
commendable contribution for this book.
Emphasis is given in such a way that the current trends of research and investigation
in the bioenergy sector can easily be worked out from the in-depth study of this book. Our
efforts will be successful if the readers dig up the expected gain out of these articles.

Editors
xvii

Contributors
Arora Richa, Biochemical Conversion Division, Sardar Swaran Singh National Institute of
Renewable Energy, Kapurthala
Bandopadhyay Rajib, Birla Institute of Technology, Mesra, Ranchi
Banerjee Chiranjib, Birla Institute of Technology, Mesra, Ranchi
Baruah Sashi Dhar, Regional Research Laboratory, Jorhat, Assam
Behera Shuvashish, Biochemical Conversion Division, Sardar Swaran Singh National
Institute of Renewable Energy, Kapurthala
Bhandarkar Upendra V., Department of Mechanical Engineering, Indian Institute of
Technology Bombay, Mumbai
Bhatia Anjana, Department of Botany, HMV College, Jalandhar
Bhavani P., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru
Bikash Bichitra, Assam Down Town University, Guwahati, Assam
Chandrashekhar B., Environmental Biotechnology Division, CSIR-National Environmental
Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra
Chatterjee Sayan, University School of Biotechnology, Guru Gobind Singh Indraprastha
University, New Delhi
Chetan A.M., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru
Chopra A.K., Department of Zoology and Environmental Sciences, Gurukula Kangri
Vishwavidyalya , Haridwar
Choudhury Nabajit Dev, Assam Down Town University, Guwahati, Assam
Dalawai Ningaraj, Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru
Dalvi Sanjaykumar N., Department of Physics, S. N. Arts, D. J. M. Commerce & B. N. S.
Science College, Sangamner, Dist. Ahmednagar, Maharashtra
Das Subrata, Department of Energy, Tezpur University, Tezpur, Assam
Dash Indira, Food and Bioprocess Technology Laboratory, National Institute of Technology,
Rourkela, Odisha
Dash Preeti Krishna, Department of Biotechnology, College of Engineering and
Technology, Biju Pattnaik University of Technology, Bhubaneswar
Deka Dhanapati, Department of Energy, Biomass Conversion Laboratory, Tezpur
University, Tezpur, Assam
Deopurkar R.L., Department of Microbiology, University of Pune, Ganeshkhind, Pune,
Maharastra
Dethose Ajay, Food and Bioprocess Technology Laboratory, National Institute of
Technology, Rourkela, Odisha
xviii

Dohare Vikas, Centre for Environmental Science and Engineering, Indian Institute of
Gadhe Abhijit, Department of Chemical Engineering, Visvesvaraya National Institute of
Technology (VNIT), Nagpur
Garg Anurag, Centre for Environmental Science and Engineering, Indian Institute of
Gera P., Dr. B. R. A. National Institute of Technology, Jalandhar
Ghangrekar M.M., Department of Civil Engineering, Indian Institute of Technology,
Kharagpur
Gohai Priyanko Protim, Department of Energy, Tezpur University, Napaam, Tezpur, Assam

Gupta Anand Kumar, University School of Biotechnology, Guru Gobind Singh
Indraprastha University, New Delhi
Gupta Pranshu, Chemical Engineering Department, University of Petroleum & Energy
Studies, Dehradun
Harinikumar K.M., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru
Hemanandh J., Department of Mechanical Engineering, Sathyabama University, Chennai
Holkar Preetam, Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra
Honkalaskar Vijay H., Centre for Technology Alternatives for Rural Areas, Indian Institute
of Technology Bombay, Mumbai
Jadhav D.A., School of Water Resources, Indian Institute of Technology, Kharagpur
Jain Mohit, University School of Biotechnology, Guru Gobind Singh Indraprastha
University, New Delhi
Jayabalan R., Food and Bioprocess Technology Laboratory, National Institute of
Jha M.K., Department of Chemical Engineering, Dr B R Ambedkar NIT, Jalandhar
Joshi Arun, Department of Chemical Engineering, Doon College of Engineering and
Technology, Dehradun
Juwar Vijay, Department of Chemical Engineering Visvesvaraya National Institute of
Kale S.P., Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research
Centre, Navi Mumbai, Maharastra
Kankia A.I., School of Biotechnology, Lovely Professional University, Phagwara, Punjab
Kar C.S., Office of the Principal CCF (Wildlife) & Chief Wildlife Warden, Bhubaneswar,
Odisha
Kataki Rupam, Department of Energy, Tezpur University, Napaam, Tezpur, Assam
xix

Kaur Navjot, Department of Botany, HMV College, Jalandhar
Kocher Gurvinder Singh, Department of Microbiology, Punjab Agricultural University,
Ludhiana, Punjab
Konwar B.K., Department of Molecular Biology & Biotechnology, School of Science,
Tezpur University, Assam
Kothari Richa, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar
University, Vidya Vihar, Lucknow
Krishnasamy Anbalagan, Department of Biotechnology, Bharathidhasan Institute of
Technology, Anna University, Tiruchirappalli, Tamil Nadu
Kumar G Sanjay, Chemical Engineering Department, University of Petroleum & Energy
Studies, Dehradun
Kumar H.B. Manoj, Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru
Kumar Sachin, Biochemical Conversion Division, Sardar Swaran Singh National Institute of
Kumar Virendra, Department of Environmental Science, Babasaheb Bhimrao Ambedkar
University, Lucknow
Madhu T., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru
Mahajan Amita, Department of Bio-chemistry, RBIEBT, Kharar
Mandal Debkumar, Centre for Environmental Science and Engineering, Indian Institute of
Meher K.K., Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra
Mohanty Kaustubha, Department of Chemical Engineering, Indian Institute of Technology
Guwahati, Guwahati
Mohapatra Sonali, Department of Biotechnology, College of Engineering and Technology,
Biju Pattnaik University of Technology, Bhubaneswar
Mukherjee Debjani, School of Biotechnology, Lovely Professional University, Phagwara,
Punjab
Narayanan K.V., Department of Mechanical Engineering, Sathyabama University, Chennai
Nirala Diwaker Prasad, Biotechnology, Genetics and Tree Improvement Division, Institute
of Forest Productivity, Lalgutwa, Ranchi
Oberoi Harinder Singh, Central Institute of Post Harvest Engineering and Technology,
Ludhiana, Punjab
Pai Padmaraj, Environmental Biotechnology Division, CSIR-National Environmental
Pal K., Thermochemical Conversion Division, Sardar Swaran Singh National Institute of
xx

Pandey A.K., Thermochemical Conversion Division, Sardar Swaran Singh National Institute
of Renewable Energy, Kapurthala
Pandey Prashant, School of Studies in Biotechnology, Jiwaji University, Gwalior- 474011,
Madhya Pradesh
Pandey R.A., Environmental Biotechnology Division, CSIR-National Environmental
Pathak Vinayak V., Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar
University, Vidya Vihar, Lucknow
Phukan Mayur M., Department of Molecular Biology & Biotechnology, School of Science,
Tezpur University, Assam
Prasad M.V.R., VAYUGRID, Bangalore
Prasad Santosh, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest
Productivity, Lalgutwa, Ranchi
Punia Rakesh, Doon College of Engineering and Technology, Dehradun
Puri Punam, Department of Life SciencesBiotechnology, Punjab Technical University,
Kapurthala
Raghuvanshi H.K., School of Biotechnology, Lovely Professional University, Phagwara,
Punjab
Rambir, Department of Chemical Engineering, Doon College of Engineering and
Technology, Dehradun
Rao A.V. Mohan, Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra
Rao R., School of Biotechnology, Lovely Professional University, Phagwara, Punjab
Rawat Rekha, Central Institute of Post Harvest Engineering and Technology, Ludhiana,
Punjab
Ray S. Ghosh, Advanced Technology Development Centre, Indian Institute of Technology,
Kharagpur
Reddy L.B., School of Biotechnology, Lovely Professional University, Phagwara, Punjab
Rengasamy Mookan, Department of Petrochemical Technology, Bharathidhasan Institute of
Sahoo Moumita, Food and Bioprocess Technology Laboratory, National Institute of
Sahu Nidhi, Environmental Biotechnology Division, CSIR-National Environmental
Saikia Madhurjya, Dibrugarh University Institute of Engineering and Technology,
Dibrugarh, Assam
xxi

Saini Jasdeep Singh, Department of Civil Engineering, Punjab Agricultural University,
Ludhiana, Punjab
Saini Vivek, Chemical Engineering Department, University of Petroleum & Energy Studies,
Dehradun
Sarma A.K., Chemical Conversion Division, Sardar Swaran Singh National Institute of
Sethi Virendra, Centre for Environmental Science and Engineering, Indian Institute of
Shadangi Krushna Prasad, Department of Chemical Engineering, Indian Institute of
Technology Guwahati, Guwahati
Shanmugam Kodhaiyolii, Department of Biotechnology, Bharathidhasan Institute of
Sharma H., School of Biotechnology, Lovely Professional University, Phagwara, Punjab
Sharma Iti, P.K. Sinha Center for Bioenergy, Indian Institute of Technology, Kharagpur
Sharma Nilesh Kumar, Biochemical Conversion Division, Sardar Swaran Singh National
Institute of Renewable Energy, Kapurthala
Sharma Rajan, Chemical Engineering Department, University of Petroleum & Energy
Studies, Dehradun
Sharma Reetika, Department of Microbiology, Punjab Agricultural University, Ludhiana,
Punjab
Shinde Vikas, Department of Microbiology, University of Pune, Ganeshkhind, Pune,
Maharastra
Singh A.D., School of Biotechnology, Lovely Professional University, Phagwara, Punjab
Singh Dheeraj, Birla Institute of Technology, Mesra, Ranchi
Singh Neetu, Department of Chemical Engineering, Dr B R Ambedkar NIT, Jalandhar
Singh Nishshesh, Chemical Engineering Department, University of Petroleum & Energy
Studies, Dehradun
Singh Richa, Biochemical Conversion Division, Sardar Swaran Singh National Institute of
Singh Yogender, Department of Food Engineering and Technology, S.L.I.E.T., Longowal,
Punjab
Singson Lhaihoichong, Department of Environmental Sciences, Babasaheb Bhimrao
Ambedkar University, Vidya Vihar, Lucknow
Sinha Animesh, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest
Productivity, Lalgutwa, Ranchi
xxii

Sonawane Shriram, Department of Chemical Engineering Visvesvaraya National Institute of
Sonawane Shriram, Department of Chemical Engineering, Visvesvaraya National Institute
of Technology (VNIT), Nagpur
Sonawane Swati R., Department of Chemistry, S. N. Arts, D. J. M. Commerce & B. N. S.
Science College, Sangamner, Dist. Ahmednagar, Maharashtra
Sooch S.S., School of Energy Studies for Agriculture, Punjab Agricultural University,
Ludhiana, Punjab
Srivastava S., School of Biotechnology, Lovely Professional University, Phagwara, Punjab
Srivatsava Neha, Central Institute of Post Harvest Engineering and Technology, Ludhiana,
Punjab
Suman, Punjab University, SSGRC, Hoshiarpur
Sundaresan Mohanraj, Department of Biotechnology, Bharathidhasan Institute of
Sut Debashis, Department of Energy, Tezpur University, Napaam, Tezpur, Assam
Swain Manas Ranjan, Department of Biotechnology, IIT Madras, Chennai
Tabassum Heena, Environmental Biotechnology Division, CSIR-National Environmental
Thakur Ashim Jyoti, Department of Chemical Sciences, Tezpur University, Tezpur, Assam
Thatoi Hrudaya Nath, Department of Biotechnology, College of Engineering and
Technology, Biju Pattnaik University of Technology, Bhubaneswar
Tiwari Avanish K., Centre for alternate energy research, University of petroleum & energy
Studies, Dehradun
Tyagi S.K., Thermochemical Conversion Division, Sardar Swaran Singh National Institute of
Varma Mahesh, Department of Chemical Engineering, Visvesvaraya National Institute of
Velan Pugalenthi, Department of Biotechnology, Bharathidhasan Institute of Technology,
Anna University, Tiruchirappalli, Tamil Nadu
Yadav Y.K., Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala

Recent Advances in Bioenergy Research, Vol. III 2014

1

Part I
Biomass and Energy Management


2

CHAPTER 1
THERMOGRAVIMETRIC CHARACTERIZATION OF WOOD
STALKS AS GASIFICATION AND PYROLYSIS FEEDSTOCK
Rakesh Punia, Sachin Kumar

Abstract
Energy is an integral part of a society and plays an important role in its socio-economic
development. A nation economic development can be assessed by the pattern of consumption
and quality of energy availability. Energy and chemicals from agriculture and regional
available wood residue can be efficiently produced by gasification and pyrolysis methods.
Indian has abundance of biomass sources. Selected physical and chemical properties of
biomass are related to thermochemical conversion, which has been observed to determine the
thermochemical conversion. The main objective of the present work is to investigate the
comparison of use of different biomass fuels and their parameters on the performance of small
scale downdraft gasifier, biomass fuels selected are locally available plant stalks such as
Prosopis juliflora (Kikkar), Eucalyptus (Sefeda), Pigeon pea (Arhar Dal), Albizia procera
(Surash), Melia sp. (Bakain) and Mulberry sp. (Sahatoot). Fuel feedstock characterisation of
selected biomass has been carried out at macroscopic as well as microscopic levels such as
determination of dry density, calorific value, proximate & ultimate analysis, and
thermogravimetric analysis (TGA). On the basis of characteristics, it is found that Melia sp.,
and Eucalyptus could be energy efficient feedstock for small scale downdraft gasifier possess
high fixed carbon content and calorific value as compared to other selected wood stalks.
Key Words: Wood stalks , Gasification, Thermogravimeter analyser, Reaction kinetics.
1.1 Introduction
To maintain the ecology, sustainable and equitable development become the critical
issues in most parts of world. The alarming population coupled with developmental activities
based on decisions for resource scarcity in many parts of India. A judicious choice of energy
utilization is required to achieve growth in a sustainable manner (Keyhani et al., 2010).
Gasification and pyrolysis can convert lignocellulosic materials to synthesis gas (syngas)
without the need for delignification. For different applications, the syngas from gasification
Recent Advances in Bioenergy Research, Vol. III ISBN 978-81-92797-2-7
!ditors" Sachin #$%ar, A.#. Sar%a, S.#. &yagi, '.#. 'adav ( SSS-NIR!-21)


3

can be further converted and separated to other chemicals (by various reforming processes) or
fuel gas or hydrogen for fuel cell (Anjireddy et al., 2011).
Gasification is a thermochemical process by which any carbonaceous feed can be
converted to gaseous products with useable heating value (primarily carbon monoxide and
hydrogen in a controlled oxidizing atmosphere). Pyrolysis in particular converts biomass into
high energy content biofuels provided that the adequate temperature and heating rate are
reached and may be used to fuel internal combustion engines and gas turbines after an
intermediate process that converts the feedstock into a liquid or gaseous biofuel (Goyal et al.,
2008; Fantozzi et al., 2003) Pyrolysis is one of the first step of all thermochemical processes
occurring in an inert atmosphere (Fantozzi et al., 2010). Energy generation from biomass is
environmental friendly and does not increase the CO
2
in the atmosphere. Biomass energy can
be generated locally and can make any country energy self-sustainable and less dependent on
foreign petroleum resources (McKendry, 2002). Interest in bioenergy has been enhanced
because it also manages the biomass wastes.
The advantage of gasification has ability to utilize a wide range of feedstocks ranging
from any plant residue, organic by-product (with protein, lignin or oil) of industry or even
municipal wastes , as compared with other bioenergy generation techniques. Gasification and
pyrolysis are efficiently viable options for processing biomass feed stocks, which cannot be
fermented to ethanol technically or economically (Kumar et al., 2008). Mathematical
modeling to predict the product gas qualities during gasification and pyrolysis requires the
reaction kinetics knowledge of biomass volatilization and its subsequent reactions.
Thermogravimetric analysis (TGA) is very useful in determining the reaction kinetics of
gasification and pyrolysis. It has been used extensively for the characterization of various
feedstocks. This method have been used by many researchers to determine the kinetics
parameter for bagasse in a nitrogen atmosphere (Nassar et al., 1996), rice husk in an oxygen
atmosphere (Mansaray and Ghaly, 1999), rapeseed straw and stalks in a nitrogen atmosphere
(Karaosmanoglu et al, 2001), forestry wastes in a nitrogen atmosphere (Lapuerta et al., 2004)
and poplar wood in a nitrogen atmosphere (Katarzyna et a.l, 2012). However, there is a lack
of kinetics information on gasification or pyrolysis of different wood stalks. This research
study surrounds to the thermochemical conversions properties of biomass and determine its
reaction kinetics in inert and oxidizing atmospheres using a thermogravimetric technique.


4

1.2 Materials and Methodology
Variety of biomass samples were collected from various accessible locations. Prosopis
juliflora, Eucalyptus, Albizia procera, Melia sp. and Mulberry sp. wood stalks were
purchased from Kapurthala timber market. Pigeon pea (Arhar Dal) stalks were collected from
SSS-NIRE (Kapurthala) campus. Biomass samples were collected from gasifier feed stock in
such a manner to represent homogeneity of biomass characteristics. Collected biomass
samples were sun dried for five days. Wood stalk dust/flakes were prepared on saw mill and
ground in grinder for analysis purpose. Ground wood samples were screened using vibratory
screen for attaining particle size of about 425 m. Moisture content was removed from
screened samples by drying in oven Broadly, biomass characterization are categorized in two
types ; macroscopic and microscopic property. Macroscopic analysis of biomass fuel
properties are proximate analysis, ultimate analysis, particle size, bulk density, calorific value,
ash fusion temperature, etc. Biomass fuel properties for microscopic analysis includes thermal
properties, chemical kinetics, and mineral data, etc.
1.2.1 Proximate analysis
The proximate analysis of the sample was done as per ASTM standards. The
parameters namely moisture content (MC) (ASTM D3172-73), volatile matter (VM) (ASTM
D3175-73) and ash content (ASTM D3174-73).
1.2.2 Ultimate analysis
The ultimate analysis of the biofuel is done with CHNS Analyzer. The modern
elemental analyzers of Elementor (Vario MICRO Cube), analysis samples from 1 mg to 800
mg solid or liquid samples.
1.2.3 Calorific value
This important characteristics of a fuel were determined by bomb calorimeter
(Toshniwal -CC01-M3). The crushed sample was compressed from the ground sample was
compacted from an original average density of 460 to 1200 kg/m
3
. The pellet was then
combusted to determine energy content.
1.2.4 Biomass density
The size and density affects the burning characteristics of biomass fuel by heating and
drying rate during combustion. It also dictates how the material is likely to behave during
subsequent thermo-chemical or biological processing as a fuel or feed stocks. Average mass

5

of is measured by Micro-balance and volume of dry biomass samples is determined by water
displacement method.
1.2.5 Kinetic study (activation energy)
In general, the microscopic characterisation of gasifier feedstock is done by
Thermogravimetric analysis (TGA), which is used to determine the kinetics parameter in
presence of oxygen or inert atmosphere. Pyrolysis is a sub-category of gasification, the
difference being this process takes place in an inert atmosphere (generally nitrogen)
Thermogravimetric analysis experiment is performed using on Perkin-Elmer (STA 6000)
equipment. The temperature of furnace and weighting system of TGA were calibrated
according to the manufacturers recommendation. Temperature calibration was performed by
measuring Curie points of alumel, nickel, perkalloy and iron.
Depending on the density of biomass, samples weight is placed in the pan of the TGA
microbalance. Approximately 20-42 mg of the wood powder were taken in crucible pan for
TGA experiment. Air is used as purge gases and all TGA experiments were conducted at a
constant purge flow rate of 20 ml/min. Residual weight of the sample and the derivative of
weight, with respect to time and temperature (differential thermogravimetry analysis, DTG),
were recorded. A program is prepared and saved in which samples were held at 30
o
C for 1
min and heated to 850
o
C at the rate of 10
o
C/min and then held at 850
o
C for 1min.
1.2.5.1 Procedure to determine parameters of reaction kinetics
To calculate chemical kinetics of reaction using the procedure of Duvvuri et al. (1975)
as applied by Mansaray et al. (1999) and Karaosmanoglu et al. (2001) which is explained as
bellow :
Global kinetics of the vitalization reaction can be written as

dx
dt
= k x
n
(1)
Where, x is the sample weight, k the reaction constant and n the order of the reaction.
Applying the Arrhenius equation,
k = Ae
-E/RT
(2)
The combined form of the above two equations (1) and (2) can be written in linear form as
ln [
-1
wo-wI

dw
dt
] = ln (A) (
L
R1
) + n ln (
w-wI
wo-wI
) , { Put x =
wo- w
wo - wI
} (3)

6

where, w
o
is the initial weight at the starting stage, w
f
is the final weight at the end of TGA
and w is the weight at any temperature, dw/dt the ratio of change in weight to change in time,
A the pre-exponential factor and R the universal gas constant (gas value).
Eq. (3) is of the form
y = B + Cx + Dz, (4)
where
y = ln[
-1
wo-wI

dw
dt
], x =
1
1
, z = ln (
w-wI
wo-wI
) ,
B = ln(A); C = (-
L
R
), D = n
The constants B, C, D were estimated by multi-linear regression. The determination of
activation energy form selected data is done by regression calculation with Analysis Tool of
MS-Excel.
1.3 Results & Discussion
Analysis results for selected biomass samples which includes Proximate Analysis,
Ultimate Analysis, Density Measurement, Calorific Value, Activation Energy are contained in
this section.
1.3.1 Proximate Analysis
Proximate analysis data (Table-1) shows that the moisture contents of analyzed
biomasses were in between 5 to 8% (by weight) which is under the range of Downdraft
Gasifier biomass feedstock. Volatile Matter and Fixed Carbon has major role in heating value
of biomass fuels. Maximum VM of Melia was found (83.79%). Maximum Fixed carbon
content was of Eucalyptus and Pigeon pea has lowest (7.44%) FC value. Ash in the biomass
was in the range of 0.7 to 4% which plays a catalyst role during biomass gasification.
Table-1: Proximate analysis data of biomass feed stocks.
Proximate analysis (Wt.%)
Biomass Moisture Content Volatile Matter Fixed Carbon Ash
Prosopis juliflora 6.32 82.46 9.02 2.52
Eucalyptus 5.93 80.53 10.82 2.67
Pigeon pea 8.27 82.53 7.44 1.53
Albizia procera 6.72 83.58 8.07 0.79
Melia (Bakain) 5.03 83.79 8.73 1.37
Mulberry 6.24 81.93 10.04 1.69

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Table-2: Results of biomass calorific value and Dry Density
Biomass Samples Calorific value (HHV)
MJ/ kg.
Dry Density
(Kg/m
3
)
Prosopis juliflora (Kikkar) 17.8038 496.711
Eucalyptus 17.4344 443.109
Pigeon pea (Arhar) 16.0138 272.509
Albizia procera 16.8509 326.300
Melia (Bakain) 17.8643 378.561
Mulberry (Sahatoot) 16.4012 251.785

On observing dry density data, it is evident that P. juliflora has substantially highest
density than other biomasses. Pigeon pea and Mulberry wood stalks have comparatively
lower dry density than other biomass samples.
1.3.2 Ultimate Analysis
Among all the biomass samples, Melia and Eucalyptus have comparatively higher
fixed carbon wt. % which indicates that they could produce syngas of high calorific value.
Nitrogen for analyzed biomass was less than 1% (by weight) of biomass. As evident from
data (Table-3), sulfur content is missing in all the samples.
Table-3: Ultimate Analysis of Biomass Samples

1.3.3 Thermogravimeter analysis (TGA)
Wood samples were characterized by thermogravimeter analyser under Nitrogen
(Inert) and Air atmosphere. The reduction in weight of biomass with steady state rise in the
reactor temperature were analysed.
Ultimate analysis (wt.%)
Biomass Samples Carbon Hydrogen Nitrogen Sulphur Oxygen
Prosopis juliflora 47.391 6.117 0.400 0.000 45.92
Eucalyptus 48.193 5.958 0.335 0.000 45.457
Pigeon pea 47.314 5.837 0.450 0.000 46.073
Albizia procera 46.431 6.851 0.470 0.000 47.044
Melia (Bakain) 48.738 6.463 0.550 0.000 44.247
Mulberry(Sahatoot) 45.285 5.963 0.095 0.000 48.221

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1.3.3.1 TGA in Nitrogen (inert) atmosphere
TGA plots of selected biomass in nitrogen atmosphere are similar to (Fig. 1). The first
stage of weight loss ranged from 30
o
C to around 125
o
C was clearly distinct from the other
stages of weight loss. It may correspond to the loss of water and light volatile compounds in
the biomass sample (Mansaray and Ghaly, 1999). Following the first stage, there was
negligible weight loss (< 0.5%) in the temperature range of 160250
o
C. The second phase of
weight loss started around 250
o
C. The derivative plot of the region between 250 and 850
o
C
showed only one observable peak (Fig. 1).

Fig. 1: Typical TGA and DTG diagram of biomass in nitrogen atmosphere
When the data between 250 and 850
o
C were used for determining parameters of
reaction kinetics, the r
2
values for the multiple-regression were less than 0.80, and the
predicted values deviated from the experimental data. This suggested that there may have
been two different reaction stages of weight loss occurring in this region (250850
o
C). Total
of three distinct stages (Fig. 1) represented the global kinetics of weight loss occurring during
TGA of biomass in inert atmosphere.
1.3.3.2 TGA in Air (Oxidizing) Atmosphere
In an air atmosphere, the TGA plots similar to (Fig. 2) clearly suggested that there
were three stages of weight loss. The first stage in the oxidizing atmosphere ranged from 25 to
115140
o
C. It was very similar to the first stage in the inert atmosphere. The weight loss

9

between the end of the first stage (130
o
C) and the start of the next stage (240
o
C) was much
less (<5%). The second stage of weight loss ranged from 240 to 350400
o
C, a very large
weight loss (~70%) at a very high rate (>10%/
o
C) as compared with the inert atmosphere.
Separation between the second and third stages in the oxidizing atmosphere was very clear.
During the third stage, which ranged from 400 to 560
o
C, there was a small amount of
weight loss (~10%) at a slower rate. The weight loss in the third stage was very much lower
as compared with the second stage and also as compared with weight loss during the third
stage in an inert atmosphere. Also, the third stage in the oxidizing atmosphere had a very
narrow temperature range as compared with the third stage in the inert atmosphere. This
suggests that the third stage in the inert and oxidizing atmospheres were different. The amount
of oxygen in the air atmosphere in our experiment was sufficient for oxidation of a small
amount of biomass particles (~1520 mg).

Fig. 2 -Typical TGA and DTG diagram of biomass in air atmosphere

1.3.3.3 Parameters of Reaction Kinetics
Kinetics of weight loss in air atmosphere at a heating rate of 10
o
C/min was similar to
that in nitrogen atmosphere . But, at higher rates of 30 and 50
o
C/min the reaction during the
second stage occurred very rapidly and activation energies were higher than activation
energies in nitrogen atmosphere.

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Collected data is studied according stages from I to III and ash point of biomass. The
first stage of weight loss corresponds to the loss of water and light volatile compounds in
analysed biomass. The low moisture content in biomass samples resulted in low weight loss
during this stage of weight loss. The second stage may correspond to the major loss (65 to
80% wt.), due to the decomposition of cellulose and hemicellulose components and partial
loss of the lignin component of biomass. Following this stage, there was a continuous and
slow weight loss from 450550 to 850
o
C, due to the thermal degradation of lignin or complex
high-molecular weight components of biomass. TGA stages shows that the performance of
eucalyptus and melia as gasifier feedstock are better than other selected biomasses. The data
between 250 and 450
o
C were used for determining parameters of reaction kinetics. Biomass
weight percent at the interval of 25
o
C are collected from main TGA data (Table 4).
Table-4 Activation Energy during second stage in an air atmosphere and N
2
Atmosphere
Activation Energy
Biomass
Air Atmosphere N
2
Atmosphere
E (kJ mol
-1
) E (kJ mol
-1
)
Prosopis juliflora 86.34 64.31
Eucalyptus 88.62 75.34
Pigeon pea 85.34 68.29
Albizia procera 86.74 66.74
Melia 78.53 68.53
Mulberry 76.35 59.57

1.4 Conclusions
TGA of wood stalks in nitrogen atmosphere indicates distinct three stages of weight
loss. A comparative study of different regionally available gasifier feed stocks i.e. Prosopis
juliflora, Eucalyptus, Albizia procera, Melia sp. and Mulberry sp.was done on the basis of
determination of dry density, proximate analysis, ultimate analysis and thermogravimetric
analysis. Proximate analysis data shows highest fixed carbon content in Eucalyptus as
compared to Mulberry, which indicates high heating value. Ultimate analysis of all the
selected biomasses was found that all the samples were free from sulfur. The carbon
percentage was higher in Melia, and Eucalyptus as compared to other biomasses. Dry density
of Prosopis juliflora and Eucalyptus were found to be highest among all the selected
biomasses. On the compilation of TGA data, it was found that activation energy of Eucalyptus

11

is highest in both the cases (inert / air) which indicates that more external energy is required to
initiate the combustion process.
Acknowledgments
One of the authors (Rakesh Punia) would like to thank to Dr. A.K. Jain, the Director
of SSS-NIRE for their permission to perform experiments at SSS-NIRE, Kapurthala.

References
1. Bhavanam A. and Sastry R.C. (2011) Biomass Gasification Processes in Downdraft
Fixed Bed Reactors: A Review. Int. J. Chem. Eng. App., 2:425-443.
2. Duvvuri M.S., Muhlenkamp S.P., Iqbal K.Z. and Welker J.R. (1975) The pyrolysis of
natural fuels. J. Fire Flamm., 6:468-477.
3. Fantozzi F., DAlessandro B. and Bidini G. (2003) IPRP Integrated pyrolysis
regenerated plant gas turbine and externally heated rotary-kiln pyrolysis as a biomass
waste energy conversion system. Influence of thermodynamic parameters. Proc. Inst.
Mech. Eng. A- J. Pow., 217:519-527.
4. Fantozzi F., Laranci P. and Bidini G. (2010) CFD simulation of biomass pyrolysis
syngas vs. natural gas in a microturbine annular combustor. Proc. ASME Turbo Expo:
Power for Land, Sea and Air, 14-18, Glasgow, UK.
5. Goyal H., Seal D. and Saxena R. (2008) Bio-fuels from thermochemical conversion of
renewable resources: a review. Renew. Sust. Energy Rev., 12:504517.
6. Karaosmanoglu F., Cift B.D. and Ergudenler A.I. (2001) Determination of reaction
kinetics of straw and stalk of rapeseed using thermogravimetric analysis. Energy
Sources, 23:767-774.
7. Keyhani A., Ghasemi-Varnamkhasti M., Khanali M. and Abbaszadeh R. (2010) An
assessment of wind energy potential as a power generation source in the capital of Iran,
Tehran. Energy, 35:188-201.
8. Kumar A., Lijun W., Dzenis Y.A., Jones D.D. and Hanna M.A. (2008)
Thermogravimetric characterization of corn stover as gasification and pyrolysis
feedstock. Biomass Bioenergy, 32:460- 467.
9. Lapuerta M., Hernandez J.J. and Rodriguez J. (2004) Kinetics of devolatilisation of
forestry wastes from thermogravimetric analysis. Biomass Bioenergy, 27:385-391.

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10. Mansaray G.K. and Ghaly A.E. (1999) Determination of kinetic parameters of rice
husks in oxygen using thermogravimetric analysis. Biomass Bioenergy, 17:19-31.
11. McKendry P. (2002) Energy production from biomass (Part I): overview of biomass.
Bioresour. Technol., 83:37-46.
12. Nassar M.M., Ashour E.A. and Wahid S.S. (1996) Thermal characteristics of bagasse. J.
App. Polymer Sci., 61: 885-890.
13. Slopiecka K., Bartocci P. and Fantozzi F. (2012) Thermogravimetric analysis and
kinetic study of poplar wood pyrolysis. App. Energy, 97: 491-497


13

CHAPTER 2
ASSESSMENT OF SOLID WASTE MANAGEMENT AND
ENERGY RECOVERY FROM WASTE MATERIALS IN
LUCKNOW ZOO: A CASE STUDY
Vinayak V. Pathak, Richa Kothari, A.K. Chopra, Lhaihoichong Singson

Abstract
Zoo is the facility in which animals are captured within enclosures, displayed to the public
and they may breed also. Thus zoo is the centers which provide entertainment, education and
protecting endangered species. The animals and the visitors inside the zoo generate large
amount of solid waste. The present work is a case study of Lucknow zoo in order to identify
the sources of solid waste generation and their sustainable management with an approach of
energy recovery. According to findings number of mammals (468), birds (100) and reptiles
(378) were present which generated 482 kg of fresh animal waste per day. On the other hand
various plant species such as Madhuca longifolia, Aegle marmelos, Poltalthia longifolia,
Cycas circinnalis, Ficus benhalensis etc were also observed which were produce 6.5 to 7 kg
of biomass per day. The selected zoo attracts more than 900,000 to 1000,000 of tourists and
visitors annually which contribute in solid waste generation. Various suggestive measures for
treatment of animal waste, plant waste and anthropogenic waste were identified for
conversion of this high organic waste in to energy rich biofuel.
Key words: Solid waste, Sustainable, Energy recovery, Biomass, Biofuel
2.1 Introduction
Wastes have been recognized as valuable sources whether it is organic or inorganic.
Various sources have been identified for the generation of waste like industrial, domestic,
agricultural or commercial etc. Animal waste generated from agricultural and commercial
practices primarily used for plant nutrient (feedstuff) and feedstock for energy production
(methane) (Morse, 1995; Williams, 1995; Gunaseelan, 1997). Animal waste are mainly
produced either in operations like dairy farming, poultry farming or in Zoo areas. Zoos are
the areas in which animals are confined within a definite boundary, where they breed and


14

displayed for the tourists, hence Zoo provide education and entertainment with protection of
endangered species (Gibson, 1980).
All animals and tourists produce large amount of waste inside the zoo premises which
consist of both organic as well as inorganic waste. Zoos having access of millions of people
have the ability to educate and communicate a number of people for sustainable lifestyles. A
serious contribution towards sustainable future can be made by the Zoo by following the
sustainability in their policies, strategies and management. A number of criteria such as
expenses, environmental impact, profitability, complexity and sponsorship are considered in
order to develop zoo as eco-development.
In India Central Zoo Authority (CZA) is the organizing body of Central government for
all zoos which is also associate member of world Association of Zoo and Aquarium (WAZA).
CZA classify zoos depending on area, number of animals and annual attendance of visitors.
CZA provide financial support to zoo and evaluate it time to time in order to provide
recognition.
Lucknow zoo at present has about 468 mammals, 378 reptiles, and many different
species of wild animals. The main attraction of this zoo is Royal Bengal tiger, White Tiger,
Lion, Wolf, Barking deer, Hog Deer, Asiatic Elephant, Giraffe, Zebra, Hill Mynahs, Giant
squirrels, Great pied hornbills, Golden pheasant, Silver Pheasant etc. Zoo not only protect
these animals but also achieve successful breeding of Swamp deer, Black buck, Hog deer,
barking deer, White Tiger, Indian wolf, and several pheasants..
A number of plants are also protected inside the zoo such as Madhuca longifolia,Aegle
marmelos, Poltalthia longifolia, Cycas circinnalis, Ficus benghalensis, tectona grandis,
Eucalyptus hybrid, Santalum album, Acacia nilotica, mangifer indica, Tamaridus indica,
cassia siamia, Delonix regia etc. Apart from being a conservation centre of various animals
it also provide various facilities such as an aquarium , nocturnal house, Botanical garden,
Museum, Jurassic Park, Solar power house etc. The Lucknow zoo attracts 9,00,000 to
1,00,00,000 of tourists due to the above-mentioned facilities which resulted in large amount
of solid waste generation.
This consists of complex waste such as polythene bags, wrappers, papers, plastic bottles
and waste from cafeteria. On the other hand animal present inside the zoo produces large
amount of organic waste. This is why the main objective of the present study is based on how

to manage solid waste problems in zoo premises with identification of conversion techniques
for waste to energy on theoretical assessment of data collected. .
Fig.1 Month wise total visitors in Lucknow Zoo (Data collected from the official website of
2.2 Materials and Methods
2.2.1 Preparation of questionnaire
In order to obtain technical infor
waste generation, its utilization questionnaire method is adapted. The questionnaire was
categorized mainly in three parts
i. Animal waste
ii. Biomass waste
iii. Anthropogenic waste

2.2.2 Identification of different routes for conversion
Various routes of conversion for waste to energy have been identified. The routes may
vary depending on the type of waste. According to the waste collected from the zoo premises,
it can be reduced or manage by using various advanced and technical processes
predication is done on the basis of data collected by the questionnaire method.

Recent Advances in Bioenergy Research, Vol. III
15
for waste to energy on theoretical assessment of data collected. .
ise total visitors in Lucknow Zoo (Data collected from the official website of
Lucknow Zoo).
Materials and Methods
Preparation of questionnaire
In order to obtain technical information regarding management of Z
utilization questionnaire method is adapted. The questionnaire was
categorized mainly in three parts:
Anthropogenic waste
Identification of different routes for conversion of waste to energy
it can be reduced or manage by using various advanced and technical processes
Vol. III 2014

ise total visitors in Lucknow Zoo (Data collected from the official website of
mation regarding management of Zoo, amount of solid
utilization questionnaire method is adapted. The questionnaire was
of waste to energy
it can be reduced or manage by using various advanced and technical processes. The

Fig. 2 Questionnaire for the animal waste
Fig. 3 Questionnaire for the Biomass waste
2.3 Results and Discussion
2.3.1 Data collection by questionnaire method
According to the data collected from the questionnaire it was found that Zoo has 468
mammals, 378 reptiles and 100 birds. These animals are the source of large amount of organic
waste which is valuable for the bio
16
Fig. 2 Questionnaire for the animal waste
Fig. 3 Questionnaire for the Biomass waste

and Discussion
Data collection by questionnaire method
waste which is valuable for the bio-energy production. Apart from the animal w
Vol. III 2014

energy production. Apart from the animal waste big

amount of plant derived waste is also collected in the Zoo as there is more than 1000 small
and big trees were present.
Anthropogenic waste was mainly consisting of non
plastic wrappers, bottles, polythene and food
the data collected from the questionnaire different alternative of energy sources can be opted
from the different type of waste materials. Thus the solid waste can be utilized in sustainable
way without harming the environment.
Fig. 4 Questionnaire
Table-1. Quantitative measurement of the Fresh animal waste in the Zoo premises
Name of the animals
Himalayan black bear
Sloth bear
Giraffe
Rhinoceros
Swamp dear
Barking dear
Hog deer
Samber deer
Spotted deer
Rabbit
Total
17
and big trees were present.
Anthropogenic waste was mainly consisting of non-biodegradable materials such as
plastic wrappers, bottles, polythene and food waste as a biodegradable waste. On the basis of
ing the environment.
Questionnaire for the anthropogenic s waste
Quantitative measurement of the Fresh animal waste in the Zoo premises
No. of animals Waste generated per day
3 1 Ibs3 (1 Kg. =2.2 Ibs )
3 1Ibs 3
2 5 Ibs 2
1 1000 Ibs1
57 3500 gm
23 3500 gm
30 3500 gm
15 3500 gm
198 3500 gm
10 2800 gm
342 482 kg/day
Vol. III 2014
biodegradable materials such as
waste as a biodegradable waste. On the basis of

s waste
Quantitative measurement of the Fresh animal waste in the Zoo premises
Waste generated per day
1 Ibs3 (1 Kg. =2.2 Ibs )
1Ibs 3
5 Ibs 2
1000 Ibs1
3500 gm
3500 gm
3500 gm
3500 gm
3500 gm
2800 gm
kg/day

18

Table-2.Quantitative measurement of plant derived waste inside the zoo premises
Sources Composition
Twigs Approx. 2 Kg/day
Dry leaves Approx. 3.5-4.0 Kg/day
Total Approximate 6.5-7.0 Kg/day

2.3.2 Potential alternative approaches for utilization of waste material
The suggestive measures based on data collected from the questionnaire can be taken
for the best use of solid waste generated inside the Zoo premises. These alternatives routes of
conversion may vary depending upon the type and composition of organic waste. Following
conversion routes are identified for the energy recovery from the waste collected from the
Zoo.
2.3.3 Animal and biomass waste
Animal and biomass waste having organic content are utilized as a feedstock in process
of biological conversion (Li et al., 2010) and thermo chemical conversion. Both waste vary
in chemical composition, physical form and quantity produced. The main factor involve the
variation of animal waste are
i. Digestive physiology of various species
ii. Composition and form of the diet
iii. Stage of growth and productivity of animal
On the other hand plant derived waste mainly differs in respect to their composition and
nature of degradability.

Fig. 4 Conversion platform for animal and biomass waste in to energy


19

a) Biological conversion
This process mainly involves biological treatment of animal and biomass waste by (i)
anaerobic digestion with full scale production of combustible biogas by using phototrophic
microorganism such as Algae, (ii) fermentative process for production of Bio-hydrogen and
(iii) Plant nutrient to support crop production.
Anaerobic digestion involves breakdown of complex organic materials and the biogas is
formed by following hydrolysis, acidogenesis, acetogenesis and methanogenesis (Van
Haandel and Van der Lubbe, 2007). The solid compound gets liquefy in acidogenesis and
converted in to acid, alcohol and volatile fatty acids.
Bio-hydrogen production is carried out by following three steps such as
photosynthetically by algae in two stage photosynthesis and H
2
production, photobiologically
by photofermentataive bacteria and by anaerobic fermentative bacteria.

Fig.5 Flow Diagram of Anaerobic Digestion Process and End Points of products

b) Thermo-chemical conversion
This is a high temperature chemical reforming process that breaks apart the bond of
organic matter and converts these intermediated in to char, syngas and highly oxygenated bio-
oil. The advantages of thermo-chemical conversion involve small footprints, efficient
nutrients recovery, no fugitive gas emission, short processing time on the order of minutes

20

and high temperature elimination of pathogens and pharmaceutically active compound
(Cantrell et al., 2007).
Pyrolysis, gasification and direct liquefaction these three processes are mainly identified
for the thermo-chemical conversion. Pyrolysis process mainly drive out in absence of oxygen
and it converts organic waste in to a mixture of char and volatile gases. Slow pyrolysis has
benefits of energy production and carbon credit generated from carbon sequestration (Tri et
al., 1996; Budenheim et al., 1994).
Direct liquefaction process includes hydrolysis of organic waste (lignocellulosic
materials) in to bio-oil. It proceeds in a pressurized environment (5-20 Mpa) and typically
occurs at low temperature.
This process convert organic portion of dry weight or biomass in to the minor by-
product and primarily non condensable permanent gases, CO, CO
2
, H
2
and low molecular
hydrocarbon gases with the help of air, oxygen or stem as a reaction medium (McKendry,
2002).
2.3.4 Anthropogenic waste
These wastes are produced due to human activities and mainly consist of fraction of
degradable and non-degradable wastes. These wastes may create serious hygienic problem as
it contains high amount of organic matter and pathogens. Vermicompsting can be applied for
the degradable waste, which enhance the soil fertility and increases crop yield. Non
biodegradable waste can be managed by incineration. This process involves burning of solid
waste in a properly designed furnace under suitable temperature and operating conditions.
Incineration provide reduction of waste to one tenth of their volume without producing
offensive gases this process not only help in the elimination of odor but also protect the wall
of incinerator.
2.3.5 Evaluation of the Biogas and Electricity production
The total amount of organic waste collected inside the zoo premises was found to be
490 kg/day (approx.). This amount of organic waste is capable to produce 25.48 m
3
/day
biogas and 25 kWh of electricity.

21

Fig. 6 Main thermo Chemical conversion Processes, their Intermediate products and
suggested End use
2.4 Conclusion
Waste management is highly important issue for Zoos as it poses as a good source of
various types of waste like animal waste, biomass and anthropogenic waste. These can be
managed using the 3 Rs (Recycling study guide, 1998) (Reduce, Reuse and Recycle of the
reduction principal of sustainability). The visitors of the zoo should be strictly prohibited from
throwing unwanted materials within the zoo premises in order to reduce the volume of waste
to great extent. From the present study area it has been found that of all the three wastes
(animal waste, biomass and anthropogenic waste) can be managed properly as per the
suggestive measures identified in the present study.

References
1. Budenheim D.L. and Wydeven T., 1994. Advances in Space Research (ISSN 0273-1
177), 14:113-123.
2. http://www.lucknowzoo.com/list_of_visitors1.html
3. Keri B. Cantrell, Thomas ucey, Kyoung S., Ro, Patrick G. hunt- Livestock waste to energy
generation opportunities, United states Department of Agriculture, ARS, coastal Plains soil,
Water and plant research center, 2611 W, lucas St. Florance, Sc29501, USA.
4. Li, R., Chen, S., Li, X., 2010. Biogas production from anaerobic co-digestion of food
waste with dairy manure in a two-phase digestion system. Appl. Biochem. Biotechnol.
160, 643654.

22

5. McKendry, P., 2002. Energy production from biomass (part 1): overview of biomass.
Bioresource Technol., 83: 3746
6. Morse, D. 1995. Environmental considerations of livestock producers. J. Anim. Sci.
73:27332740.
7. P. W. Gibson (Bienergy Organizers, Inc., Baltimore, MD), Baltimore Zoo Digester
Project: Final
8. Recycling Study Guide, Wisconsin Department of Natura Resources, January, 1988.
9. Report, DOE/R3/06058T1, U.S. Department of Energy, 1980.
10. Tri, T.O., Edeen, MA., and Henninger, D.L., SAE 26th International Conference on
Environmental Systems, Monterey, CA. Paper #961592, 8p. July 8-1 1, 1996.
11. V. N. Gunaseelan, Anaerobic Digestion of Biomass for Methane Production: A
Review, Biomass and Bioenergy 13, 83114 (1997).
12. Van Haandel, A.C., van der Lubbe, J., 2007. Handbook biological waste water treatment:
design and optimisation of activated sludge systems, rst ed. Quist, Leidschendam.
13. Williams, P.E.V. 1995. Animal production and European pollution problems. Anim. Feed
Sci. Technol. 32:106115.


23

CHAPTER 3
BIOPROSPECTION OF EUPHORBIA COTINIFOLIA FOR
BIOFUEL: CHROMATOGRAPHY STUDY
Punam Puri, Amita Mahajan, Anjana Bhatia

& Navjot Kaur

Abstract
Biodiesel have been receiving attention in recent years to overcome energy crisis. India is rich
in biodiversity and known for vast treasure of knowledge about use of plants for various
purposes. Many Euphorbiaceae species have been proposed as potential biofuel crops.
E.cotinifolia as one of the potential biofuel crop in future. In this research paper the focus will
be on the chemical constitutions that have been identified from Euphorbia cotinifolia species.
With different modes of chromatography, different lipids were determined. TLC in the
adsorption mode (silica gel), the principle application in lipid analysis is for the separation of
different lipid classes from plant tissues. It is a relatively easy matter to resolve each of the
main simple lipids from a tissue in one step, i.e. cholesterol esters, triglycerides, free fatty
acids, cholesterol and diacylglycerols, using mobile phases consisting of a mixture of hexane
and diethyl ether, with a little formic acid to ensure that the free acids migrate successfully.
The aim of the study was to investigate the biochemical constituents and thin layer
chromatography (TLC) of the ethanol and acetone extracts of Euphorbia cotinifolia.
Keywords: - Euphorbia cotinifolia, chromatography, lipids analysis, complex lipids.
3.1 Introduction
Renewable energy is an alternative solution for fossil fuel because it is clean and
environmentally safe. Professor Melvin Calvin (Calvin, 1977) revived the idea that
hydrocarbon-producing plants could be used as future oil and other chemical sources. He also
gave the energy farming concept. The plant families mainly Euphorbiaceae and
Asclepiadaceae were screened for assessing their suitability as a source of low molecular
weight (mw) and non-polar petroleum-like hydrocarbons. Air-dried plant materials were
successively extracted with acetone and benzene, and the extracts were analyzed
spectroscopically for yield of rubber, wax, glycerides, isoprenoides and other terpenoides. In
the Euphorbiaceae family, the genus Euphorbia comprises 2000 species ranging from annuals


24

to trees and is subdivided into many subgenera and sections. This family is one of the largest
families in the plant world. All contain latex and have unique flower structures (Barla et al.,
2006; Chaudhry et al., 2001; Jassbi et al., 2006). In the search for new plants with a high
potential for the production of chemicals and liquid fuels as alternative energy sources,
several Euphorbia species have been previously examined for possible economic utilisation
(Zarrouk and Cherif, 1983; Hemmers and Glz, 1986; Villalobos and Correal, 1992). This
genus has been investigated in view of different specialties, like more energy content, as
alternative source of hydrocarbons, laticifers, phytochemicals and systematic (4-10). Plants of
the genus Euphorbia are well known for their chemical diversity of their isoprenoid
constituents. An analytical screening program has been conducted by the USDA [1115] to
evaluate and identify plant species as source of high energy, easily extractable compounds
suitable for fuel, chemicals and petroleum-sparing chemical feedstock. Plant families that
yielded more than one promising species were Anacardiaceae, Asclepiadaceae,
Caprifoliaceae, Compositae, Eupforbiaceae and Labiaceae. This research emphasized on
phytochemical screening of latex extract of E.Cotinifolia by chromatography techniques.
Chromatography is a method for separating the components of a mixture by differential
adsorption between stationary phases and a mobile (moving) phase. Thin-layer
chromatography and column chromatography are different types of liquid chromatography. A
column (or other support for TLC, see below) holds the stationary phase and the mobile phase
carries the sample through it. Sample components that partition strongly into the stationary
phase spend a greater amount of time in the column and are separated from components that
stay predominantly in the mobile phase and pass through the column faster. As the
components elute from the column they can be quantified by a detector and/or collected for
further analysis. An analytical instrument can be combined with a separation method for on-
line analysis (16-18).
3.2 Objectives
The general project objective is to evaluate the potential of integrating biofuel raw
material production in wasteland, adapted technology and their implications on energy access,
ecological sustainability, food security, economic and social efforts for wellbeing for future
generation.The present proposal deals with a plant of Euphorbia cotinifolia which will be
taken up for fatty acid and hydrocarbon estimation. In this research paper main objectives
are:-

25

i) Extraction of oil and different lipids polar as well as non polar lipids with respective
solvents by column chromatography technique.
ii) To study phyto-chemical screening of plant extracts of E.cotinifolia.
iii) To find out the same compound in the known and unknown sample.
iv) Further analysis of biocrude oil (viscosity, density, cetane number, distillation range
water content, discussion of fuel properties of triglycerides etc.).
3.3 Methodology
3.3.1 Materials and methods
Biocrude of E. cotinifolia of acetone extract had determined through soxhlet extraction
method.
3.3.2 Phytochemical screening of plant extracts E.Cotinifolia
The latex acetone extract of E.Cotinifolia was analyzed by thin layer chromatography
(TLC) and column chromatography.
3.3.3 Method for Thin layer chromatography
TLC on silica gel G plates, on Silica Gel G plates (Loba chemie Pvt.Ltd.107,
Wodehouse road, Mumbai, India).Prepared silica gel slurry .Silica gel plate of layer thickness
0.25 mm kept in oven activation for 2-3 hrs at 70
0
C. Sample of acetone extract of
E.Cotinifolia and standard oleic acid were loaded and allowed to run in developing solvent
Petroleum Ether: Diethyl Ether: Acetone (7:3:0.1), spray reagent concentrated sulphuric acid,
p-anisaldehyde and glacial acetic acid (2:1:100). After development, the phytocompounds
plates were transferred into iodine chamber (resublimed iodine, Avarice laboratories Pvt. Ltd.,
India).
3.3.4 Method for Column chromatography
Weighed 5gm silica gel 60-120 (Avarice laboratories Pvt. Ltd., India) and made slurry
in water. Poured the gel into the column tightly so that no air spaces were left. This left a
space of 4-5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column
securely to a ring stand .5ml sample of 3 mg acetone extract was loaded in the silica gel filled
column. Once the sample was in the column, fresh eluting solvent was added to the top and
we were ready to begin the elution process. Only force the solvent to the very top of the silica:

26

do not let the silica go dry. Add fresh solvent as necessary. The following solvent system ran
in the column chromatography were:-
1) Hexane: petroleum ether (90:10) respectively.
2) Hexane: petroleum ether (50:50) resp.
3) Petroleum ether: diethyl ether (50:50) resp.
4) Diethyl ether: acetone (50:50) resp.
5) Acetone: methanol (50:50) resp.
6) Methanol 100%
The first solvent running in the column was hexane and petroleum ether in (90:10
ratios) respectively and the total quantity was 100 ml. There was appearance of 3 colored
bands in the column. The colored bands were travel down the column as the compound was
eluted. As soon as the colored compound began to elute, the collection beaker was changed.
The colored fractions were collected separately. Similarly all the above solvents ran in the
column and collected different fractions from the running column.
3.4 Results
Results of TLC
Fig. 1 Biocrude of plant extract spp.
E.Cotinifolia

Fig. 2 Performing TLC

27

Fig 3. Results of TLC Fig 4. Results of TLC

Results of column chromatography

Fig. 5. Shows loaded sample in coloumn
chromatography
Fig 6. Shows band formation by using
different solvents running in the column
chromatography.

28

Fig 7. Shows different colored band
formation by using solvent hexane:
petroleum ether (90:10) respectively.

Fig 8. Shows band is travelling down in the
column.

Fig 9. Shows oil sample was obtained by
running first solvent i.e. hexane: petroleum
ether (90:10)

Fig 10. Shows five fractions in
eppendorf vials were obtained from
respective solvents.


29

3.5 Discussion
The results obtained from TLC and column chromatographies were good. In TLC
standard sample was oleic acid travel with acetone extract of biocrude of E. Cotinifolia.in the
results arrows shows that there was a same compound in E. Cotinifolia which were present in
the known sample. In fig. 4, TLC results shows that the standard oleic ran with oil sample
extracted during column chromatography. The good results were obtained.
In column chromatography the results were also good and surprising we got oil in very
small fractions from 3 mg sample during running the solvent hexane and petroleum ether
(90:10) and other five fractions were collected from respective solvents. In future we will do
the triglycerides analyses (viscosity, density, cetane number, distillation range water content
discussion of fuel properties of triglycerides etc.). The sample will be forward to SAIF Centre
Chandigarh for IR, GC/MS AND NMR data.
3.6 Conclusion
The bio-energy system makes a significant contribution to the worlds growing energy
needs. The renewable sources would only be able to compete with the fossil fuel resources, if
special plant crops containing energy-producing, hydrocarbon-like material are breed and
cultivated. A great advantage of utilization of such plants is by replacing the current use of the
traditional food crops for fuel production and providing the biodiesel industry with a more
consistent "green" supply. Large scale experiments would be required to analyses of the
different classes of secondary metabolites isolated from this plant. In this paper more
emphasize on phyto-chemical analysis of compounds and extraction of components from
different running solvents system and all these extractions further analysed by different
techniques. Hope this will be the one of the future petrocrop in the world.

References
1. Calvin M. Chem Eng News 1978;20:316.
2. Barla A, Biraman H, Kultur S, et al. Secondary metabolites from Euphorbia
helioscopia and their Vasodepressor activity.Turk J Chem, 30, 2006, 325- 332.
3. Zarrouk M. and Cherif A. (1983), Lipid contents of Biol. Plant. 42, 417422.
4. Kalita D, Saikia CN (2004). Chemical constituents and energy content of some latex
bearing plants, Bioresource Technology 92 (3), 219-227.

30

5. Monacelli B, Valletta A, Rascio N, Moro I, Pasqua G (2005). Laticifers in
Camptotheca acuminata Decne: distribution and structure. Protoplasma 226 (3-4),
155-161.
6. Bruni, R, Muzzoli, M, Ballero, M, Loi, MC, Fantin,0G, Poli, F, Sacchetti, G. (2004).
Tocopherols,Fatty Acid acids and sterols in seeds of four Sardinian wild
7. Mallavadhani UV, Satyanarayana KVS, Mahapatra A, et al. (2006). Development of
diagnostic, microscopic and chemical markers of some Euphorbia latexes Journal of
Integratve Plant Biology 48 (9), 1115-1121.
8. Shi HM, Williams ID, Sung HHY, et al. (2005). Cytotoxic diterpenoids from the roots
of Euphorbia ebracteolata, Planta Medca 71 (4), 349-354.
9. Jiao, W, Mao, ZH; Dong, WW, et al. (). Euphorbia factor L-8: a diterpenoid from the
seeds of Euphorbia lathyris. Acta Crystallographica Section E-Structure Reports
Online, 64, (03).
10. Suarez-Cervera M, Gillespie L, Arcalis E, L Thomas A., Lobraeau Callen D.,
Seoane Camba JA., (2001). Taxonomic significance of sporoderm structure in pollen
of Euphorbiaceae: Tribes Plukenetieae and Euphorbiceae. Grana 40 (1-2), 78-104.
11. Abbott TP, Patterson RE, Tjark LW, Palmer DM, Bogby MO. Econ Bot 1990;44:278
84.
12. Bagby MO, Buchanan RA, Otey FH. In: Klass DL, editor. Biomass as a non fossil fuel
source. ACS Symposium. Series, vol. 144. 1981. p. 12536.
13. Campbell TA. Econ Bot 1983; 37:17480.
14. Carr ME, Bagby MO, Roth WB. J Amer Oil Chem Soc 1986;63:14604.
15. Seiler GJ, Carr ME, Bagby MO. Econ Bot 1991;45:414.
16. Archer J. P. Martin (1952). "The development of partition chromatography". Nobel
Lecture, December 12, 1952. Nobel Lectures, Chemistry 1942-1962, Elsevier
Publishing Company, Amsterdam, 1964.
17. Ettre, L. S. (2001). "The Predawn of Paper Chromatography". Chromatographia, vol.
54, pp. 409-414.
18. Frederick Sanger (1988). "Sequences, Sequences, and Sequences". Annual Review of
Biochemistry, vol. 57 (1988), pp. 1-28


31

CHAPTER 4
COST EFFECTIVE ELECTRICAL POWER GENERATION IN
PUNJAB USING AGRICUTURAL BIOMASS
Suman

Abstract
Energy is the key input to drive and improve the life cycle. The impact of the traditional fossil
fuels on our environment and the fact that these are fast depleting sources of energy, have
encouraged the need to find alternative energy sources to fossil fuels. Biomass can be burned
for fuel by itself or co-fired with other fuels. But in recent years Biomass and Coal co-fired
based systems are receiving more attention due to high Power Generation Efficiency and
reduced Green House Gas (GHG) emissions. This paper critically analyzes the scope,
potential and implementation of agricultural -Biomass conversion to Energy in Punjab
context. Brief descriptions of potential conversion routes have been included, with their
possible and existing scope of implementation. As far as possible, the most recent statistical
data have been reported from various sources. The discussion reveals that a large potential
exists for the Biomass feed-stocks from the various kinds of waste Biomass. The analysis to
identify irreversibility and the ways to improve the performance of Power Generation systems
is discussed. The Energy generated from various kinds of Biomass products is analyzed and
its role to improve the Power Generation systems is also presented.
Keywords: Biomass, Electrical energy, Efficiency, Greenhouse gas (GHG), Green Economy,
Power Generation
4.1 Introduction
In recent years, the World is facing Energy crisis, Economic, Green & clean
Environmental problems. A lot of research efforts are put to find economically viable and
sustainable energy resources to reduce this energy crisis with green and clean environment.
With growing population, improvement in the living standard of the humanity,
industrialization of the developing countries, the Global demand for energy is expected to
increase. India rank fifth in the world in total energy consumption (with installed capacity
228.722 GW up to September 2013). Coming to Power production in the country, India ranks


32

sixth in the world with increased installed power capacity from 1362MWh to 855.3 billion
kWh (up to 2012) since independence [6]. This achievement is impressive but not sufficient.
The country still encounters peak and energy shortage of 9 % & -8.7 % respectively (up to
2013). The major sources which meet the energy requirement of India are coal and oil. The
use of these fuels is a problem because of the reasons: a) The natural formation of Coal and
Oil is a very slow process which takes long time .b) Emission of Green house gases. c) These
are fast depleting sources of Energy. Moreover, India is dependent on the imports for Oil
requirements. In 200405, 72% of Indias total oil consumption was dependent on the imports
[2]. This figure reached to 76.5% during 200910, 78% for 2001011, and the tentative figure
for 201112 is 80.5% [3]. These imports are increasing year after year with the growing
economy of the country and contribute in continuous increase of the import bills. By 2025, it
will be importing 90% of its crude oil from OPEC countries. Therefore, Utilization of
renewable energy sources is one of the best ways to meet the objectives as : a) These are the
energy sources that will never run out. b) These sources are Environmental friendly means
reduce Green house effect and provide clean Environment. c) Social cost benefits. The
major Renewable sources of Energy available freely are Solar energy, Wind energy, Small
Hydropower, Biomass, Biogas, and Energy recovery from Municipal and Industrial wastes.
4.2 Status of Bio-energy Resources in Punjab
Indias energy basket has a mix of all the resources available including renewable.
Biomass contributes as the worlds fourth largest energy source up to 14% and in developing
countries it can be as high as 35% of the primary energy. Punjab the Grain Bowl of India is
the major agriculture state of the country. Agricultural biomass has immense potential for
power production in Punjab. Punjab has made tremendous progress not only in the agriculture
sector but in the industrial, transport and household sectors. This has increased energy
demand significantly. This state does not have its own resources of conventional fuels such as
coal, petroleum products for electricity energy. The state has to depend on neighboring states
for petroleum products and on the far-off states for coal. But the state has plenty of renewable
energy sources, such as biomass, wind and solar energy, which can be exploited to provide
sustainable energy base for socio-economic development. Table 1: shows the various type of
biomass available in Punjab [3].
Punjab "Granary of India is historically considered to be one of the most fertile areas
on Earth. The region is ideal for growing rice, wheat, cotton, sugarcane, maize and

33

vegetables covering nearly 1.5 percent of India's land. Today the state produces nearly Indias
11% rice, 22% wheat, 18 % cotton and 3 % sugarcane .In worldwide terms; this represents
1/30th or 3% of the world's production of these crops, so Punjab produces worlds 1% of
cotton, 2% of its wheat, 2% rice and 0.3% of sugarcane. Table2: Shows production of major
crops during the recent years with area in Punjab & it is clear that from the last few years
production of major crops increases which result in increase in the bio waste product [5].
Table 1: Type of Biomass
S.No. Type of biomass Name of crop
1 Straw Wheat ,Paddy, Barley, Pulses
2 Stalk Cotton, Maize, Rapeseed &mustard
3 Bagasse Tops & leaves Sugarcane Sugarcane
4 Cobs Maize
5 Husk Paddy

Table-2: Production of Major Crops during the Recent Years

Year
Type of
Bio-
Mass
2009- 10 2010-11 2011-12 2012-13 2013-14(E)

Area
(lac
ha.)
Production
(lac MT)
Area
(lac
ha.)
Production
(lac MT)
Area
(lac
ha.)
Production
(lac MT)
Area
(lac
ha.)
Production
(lac MT)
Area
(lac
ha.)
Production
(lac MT)
Rice 28.02 112.36 28.31 108.37 28.18 105.42 28.45 113.69 27.50 110.00
Sugar
cane
0.60 40.56 0.70 49.04 0.80 56.53 0.82 56.73 0.95 66.50
Wheat 34.02 151.69 35.10 164.72 32.03 125 34.52 140.60 35 161.02
cotton 5.11 20.06 4.83 18.22 5.15 16.21 4.81 16.44 5.20 19.58
Maize 1.39 4.75 1.33 4.91 1.26 5.02 1.29 4.71 1.50 5.40


34

On the basis of survey of ratios of various major crop residues for the year 2012-2013,
the net production of residue could be around 481 Lac MT ,as described in greater detail in
Table 3.
Table -3: Estimation of Biomass Production in Punjab (Crop Wise Data)
Crop Main Crop Production
(Lac MT)
Type of
Residue
Crop to
Residue
ratio
Residue
Quantity
(Lac MT)
Rice 113.69 Husk 0.3 34.107
Straw 1.3 147.797
Wheat 140.60 Straw 1.5 210.9
Sugarcane 56.73 Bagasse 0.3 17.019
Tops & leaves 0.09 5.105
Cotton 16.44 Stalk 3.5 57.54
Gin Waste 0.1 1.644
Maize 4.71 Stalk+Cobs 1.5 7.065
Grand Total 481.177

Further studies indicate that about 15-20% of the agriculture residue is available for
power generation rest is used for other purposes such as cooking & cattle feed. So we have
nearly 100 Lac MT crop residue is available for Power Generation [1].
4.3 Power Consumption in Punjab
The Total Demand of Electricity in Punjab is 48724 MU. The availability of Energy in
Punjab is approximately 46119 MU, facing energy shortage of 5.2%. Total energy in Punjab
state is provided by the PSPCL with its own Thermal Plants and Hydro Plants. Electricity
demand of Punjab will vary with changes in weather. On an average, the demand of power in
Punjab will vary between 1,039LU to 2,072LU while the availability of power will also vary
between 873LU to 1,584LU during different months of the year [4].
The variation in annual demand and energy requirement for the year April 2012 to
March 2013 are given in Table 4. The common pool projects are the Bhakra Nangal Complex,
the Dehar Power Plant & the Pong Power Plant. Punjab shares about 51% of the Power
generated from the Bhakra Nangal Complex. 48% from the Power generated at the Pong

35

Project. By including this share of generation Punjab is still deficit with 2600 MU Power.
According to PSPCL estimates, the power-supply gap will vary between 4% to 31% the entire
year.
Table -4: Month Wise Power Supply Position of Punjab in 2012-13
Table -5: Anticipated Power Supply Position in the Punjab during 2013-14
Energy Peak
Requirement
(MU)
Availability
(MU)
Surplus(+)/Deficit(-)
(MU)
Requirement
(MU)
Availability
(MU)
Surplus(+)/Deficit
(-)
(MU)
50850 40819 -10031 -19.7 12200 9075 -3125 -25.6

Year Demand Energy
Peak
Demand
Demand
Met
Surplus
(+) /
Deficit (-)
(%)
Surplus
/Deficit
Energy
requirement
Availability Surplus(
+)
/Deficit
(-)
(%)
Surplus/
Deficit
April -12 6391 5246 -1145 -17.9 3031 2948 -83 -2.7
May -12 7236 6091 -1145 -15.8 3763 3651 -112 -3.0
June -12 10474 8452 -2022 -19.3 5437 5053 -384 -7.1
July -12 11520 8073 -3447 -29.9 6611 5867 -744 -11.3
Aug-12 9114 8751 -363 -4.0 5923 5374 -549 -9.3
Sep-12 8147 8147 0 0.0 4745 4622 -123 -2.6
Oct-12 8441 6860 -1581 -18.7 3813 3671 -142 -3.7
Nov-12 5676 4502 -1174 -20.7 3040 2941 -99 -3.3
Dec-12 5336 5336 0 0.0 2517 2362 -155 -6.2
Jan-13 5797 5197 -600 -10.4 3055 2938 -117 -3.8
Feb-13 5197 5018 -179 -3.4 3917 3844 -73 -1.9
Mar -13 5264 5264 0 0.0 2872 2848 -24 -0.8

36

The studies carried out for formulating the anticipated power supply in Punjab for the
next year 2013-14, as shown in Table 5. indicate that there would be Energy shortage of
19.7% and Peak shortage Of 25.6% in Punjab.
4.4 Existing Technologies for Biomass Conversion
Due to technological developments and cost reductions, renewable solar, hydro, wind
and biomass energy are gaining momentum across the globe. There are a variety of processes
and technologies that convert biomass into heat, steam, electricity, and other types of fuel &
products. Some of them are depicted in Table 6 [2].
Table 6: Waste Agricultural Biomass to Energy Technology
S.No Type of
Technology
Examples of Types of
Waste Handled
Byproducts Applications
1 Direct
Combustion
Crop residues such as
wheat straw, rice straw,
rice husk, Bagasse
Carbon Dioxide, Water
& Heat
Power Generation ,
Heating , Cooking
2 Gasification Crop residues such as
rice husk
Syngas, Heat, Some
CO
2
and H
2
O
Power Generation ,
Heating , Cooking ,
Transportation
3 Pyrolysis Crop residues such as
rice husk
Bio- Ethanol Power Generation ,
Transportation
4 Fermentation Sugarcane & starch
substrates like wheat,
maize, sugar beet
Solids (charcoal),
Liquids (Pyrolysis oils)
and a mix of
Combustible gases
Power Generation ,
Transportation ,
Heating
5 Esterification Rape-seed Glycerine and
RME(RapeMethyl
Ester)
Power Generation ,
Transportation

Above mentioned technologies which are already installed must be upgraded keeping
requirements in mind while those which are presently running Global like Fermentation,
Esterification. Brazil recovered from oil crisis because of development of Cars powered by

37

100% Ethanol or Petrol or combination of both, such technologies are awaited to be modeled
for Punjabs Energy Policy.
4.5 Biomass as a Coal Substitute
Biomass Power technologies compete in niche applications as well as in direct
competition with Conventional Electricity sources in Centralized Electricity supply. In large
scale grid based applications, cost is the primary determinant of competitiveness. A power
plant with the capability of producing 8MW of electricity could cost up to 40 crore INR.
While annual maintenance (done for 1week twice a year) costs 50 Lakhs INR. Variable
expenses are related to price of Biomass cost (approx. 3500-5500 INR per metric ton) is
highly variable, depending upon the source, location etc while other expenses include
manpower wages [1].
Operating life of Biomass Power plant lies between 25 to 30 years. since the cost of
setting a biomass plant is high as compared to thermal plant but it has many advantages over
thermal power plant such as--a) Biofuels can be transported and store and allow for heat and
power generation on demand. b) The energy balance of biomass plants indicates that biomass
energy is 10 to 30 times greater than the energy input for fuel production and transport. c)
Accessibility in rural areas where commercial fuels and centralized electric grid are not
available. d) Greater employment for local populations. 5) Restoration of deforested and
degraded lands by energy plantations. e) Near-zero fuel costs (paid in local currency),
commercial use of a waste product, decentralized supply and increased fuel efficiency leading
to an increase in the economic. f) Cost of electricity per unit from biomass power plant is
lower than coal plan.
4.6 Environmental Criteria
Expanding the share of Renewable Energy in its Energy mix is one of the key pillars of
Indias low-carbon development strategy. The Biomass fuels are more suitable & promising
than coal due to its low carbon, sulphur and nitrogen content as depicted in Table 7. Since
CO
2
and acidification of SO
2
& NO
2
are primarily responsible for global warming & coal
contain maximum value of these elements (Carbon, Nitrogen & Sulphur) as compared to
other Biomass.[1] So coal contributes more towards the Global warming. Moreover
depending upon the content of Carbon & Sulphur there is Environmental taxes (High Tax &
Low Tax). High tax scenario with $50 per ton of carbon tax and $400 per ton of Sulphur
dioxide tax. Low tax scenario with $25 per ton of carbon tax and $200 per ton of Sulphur

38

dioxide tax. The cost of delivered Electricity under the Low tax and High tax cases for Coal
Power increases by 1.4 and 2.8 cents/kWh, respectively.
Table -7: Estimated Analysis:(Where C-carbon, H-hydrogen,O-oxygen, N-nitrogen,S-
sulphur)
C H O N S Ash content (%)
Coal 75 5 8 1.5 0.5 10
Rice husk 38.35 5.08 36.24 0.56 0.16 14.9
Wheat straw 48.54 5.73 40.71 0.81 0.17 8.5
Rice straw 43.36 5.44 39.03 0.87 0.10 19.2
Maize(stalk+cob) 49 4.9 - 0.6 -
Bagasse(dry basis) 49 6.5 42.7 0.2 0.1 1.5
Cotton stalk 51 4.9 43.87 1.00 - 6.68

Further Biomass fuels have less reactive character as compared to Coal & Cogeneration
applications in agriculture processing industries typically achieve fuel efficiency of 40 to 45%
compared to 30% efficiency of the conventional technologies . Although the conversion of
Biomass to Electricity in itself does not emit more CO
2
than is captured by the Biomass
through photosynthesis.
This analysis suggests that under, these advantages, together with more efficient and
versatile Biomass Electricity Generation with Modern Technologies, have led to the transition
of re-emergence of Biomass as a competitive and Sustainable Energy option in the Future
Energy Scenarios.
4.7 Conclusion
Significant conclusions of this paper are as follows:
a) Punjab has abundant capacity to produce reliable, price competitive and ecologically
sustainable Bio-energy to meet the energy demand of domestic and commercial sector. A
number of such Power Generation project have not only solved the rural electrification
problem for the remote villages, where infrastructural costs could have been quite high
for conventional electrification, but also the power generation cost has also been
relatively low.

39

b) In Biomass, Carbon, Nitrogen & Sulphur contents are low, which favours in lesser or
Zero Global warming. Moreover their quantity decides the Environmental tax, so for
biofuel we have to pay less tax as compared to Coal. Further, it is analyzed that
replacement of each KWh of Coal based electricity by Biomass-based electricity is
likely to reduce CO
2
by 1Kg.
c) Biomass based decentralized generation is likely to generate direct or indirect & skilled
or unskilled employment to about 84 people in rural areas.
d) Biomass based power plants helps in reducing the bio-waste disposal problem in effective
way.
e) Cogeneration applications in agriculture processing industries achieve fuel efficiency up
to 40 to 45% as compared to 30% efficiency of the conventional technologies.
Apart from having so many Economical & Environmental benefits, it also opens a new
door for future innovations in our Country.

Refereences
1. Buljit Buragohain, Pinkeswar Mahanta & Vijayanand S. Moholkal (2010) Biomass for
decentralized power generation :The India perspective . Science Direct ,14:73-92
2. Sara Schuman & Alvin Lin(2012)Chinas Renewable Energy Law & its impact on
renewable power in China. Energy policy ,51:89-101
3. Jagtar Singh , B.S Panesar & S.K. Sharma (2008) Energy Potential through
agricultural biomass using geographical information system-A case study in Punjab.
Science Direct , 14:301-307
4. Load Generation Balance report 2013-2014 by Central Electricity Authority
5. Department of Agricultural Punjab (2013) National Conference on Agricultural for
Kharif Compaign.
6. K.S.Sidhu .(2006) Director of Punjab state Electricity Board. Non Conventional
energy resources


40

CHAPTER 5
DEVELOPMENT OF QUALITY TESTING
METHODOLOGIES OF FUEL BRIQUETTES
Madhurjya Saikia and Bichitra Bikash

Abstract
Biomass material such as rice straw, banana leaves and teak leaves (Tectona grandis) could
be densified by means of wet briquetting process. Wet briquetting is a process of
decomposing biomass material up to a desired level under controlled environment in order to
pressurize to wet briquettes at a lower pressure. Upon drying these wet briquettes could be
used as solid fuels. This study is aimed at to develop methodologies to measure quality and
handling characteristics of these briquettes and burning characteristics as well.
Key words: Biomass, briquettes, durability, shear strength, impact resistance.
5.1 Introduction
India produces yearly a large amount of agro residue such as rice straw, rice husk,
coffee husk, jute stick, coir pith, bagasse, groundnut shells etc. Some part of this agro residue
is used as animal fodder. A large amount of agro residue is left on the paddy fields to be burnt
or decomposed [Ponnamperuma et al., 1983]. Both ways are means of pollution to
environment, as field burning of agro residue emits a lot of GHGs to environment and
decomposition of the agro residue too produces methane gas which is considered of the GHGs
[Campbell et al., 2002; Sokhansanj et al., 2006]. Instead of letting these agro residues to be
burnt or decomposed, these could be converted to densified forms by briquetting method
[Stanely, 2003]. This will mitigate the problems of pollution while at the same time we would
be successful to trap this energy resource for industrial purposes. Briquettes are far better to
handle rather than loose biomass.
Biomass briquetting is the densification of loose biomass material to produce compact
solid composites of different sizes with the application of pressure. Three different types of
densification technologies are currently in use [Saikia and Baruah, 2013]. The first, called
pyrolizing technology relies on partial pyrolysis of biomass, which is added with binder and


41

then made into briquettes by casting and pressing. The second technology is direct extrusion
type, where the biomass is dried and directly compacted with high heat and pressure (Grover
and Mishra, 1994). The last type is called wet briquetting in which decomposition is used in
order to breakdown the fibers. On pressing and drying, briquettes are ready for direct burning
or gasification. These briquettes are also known as fuel briquettes [Stanley, 2003, Chou et al.,
2010, Saikia et al., 2013].
These fuel briquettes are the newest edition of briquettes manufactured at low pressure
unlike the others. As these briquettes are produced at relatively low pressure, a series of
quality test such as durability, compressive strength, shear strength, impact test and
combustion tests are needed to be done so that they could be more competitive with existing
types.
5.2 Parameters of Quality Assessment
Durability, shear strength and impact resistance of briquettes are the basic parameters to
assess the quality of briquettes in terms of handling and transportability of fuel briquettes
(Grover and Mishra, 1994). .
5.2.1 Durability
Durability of briquettes gives the mechanical handling of the solid fuel (Chou, 2009).
This is measured by standard procedure ASAE S269.4 [Kaylyan et al]. To measure durability,
100 g of sample is taken and sample is tumbled at 50 rpm for 10 minutes in a dust tight
enclosure of size 300mm300mm. Metal cloth of aperture size 4mm is used to retain
crumbled briquettes after tumbling.
Briquettes durability index in % given by=
WcIght oI BrIqucttc sampIc IcIt aItcr tumbIIng,g
OrIgInaI wcIght oI brIqucttc sampIc,g
100
Durability test set up: Working principle: The test set up is fabricated according to
specification of ASAE S269.4 (Temmerman et al., 2006). The set up consists of a box 300
mm 300 mm 125 mm mounted on a frame. The box can be rotated by wooden handle or a
motor at 50 rpm for 10 minutes which simulates the probable conditions of briquettes under
transport by truck or conveyer belt into furnace. Figure below shows a durability measuring
test set up made according to specification.
5.2.2 Shear Strength Test
To measure shear strength, a simple test is done. Briquette is sheared between two
planes and shear force developed is the shear strength of briquette (Saikia and Baruah, 2013).

42

Fig. 1 Durability test set up

Fig. 2 Shear strength test set up

Shear strength test set up: To measure shear strength, shearing test set up has been
fabricated. The instrument consists of three wooden plates. The middle plate with a central
cylindrical hole of 45 mm diameter and 30 mm thickness slides over the bottom plate. In the
top plate, a cylindrical hole of same diameter as that of moving plate with 20 mm thickness is
being provided in such a way that it coincides with the one that is provided in the movable
plate when this plate is fully inserted between top and non moving bottom plate. The movable
plate is connected to dead weights.
Shear strength, kPa =
2F
D
2

Where F= Force causing shear in briquette, kN
D= diameter of briquette, m

5.2.3 Impact Resistance Test
This test simulates the forces encountered during emptying of densified products from
trucks onto ground, or from chutes into bins. Drop tests can be used to determine the safe
height of briquette production during mass production (Debdoubi A. et al., 2005). ASTM
D440-86 method is used to determine impact resistance index (Kaliyan N. and Morey R.V.,
2008).. In the drop test, briquettes are dropped twice from a height 1.83 m onto a concrete
floor. An impact resistance index (IRI) is calculated.
IRI =
100N
n

Where, N= Number of drops, n= Total number of pieces. The upper limit of IRI is 200 which
would result if the briquettes are not broken even after dropping twice.

43

5.3 Parameters of Combustion Characteristics of Briquettes
Proximate analysis and combustion rates of briquettes were done to assess effectiveness
of briquettes as cooking fuel.
5.3.1 Proximate Analysis
Moisture Content: The moisture content is determined according to the method described in
the Forestry Hand Book (Wenger, 1984). 10 g of sample is taken immediately upon sampling
and then air dried. This air- dried sample is taken immediately in an aluminum moisture box
and kept in an oven heated at 105 3 C until constant weight is obtained. The difference of
the oven dry weight of the sample and the fresh weight of the sample is used to determine the
percentage of moisture content as follows:
Moisture content, % =
Frcsh wcIght-Ovcn dry wcIght
Frcsh wcIght
100
Ash content: For determination of ash content, ASTM Test No. D-271-48 is followed. At
first, an empty 25 ml. silica crucible as well as the sample is heated in a moisture oven.
Sample is weighed accurately to 2 g. The sample in the crucible is kept in muffle furnace with
the lid cover. Muffle furnace temperature is set at 550 50C and kept for 6 hours. After 6
hours of burning crucible is removed from the furnace and placed in a desiccators and
weighed accurately. Percentage of ash content was calculated as follows:
Ash content, % =
Weight of ash
Weight of sample
100
Volatile matter: Volatile matter of samples is determined by ASTM Test No. D- 271- 48. A
clean platinum crucible of 10 ml. capacity is taken and heated in a furnace at 950C for 2
minutes and cooled in desiccators for 15 minutes. Crucible weighed to nearest 0.1 mg.
Sample filled crucible is weighed and heated in a furnace at 950C for 2 minutes. After
volatile matter escaped, the crucible is removed from furnace and cooled in air 2 to 5 minutes
and then in desiccators for 15 minutes. The percentage of weight loss of the samples is
reported as volatile matter as follows:
VM=
Wt. loss of dry sample
Net wt.of dry sample
100
VM=
Wt. loss of wet sample 100
Gross wt. of wet sample
100- percent moisture
100 (Wet samples)
Determination of fixed carbon content
Fixed carbon (FC) is determined by ASTM Test No. D- 271- 48
FC (on dry basis) = 100- (% of volatile matter +% of ash)

44

FC (on wet basis) = 100 (% of volatile matter + % of ash + % of moisture)
5.3.2 Calorific Value
Calorific value is determined by using Automatic Microprocessor Calorimeter (5E-
AC/ML) (make- Changsha Kaiyuan, China) (Saikia M. and Baruah D., 2013). The briquettes
material is nicely ground and pellets of 10 mm diameter are prepared from grounded material.
The pellets are placed inside a crucible so that tungsten wire touches the pellet. The calorific
value is analyzed by the Automatic Microprocessor Calorimeter or auto bomb calorimeter.
The system has facilities such as water cycling system, automatic water feeding, temperature
adjusting with a PT 500 temperature sensor, zero drift bridge temperature circuit to ensure
that temperature resolutions reach 0.0001 , auto-diagnose, remote data transfer and auto-
weight entry.

Fig. 3 Auto bomb calorimeter for proximate analysis
5.3.3 Combustion Rate
Combustion rate or burning rate is the mass loss per unit time. The briquettes are dried
at 105C so that it does not affect on combustion or burning. Dried briquette is placed on a
steel wire mesh grid resting on three supports allowing free flow of air

(Chaney J. O.et al. ,
2010) Now the whole system is placed on mass balance. Briquette is ignited from top and
mass loss data is taken at an interval of 30 seconds.


45

Fig. 4 Combustion rate determination set up for briquettes
5.4 Conclusion
With the help of this study we aim to standardize the practice of briquette making for
the commercial purpose. Generally, there are very few literatures on the standardization of
quality testing methodologies of briquettes and burning characteristics as well. Durability,
shear strength and impact resistance of briquettes could be determined easily as discussed in
the section II in order to assess the quality of briquettes in terms of handling and
transportability. Higher the durability, impact resistance and shear strength, higher will be the
handling characteristics. But we need to reach at an optimum value of these indexes in order
to produce quality briquettes at a cost effective way as quality always adds cost to production.
Similarly, we can also asses the burning rates of briquettes in room condition as discussed in
the section III. This generally helps to build a rough idea of performance of briquettes in
actual condition. Moreover, with the knowledge of burning rates, we can further manipulate
parameters of briquette manufacturing such a density and porosity of briquettes in order to
obtain a desired level of burning rate. Apart from that proximate analysis and calorific value
tests will so help us to give answer to some of the questions regarding the fuel such as
whether it produces too much harmful fly ash and unwanted gases which are general indoor
air pollutants many households and effectiveness of the fuel in terms of heat value. A fuel
without heat value would be useless as a lot of fuel will be needed during use for its lower
heat value.

References
1. Stanley R. (2003). Fuel Briquette making, Legacy Foundation.
Steel wire mesh
Three leg support
Electronic mass
balance
Briquette
Stop watch

46

2. Kaliyan N. and Morey R.V. (2008). Factors affecting strength and durability of
densified of biomass products, Biomass and Bioenergy, Vol 33, pg 337-359.
3. Wenger (1984). Forestry Hand Book.
4. Chaney J. O., Clifford M. J., Wilson R. An experimental study of the combustion
characteristics of low-density biomass briquettes. Biomass magazine 2010, Vol 1.
5. Grover P.D. and Mishra S.K. (1994). Development of an appropriate biomass
briquetting technology suitable for production and use in developing countries Energy
for Sustainable Development, Vol 1.
6. Faborod M. 0 (1987).Optimizing the Compression/Briquetting of Fibrous Agricultural
materials, Journal of agricultural. Engineering Resources, Vol 38, pg 245-262.
7. Chen L. (2010). The development of agro-residue densified fuel in China based on
energetics analysis, Waste Management, Vol 30, pg 808813.
8. Suramaythangkoor T. and Gheewala S.H. (2010). Potential alternatives of heat and
power technology application using rice straw in Thailand, Applied Energy, Vol 87, pg
128133.
9. Chou CS, Lin S.H., Peng C.C. and Lu W.C. (2010).The optimum conditions for preparing
solid fuel briquette of rice straw by a piston-mold process using the Taguchi method,
Fuel Processing Technology, Vol 90, pg 10411046.
10. Mania S., Tabil L.G. and Sokhansanj S. (2006). Effects of compressive force, particle
size and moisture content on mechanical properties of biomass pellets from grasses,
Biomass and Bioenergy, Vol 30, pg 648654.
11. Chou C. S. (2009). Preparation and characterization of solid biomass fuel made from
rice straw and rice bran, Fuel Processing Technology, Vol 90, pg 980987.
12. Singh R.N. (2004). Equilibrium moisture content of biomass briquettes, Biomass and
Bioenergy, Vol 26, pg 251 253.
13. Demirba A. (1997). Evaluation of biomass residue briquetting waste paper and wheat
straw mixtures, Fuel Processing Technology Vol 55, pg 175183.
14. Parikh Jigisha, Channiwala S.A. and Ghosal G.K. (2005). A correlation for calculating
HHV from proximate analysis of solid fuels, Fuel, Vol 84 (2005), pg 487494.
15. Bamgboye I. and Bolufawi S.J. (2009). Physical Characteristics of Briquettes from
Guinea Corn (sorghum bi-color) Residue, Agricultural Engineering International: the
CIGR Ejournal.
16. Husain Z., Zainac Z. and Abdullah Z. (2002). Briquetting of palm Fiber and shell from
the processing of palm nuts to palm oil, Biomass and Bioenergy, Vol 22, pg 505 509.

47

17. Kumar N., Patel K., Kumar R.N. and Bhoi1 R.K. (2009). An assessment of Indian fuel
wood with regards to properties and environmental impact, Asian Journal on Energy
and Environment, Vol 10(02), pg 99-107
18. Saikia M. and Baruah D. (2013). Analysis of Physical Properties of Biomass Briquettes
Prepared by Wet Briquetting Method, International Journal of Engineering Research
and Development Volume 6, Issue 5 (March 2013), PP. 12-14.
19. Bikash B. Bhowmik R. and Saikia M. (2013). Challenges of Wet Briquetting from
Locally Available Biomass with Special Reference to Assam, International Journal of
Computational Engineering Research , Vol, 03, Issue, 7.

Recent Advances in Bioenergy Research Vol. III 2014

48

Part II
Thermochemical Conversion


49

CHAPTER 6
THERMAL AND CATALYTIC CRACKING OF NON-EDIBLE
OIL SEEDS TO LIQUID FUEL
Krushna Prasad Shadangi and Kaustubha Mohanty

Abstract
Verities of edible and non-edible oil containing seeds are available in nature. Out of these, the
seeds containing edible oil habitually fulfill our food requirements. Hence edible seeds should
not be use as a feedstock for production of fuel as it will directly affect the food chain. The
seeds that are less edible or totally non-edible can be a considered as a feed stock for pyrolysis
for the production of bio-oil. The quality and yield of pyrolytic product depends on biomass
composition which include cellulose, hemicelluloses and lignin, oil and extractives. Higher
amount of cellulose and extractive content enhances the yield of oil whereas the presence of
lignin results in char during pyrolysis. The product yield and its quality is a function of
reactor type, physical and chemical properties of biomass, final pyrolysis temperature, gas
residence time in the reactor, pressure, ambient gas composition and catalyst types. The high
viscosity, acid and water content of the thermal pyrolytic oil as well as low stability retards its
use as an alternative fuel which can be overcome by cracking in presence of a suitable
catalyst. Literatures revealed that catalytic cracking of non-edible oil seeds such as mahua,
karanja, castor in presence of zeolite, Al
2
O
3
, CaO, Kaolin, Criterion-424 and BP 3189
enhanced the yield of pyrolytic oil and the fuel quality by altering the pH, reducing the water
content and viscosity. It has been observed that the catalytic activity varies with the types of
feedstock and its composition. Moreover non-edible seeds yield more oil compared to other
feedstocks and has closer fuel properties to that of diesel which indicates their suitability as an
alternative fuel for diesel engine.
Keywords: Non-edible seeds; Thermal pyrolysis; Catalytic pyrolysis; Fuel properties
6.1 Introduction
The depletion of the fossil fuel, increasing prices along with environmental
degradation is a major global problem. The utilization of energy also increased due to the
rapid industrialization and growth of population. Present sources of energy are not enough to



50

prevail over the increasing needs. Since 1973 the energy resources have been doubled in
developed countries but the requirement is still higher. The major demand of energy is
fulfilled by the conventional energy resources such as coal, petroleum and natural gas. It was
estimated that these oil sources might be depleted by 2050. The process of generating energy
from these sources causes atmospheric pollution and creates problems such as global
warming, acid rain etc. In view of increase in the energy demand as well as inherent problems
associated with environmental pollution the attention of energy researchers has shifted to non-
conventional energy sources such as wind, solar, tidal and biomass etc. Biomass is available
in abundance which is renewable, cheap and can be converted it to energy in the form of
liquid, solid and gaseous fuel. Natural biomass and its derivatives are the major sources of
biomass which include wood, wood wastes, agricultural crops, crop wastes and residues,
agricultural processing wastes, mill wood wastes and urban organic wastes. Energy crops are
another and important resources which include short rotation woody crops, herbaceous woody
crops, grasses, starch crops (corn, wheat and barley), sugar crops (cane and beet) and oil seed
crops (such as soyabean, sunflower, safflower, mahua, karanja, neem, linseed, niger seed,
guava, castor and other edible and non-edible seeds). One of the suitable and efficient
methods for production of energy such as oil, gas and char is pyrolysis. Pyrolysis of wood by-
products has been a standard method for the production of energy for decades. Improvements
and modifications have resulted in a more efficient and profitable biomass pyrolysis process
that yields solid (bio-char), liquid (bio-oil and fine chemicals) and gaseous products (syn gas)
(Goyal et al., 2006). Pyrolytic liquid is usually separated in to two phases, one is organic rich
phase (the oil phase) and the other contains water and water soluble compounds (the aqueous
phase). Pyrolytic liquid is being looked for as an alternate sustainable liquid fuel substitute for
diesel and jet engines (the oil phase) as well as a source for commodity and fine chemicals
(the aqueous phase). The oil phase is typically known as pyrolytic oil or bio-oil. The solid
product known as bio-char is traditionally used as a soil enhancer that can hold carbon, boost
food security, and increase soil biodiversity, and discourage deforestation. Bio-char also
improves water quality and quantity by increasing the retention of nutrients and
agrochemicals in soil for plant and crop utilization (Goyal et al., 2006). The product yield and
quality of pyrolytic oil varies with type of feedstocks, their composition. Literatures revealed
that less lignin and more extractive content in the biomass resulted in higher yield as well as
better quality pyrolytic oil. Among the plant derivatives, seeds contain less lignin and more
extractives in the form of oil. Several edible and non-edible seeds exist in the world. Since the
edible seeds come under the food chain, hence should not be used for the production of fuel.

51

Therefore, it is always better to focus on non-edible oil seeds as feedstock to produce liquid
fuel as they are outside the food chain.
In this work, the use of non-edible seeds for the production of liquid fuel following
thermal and catalytic pyrolysis is reported. Various process parameters that affect the yield as
well as fuel properties of pyrolytic oil are discussed here. The role of catalyst during pyrolysis
on the yield and quality of pyrolytic oil along with some drawbacks of thermal pyrolytic oil
are also reported.
6.2 Pyrolysis and its Types
Biomass pyrolysis simply means the heating of selected biomass at the elevated
temperature in absence of oxygen. During pyrolysis of biomass, the biomass undergoes
various phases such as dehydration and depolymerization. Initially, within 100-200
o
C most
of the moisture and water gets eliminated and at the elevated temperature depolymerization of
such chemical bonds associated with extractives hemicelluloses, cellulose and lignin takes
place respectively. At the lower temperature the weaker chemical bonds and at higher
temperature stronger chemical bonds breaks and the long chain chemical bond breaks to form
short chain compounds (Singh and Shadangi, 2011). Pyrolysis can be carried out thermally
where the operating parameter is only temperature and known as thermal pyrolysis. Pyrolysis
in presence of catalyst is termed as catalytic pyrolysis. To overcome the drawbacks of thermal
process, pyrolysis is carried out in presence of several catalysts which is discussed latter.
6.3 Process parameters that affect the yield
6.3.1 Effect of temperature on products yields
One of the most important parameters that affect the yield of pyrolytic products is
temperature. The yield of pyrolytic liquid, gas and char varies with final pyrolysis
temperature. During non-edible seed pyrolysis the foremost aim is to produce high yield of
pyrolytic liquid/oil other than that of char and non-condensable gas. However, char and non-
condensable gases are the other by-products of biomass pyrolysis. The temperature at which
the yield of liquid is highest is considered as the optimum temperature. The thermal pyrolysis
of Pistacia khinjuk seed (Onay, 2007 a), rapeseed (Onay and Kockar, 2004), safflower seed
(Beis et al., 2002, Onay, 2007 b), pomegranate seed (Ucar and Karagoz, 2009), cherry seed
(Dumanet et al., 2010), rape seed (Sensoz et al., 2000), jatropha seed (Figueiredo et al., 2011),
tamarind Seed (Kader et al., 2011), mahogany seed (Kader et al., 2012), castor seed (Singh
and Shadangi, 2011), neem seed (Nayan et al., 2013), mahua (Shadangi and Mohanty, 2014 a)

52

and karanja seed (Shadangi and Mohanty, 2014 b) was carried out and the effect of
temperature parameter on yield is reported in Table 1. It was found that non-edible oil seeds
can be used as a suitable feed stock for the production of alternative fuel by pyrolysis. Table 1
also provides the optimum temperature for maximum yield of pyrolytic liquid of several non-
edible oil seeds. It was observed that the degradation temperature and the optimum
temperature for maximum liquid yield varied with the types of feedstock. The yield of
pyrolytic liquid increased with increasing temperature, whereas the yield decreased after a
certain temperature. The temperature beyond which the yield of liquid starts decreasing is
termed as optimum temperature for maximum liquid yield. Several researchers reported
similar findings for the decrease in the liquid products. At higher temperatures, the secondary
decomposition of char may also produce non-condensable gaseous products which would also
contribute to increase in gas yield with increase in pyrolysis temperature.
6.3.2 Effect of heating rate on yield
During pyrolysis the feed biomass is heated from room temperature to the
depolymerization temperature. Hence the required rate of heating plays an important role for
maximum liquid yield. Lower the heating rate, the yield of char is more and liquid is less
during pyrolysis. Higher heating rate breaks the heat and mass transfer limitation during
pyrolysis and resulted in maximum yield of oil. Onay (2007 a) reported that the yield of
pistacia khinjuk seed pyrolytic oil increased by 25 % when the rate of heating raised from 5
o
C min
-1
to 300
o
C min
-1
. Similar results were also obtained by Onay (2007 b) during
pyrolysis of safflower seed, rape seed (Onay and Kockar, 2004) and safflower seed (Beis et
al., 2002).
6.3.3 Effect of sweeping gas flow rate on yield
Biomass pyrolysis may be carried out in an inert atmosphere or without flowing of any
sweep gas. The yield of pyrolytic liquid does increase with supply of any sweep gas. In
general nitrogen is used as a sweep gas to maintain inert atmosphere during the process. The
important role of flow of sweep gas during pyrolysis is that it helps in reducing the formation
of char, which is controlled by mass transfer of tar molecules into the light gas species. Onay
and Kockar (2004) pyrolyzed rape seed in a fixed bed reactor with and without sweeping gas
atmosphere and observed that the oil yield increased from 41.4 % 51.7%. Onay (2007 a) and
Onay and Kockar (2004) has reported that the effect of nitrogen flow rate on the liquid yield
was negligible if nitrogen flow rate exceed more than 100 cm
3
min
-1
. Similar conclusions

53

about the use of sweep gas flow rate are also reported by Kader et al. (2011) during pyrolysis
of mahogany seed and during pyrolysis of pistacia khinjuk seed (Onay, 2007 a).
Table 1. Thermal pyrolysis of non-edible seeds
Seeds
P
i
s
t
a
c
i
a

k
h
i
n
j
u
k

R
a
p
e
s
e
e
d

S
a
f
f
l
o
w
e
r

S
a
f
f
l
o
w
e
r

C
h
e
r
r
y

R
a
p
e
s
e
e
d

J
a
t
r
o
p
h
a

T
a
m
a
r
i
n
d

M
a
h
o
g
a
n
y

C
a
s
t
o
r

N
e
e
m

M
a
h
u
a

K
a
r
a
n
j
a

P
o
m
e
g
r
a
n
a
t
e

Process Thermal pyrolysis
N
2
flow
rate, cm
3
min
-1

100 200 25 No 500 600 500 No 30
Reactor
type
Fixed-bed
C
o
n
t
i
n
u
o
u
s

Fixed-bed Semi-batch reactor
Heating
rate,
o
C min
-1

300 5 40 10 At final
temp.
25 5
Temp.,
o
C 600 550 600 500 500 500 420 400 550 550 47
5
525 550 600
Total
liquid
yield, %
57.6 68 54 44 44 46.
1
23 45 49 64.4 38 57.7
5
55.17 54.2
Char
yield, %
15.2 13.5 17.
2
20.
5
16.
5
20.
44
- 40 35 20.9
3
30 21.5
5
19.81 29.28

6.3.4 Effect of particle size on yield
The yield of pyrolytic liquid during pyrolysis is also affected by the particle size of the
feedstock. Larger feed size decreased the heat transfer rate and increased the pyrolysis time
which is liable for the formation of more char. In general, lower particle size of feed materials
enhances the pyrolytic yield of liquid. Kader et al. (2011) experimented pyrolysis on tamarind
seed and reported that liquid yield first increased up to a maximum value for feed size of 1.07
cm
3
and subsequently decreased for larger feed size above 1.07 cm
3
. Pyrolysis experiments
performed by Onay and Kockar (2004) on rapeseed, Kader et al. (2012) on mahogany seed
and Onay et al. (2007b) on safflower seed suggested that particle size more than 1mm
decreased the yield of pyrolytic oil by producing more char.
6.4 Fuel properties of Seed Pyrolytic Oil
6.4.1 Calorific Value

54

The calorific values of several non-edible seeds are presented in Table 2. This
confirmed that non-edible seed pyrolytic oils have higher calorific value and closer to diesel.
The calorific value of pyrolytic oil is a function of feedstock, operating conditions and the
process used. Zhang et al. (2007) reported that non-edible seed pyrolytic oil having more
heating value in comparison with wood pyrolytic oil.
Table 2. Fuel properties of seed pyrolytic oil
Seeds Calorific value, MJ kg
-1
Viscosity, cSt References
Pistacia khinjuk 39.84 -- Onay, 2007 a
Rapeseed 38.4 43 (50
o
C) Onay and Kockar, 2004
Safflower 41 33 (50
o
C) Onay, 2007 b
Cherry 38.4 -- Duman et al., 2011
Tamarind 19.1 6.51 (30
o
C) Kader et al., 2011
Mahogany 32.4 3.81 (26
o
C) Kader et al., 2012
Castor 35.64 83.19 (25
o
C) Singh and Shadangi, 2011
Neem 32.49 22.6 (40
o
C) Nayan et al., 2013
Mahua 41 33.97(25
o
C) Shadangi and Mohanty, 2014 a
Karanja 37.65 51.67(25
o
C) Shadangi and Mohanty, 2014 b
Diesel 45-47 3.68 (40
o
C)
4.57 (25
o
C)
--

6.4.2 Viscosity
High viscosity is one of the principal drawbacks of seed pyrolytic oils. Viscosity of
the pyrolytic oil is concerned with water content and water insoluble compounds. The higher
water content and less water insoluble components in the pyrolytic oil reduce the viscosity of
the pyrolytic oil. Table 2 shows the viscosities of various non-edible seed thermal pyrolytic
oils. It is concluded that viscosity of pyrolytic oil varies with biomass types and the process
type. The pyrolytic oil viscosity is much more higher compared to conventional diesel. Thus,
the direct use of thermal pyrolytic oil in combustion engine is not acceptable and it needs
upgradation to match the fuel properties.
6.4.3 pH of pyrolytic oil
Pyrolytic oil is acidic in nature having high pH value ranging from 3-5. The high acid
content is due to the presence of carboxylic acid, acetic acid and formic acid in the pyrolytic
oils. These acids are formed by the depolymerization of cellulose and hemicelluloses. High
cellulose content in the biomass produces more acid in the pyrolytic oil during pyrolysis. The
presence of various acids in the pyrolytic oil also reduces the thermal stability during storage.
The direct use of seed pyrolytic oil as a transportation fuel may damage the engine. Table 3

55

confirmed that the non-edible oil seed pyrolytic oils are highly acidic in nature and also varies
with the feedstocks.
6.4.4 Water content
The presence of water content in pyrolytic oil reduces the heating value and the flame
temperature. Less water content decreases the viscosity which enhances the atomization
towards complete combustion and reduces the harmful emissions such as SOx and NOx. It is
confirmed from the literature that non-edible seed pyrolytic oil contains very less water in it
which can be observed from Table 3. It was reported that the water content of pyrolytic oil
varies from 0.33 % to 35 %. Since water is insoluble with organic pyrolytic oil most of the
produced water and the water soluble chemicals collected separately as aqueous pyrolytic
liquid (Shadangi and Mohanty, 2014 a, b). This confirmed that the non-edible seed pyrolytic
oil can be accepted as a future alternative fuel.
6.4.5 Pour point
Higher pour point is one of the disadvantages of pyrolytic oil. The pour point of seed
pyrolytic oil varies from 5-27
o
C. Table 3 shows the pour point of several non-edible seed
pyrolytic oils. High pour point of pyrolytic oil reduces the flow ability in winter especially in
low temperature regions due to the formation of crystals which clogs the filter and reduces the
efficiency of the combustion engines.
Table 3. pH, water and pour point of some non-edible seed pyrolytic oil
Pyrolytic oil pH Water content, % Pour point,
o
C References
Neem 3.9 30-35 11 Nayan et al., 2013
Mahua 4.86 0.33 26.63 Shadangi and Mohanty, 2014 a
Karanja 4.05 1.33 12.05 Shadangi and Mohanty, 2014 b
Castor 3.7 -- 5 Singh and Shadangi, 2011

6.5 Catalytic Pyrolysis of Non-edible Seeds
6.5.1 Effect of Catalyst on Yield
Theoretically catalyst has an effect on the rate of reaction. The catalytic effect
enhances the rate of reaction which increases the conversion during pyrolysis. It might have
positive or negative impact. The influence of various catalysts on the pyrolytic yield of non-
edible oil seeds were studied by different researchers. Catalytic pyrolysis of Pistacia khinjuk
seed was carried out by Onay (2007 a) using BP3189 and Criterion-424 as catalyst and a
liquid yield of 66.5% and 69.2% was found for the two catalysts respectively while it was

56

only 57.6% without catalyst. Shadangi and Mohanty (2014 a,b) studied the catalytic pyrolysis
of Mahua and Karanja seed using CaO, Al
2
O
3
and Kaolin and observed little increase in the
yield of pyrolytic oil (4-6%) over thermal pyrolysis.
6.5.2 Effect of catalyst on fuel properties of pyrolytic oil
Literature revealed that the use of catalyst altered the physical properties of non-edible
seed pyrolytic oils. The catalyst pyrolysis decreases the viscosity, increases the calorific value
and decrease the acidity of pyrolytic oil. Shadangi and Mohanty (2014 a) reported that the use
of CaO catalyst increased the calorific value from 41 to 43.15 MJ kg
-
1, reduced the viscosity
from 0.033 to 0.018 Pa s and alter the pH acidic to basic (4.86 to 8.58) for Mahua pyrolytic
oil. The effect of catalyst Al
2
O
3
, CaO and Kaolin on pyrolysis of karanja seed was studied by
Shadangi and Mohanty (2014 b) and it was reported that CaO at 8:1 ratio produced less
viscous (0.019629 Pa-s) pyrolytic oil compared to other catalysts. The calorific value
increased for all the three catalytic pyrolysis with CaO (40.42 MJ kg
-1
), Al
2
O
3
(41.21 MJ kg
-1
)
and Kaolin (39.04 MJ kg
-1
) compared to thermal pyrolysis (37.65 MJ kg
-1
).
6.6 Conclusion
The study of thermal and catalytic pyrolysis of non-edible seed confirmed that the
pyrolytic oil can be used as an alternative fuel. Non-edible seed produced more organic liquid
in comparison with other feed stocks due to its high extractive content. The thermal pyrolytic
oil is highly acidic in nature and very viscous and hence is not suitable for direct use in diesel
engine. Catalytic pyrolysis is one of the suitable processes which altered the pH, reduces the
viscosity and enhances the calorific value. Hence, catalytic pyrolytic oil from seeds will be
suitable as an alternative fuel. However, more emphasis should be given on the effect of
various catalysts to found out the most suitable catalyst which will enhance the fuel properties
and stability of pyrolytic oils.

References
1. Beis S.H., Onay O., Kockar O.M. (2002) Fixed-bed pyrolysis of safflower seed:
Influence of pyrolysis parameters on product yields compositions. Renewable Energy,
26: 2132.
2. Duman G., Okutucu C., Ucar S., Stahl R., Yanik J. (2011) The slow and fast pyrolysis
of cherry seed. Bioresource Technology, 102: 1869-1878.

57

3. Figueiredo M.K.K., Romeiro G.A., Silva R.V.S., Pinto P.A., Damasceno R.N., DAvila
L.A., Amanda P. (2011) Pyrolysis Oil from the Fruit and Cake of Jatropha curcas:
Produced Using a Low Temperature Conversion (LTC) Process: Analysis of a Pyrolysis
Oil-Diesel Blend. EPE., 3: 332-338.
4. Kader Md A., Islam M.R., Joardder M.U.H. (2011) Fast Pyrolysis for Better Utilization
of Tamarind Seed from Renewable Energy Point of View. Proceedings of the
International Conference on Mechanical Engineering and Renewable Energy.
Chittagong, Bangladesh., 22- 24.
5. Kader M.A., Joardder M.U.H., Islam M.R., Das B.K., Hasan M. (2012) Production of
Liquid Fuel and Activated Carbon from Mahogany Seed by Using Pyrolysis
Technology. In Green Chemistry for Sustainable Development. Bangladesh, Jessore:
July 14. Id code: 53696.
6. Nayan N.K., Kumar S., Singh R.K. (2013) Production of the liquid fuel by thermal
pyrolysis of neem seed. Fuel, 103: 437443.
7. Onay O. and Kockar O.M. (2004) Fixed-bed pyrolysis of rapeseed (Brassica napus L.).
Biomass and Bioenergy, 26: 289299.
8. Onay O. (2007a) Fast and catalytic pyrolysis of Pistacia khinjuk seed in a well-swept
fixed bed reactor. Fuel, 86: 14521460.
9. Onay O. (2007b) Influence of pyrolysis temperature and heating rate on the production
of bio-oil and char from safflower seed by pyrolysis, using a well-swept fixed-bed
reactor. Fuel Process Technology, 88: 523531.
10. Shadangi K.P., and Mohanty K. (2014a) Thermal and catalytic pyrolysis of Karanja
seed to produce liquid fuel. Fuel, 115: 434442.
11. Shadangi K.P., and Mohanty K.. (2014b) Comparison of yield and fuel properties of
thermal and catalytic Mahua seed pyrolytic oil. Fuel, 117 (30): 372380.
12. Singh R.K., and Shadangi K.P. (2011) Liquid fuel from castor seeds by pyrolysis. Fuel,
90: 25382544.
13. Sensoz S., Angn D., Yorgun S. (2000) Influence of particle size on the pyrolysis of
rapeseed (Brassica napus L.): fuel properties of bio-oil. Biomass and Bioenergy, 19:
271-279.
14. Uar S. and Karagz S. (2009) The slow pyrolysis of pomegranate seeds: The effect of
temperature on the product yields and bio-oil properties, J. Analytical Applied
Pyrolysis, 84: 151-156.


58

CHAPTER 7
EVALUATION OF MICRO GASIFIER COOKSTOVE
PERFORMANCE WITH HANDMADE BIOMASS PELLETS
USING REGION-SPECIFIC FUELS AND ASSESSMENT OF
DEPLOYMENT POTENTIAL
Debkumar Mandal, Vikas Dohare, Vijay H. Honkalaskar, Anurag Garg, Upendra V.
Bhandarkar

and Virendra Sethi

Abstract
In rural areas of developing countries, large populations depend on traditional biomass such
as wood, crop residues, and cattle-dung for cooking. Emissions from these activities have
been reported to cause regional environmental impacts and global climate change. While
engineered cook stove designs could be considered as a solution for reducing emissions, it is
not independent of the region and season specific availability of fuels. A design solution
based on gasification using engineered solid fuel from agricultural residues/coal powder is a
relatively new development, and accounts for both the design of the cookstove and the
physical property of the fuel. Work is needed to fine tune these gasifier cookstoves, quantify
emissions and fuel consumption with emphasis on the utilisation of region-specific fuels.
Micro-gasifier type cookstove have become more popular among small scale
commercial operations where replacement of LPG with biomass pellets is economically
profitable. However, in rural areas, where wood/agricultural residue is freely available as a
natural resource, purchase of commercial pellet-based fuel is not affordable. The purpose of
the study was to determine how locally available biomass may be used to take advantage of
the micro-gasifier cookstove design. Further, it is also important to understand the
behavioural and cultural obstacles for deployment of these stoves, and the suitability for
ensuring reduction in indoor kitchen exposure. A field campaign was carried out in
Gawandwadi village in Maharashtra, to understand deployability based on cooking practices
and fuel availability. Experiments were conducted using local biomass residues (wheat husk,
rice husk and saw dust) with locally available binding materials (cow dung, wheat flour and
rice flour) in different proportions for making handmade pellets. Spherical balls were made



59

by pressing mixture (biomass residues with binding material) by hand. A simple sewai
making home machine was modified and also used for extrusion for making disk shaped
pellets. Standard Water Boiling Test was conducted to evaluate the performance of the
handmade pellets in micro-gasifier stove. Besides, locally available fuel woods in small
pieces were also used and the results were compared with the handmade pellets. The
evaluation was done on the basis of Input power (kW), fuel required per output power
(kg/kWh) and Efficiency (%). The efficiency and input power of the handmade pellets
ranged between 33% to 57% and 2.4 kW to 3.1 kW respectively.
Keywords: Micro-gasifier coookstove, Biomass, Handmade pellets
7.1. Introduction
Three billion people across the world use biomass for cooking and heating purposes.In
India, 82% of the population use solid fuels for cooking, of which rural and urban population
accounts for 88 % and 24.6% respectively (URL 1).The global use of biomass fuels from
traditional stoves (three stone, open fire) has been linked to adverse health effects, climate
change, and deforestation. In India, forest area grew by ~2% annually, but afforestation
quality is poor and deforestation persists due to urbanization across many parts in the country.
Encouraging use of non-solid fuel in improved cookstoves (such as, micro-gasifier cookstove)
can play a crucial role in preventing land degradation and deforestation, particularly in areas
with negligible or negative afforestation rates.Improved cooking stoves have been shown to
reduce the amount of fuel used to cook food and the air pollution produced in kitchens.58% of
Indian population relies on fuel wood and 11% uses cow-dung for cooking purposes. On the
other hand only 0.4% uses other fuels which include agricultural residues for cooking (URL
1).
The traditional cookstoves used in the rural areas lead to the emission of unburnt gases
such as CO,a toxic air pollutant in indoor air in rural as well as urban areas. The CO emission
factors ranged widely from 3.010
-2
g/Kg for coal gas/traditional stoves to 2.8 10
2
g/Kg for
the charcoal/ Angethi stoves (Zhang et al., 1999). Besides this, at a temperature of around
800-1000C particulate matter (PM) is dominated by soot (carbon aggregate) in conventional
cookstoves (Bolling et al., 2009). The CO standards for residential and agricultural areas are 2
mg m
-3
for an 8 hour average, or 16 h-mg m
-3
exposure equivalent according to the WHO
guideline. These could be easily exceeded by CO exposure caused by traditional cookstoves
using biomass (Zhang et al., 1999).

60

To reduce these emission of CO and PM, improvements have been made in the
combustion devices by studying the combustion pattern of the raw biomass and its processed
form (in form of briquettes and pellets) so that less amount of gases and PM are released. A
gasifier stove is such an attempt in which the processed forms of biomass (i.e. briquettes or
pellets) are used. While studying the combustion pattern of fuel in many stoves it has been
found that if the ratio of fuel and air is maintained at certainlevel then clean combustion can
be achieved (Mukunda, 2011). Many of the fundamental phenomena observed in biomass
combustion, which are most relevant to cookstoves, have been extensively studied and mainly
focus on understanding how wood burns. It includes modelling of air-flow rates both on the
solid and gas phases and heat transfer (Burnham-Slipper, 2008 and Mukunda, 2011).
The present work deals with the fan based gasifier stove, Oorja, built by BP, India.
The main focus of the work is the issue of using different handmade pellets or briquettes
(using powdery biomass) and to check their adaptability for the Oorjagasifier stove.The Oorja
stove has two modes of air flow rates i.e. low and high modes. In the present study, the
biomass handmade pellets (using commonly available biomass) have been tested mainly
using the high mode. For most existing stoves, whether traditional stoves such as the three
stone fire or other stoves developed over the last two decades, the efficiency (determined
using water boiling tests) is between 15 to 35%. In contrast, the water boiling efficiency of the
Oorja stove (used with pellets designed for Oorja) has been found to be up to 60%
(Varunkumar, 2012).
7.2. Materials and Methods
Table 1 shows the equipment and materials used for this study. Several combinations
of biomass-binders were used to hand-make pellets and compacted briquettes. This section
describes the method followed for making of the region specific fuel to adapt in and Oorja
(gasification type) domestic scale stove.
7.2.1 Process for Making the Handmade Pellets
The making of the pellet without any kind of sophisticated pellet making machine
using the locally available biomass and the binder was done in three steps, which are
described in the following sub sections.
a) Preparing the mixture of the biomass and the binder
Preparing the mixture for the pellet has been the most important step in the process to
ensure maximum energy density, by maximising the amount of biomass with the minimum

61

amount of binder. The local binder used in the preparation were cowdung, wheat flour and
rice flour while the biomass used were saw dust, rice husk and wheat husk. Sophisticated
pellet making machine (designed especially for the manufacturing of the pellets that are
commercially available in the market) generally the binder is not required as the lignin present
in the biomass, when compressed, at high temperature acts as the binder. For such cases high
degree of the compaction is needed which cannot be achieved by the hand pressing of the
mixture to make the pellets.
Table 1 Equipment and materials used in the present study

S.No

Materials
Required

Quantity

Remarks

References

1
Biomass fuel 4 types Oorja pellets, Rice Husk,
Wheat Husk and Saw Dust
As per the requirement of the
experiment

2
Binders 3 types Cow Dung, Rice Flour and
Wheat Flour
As per the requirement of the
experiment
3 Thermocouple 2 K- type Thermocouple to
measure temperature up to
1200 C
Varunkumar (2012)
4 Thermometer
1
Thermometer of range up to
100C
Water Boiling Test version
4.1.2 (URL 2)
5 Cooking vessel 2 3 L Aluminium vessel Water Boiling Test version
4.1.2 (URL 2)
6 Hood 1 Hood including exhaust Water Boiling Test version
4.1.2 (URL 2)
7 Weighing
Balance
1 Range 0-50 kg (accuracy in
grams)
As per the requirement for the
experiment
8 Stove 1 Oorja pellet gasifier Stove As per the requirement for the
experiment
9 Stirrer
1
For stirring the water for
constant mixing
Water Boiling Test version
4.1.2 and Varunkumar(2011)
10 Kerosene 2 l To ignite the stove As per the requirement for the
experiment
11

Bomb
Calorimeter
1 To determine the calorific
value of the pellets
As per the need for the
experiment

During the making of the pellet, it was first determined as to what amount of
minimum amount of binder could hold the maximum amount of biomass. Different binders
were mixed in equal proportions with the biomass, and its binding capacity was checked. The
biomass was then added continually until the binder could not hold more of the biomass. The
composition, in which the binder cannot hold more of the biomass was determined, was then
used as the mixture for the pellet making. Binding capacity of different binders for different
biomass varied, in which the cow dung was found to have the minimum binding capacity and
wheat flour with the maximum binding capacity. But, since the cow dung is available freely it

62

was preferred over the other binders, even though the binding property was low as compared
with the wheat and the rice flour.
Table 2 Different types of fuels and binders used for the pellet making
S.No. Biomass Binders
1 Saw dust Cow dung
2 Rice husk Rice flour
3 Wheat husk Wheat flour

b) Compaction of the mixture for the making of the pellets
The compaction of the mixture was the second step in the making of the pellets
making by hand pressing into small balls as shown in Fig 1(a). Besides the hand pressing
technique a commonly available snack making machine called as sevai machine was used.
This is a hollow cylinder fixed with a piston. The top of the piston is provided with a rotating
shaft. When this shaft is rotated the piston moves downwards and presses the mixture filled in
the cylinder. At a certain point shaft, cannot be rotated, at that particular point the piston is
removed and then compacted form of the mixture is recovered in the form of the discs as
shown in the Figure 1 (b). This compacted form of the mixture, which can also be called as
pellets are then put for drying.
c) Drying of the pellets
These wet pellets are dried until the moisture content of the pellet reaches ~12 % or
below (needed for the gasification of the pellets). The drying can either be done either in the
sun for over a week or they can also be dried in the hot air oven for 48 to 72 hours at the
temperature of 45C. Figure 1 (a) and (b) shows the different pellets made by hand pressing
and using household sewai machine.
Table 3 Different types of the pellets and their composition (notation is indicated at the
bottom of the table)
S. No. Pellets Composition
1 CD+RH+SD (Balls) 400 g cow dung + 100 g rice husk + 100 g saw dust
2 CD+SD (Balls) 500 g cow dung + 60 g saw dust
3 SD+WF (Balls) 250 g Saw Dust + 270 g Wheat Flour + 375 g water
4 CD+SD+WH (Briquette) 1120 g Cow Dung + 245 g Saw Dust + 180 g Wheat Husk
5
CD+SD+WH+WF
(Briquette)
650 g Cow Dung + 200 g Saw Dust + 150 g Wheat Husk +
75 g Wheat Flour
6 WH+RF (Briquette) 340 g Wheat Husk + 110 g Rice Flour + 600 g Water
CD = Cow Dung, SD =Saw Dust, RH = Rice Husk, WF = Wheat Flour, WH = Wheat Husk and
RF= Rice Flour

63

Figure 1(a) Disk shaped pellets made using the sewai machine

Figure 1(b) Ball shaped pellets made using hand pressing (a) Cow dung + Rice husk + Saw
dust (b) Cow dung + Saw dust, (c) Saw dust + Wheat flour and (d) Wheat husk + Wheat
flour
Stove Performance with Different Types of Handmade Biomass Pellets with the Oorja
Gasifier Stove
To evaluate the performance of the different compositions of the handmade biomass
pellets, the following parameters are discussed:
1. Determination of the calorific value
2. The input power for different types of pellets in Oorja stove
3. Specific fuel consumption for each fuel
4. Efficiency of the stove
The calorific values and densities of the fuels are shown in the following Table 4.


64

Table 4 Calorific values and the densities of the different biomass fuels
Biomass fuels Calorific Value (MJ/Kg) Density (Kg/m
3
)
CD+RH+SD (Balls) 15.66 244.3
CD+SD (Balls) 16.13 270.5
SD+WF (Balls) 15.4 423.2
CD+SD+WH (Briquette) 16.04 307.9
CD+SD+WH+WF (Briquette) 15.34 275.4
WH+RF (Briquette) 17.02 566.0
Oorja pellet 16.04 ___
Babool wood 17.3 ___
Ain wood 16.8 ___

To conduct the water boiling test, the standard 3 litrewater boiling test protocols were
taken into accounts with slight modification to test the biomass handmade pellets on high
mode in the domestic gasifier Oorja stove. Tests were conducted in a way only a batch
process was considered.The operational procedure does not require refuelling. Table 4 and 5
shows the result of water boiling tests and the performance of the stove respectively, for
biomass pellets.
Figure 2 plots the temperature of the boiling water as a function of time for each fuel.
From the plot, we can observe that Oorja pellets take only 15 minutes to boil the water and
complete the test in 49 minutes for one batch process, which is comparatively faster than the
other types of pellets. On the other hand, the small pieces of Babool wood takes only 10 min
to boil the water which is comparatively faster than the other types of biomass fuels but lasts
only 26 minutes for one batch process. For the other pellets, the water did not even reach the
boiling temperature, except for the ball shaped pellets made from Saw Dust and Wheat Flour
that took 50 minutes to reach the temperature of 99C.

65

Table 5 Result of Water Boiling Test (3 L Water) in Oorja Stove
Parameters CD+SD
(Balls)
CD+RH+
SD (Balls)
SD+WF
(Balls)
CD+SD+WH
(Briquette)
CD+SD+
WH+WF
(Briquette)
WH+RF
(Briquette)
Oorja
Pellet
(not
handmade)
Ain
wood
Small
Babool
pieces
Large
Babool
pieces
Room Temp
(C)
31 29 27 28 27 27 26 26 27 28
Initial water
Temp ( C)
31 29 27 28 27 27 26 26 27 28
Final Water
Temp (C)
58 69 96 63 89 78 99 (15 min,
Cold Start.)
99 (14 min,
Hot Start.)
96 (20 min,
Simmering)
89 100 (10
min, Cold
Start.)
62 (16
min, Hot
Start.)
100
Mass of
Pellet (g)
140 150 575 380 440 210 595 335 365 385
Duration of
Burning
(min.)
12 14 50 15 41 24 49 10 26 15
CD = Cow Dung, SD =Saw Dust, RH = Rice Husk, WF = Wheat Flour, WH = Wheat Husk and RF= Rice Flour

66

Table 6 Stove performance with different biomass fuels on Oorja stove
Sl. No Pellets
Input Power
(kW)
Fuel required per
output power
(Kg/kWhr)
Efficiency (%)
1 CD+RH+SD (Balls) 3.1 0.44 54.1
2 CD+SD (Balls) 2.8 0.47 57.1
3 SD+WF (Balls) 2.9 0.73 42.9
4 CD+SD+WH (Briquette) 6.7 1.75 33.1
5
CD+SD+WH+WF
(Briquette)
2.7 0.92 40.0
6 WH+RF (Briquette) 2.5 0.62 48.8
7 Oorja Pellet 2.2 1.17 32.3
8 Babool small wood pieces 3.7 0.59 58.9
9 Babool large wood pieces 6.7 0.87 37.5
10 Ain wood 8.1 1.35 24.9

Figure 2 Comparision of Temperature vs. Time profiles for different fuels in the Oorja stove
during the Water Boiling Test

0
20
40
60
80
100
120
0 20 40 60
T
e
m
p

(
C
)
Time (min)
WBT of Biomass Fuels in Oorja
CD+RH+SD(Balls)
SD+WF (Balls)
CD+SD+WH
(Briquette)
CD+SD+WH+WF
(Briquette)
WH+RF(Briquette)
Oorja (Karjat)
sall !a!ool "ie#es

67

7.4 Conclusions
In this present work the analysis of gasification behaviour of the handmade pellets
made from different locally available biomass was carried out to address the issue of how
region specific fuel can be used for the clean combustion process, i.e., the gasification in the
already available Oorja pellet gasifier stove. It also focussed on what changes can be made in
the existing stove and what could be the alternate fuel that can be used by the rural people
without having to buy the commercially available pellets. During the study, it was found that
if the pellets are more densified with some better technique, the locally available biomass
could be pelletized and used in the forced draft gasifier stoves like Oorja.

References
1. Bolling, A.K., Pagels, J., Yttri, K. E., Barregard.,Sallesten, G., Schwarze, P.E., and
Boman, C. (2009) Health Effect of Residential Wood Smoke particles: The
Importance of Combustion Condition and Physiochemical Particle Properties, Particle
and Fibre Technology, 6:29.
2. Burnham-Slipper, H., Clifford, M.J. and Pickering, S.J. (2007) A simplifiedwood
combustion model for use in the simulation of cooking fires, in 5th International
Conference on Heat Transfer, Fluid Mechanics and Thermodynamics.
3. Mukunda, H.S. (2011) Understanding Clean Energy and Fuels from Biomass, Wiley
India.
4. Varunkumar, S (2012) PhD Thesis- Packed bed gasification-combustion in biomass
domestic stove and combustion system, IISc, Bangalore.
5. Zhang, J., Smith, K.R., Uma, R., Ma, Y., Kishore, V.V.N., Lata, K., Khalil, M.A.K.,
Rasmussen, R.A. and Thorneloe, S.T. (1999) Carbon monoxidefromcookstoves in
developing countries: 1. emission factors, Chemosphere: Global Change Science, 1(1-
30: 353366.
Web References
6. http://www.cleancookstoves.org/ (Last accessed on 17
th
November, 2013).
7. www.aprovecho.org/lab/component/rubberdoc/doc/231/raw (Last accessed on 15
th

September, 2013).


68

CHAPTER 8
PRODUCTION OF HYDROCARBON LIQUID BY
PYROLYSIS OF CAMELLIA SINENSIS (TEA) SEED
DEOILED CAKE AND CHARACTERIZATION OF
PRODUCTS
Nabajit Dev Choudhury, Priyanko Protim Gohai,

Bichitra Bikash, Sashi Dhar Baruah and
Rupam Kataki

Abstract
Increasing demand of the liquid fuel can be partially fulfilled by utilization of the bio-wastes
for biofuel production through pyrolysis. In this line, the present work aims to explore
Camellia Sinesis de-oiled cake (CSDC) for bio-fuel production. CSDC was pyrolysed for
obtaining liquid biofuel. The thermal pyrolysis of CSDC was carried out in a fixed-bed
reactor made up of stainless steel at temperature range from 400-600
o
C and at heating rate
30
0
Cmin
-1
. and at 150 mlmin
-1
nitrogen gas flow rate to determine the yield and
characteristic of the liquid and solid product. The maximum bio-oil yield was 27.38% at
500
0
C. The chemical composition of the bio-oil was investigated using FTIR and GC/MS.
Results showed that the bio-oil obtained from deoiled cake of CSDC is a valuable source of
fuel and chemicals.
Keywords: Pyrolysis, Deoiled cake, Bio-oil, FTIR, GC-MS.
8.1 Introduction
Increasing energy consumption, depleting conventional fossil fuel and environmental
consideration intensified the research on renewable energy particularly on utilization of
biomass for the production relatively clean fuel due to its easy availability, easy process
ability and environment friendly nature in contrast to the fossil fuel. Biomass act as a sink for
greenhouse gases as it absorbs CO
2
during its cultivation. Biomass therefore can be
considered as an alternative clean development mechanism (CDM) option for reducing
greenhouse gas emission [Mulligan et al., 2010]. The different thermo-chemical and bio-



69

chemical process such as pyrolysis, gasification, liquification and anaerobic digestion; are
used to convert biomass to fuel. Among these processes pyrolysis is one of the suitable
thermo chemical conversion processes to get maximum liquid product from biomass. Any
type of biomass can give liquid fuel after pyrolysis which can be used as energy fuel and also
for production of different chemicals [Bridgwater and Peacocke, 2004]. Pyrolysis is a
conversion technique in which biomass is heated to a desired temperature in an oxygen or air
free atmosphere to yield solid, liquid and gaseous products. There are many parameters that
influence the yield and quality of the products obtained through pyrolysis. These parameters
can be divided into processs parameters and non process parameters. The feedstocks
properties such as particle size, fixed carbon, cellulose, hemicelluloses, lignin, ash and
mineral content are non process parameters and heating rate, temperature, residence time,
sweeping gas type and flow rate, raction time and type of catalysts used are process
paremeters. The advantage of the pyrolysis process is that the process parameters can be
controlled to maximize the production of either solid char, liquid or gaseous products.
Ususally, fast pyrolysis with high temperature and longer residence time favour conversion of
biomass into uncondensable gaseous product and moderate temperature with short residence
time favour the production of bio-oil. Slow pyrolysis is preferred when solid char is desirable
product.
Deoiled cake of non-edible oil seeds can serve as potential feedstocks for pyrolysis to
produce fuels including liquid and gas which can be used as substitute for petroleum or
natural gas for internal combustion engines, power station and heat supplies. These biofuel
are environment bening in contrast to fossil fuel as they contain low nitrogen and sulphur
content. Many researchers have done pyrolysis experiment on different de-oiled cakes such
as Jatropha [Raja et al., 2010], Soybean [Uzun et al., 2006], rapeseed cake [Ozcimen et al.,
2004] etc. In present study, a new source of de-oiled cake, Camellia Sinesis de-oiled cake
(CSDC) was taken and pyrolysis experiment was done. The objective of th present work is to
determine the maximum bio-oil production condition in fixed bed reactor. The present work
also reports on the characterization of the bio-oil by FTIR and GC-MS for chemical
compositon.Thermogravimetric analysis (TGA) and was used to determine the thermal
behavior of CSDC. The physical properties of the bio-oil such as kinematic viscosity, flash
point, acid number, and pH were also determined.


70

8.2.1 Materials
In this study, the sample (Fig 1) was collected from Biomass Conversion &
Gasification Laboratory of Department of Energy, Tezpur University, India, after lipid
extraction with mechanical oil expeller [Malnad type oil expeller (Indus)]. CSDC were
ground using a Wiley mill to pass a 0.4 mm (40 mesh) screen (as per TAPPI T257 Om- 85
methods) to fine particles (420 micron) in order to eliminate heat transfer effects during
pyrolysis and then samples were oven dried and kept in a desiccator. The proximate and
ultimate analyses data for CSDC are given in Table 1.

Figure 1: Camellia Sinesis amd CSDC
8.2.2 Characterization of feed stocks and biochar
The proximate analysis of tea seed deolied cake and biochar obtained after pyrolysis
were done by ASTM D 3173-75 and ultimate analysis was done using CHN analyzer (Perkin
Elmer, 2400Series-II). The percentage of oxygen was determined by means of difference.
Higher heating value (HHV) was determined by 5E-1AC/ML, Auto bomb calorimeter
according to ASTM D2015. The thermal behavior of CSDC to 900
o
C at different heating
rates of 20
o
C/min were studied non-isothermally using Pyris Diamond TG/DTA analyzer
(PERKIN ELMER). A high purity N
2
gas (99.99%) was used as a carrier gas at a flow rate of
100 ml min
-1
.
8.2.3 Pyrolysis set-up
The schematic diagram of the experimental setup is shown in Fig 2. The pyrolysis
setup consists of fixed bed reactor made of stainless steel with a length of 48 cm and an
internal diameter of 3 cm, equipped with an inert gas (nitrogen) connection. The reactor was
heated externally by an electric furnace, with the temperature being controlled by a NiCrNi

71

thermocouple fitted inside the reactor. The thermocouple was connected to a PID controller for
controlling the temperature and heating rate. Before the experiments, the reactor was purged by
nitrogen gas for 10 min at a flow rate of 30 ml min
-1
to remove the air inside. Then, nitrogen flow
rate was set to the desired value. A 10g of deoiled cake were loaded for each run of experiment.
The liquid portion was recovered with diethylether washing. The aqueous phase was separated
from oil phase with a separating funnel. The bio-oil and solvent mixture was passed over dry
sodium sulphate to make it water free and then the solvent was evaporated from bio-oil by rotary
evaporator and the residual was weighed as bio-oil. The residual solid in the reactor was weighed
as char. The gas yield was calculated from the material balance. The reactions were carried out at
different temperatures ranging from 400 600
o
C.

Figure 2: Schematic diagram of experimental unit for pyrolysis
8.2.4 Characterization of bio-oil
Fourier Transformer Infrared spectroscopy (FTIR) of the pyrolytic oil obtained at
maximum yielding condition was taken with a Nicolet Impact I-410 model Fourier Transform
Infrared Spectrometer to chemical composition of the bio-oil. The components of the bio-oil
were analyzed using Perkin Elmer Clarus680 GC/600C MS. A capillary column coated with
a 0.25 m film of DB-5 with length of 30 m and diameter 0.25 mm was used. The GC was
equipped with a split injector at 200
0
C with a split ratio of 1:10. Helium gas of 99.995%
purity was used as carrier gas at flow rate of 1.51 ml min
-1
. The oven initial temperature was
set to 70
o
C for 2 min and then increased to 290
o
C at a rate of 10
o
Cmin
-1
and maintained for 7
Gas
Thermocouple
N2 cylinder
F F
R
C
V 1 V 2
FC
Liquid product
collector
F- Furnace
R- Reactor
C- Condenser
V- Valve
FC- Flow controller


72

min. All the compounds were identified by means of the NIST library. Mass spectrometer
was operated at an interface temperature of 200
o
C with ion source temperature of 180
o
C of
range 401000 m/z.
The physical properties such as density, kinematic viscosity , flash point and calorific
value were determined using standard test methods.
8.3 Result and Discussion
8.3.1 Characterization of feedstock and obtained biochar
The proximate and ultimate analysis of CSDC and biochar are shown in the Table 1.
The volatile matters and ash content of the sample in proximate analysis was found to be
80.7% and 5.34% respectively. Low ash content of deoiled cake indiacate that the sample is
good candidate for thermochemical conversion process. The volatile matter of the sample
was 80.7% which is reduced to 22.72% after pyrolysis. It indicates that a large portion of the
sample was converted to condensable and incondensable gases. As a result of significance
decrease in the volatilie matters the fixed carbon content of the solid material increased which
indicates relatively less liberation of the fixed carbon during pyrolysis. Moisture content play
an important role in selection of the conversion process. Sample with less moisture content
are suitable for thermal conversion while those with high moisture content are more suitable
for biochemical conversion process such as fermentation. In this regard, tea seed deoiled cake
with moisture content 4.61 % is a potential candidate for thermal conversion. Ultimate
analysis presented in the Table1 showed a significance increase in carbon content of biochar
whereas its oxygen content decreased in comparison to the oxygen content of the raw
material.
8.3.2 TGA and DTG analysis of CSDC
Thermogravimetric analysis (TGA) technique is applied in determination of thermal
stability of the sample in various ranges of temperatures. The TGA plot of CSDC at heating
rate of 20
0
C min
-1
under nitrogen atmosphere is shown in Fig 3(a). The TGA of the sample
shows that the temperature range from 133- 363
o
C associates with maximum weight loss. In
this region this zone of TGA can be referred to as active pyrolytic zone. The intial weight loss
in the temperature range 43- 133
o
C represents the evaporation of moisture contents physically
absorbed in the deoiled cake. There is also possibility of loss of some light volatile matters.
During the final stage of decomposition starts at 363
o
C, the rate of weight loss is very slow.
In initial stage 7.24%, in the active pyrolytic zone or the second stage of decomposition,

73

52.76 % and in the final stage 40% weight loss was observed. During the Second stage, the
intermolecular associations and weaker chemical bonds are destroyed [Jinno et al., 2004;
Chan et al., 2009]. The side aliphatic chains may be broken and some small gaseous
molecules are produced at the lower temperature [Biswal et al., 2013]. During the Third stage
at higher temperature chemical bonds are broken and the parent molecular skeletons are
destroyed. As a result, the larger molecule decomposes to form smaller molecules.
DTG curve (Fig 3(b)) shows that there is only one major peak at 294
o
C which was
present in active pyrolytic zone at temperature range 133-363
o
C.
Table 1: Proximate and ultimate analysis of CSDC
Properties Tea seed deoiled
cake
Biochar obtained
at 500
0
C
Rapeseed char
(Ucar et al., 2008)
Proximate analysis (wt%)
Moisture 4.34 0.27 6.23 0.34 -
Volatile matter 80.22 0.48 22.45 0.27 20.01
Fixed Carbon 10.66 0.41 38.46 0.02 16.41
Ash 4.78 0.66 9.78 0.41 18.54
Ultimate analysis (wt%)
Carbon 47.63 76.35 56.48
Hydrogen 7.34 3.76 3.22
Nitrogen 3.48 5.67 7.32
Oxygen 41.55 14.22 32.25
Sulphur - - 0.23
H/C molar ratio (on ash
free basis)
1.849 0.591 0.68
Emperical formula CH
1.849
N
0.062
O
0.654
CH
0.591
N
0.063
O
0.139
CH
0.08
N
0.114
O
0.43
S
0.001
Gross calorific value
(MJ/kg)
19.65 27.92 23.88

8.3.3 Effect of temperature on the product distribution
The pyrolysis of the tea seed deoiled cake yielded three different products viz. liquid,
gas and solid residue (biochar). Results of mass balance of pyrolytic decomposition products
were presented in Table 2. The bio-oil yield increased from 22.25% to 27.38% with increased
in temperatures from 400-500
o
C but decreased from 27.38% to 23.74% in temperature range
500-600
o
C. The maximum char yield 37.43% was obtained at temperature 400
o
C. The yield
of char decreased with increasing temperature. The decrease in char yield with increase in
temperature may be due to the higher decomposition of the biomass sample in higher
pyrolytic temperature or may be due secondary decomposition of char residue [Horn and
William, 1996]. The yield of gas increased with increase in temperatures. This is may be due

74

to the secondary decomposition of the char and secondary cracking of the pyrolysis may
enrich the content of the gas product at higher temperature.
0 200 400 600 800 1000
0
20
40
60
80
100
W
e
i
g
h
t

r
e
m
a
i
n
i
n
g

(
%
)
Temperature (
0
C)
(a)

100 200 300 400 500 600 700 800 900
100.0
100.2
100.4
100.6
100.8
101.0
101.2
101.4
D
e
r
i
v
a
t
i
v
e

Y
1

(
m
g
/
m
i
n
)
Temperature (
0
C)
(b)

Figure 3: (a) TGA and (b) DTG plot of Camellia Sinesis deolied cake
Table 2: Effect temperatures on product distribution of pyrolysis
Temperature
(
o
C)
Biochar
(wt%)
Bio-oil (wt%) Aquous Phase
(wt%)
Gas (wt%)
400 37.43 22.25 18.87
20.31
450 32.23 25.46 17.81
23.67
500 29.13 27.38 17.36
25.43
550 27.67 24.51 16.54
30.67
600 25.47 23.74 15.23
35.28

8.3.4 FTIR of the bio-oil sample obtained at 500
o
C
Biomass pyrolytic oil is composed of wide range of complex organic compound.
FTIR analysis was performed to investigate the chemical structure of the bio-oil sample.
Figure 4 shows the FTIR spectra of tea seed deoiled cake. The O-H stretching vibrations at

75

frequency 3379 cm
-1
indicates the presence of alcohol or phenols. The presence of alkanes is
detected at peaks around 2851 cm
-1
and 2930 cm
-1
with C-H stretching vibrations. C=O
stretching vibrations cause band at 1717 cm
-1
. The presence of alkenes was detected by C=C
stretching vibrations at 1610 cm
-1
. The peaks in the range of 9501300 cm
-1
show the
presence of CO stretching vibrations present in alcohol or ester. the C-H bending vibrations
frequency 812 cm
-1
indicates the presence of phenyl ring substitution bands. The results were
found consistent when compared with the results of GC-MS.
4000 3500 3000 2500 2000 1500 1000 500
20
30
40
50
60
70
80
90
100
110
%

T
r
a
n
s
m
i
t
t
a
n
c
e
Wavenumber (cm
-1
)
O-H
C-H
C=O,C=C
C-O,C-H

Figure 4: FTIR of CSDC bio-oil
8.3.5 GC-MS of the bio-oil sample
The GC-MS analysis (Fig.5) of the oil sample is summarized in Table 3. More than
40 peaks are displayed in the GC/MS chromatograph but because of the complex nature the
perfect separation of all the peaks are not possible and also the depending on strength of MS
library, 10 peaks are evaluated. Comparing the mass spectra fragmentation pattern with
Perkin Elmer NITS library and published data, the highest likelihood of compounds
identification were obtained. The carbon distribution of the identified compounds were in the
range of C
5
-C
29.

Figure 5: GC-MS of CSDC bio-oil
Scan
TI
1.78e
I-(b)_15-5-
0
%
10
10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0
Tim
8.5
9.9
10.1
11.3
14.6
16.4
21.2
21.9
22.5
23.9
24.4

76

Table 3: Compound identified by GC-MS of CSDC bio-oil
S.No. T
R
(min) Tentative compounds Empirical
formulae
1 8.57 2-PYRIDINECARBOXYLIC ACID, METHYL ESTER C
7
H
7
NO
2

2 9.93 DECANOIC ACID, 2-METHYL- C
11
H
22
O
2

3 10.14 3-FURANMETHANOL C
5
H
6
O
2

4 11.37 FURAN, 2,4-DIMETHYL- C
6
H
8
O
5 14.67 MEQUINOL C
7
H
8
O
2

6 16.47 PHENOL, 2-METHOXY-4-METHYL- C
8
H
10
O
2

7 21.28 N-TETRACOSANOL-1 C
24
H
50
O
8 21.94 1-HEPTACOSANOL C
27
H
56
O
9 22.53 METHYL OCTACOSANOATE C
29
H
58
O
2

10 23.97 CYCLOTRISILOXANE, HEXAMETHYL- C
6
H
18
O
3
Si
3

8.3.6 Physical properties of the bio-oil
Table 4 shows the results of the elemental analysis of the CSDC bio-oil. The result
shows that the bio-oil has higher heating value of 32.25 KJ/kg which is higher than some
bio-oils produced from different de-oiled cake such as Neem cake bio-oil (30 MJ/kg) [Volli
et al., 2012], Mustard de-oiled cake (25.2 MJ/kg) [Volli et al., 2012].
Table 4: Ultimate analysis of bio-oil
PARAMETERS CSDC BIO- OIL
C 71.35
H 8.05
N 4.65
O 15.95
H/C 1.354
O/C 0.167
EMPERICAL FORMULA
Higher Heating Value(MJ/kg)
CH
1.354
N
0.055
O
0.167
32.25

The comparison of the various necessary properties of the oil obtained from the
mustard de-oiled cake and diesel is shown in Table 5. The viscosity of the bio-oil is
comparatively higher than that of diesel which may lead to poor atomization and incomplete
combustion. Therefore the bio-oil is not suitable for direct use as a engine fuel but can be use
in moder diesel engine by blending with the diesel. The higher heating value of bio-oil is also
lower than that of diesel engine.


77

Table 5: Fuel properties of of CSDC pyrolyitic oil
Properties Standard test method Pyrolytic oil Diesel [Tuttle
et al. 2004]
Kinematic viscosity, 40
0
C ASTM D 445 28 2-5.5
Flash point ASTM D 93 62 53-80
P
H
P
H
meter 3.24 -
Acid Number ASTM D664 33.56 -
Higher heating value (MJ/kg) - 32.25 42-45
Appearance

- Dark brown
Chemical formula Identified C
5
-C
29
C
8
-C
25

8.4 Conclusion
Pyrolysis of Camellia Sinesis de-oiled cake was carried out in a fixed-bed reactor
made up of stainless steel at temperature range from 400
o
C to 600
o
C and at a rate of 30
o
C
min
-1
to produce bio-fuel. The maximum yield of oil is 27.38% on wt. % basis for Camellia
Sinesis de-oiled cake, was obtained at a temperature of 500
o
C. The fuel and chemical analysis
of bio-oil reveals that these pyrolytic oils can be used as fuel and as a source of chemicals.
The carbon distribution in the chemical identified in bio-oil is in range C
5
-C
29.

References
1. Biswal B., Kumar S. and Singh R.K.(2013).Production of hydrocarbon liquid by thermal
pyrolysis of paper cup waste. Journal of Waste Management, URL:
http://dx.doi.org/10.1155/2013/731858.
2. Bridgwater A.V. and Peacocke G.V.C., (2004).Fast pyrolysis: Fast pyrolysis processes
for biomass. Renewable and Sustainable Energy Reviews, 4:41-73.
3. Chan P.W., Atrey A., Howard R.B. (2009). Determination of pyrolysis temperature for
charring materials. Proceedings of the combustion Institute, 32: 2471-2479.
4. Horne P.A., Williams P.T. (1996). Influence of temperature on the products from the
flash pyrolysis of biomass. Fuel, 75: 1051-1059.
5. Jinno D., Gupta A.K.(2004). Determination of chemical kinetic parameters of surrogate
solid wastes. J.Eng. Gas. Turb. Power, 126: 126-685.
6. Mulligan C.J., Strezov L., Strezov V., (2010). Thermal decomposition of wheat straw
and mallee residue under pyrolysis condition. Energy and fuel, 24:46-52.

78

7. Ozcimen D, Karaosmanoglu F. (2004) Production and characterization of bio-oil and
biochar from rapeseed cake. Renew Energy; 29:77987.
8. Raja S.A., Kennedy Z.R., Pillai B.C., Lee C.L.R.(2010). Flash pyrolysis of jatropha oil
cake in electrically heated fluidized bed reactor. Energy; 35:281923.
9. Tuttle J., Kuegelgen V., (2004). Biodiesel Handling and Use Guidelines, Third edition in
National Renewable Energy Laboratory.
10. Ucar S., Ozkan A.R. (2008). Characterization of products from the pyrolysis of rapeseed
oil cake. Bioresour Technol; 99:87716.
11. Uzun BB, Putun AE, Putun E. (2006) Fast pyrolysis of soybean cake: product yields and
compositions. Bioresour Technol; 97:56976.
12. Volli V., Singh R.K. (2012). Production of bio-oil from de-oiled cakes by thermal
pyrolysis. Fuel, 96: 579-585.


79

CHAPTER 9
COMPARATIVE STUDY OF DIFFERENT BIOMASS
COOKSTOVE MODEL: AN EXPERIMENTAL STUDY
K Pal, A K Pandey, P Gera and S K Tyagi
Abstract
This article presents the comparative experimental study of five different types of improved
biomass cookstoves models based on their thermal efficiency, power output rating and
emission reduction potential. All the cookstoves models are design and fabricated in the
laboratory based on the gasification principle except NIRE-02, which is the modified form of
the traditional cookstove. The performance of each cookstoves were evaluated following
water boiling test as per Bureau of Indian Standard (BIS) protocols, whereas the emission
reduction was calculated based upon the clean development mechanism (CDM) of United
Nation Framework Convention on Climate Change (UNFCCC). The overall performances of
these models were found to be much better than that of the traditional cookstoves being used
by the majority of population around the globe. The emission reductions potential from these
models were found to be between 2.0-3.0 tons per household annually, which not only shows
the good agreement with the experimental values available in the literature but also represent
a high potential for disseminating these cookstoves through CDM in the rural and remote area
of the country, especially, where woody biomass is the major consumption as the cooking
fuel.
Keywords: Energy efficiency, Improved biomass cookstove, Clean development mechanism.
9.1 Introduction
The past evidences of fire have been found as old as four lakh years i.e. during the
first ice age (Bronowski, 1973). However at that time fire was used for roast the meat.
Mastery of fire is considered to be an important step toward human development which took
off only about 12,000 years ago (FOA, 1993). A three stone fire arrangement was first time
was used by people of ancient time for cooking their food. The use of three stone fire
arrangement has many health and environment problems like exposure to heat as well as fire
hazard beside the problem of poor thermal efficiency. The requirement for a better cookstove



80

arose which gave rise to U-shaped mud cookstove/traditional cookstove. The traditional
cookstoves solved many of the technical problems such as, enhancement of thermal
efficiency by many folds. However, the health, socio-economic and cultural problems were
not solved completely.
The development of efficient cookstoves at International level was started in the
1940s (Anhalt and Holanda, 2013). However, in India the efforts for making improved
biomass cookstoves started during 1950s with the main objective to improve the design of
biomass-fired stoves and the widespread R&D started in 1970s. Later on, improved
cookstove programs (ICPs) were considered as a solution to the fuel wood crisis and to
reduce deforestation. A model of improved multiport stove was introduced by Raju in 1953
which was one of the high-mass and shielded-fire type and had a chimney to remove smoke
and adjustable metal dampers to regulate the fire. Singer (1961) in Indonesia conducted
cookstove efficiency tests on high mass mud stove with the main objective to increase
efficiency and save fuel, without considering the socio-economic and cultural aspects.
However, in 1990s the focus shifted more on the issues involving the Indoor air pollution and
its effect on human health (World Bank, 2011).
In addition to above factors, the comfort of cooking, smoke free kitchens,
convenience and safety of stove users were tested and considered to be the important aspects
as compared to the fuel savings. Smith et al., (2000) stated that the burning of biomass fuels
emits high levels of smoke, containing hazardous pollutants such as, respirable suspended
particulate matter (RSPM), carbon dioxide (CO
2
), carbon monoxide (CO), nitrogen oxides
(NO
x
), sulfur oxides (SO
x
) and some cancer causing organic compounds like Benzopyrene,
benzene and 1,3-Butadiene. Fullerton et al., (2008) found that different types of health risks
were associated with the indoor air pollution such as, respiratory infections like pneumonia,
tuberculosis, chronic obstructive pulmonary disease, birth defects, cataracts, cardiovascular
events which cause serious problems in the women and child. Kleeman et al., (2000) studied
the adverse health impacts of the particle size distribution and result of deposition of those
pollutants in different areas of lungs.
Keeping the above facts in mind, Ministry of New and Renewable Energy,
Government of India has initiated a National level program to develop next-generation
cleaner cookstoves and deploy them to the households where traditional cookstoves being
used for cooking and heating applications. The initiative has set itself the lofty aim of

81

providing energy service comparable to clean sources like LPG without changing the fuels.
Based on National surveys, published literature and assessments, and measurements of
cookstove performance around the globe, it was found that about 570,000 premature deaths
in poor women and children and over 4% of India's estimated greenhouse emissions could be
avoided if such an initiative were in place. Although, the current advanced biomass stoves
show substantial emissions reductions over the traditional stoves but there is a lot to be done
to reach the LPG-like emission levels.
WHO (2011) has estimated that every year indoor air pollution (IAP) is responsible
for the death of 1.8 million people around the globe which comes out to be one death every
20 seconds. It was further estimated that the exposure to smoke from simple act of cooking
constitutes the fifth worst risk factor for disease in the developing countries and causes
almost two million premature deaths per year, exceeding the deaths attributable to malaria or
tuberculosis (WHO, 2006). Ramanathan and Carmichael, (2008) recently found that the black
carbon is playing a major role in the global warming.
The Global Alliance for Clean Cookstoves (GACC), a new publicprivate partnership
led by the UN Foundation was established to create a thriving global market for clean and
efficient household cooking solutions (Global Alliance for Clean Cookstoves, 2011). The newly
cookstoves which are known as advanced biomass cookstoves are based on better design
principles; they have the better combustion efficiency and thus, reduce the fuel consumption
to a greater extent. These cookstoves can deal with both the emissions and health issues
resulting from cooking with open fires or traditional biomass cookstoves. These cookstoves
have the ability to get carbon credits (UNFCCC, 2013), not only because of their contribution
to climate-change mitigation but also they yield major co-benefits in terms of energy access
for the poor people. Although, the current advanced biomass stoves show substantial
emissions reductions over the traditional stoves, yet more improvements are needed to reach
the LPG-like emission levels.
9.2 Mathematical Modeling
An improved biomass cooking stoves have the ability to reduce indoor air pollution,
deforestation, climate change, and improve quality of life of rural peoples on a global scale.
The better design of these cookstoves can significantly impact their performance and
emissions. Although these improved biomass stoves have been studied for a long time
however, a theoretical understanding of their operating behavior and the development of

82

engineering tools for an improved cookstove based on natural convection is still lacking. This
section presents the mathematical modeling of improved biomass cookstove based on various
performance parameters as below:
9.2.1 Side feeding wood-burning cookstove
Agenbroad et al. (2011a) developed a simplified model for the understanding the
fundamental operating behaviour of these natural convection based biomass cookstoves. This
model was developed utilize the dimensional form of two equation system. This has been
further developed into a dimensionless form at a later stage (Agenbroad et al. 2011b). A
simplified model of the fundamental stove has been developed for predicting bulk flow rate,
temperature, and excess air ratio, based on stove geometry including chimney height,
chimney area, viscous and heat release losses and the operating firepower. The stove operator
decided the operating firepower of the stove by controlling the fuel feed rate, excess air ratio.
The resulting bulk flow rate, temperature, and excess air ratio etc. are the fundamental inputs
for stove performance. The processes are categorized into two basic and fundamental, (a)
heat addition from combustion, and (b) kinetic energy addition due to the chimney effect, and
the details are given as below:
9.2.2 Heat addition from combustion
The heat of combustion increased the temperature and decreased the density of bulk
flow passed over there. If we assume that the heat addition is efficient and instantaneous and
the system is isobaric with no mechanical work done on/by the system, having ideal gas
behavior, with constant potential and kinetic energies. The heat addition from the combustion
showed an enthalpy increase (h
C
to h
H
) distributed in the mass flow rate (
A m
.
). The increased
bulk flow temperature is calculated for a given mass flow rate
A m
.
, heat release rate
in
Q
.
, using
the constant pressure specific heat of air (C
p
), as below (Kumar et al., 2013):
) (
,
.
) (
.
) (
. .
C
T
H
T
avg p
C A m
H
T
c
T
dT T
p
C A m
H
h
C
h A m
in
Q = = =
(1)

where T is the bulk flow temperature and the subscripts H and C denote the hot and cold
conditions, respectively.
9.2.3 Kinetic energy addition due to the chimney effect
The air flow through the stove depends on the chimney effect resulting from the
buoyant force of the decreased density of air after heat addition of combustion. The decreased

83

density of air in the chimney creates a lower pressure as compare to the ambient pressure. In
a fluid, the relationship between pressure variation with depth can be determined using the
hydrostatic equation and the net pressure difference (P) as given below (Kumar et al.,
2013):
= dh h g P ) ( (2)
where g is the acceleration due to gravity, and is the density of medium which is a function
of chimney height (h). The pressure can be calculated as below (Kumar et al., 2013):

= + + =

2
3
2
3
3
1
3
3 2 1
) ( ) ) ( ( ) ( dh h g h g dh h g P dh g P P
amb amb
(3)
If chimney walls are assumed to be adiabatic, than the temperature and density,
of the flue
gases in the chimney (T
H
and
H
) remain constant, so Eq. (3) can be simplifies which
determined the chimney effect due to the pressure difference as (Kumar et al., 2013):
) (
2 1
H amb
gh P =

(4)
The gain in kinetic energy from the stagnant ambient air can be calculated using the integral
form of Bernoulli's equation for the compressible flow, as (Kumar et al., 2013):
2
2
2
1
) (
2 1
v gh P
H H amb
= =
(5)
where
H
is the density of hot gas and
2
v
is the velocity use for calculating the kinetic
energy. Again assuming ideal gas behavior of the flue gases, the density is related to the
temperature by the ideal gas law. Using ideal gas equations and solving Eq. (5) for volume
and mass flow rate gives (Kumar et al., 2013):
2
.
2
1
) (
=
A
V
gh
H H amb

(6)
H
H amb
gh CA V

= 2
.
(7)
amb
amb H
T
T T
gh CA V

= 2
.
(8)
amb
amb H
H s
A
T
T T
gh
T R
P
CA m

= 2
1
.
(9)

84

where C is the loss coefficient introduce to account for uncertainties and inefficiencies in the
chimney effect including viscous losses, chimney wall heat transfer, and the unrealistic ideal
point heat addition at state point 2 and its range is 0 C 1. The mass flow rate of fuel (
F
m
.
)
in the cookstove model can be calculated from the firepower (
in
Q
.
) and the heating value (HV)
of the fuel as (Kumar et al., 2013):
LHV
Q
m
F
.
.
= (10)
The air fuel ratio (AFR) and excess air ratio (EAR) can be determined as below (Kumar et
al., 2013):
.
.
F
A
m
m
AFR = (11)
AFR
AFR
stoich
=
(12)

=
% 100 ). 1 (
%EAR
(13)
where AFR
stoich
is the air furl ratio (AFR) for stoichiometric combustion. The lower heating
value (LHV) is used because the latent heat of the water vapor is significant, and seldom
recovered.
9.2.4 Dimensionless chimney effect equation
The advantages of working in the dimensionless form include the scale similarity and
reducing the number of independent parameters for experimentation independent of stove
geometry. The dimensionless temperature form can be determined by inspection and is given
as below (Kumar et al., 2013):
amb
amb H
T
T T
T

*
(14)
Using Eq. (14) and Eq. (9), the mass flow rate of air in the cookstove model can be
expressed as given below (Kumar et al., 2013):
) 1 (
2
*
*
.
+
=
T
ghT
T R
P
CA m
amb
A
(15)

85

Substituting P/RT
amb
as the ambient density
amb
in the above equation and rearranging it as
(Kumar et al., 2013):
1
2
*
*
.
+
=
T
T
gh CA
m
amb
A
(16)
This dimensionless mass flow rate can also be defined as the ratio of the actual mass
flow rate to the characteristic natural convection and from Eq. (16), the dimensionless mass
flow rate of air for the given geometry given as below (Kumar et al., 2013):
gh
amb
CA
A m
A m
.
*
.

(17)
Using the dimensionless mass flow rate
*
.
A m of Eq. (17), the final form of chimney
effect equation is given as below (Kumar et al., 2013):
1
2
*
*
*
.
+
=
T
T
mA
(18)
9.2.5 Dimensionless heat addition equation
The mass burn rate of fuel is used instead of the firepower. The relation between the
firepower, mass burn rate and heating value is given as below (Kumar et al., 2013):
HV m Q F
in
. .
=
(19)
Substituting Eq. (19) and Eq. (14) into Eq. (1) and rearranging it, a dimensionless heating
value group (HV
*
) is formed as (Kumar et al., 2013):
*
. .
T m
T C
HV
m A
amb p
F =
(20)
amb p
T C
HV
HV
*
(21)
This group defined as the ratio of the combustion heating energy to the initial thermal
energy of the flow. Substituting the dimensionless mass air flow rate as defined in Eq. (17),
into Eq. (20) which gives the relation between dimensionless heating value, dimensionless
mass flow rate of air and dimensionless temperature as (Kumar et al., 2013):

86

* *
.
*
.
T gh
amb
CA
A
m HV
F
m =

(22)
A dimensionless mass burn rate similar to the dimensionless air flow rate of Eq. (17) is given
as below (Kumar et al., 2013):
gh CA
m
m
amb
F
F
.
*
.
(23)
Using the dimensionless mass burn rate
*
.
F m , the final form of the dimensionless heat addition
equation becomes (Kumar et al., 2013):
* *
.
* *
.
T m HV m A F =
(24)
9.2.6 Air/fuel ratio from dimensionless model
The air/fuel ratio (AFR) is defined as the ratio of mass flow rate of air to the mass
burn rate of fuel and can be given by rearranging Eq. (24) as shown below (Kumar et al.,
2013):
*
*
*
.
*
.
T
HV
F
m
A
m
AFR = =

(25)
The dimensionless heating value (HV
*
) is considered as remain constant throughout the stove
operation. From the above equation a simple inverse linear relationship can be seen between
dimensionless temperature (T
*
)

and the air fuel ratio (AFR).
A comparative study on modified traditional cookstove and three different improved
biomass cookstoves (NIRE-03, NIRE-05, NIRE-06) has been presented in this research
article. These improved cookstove work on the principle of down-draft gasifier where
pyrolysis, gasification and combustion of biomass are taking place simultaneously. These
cookstoves were made of mild steel whereas for insulation clay and wheat straw mixture was
used. The line diagram of NIRE-02, NIRE-03, NIRE-05 and NIRE-06 are shown in the figure
1-4.
9.3.1 Materials Use
The following instruments/equipments have been used during the experiments:
(a) Platform balance

87

(b) Glass cylinders for measuring water
(c) Aluminium vessels with lids of proper volumes as per BIS standard
(d) Kerosene oil to ignite the process
(e) Match stick
(f) Stopwatch
(g) Thermometer/Thermo-couple
(h) Bomb calorimeter
(i) Wood fuel in proper size

Space for flame
Air
200
260
190
200
40
35
35
260
Grate
Platform
Fig. 1: Line diagram of the modified traditional Cookstove, (NIRE-02)

88

Fig. 3: Line diagram of improved cookstove (NIRE-05)
410
25
253
Primary air inlet
50
60
50
125
Wick
Handle
Grate
Pot support
Secondary air inlet
25
15
Insulation
23
50
Top view
Bottom view
Handle for primary
Air adjustment
Handle for primary
Air adjustment
25
410
Bottom view
Primary air inlet
Secondary air inlet
50
50
60
50
150
Wick
Handle
Grate
Pot support
25
15
Insulation
23
Top view
253

89

9.3.2 Methods
As per the Bureau of Indian Standards (BIS, 2013) water boiling test of all above
mentioned four cook stoves were performed for the measurement of thermal efficiency.
According to the BIS protocol stepwise procedure was follow for cookstove testing. At the
time of experimental run, the temperature of water, flame and cookstove body was measured
with the help of digital temperature sensor whereas the temperature of pot, plate and ambient
was measured with mercury-glass thermometer. The measured value of thermal efficiency of
each cookstove is compared with the open three stone fire cookstove which generally have
the thermal efficiency 8-10%. The stepwise procedure of fuel preparation, burning capacity
rate and water boiling test is as follow:
a) Fuel Preparation
The fuel wood cut from the same log into pieces of 3x3 cm square cross-section and
length of half the diameter/length of combustion chamber so as to be housed inside the
combustion chamber. The fuel pieces shall be oven dried by the following method (BIS,
2013):
a) Weigh total quantity of wood (say 'M' kg.).
b) Pick up one piece and mark 'X' by engraving and take its mass (say 'm' g.).
230
Secondary air inlet
Handle for primary
Air adjustment
Primary air inlet
25
50
60
410
50
110
Wick
Handle
Grate
Pot support
25
15
Insulation
45
50
Air gap
Bottom view
Top view

90

c) Raise the temperature of oven up to 105 C.
d) Stack the wood pieces in a honey comb fashion inside the oven.
e) Maintain the oven temperature at 105 C.
f) After 6 hours, remove the marked 'X' piece, weigh it and note reduction in mass from
'm' g, if any. If reduction is observed put the marked piece in the oven again and
repeat the weighing of 'X' marked piece after every subsequent 6 hours period till the
mass is constant and no further reduction in mass is observed.
g) At this stage, weigh the total quantity of wood and note loss of mass from 'M' kg.
h) Determine the calorific value of the prepared wood with the help of bomb calorimeter.
b) Burning Capacity Rate
Fuel burn per hour is known as the burning capacity rate of a cookstove, which can be
calculated as: Stack the combustion chamber with test fuel in honey comb fashion up to 3/4
of the height or in a pattern recommended by the manufacturer. Sprinkle 10 to 15 ml. of
kerosene on the fuel from the top of chulha/fire box mouth. Weigh the chulha with fuel; let
the mass be M
1
kg. After half an hour of lighting weigh the chulha again and let the mass be
M
2
kg. If the calorific value (CV) of the test fuel in kcal/kg then calculate the burning
capacity of the chulha as heat input per hour as follows (BIS, 2013):
Heat input: per hour = 2 (M
1
- M
2
) x CV kcal/h (26)
c) Water Boiling Test
a) Take the test fuel according to burning capacity rating for one hour. Let the mass be
'X' kg.
b) Stack the first lot of test fuel in the combustion chamber in honey comb fashion or as
indicated by the manufacturer.
c) Select and weigh the vessel with the lid in accordance with the table above. A
minimum of two such vessels in a set will be required. Put the recommended quantity
of water at 23 + 2 C (T
1
).
d) Sprinkle measured quantity 'X' ml. (say 10 - 15 ml.) of kerosene for easy lighting on
the test fuel and light. Simultaneously start the stop watch.
e) Feeding of fresh test fuel lot shall be done after every 15 minutes.
f) The water in the vessel shall be allowed to warm steadily till it reaches a temperature
of about 93 C, then stirring is commenced and continued until the temperature of
water reaches 5 C below boiling point at a place. Note down time taken to heat the

91

water up to final temperature (less than 5 C below the boiling point) T
2
C.
g) Remove the vessel of from the chulha and put the second vessel immediately on the
chulha. Prepare first vessel for subsequent heating.
h) Repeat the experiment by alternatively putting the two vessels taken till there is no
visible flame in the combustion chamber of the chulha. Note down the temperature of
the water in the last vessel.
9.4 Analysis of Cookstove
Improved biomass cookstoves can reduce indoor air pollution, deforestation, climate
change, and therefore, the quality of life can be improved on a universal scale. The better
design of these cookstoves can significantly impact their performance and emissions.
Although these improved biomass cookstoves have been studied for a long time however, a
theoretical understanding of their operating behaviour and the development of engineering
tools for an improved cookstove based on natural convection is still missing.
9.4.1 Thermal efficiency
Thermal efficiency of a cookstove may be defined as the ratio of heat utilized to the
heat produced by complete combustion of a given quantity of fuel based on the net calorific
value of the fuel and this can be written as below [Kumar et al., 2013, Kishore and Ramana,
2002, Tyagi et al., 2013, BIS, 2013].

100
Produced Heat
Utilized Heat
efficiency Thermal =
(2)
Heat utilized= )} T - 8)(T 4.186 w + 0.896 )(W T - 8)(T 4.186 w + 0.896 1)(W - {(n
1 3 1 2
kJ
Heat produced = /1000)} c (V + ) c {(X 8 4.186
2 1
kJ

100
)} 1000 / ( + ) c {(X 8 4.186
)} T - 8)(T 4.186 w + 0.896 )(W T - 8)(T 4.186 w + 0.896 1)(W {(n-
2 1
1 3 1 2

=
c V

where w is the mass of water in vessel, W is the mass of vessel complete with lid and stirrer,
X is the mass of fuel consumed, c
1
is the calorific value of wood, V is the volume of kerosene
consumed, c
2
is the calorific value of kerosene, is the density of kerosene, T
1
is the initial
temperature of water, T
2
is the final temperature of water, T
3
is the final temperature of water
in last vessel at the completion of test, in C; and n is the total number of vessels used.

92

9.4.2 Exergy efficiency
Exergy efficiency may be defined as the ratio of output exergy to the input exergy and
given by [Dincer, 2007, Tyagi et al., 2013]:
100
c d x )
T
T
(1 c m
)
T
T
)(1 T (T C m )
T
T
)(1 T (T C m
100
E
E
100
input Exergy
output Exergy
2
fuel
a
1 wd
fp
a
ip fp 1 pA pot
fw
a
iw fw p w
in
o
+
+
=
= =
x
x
(3)
where m
w
is the mass water in the pot, C
p
is the specific heat of water, T
fw
is the final
temperature of water, T
iw
is the initial temperature of water, m
pot
is the mass of pot, C
pA1
is
the specific heat of Aluminium pot, T
fp
is the final temperature of pot, T
ip
is the initial
temperature of pot, m
wd
is the mass of wood, c
1
is the calorific value of wood, T
a
is the
ambient temperature, T
fuel
is the flame temperature, theoretical efficiency, x is the volume
of kerosene, d is the density of kerosene and c
2
is the calorific value of kerosene.
9.4.3 Power Output Rating
The power output rating of a cookstove is a measure of total useful energy produced
during one hour burning of fuel wood. It shall be calculated as follows (BIS, 2013):
kW ,
100 3600
CV
rating output Power

=
m
(4)
where m is the quantity of fuel wood burnt per hour, CV is the calorific value of fuel wood
and is the thermal efficiency of the cookstove, as calculated above.
9.4.4 Emission reduction calculations
The emission reduction from each cookstove is based upon fraction of biomass that
can be saved during the project year and the calorific value of the biomass used and this can
be calculated according to the formula given below (Global Alliance for Clean Cookstoves,
2011):
fossilfuel projected biomass y NRB savings y y
EF NCV f B ER
_ , ,
=
(5)
where
y
ER

is emission reductions during the year y in tCO
2
,
savings y
B
,

is quantity of woody
biomass that is saved in tonnes,
y NRB
f
,
is the fraction of woody biomass saved by the project

93

activity in year y that can be established as non-renewable biomass,
biomass
NCV is the net
calorific value of the non-renewable woody biomass that is substituted (IPCC default for
wood fuel, 0.015 Tj/tonne),
fossilfuel projected
EF
_
is emission factor for the substitution of non-
renewable woody biomass by similar consumers, use of value of 81.6 tCO
2
/TJ and
savings y
B
,

can be calculated using the following formula:
=
new
old
old savings y
B B
1 .
,
(6)
Where B
old
is Quantity of woody biomass used in the absence of the project activity in
tonnes,
old
is the efficiency of the baseline system being replaced, measured using
representative Sampling methods or based on referenced literature values (fraction), use
weighted Average values if more than one type of system is being replaced; a default value of
0.10 may be optionally used if the replaced system is the three stone fire or a Conventional
system with no improved combustion air supply or flue gas Ventilation system i.e., without a
grate or a chimney; for other types of systems a Default value of 0.2 may be optionally used
and
new
is the efficiency of the system being deployed as part of the project activity
(fraction), as Determined using the water boiling test (WBT) protocol. Use weighted average
Values if more than one type of system is being introduced by the project activity.
Based on the biomass characteristics, the performance of different cookstoves has
been carried out using burning capacity rate, power output, thermal efficiency and carbon
emission reduction analysis at a typical location in India, while the discussion of results is
given as below:
Figure 5 shows the burning capacity rate and power output rating of the different
model of cookstoves. As shown from the figure the burning rate and power output of NIRE-
02 are 3.6 kg/hr and 4.80 kW respectively. The burning rate and power output of NIRE-03
are 2.42 kg/hr and 3.45 kW respectively. NIRE-05 has the burning rate 3.22 kg/hr with 5.23
kW power output rating and NIRE-06 has the value of these quantities 2.32 kg/hr and 3.22
kW respectively. The maximum power output is found with NIRE-05 with 3.22 kg/hr
burning rate which is the highest among all the other improved biomass cookstoves (NIRE-
03 and NIRE-06) and also from the modified traditional cookstove. These results are shows
that the combustion bf biomass in NIRE-05 is better than that of the other cookstove models.

94

Fig-5: Burning rate and power output of different cookstove model.

On the basis of results obtained from the water boiling test the thermal efficiency,
exergy efficiency and emission reduction of different cookstove models was calculated using
the equation (2)-(5) and the results obtained are presented in Fig 6. The thermal efficiency,
exergy efficiency and emission reduction potential of modified traditional cookstove (NIRE-
02) was found to be 25.31%, 1.86% and 2.08 tCO
2
/year respectively. The performance of this
modified model (NIRE-02) is two to three folds higher that of the traditional three stone fire.
This cookstove model also has the good agreement with the carbon emission reduction as
shown in the figure. However the thermal efficiency of improved cookstove models NIRE-
03, NIRE-05 and NIRE-06 are 35.19%, 37.47%, and 32.26 percent respectively, exergy
efficiency 6.17%, 6.23% and 5.45% respectively and emission reduction potential from
different model was 2.46, 2.52, and 2.37 tCO
2
/year respectively. Among all the improved
cookstove models, NIRE-05 has the maximum value of thermal efficiency, exergy efficiency
and as well as the CO
2
emission reduction potential. This is because of the fact that the
burning of the fuel wood is very good in this cookstove with higher power output and
minimum heat loss into the environment. Based on the results obtained from different
cookstove models it is found that the NIRE-05 cookstove is a best designed model which is
steadied in this present study.
3.6
2.42
3.22
2.32
4.80
3.45
5.23
3.22
NIRE-02 NIRE-03 NIRE-05 NIRE-06
Cookstove Model
Burning Rate(kg/hr) Power Output(kW)

95

Fig-6: Thermal efficiency, Exergy efficiency and emission reduction of different cookstove
at burning capacity rate.

9. 6 Conclusions
Based upon the analysis of experimental data following conclusions are drawn:
Based on the experimental observations the energy and exergy efficiency and as
well as the emission reduction potential for NIRE-02 model was found to be
best when both grate and top space was provided.
The thermal efficiency 25.31% of modified traditional cookstove NIRE-02 was
found which is around two-three times higher than that of the open three stone
fire.
The performance and the CO
2
emission reduction potential of NIRE-05 model is
found to be higher than that of all other models for all set of operating
parameters.
NIRE-05 model performs best and gives a large amount of emission reduction
value i.e. 2.52 tonnes of CO2 reduced per household per year which means if
approximately 10,000 NIRE-5 cookstove are provided to the consumers it will
save approximately 25,200 tonnes of CO2 in one year.
Also energy efficiency was found to be always higher than that of exergy
efficiency which is due to the energy gained by the hot water at that particular
temperature.
25.31
35.19
37.47
32.26
1.86
6.17 6.23
5.45
2.08
2.46 2.52 2.37
NIRE-02 NIRE-03 NIRE-05 NIRE-06
Cookstove Model
Thermal Efficiency, (%) Exergy Effficiency ,(%) Emission Reduction (tCO2/year)

96

Acknowledgment
Authors gratefully acknowledge the financial assessment provided by Sardar Swaran
Singh National Institute of Renewable Energy, Kapurthala and Ministry of New and
Renewable Energy, New Delhi.

References
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2. Agenbroad J, DeFoort M, Kirkpatrick A, Kreutzer C, A simplified model for
understanding natural convection driven biomass cooking stovesPart 1: Setup and
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3. Agenbroad J, DeFoort M, Kirkpatrick A, Kreutzer C, A simplified model for
understanding natural convection driven biomass cooking stoves-Part 2: With cook
piece operation and the dimensionless form, Energy for Sustainable Development
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4. Bronowski J. (1973) The Ascent of Man, Little, Brow and Co., Boston.
5. Bureau of Indian Standards. www.bis.org.in. Accessed 2013.
6. Fullerton D.G., Brucec N. and Gordona S.B. (2008) Indoor air pollution from
biomass fuel smoke is a major health concern in the developing world, Transactions
of the Royal Society of Tropical Medicine and Hygiene; 102: 843-851.
7. FAO, (1993) Improved solid biomass burning cookstoves: A development manual,
Regional Wood Energy Development Program in Asia, file document 44; accessed
from internet from wgbis.ces.iisc.ernet.in/energy/HC270799/RWEDP/acrobat/fd44.
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8. Global Alliance for Clean Cookstoves (2011) (http://www.cleancookstoves.org)
9. Household cookstove, Environment, health and climate change. The world bank,
2011 Accessed from internet on 13.09.13 climatechange.worldbank.org//
Household%20Cookstoves-web.pdf
10. http://cdm.unfccc.int/ accessed from internet on 30-08-13.
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development (Elsevier).

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12. Kleeman M.J., Schauer J.J. and Cass G.R. (2000) Size and composition distribution
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13. Kumar M., Kumar S. and Tyagi S.K. (2013) Design, development and technological
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14. Raju S.P. (1957) Smokeless Kitchens for the millions, Rev. edn., The Christian
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15. Ramanathan V, and Carmichael G (2008) Global and regional climate changes due
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16. Singer H. (1961) Improvement of fuel wood cooking stoves and economy in fuel
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17. Tyagi S.K., Pandey A.K., Sahu S., Bajala V. and Rajput J.P.S. (2013) Experimental
study and performance evaluation of various cookstove models based on energy and
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18. Smith KR, Uma R, Kishnore VVN, Zhang J, Joshi V and Khalil MAK (2000)
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20. WHO (2006) Fuel for Life. Household Energy and Health, Geneva.


98

Part III
Biochemical Conversion


99

CHAPTER 10
BIOPROSPECTING HALOTOLERANT CELLULASE FROM
SALINE ENVIRONMENT OF BHITARKANIKA NATIONAL
PARK, ODISHA
Dash Indira, Sahoo Moumita, Dethose Ajay, C. S. Kar, R. Jayabalan

Abstract
Research interest on biofuels gained much attention with depleting fossil fuel reserves and
growing concern on environment. Bioethanol and biodiesel from biomass will fulfil the future
energy needs. Lignocellulosic and algal biomass are sustainable and cost-effective resources for
bioethanol production. However, algal biomass is preferred over lignocellulosic biomass due to
its high cost of pre-treatment and production of fermentation inhibitors. Systems, which use non-
potable water for biofuel production, are considered due to the future threats on availability of
potable water. Halotolerant enzymes are one of the promising candidates to be used in seawater
based systems. Halotolerant enzymes can also be used for saccharifying the biomass, which are
pre-treated with ionic liquids. Odisha being a coastal state with a coastline of 450 Km and with a
mangrove ecosystem has potential to evidence halotolerant microorganisms, which can produce
salt tolerant enzymes. In the present study five isolates were confirmed for cellulase production
using CMC agar plates and Congo red assay. Activity of FP-endoglucanase produced by the
isolates was assayed by determining the amount of reducing sugar formed from Cellulose by
methods recommended by IUPAC. Endoglucanase produced by halotolerant microorganisms,
named as BHK1, BHK2, BHK3, BHK4 and BHK5 which were isolated from soil and water
samples were having optimal pH 5.0, 4.0, 5.0, 5.0 and 6.0 and optimal temperature of action at
65C, 45C, 65C, 55C and 65C respectively. Endoglucanase activity was found to be
enhanced several folds by use of 5 mM MnSO
4
as cofactor. Identification of microorganisms and
further characterization of the endoglucanase are in progress.
Keywords: Cellulase, halotolerant, optimal temperature, optimal pH, cofactor.



100

10.1 Introduction
In 2008, 88% of global energy consumption was dependent on fossil fuels (Brennan and
Owende, 2010). Fossil fuel however, now accepted as unsustainable resources due to its
depletion and increasing environmental concern due to accumulation of green house gases
(Schenk et al., 2008). To overcome the dependence on fossil fuels, maintenances of sustainable
economy and for global environmental concern, it is necessary to focus over and promote
renewable resources of energy (Brennan and Owende, 2010; Prasad et al., 2001 a,b; Singh et al.,
2010 a,b). Biomass is one of the most promising renewable resources for the production of
biofuels, such as bioethanol (John et al., 2011) and biodiesel (Ho et al., 2010). Most of the
bioethanol production across the globe comes from sugar and starch crops and from
lignocellulosic biomass. However, with increasing demand of food, decreasing water availability
and cost involved in pre-treatment of lignocellulosic biomass, agriculture crops are losing
importance. By overcoming the drawbacks of lignocellulosic and sugar rich biomass, microalgae
has gained the title of third generation feedstock for biofuel production (Nigam and Singh, 2011).
Microalgal biomass rich in carbohydrate forms an excellent substrate for bioethanol production.
It grows faster and fixes carbon dioxide at higher rate than terrestrial plants (Ho et al., 2012),
also, rich in starch and cellulose and lacks lignin, which makes monosaccharide conversion
easier (Ho et al., 2012, John et al., 2011). The carbohydrates in the form of starch or cellulose or
complex polysaccharides are not fermentable and therefore are hydrolyzed to fermentable sugar
prior to fermentation (Hagerdal-Hahn et al., 2007). Acid or alkali hydrolysis are common
chemical methods used as they are faster, cheaper and easier however, they lead to release of
components that acts as fermentation inhibitors and as a result hampers the fermentation process
(Harun et al., 2010; Girio et al., 2010; Moxley and Zhang, 2007). Enzymatic hydrolysis process
is amiable to environment and gives higher glucose yield without production of fermentation
inhibitors. Cellulases are the key enzymes of saccaharification of both lignocellulosic and algal
biomass. However, it is interestingly observed that in saline system, which is independent of
fresh water, cellulase and other glucoamylases are completely inhibited thereby making them
unsuitable for industrial usage (Matsumoto et al., 2003). There is expanding knowledge on
halophilic enzymes and organisms capable of effectively treating marine biomass. A recent
review on industrial and environmental applications of halophilic microorganisms states that

101

demand of salt tolerant enzymes and microorganisms is still limited (Oren, 2010), nevertheless,
continuous efforts in this field and favourable results may change the scenario. Potential salt
tolerant enzymes have been isolated from hypersaline microorganisms, which basically includes
the halophilic -amylase from Haloarcula hispanica (Vasisht et al., 2005), glucoamylases from
marine yeast Aureobasidium pullulans N13d (Oren et al., 2006), thermophilic and halophilic
amyloglucosidase from Halobacterium sodomense with optimal temperature between 66C to
76C and optimal activity between 8% to 22% of NaCl (Duan et al., 2006). An alkali-
halotolerant cellulase from Bacillus flexus NT, isolated from Ulva lactuca with residual activity
of about 70% at 15% NaCl concentration (Trivedi et al., 2010). Reports suggests that
commercially available accellerase-1500 (cocktail of different glycosidases) performs
depolymerisation of cellulose and avicel in reaction media containing 1X, 2X and 4X
concentration of seawater (Grande et al., 2012) . Regarding the fermentation of marine algal
biomass, minimal progress reported in the past 30 years after the reports of Clostridium
pasteurianum fermenting Dunaliella species to produce butanol, 1, 3-propanediol and ethanol in
presence of 10% NaCl. To overcome the salinity issues micro algae is either washed in deionised
water or grown in a less saline medium however, the salinity problem during fermentation can be
encountered by using halotolerant yeast. Citeromyces siamensis is novel halotolerant yeast
isolated from dry salted squid and fermented soybeans in Thailand (Nagatsuka et al., 2002)
which is tolerant to higher concentration of cations (3M NaCl and 0.8M LiCl) and osmotic
pressure (60% glucose). Pichia sorbitophila is also reported halotolerant yeast, which can grow
in 4M NaCl concentration when glucose and glycerol are sole carbon source (Lages and Lucas,
1995). The present work aimed to determine the effect of temperature, pH and metal ions on the
cellulase produced by salt tolerant cellulolytic bacteria isolated from Bhitarkanika National Park,
Odisha.
10.2 Materials and methods
10.2.1 Isolation of cellulase producing bacteria
Soil and water samples were collected from Bhitarkanika National Park, Odisha. Samples
were serially diluted and inoculated in Trypticase soy agar (TSA) media (15 g Tryptone, 5 g
Soytone, 5 g Sodium Chloride, 15 g agar/ 1 L Distilled water) and incubated at 37C for 24
hours. Individual colonies were obtained by streaking the culture on same media. Confirmation

102

of cellulose-degrading ability of bacterial isolates was performed by streaking on the CMC agar
media (5 g CMC, 1 g NaNO
3
, 1 g K2HPO
4
, 1 g KCl, 0.5 g MgSO
4
, 0.5 g yeast extract, 1 g
glucose, 17 g agar/ 1L Distilled water). The use of Congo red (1 mg/ml) as an indicator for
cellulose degradation in an agar medium and 1 M NaCl as destaining solution provides the basis
for a rapid and sensitive screening test for cellulolytic bacteria (CB). Colonies showing
discoloration of Congo red was taken as positive cellulose-degrading bacterial colonies (Lu et al.,
2004) and only these were taken for further study.
10.2.2 Cellulase enzyme production
The selected cellulolytic bacterial isolates were cultured at 37C at 150 rpm in 100 ml of
enzyme production media composed of 0.1 g NaNO
3
, 0.1 g KH
2
PO
4
, 0.0.1 g KCl, 0.5 MgSO
4
,
0.5 g yeast extract , 0.1 g glucose, 0.5 g filter paper at pH 6.87.2. Broth culture after three days
of incubation period was subjected to centrifugation at 5000 rpm for 15 min at 4C. Supernatant
was collected and stored as crude enzyme preparation at 4C for further enzyme assays (Tailliez
et al., 1989).
10.2.3 Cellulase assay
Filter paper cellulase activity was assayed by measuring the amount of reducing sugar
formed from soluble form of cellulose, CMC. Determination of enzyme activity is measured
using methods suggested by International Union of Pure and Applied Chemistry (IUPAC)
(Ghose, 1987). In these tests, 0.5 ml of crude enzyme extract is incubated with 0.5 ml of 2%
CMC in 10 mM citrate buffer at different temperatures and pH and the amount of reducing
sugars formed were estimated spectrophotometrically with 3,5-dinitrosalicylic acid using glucose
as standards (Miller, 1959). Then enzymatic activities of total endoglucanase were defined in
units. One unit of enzymatic activity is defined as the amount of enzyme that releases one
micromole reducing sugars (measured as glucose) per minute.
10.2.3.1 Effect of temperature on cellulase activity
The crude enzyme extract along with the substrate (2% CMC in 10 mM citrate buffer)
was incubated for 15 min at different temperatures such as 30, 40, 50, 60 and 70C to study their
effect on cellulase activity and assayed spectrophotometrically for their effect of enzyme activity.
10.2.3.2 Effect of pH on cellulase activity

103

The pH of the test solution was adjusted to different pH range of 3.0, 4.0, 5.0, 6.0 and 7.0
with phosphate buffer (10 mM Na
2
HPO
4
.2H
2
O, 1.8 mM KH
2
PO
4
) at their respective optimal
temperature for 15 minutes. The test samples were then assayed to study their effect on enzyme
activity.
10.2.3.3 Effect of metals on cellulase activity
After getting optimum temperature and pH value different metal ions such as MgCl
2
,
ZnCl
2
, MnCl
2
, FeCl
2
, CoCl
2
, EDTA and PMSF were subjected to study their effect on enzyme
activity. They all were taken in a concentration of (5 mM) and assayed.
10.3 Result and discussion
10.3.1 Isolation and screening of cellulase producing bacteria
Five strains of cellulolytic bacteria were isolated from soil and water samples from
Bhitarkanika National Park, Odisha, India. Three bacterial isolates from soil (BHK1, BHK2,
BHK3) and two from water (BHK4 and BHK5) were confirmed for cellulase production on
CMC agar plates on aerobic incubation of 48 hours at 37C by production of clear zone which
was identified using Congo red assay (Figure1).
10.3.2 Production of cellulase
All the five isolates (BHK1, BHK2, BHK3, BHK4 and BHK5) were cultured on enzyme
production medium containing filter paper as sole cellulosic substrate. All the isolates showed
cellulase activity on filter paper ranging between 0.93 EU/ml to 0.98 EU/ml. Highest cellulolytic
potential was exhibited by BHK1 with 0.98 EU/ml whereas lowest was recorded in BHK4 with
0.93 EU/ml (table 1).
10.3.3 Cellulase assay
Cell free supernatant collected from culture of BHK1, BHK2, BHK3, BHK4 and BHK5
was used as crude enzyme extract for determination of effect of temperature, pH and metal ions.
10.3.3.1 Effect of temperature on cellulase activity
Crude enzyme extract from all the five isolates (BHK1, BHK2, BHK3, BHK4 and
BHK5) were assayed at temperature ranging between 30C to 70C for 15 minutes. Maximum
enzyme activity was recorded by BHK1 at 65C, BHK2 at 45C, BHK3 at 65C, BHK4 at 55C

104

and BHK5 at 65C (Figure 3a). The above result in correlation with the fact that most of the
cellulases have optimal temperature of action at 55C.

Figure1: Clear zone formed on CMC agar plates by BHK1, BHK2, BHK3, BHK4 and BHK5
after 48 hours of incubation and stained with Congo red confirming the presence of extracellular
cellulase.
Table 1: Production of cellulase by BHK1, BHK2, BHK3, BHK4 and BHK5 in enzyme
production medium after 72 hours of incubation.
Cellulolytic bacterial strains Amount of cellulase produced after 72 hours
incubation (EU/ml/min)
BHK 1 0.98
BHK 2 0.95
BHK 3 0.96
BHK 4 0.93
BHK 5 0.93


105

10.3.3.2 Effect of pH on cellulase activity
Crude enzyme extract from all the isolates (BHK1, BHK2, BHK3, BHK4 and BHK5)
were assayed in pH ranging between 3.0 to 7.0. Optimal pH of action for all the five isolates
recorded as BHK1 at pH 5.0, BHK2 at pH 4.0, BHK3 at pH 5.0, BHK4 at pH 5.0 and BHK5 at
pH 6.0 (Figure3.b). Optimal pH of action ranging between 5.0 to 6.0 in most cases may be due to
the fact that all the isolates were from estuarine environment and not from purely saline sources.
10.3.3.3 Effect of metal ions on cellulase activity
With optimal temperature and pH value obtained from the above five cellulolytic isolates
(BHK1, BHK2, BHK3, BHK4 and BHK5) were assayed for effect of metal ions (5 mM
concentration each Of MgCl
2
, ZnCl
2
, MnCl
2
, FeCl
2
, CoCl
2
, EDTA and PMSF). In all cases,
enzyme activity is recorded maximum in presence of Mn
2+
. Other ions (Mg
2+
, Zn
2+
, Fe
2+
, Co
2+
,

EDTA and PMSF) did not show any substantial effect on the enzyme activity except Co
2+
in
BHK1, which enhanced the enzyme activity but not to such extent as Mg
2+
(Figure 3.c and d). It
may be inferred that Mn
2+
may act as cofactor or enhancer of enzyme activity in these filter paper
cellulases isolated from cellulolytic bacteria isolated from Bhitarkanika National Park, Odisha.
10.4 Conclusion
The present study focused on screening of cellulase producing organisms from saline
environment of Bhitarkanika National Park, Odisha. Five isolates tested positive for production
of extracellular cellulase and utilized for production of cellulase using suitable enzyme
production media with filter paper as raw source of cellulose. Crude enzyme so produced were
analysed for effect of temperature, pH and metal ions on their activity. All the potential cellulase-
producing organisms had enzyme ranging between 0.93EU/ml/min to 0.98 EU/ml/min. Optimal
temperature of enzyme action is between 45C to 65C suggests that the enzymes work at higher
temperature and at thermotolerant in nature. Optimal pH of action in the range 5.0 to 6.0 suggests
that strains from less saline environment. Endoglucanase activity was found to be enhanced
several folds in presence of 5 mM Mn
2+
suggesting its role as cofactor or enhancer molecule.
Identification of microorganisms and further characterization of the endoglucanase are in
progress. A further study also includes isolation of potential halotolerant cellulolytic bacteria
from more saline environment for their utilization in seawater-based medium.

106

(a) (b)

(b) (d)
Figure 3. (a) Effect of Temperature on enzyme action, (b) Effect of pH on enzyme action, (c)
Effect of metal ions on enzyme action and (d) Comparative effect of Mn
2+
on different strains of
cellulolytic bacteria.
Acknowledgement
Authors are very much thankful to MHRD and National institute of Technology,
Rourkela for the financial support for providing all research facilities and Office of the Principal
CCF (Wildlife) & Chief Wildlife Warden, Odisha for authorization of permission for sample
collection from Bhitarkanika National Park, Odisha.


107

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Kruse O., Hankamer B. (2008) Second generation biofuels: high-efficiency microalgae
for biodiesel production. Bioenergy Res., 1: 2043.
3. Prasad S., Singh A., Jain N., Joshi H. C.( 2007) Ethanol production from sweet sorghum
syrup forutilization as automotive fuel in India. Energy Fuels., 21: 24152420.
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industrial and urban residues. Resour Conserv Recy., 50: 139.
5. Singh A., Pant D., Korres N. E., Nizami A. S., Prasad S., Murphy J. D. (2010) Key issues
in life cycle assessment of ethanol production from lignocellulosic biomass: challenges
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6. Singh A., Smyth B. M., Murphy J. D. (2010) A biofuel strategy for Ireland with an
emphasis on production of biomethane and minimization of land take. Renew. Sustain.
Energy Rev., 14: 277288.
7. John R.P., Anisha G.S., Nampoothiri K.M., Pandey A. (2011) Micro and macroalgal
biomass: a renewable source for bioethanol. Bioresour. Technol., 102 (1): 186 193.
8. Ho S.-H., Chen W.-M., Chang J.-S. (2010) Scenedesmus obliquus CNW-N as a potential
candidate for CO 2 mitigation and biodiesel production. Bioresour. Technol., 101 (22):
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9. Nigam P.S. and Singh A. (2011) Production of liquid biofuels from renewable resources.
Prog. Energ. Combust., 37 (1): 5268.
10. Ho S.-H., Chen C.-Y., Chang J.-S. (2012) Effect of light intensity and nitrogen starvation
on CO 2 fixation and lipid/carbohydrate production of an indigenous microalga
Scenedesmus obliquus CNW-N. Bioresour. Technol., 113: 244252.

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14. Moxley G. and Zhang Y.H.P. (2007) More accurate determination of acid-labile
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22. Lages F., Lucas C. (1995) Characterization of a glycerol/H+ symport in the halotolerant
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110

CHAPTER 11
ISOLATION AND MOLECULAR CHARACTERIZATION OF
CELLULOLYTIC FUNGI USED FOR CONVERSION OF
SUGARCANE BIOMASS FOR BIOETHANOL PRODUCTION
Chetan, A. M., Harinikumar, K. M., Bhavani, P., Manoj Kumar, H. B., Madhu T., Ningaraj
Dalawai

Abstract
One of the major alternatives to fossil fuels that received major attention is bioethanol derived
from biomass. The cellulosic material are potential sources of ethanol. Cellulolytic fungi are
capable of degrading cellulose to smaller sugar components like glucose units. The aim of this
research was to isolate and screen fungi capable of producing cellulases and to convert pre-
treated lignocellulosic material to fermentable sugars for the production of ethanol using
Saccharomyces cerevisiae. The lignocellulosic material such as sugarcane bagasse and sugarcane
trash were used as substrates for ethanol production. Fungi were isolated from soil and compost
samples collected from various regions. The pure cultures were screened for the ability to
degrade cellulose. The cellulolytic activity was determined by measuring the clearing zone
created by the fungi. The cultures were further characterized using five random primers. The
fungi capable to produce cellulases were identified as Aspergillus niger, Aspergillus fumigatus,
Trichoderma viride, Trichoderma harzianum and Trichoderma reesei based on colony
characters, microscopic observation and identification at molecular level based on DNA coding
for 18S rRNA. The substrates were powdered and pretreated with fungal isolates using Mandels
and Reese media. The substrates were used as a carbon source. Sugarcane bagasse and trash
treated with Trichoderma reesei have shown highest concentration of reducing sugars of
45.95mg/g and 40.56mg/g respectively and ethanol yield of 11.56g/l and 10.92g/l respectively.
From this study the fungal cultures having the potential to degrade cellulosic material were
identified and they can be used for bioethanol production from lignocellulosic wastes.



111

11.1 Introduction
The growth of population and the associated demand for fuel and food coupled with more
restrictive environmental regulations have intensified the research and development of renewable
energy feedstocks to substitute for and/or to complement fossil fuel sources (Pereira Jr. et al.,
2008). The transfer of crude oil-based refinery to biomass-based biorefinery has attracted strong
scientific interest which focuses on the development of cellulosic ethanol as an alternative
transportation fuel to petroleum fuel (Zheng, et al., 2009). In India, ethanol is primarily produced
from molasses which is a byproduct from sugar mills using Saccharomyces cerevisiae strains.
Sugarcane bagasse and trash, the left-over residue of leaves and tops, can be converted to ethanol
by enzymatic or acid catalyzed hydrolysis and fermentation of the released monosaccharides
(Michael et al., 2006). Sugarcane, an important cash crop, is grown over an area of 4.2 million
hectares in India with an average productivity of 70 tons ha1. Sugarcane converts approximately
2% of solar energy into chemical bonds of carbohydrates where in two third of these
carbohydrates are in the form of lignocelluloses. These lignocellulosic material are a potential
source of bioethanol production. There are three major steps to be employed in the conversion of
lignocellulosic to Bioethanol which are pretreatment for lignin breakdown, hydrolysis and
fermentation for Bioethanol production. The most challenging part is the hydrolysis process in
order to obtain the reducing sugars. Hydrolysis of lignocellulosic can be done in two ways, either
by using enzymatic or chemical methods. However, enzymatic hydrolysis is more environmental
friendly as compared to chemical hydrolysis. Although, the costs of using commercial enzymes
are expensive, however this problem can be overcome by using cellulose degrading organisms
(Zainan et al., 2011). This research work has been carried out in order to produce bio-ethanol
from lignocellulolytic wastes by using cellulolytic fungi.
11.2 Material and Methods
Present investigation was carried out to isolate and identify cellulolytic fungi for the
conversion of lignocellulolytic biomass into Bio-ethanol and to characterize them using ITS
primers (Internal transcribed spacer) and RAPD primers.
11.2.1 Isolation


112

The soil and compost samples were collected from various regions of Mandya. The
samples were inoculated on potato dextrose agar (PDA) medium. For the isolation of fungi,
dilution plate method was used. All the samples (i.e., three soil samples and two compost
samples) were serially diluted and the dilutions 10-2, 10-3, 10-4 were plated using spread plate
method.
11.2.2 Screening

From the various isolates, screening for cellulolytic fungi was made using selective media
(Mandels' and Reese agar medium) procedures to select the potential isolates that could
saccharify lignocellulosic wastes. Fungi determined to be cellulolytic were then cultured in
Mandels salt medium supplemented with cellulose. (Mandels & Reese, 1957). Cellulolytic fungi
create a clearing zone around the colony on the agar.
11.2.3 Molecular characterization

Molecular characterization was done using five random primers OPA-1; OPD-6; OPA-4;
A-5 and AA-11.
11.2.4 Extraction of DNA

DNA extraction protocol was followed according to Chakraborty et al., (2010). Isolation
of fungal genomic DNA was done by growing the fungi for 4 days. The quality and quantity of
DNA was analyzed both spectrophotometrically and in 0.8% agarose gel. The DNA from all
isolates produced clear sharp bands, indicating good quality of DNA.
11.2.5 PCR Amplification of ITS Region of fungal Isolates:

All fungal isolates were taken up for ITS-PCR amplification. Genomic DNA was
amplified by mixing the template DNA (50 ng), with the polymerase reaction buffer, dNTP mix,
primers and Taq polymerase. Polymerase Chain Reaction was performed in a total volume of 50
l, containing Sample ~50ng of gDNA, 100ng of Forward Primer, 100ng of Reverse Primer, 2l
of 10mM dNTPs mix, 5l of 10X Taq Pol. Buffer, 3U Taq Polymerase enzyme, PCR grade water
to make the volume upto 50l. PCR was programmed with an initial denaturing at 94C for 5
min. followed by 30 cycles of denaturation at 94 C for 30 sec, annealing at 52C for 30 sec and
extension at 72C for 1 min and the final extension at 72 C for 3 min in a Thermocycler. After

113

PCR, all the amplified products were ran on 1.5% Agarose gel with 1X TAE buffer. Sequencing
was done at Amnion biosciences using ABI3730xl sequencer.
11.2.6 RAPD of Trichoderma Isolates:

For RAPD, six random primers i.e. OPA-1; OPD-6; OPA-4; A-5; AA-04 and AA- 11
were selected (Table-1). Polymerase Chain Reaction was performed in a total volume of 50 l,
containing Sample ~25ng of gDNA, 400ng RAPD Primer, 2l of 10mM dNTPs mix, 5l of 10X
Taq Pol. Buffer, 3U Taq Polymerase enzyme, PCR grade water to make the volume upto 50l.
PCR was programmed with an initial denaturing at 94C for 5 min. followed by 45cycles of
denaturation at 94C for 1 min, annealing at 38C for 1 min and extension at 72C for 2 min and
the final extension at 72C for 5 min in a Thermocycler. After PCR, all the amplified products
were ran on 1.5% Agarose gel with 1X TAE buffer.
11.2.7 Scoring the data

The image of the gel the ribosomal RNA genes (rDNA) possess electrophoresis was
documented through gel documentation system and analysis software. All reproducible
polymorphic bands were scored and analysed following UPGMA cluster analysis protocol. The
RAPD patterns of each isolate was evaluated, assigning character state 1 to indicate the
presence of band in the gel and 0 for its absence in the gel. The scored band data (Presence or
absence) was subjected to cluster analysis-using STATISTICA.
Production of Bio-ethanol from Pretreated Sugarcane Biomass Using Saccharomyces
Cerevisiae

The two substrates, sugarcane bagasse and sugarcane trash were powered and pretreated
with the fungal cultures using Mandels' and Reese medium using the substrates as carbon source.
Culture conditions: 10g /l of each residue was taken in conical flask containing 200ml of
Mandles medium. The conical flasks were plugged with cotton and sterilized at 15lbs per
sq.inch for 20 minutes. Each flask was inoculated with 4-5 discs of different fungi. These flasks
were incubated at room temperature for 5days on an orbital shaker. After five days mycelium was
separated by filtration through Whatman filter paper No.1. The filtrate was used for further
studies (Kader et al., 1999).


114

Determination of total carbohydrate: The carbohydrate content of untreated and pretreated raw
material in the culture broth was measured by phenol sulphuric acid method with glucose as
standard (Dubois et al., 1956; Krishnaveni et al., 1984).
Determination of reducing sugars: Reducing sugars in untreated and pretreated raw material in
the culture broth were determined by dinitrosalicylic acid (DNS) method with glucose as
standard (Miller, 1972).
Fermentation: Culture filtrate was further inoculated with Saccharomyces cereviseae strain and
allowed for fermentation for seven days. After fermentation it was filtered and ethanol content
was determined.
Ethanol estimation: The ethanol was estimated by Colorimetric method as described by Caputi
et al., (1968).
11.3 Result and discussion
Nine fungal isolates were obtained using Potato Dextrose Agar medium from the soil and
compost samples collected. Among them five isolates were identified and pure cultures were
maintained on PDA plates. The pure fungal isolates were screened for cellulolytic ability.
Highest cellulolytic activity was detected in three isolates of Trichoderma sp. Zone of clearance
was highest in Trichoderma reesei (2.10mm) followed by Trichoderma viride (2.00mm) and
Trichoderma harzianum (1.60mm). The enzymatic activity was considerably low in other fungi
such as Aspergillus sp the clearance zone was lesser in Aspergillus niger (1.50mm) and the least
was Aspergillus fumigatus (0.80mm) . The screened fungal strains were used for further studies.
The DNA isolated from the desired cellulose producing fungi tentatively identified as species of
Apergillus and Trichoderma species. When checked for purity exhibited that the DNA isolated
from the two sources was pure. The same DNA samples, when run on an agarose gel also,
confirmed to be pure as the bands of DNA are single and distinct. Traces of contaminants were
found when observed under the Gel doc and photographed. In the present study we focused on
the ITS region of ribosomal genes for identifying fungal species. ITS region of rDNA was
amplified using genes specific ITS-1 and ITS-4 Primers. Amplified products were of size in the
range 100bp to 180bp. The results are in accordance with Chakroborthy et al., (2010) who

115

studied the identification and genetic variability of fungal isolates. The ITS PCR has helped to
detect polymorphism at ITS region of rDNA among fungal isolates.
In this study, a set of 5 accessions were genotyped with 5 RAPD primers as these primers
are considered as superior for assessing genetic diversity. The genotypes were grouped into two
major clusters, cluster II was the largest with 3 genotypes i.e. Trichderma reesei, Trichoderma
viride and Trichoderma harzianum indicating that the three species are genetically closely related
followed by cluster I with 2 genotypes i.e. Aspergillus niger and Aspergillus fumigatus indicating
that the two species are closely related. The results obtained in the present study are also in
accordance with the cluster analysis results obtained by Chakraborthy et al., (2010). Total sugar,
reducing sugar and nonreducing sugar content of fungal pretreated and untreated samples were
estimated. The results obtained are presented in Table 1. Autoclaving for sterilization has
affected and resulted in increase in sugar content. With fungal treatment still increase in the yield
of sugars was observed. The treated samples were subjected to distillation and the ethanol thus
obtained was estimated using potassium dichromate method and the concentration of ethanol
obtained is presented in Table 2. The results obtained were analyzed statistically using
completely randomized design. In Tables 1 and 2 mean values assigned with same superscript(s)
do not differ significantly (P=0.05). Among the two substrates sugarcane bagasse pretretaed with
Trichoderma reesei has given maximum reducing sugar content (45.95 mg/g) followed by
sugarcane trash pretreated with the same culture (40.56 mg/g). The substrates pretreated with
Trichoderma viride and Trichoderma harzianum have moderately increased the sugar content.
Pretreatment with Aspergillus niger and Aspergillus fumigatus have shown comparatively lesser
results. Among the two substrates sugarcane bagasse pretretaed with Trichoderma reesei has
given maximum ethanol yield (11.56g/l) followed by sugarcane trash pretreated with the same
culture 10.92g/l. The substrates pretreated with Trichoderma viride and Trichoderma harzianum
have moderately increased the sugar content. Pretreatment with Aspergillus niger and Aspergillus
fumigatus have shown comparatively lesser results.

116

Fig (a) Fig (b) Fig(c)


117

Fig (d) Fig (e) Fig (f)

Fig a: genomic DNA; Fig (b-f) Gel picture RAPD analysis of fungal isolates with OPA-1, OPD-6, OPA-4, A-5, AA-11. Lane TH:
Trichoderma harzianum, Lane M: 100bp DNA ladder (100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp)
TV: Trichoderma viride, TR: Trichoderma reesei, AF: Aspergillus fumigatus, AN: Aspergillus niger


118

Table: 1: Concentration of reducing sugars, non reducing sugars and total sugars of the hydrolysates
Sl no. Cultures
Sugarcane bagasse Sugarcane trash
Reducing
sugar
(mg/g)
Non
reducing
sugar
(mg/g)
Total sugar
(mg/g)
Reducing
sugar
(mg/g)
Non
reducing
sugar
(mg/g)
Total sugar
(mg/g)
Control Untreated 0.98
f
1.27
e
2.25
f
0.88
f
1.15
e
2.030
f

1. Aspergillus niger 35.45
d
23.55
c
59.00
d
31.12
d
20.28
c
51.40
d

2. Aspergillus fumigatus 33.32
e
21.02
d
54.34
e
28.19
e
18.82
d
47.01
e

3. Trichoderma viride 39.22
b
30.90
a
70.12
b
38.54
b
28.89
b
67.43
b

4. Trichoderma harzianum 37.98
c
28.00
b
65.98
c
32.22
c
29.32
b
61.54
c

5. Trichoderma reesei 45.95
a
30.05
a
76.00
a
40.56
a
31.34
a
71.90
a

SEm 0.212 0.220 0.107 0.171 0.189 0.082
CD at 1% 0.841 0.872 0.426 0.677 0.747 0.320


Tree Diagram for 5 Variables
Unweighted pair-group average
Squared Euclidean distances
Linkage Distance
TV
TH
TR
AN
AF
0 2 4 6 8 10 12 14 16 18

Table 2: Concentration of Ethanol obtained from hydrolysates of Sugarcane Biomass
Sl no. Cultures
Ethanol (g/l)
Sugarcane bagasse Sugarcane trash
1. Untreated 1.56
f
1.20
f
2. Aspergillus niger 7.32
d
6.58
d
3. Aspergillus fumigatus 6.10
e
5.86
e
4. Trichoderma viride 10.15
b
9.78
b
5. Trichoderma harzianum 9.01
c
8.77
c
6. Trichoderma reesei 11.56
a
10.92
a
SEm 0.112 0.066
CD at 1% 0.440 0.261


120

References
1. Caputi A, Ueda JM, Brown T. (1968) Spectrophotometric determination of chromic
complex formed during oxidation of alcohol. AJEV. 19:160-165.
2. Chakraborty BN, Chakraborty U, Saha A, Dey PL, Sunar K. (2010) Molecular
Characterization of Trichoderma viride and Trichoderma harzianum Isolated from Soils
of North Bengal Based on rDNA Markers and Analysis of Their PCR-RAPD Profiles.,
Global J. Biotech. Biochem. 5(1): 55-61.
3. Dubois M., Gilles KA, Hamilton JK, Rebers PA, Smith F. (1956). Anal. Chem., 26:350-
351.
4. Kader AJ, Omar O, Feng LS. (1999) Isolation of cellulolytic fungi from the Barino
Highlands, Sarawak. ARBEC.
5. Krishnaveni S, Balasubramanian T, Sadasivam S. (1984) Carbohydrates. Food Chem.,
15:229.
6. Mandels M, Reese ET. (1957) Induction of cellulase in fungi by cellobiose. J.
Bacteriology. 73:816- 826.
7. Michael S, Carlos M. (2006) Production of fuel ethanol from sugarcane bagasse and
sugarcane trash. Congress on Sugar and Sugar Cane Derivatives, Havana, Cuba, 19 -
22.
8. Miller GL. (1972) Carbohydrates. Anal. Chem.31:426.
9. Pereira JR, Nei; Couto, Maria Antonieta PG, Santa Anna, Lidia Maria M. (2008)Series
on biotechnology: Biomass of lignocellulosic composition for fuel ethanol production
within the context of biorefinery. Rio de Janeiro, Amigadigital Press, 47 p. ISBN 978-
85-903967-3-4.
10. Zainan NH, Md. Alam Z, Maan Fahmi al-Khatib. (2011) Production of sugar by
hydrolysis of empty fruit bunches using palm oil mill effluent (POME) based cellulases:
Optimization study. Afr. J. Biotechnol. 10(81):18722-18727.
11. Zheng Y, Pan Z, Zhang R. (2009) Overview of biomass pretreatment for cellulosic
ethanol production. Int. J.Agric and Biolo. Eng. 2(3):51-5.


121

CHAPTER 12
APPLICATION OF THERMOSTABLE CELLULASE IN
BIOETHANOL PRODUCTION FROM LIGNOCELLULOSIC
WASTE
Neha Srivatsava, Rekha Rawat and Harinder Singh Oberoi

Abstract
Lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical,
chemical, biological pretreatment operations and slow enzymatic hydrolysis makes the overall
process less economical than presently available fossil fuels. Cellulase is a group of enzymes,
which can be classified into several types based on the reactions they catalyze, including
endoglucanase (EG) or carboxymethyl cellulase (CMCase), exoglucanase or cellobiohydrolase
(CBH), cellobiase or -glucosidase. Auxilliary enzymes like -xylosidase, -L-
arabinofuranosidase, and feruloyl esterase if present along with xylanases help in complete
conversion of hemicellulose to sugars like xylose and arabinose. The synergistic action of
these enzymes plays an important role in the hydrolysis of cellulosic and hemicellulosic
fractions. It is therefore desirable to have a consortium of all the cellulase and xylanase
components for effective hydrolysis of cellulosic biomass. The use of thermostable cellulases
for hydrolysis of cellulosic biomass have significant advantages, such as (i) improved
hydrolysis of cellulosic substrates because of the higher rate of reaction, (ii) higher mass-
transfer rates leading to improved substrate solubility, (iii) lowered risk of contamination, and
(iv) increased flexibility with respect to process design, thereby improving the overall
economics of the process. Therefore, research on developing thermostable cellulase
consortium for hydrolysis of cellulosic biomass is gaining momentum. Thus, the current
chapter covers sources and application of thermostable enzyme along with their characteristic
features. Limitations and possible approaches are also discussed.
Keywords: Bioethanol, Lignocellulosic biomass, Cellulases, Thermostability, Protein
engineering, Hydrolysis



122

12.1 Introduction
The production and utilization of bio-ethanol is gaining attention worldwide because
of many advantages like reducing global warming and improving global energy crises
(Chovau et al., 2013). Ethanol can be produced by fermentation of sugars from agro-industrial
waste materials. Lignocellulosic biomass is known as the most abundant polymer and
renewable source of energy which is finally converted into glucose and soluble sugars in
ethanol production process (Reese and Mandels, 1984). Lignocellulosic agricultural biomass
is used as substrate for the production of second generation biofuels. Lignocellulosic biomass
is a complex which is composed of cellulose, hemicellulose and lignin and the conversion
efficiency of lignocellulosic biomass into biofules depends upon the lignin content and degree
of polymerization (DP) in cellulose and hemicellulose (Oberoi et al., 2012). Cellulose and
hemicellulose are regarded as the potential sources of sugars for second generation biofuel
production and cover around two-third of the lignocellulosic biomass (Hamelinck et al.,
2005). Different pretreatment methods are applied to increase the accessibility of cellulosic
substrate which helps to open the lignin sheath (Alvira et al., 2010). Lignocellulose-degrading
enzymes, such as cellulases and hemicellulases are used to release fermentable sugars after
pretreatment of biomass.
Progressive research on cellulase enzymes started in the early 1950s due to their
potential in conversion of lignocellulosic biomass, the most abundant polymer and renewable
source of energy which is finally converted into glucose and soluble sugars in ethanol
production process (Reese and Mandels, 1984). Continued research on cellulases and
hemicellulases revealed their industrial potential in different sectors and industries, such as
bioenergy, food, brewery and wine, animal feed, textile and laundry, pulp and paper,
agriculture. Cellulases is a complex enzyme system which is divided into three major groups:
endo-1,4--D-glucanases (EC 3.2.1.4) which cleaves -linkages at random, commonly in the
amorphous parts of cellulose; exo-1,4--D-glucanases (EC 3.2.1.91) (or cellobiohydrolase)
which liberates cellobiose from the non-reducing or the reducing end, in general from the
crystalline parts of cellulose; and -glucosidases (EC3.2.1.21) which releases glucose from
cellobiose and short-chain cello-oligosaccharides. Thermostable enzymes have number of
commercial applications as the paper processing industries are always interested in such type
of cellulases which can withstand higher temperatures. In addition, one of the most important
applications of thermostable cellulase is in the bioconversion of cellulosic biomass into

123

fermentable sugars for bioethanol production at elevated temperature. Generally, enzymatic
hydrolysis reactions are carried out at 45
o
C-50
o
C which shows slow enzymatic hydrolysis
rates, low yield of sugars, and incomplete hydrolysis, more amounts of enzyme requirement
and is very sensitive to microbial contamination. These limitations could be resolved by using
thermostable enzymes (Yeoman et al., 2010; Viikari et al., 2007). Figure.1 is showing
possible advantages of thermostable cellulases in bioconversion of lignocellulosic waste.
Therefore, the current chapter focuses on thermostable cellulase enzymes and their
applications in biofuels production.

Figure-1 Application of thermostable cellulases in biofules production
12.2 Bioethanol production: Current production status and Challenges
The production of biofules has reached 105 billion liters in 2010 and increased up to
17% from the year 2009. Biofuels contributed about 2.7% of worlds fuel road transportation

Application of thermostable
cellulases in biofuels production
Complete
hydrolysis

Economically
viable
High reaction
rate
Less risk of
contamination
Fungi and bacteria are
the best source of
production of
thermostable cellulases

Less time
required
for
hydrolysis
reaction
High Sugars yield
Pretreatment
step can be
avoided

124

in which bioethanol and biodiesel are the most prominent. In 2010, global bioethanol
production reached about 86 billion liters. United States and Brazil are the top producers of
ethanol in the world accounting for 90% of the total global production (Kocar and Civas
2013). USA produces more fuel ethanol than any other country; Brazil is the second largest
producer of ethanol in the world. The US and Brazil put together produced a little over 86%
of the worlds fuel ethanol in 2010. Although production of bioethanol has improved using
new technologies, there are still some challenges that need further investigations. For
example, the cost of cellulase and ethanol distillation using lignocellulosic biomass as
substrate, account for 30 to 50% and 20% of the total cost, respectively. To achieve the
commercialization of cellulosic ethanol, a number of technological break throughs as well as
cost reductions in all the process steps are required (Chen and Qiu, 2010).
12.3 Microorganism for thermostable cellulase production
Thermophilic microorganisms are potential sources of highly active and thermostable
enzymes (Zambare et al., 2011; Liang et al., 2011; Yeoman et al., 2010). However, many
mesophilic microorganisms are also known for significant production of thermostable
cellulases (Gao et al., 2008; Lee et al., 2010). Numbers of bacteria and fungi have been
reported to produce thermostable cellulases. Fungi are the most studied organisms with
respect to degradation of cellulose and production of cellulolytic enzymes because bacteria
degrade cellulosic biomass slowly due to lack of penetrating ability like fungi (Swaroopa et
al., 2004). Thermophilic and mesophilic fungal genera belonging to Aspergillus, Rhizopus,
Trichoderma, Sclerotium and Sporotrichum thermophile etc. (Barnard et al., 2010) are known
for the production of cellulases. Synthesis of heat shock protein (HSPs) is a common
phenomenon for production of thermostable enzymes. In presence of cycloheximide, the
ability to produce thermostable enzyme and acquired thermo-tolerance is lost (Maheshwari et
al., 2000). There are number of reports available showing HSPs synthesis in thermophillic
microbes along with rapid breakdown of pulse label proteins (Trent et al., 1994, Maheshwari
et al., 2000). Thermophilic microorganisms have specialised protein known as chaperonins
which help protein to refold and retain their native form and store their function even after
denaturation. The cell membranes of thermophiles are made up of saturated fatty acids which
create hydrophobic environment for cell and keeps it rigid so that it can survive at higher
temperatures (Haki and Rakshit, 2000). The DNA of thermophiles has reverse gyrase which
forms positive supercoils and hence enhances melting point. Besides this, thermophiles also

125

have electrostatic, disulphide bridge and hydrophobic interactions in their cell membranes like
thermotolerants which enhance the tolerant capacity of thermophiles at extremely higher
temperatures (Kumar and Nussinov, 2001). Many thermophilic fungal species have been
reported to produce cellobiose dehydrogenase and glycoside hydrolase 61 (GH61) family of
proteins (Dimarogona et al., 2012), addition of which enhance cellulase performance in
lignocellulose hydrolysis (Barakat et al., 2012; Harris et al., 2010). Thermophilic fungi
produce multiple forms of the cellulase components like mesophilic fungi. However, two
different strains of T. aurantiacus produced one form each of endoglucanase, exoglucanase,
and -glucosidase, but the forms from the two strains had somewhat different properties
(Khandke et al., 1989). Several thermophilic bacteria, belonging to the genera Bacillus,
Geobacillus, Caldibacillus, Acidothermus, Caldocellum, and Clostridium produce
thermostable cellulases. Hyperthermostable lignocellulolytic enzymes (optima above 80
o
C)
have been isolated from Thermotoga anaerocellum (Evans et al., 2000). Most of the studies
reported that hyperthermophilic microbes did not degrade crystalline cellulose, when
temperatures increased beyond 75
o
C due to the lack of carbohydrate-binding modules (CBM)
(Bhalla et al., 2013, Graham et al., 2011; Maki et al., 2009) while presence of a multi-domain
hyperthermophilic cellulase in an archaeal enrichment, allowed maximum deconstruction of
lignocellulosic biomass beyond 90
o
C (Bhalla et al., 2013, Graham et al., 2011).
12.4 Thermostable enzymes
12.4.1 Thermostable cellulases
Cellulases are enzymes that catalyze the depolymerization of cellulose and work
synergistically to efficiently hydrolyze substrate. Endoglucanases and exoglucanases are
commonly referred to as cellulases (Blumer-Schuette et al., 2008). Endoglucanases having
carbohydrate-binding modules (CBM) are considered as primary cellulases and are
responsible for efficient utilization of crystalline cellulose. Trichoderma and Aspergillus sp.
are referred to as model for significant cellulase production. Several fungi and bacteria have
been reported earlier for the production of thermostable cellulases (Evans et al., 2000; Bok et
al., 1998) (Table-1). Thermostable enzymes are stable and active at high temperatures which
are higher than the optimum growth temperatures of the microorganisms. Endoglucanases (30
to 100 kDa) of thermophilic fungi are thermostable, with optimal activity between 55 and
80C at pH 5.0 to 5.5 and with carbohydrate contents varying from 2 to 50%. Exoglucanases
(40 to 70 kDa) are optimally active at 50 to 75C and are thermostable (Maheshwari et al.,

126

2000). Combination of thermostable enzyme with a wide pH range makes them suitable
candidates for bioprocessing. Hydrolysis of substrate with thermostable enzyme increases the
rate of reaction, decreases viscosity, increases diffusion coefficient, increases bioavailability
of organic compounds and the solubility of the substrate, resulting in complete hydrolysis and
less risk of contamination at elevated temperatures. Such types of enzymes can also be used
for models studies for the understanding of temperature stability and activity, which is helpful
for protein engineering (Haki and Rakhsit, 2003).
Table- Thermostable cellulases from different microorganisms
Organism Optimum
temperature
Stability References
Fusarium proliferatum 55
o
C 50% (Badal, 2002)
Teheromyces lanuginosus (wild
and mutant)
70
o
C 50% (Bakalova et al. 2002)
Bacillus subtilis 70
o
C 50% (Mawadza et al., 2000)
Thermotoga neapoltana
(EndocellulaseA)
95
o
C 50% (Bok et al., 1998)
Alicyclobacillus
acidocaldarius ATCC27009
(Endoglucanases)
80
o
C 60% (Eckert and
Schneider, 2003)
E. coli expressing endoglucanase
gene from
Clostridium thermocellum
80
o
C 50% (Zverlov et al., 2005)
gene from
Geobacillus sp. 70PC53
65
o
C 80% (Ng et al., 2009)
Geobacillus sp. WSUCF1
(Endoglucanases)
70
o
C 50% (Rastogi et al., 2010)
gene from
Thermoanaerobacter
tengcongensis MB4
80
o
C 50% (Liang et al., 2011)
Aspergillus fumigates SK1 60
o
C 50% (Ang et al., 2013)

12.4.2 Thermostable xylanases
Xylan is the second most abundant polysaccharide in nature after cellulose. Complete
degradation of xylan needs the combined action of a group of hydrolytic enzymes: the
endoxylanases (EC 3.2.1.8), which cleaves -1,4-linked xylose randomly (xylan backbone);
the -xylosidases (EC 3.2.1.37), which converts xylobiose to monomeric xylose units ; and
the various side-branch splitting enzymes such as -glucuronidase and -arabinosidase, acetyl
xylan esterase, and acetyl esterase, which liberate other sugars (glucuronic acid arabinose) that

127

are attached as branches to the backbone (Bhalla et al., 2013, Biely., 1985). Thermostable
endo--1,4 xylanases (referred to as endoxylanases) produced by thermophilic and
hyperthermophilic bacteria such as Thermotaga, Acidothermus, Cellulomonas, Paenibacillus
,Thermoanaerobacterium, Actinomadura have been reported and are receiving considerable
attention because of their application in bio- bleaching of pulp in the paper industry, wherein
the enzymatic removal of xylan from lignin-carbohydrate complexes facilitates the leaching of
lignin from the fibre cell wall, avoiding the need for chlorine for pulp bleaching in the
brightening process. They also have applications in the pre-treatment of animal feed to
improve its digestibility. Production of xylnase has also been reported from marine algae,
protozoa, snails and insects but bacteria and fungi are the major producers of thermostable
xylanases (Collins et al., 2005). According to Brienzo et al., (2009), xylanses obtained from
bacteria are more diverse than fungi. Fungi including Laetiporus sulphureus (Lee et al., 2009),
Talaromyces thermophiles (Maalej et al., 2009), Thermomyces lanuginosus (Singh et al.,
2003), Rhizomucor miehei (Fawzi, 2011) produce thermostable xylanases. Thermostable and
alkalistable xylanases have also been reported from many fungi and bacteria, such as
thermoalkalophilic xylanase obtained from Enterobacter sp. MTCC 5112 retained its 90% of
enzyme activity after 0.66 h at 80
o
C and pH 9, and 85% and 64% of its activity after 18 h at
60 and 70
o
C, respectively, showing its potential for industrial applications (Bhalla et al., 2013,
Khandeparkar Bhosale, 2006).
12.4.3 Thermostable endoglucanses
Endoglucanase is responsible for formation of cellobiose (oligosaccharides) from
cellulose which is finally converted into glucose molecules by the action of -glucosidase.
Several studies have reported production of thermostable endoglucanase production from
mesophillic and thermophillic microorganisms including fungi and bacteria both. Generally,
endoglucanases show high thermal stability, for example endoglucanases from T. aurantiacus
shows thermal stability at 70
o
C with half life of 98 h, (Gomes et al., 2000). In one of the
study, Parry et al. (2002) discussed about an endoglucanase with a molecular weight of 34
kDa (based on SDS-PAGE) along with a pI of 3.7. Endoglucanases, broadly with molecular
weight ranging from 30 to 100 kDa obtained from thermophilic fungi are thermostable, with
optimal activity between 55 and 80C at pH 5.0 to 5.5 and with carbohydrate contents varying
from 2 to 50% (Maheshwari et al., 2000). Exoglucanases having molecular weight in the
range of 40 to 70 kDa, are optimally active at 50 to 75C and are thermostable; these enzymes
fall into the category of glycoproteins (Brizeno et al., 2009).

128

12.4.4 Thermostable -glucosidase
Conversion of cellooligosacchrides into glucose is done by the help of enzyme -
glucosidase (BGL). Several hyperthermophilic bacteria (Dion et al., 1999) such as
archaeabacteria (Kim et al., 2009) as well as fungi (Parry et al., 2001) are known for the
production of significant thermostable BGL which shows maximum optimal temperatures of
88, 90, and 80C, respectively (Handelsman J., 2005). In one of the study, Duex et al., (2012)
reported thermostability of BGL at 66
o
C. Now days, with the help of metagenomics, complete
microbial genomes may be screened and isolated directly from the natural environments (Feng
et al., 2009; 2010, Jiang et al., 2011). Number of BGL genes have been isolated by employing
metagenomic libraries of different samples, including rabbit cecum, marine samples (Jiang et
al., 2009), soil (Jiang et al., 2009, 2011), and termite gut (Scharf et al., 2010, Matteotti et al.,
2011).
12.5 Application of thermostable cellulases
The significant industrial importance of cellulases lies mainly in the bioenergy
development, textile, and detergent and cellulosic pulp paper industries. In the present
available industrial processes, cellulolytic enzymes are used in: (i) clarification of juices and
wines; (ii) detergents causing colour brightening and softening; (iii) pretreatment of biomass
to improve nutritional quality of forage; and (iv) pretreatment of industrial wastes (Bhalla et
al., 2013, Brienzo et al., 2009, Haki and Rakshit, 2003, Bhat, 2000).
Currently, lignocellulosic biomass conversion into fermentable sugar is one of the
main thrust area for the production of biofuels. Some studies have shown that a long reaction
time is required for complete hydrolysis when cellulases do not have this property (Sassner et
al., 2008; Zhu et al., 2008; Borjesson et al., 2007). The use of thermostable enzymes like those
produced from T. aurantiacus help in improving the hydrolysis process. In one of the study,
Ang et al., (2013), reported optimum hydrolysis temperature of 60
o
C by using thermostable
enzyme obtained from fungus Aspergillus fumigates SK1. It is therefore important to have a
combination of all the forms of cellulases like exo-, endo-glucanases and -glucosidases in a
consortium along with the xylanases and accessory enzymes capable of hydrolysis of biomass
at elevated temperatures, thereby giving a characteristic advantage to such a consortium.
Development of this kind of consortium is the need of an hour in improving the sugar
productivity and decreasing the enzyme cost in bioethanol production from lignocellulosic
biomass.

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12.6 Concluding remarks
The remarkable progress has been done in the production and development of
thermostable cellulases and has attracted worldwide attention for further research. Although
thermostable enzymes have many advantageous but for industrial scale processes, final
selection depends on the many factors such as energy consumption for overall process,
production cost, enzyme efficiency and environmental issues of the complete process of
lignocelluloses biomass conversion. Cultivation of thermophiles on commercial-scale for the
production of thermostable enzyme is still an economical challenge because of low cell yield
and might even increase the overall production cost. Based on this, there is need of more
research for development and optimization of thermophilic processes of lignocellulosic
biomass conversion to gain an economical industrial biofuel production comparable to
existing processes.
Acknowledgements
Authors thankfully acknowledge the financial assistance received from the NAIP sub-project
(4183) funded by World Bank through Indian Council of Agricultural Research, Government
of India for conducting a part of the study reported in this book chapter.

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CHAPTER 13
ENDOGLUCANASES: CHARACTERIZATION AND ITS
ROLE IN BIOCONVERSION OF CELLULOSIC BIOMASS
Rekha Rawat, Neha Srivastava, Harinder Singh Oberoi

Abstract
The growing global energy demand and negative environmental impacts created by growing
greenhouse gas emissions from fossil fuels have forced the global scientific community to
intensify research on the use of cellulases to perform enzymatic hydrolysis of the
lignocellulosic materials for the production of bioethanol. Endoglucanases are the major
component of cellulase enzyme involved in the initial stages of cellulose breakdown. These
are classified into11 glycoside hydrolase (GH) families, including GH5, 6, 7, 8, 9, 12, 44, 45,
48, 51, and 74 on the basis of different sequences, specificity and tertiary structure. The
application of this enzyme in biofuel industry requires identification of highly stable enzymes
that are able to perform at extreme values of pH and temperature. Several bacteria and fungi
are known to produce thermoacidophilic as well as thermoalkalophilic endoglucanases. The
present chapter focuses on the importance of extremophilic endoglucanases in order to
improve the existing biomass conversion processes. In addition to this, structural information,
protein dynamics and models for thermostable endogucanases are also discussed.
Key words: Endoglucanase, Structure, Classification, Catalytic Mechanism, Thermostability.
13.1 Introduction
Biofuels are drawing increasing attention worldwide as substitutes to petroleum-
derived transportation fuels to help address energy cost, energy security and global warming
concerns associated with liquid fossil fuels (Gray et al., 2006; Lee, 2011). It refers to energy
obtained from biomass which is the biodegradable fraction of products, or waste and residues
from agriculture, forestry and related industries, as well as the biodegradable fraction of
industrial and municipal waste. Lignocellulosic biomass is the most abundant renewable
organic material on earth which is composed of cellulose, hemicellulose and lignin. Cellulose,
which is an unbranched linear homopolymer of glucose molecules with (1-4) linkages,


136

generally accounts for 20- 45% of plant biomass. Cellulose is the main source of sugars for
biofuel production (Hamelinck et al., 2005).
Processing of lignocellulosic biomass into biofuel consists of four major unit
operations: pretreatment, hydrolysis, fermentation, and product recovery. A key step in the
production of biofuel is the hydrolysis of biomass into fermentable sugars that is facilitated by
cellulase enzyme. Cellulases comprise of three enzymes namely endo-1,4--glucanase (also
referred to as carboxymethylcellulase or CMCase; EC 3.2.1.4), exo-1,4--glucanase or
cellobiohydrolases (EC 3.2.1.91) and -glucosidase (EC 3.2.1.21) that synergistically convert
cellulose into soluble sugars and glucose (Lynd et al., 2002). Among these, endoglucanases
are cellulases that act synergistically in the initial attack on cellulose as they hydrolyze the -
1,4 glycosidic bonds. The by-product is subsequently catalyzed by other enzymes, thereby
making endoglucanases crucial for the bioprocessing of plant biomass (Bhat, 2000).
Therefore, this chapter presents information about structure, mechanism of cellulose
hydrolysis and applications of endoglucanases. In addition, importance of thermostable
endoglucanases and factors affecting thermostability are also discussed.
13.2 Mechanism of cellulolysis
The hydrolysis of insoluble cellulose requires the synergistic action of three types of
enzyme: endoglucanase (1,4--D-glucan-4-glucanohydrolase; EC 3.2.1.4), exoglucanase (1,4-
-D-glucan cellobiohydrolase; cellobiohydrolase; EC 3.2.1.91) and -glucosidase (-
glucosideglucohydrolase; cellobiase; EC 3.2.1.21) as mentioned previously. Endoglucanases
initiate cellulose hydrolysis by cleaving the cellulose polymer exposing both the reducing and
non-reducing ends, while cellobiohydrolases acts upon these reducing and non-reducing ends
to liberate cello-oligosaccharides and cellobiose units, and -glucosidases cleave the
cellobiose to liberate glucose, thereby completing the hydrolysis process (Bhat and Bhat
1997). Endoglucanases are classified as endo-acting cellulases because they are thought to
cleave -1,4-glycosidic bonds internally only and appear to have cleft-shaped open active
sites. They are typically active on the more soluble amorphous region of the cellulose crystal,
increase the concentration of chain ends and significantly decrease degree of polymerization
by attacking interior portions of cellulose molecules (Tomas et al., 2009). On the other hand,
cellobiohydrolases are classified as exo-acting cellulases as they cleave -1,4-glycosidic bonds
from chain ends and have a tunnel-shaped closed active site that retains a single glucan chain
and prevents it from re-adhering to the cellulose crystal (Divne et al., 1998). They are usually


137

active on the crystalline regions of cellulose; shorten degree of polymerization incrementally
by binding to the chain ends releasing mainly cellobiose units. Thus, endoglucanase activity is
thought to be primarily responsible for chemical changes in solid-phase cellulose that occur
over the course of hydrolysis, but plays a minor role in solubilization relative to exoglucanase,
while exoglucanase activity is thought to be primarily responsible for solubilization but plays
a minor role in changing the chemical properties of residual cellulose.
13.3 Classification of Endoglucanases
Based on sequence and 3-dimensional structure, endoglucanases (EGs) are grouped
into 11 glycoside hydrolase (GH) families, including GH5, 6, 7, 8, 9, 12, 44, 45, 48, 51, and
74 (Cantarel et al., 2009). Glycoside hydrolases (EC 3.2.1.) are a widespread group of
enzymes which hydrolyse the glycosidic bond between two or more carbohydrates or between
a carbohydrate and a non-carbohydrate moiety. The International Union of Biochemistry and
Molecular Biology (IUBMB) Enzyme nomenclature of glycoside hydrolases is based on their
substrate specificity and occasionally on their molecular mechanism; such a classification
does not reflect the structural features of these enzymes. A classification of glycoside
hydrolases in families based on amino acid sequence similarities has been proposed a few
years ago. Because there is a direct relationship between sequence and folding similarities,
such a classification:
(i) Reflects the structural features of these enzymes better than their sole substrate specificity,
(ii) Helps to reveal the evolutionary relationships between these enzymes, (iii) provides a
convenient tool to derive mechanistic information (Henrissat, 1991; Henrissat and Bairoch,
1993). The details of each family are shown in the table 1.
13.4 Structure of endoglucanases
Endoglucanase is made up of different domains. The main domain contains the large,
globular catalytic domain which expresses the active site. A loop of the protein chain forms a
tunnel that encloses the active site. It is attached at the O-glycosylated B block hinge region of
the catalytic domain to the smaller, globular cellulose binding domain (CBM) at its C-
terminal A block by a linker peptide (Nimlos, et al., 2007).The overall shape of the complex
looks like a tadpole, with the A and B blocks forming the extended tail and the catalytic
domain forming the head (Pilz, et al., 1990). These structural domains contain three types of
structure folds depending upon the endoglucanases (Yennamalli et al., 2011).


138

Table 1: Classification of different endoglucanases and their properties
Family Mechanism Structure
Catalytic
Nucleophile/Base
Catalytic proton
donor
GH5 Retaining (/)
8
Glu Glu
GH6 Inverting - Asp Asp
GH7 Retaining -jelly roll Glu Glu
GH8 Inverting (/)
6
Asp Glu
GH9 Inverting (/)
6
Asp Glu
GH12 Retaining -jelly roll Glu Glu
GH44 Retaining (/)
8
Glu Glu
GH48 Inverting (/)
6
- Glu
GH51 Retaining (/)
8
Glu Glu

13.4.1 (/)
8
fold
This type of fold has an alternating pattern of eight and subunits in a single
domain, such that the eight parallel strands on the inside are protected by eight helices on
the outside. This very common fold has been reported to exhibit the highest diversity of
enzymatic functions (Wierenga, 2001).
13.4.2 -jelly roll fold
This type of fold consists of 15 -strands in two twisted antiparallel -sheets, named A
and B, that pack against each other. -sheet A contains six antiparallel -strands forming the
back, convex surface while -sheet B contains nine anti-parallel -strands arranged to form
the front, concave binding surface (Sandgren et al., 2005). Additionally two -helices pack
against the back side of -sheet B.
13.4.3 (/)
6
fold


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The substrate binding cleft in this type of fold has a tunnel shape that is formed at the
N-termini of six central, parallel -helices. These six helices are surrounded by six external -
helices (Alzari et al., 1996).
13.5 Mechanism of cellulose hydrolysis by endoglucanases
Endoglucanases uses two types of catalytic mechanisms for hydrolysis of glycosidic
bonds of cellulose:
13.5.1 Retaining mechanism
In this type of mechanism, the stereomeric configuration of the anomeric carbon is
retained in the configuration after hydrolysis. A pair of Glu amino acids, act as the catalytic
residues: one as a nucleophile and the other as an acid-base donor. The first step in this double
displacement mechanism is glycosylation, where one residue plays the role of a nucleophile,
attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme
intermediate. At the same time the other residue functions as an acid catalyst and protonates
the glycosidic oxygen as the bond cleaves. The second step in this mechanism is
deglycosylation step, where water molecule acts as a nucleophile and the first residues
carboxylic group acts as a base. Once deprotonated, the water molecule is an activated
nucleophile that then hydrolyzes the glycosyl-enzyme intermediate leading to a break in the
polymer (Yennamalli et al., 2011).
13.5.2 Inverting mechanism
In this type of mechanism, the configuration of the anomeric carbon is inverted; i.e.,
hydrolysis of -glycosidic bond leads to -configuration of carbon and vice versa. Contrary to
retaining method, this method involves single displacement mechanism. The reaction typically
occurs with general acid and general base assistance from two amino acid side chains,
normally glutamic or aspartic acids (Guimaraes et al., 2002; Guerin et al., 2002) and a water
molecule acts as a nucleophile. Utilizing the water molecule on the opposite side of the sugar
ring to stabilize the transition, these residues catalyze the glycosylation or deglycosylation in
one step. Unlike the retaining mechanism, this mechanism does not involve the glycosyl-
enzyme intermediate.
13.6 Microbial sources of endoglucanase enzyme


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A variety of microorganisms including fungi, bacteria and actinomycetes produce
endoglucanases for utilizing cellulose as a source of carbon and energy. Among them, most
emphasis has been placed on fungi and bacteria because of their ability to produce plentiful
amounts of endoglucanses. The list of microbes having potential for endoglucanase
production is given in Table 2.
13.7 Application of endoglucanases
Microbial endoglucanases can be used for the production of bioenergy and other value
added products and chemicals with great potential.
Table 2: Most extensively studied microbes employed for endoglucanase production
Group Organisms
Fungi Agaricus bisporus, Aspergillus niger, A. fumigatus, A. oryzae, A. terreus, A.
wentii, A.aculeatus, A. awamori, Chaetomium cellulyticum; C.
thermophilum, Daldinia eschscholzii, Fomitopsis sp., Fusarium solani, F.
oxysporum, Humicola insolens, H. grisea, Macrophomina phaseolina,
Melanocarpus albomyces, Mucor circinelloides, Paecilomyces inflatus,
Penicillium pinophilum, P. chrysogenum, P. occitanis, P. purpurogenum,
Phanerochaete chrysosporium, Phlebia gigantean, Pleurotus ostreatus,
Pyrenochaeta lycopersci, Rhizopus stolonifer, R. oryzae, Schizophyllum
commune, Trametes versicolor, Trichoderma reesei, T. atroviride, T.viridae,
T. harzianum, Thermoascus aurantiacus
Bacteria Acetivibrio cellulolyticus, Acinetobacter junii, A. amitratus, Anoxybacillus
sp., Bacillus subtilis, B. pumilus, B.amyloliquefaciens, B. licheniformis, B.
circulan, B. flexus, Bacillus agaradhaerens, Bacteriodes cellulosolvens,
Butyrivibrio fibrisolvens, Cellvibrio gilvus, Clostridium thermocellum; C.
cellulolyticum; C. acetobutylium; C. cellulofermentans, C. cellulovorans,
Eubacterium cellulolyticum, Geobacillus sp., Fibrobacter succinogenes,
Microbispora bispora, Paenibacillus campinasensis, P. polymyxa,
Pectobacterium chrysanthemi, Pseudomonas fluorescens, Rhodothermus
marinus, Ruminococcus albus, R. succinogenes, Thermotoga maritime
Actinomycetes Cellulomonas fimi, C. flavigena, C. cellulans, C. uda, Streptomyces
cellulyticus, S. aureofaciens, Thermomonospora fusca, T. curvata,


141

Thermobifida fusca, T. cellulolytica
Kuhad et al., 2011
13.7.1 Biofuel industry
Bioconversion of lignocellulosic biomass into the fermentable sugars is the most
important application of endoglucanases. Bioconversion is carried out either enzymatically or
chemically using sulfuric or other acids (Wyman, 1999). However, when sulfuric acid is used,
it is necessary to remove the residual sulfuric acid from the hydrolyzing solution prior to yeast
fermentation. Furthermore, it produces toxic compounds that inhibit fermentation. The
advantages of enzymatic hydrolysis are the lower requirements for cooling water, gas,
electricity and disposal costs and no corrosion issues for equipment together with lower
environmental pollution (Sun and Cheng, 2002).
13.7.2 Textile and laundry
Endocellulases play a key role in textile and laundry because of their ability to modify
cellulosic fibres in order to improve the quality of fabrics. They especially help in biostoning
and biopolishing of cotton and fabrics. In bio-stoning of denim fabrics, endoglucanases
remove excess dye from denim fabrics and restore the softness of cotton fabrics without
damaging the fibre. Similarly, in biopolishing, they facilitate the removal of excess
microfibrils from the surface of cotton and non-denim fabrics. In addition to this, they
improve the detergent performance and allow the removal of small, fuzzy fibrils from fabric
surfaces and restore the appearance and color brightness (Galante et al., 1998a; Godfrey,
1996; Kumar et al., 1996).
13.7.3 Paper and Pulp industry
Paper and pulp industry also require endoglucanases for substantial energy savings
during mechanical pulping, improvements in hand-sheet strength properties and deinking of
fibre surface for improving brightness and freeness of the pulp by partial hydrolysis of
carbohydrate molecules (Akhtar, 1994; Leatham et al., 1990; Prasad et al., 1993).
13.7.4 Wine and Brewery Industry
Endoglucanases are also vital for fermentation processes to produce alcoholic
beverages including beers and wines. They improve color extraction, skin maceration, must
clarification, filtration, and finally the wine quality and stability. These enzymes can improve
both quality and yields of the fermented products (Singh et al., 2007; Galante et al., 1998b).


142

13.7.5 Other applications
Endoglucanases also find applications in clarification of fruit and vegetable juices, oil
extraction, and in improving the nutritive quality of bakery products and animal feed (Bhat,
2000). The enzymes are used to improve cloud stability and texture and decrease viscosity of
the nectars and purees from tropical fruits, thus clarify the juices (Singh et al., 2007). The
enzyme causes degradation of -glucan in feed which lowers the viscosity of the intestinal
contents and thus, improves the quality of the feed (Bedford, 1995).
13.8. Significance of thermostable endoglucanases
Currently, the bioconversion of lignocellulosic biomass into fermentable sugar is the
major concern for the production of biofuel. Thermostable endoglucanases are economically
important because they are able to contribute to the hydrolysis of cellulose at higher
temperatures compared to their mesostable homologs, reducing the number of processing
steps during biofuel conversion. Thus, lignocellulosic conversion using thermostable
endoglucanses have attracted much attention because of their several potential advantages
such as (i) higher reaction rates due to the increased solubility of substrates; (ii) higher
productivity as hydrolysis time is shortened; (iii) lessen the amount of enzyme needed; (iv)
decreased risk of contamination; (v) facilitated recovery of volatile products; (vi) decreased
cost of energy for cooling as the thermostable enzymes can be used directly after the heating
step without a pre-cooling step and (vii) loss of enzyme activity is low during processing, at
higher temperature used during pre-treatments (Zhang et al., 2011; Viikari et al., 2007; Bhalla
et al., 2013).
13.9 Factors responsible for thermal stability
Thermostability is ability of an enzyme to maintain its active structural conformation
at higher temperature for a prolonged incubation period (Bhalla et al., 2013).There are
multiple factors that are responsible for increased thermostability of enzyme.
13.9.1 Amino acid composition
Positively charged residues (Lys, Arg and Glu) on the solvent accessible surface are
more significant in thermophiles than in mesophiles (Glyakina et al., 2007). Kumar et al.,
(2000) reported that Arg and Tyr are significantly higher whereas Cys and Ser are
significantly lower in thermophilic proteins. It was reported that a decrease in the number of
Gly residues in thermophilic proteins leads to greater stability at higher temperatures (Panasik


143

et al., 2000). Yennamalli et al., (2011) observed that amino acids Arg and Met are statistically
significant among thermophilic proteins, whereas Gln and Ser are statistically significant
among mesophilic proteins.
13.9.2 Intramolecular interactions
For the thermophiles, only ionic interactions were significant, whereas for mesophiles,
no intramolecular interactions were significantly different from thermophiles (Yennamalli et
al., 2011). Comparison of intramolecular interactions showed that cation- interactions are
highly significant in imparting thermophilicity (Chakravarty and Varadarajan, 2002).
13.9.3 Fold specificity:
Recently, Yennamalli et al., (2011) conducted the study on thermostable
endoglucanases and demonstrated fold specificity as a key factor for controlling
thermostability in endoglucanases. For the (/)
8
fold, Arg and Pro (significant in
thermophiles) are replaced by polar amino acids whereas Leu is primarily replaced with
aromatic amino acids in the mesophilic counterpart. The absence of arginine amino acids
leads to a loss of ionic interactions in mesophiles, rendering them enzymatically inactive at
higher temperatures. In the -jelly roll fold, the amino acids Glu, Arg, and His are substituted
with polar, hydrophobic amino acids. Substitution to Pro is higher for Arg indicating the
potential for fewer salt-bridges in mesophiles whereas number of salt bridges is high in
thermophiles. For the Ser and Thr positions (significant among mesophiles) the thermophilic
protein has hydrophobic, acidic, and basic amino acids substituted. In the (/)
6
fold Glu and
Val are replaced with polar amino acids and to a lesser extent with other amino acid groups.
Gln (significant in mesophiles) is substituted to a large extent by hydrophobic, acidic and to a
lesser extent with basic amino acid groups in thermophiles indicating that in the thermophilic
protein these substitutions contribute towards more intramolecular interactions and extend
stability to proteins at higher temperatures.
13.9.4 Other factors
These include a decrease in loop length and a concomitant increase in secondary
structure, an increase in aromatic stacking, increased hydrophobic interactions, increased
metal-binding capacity, and increased oligomerization (Yano and Poulas, 2003). Gromiha et
al., (1999) found Gibbs free energy change of hydration, long-range non-bonded energy, -
strand tendency and average long-range contacts as factors responsible for imparting


144

thermostability. Several studies implicated partial, reversible folding of the protein as a factor
responsible for thermostability at high temperatures (Uversky, 2009).
13.10 Conclusion
Endoglucanases, also called primary cellulases, are synergistically involved in the first
stage of cellulose breakdown-a vital step in the bioprocessing of lignocellulosic biomass into
biofuel and other industrial bioprocesses. Despite possessing several advantages,
lignocellulosic biofuel has not yet been well implemented on a commercial scale because of
high costs of cellulolytic enzymes and lack of robust cellulases that can function efficiently at
high temperature. Thus, understanding the structure of enzyme, mechanism of reaction and
basis for thermostability helps in engineering the protein for enhanced activity. It can lead to
more cost effective processes for biofuels and other industrial applications by designing a
more efficient endoglucanase enzyme.
Acknowledgements
Authors thankfully acknowledge the financial assistance received from the NAIP sub-
project funded by the World Bank through the Indian Council of Agricultural Research
(ICAR) Government of India (PR/8488/PBD/26/68/2006) for conducting a part of the study
reported in this book chapter.

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149

CHAPTER 14
COMPARATIVE STUDY OF FERMENTATION EFFICIENCY
FOR BIO-ETHANOL PRODUCTION BY ISOLATES
Richa Arora, Shuvashish Behera, Sachin Kumar

Abstract
Recent production of bio-ethanol through microbial fermentation as an alternative source has
renewed its research interest because of the increase in the fuel price and environmental
concern. Yeast strains are commonly associated with the ethanol production potential in sugar
rich environments. In the present study, isolation of various yeast strains were carried out from
different soil samples collected from dumping sites of sugar-mills. A total of four yeast strains
were isolated with the ethanol producing ability, which were used for the further study. An
attempt has been made to evaluate the pattern of sugar utilization and ethanol yield by the
yeast strains using the salt medium. The results obtained in this study showed a range of
ethanol production between 5.0 0.2 and 22.0 0.4 in all the strains. Two isolates NIRE K1
and NIRE K3 showed the highest ethanol yield of 0.49 and 0.41, respectively after 40 h of
incubation at 45
o
C. This study revealed the characteristics of the isolate NIRE K1 allow it to
ferment glucose efficiently to ethanol and have the potential to develop a bioprocess for
bioethanol production.
Key words: Bio-ethanol; Fermentation; Ethanol Yield; Thermotolerant.
14.1 Introduction
The global rise in energy consumption, predicted increase in energy demands in the
near future, the depletion of low extraction cost fossil fuel reserves, and climate change are
forcing the search for new and alternative energy sources (Agbor et al., 2011). A concern
about energy security, the environmental impact of energy production has led to the
implementation of policies designed to encourage the production and use of renewable
bioenergy (Glithero et al., 2013).



150

An alternative fuel must be technically feasible, economically competitive,
environmentally acceptable, and readily available. Numerous potential alternative fuels have
been proposed, including bioethanol, biodiesel, methanol, hydrogen, natural gas, liquefied
petroleum gas (LPG), FischerTropsch fuel, electricity, and solar fuels (Limayem and Ricke,
2012). Bioethanol has been considered in all over the world as an alternative renewable fuel
with the largest potential to replace fossil derived fuels, responsible for a significant fraction
of greenhouse gas emissions (Dias et al., 2013).
Current bioethanol research focus on lignocellulosic feedstocks due to its abundance
and renewability; especially in relation to reduce the cost and increase the efficiency of the key
steps in the lignocelluloses-to-bioethanol process (e.g. lignocellulosic pre-treatment,
enzymatic hydrolysis and fermentation) (Saratale and Oh, 2012; Mathew et al., 2013;
Matsushita et al., 2013). The main advantage of the production of second-generation biofuels
from lignocellulosic biomass is to reduce the limitation between food versus fuel competition
associated with first generation biofuels (Nigam and Singh, 2011; Singh et al., 2010).
Most of the potential ethanologens that are in industrial use belong to mesophillic
group (28-37
o
C). However, the bioethanol production from lignocellulosic biomass by
thermophillic/ thermotolerant species have some process advantages over mesophiles due to
high growth temperatures, require less energy for mixing and product recovery, higher
saccharification and fermentation rates, minimized contamination risk, lower costs of
pumping and stirring and no aeration and cooling problems (Georgieva&Ahring, 2007; Oberoi
et al., 2010; Frock & Kelly, 2012).
Considering the above, this study was carried out to compare the performance of the
thermotolerant yeast isolates for ethanol production. Further, the growth and fermentation
parameters of the isolates during fermentation were compared.
14.2.1 Microorganisms and culture conditions
Microorganisms were isolated from soil samples collected from dumping sites of
crushed sugarcane bagasse in Sugar Mills at 45
o
C. Four yeast isolates were compared for high
ethanol production rate and higher sugar consumption rate.
For growing the isolated strains, salt medium (SM) was used in g l
-1
, di-sodium hydrogen
ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate,


151

2.0; yeast extract, 1.0; glucose, 10.0 at pH 5.0. The cells were grown in 250-ml flasks in a
shaker at 45
o
C and 150 rpm for 24 h.
14.2.2 Fermentation conditions
The medium for fermentation was the same as that for the growth medium, except for
glucose 50 g l
-1
. Fermentation was carried out in 250-ml capped flasks at 100 rpm at 45
o
C.
14.2.3Analytical methods
At 4h intervals, fermented broths (in triplicate) were removed and the contents were
analyzed for biomass, sugar and ethanol. Glucose was analyzed by high-performance liquid
chromatography (HPLC) using Hi-Plex H column at 57
o
C with 1mM H
2
SO
4
as the mobile
carrier at a flow rate 0.7 ml min
-1
and detected by refractive index detector. Ethanol was
analyzed by gas chromatography using Nucon 5765 with a 2-m-long and 1/8-in. diameter
Porapak-Q column with mesh range 80/100. The sample was injected at an inlet temperature
150
o
C, and flame ionization detector 250
o
C using nitrogen gas as a carrier.
Dry cell weight (DCW) was determined in the broth by centrifuging 1 ml of broth in
pre-dry weighted Eppendorf tube using Eppendorf centrifuge 5430 R at 10,000 rpm for 5 min,
followed by washing twice with distilled water and drying in a vacuum oven at 80
o
C to a
constant weight.
The cultural characteristics of the isolates are shown in Table 1. In case of isolates
NIRE K1 and NIRE K3, the concentration of sugar dropped down in 24 h, with concomitant
production of ethanol; thereafter, the decline was gradual. At the end of 40 h of incubation,
the residual sugar concentration reached close to 5 g l
-1
with the ethanol concentration was 22
g l
-1
.
In contrast to NIRE K1 and NIRE K3, there was a marginal difference in sugar
consumption albeit, there was no ethanol produced in 20 h NIRE K4 and NIRE K5. The
marginal decrease in sugar concentration might be due to their utilization for growth and
metabolism by NIRE K4 and NIRE K5. After 24 h, there was a gradual increase in ethanol
concentration over the incubation period with simultaneous decrease in sugar concentration.
At the end of 40 h of incubation, the ethanol production from NIRE K4 and NIRE K5 reached


152

8 and 5 g l
-1
, respectively. The pattern of sugar utilization and ethanol formation among the
strains is shown in Fig. 1.
The initial DCW of NIRE K1 and NIRE K3 was kept at 2.7 g l
-1
and 2.8 g l
-1
, which
was 2.6 g l
-1
and 2.8 g l
-1
after fermentation, and shows almost constant DCW throughout the
process, respectively. No significant change was observed during fermentation in the cell-
mass concentration, which means the ethanol formation is non-growth associated when using
NIRE K1 and NIRE K3.
Table 1 Cultural characteristic of four yeast isolates

Fig. 1Comparison of ethanol production between free cells of four yeast isolates at 45
o
C
Isolate
Colour Margin Shape Opacity Elevation
NIRE-K1 White Entire
Circular
Transparent Flat
NIRE-K3 White Crenate
Circular Opaque
Raised
NIRE-K4
White
Crenate
Circular Opaque
Flat
NIRE-K5 Creamish yellow Undulate Irregular
Opaque
Umbonate


153

However, in case of isolates NIRE K4 and NIRE K5, the initial DCW was kept at 2.5
g l
-1
, which was 1.7 and 1.3 g l
-1
after fermentation, respectively. The DCW declined slightly
when ethanol concentration increased in broth.
Table 2 shows the fermentation parameters evaluated among the isolates. The
maximum ethanol concentration was 22 0.4 g l
-1
on initial glucose concentration of 50 g l
-1
with 89% of sugar conversion and ethanol yield of 96% of theoretical yield in 40 h. Banat et
al., 1992 reported the maximum ethanol concentration of 7.2% (w/v) with ethanol yield of
98% of theoretical yield and ethanol productivity 1.71 g l
-1
h
-1
on 140 g l
-1
glucose by K.
marxianusIMB2 at 45
o
C. Cazetta et al., 2007 achieved an ethanol concentration of 55.57 g l
-1
with an ethanol yield of 63.03% of theoretical yield and productivity of 1.16 g l
-1
h
-1
on
molasses containing 200g l
-1
reducing sugar in 48 h at 30
o
C by using Zymomonasmobilis.
Table 2 Growth and Fermentation kinetics of free cells of four yeast isolates at 45
o
C in Salt
Medium
Parameters evaluated NIRE K1 NIRE K3 NIRE K4 NIRE K5
Final ethanol (P, g l
-1
) 22.0 0.4* 12.0 0.4 8.0 0.1 5.0 0.2
Ethanol Yield (Yp/s, g g
-1
) 0.49 0.41 0.21 0.17
Sugar utilisation (g l
-1
) 44.90 0.04 29.27 0.05 38.10 0.07 29.41 0.03
Conversion rate (%) into
ethanol
89.80 58.54 76.19 58.82
Maximum specific growth
rate, h
-1

0.51 0.43 0.16 0.14
*mean standard deviation
The ethanol yield (Y
p/s
= 0.49) obtained with the free cells of NIRE K1 was more than
that of free cells of NIRE K3 (Y
p/s
= 0.41) followed by NIRE K4 (Y
p/s
= 0.21) and NIRE K5
(0.17). Kumar et al., 2009 reported ethanol yield of 90% of theoretical yield on glucose by
Kluyveromyces sp. IIPE453 at 50
o
C and concluded thatthe strain has the ability to convert
hexose sugars to cell mass as well as ethanol during the growth phase. Similar effect was
found in our studies where NIRE K1 and NIRE K4 follow the Crabtree rather than the Pasteur
Effect. In this study, isolate NIRE K1 was found to be more efficient than the other 3 isolates
in ethanol production.


154

14.4 Conclusion
The new isolated thermotolerant yeast strain NIRE K1 has shown the good
consumption of sugar for ethanol fermentation. The results showed that ethanol production by
isolate NIRE K1 was the highest in comparison to other isolates. For all the isolates, the peak
ethanol concentration was obtained after 40 h of fermentation. This study revealed the
characteristics of the isolate NIRE K1 allow it to ferment glucose efficiently to ethanol. The
yield could be further increased after optimization of the fermentation parameters andhave the
potential to develop a bioprocess for bioethanol production.
Acknowledgments
The authors are thankful to Dr. Y. K. Yadav, Director, SSS-NIRE for all the possible
support and encouragement. The authors also acknowledge MNRE, Govt. of India for
providing financial assistanceship.

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mahula (Madhuca latifolia L.) flowers by solid-state fermentation. Appli. Ener., 86:640-
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8. Narendranath N.V. and Power, R. (2005) Relationship between pH and medium
dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces
cerevisiae during ethanol production. Appl. Environ. Microbiol., 71:2239-2243.
9. Sakaguchi K., Matsui M. and Mizukami F. (2005) Applications of zeolite inorganic
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67 : 306311.
10. Sakai Y., Tamiya Y. and Takahashi F. (1994) Enhancement of ethanol formation by
immobilized yeast containing iron powder or Ba-ferrite due to eddy current or
hysteresis. J. Ferment. Bioeng., 77: 169172.
11. Swain M.R., Kar S., Sahoo A.K. and Ray, R.C.( 2007) Ethanol fermentation of mahula
(Madhuca latifolia L.) flowers using free and immobilized yeast Saccharomyces
cerevisiae. Microbiol. Res.,162:16293-16298 .
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CHAPTER 15
SWEET SORGHUM - AN IDEAL FEEDSTOCK FOR
BIOETHANOL PRODUCTION
Reetika Sharma1, Gurvinder Singh Kocher1 and Harinder Singh Oberoi

Abstract
Sweet sorghum can serve as a potential feedstock for ethanol production, largely because of
its ability to grow under hot and dry conditions; short duration, so that two crops could be
taken in a year in arid regions; low water requirement and high biomass yield. Since sweet
sorghum juice cannot be crystallized as cane sugar, it could be converted to ethanol through
fermentation. The sugar content in different varieties of sweet sorghum varies from 14-22 %.
After juice extraction, the bagasse could also be used for ethanol production. Although, the
ethanol production process from lignocellulosic biomass is cost and energy intensive, sweet
sorghum bagasse (SSB) contains relatively higher cellulose content (40 % or higher) as
compared to rice straw, sugarcane bagasse or wheat straw. This means that there is an
opportunity to obtain higher glucose concentration, which is essentially required for ethanol
production. In addition, relatively lower concentrations of lignin and ash in SSB in
comparison to other agricultural residues makes it easily amenable to pretreatment and
hydrolysis processes. Therefore, higher ethanol concentration obtained using both sweet
sorghum juice and bagasse could reduce the cost and energy required during downstream
processing, thereby making sweet sorghum as an ideal and cost-effective feedstock for
bioethanol production for its use as biofuel.
Key words: Lignocellulosic biomass, Sweet sorghum, Pretreatment, Hydrolysis,
Fermentation, Ethanol.
15.1 Introduction
Fossil fuel limitations and constraints on carbon dioxide emissions have a high impact
in the market of bioethanol, which is the most commonly used biofuel for petrol substitution
in the world (Taherzadeh and Karimi, 2008). Ethanol can be produced from a variety of feed
stocks such as saccharine materials, starchy materials and many types of lignocellulosic


157

wastes (Sanchez and Cardona, 2008). Lignocellulosic biomass is considered a future
alternative as raw material for bioethanol production, because it is more abundantly available
and is much less expensive than food crops, especially when waste streams are used
(Hamelink et al., 2005; Prasad et al., 2007b). Furthermore, the use of lignocellulosic biomass
is more attractive in terms of energy balances and green house gas emissions (Taherzadeh and
Karimi, 2007).
Sweet sorghum (Sorghum bicolor (L.) Moench) represents an analogous crop to
sugarcane with similar accumulation of sucrose but with a higher agronomic stability to
temperature fluctuations, less water requirement and better tolerance to salinity, alkalinity and
drought (Prasad et al., 2007a; Almodares and Hadi, 2009; Goshadrou et al., 2011). It contains
43.6 - 58.2 % soluble sucrose, glucose and fructose in the stalk (Billa et al., 1997; Dolciotti et
al., 1998; Amaducci et al., 2004; Antonopoulou et al., 2008) and 22.6 - 47.8 % insoluble
cellulose and hemicellulose (Dolciotti et al., 1998; Rattunde et al., 2001; Antonopoulos et al.,
2008). In addition, it is an annual crop with a typical growing season of 3-5 months instead of
9-12 months required by sugarcane. Additionally, the sweet sorghum bagasse has a
comparatively higher nutritional value for ruminants because of its more favorable fiber
composition and is a better alternative for further hydrolysis and fermentation (Almodares and
Hadi, 2009). Because of its agronomic flexibility and productivity, sweet sorghum is viewed
as a viable feedstock option for ethanol production in some regions of the world. Sweet
sorghums have potential for specific tropical, subtropical and arid regions of the US, Mexico,
China, India, Southern Africa and other developing countries where the use of maize and
other crops for ethanol production is not feasible due to either economic, agronomic or social
considerations (Reddy et al., 2005; Chuck-Hernandez et al., 2009; Wu et al., 2010; Zhang et
al., 2010).
Sweet sorghum has a high a ratio of energy output to fossil energy input in comparison
to sugarcane, sugar beet, maize and wheat and its fermentation efficiency has been reported
higher than 90 % (Almodares and Hadi, 2009; Wu et al., 2010). Lastly, sorghum is known as
one of the most variable crops in terms of genetic resources and germplasm that allows the
breeding and development of new cultivars adapted to different regions around the globe
(Zhang et al., 2010). For all these reasons, bioethanol produced from sweet sorghum presents
a high environmental, economic and energetically sustainable biofuel which ascribes GHGs
saving upto 70-71 % (Liu et al., 2008).


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Despite these advantages, utilization of sweet sorghum as a potential energy crop
presents some major technical challenges which must be resolved before sweet sorghum is
widely planted to provide feedstock for ethanol biorefineries. These include, its high
concentration of soluble sugars, due to which it can be easily contaminated, limiting its
storage stability. Secondly, the lignocellulosic stalk cannot be easily hydrolyzed enzymatically
to fermentable sugars due to the presence of free sugars inhibiting the hydrolytic enzymes
action; thus, the sugars in the stalk should be removed before the enzymatic hydrolysis of the
cellulosic part of the plant (Taherzadeh and Karimi, 2007; Molaverdi et al., 2013). Short
harvest periods also increase the capital cost involved in building a central processing plant
that may be operated only seasonally. Finally, the common practice of utilizing sweet
sorghum either involves a stage of sugars extraction and separate utilization of soluble sugars
and fiber fraction, which exhibits some technical difficulties, or involves a solid state
fermentation, which makes fermentation process and ethanol extraction more difficult,
leading to increased cost (Mei et al., 2009; Kundiyana et al., 2010, Wu et al., 2010, Matsakas
and Christakopoulos, 2013). Moreover, fermentation performance of sweet sorghum can be
affected by the microorganism used, bioreactor configuration, free amino nitrogen, sugar
content and composition of juices (Lui and Shen, 2008; Laopaiboon et al., 2009; Chohnan et
al., 2011). For this reason, evaluation of different sweet sorghum cultivars for their bioethanol
production potential is critically important (Zhao et al., 2009; Davilla-Gomez et al., 2011).
15.2 Origin and biology of sweet sorghum
Cultivated sorghum (Sorghum bicolor spp. bicolor L. Moench) is in the sub-genus
Sorghum and originates from semi-arid regions of Africa. However, due to its adaptive
capacity, now- a -days it is cultivated on a wide spectrum of climates in every continent
(Saballos 2008). Sweet sorghum varieties belong to the grain, forage and broomcorn
sorghums. These names describe well the diversity of phenotypes, as well as the aim and
direction of selection. Sweet sorghum has been selected for accumulation of high amount of
sucrose in the stem (Murray et al., 2009). Sweet sorghum belongs to C4 crop with high
photosynthetic efficiency and high productivity. The plant grows to a height of about 14 feet.
Seeds are produced by self-pollination from the panicle at the top of the plant and contains the
male and female inflorescences. The plant lodging is more likely to occur in high population
fields because stalks become smaller in diameter due to competition. The plant can also be
blown down in strong winds due to its height (Nahar, 2011).


159

15.3 Cultivation and harvesting of sweet sorghum
Cultivation of sweet sorghum possesses the following advantages arising from the
physiology and biochemistry of C4 plants that usually generate high biomass yield with minimal
inputs (Rooney et al., 2007; Saballos, 2008; Byrt et al., 2011):
1. High conversion efficiency of light into biomass (biomass yields competing with
switchgrass and miscanthus) resulting in high sugar, and thus ethanol yields;
2. High water use efficiency and thus low water requirement that is 25% of that needed for
sugar cane and 50-66% needed for maize production (no irrigation);
3. Drought tolerance - even though drought leads to reduction in plant growth, enhanced
accumulation of sucrose and starch was observed in drought stressed stems and thus
resulting in equal sugar yields (Massacci et al., 1996);
4. Reduced demand for fertilizer due to the high leaf nitrogen use efficiency and large
fibrous root system;
5. Modest demand for soil quality (that are not appropriate for corn or wheat)
6. High tolerance towards salinity and water-logging;
7. Pest and disease management is less complex,
8. Greater tolerance towards climate changes (e.g. temperature extremities, droughts).
9. Its cultivation is also possible on marginal lands and therefore, it can contribute to
sustainable ways to produce bioethanol, for instance, avoiding the food versus fuel
debate often related to bioethanol production (Prasad et al., 2009; Allen et al., 2011);
10. Its untapped genetic diversity as presented by its viability in almost every climate
condition, carries enormous potential for further breeding (Rooney et al., 2007; Saballos,
2008).
Cultivation Technology: The plant can be grown on soils ranging from heavy clay to light
sand. Loam and sandy loam soils generally allow the best syrup production. Good surface
drainage is preferred although sweet along sorghum can withstand long waterlogged condition;
clay loam is preferred with soil acidity not lower than pH 6.
Sowing: Sowing can be done on ridges or in furrows at a spacing of 60 cm between rows and
15 cm between plants. Three to four seeds are dibbled in each planting hole and the seedlings
are to be eventually thinned to one per hole. Sweet sorghum is not suitable for high density;
recommended density is about 7000 plants/ha. The plant is ideally shown during June to


160

September, when soil can hold much water (deep).The crop does not prefer high rainfall as high
soils moisture or continuous heavy rain after flowering may decrease sugar content in plants.
Setting of Furrow: Two planting seasons are possible for sweet sorghum. During the wet
season, furrows are set 100 cm apart while in the dry season planting are set about 75 cm apart.
Fertilizer Application: The plant needs adequate nutrients to produce good yields. Quality of
syrup is also affected by the fertilizer applications. The recommended dose of fertilizer for sweet
sorghum is 80 kg of nitrogen, 60 kg of phosphorous and 40 kg of potassium per hectare. Half of
N and whole of P and K are applied as basal dose. Remaining N is top-dressed during 25-35
days after germination, following weeding and inter-cultivation. Nitrogen fertilizer should not
be applied in the field when sweet sorghum is grown immediately after a legume crop, as the
soil contains nitrogen.
Intercropping: Sweet sorghum is suitable to intercropping with early maturing crops for its
characteristics in growth and development. Sweet sorghum seedlings develop slowly at their
early stage. It can be intercropped with potato, maize, wheat etc. Adaptation and Yield: It is
relatively inexpensive to grow high yield sweet sorghum plants and can be used to produce a
range of high value added products like ethanol, energy and dried grains. It can produce
approximately 30 tons/ ha per year of biomass on low quality soils with low inputs of fertilizer,
half of that required by sugar beet and a third of the requirement for sugarcane or corn.
Harvesting: The plant varieties mature between 115-125 days after plantation. To obtain high-
quality syrup and high yields, the crop should be harvested when the seed is in the soft dough
stage. Stalks can be harvested either along with the grain, or 4-5 weeks after the grain harvest.
The ear-head and peduncle (between the base of the seed head and the top node) should be
removed before processing the stalks. Ear heads may be dried and threshed so the seeds can be
used for the next year's crop.
15.4 Inherent advantages of sweet sorghum
Sorghum is an annual crop with a lot of varieties (at present estimated at about 4,600
approximately), and some of these have a very high sugar content in the stems. Its bioenergy
applications are numerous: sweet sorghum can be used to produce ethanol, but alternatively
also biogas through anaerobic digestion, fiber sorghum (pelletized or not) can supply
combined heat and power (CHP) plants, grain sorghum can be employed for food, feed and
energy needs of small isolated communities. Grain sorghum is not so suitable for the ethanol


161

production, because in these varieties, sugars are prevalently polymerized to starch, which
then require hydrolysis before the alcoholic fermentation. Sweet sorghum is a sugar cane-like
C
4
plant, containing juice in the stem with large amount of sucrose (11-23 % brix, depending
on variety and conditions) that can be effectively extracted by squeezing and thereafter,
readily fermented to ethanol by yeast (Almodares and Hadi, 2009, Byrt et al., 2011). While
it is not well suited to the production of refined sugar, sweet sorghum has multiple inherent
advantages which have been represented in Table 1. Most important, sweet sorghum is a seed-
propagated annual crop, i.e., a crop established through sowing seed and which matures and is
harvested in a single season. These key characteristic impacts both its fit within current
production cycles as well as the pace of scale-up and on-going improvements to the varieties
themselves. In addition, sweet sorghum can be utilized in rotation with other annual crops,
and potentially, with sugarcane itself, where sweet sorghum could be sown on fallow
sugarcane land, hectares destined for rotation and land where sugarcane yields are limited due
to marginal soils. This flexibility is due to the sorghum plants natural hardiness and rapid
growth.
Table 1: Inherent advantages of sweet sorghum over sugarcane
Sugarcane Sweet sorghum
Sugar quality Sucrose Mixed sugars
Establishment cost Vegetative propagation Seed propagation
Sugar yield (% fw) 13-15 8-13
Input requirements Limited by water, nitrogen 50% water, 60% nitrogen
Scale-up time Vegetative propagation Seed propagation
Biomass yield (tons/ha) 70-90 tons/ha 60-100 tons/ha
Marginal land Limited yield Significant yield
Season extension 12-18 months 70-120 days
Product development Perennial, 10-16 years Annual, 3-5 years
Source: Wu et al., 2008

Traditional uses of the juice include production of syrup, alcoholic beverages,
crystalline sugar and in some regions stalks are also consumed fresh (Saballos, 2008). The


162

leftover, built up of lignocellulose, is called bagasse. The main advantage of sweet sorghum
bagasse over other cellulosic biomass is relatively higher cellulose and low ash content (Table
2). Low ash content facilitates the use of milder and low energy intensive pretreatment, while
higher cellulose content creates a possibility of obtaining higher quantity of glucose (Gao et
al., 2010).
Table 2: Comparative compositional analysis of sweet sorghum bagasse with other agro
residues
Agro Residue Cellulose
(%)
Hemicellulose
(%)
Lignin
(%)
Ash (%) Reference
Kinnow pulp 8.820.70 4.440.55 3.710.40 5.930.34 Oberoi et al.,
2010
Sunflower
Stalks
32.561.65 20.730.66 14.360.56 6.032.46 Daz et al., 2011
Soybean hulls 33.493.18 17.150.04 9.880.01 4.710.07 Brijwani et al.,
2011
Wheat bran 7.570.17 31.190.30 4.060.09 6.530.01 Brijwani et al.,
2010
Sugarcane
bagasse
35.220.91 24.520.63 22.280.14 3.710.31 Rezende et al.,
2011
Sweet
sorghum
bagasse
44.6 0.13 27.10.23 20.70.21 0.40.11 Kim and Day,
2011
Sorghum
bagasse
40.41.01 35.50.91 22.30.43 0.20.01 Dogaris et al.,
2009

Besides these uses, another possibility is gaining growing attention i.e. to cultivate it
as energy crop for bioethanol production (Almodares and Hadi 2009; Byrt et al., 2011;
Ratnavathi et al., 2011). Ethanol yields of 2100-8000 L/ha have been reported with one
harvest annually that significantly exceed the ethanol yields from starchy materials, such as


163

corn and wheat grains (Byrt et al., 2011; Balat and Balat, 2009). Furthermore, when
comparing the energy performance of wheat and sweet sorghum monocultures, a significantly
higher net energy gain for sweet sorghum was demonstrated in Northern Italy (Monti and
Venturi, 2003). In Asia (China, India and the Philippines) and South America fermentation of
sweet sorghum juice is carried out on a small to medium scale (Saballos, 2008). Contrarily,
the conversion of sweet sorghum bagasse to ethanol is still in an experimental phase
(Ballesteros et al., 2004; Sipos et al., 2009; Li et al., 2010; Yu et al., 2010; Shen et al.,
2011; Ratnavathi et al., 2011). Economic evaluation of a sweet sorghum biorefinery for
ethanol production from bagasse has been studied under North Chinese circumstances
(Gnansounou et al., 2005), while the economy of juice processing has been investigated
more deeply, for example, in Indian context (Prasad et al., 2007), upper Midwest of the USA
(Bennett and Anex, 2009), Zimbabwe (Woods, 2001) and Inner Mongolia (Wang and Liu,
2009).
15.5 Technical hurdles
Under moderate climate, the technological difficulty of sweet sorghum processing is
the short harvest period making the juice available only for 1-2 months annually. Due to this
reason, the juice cannot be stored because the microbes including its natural microbial flora
degrade the easily fermentable sugar content. Without any preservation, up to 12-30 % of the
fermentable sugar content can be lost in 3 days and 40-50 % in a week at room temperature
(Daeschel et al., 1981; Wu et al., 2010). Many methods have been proposed to elongate the
availability of the juice, for instance: refrigerating (Wu et al., 2010), evaporation (Hodr et
al., 2008), ensiling the whole stalks (Bennett and Anex, 2009), proper harvest and
processing method (Lingle et al., 2012) and lowering the pH with the addition of different
acids (Hodr et al., 2008). But these alternatives lead to elevated energy demands and/or
chemicals needs, thereby, influencing the overall process economy. The reports of Bennet
and Anex (2009) and Gnansounou et al. (2005) suggested that fermentation of sweet
sorghum juice under moderate climate could only be a complementary process, for example,
in a biorefinery concept.
15.6 Bioethanol production from sweet sorghum
Sweet sorghum is being considered as a SMART crop as it offers triple benefits of
F, i.e., food, fodder and fuel, without significant tradeoffs in any of these uses in the


164

production cycle. Its relatively short vegetation cycle allows sweet sorghum to be grown in
double cropping systems based on water availability, which in turn can lead to greater agro-
biodiversity and a reduced demand for fertilizers and pesticides (Nahar, 2011). Sorghum has
the potential to be an excellent diversified biofuel crop able to fill the needs of multiple
bioenergy conversion process across many environments with reduced energy requirements.
Besides environmental advantages, sorghum is one of the more acquiescent plant to genetic
modifications because it is highly variable in terms of genetic resources and germplasms. This
facilitates plant breeding and development of new cultivars adapted to different regions
around the globe (Ratnavathi et al., 2011). Overall scheme for production of bioethanol from
sweet sorghum involving various steps which include pretreatment, enzymatic hydrolysis and
various fermentation methods is presented in Figure 1.
For the effective conversion of lignocellulosic material into ethanol, the following three major
steps are involved:
1. A thermo-chemical pretreatment is a pre-processing step that improves the access of
enzymes to cellulose and hemicellulose.
2. Enzymatic saccharification that breaks down cellulose and hemicellulose into simple
sugars.
3. Fermentation of released sugars by specialized organisms.
It is clear from the figure that sweet sorghum bagasse (SSB) can be used as an ideal
substrate for production of cellulases which can further be used for saccharification of
pretreated biomass. Pretreatment is an important step for cellulose conversion processes, and
is one of the important factors affecting ethanol production from lignocellulosics. It is
essential for causing alterations in the structure of cellulosic biomass in order to facilitate the
enzymatic saccharification process. The goal here is to break the lignin seal and disrupt the
crystalline structure of cellulose (Agbor et al., 2011; Zhang and Shahbazi, 2011).
Once the cellulose and hemicellulose fractions are exposed after pretreatment and are
accessible to the action of cellulases and xylanases, a mixture of hexose as well as pentose
sugars are produced (Sun and Cheng, 2002). The sugars thus produced are fermented by the
fermenting micro-organisms, such as Saccharomyces cerevisiae, Pichia kudriavzevii or a
combination of hexose and pentose fermenting microbial strains to exploit the full potential of
the sugars, thus produced. However, in absence of robust pentose fermenting micro-


165

organisms, generally the alcohol yields from the mixed sugar streams are low. Table 3
represents the different microorganisms used in the fermentation for ethanol production from
sweet sorghum along with their ethanol yields. However, the biological/biochemical route for
ethanol production from lignocellulosic biomass is a preferred method, because it is less
energy intensive as compared to the thermo-chemical conversion method and the
infrastructural and capital requirements are relatively lower.

Figure 1: Flow chart for the bioethanol production from sweet sorghum (Ratnavathi et al.,
2011)
In one of our studies on production of ethanol from sweet sorghum bagasse, we found
the ethanol concentration in the range of 35-40 g/l using crude cellulases and thermotolerant
Pichia kudriavzevii strain (unpublished data).
15.7 Energy ratio and environmental sustainability
The available data about the European Union (EU) model for the ethanol produced
from all components of sweet sorghum crop suggest an output/input ratio of 1.7 -7.3
(depending on the strategy chosen for the by-products exploitation), a high greenhouse gases
(GHGs) saving in accordance with the RES Directive and a low water footprint. The main
environmental advantage relative to the use of bioethanol in substitution of gasoline and/or


166

the use as bio-ETBE (ethyl tert-butyl ether) instead of fossil antiknocks is the abatement of
the transport sector contribution to the GHG emissions. For bioethanol produced using sweet
sorghum, this assertion assumes an absolute validity because in this case, the saving
calculated with the methodology indicated by EU is 71% which is one of the most virtuous
values among the attainable ones. The use of sweet sorghum for bioethanol production
conciliates the production of sustainable bioethanol applying already mature technologies
with an involvement of the agricultural sector in the pathway.
Table 3: Micro-organisms used in the fermentation for ethanol production from sweet
sorghum
Sr. No. Micro-organisms used in the
fermentation of:
Ethanol yield Reference
Juice

1
Saccharomyces cerevisiae CFTR 01 and SG 0.39-0.48 (g/g) Ratnavathi et al., 2010
2
Fermax yeast (Saccharomyces cerevisiae) 77.07-79.58(g/l) Kundiyana et al., 2010
3
Super start yeast (Saccharomyces cerevisiae) 73.18-76.95(g/l) Kundiyana et al., 2010
4
Saccharomyces cerevisiae TISTR 5048 0.42-0.48 (g/g) Laopaiboon et
al.,2007
5
Saccharomyces Strains 29-87% (sugar
conversion
effienciency)
Bulawayo et al., 1996
6
Saccharomyces cerevisiae (Nanayang) 91.61 % Liu et al., 2008
7
Saccharomyces cerevisiae 0.42-0.45 (g/g) Balint-Sipos et al.,
2009
Bagasse

8
Saccharomyces cerevisiae Ethanol conc. -
42.3 (g/l)
Li et al., 2010
9
Saccharomyces cerevisiae 0.147 (g/g) Ban et al., 2008
Source: Ratnavathi et al., 2011

15.8 Small-medium scale bioethanol production plant from sweet sorghum
Sweet sorghum could favour the diffusion of the bioethanol pathway in the local
agricultural sector, because the creation of small-medium plants involves the plant building
near the fields where the biomass is harvested (max 50 km approximately). This choice


167

allows an increase of sustainability of the bioethanol production, because the impacts of the
long distance transportation is reduced. The size of small-medium plants does not exceed
15,000 t/y of ethanol capacity. Table 4 represents the comparative theoretical ethanol
production from sweet sorghum with other feedstocks. There are many criteria to select a
suitable bioethanol production technology, such as plant scale, investment and operation cost,
management operations, and conversion yield, which have a reflecting effect on the
production cost. Another important criterion is the energy balance that shows the efficiency of
processing energy used.
Pilot studies in India have indicated that ethanol production from sweet sorghum is
cost-effective (Dayakar et al., 2004). The International Crops Research Institute for the Semi-
Arid Tropics (ICRISAT) located at Patancheru in Andhra Pradesh, India, has developed
several improved sweet sorghum lines with high stalk sugar content and a few of these lines
are being tested in pilot studies for sweet sorghum-based ethanol production in India, of
which, SSV 84, SSV 74 and NSSH 104 have been released (Reddy et al., 2008). Further,
sweet sorghum bagasse (SSB) has a higher biological value than the bagasse from sugarcane
when used as feed for cattle, as it is rich in micronutrients and minerals and is as good as
stover in terms of digestibility (Panwar et al., 2000). In addition to the above characteristic
features, short duration of this crop and a yield in the range of 54-69 tons/ha renders SSB an
important and readily available substrate for ethanol production (Almodares and Hadi, 2009).
Thus, sweet sorghum represents a potential opportunity to improve the economic and
environmental sustainability of the bioethanol pathway.
Table 4: Theoretical ethanol production from multiple feedstocks (kg/ha, dry basis)
(Adapted from Kim and Day, 2011)
Component Sugarcane Energy cane Sweet sorghum
Feed stock 70,000 100,000 60,000
Juice
a
0 53,600 43,140
Fiber 9,450 26,700 7,800
Sugar
Monomeric sugar from juice 0 5,253 5,091
Glucose from cellulose
b
4,368 12,846 3,865
Xylose from hemicellulose
c
2,695 7,221 2,402
Ethanol


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Ethanol from juice
d
0 2,684 2,601
Ethanol from cellulose
d
2,232 6,564 1,975
Ethanol from hemicellulose
d
1,377 3,690 1,227
Total ethanol 3,609 12,938 5,804
a
Juice is wet kilograms
b
Glucose (kg)= Glucan (kg) 1.11; 1.11 is a conversion factor considering water addition during hydrolysis
c
Xylose (kg) = Xylan (kg) 1.14; 1.14 is a conversion factor considering water addition during hydrolysis
d
Ethanol (kg) = Glucose, fructose, sucrose or xylose (kg) 0.511; 0.511 is a conversion factor for sugar to ethanol
based on stoichiometric biochemistry of yeast.
15.9 Conclusions
Sweet sorghum has distinct advantages over the other lignocellulosic biomass for
bioethanol production because of some of the inherent characteristics of this crop. Higher
biomass yield, ability of the crop to grow under drought and stress conditions, ability to get
two crops in a year makes sweet sorghum as an attractive contestant for ethanol production.
The juice extracted from sweet sorghum could be used directly for bioethanol production
through fermentation and the sweet sorghum bagasse could be converted to ethanol through a
series of steps, like, size reduction, pretreatment, hydrolysis and fermentation. Therefore,
combining the ethanol yields from both the juice as well as bagasse confers a distinct
advantage to sweet sorghum over other lignocellulosic biomass, such as rice straw, cotton
stalks, bagasse, etc.
Acknowledgements
Authors thankfully acknowledge the financial assistance received from the project
funded by the Department of Biotechnology, Government of India
((PR/8488/PBD/26/68/2006) for conducting a part of the study reported in this book chapter.

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175

CHAPTER 16
FERMENTATION OF GLUCOSE AND XYLOSE SUGAR
FOR THE PRODUCTION OF ETHANOL AND XYLITOL BY
THE NEWLY ISOLATED NIRE-GX1 YEAST
Shuvashish Behera

, Richa Arora

, Nilesh Kumar Sharma and Sachin Kumar

Abstract
Hemicellulose is the second most abundant polysaccharide after the cellulose available on the
earth in the form of lignocelluloses. The hemicellulose must utilized for the cost-efficient
production of ethanol. Xylan, the major component in the hemicellulose in plant biomass,
yields mainly xylose as pentose sugars on hydrolysis. The progress in fermentation of pentose
sugars has gone on slow pace as there are few microorganisms known, which are capable of
pentose metabolism. Therefore, this study was carried out to isolate and screen the yeasts from
soil samples collected from different dumping sites for the production of ethanol using
glucose and xylose sugar. About 16 yeast strains showed positive results in ethanol production
from glucose sugar. Four isolates designated NIRE-GX1, NIRE-GX2, NIRE-GX3, NIRE-
GX4 showed positive result in ethanol production from both glucose and xylose sugars.
Further study was carried out using the isolate NIRE-GX1 yeast, which showed more growth
and fermentation efficiency at a temperature of 40
o
C on both the sugars. Anaerobic batch
fermentations were carried out using the yeast strain from the individual glucose and xylose
sugar separately and further mixture of both at 40
o
C temperature. The strain also showed
xylitol production from xylose. The strain showed maximum ethanol concentration of 7.1
0.6 g l
-1
with complete utilization of glucose (20 g l
-1
) in 24 h. However, in case of xylose
fermentation, the strain showed maximum ethanol concentration of 0.8 0.08 g l
-1
as well as
xylitol concentration of 0.64 0.3 g l
-1
in 72 h on initial xylose concentration of 20 g l
-1
. The
strain was capable of simultaneously using glucose and xylose in a mixture of glucose
concentration of 14 g l
-1
and xylose concentration of 6 g l
-1
, achieving maximum ethanol and
xylitol concentration of 5.3 0.5 g l
-1
and 0.95 0.32 g l
-1
, respectively in 72 h.
Key Words: Bio-ethanol; Fermentation; Glucose; Xylose.


176

16.1 Introduction
The largest potential feedstock for ethanol is lignocellulosic biomass, which includes
materials such as agricultural residues (corn stover, crop straws, sugarcane bagasse),
herbaceous crops, short rotation woody crops, forestry residues, waste paper and other plant
wastes (Kim and Dale, 2005). It varies among plant species but generally consists of cellulose
(4050%), hemicelluloses (2530%), and lignin (1020%) (Wiselogel et al., 1996). Complete
hydrolysis of cellulose and hemicellulose results in glucose and xylose respectively, which can
be ferment to ethanol by several microorganisms (Alvira et al., 2010; Sims et al., 2010).
Cellulose contains glucose and hemicellulose contains mainly pentoses, such as a xylose,
which are not fermentable by the natural industrial strains and can add up to 25% of the
constitution of lignocellulosic materials (Schell et al., 2004; Olofsson et al., 2008).
Yeasts that produce ethanol from D-xylose have been isolated from various locations,
including tree exudates (Ipsit et al., 2013), wood-boring insects (Suh et al., 2003), decaying
wood (Cadete et al., 2009), rotten fruit and tree bark (Rao et al., 2008). There are also several
yeast species such as Candida shehatae, Pachysolen tannophilus, Brettanomyces
naardenensis, C. tenuis, Pichia segobiensis, C. lyxosophila, C. intermedia, C. jeffriesii,
Spathaspora passalidarum, Spathaspora arborariae, C. prachuapensis, and Scheffersomyces
stipitis which has been reported as xylose fermenting yeasts (Barnett et al., 2000; Nguyen et
al., 2006; Cadete et al., 2009; Nitiyon et al., 2011). Candida and Pichia are the two naturally
occurring best ethanol producing organism from pentose. However, those yeast strains are
neither well adapted to ethanol production nor can tolerate to by-products of lignocellulosic
hydrolysates (Jeffries, 2006; Hahn-Hgerdal et al., 2007). Although Saccharomyces cerevisiae
combines several desired attributes such as high ethanol tolerance, general robustness and
operation experience which favored for industrial scale of ethanol production; the success is
restrained by the organisms inability to naturally ferment pentose sugars (Helle et al., 2004;
Hahn-Hgerdal et al., 2007).
In a study, cocultivation of S. cerevisiae and S. stipitis strains to co-ferment glucose
and xylose was unsatisfactory, due to their difference in fermenting condition and ethanol
tolerance (Agbogbo et al., 2006). However, S. stipitis strain prefers to ferment glucose rather
than xylose having lower ethanol tolerance than S. cerevisiae strain (Watanabe et al., 2007).
Further, in order to assimilate xylose, some strains produce xylitol as the intermediate product
with the production of ethanol. Despite the existence of these microorganisms, it is still


177

challenging to reach high yields of ethanol from pentose sugars on a large scale (Hahn-
Hagerdal and Pamment, 2004) because no microorganisms that robustly convert pentose
sugars into ethanol at high yields while withstanding fermentation inhibitors have been
identified (Chandel et al., 2011). Therefore, this study was carried out for a new search of
yeasts from soil samples collected from different dumping sites for the production of ethanol
using glucose and xylose sugar. Finally fermentation of both glucose and xylose sugars were
carried out using NIRE-GX1 yeast for the production of ethanol.
16.2.1 Sample collection
Samples for isolation of ethanol producing yeasts were obtained from the dumpyard at
Jalandhar, Punjab and from Wahid Sandhar Sugars Ltd., Punjab. Exploration for collecting
potential samples was carried out by walking-through within 60-90 min for each site. Soil
samples were put into plastic or polythene bags and finally sample details were recorded.
16.2.2 Isolation of yeast
To isolate yeast, 1g of each soil samples was suspended in 10 ml of sterile water by
vortexing for 2 minute on maximum speed, followed by a 10x serial dilution. About 0.1 ml of
each dilution in the series was spread onto the surface of yeast extract-peptone-dextrose
(YPD) phytagel (yeast extract, 1%; peptone, 2%; dextrose, 2%; phytagel, 1.5%; ampicillin, 50
mg/ml; water, 1000ml; pH, 5.5) plates and incubated at 40
o
C for 24 hour. For isolation of
xylose utilizing yeasts, xylose sugar (2%) was added in replacement of dextrose to the above
medium. Various colonies were selected based on their morphology, size and color appear on
the phytagel plates. The selected colonies were then subcultured on to separate phytagel plates
to ensure their purity.
16.2.3 Microorganism and culture condition
The yeast culture was maintained on yeast extract-peptone (YP) medium (g/l): yeast
extract, 1%; peptone, 2%; phytagel, 1.5%; ampicillin, 50 mg/ml; water, 1000ml; pH, 5.5 and
sugar (glucose and xylose) was added according to the use by the isolates. The culture was
stored at 40.5
o
C for further use.
16.2.4 Preparation of inoculum


178

The inoculum was prepared in 100 ml YP growth medium (as mentioned above but
without phytagel) containing glucose and xylose as the sugar source, taken in sterilized (at
121
o
C for 20 min) 500 mL erlenmeyer flask and cotton plugged. The flask was inoculated
with a loopful of the yeast culture and incubated for 24 h at 40
o
C at 120 rpm in an orbital
shaker incubator (Remi Pvt, Ltd, Bombay, India). The cells grown after 24 h were acts as the
inoculum for the fermentation medium.
16.2.5 Fermentation medium
Fermentation medium was prepared using salt medium (SM) in gl
-1
: di-sodium
hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium
sulphate, 2.0; yeast extract, 1.0; glucose, 10.0 at pH 5.5. The cells from the YP medium was
centrifuged at 7500 rpm for 10 min and added in the fermentation medium. The cells were
grown in 250-ml capped flasks in a shaker at 40
o
C and 120 rpm for 72 h.
16.2.6 Analytical methods
At 24 h interval, fermented broths (in triplicate flasks) were removed and the contents
were analyzed for total sugar, ethanol and xylitol production. Glucose was analyzed by high-
performance liquid chromatography (HPLC) using Hi-Plex H column at 57
o
C with 1mM
H
2
SO
4
as the mobile carrier at a flow rate 0.7 ml min
-1
and detected by refractive index
detector. The pH was measured by a pH meter (Systronics, Ahmadabad, India) using glass
electrode. The fermentation kinetics was studied as per the formulae described below (Bailey
and Ollis, 1986)
16.3 Results and discussion
About 16 yeast strains showed positive results in ethanol production from glucose
sugar. Four isolates designated NIRE-GX1, NIRE-GX2, NIRE-GX3, NIRE-GX4 showed
positive result in ethanol production from both glucose and xylose sugars. The isolated strains
were carefully identified by morphological characteristics including color of the colony and
growth pattern studies with the help of microscope. Strain selection was based on anaerobic
growth on xylose as well as on high xylose uptake rate. In fermentation studies on defined
media the resulting NIRE-GX1 yeast was shown to possess the ability to grow on xylose
under anaerobic conditions and to ferment both xylose and glucose to ethanol in good yield
and productivity.


179

Therefore further study was carried out using the isolate NIRE-GX1 yeast, which
showed more growth and fermentation efficiency at a temperature of 40
o
C on both the sugars.
Anaerobic batch fermentations were carried out using the yeast strain from the individual
glucose and xylose sugar separately and further mixture of both at 40
o
C temperature. The
strain also showed xylitol production from xylose. The strain showed maximum ethanol
concentration of 7.1 0.6 g l
-1
with complete utilization of glucose (20 g l
-1
) in 24 h.
However, in case of xylose fermentation, the strain showed maximum ethanol concentration
of 0.8 0.08 g l
-1
as well as xylitol concentration of 0.64 0.3 g l
-1
in 72 h on initial xylose
concentration of 20 g l
-1
. The strain was capable of simultaneously using glucose and xylose in
a mixture of glucose concentration of 14 g l
-1
and xylose concentration of 6 g l
-1
, achieving
maximum ethanol and xylitol concentration of 5.3 0.5 g l
-1
and 0.95 0.32 g l
-1
, respectively
in 72 h (Table 1). Nigam et. al. (1985) found maximum ethanol concentration of 8.8, 10.9 and
9.8 g l
-1
with initial D-xylose sugar concentration of 40, 60 and 80 g l
-1
.
Table 1. Ethanol production from glucose and xylose sugar using yeast isolate NIRE-GX1
Glucose Fermentation Xylose Fermentation Fermentation of mixture of sugar
Time (h) pH Sugar Consumed
(g l
-1
)
Ethanol (g
l
-1
)
pH Sugar
Consumed
(g l
-1
)
Ethanol
(g l
-1
)
Xylitol
(g l
-1
)
pH Sugar Consumed
(g l
-1
)
Ethanol (g
l
-1
)
Xylitol
(g l
-1
)
24 2.93 19.980.04 7.30.06 3.48
7.260.2 0.120.03 0.470.1
3.08
16.751.4 4.430.1 0.790.05
48 - - - 3.39
8.381.2 0.20.04 0.60.05
3.06
17.070.2 4.970.3 0.850.2
72 - - - 3.19
9.30.1 0.80.06 0.640.3
3.03
17.120.8 5.30.5 0.950.32

The growth and fermentation kinetics of free cells in presence of individual and mixed
of sugars were also studied (Table 2). The ethanol concentration (P) obtained with glucose
fermentation with NIRE-GX1 yeast (7.30.06 g l
-1
) was 89.04 % more than that of xylose
fermentation (0.80.06 l
-1
), where as the volumetric substrate uptake (Qs) was found to be
84.34 % more in case of glucose fermentation (0.83 g l
-1
h
-1
) over xylose fermentation (0.13 g
l
-1
h
-1
). The ethanol yield (Yp/s= 0.37 g g
-1
) and volumetric product productivity (Qp=0.3 g l
-1

h
-1
) obtained with glucose fermentation was found to be 75.68 and 96.7 %, respectively higher
than that of Yp/s (0.09 g g
-1
) and Qp (0.01 g l
-1
h
-1
) of xylose fermentation. However, the final
biomass (X) concentration in case of xylose fermentation (1.5 0.2 g l
-1
) was considerably
lower than glucose fermentation (1.2 0.2 g l
-1
), which is useful during product separation
and purification process (Diderich et al. 1999). Cadete1 et al. (2012) reported that the newly
isolated S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and


180

productivities (0.62 g l
-1
h
-1
to 0.75 g l
-1
h
-1
). Also another strain Candida amazonensis
exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g g
-1
to 0.59 g g
-1
), with concentrations up to 25.2 g l
-1
.
Table 2. Growth & Fermentation Kinetics of Ethanol Production

Glucose
fermentation
Xylose
fermentation
Fermentation of
mixed sugar
Final ethanol (P, g l
-1
) 7.30.06 0.80.06 5.30.5
Final biomass concentration (X, g
l
-1
)
1.90.2 1.51.2 1.80.7
Ethanol yield (Yp/s, g g
-1
) 0.37 0.09 0.3
Volumetric substrate uptake (Qs, g
l
-1
h
-1
)
0.83 0.13 0.24
Volumetric product productivity
(Qp, g l
-1
h
-1
)
0.3 0.01 0.07

16.4 Conclusion
Efficient ethanol production from xylose is crucial for Bioethanol production from
lignocellulosic biomass. We attempted to enhance the ethanol productivity of the xylose-
fermenting yeast NIRE-GX1. The strain showed utilization both hexose and pentose sugar for
the production of bioethanol with a low amount of xylitol. Further studies like pentose sugar
uptake, presence of sugar transporter and inhibitor tolerance are required to increase the
uptake rate of pentose sugar.
Acknowledgment
We greatly acknowledge the Ministry of New and Renewable Energy, New Delhi,
Govt. of India for providing funds to carry out research work.

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2. Alvira P., Tomas-Pejo E., Ballesteros M. and Negro M.J. (2010) Pretreatment
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3. Bailey J.E. and Ollis D.F. (1986) Biochemical engineering fundamentals. McGraw-Hill.,
New York.
4. Barnett J.A., Payne R.W. and Yarrow D. (2000) Yeast Characteristics and
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5. Cadete R.M., Santos R.O., Melo M.A., Mouro A. and Goncalves D.L. (2009)
Spathaspora arborariae sp. nov., a D-xylose-fermenting yeast species isolated from
rotting wood in Brazil. FEMS Yeast Res., 9:13381342.
6. Cadete1 R.M., Melo1 M.A., Dussan K.J., Rodrigues R.C.L.B., Silva S.S., Zilli J.E.,
Vital M.J.S., Gomes F.C.O., Lachance M.A. and Rosa1 C.A. (2012) Diversity and
physiological characterization of D-xylose-fermenting yeasts isolated from the
Brazilian Amazonian forest. Plos One, 7:1-11.
7. Diderich J.A., Schepper M., Van H.P., Luttick M.A., Van D.J.P. and Pronk J.T. (1999)
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10. Ipsit H., Bidisha S., Anindita R. and Subhra K.M. (2012) Ethanol production from
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183

CHAPTER 17
COMPARATIVE BIOETHANOL PRODUCTION BY S.
cerevisiae AND Z. mobilis FROM SACCHARIFIED SWEET
POTATO ROOT FLOUR (Ipomoea batata L) USING
IMMOBILIZED - AMYLASES AND GLUCOAMYLASE
Preeti Krishna Dasha, Sonali Mohapatraa , Manas Ranjan Swainb, Hrudaya Nath Thatoi

Abstract
Sweet potato (Ipomoea batatas L) represents an important biomass resource for fuel alcohol
production, because of its chemical composition which has a dry matter content of 30-40%
and high density of starch (70%), compared to other forms of biomass and thus promises as an
alternative bioresource for the production of ethanol through fermentation. The starch present
in sweet potato can be hydrolysed to simple sugars and can then be used by microorganisms in
fermentation process. Fermentation, by using immobilized enzyme is an advanced technology
which has several benefits as compared to free cell culture which include enhanced ethanol
yield, easy to separate, reduced percentage of contamination, better operational stability and
viability of enzyme actions for several cycles of fermentation. In this experiment batch
fermentation is carried out in flask condition to compare the bioethanol production by
Saccharomyces cerevisiae (S. cerevisiae) and Zymomonas mobilis (Z. mobilis) from
saccharified sweet potato root flour (SPRF) using immobilized -amylase and glucoamylase.
The ethanol yields were 485.5 g/ kg and 448.7 g/ kg of SPRF for S. cerevisiae and Z. mobilis
respectively after an incubation period of 96 hours maintained at pH 4.5 and 30
o
C
temperature. The study demonstrated that S. cerevisiae can produce 10.6% higher bioethanol
in comparison to Z. mobilis. The highest ethanol concentration was obtained after 96 hour of
fermentation. Thus, it was evident from the present study that Z.mobilis does not appear to be
an efficient microbial source for commercial bioethanol production, attributed to its lower
tolerance to temperature, ethanol and utilization of substrate range compared to S. cerevisiae.
Thus the use of S. cerevisiae has great scope for bioethanol production under enzyme
immobilization condition having potential for commercial application.


184

Keywords: Sweet potato, Saccharification, Immobilization, S. cerevisiae, Z. mobilis.
17.1 Introduction
The uses of ethanol as an alternative motor fuel has been steadily increasing around
the world for a number of reasons such as it decreases dependence on foreign oil, reduces
trade deficits, creates jobs in rural areas, reduces air pollution by replaceing aromatic and
sulfur-content in gasoline, it also reduce nitrogen oxide (NOx) emission to improve air quality
which can reduce urban smog and reduces global climate change carbon dioxide buildup
(Wheals et al., 1999). The high oxygen content in bioethanol could reduce the generation of
known hazardous volatile organic compounds (VOCs) and carbon monoxide in vehicle
exhaust (Wyman, 1996; Yoon et al., 2009).
Establishment of ethanol industry requires sufficient and cheap feedstock in order to
reduce the costs of production that has been recognized as a critical point (Cardona and
Sanchez, 2007). However, the transformation of some conventional raw materials (like corn,
wheat and rice etc) is not feasible, due to food security issues (Lin and Tanaka, 2006). With
high concentrations of starch, tuber crops are considered one of the most important sources for
bioethanol. The tuber crops viz. potato, sweet potato, cassava are most promising feed stock
due to their economic viability and availability. Sweet potato (Ipomoea batata L.) is one of
the most important starch producing crops grown worldwide. The dry matter content in sweet
potato ranges from 21 to 30% of which about 80% starch (Zhang and Oates, 1999). Starch is a
complex carbohydrate which needs conversion into simpler sugars before being converted into
ethanol. Starchy materials required to be converted to glucose monomers by saccharification
process which can be achieved by enzyme treatments (by amylases and glucoamylases). After
conversion to simpler monomers (glucose) it is fermented to produce ethanol with help of
ethanol producing microorganisms (Lin and Tanaka, 2006).
Saccharomyces cerevisiae and Zymomonas mobilis are usually the first choice for
industrial ethanol production, because of their good fermentative capacity, high tolerance to
ethanol and the capacity to grow rapidly under the anaerobic conditions that are
characteristically established in large scale fermentation vessels (Agbogo and Kelly, 2008).
The concept of immobilization provides a promising strategy for the use of enzymes in a
bioreactor for easy scale up an industrial biomass conversion. Day by day number of
applications of immobilized industrial important enzymes are increasing steadily.


185

Immobilized enzymes could be employed in the bioethanol production with the aim of
reducing the production cost by reusing the enzymes.
Considering the above facts, the present study is carried out to compare the bioethanol
production by S. cerevisiae and Z. mobilis from saccharified sweet potato root flour (SPRF)
using immobilized -amylase and glucoamylase.
17.2.1 Sweet potato
Freshly harvested and sweet potato (SP) roots (var. ST-13) (starch, 178 g/kg; total
sugar, 25 g/kg dry weight basis) were collected from the experimental farm of Regional
Centre of Central Tuber Crops Research Institute (CTCRI), Bhubaneswar during the month of
November, 2011 and used within 24 h after harvest. The fresh SP roots were chipped
manually, dried at 60 C to reduce the moisture level to ~ 12 % and finally grinded to flour by
dry milling and consist particles with diameter 0.2- 1.7 mm (95 % or more particles pass
through a 1.70 mm sieve).
17.2.2 Microorganisms and culture conditions
The Z. mobilis MTTC 92 strain was maintained on ZS (Z. mobilis specific) medium
[(g/L) yeast extract ,10; glucose, 20; MgCl
2,
10; NH
4
SO
4
,10 ;

KH
2
PO
4,
10; agar, 15 and pH
6-6.5] and S. cerevisiae (CET) was maintained on malt-extract-yeast extract-glucose-peptone
(MYGP) medium [(g/L): malt extract,3; yeast extract,5; peptone, 5; glucose, 20; agar, 15; pH
5.5 ]. Both the cultures were stored at 4
0
C for further use. A loop of microbial culture (Z.
mobilis and S. cerevisiae) was inoculated to a 250 ml Erlenmeyer flask which contained 100
ml sterile respective medium (as mentioned above) and were incubated at 30
0
C in a rotary
shaker (120 rpm) for 24 h. The liquid culture had initial cell density (310
9
colony forming
unit (CFU)/ml).
17.2.3 Enzymatic saccharification of SPRF
SPRF (Sweet Potato Root Flour) (10%) slurry was prepared in 250 ml Erlenmeyer
flasks with a working volume of 100 ml by adding tap water in a ratio of 1:10 for experiment.
In first step the slurry was dextrinized by addition of 32 l of immobilized Palkolase- HT at
pH 5.5 and 90C for 1hour and then slurry was cooled down to room temperature. In second


186

step, immobilized Palkodex (329.7 l) was added to the dextrinized slurry at pH 4.5 and
incubated for 24 h at 60 C for saccharification.
17.2.4 Immobilization of Enzymes
The immobilized beads of amylase were prepared with 0.3% sodium alginate, 0.5%
glutaraldehyde, 0.3M CaCl
2
solution and 0.2% starch concentration where as for glucoamylase
0.3% sodium alginate with 0.5% glutaraldehyde in 3M CaCl
2
solution with starch
concentration of 0.2% were required.
17.2.5 Ethanol fermentation from saccharified SPRF
Ethanol fermentation was conducted by S. cerevisiae and Z. mobilis under anerobic
condition in an Erlenmeyer flask sealed with rubber stopper equipped with opening for CO
2

venting. The immobilized enzyme hydrolysed SPRF (100ml) was inoculated with freshly
harvested Z. mobilis and S. cerevisiae starter cultures at [10 % v/v (310
9
CFU)/ml]
aseptically. The fermentation medium containing flasks (n=3) were incubated in an incubator-
cum shaker at 302
0
C for 120 h with a constant shaking (100 rpm). The fermented broth was
distilled to recover ethanol using alcohol distillation apparatus (Borosil Glass Works Ltd.,
Mumbai, India).
17.2.6 Study of Fermentation parameter
(1) Incubation period: The enzyme enzyme hydrolyzed sweet potato slurry was inoculated
with 10 % (v/v) starter culture with pH 4.5 and incubated for 24-120h.
(2) Initial medium pH: The fermentation medium (saccharified 10% SPRF slurry) with pH
(3- 5.5) was inoculated with 10 % (v/v) starter culture and incubated for 96 h at 30
0
C.
17.2.7 Analytical techniques
At appropriate time intervals, fermentation medium (in triplicates) were removed and
contents were analysed for sugar and ethanol. The ethanol concentration was determined
based on the density of the alcohol distillates obtained from the fermentation broth. Ethanol
concentration of the fermentation liquid was determined by measuring the specific gravity of
the distillate according to the procedure described by Amerine and Ough (1984). The pH was
measured using a pH meter (Systronics, Ahmadabad, India) fitted with a glass electrode. The
total sugars were assayed by Dinitrosalicylic acid and Anthrones method (1999).
Fermentation kinetics was calculated using the formulae by Bailey and Ollis (1986).


187

17.2.8 Population count
Yeast and bacterial populations in the fermented mash were calculated by serially
diluting the substrate (fermented SPRF slurry) in sterile distilled water and plating in suitable
dilution (10
4
-10
5
) on Petri plates containing ZSM and MYGP medium for Z. mobilis
(bacteria) and S. cerevisiae (yeast) respectively.
17.2.9 Calculations
The maximum theoretical ethanol yield from sugar was calculated according to the
stoichiometric relation represented by Eq. (1), i.e. 100 g of hexose produce 51.1 g of ethanol
and 48.9 g of CO
2.
Ethanol yields over total initial sugar (Y
1
) and average ethanol productivity
rate (Y
2
) were calculated according to Eqs. (2) and (3).
C
2
H
12
O
6
2CH
3
CH
2
OH + 2CO
2
(1)
Y
1
= ethanol produced in fermentation/ ethanol produced in theoretical x 100% (2)
Y
2
= Final ethanol concentration/fermentation time (3)
17.3.1 Effect of incubation period
In this study, the immobilized enzyme liquefied SPRF hydrolysate was used for
subsequent fermentation (submerged) by yeast, S. cerevisiae and bacteria, Z. mobilis. A 10%
inoculums was added and cultivated at 30 C for both the organisms and the comparison of
sugar utilization and ethanol production profile of the two organisms are given in (Figure1 a
and b) respectively. After 96 h S. cerevisiae and Z. mobilis produced 487.1 and 445.3g/kg of
ethanol respectively. Both the organisms S. cerevisiae and Z. mobilis utilises 88.4 %, 84.7 %
total sugar and 94.3%, 86.7% of reducing sugar respectively. Z. mobilis utilises less sugar
content and produce less amount of ethanol with respect to Saccharomyces cerevisiae and
produces 10.4% less bioethanol with comparison to S. cerevisiae. The ethanol productivity
(1.95g/L/h, 1.77g/L/h), ethanol yield (0.614g/g, 0.573g/g) and final ethanol efficiency (98.7%,
94.4%) were obtained by S. cerevisiae and Z. mobilis respectively at optimum incubation
period (96 h).
In case of S. cerevisiae the concentration of total sugar decreased rapidly within 24 h
(73.08% over initial content) of fermentation with concomitant production of ethanol (231.3
g/kg). Thereafter the decline of total sugar was gradual in between 24 and 96 h and then


188

decreases. After the end of 96 h incubation period, the residual total and reducing sugar
concentration was 100 g/kg and 47 g/kg respectively in culture broth. Similar ethanol
production and sugar utilization were observed for bacterium (Z. mobilis) but after 24 h but
the bioethanol production is 10.5% low in comparison to S. cerevisiae. The result shows that
Z. mobilis was significantly less efficient than S. cerevisiae (7.3%) [Fishers LSD test, P<0.05,
LSD between treatments is 0.65] in converting sugar into ethanol at least in the case of SPRF
hydrolysate.
A similar finding on bioethanol production from mahula flower using S. cerevisiae and
Z. mobilis was reported by Behera et al., (2007). The S. cerevisiae strain showed 10.5% more
final ethanol production in comparison to Z. mobilis. Ethanol productivity, volumetric product
productivity and sugar to ethanol conversion rate (%) obtained by S. cerevisiae were found to
be 68.5%, 3.08%, 134% higher than Z. mobilis respectively after 96h of fermentation. In
another study for bioethnaol production from the molasses reported higher bioethanol
production by S. cerevisiae than Z. mobilis at sugar concentration above 15% (v/v) (Bansal
and Singh 2003).
17.3.2 Effect of pH
The effect of initial pH of SPRF hydrolysate on bioethanol fermentation is shown in
(Figure 2 a and b). The ethanol productivity increased up to 4.5 and decreased marginally
above 4.5. The maximum of ethanol concentration 477.5 and 449.3 g/kg were for S. cerevisiae
and Z. mobilis respectively. Ethanol productivity (2.001g), ethanol yield (0.632g/g) and final
ethanol efficiency (97.3%) were obtained in culture grown at pH 4.5. In the earlier study,
Roukas (1994) has studied the effect of pH on ethanol production from carob pod by S.
cerevisiae and found that the maximum ethanol concentration, ethanol yield and fermentation
efficiency were obtained at pH 4.5. Swain et al., (2006) previously reported optimum pH 5.5
for bioethanol production from mahula flowers using free cells of S. cerevisiae in submerged
fermentation. Yeast has a pH optimum between 4.0 and 6.0 although it can grow in a long pH
range 2.5 to 8.5 (Narendranath and Power, 2005).
In contrast to S. cerevisiae, a similar pattern of bioethanol and sugar utilization was
observed from Z. mobilis (Figure 2 b).The maximum ethanol concentration 429.3 g/kg was
obtained at medium. Ethanol productivity (1.788g), ethanol yield (0.591g/g) and final ethanol
efficiency (93.7%) were obtained in culture grown at pH 4.5. Maiti et al., (23) reported


189

slightly higher pH 5.13 for optimum bioethanol production from sugar cane molasses. But in
another study, Z. mobilis produced optimum ethanol from mahula flower at pH 6.5 (Swain et
al., 2007). Both the organism S. cerevisiae and Z. mobilis utilised 87.1%, 83.1% total sugar
and 93.5, 86.4 % reducing sugars respectively at optimum pH 4.5. However, the optimum
ethanol produced by S. cerevisiae is 10.8% higher [Fishers LSD test, P<0.05, LSD between
treatments is 0.30] than the Z. mobilis.

Figure (1): A comparative study for role of incubation period on ethanol concentration and
sugar consumption (total and reducing sugar) by S. cerevisiae [a] and Z. mobilis [b]
17.4 Importance of enzyme Immobilization
In this experiment the advantage of using enzyme immobilized beads was that the used
beads survived and were active on the support used for immobilization for four cycles of
fermentation, which could save considerable time and energy. In this study, the enzyme
A
B


190

immobilized beads were successfully recycled for four more times without much affecting the
final ethanol concentration. The cells not only survived but were also active physiologically
yielding ethanol 4850.02, 4830.08, 4780.12 and 4680.12 and 448.60.04, 4440.02,
436.20.06 and 428.20.06 g/kg of SPRF in 1
st
, 2
nd
, 3
rd
and 4
th
cycles of 96 h fermentation,
from S. cerevisiae and Z. mobilis, respectively. Both of this experiment bioethanol production
remains constant. The ethanol production is 10.6% higher in S. cerevisiae than that of Z.
mobilis.

Figure (2): A comparative study for effect of fermentation medium pH on ethanol
concentration and sugar consumption (total and reducing sugar) by S. cerevisiae [a] and Z.
mobilis [b].
B
A


191

17.5 Conclusion
The present experiment revealed that, enzyme immobilization is an advanced
technique for bioethanol production. In the present study, S. cerevisiae was found to be a
promising microbial source for bioethanol production from immobilized enzyme saccharified
sweet potato flour as substrate in comparison to Z. mobilis. The study demonstrated that S.
cerevisiae can be able to produce 10.6% higher bioethanol than Z. mobilis, the peak ethanol
concentration was obtained after 96 hour of fermentation. Further, it is evident from the
present study Zymomonas does not appear to be an efficient microbial source for commercial
bioethanol production, attributed to its lower tolerance to temperature, ethanol and utilization
of substrate range compared to S. cerevisiae.
Acknowledgements
The Department of Science and Technology, Govt. of Odisha is gratefully
acknowledged for the financial support to carry out this work in the form of the research
project (Project no.3897/ST, dated 07/08/10). The authors are thankful to the M/s Maps
Enzymes Ltd, Ahamadabad, India for providing the enzymes. We thank the Principal, College
of Engineering and Technology for providing necessary facilities for this research work.

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194

CHAPTER 18
GENETIC MODIFICATION FOR SIMULTANEOUS
UTILIZATION OF GLUCOSE & XYLOSE BY YEAST
Nilesh Kumar Sharma, Shuvashish Behera, Sachin Kumar

Abstract
In recent era, escalating utilization of ethanol as a blend in gasoline has increased the demand
in the world market due to its valuable contribution to the energy security and environment
protection. In recent decades, the emphasis has been given on the agro-based bioethanol
production due to shortage of conventional raw materials such as molasses. The major sugars
present in any lignocellulosic biomass are glucose and xylose and other sugars such as
mannose, galactose, arabinose are found at lower level. Xylose is the second major
fermentable sugar in the lignocellulosic hydrolysates after saccharification. For economical
production of agro-based ethanol, it is recommend to utilize both pentose and hexose sugars,
present in the lignocelluloses, simultaneously. The conventional ethanologens such as
Saccharomyces cerevisiae are not able to utilize both glucose and xylose for ethanol
production. Some pentose utilizing ethanologens such as Scheffersomyces stipitis, Candida
shehatae and Pachysolen tannophylus have also been used for ethanol fermentation. But, they
could not be commercialized due to inefficient in ethanol yield, glucose utilization, etc.
However, there are some reported yeasts such as Kluyveromyces sp., which are able to utilize
both pentose and hexose sugars simultaneously. But, they utilize pentose sugars for higher
yield of xylitol as compared to ethanol. We have isolated a yeast of same characteristics in our
laboratory. As the yeast has capability of utilizing both pentose and hexose sugars
simultaneously, the ethanol yield on xylose can be increased by using metabolic tools.
Keeping in mind, the proposed study is focused on the methods to identify the metabolic
inefficiency and probable solution including metabolic pathway identification, metabolic flux
balance, toxicity tolerance and solution to feedback inhibition.
Key words: Metabolic engineering, Pathway modelling, Xylose metabolism, metabolic flux
balance.


195

18.1 Introduction
Rising concerns over the cost of petroleum and the prospect of global warming are
driving the development of technologies for the production of alternative fuels such as ethanol
(Jeffries and Jin, 2004; Ragauskas et al., 2006; Behera et al., 2012). Currently, bioethanol is
produced mainly from sugar-containing or starchy biomass such as sugarcane and corn as the
raw material. However, lignocellulosic biomass, such as woods and agricultural residues, is
also an attractive feedstock for bioethanol production because of its large amount of
potentially fermentable sugars (Matsushika et al., 2009).
The main component of lignocellulosic biomasses is glucose, a hexose sugar derived
from cellulose and hemicellulose. Xylose is the second most abundant carbohydrate in nature
and its commercial fermentation to ethanol could provide an alternative fuel source for the
future (Cadete et al., 2012). Fermentation of the available sugars in cellulosic biomass
presents a unique challenge because of the presence of other sugars such as xylose and
arabinose (pentose sugars). The processes that efficiently utilize both the sugar component of
lignocellulose could significantly decrease the cost of bioethanol production (Bhatia et al.,
2012). The larger sizes, thicker cell walls, better growth at low pH, less stringent nutritional
requirements, and greater resistance to contamination give yeasts advantages over bacteria for
commercial fermentations. One of the most effective ethanol-producing yeast for hexose
sugars including glucose, mannose, and galactose is Saccharomyces cerevisiae, a yeast with
high ethanol productivity, high tolerance to ethanol, and tolerance to inhibitory compounds
present in the hydrolysate of lignocellulosic biomass (Kumar et al., 2009; Goshim et al.,
2013). However, it does not naturally use xylose as a substrate, however, and must be
engineered to both transport and ferment xylose. In contrast to the efficient glucose
fermentation in yeast, xylose fermentation has been challenging because only a few ethanol-
producing microorganisms can readily ferment xylose, though many microorganisms.
Developing an efficient organism to ferment the pentose sugars has been pursued for the past
few decades. Microbes such as yeasts and bacteria are essential for the fermentation of xylose
(Dien et al., 2003; Jeffries and Jin, 2004) (Table 1). Organisms to ferment the pentose sugars
in lignocellulosic biomass can be divided into two subgroups, namely naturally occurring and
genetically engineered microorganisms. The naturally-occurring microorganisms include
Pichia stipitis, Candida shehatae, and Pachysolen tannophilus. But the rate and yield of
ethanol production from xylose in these xylose-utilizing yeast strains are considerably low


196

compared to their glucose fermentation (Kahr et al., 2011; Villanueva et al., 2012).
Genetically-engineered organisms with pentose fermenting capabilities include
Saccharomyces cerevisiae, E. coli, Zymomonas mobilis (Agbogbo and Kelly, 2008).
Table 1. Comparative analysis of ethanol yield with different xylose utilizing strains.
Source: (Lisbeth et al., 1996)
A number of different approaches have been used to engineer yeasts for both transport
and ferment xylose, including modeling, flux analysis and expression analysis followed by the
targeted deletion or altered expression of key genes. Therefore this review is based on the
details about the genetic engineering of yeasts, problems, solutions and its future perspective.
18.2 Necessity of pentose (C5) sugar fermenting organisms
Many researchers are interested in economical production of fuel-grade ethanol from
cellulosic materials. They generally consist of 40% cellulose, 30% hemicellulose and 20%
lignin as main components and may be considered as a potential feedstock for the production
Strain Xylose ( g l
-1
)

Ethanol ( g l
-1
)

Yield (g g
-1
)

Candida blankii ATCC 18735 50 5.1 0.10
C. famata 20 3.9 0.20
C. fructus JCM-1513 20 4.7 0.24
C. guilliermondii ATCC 22017 40 4.5 0.11
C. shehatae CSIR-Y492 90 26.2 0.29
C. tenius CBS 4435 (11) d 20 6.4 0.32
C. tropicalis KY 5014 (2) 20 2.8 0.14
Clavispora sp. UWO(PS) 83-877-1 (11) 20 5.9 0.30
Kluyveromyces cellobiovorus KV 5199 (3) 20 4.4 0.22
K. marxianus 20 5.6 0.28
P. tannophilus RL 171 20 6.2 0.31
Pachysolen tannophilus NRRL Y-2460 50 13.8 0.28
Pichia segobiensis CBS 6857 20 5.0 0.25
P. stipitis CBS 5773 (5) 20 5.9 0.30
P. stipitis CBS 5776 50 22.3 0.45
Schizosaccharomyces pombe ATCC
2478(8)
50 5.0 0.10


197

of ethanol by microbial fermentations (Enari et al., 1984). Hemicellulose constitutes up to
35% of hardwood species and other woody angiosperms by dry weight, with the aldopentose
D-xylose as major constituent of xylan, amounting up to 25% of dry biomass. The hydrolysis
of cellulose and hemicelluloses, the major constituents of plant materials, yields a mixture of
sugars in which D-glucose and D-xylose are the major components. The economic feasibility
of ethanol production from these materials depends on the ability of microorganisms to fully
convert the available carbon sources. Industrial yeast strains normally ferment hexoses, but D-
xylose remains unutilized (Lin and Tanaka, 2006).
Efficient ethanol production from xylose is crucial for bioethanol production from
lignocellulosic biomass. An economic analysis of xylose fermentation concluded that a fixed
substrate cost, the yield and final concentration of ethanol are the most important factors in the
cost of ethanol production (Hinman et al., 1989). For ethanol production from xylose to be
commercially viable, it was suggested that a microorganism should be capable of producing
50 to 60 g/L ethanol within 36 h with a yield of at least 0.4 g ethanol/g sugar. Developing an
efficient organism to ferment the pentose sugars has been pursued for the past few decades.
There are different organisms responsible for the production of different yield of ethanol from
pentose sugar which is depicted in (Table 1).
18.3 Problems with pentose (C5) sugar fermenting yeast
The general requirements of an organism for ethanol production from pentose sugar
hydrolysate should be high ethanol yield, high productivity, good tolerance against inhibitors
as well as high ethanol concentrations and ability to ferment at relatively low pH. S. cerevisiae
is one of the most commonly used yeasts for ethanol fermentation using glucose. However, it
does not have the ability of fermenting pentose sugars. In yeasts, the conversion is carried out
by two oxidoreductases; xylose reductase (XR) and xylitol dehydrogenase. XR is NADPH
cofactor specific whereas XDH is NAD+ cofactor specific. The difference in cofactor
preference of XR and XDH leads to the formation of xylitol under anaerobic conditions.
Xylitol is therefore a byproduct in ethanol fermentation and its production reduces the final
ethanol yield. However, one of the key problems in utilizing xylose for ethanol production is
the impairment of the redox balance that arises from the different coenzyme specificities in
the xylose metabolic pathway (Jeppsson et al., 2003). P. stipitis is one of the few types of
yeast that is able to ferment xylose to ethanol under anaerobic conditions because it possesses
both NADH and NADPH specific XR cofactor.


198

Generation of an NADH dependent XR by protein engineering could resolve this
problem. Decreasing the NADPH-preferring activity of Scheffersomyces stipitis XR (PsXR)
might therefore increase ethanol production in S. cerevisiae (Watanabe et al., 2007, Zhang et
al., 2013). The choice to produce ethanol or cell mass by P. stipitis depends on the O
2
supply
to the cells (Agbogbo and Kelly, 2008, Zhang et al., 2013). The benefit of XR using NADH is
that there is a total cofactor balance when this cofactor is used, and therefore no xylitol is
produced. Kinetic studies indicate that NADPH is the preferred coenzyme because its affinity
is about double the value for NADH (Agbogbo and Kelly, 2008). Under anaerobic conditions,
xylose fermentation by P. stipitis must proceed by NADH-linked XR for a total cofactor
balance. The ability of P. stipitis and P. tannophilus to use NADH for XR provides these
yeasts with the ability to produce less xylitol in xylose conversion compared to other xylose
fermenting yeasts under anaerobic conditions (Agbogbo and Kelly, 2008).
18.4 Need of Genetic Engineering for xylose fermentation
In yeasts, filamentous fungi and other eukaryotes, conversion of D-xylose to D-
xylulose via a two-step reduction and oxidation, which are mediated by XR and XDH,
respectively. The cofactor requirements of these two reactions affect cellular demands for
oxygen, as explained in the text. S. cerevisiae can grow on xylulose, an isomer of xylose but
not in xylose. Therefore, the cloning of XR and XDH into S. cerevisiae would allow xylose to
be used as a substrate (Katahira et al., 2006; Hahn-Hagerdal et al., 2007). The yeast S.
cerevisiae has been extensively engineered for ethanolic fermentation of the pentose sugar
xylose either by introducing genes encoding XR and XDH, or by introducing the gene
encoding xylose isomerase (XI) (Hahn-Hgerdal et al., 2007; Van Vleet and Jeffries, 2009;
Matsushika et al., 2009) (Table 2).
Still xylose fermentation with recombinant S. cerevisiae is significantly less efficient
than hexose fermentation. Among others this has been attributed to the difference in cofactor
preference of XR and XDH, which results in xylose to xylitol conversion rather than ethanolic
fermentation. Protein engineering to alter the coenzyme specificities of XR and XDH is a
useful strategy for the development of yeast strains with improved xylose fermentation (Hou
et al., 2007; Klimacek et al., 2010; Krahulec et al., 2010). By changing one residue in the
putative phosphate pocket that binds NADPH, the resulting enzyme exhibited an
NADH/NADPH activity ratio of 23:1 (Watanabe et al., 2007; Van Vleet and Jeffries, 2009).


199

Genetic engineering can also improve the fermentative activities of some native
xylose-metabolizing yeast such as S. stipitis. A number of different approaches have been
used to engineer yeasts for this purpose, including modeling, flux analysis and expression
analysis followed by the targeted deletion or altered expression of key genes. P. stipitis is
engineered by alteration in XR with altered coenzyme preference improves ethanolic xylose
fermentation (Liang et al., 2007, Bengtsson et al., 2009), some system biological approach has
also done including central carbon metabolic network of P. stipitis has been reconstructed on
the basis of Flux Balance Analysis (FBA) and Principal Component Analysis (PCA). Rather
than P. stipitis many approaches has been done on other xylose fermenting yeast like in
cloning and characterization of xyl1 gene, which encoding NADH preferring XR from
Candida perapsilosis, to Candida tropicalis. (Lee et al., 2003). Mutated Candida tropicalis
strains showing more XR activity in comparison with wild type (Rao et al., 2006) some
substantial factor also applied to increases activity of XR (Liu et al., 2012, Verduyn et al.,
1985). XDH obtained same interest as of XR. XDH were experimented by so many
approaches simultaneously as XR including change in substantial factor like temperature,
NADPH availability, UV treatment etc (Hughes et al., 2012). The genetic engineering
approach has been applied in different strains of microorganisms for xylose utilization which
is shown in Table 3.
18.5 Conclusion and future prospects
More than a decade of research has been devoted to the development of strains for
efficient pentose fermentation. The majority of the studies conducted used metabolic
engineering through a rational selection of genes to be manipulated for the development of
novel pentose-fermenting strains of yeast with varying levels of success. The increased
knowledge about pentose metabolism, substrate binding and cofactor specificity of pentose
assimilating enzymes and sugar transport has contributed to the improvement of yeast for
bioconversion of pentose sugars to bioethanol. However, the mechanisms by which pentose-
fermenting yeasts can accomplish an increased rate of ethanol production are still not fully
understood and the simultaneous co-fermentation of hexose and pentose sugars still
constitutes a major strain engineering challenge. More emphasis on host genome and
regulatory structure must occur in future projects to understand the full effect of biological
complexity on a pathway. In this regard, classic approaches combined with next-generation
technologies may be combined to allow simultaneous optimization of all steps in a pathway.


200

The idealized approach combines the power of many approaches including pathway
engineering, directed evolution, evolutionary engineering, and combinatorial genetics to
harness this cellular complexity.
Table 2. Genetic engineering of Sacharomyces cerevisae in xylose utilization for ethanol
production.

****** =100%, ****=80%, ***=60%, **=40%, *=20%

Gene from different
strain
NADPH
Afinity
Redox
Imblance
Ethenol
Production
Xylitol
production
Reference
XR
(P. stipitis)

*

***

*

***

Watanabe et al., 2007
Karhumaa et al.,, 2005
Matsushika et al., 2009
Bengtsson et al., 2009
XR
(P. stipitis)

**

**

*

**

Jeppsson et al., 2003
Bettiga et al., 2008
Krahulec et al., 2010
Runquist et al., 2010
XR
(C. tenuis)

**

**
**

**

Bengtsson et al., 2009,
Petschacher and
Nidetzky, 2008
XR
(P. stipitis)

**

** **
*

Zeng et al., 2009
Khattab et al., 2011a
Lee et al., 2012
XDH
(P. Stipitis)
*

***

*

***

Karhumaa et al., 2007
Bettiga et al., 2008
Bengtsson et al., 2009
XDH
(S. stipitis+
T. brockii)
**

***

*

***

Watanabe et al., 2007

XDH
(P. stipitis)
**

**

**
*

Khattab et al., 2011b
Gene from different
strain
Active at
higher temp
Ethanol

Reference
XI
(Piromyces sp.)
No

*

Kuyper et al., 2003

XI
(T. thermophilus)
Yes

* *

Lonn and co-workers, 2002

XI
(Piromyces sp.)
Yes

* *

Brat et al., 2009

XI
(C. phytofermentans)
Yes

* *

Brat et al., 2009


201

Table 3. Comparative analysis of experimental study on xylose utilizing strains.
Strain
Xylose
Utilization
Ethanol
production
Redox flux
Imbalance
Xylitol
production
Reference
Xylose Reductase (XR)
Scheffersomyces
stipitis
*** ** ** **
Liang et al.,
2007,
Bengtsson et al.,
2009,
Peng et al., 2012
Candida
tropicalis
** ** ** **
Lee et al., 2003,
Rao et al., 2006
Pachysolen
tannophilus
*** * ** ***
Sanchez et al.,
2004
Verduyn et al.,
1985
Liu et al., 2012
Xylitol Dehydrogenase (XDH)
Scheffersomyces
stipitis
*** * *** **
Bicho et al.,
1988,
Slininger et al.,
2011
Candida
tropicalis
* ** ** **
Walther et al.,
2001,
Lima et al., 2006
Kluyveromyces
sp.
* ** ** **
Fonseca et al.,
2008,
Lulu et al., 2013
Xylose Isomerase (XI)
Kluyveromyces
marxianus
** ** - -
Wang et al.,
2013
Hansenula
polymorpha
** ** - -
Dmytruk et al.,
2008
Piromyces ** *** - - Zhou et al., 2012
****** =100%, ****=80%, ***=60%, **=40%, *=20%
Acknowledgment
Authors are very much thankful to Ministry of New and Renewable Energy, New
Delhi, Govt. of India for the financial support and providing all research facilities to carry out
research work.


202

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evolutionary engineering enable rapid xylose utilization and ethanol production by
Saccharomyces cerevisiae. Metab. Eng. 2012, 14:611622.


208

CHAPTER 19
TO OPTIMIZE THE PROCESS OF ALCOHOL PRODUCTION
FROM BANANA PEEL
Mohit Jain, Anand Kumar Gupta, Sayan Chatterjee

Abstract
With day by day increase in demand of fuel and price hike, has forced the world to use an
alternative source of fuel. There are two global biorenewable transportation fuels that might
replace oil derived gasoline and diesel fuel. These are bioethanol and biodiesel. Even in few
countries like Brazil, United States they are in use as a fuel alternative since 2006. Currently
the popular raw materials for preparation of biofuel are corn, sweet potato, potato, sugarcane
molasses etc. The aim of our study was to use banana waste as a raw material for the
production of alcohol which also targets another global problem of waste disposal. Using
banana peel for alcohol production provides a way to use waste material efficiently in addition
to that fuel produce through banana waste is ecofriendly, cheap and easily renewable. MTCC
178 strain of Saccharomyces cerevisiae yeast was used for fermentation to produce alcohol.
To optimize the production of alcohol from banana peel different conditions i.e. temperature,
pH and yeast concentration were varied with the help of Response Surface Methodology
(RSM). The results obtained from the design expert software shows that the production of
alcohol from the banana peel was optimized at 35.78
o
C temperature, 3.81 pH, 9.57 yeast
concentration and the yield of alcohol obtained was 7.43% v/v.
Keywords: Optimization, Fermentation, Banana peel, Response surface methodology (RSM),
bioethanol.
19.1 Introduction
Today our world has been surrounded with many problems and one of the most
important among them is fuel crisis. In recent years, with rising problems such as uncertain
fuel supply and efforts to reduce carbon dioxide emissions lead us to think about an
alternative source of fuel. There are two global biorenewable transportation fuels that might
replace oil derived gasoline and diesel fuel. These are bioethanol and biodiesel. From ages


209

humans have been producing ethanol. According to the time flow, the area of ethanol has been
extending dramatically. The very first time, ethanol existed only in alcoholic drinks. After
establishment of some purification methods, the usage of ethanol highly extended and after
reviewing current scenario ethanol becomes highly attractive again.
Bioethanol is a high-octane fuel which was used primarily as a gasoline additive and
extender. It is seen as a good fuel alternative because the source crops such as sugarcane
molasses, corn etc can be grown renewably and in most climates around the world. In addition
bioethanol can be used as CO
2
neutral i.e. during the growing phase of the source crop, CO
2

absorbed by the plant and oxygen released in the equivalent volume to that CO
2
produced in
the combustion of the fuel. This creates an obvious advantage over fossil fuels which only
emit CO
2
as well as other poisonous emissions (European Renewable Energy Council, 2006).
Only in the last few years due to rising environmental concerns and to the periodic
crises in some of the larger oil exporting countries, has made bioethanol a viable and realistic
alternative in the energy market. The United States produced 5.6 billion gallons per year as on
February 2007 (Renewable Fuels Association, 2007) from 3.4 billion gallons in 2004. In
2009, Brazil, produced 7.65 billion gallons of ethanol (Renewable energy world.com, 2009)
up from 4.0 billion gallons in 2004. Production of ethanol in Brazil utilizes sugarcane
molasses as a primary feedstock. Currently, corn is the primary feedstock being used in the
production process.
Fruits are among the most important foods of mankind as they are not only nutritive
but are also vital for the maintenance of health. Fruits both in fresh as well as in processed
form not only improve the quality of our diet but also provide essential ingredients like
vitamins, minerals, carbohydrates etc. India is the largest producer of fruits in the world.
Among the fruit crops, banana occupies the fourth world rank of the most significant
foodstuffs after rice, corn and milk. According to India Agricultural Research Data Book
2004, the estimated fruit and vegetable production in India was 150 million tones and the total
waste generated was 50 million tones. The extent of total losses in these commodities is
approximately estimated as 20-30% of the total production, amounting to a loss of Rs. 30,000
crore per annum.
Peels are the major by-products obtained during the processing of various fruits and
these were shown to be a good source of various bioactive compounds which posses various


210

beneficial effects. But, significant quantities of fruit peels (20- 30% for banana) are discarded
as waste According to FAO (FAOSTAT, 2003), the total waste generated from fruits was
estimated as 3.36 million tones (MT) out of the total production of 16.8 MT and particularly
for banana it was 6.4 MT. The solid wastes generated by fruit processing industries can serve
as potential raw materials for the production of secondary metabolites of industrial
significance by microorganisms such as bioethanol production and this could also be an
attractive alternate for disposal of the polluting residues (Wyman, 2001).
19.1.1 Response surface methodology
RSM method was introduced by G.E.P. Box and K.B. Wilson in 1951. Whereas Mead
and Pike stated origin of RSM was in 1930s with use of Response Curves (Myers et al., 1989).
Response surface methodology (RSM) is a collection of mathematical and statistical
techniques for empirical model building. It uses quantitative data from appropriate
experiments to simultaneously determine and solve multivariate equations thus providing an
optimum solution (Daramola et al., 2007). The objective of RSM is to optimize a response
(output variable) which is influenced by several independent variables (input variables) in
addition to that it aims at reducing the cost of expensive analysis methods. Response surface
methodology gives advantage over other methods as it reduces the number of experimental
trials need to evaluate multiple parameters, it is less laborious and less time-consuming.
RSM plays important role in designing, formulating, developing, and analyzing new
scientific studying and products. It is also efficient in improving existing studies and products.
The most common applications of RSM are in Industrial, Biological and Clinical Science,
Social Science, Food Science, and Physical and Engineering Sciences. It has been successfully
applied to optimize alcoholic fermentation and other fermentation media (Maddox &
Reichert, 1977; Zertuche & Zall, 1985; Coteron et al., 1993; Chen, 1996; Sunitha et al., 1998;
Ambati & Ayyanna, 2001; Ratnam, 2001; Ratnam et al., 2003).
19.2 Materials used
Musa acuminate variety of banana was procured from the local vendor in Delhi market
and banana peel was obtained. The culture used for fermentation was Saccharomyces
cerevisiae strain MTCC 178 was procured from Microbial Type Culture Collection (MTCC),
Institute of Microbial Technology (IMTECH), Chandigarh, India. Commercial cellulase and


211

pectinase enzymes were obtained from Sigma company. Alcohol refractometer (0-80%) which
was ordered online through university portal.
19.3 Methodology
19.3.1 Enzyme Stock preparation
A stock of cellulase and pectinase enzyme was prepared in phosphate buffer. As
optimum working pH for pectinase enzyme is 3.4 so 100ml phosphate buffer (1M) was
prepared by mixing monobasic (KH
2
PO
4
) and dibasic (K
2
HPO
4
) potassium phosphate salts in
a particular ratio i.e. 13.795g and .008g respectively. Similarly the optimum working pH for
cellulase enzyme is 4.8 so monobasic and dibasic salts were mixed in a ratio of 13.68g and
.228g respectively.
19.3.2 Revival and Sub-culturing of yeast
Yeast strain MTCC 178 was initially in freeze dried form in an ampoule. After
procuring sample the content from the ampoule was poured into test tubes with 1.8 cm3
physiological solution. In order to activate the yeast cells, the test tube was put in a thermostat
at 30
0
C for 30 minutes time sufficient to restore their viability. For culturing and sub-
culturing of yeast strain a YPD media was used. For 100ml seed media yeast extract, dextrose,
peptone and agar were mixed in an amount i.e. 1g, 2g, 2g and 2g respectively and volume was
makeup to 100ml using distilled water.
19.3.3 Experimental design
The statistical analyses were performed according to the response surface method
using Design Expert 8.0.7.0 (Stat-Ease Inc., 2009) trial version software. Central composite
experimental design (CCD) (Box & Wilson, 1951) was employed to study the combined
effect of three variables namely temperature, pH and inoculum size. The response variable i.e.
alcohol content (%) was measured timely. A CCD has three groups of design points two-level
factorial or fractional factorial design points, axial points (sometimes called "star" points) and
center points.
19.3.4 Processing of Banana peel
Banana was thoroughly washed with running water and then with distilled water to
remove any dirt that could be on it. The peel was removed and cut into small pieces with
sterilized blade and it was left overnight in hot air oven at 65
o
C to dry it completely. The dried


212

peel was then subjected to grinding in a mixer grinder machine. Then banana peel extract was
treated with pectinase and cellulase enzyme. After treating with enzymes a clarified juice was
obtained which works as an production media for anaerobic fermentation.
19.4 Results & Discussion
Table 1 shows the run sheet prepared through Design Expert Software using Central
Composite Design. It provides 18 sets of 3 parameters i.e. temperature, pH and inoculum
concentration according to which experimental trials have been performed to find out the
response factor i.e. alcohol concentration for each set of trials. In relation to actual values
RSM also provided the coded values. Table 2 shows the minimum and maximum range
values for the parameters were coded as -1 & +1, mean value was coded as 0 and minimum
and maximum extreme values were coded as - & + , where =1.68179. The second order
polynomial equation obtained from the analysis of multiple regression for estimation of
alcohol concentration in terms of coded value was: alcohol = +7.40 +0.10*A +0.029*B
+0.14*C +0.0000*A*B +0.0000*A*C -0.050*B*C -0.17*A
2
-0.21B
2
-0.071C
2
.
TABLE 1: Run sheet prepared by Design Expert Software
Run Temperature
(
o
C)
pH Inoculum conc.
(%)
Alcohol conc.
(%)
1 37 3.33 5.0 6.8
2 23 3.33 10.0 7.0
3 30 3.83 7.5 7.4
4 30 3.83 3.3 7.0
5 37 3.33 10.0 7.2
6 30 3.83 11.7 7.4
7 30 4.67 7.5 6.8
8 37 4.33 5.0 7.0
9 30 3.83 7.5 7.4
10 30 3.83 7.5 7.4
11 30 3.83 7.5 7.4
12 37 4.33 10.0 7.2
13 23 4.33 5.0 6.8
14 30 3.83 7.5 7.4
15 23 3.33 5.0 6.6
16 30 2.99 7.5 6.8
17 30 3.83 7.5 7.4
18 23 4.33 10.0 7.0

Table 3 shows the design matrix evaluation for the quadratic model suggested by the
response surface methodology central composite design where A, B & C represents
temperature, pH and inoculum concentration respectively.


213

Variance inflation factor (VIF) is generally the reciprocal of tolerance in
computational language. In other terms it explains about the severity of multicollinearity
which have a direct influence on the analysis of regression. As VIF increases the severity of
multicollinearity increases leading to poor estimation of model. The ideal value for VIF is 1.
Similarly Ri-squared value indicates about the correlation between the parameters and its
higher value possibly leads to poor model prediction.
TABLE 2: Showing the coded values for the parameters
Run Temperature
(
o
C)
pH Inoculum conc.
(%)
Alcohol conc.
(%)
1 1 -1 -1 6.8
2 -1 -1 1 7.0
3 0 0 0 7.4
4 0 0 - 7.0
5 1 -1 1 7.2
6 0 0 7.4
7 0 0 6.8
8 1 1 -1 7.0
9 0 0 0 7.4
10 0 0 0 7.4
11 0 0 0 7.4
12 1 1 1 7.2
13 -1 1 -1 6.8
14 0 0 0 7.4
15 -1 -1 -1 6.6
16 0 - 0 6.8
17 0 0 0 7.4
18 -1 1 1 7.0

TABLE 3: Evaluation of model generated through Response surface
Term Standard Error VIF Ri-squared
A 0.27 1.00 0.0000
B 0.27 1.00 0.0000
C 0.27 1.00 0.0000
AB 0.35 1.00 0.0000
AC 0.35 1.00 0.0000
BC 0.35 1.00 0.0000
A
2
0.26 1.02 0.0179
B
2
0.26 1.02 0.0179
C
2
0.26 1.02 0.0179


214

Table 4 shows the overall effect of process parameters on the alcohol response. It
indicates that temperature (F=55.72, p<0.0001) and inoculum concentration (F=178.88,
p<0.0001) had a significant effect on alcohol response whereas pH (F=8.16, p=0.0213) did
not had a significant effect on alcohol concentration. Through the software it has been
predicted that at 35.78
o
C temperature, 9.57% inoculum concentration and 3.81 pH alcohol
response was optimized and its value is 7.43391%. Figure 1 shows the 3D graph plotted
between two independent variables and a dependent variable or a response variable i.e.
alcohol is a response variable and two independent variables were pH and temperature and
inoculum concentration was constant at 9.57%.
TABLE 4: ANOVA result for response surface quadratic model
Source Sum of
squares
df* Mean
squares
F value p-value
(prob > F)
Model 1.29 9 0.14 99.55 <0.0001
A 0.080 1 0.080 55.72 <0.0001
B 0.012 1 0.012 8.16 0.0213
C 0.26 1 0.26 178.88 <0.0001
AB 0.000 1 0.000 0.000 1.0000
AC 0.000 1 0.000 0.000 1.0000
BC 0.020 1 0.020 13.93 0.0058
A
2
0.110 1 0.110 73.64 <0.0001
B
2
0.54 1 0.54 376.14 <0.0001
C
2
0.060 1 0.060 41.79 0.0002

* degree of freedom


215

Figure 1: 3 Dimensional representation of optimized results
19.5 Conclusion
The energy demands of the growing population can be somewhat fulfilled if steps are
taken to make use of the alternative renewable energy sources and their production is
optimized so that input cost incurred during their production can be minimized, and the raw
materials are fully utilized. Using agricultural waste for production of industrially important
metabolites also eliminates the risk associated with the disposal of such substances and
minimizes the chances of polluting the environment.
Through the help of Response Surface Methodology the effect of many independent
variables can be studied on the response variable with minimum experimental trials.
Acknowledgement
We would like to acknowledge Guru Gobind Singh Indraprastha University, Dwarka,
New Delhi; for providing facilities to carry out our experimental work and also like to
acknowledge the Dean of our department (University School of Biotechnology) Prof. P.C.
Sharma for his help and support throughout the project.


216

References
1. Ambati P. & Ayyanna C. (2001) Optimizing medium constituents and fermentation
conditions for citric acid production from Palmyra jaggery using response surface
method. World Journal of Microbiology and Biotechnology, 17, 331335.
2. Box G. E. P. and Wilson K.B. (1951) On the Experimental Attainment of Optimum
Conditions (with discussion). Journal of the Royal Statistical Society Series B, 13(1),
145.
3. Chen H.C. (1996) Optimizing the concentrations of carbon, nitrogen and phosphorus
in citric acid fermentation with response surface method. Food Biotechnology, 10, 13
27.
4. Coteron A., Sanchez M., Martinez M. & Aracil J. (1993) Optimization of the synthesis
of an analogue of jojoba oil using fully central composite design. Canadian Journal of
Chemical Engineering, 71, 485488.
5. Daramola M. O., Keesman K. J. and Spenkelink F. (2007) Process modeling of
ultrafilteration units: An RSM approach. Journal of Applied Science, 7, 3687-3695.
6. Design Expert version 8.0.7.0, (2009) Stat-Ease Inc., Minneapolis, MN, USA.
(http://www.statease.com/news/news0912.pdf)
7. European Renewable Energy Council. (2006) Bioethanol production and use,
European biomass industry association, Brussels.
(http://www.erec.org/fileadmin/erec_docs/Projcet_Documents/RESTMAC/Brochure5
_Bioethanol_low_res.pdf)
8. Food and Agricultural Organization of the Unites Nations. (2003) FAOSTAT statistics
data base, Agriculture, Rome, Italy..
9. Maddox I.S. & Reichert S.H. (1977) Use of response surface methodology for the
rapid optimization of microbiological media. Journal of Applied Bacteriology, 43,
197204.
10. Myers R. H., Khuri A. I. and Carter W. H. Jr. (1989) Response surface methodology,
Techno metrics, 31(2): 137-153.
11. Ratnam B.V.V. (2001) Studies on physico-chemical and nutritional parameters for the
production of ethanol from Palmyra jaggery by submerged fermentation using
Saccharomyces cerevisiae. PhD Thesis, Andhra University, Visakhapatnam, AP, India.


217

12. Ratnam B.V.V., Rao M. N., Rao M. D., Rao S. S. & Ayyanna C. (2003) Optimization
of fermentation conditions for the production of ethanol from sago starch using
response methodology. World Journal of Microbiology and Biotechnology, 19, 523
526.
13. Renewable energy world.com (2009). (http://www.renewableenergyworld.com/
rea/news/article/2009/09/brazils-2009-ethanol-production-set-to-break-records)
14. Renewable Fuels Association. (2007) Ethanol Biorefinery Locations, updated Feb.
12. (http://www.energyfuturecoalition.org/biofuels/fact_ethanol.htm)
15. Sunitha I., Subba Rao M.V. & Ayyanna C. (1998) Optimization of medium
constituents and fermentation conditions for the production of L-glutamic acid by the
co-immobilized whole cells of Micrococus glutanicus and Pseudomonas reptilivora.
Bioprocess Engineering, 18, 353359.
16. Wyman, Ch.E. (2001). Twenty years of trials, tribulations, and research progress in
bioethanol technology. Appl. Biochem. Biotech., 91: 5
17. Zertuche L. & Zall R.R. (1985) Optimizing alcohol production from whey using
computer technology. Biotechnology and Bioengineering, 27, 547554.

218

CHAPTER 20
COMMERCIAL PRODUCTION OF BIO-CNG & ORGANIC
MANURE FROM PRESSMUD BIOMETHANATION
Preetam Holkar, A.V. Mohan Rao and K.K. Meher

Abstract
Increasing demand for fossil fuels and escalating prices of chemical fertilizers has been the
prime source of motivation for this project. The concept of anaerobic digestion is a promising
eco-friendly solution for the treatment of organic biomass, which leads to bioenergy production.
Press mud is a byproduct of sugar making process from sugar cane and accounts for 3.5 to 4
% of the total sugarcane crushed. About 5.2 million tons of press mud is produced in our
country every year. Generally, press mud is used as manure in agriculture after composting.
There are no reports on large commercial scale biomethanation of press mud. SREL has
established first commercial BIO-CBG and organic manure plant of 100 TPD capacity of
press mud feed at Warnanagar, in India. This plant is constructed with Indo-German technical
inputs and is in operation for the last 15 months. It is generation capacity is about 7000 to
7,500 kg of BIO-CNG and 40 to 45 tons of organic manure per day. Calorific value of BIO-
CNG and commercial CNG are comparable. While BIO-CNG is being supplied as industrial
fuel, organic manure as spectrum digestate (liquid) and spectrum soil conditioner are used
in agriculture. Application of these products improved soil physical properties, soil health,
moisture retaining capacity etc. Horticulture crops recorded 15 to 20% higher yields.
Key words: Biomethanation, Pressmud, BIO-CNG, Organic manure.
20.1 Introduction
In the current energy scenario, growing gap between demand and supply, increasing
prices of fossil fuels in international markets are putting tremendous pressure on the
economies of many countries. This gap can be reduced with the energy generation from
renewable resources. Biomass is one of the feasible renewable energy sources. In the recent
years interest in bio energy has increased and it represents approximately 14% of world final
energy consumption (1). Large quantities, about 150 million tones of biomass wastes such as
household and agro processing wastes are generated annually in India and are disposed in a


219

dispersed manner. Dumping results in serious environmental consequences such as air and
water pollution. In India biomass based power generation is developing into an industry. The
two promising pathways for biomass energy conversion are biological, Anaerobic Digestion
(AD) route to generate biogas fuel and Thermo-Chemical (TC), biomass gasification route to
generate producer gas fuel. Biogas production technology has received attention worldwide
and remains attractive and promising in meeting future energy demands.
Globally, India is the second largest producer of sugarcane producing over 350 million
tons of sugar per year. In India Uttar Pradesh (37.2 %) is the biggest producer of sugarcane
followed by Maharashtra (2) with 23.5% (2011-12). Sugar industry (more than 500 mills) is
one of the major agro-industry in India. Several co-products of immense value are generated
during sugar making. Press mud is one such product which accounts for about 3.5 4.0 % of
the total sugarcane crushed. About 5.2 million tons of press mud is produced in our country
every year. It is a soft, spongy, amorphous and dark brown material containing 30 to 35 % dry
matter out of that 80% organic matter hence, its a very good feed stock for biogas generation.
There are very few reports on the anaerobic digestion of press mud, no large scale
biomethanation plants based on Press mud are reported. SREL has established first
commercial BIO-CNG and organic manure plant based on 100 TPD press mud feed capacity
at Warnanagar, Maharashtra in India. Press mud is selected as feed material due to its
abundant availability in sugar mills and tremendous scope for the expansion. During 2011-12
Warana sugar factory crushed about 13.8 lakh tons of sugar cane and generated about 42,000
tons of press mud. It contains rich organic matter, organic carbon, sugar, protein, enzymes,
micro (N, P, and K) and macro (Zn, Fe, Mg, Mn, Cu etc) nutrients and microbes (Yaduvanshi
and Yadav, 1990; Ranganathan and Parthasarathi, 1999; Parthasarathi and Ranganathan,
1998, 1999, 2000). The composition of the press mud indicates that it is a good source for
extracting bioenergy (Table-1).
Press mud is procured from Warna sugar factory during the sugarcane crushing season
and used directly as it comes from the factory, during off season press mud stored in yard is
used. Total holding capacity of the storage yard is 18000 tons. Press mud is conveyed to
the feed tank by a conveyer belt. During off season other feed stocks such as cattle dung,
poultry waste, milk effluents etc are added as when needed.


220

20.2 Feed preparation
Press mud is mixed with the digesting slurry taken from digester to prepare feed slurry
by constantly mixing it with an agitator to have uniform feed slurry. Fresh water is added
to have 10 to 12% solids in the feed slurry. This slurry is pumped back into the respective
digesters using feed pump. Daily data on feed and effluent slurry pH, TS, VS, VFA,
Alkalinity, Temperature and gas composition etc are collected.
. Table-1: Press mud composition (karan et al.)
S.No Componenet %
1 Moisture 70
2 Volatile matter 76.6
3 C:N 14
4 Sugar 5.7
5 Cellulose 11.4
6 Hemi cellulose 9.3
7 Protein 15.5
8 Lignin 9.3
9 Wax 8.4

20.3 CSTR Digesters
Three CSTR digesters, each of 3400 cum capacity are built, these are equipped with
double layered gas capturing system to hold about 1000 cum of biogas in each. All the three
digesters are of the same design and are interconnected through pipes at the upper gas storage
area to have equal gas pressure. Hot water pipes are fitted in each digester. Hot water (60
o
C)
from the generator is circulated through these pipes [Fig.1] for maintaining the optimum
digester temperature. Each digester is also provided with four agitators for mixing the digester
contents. All the digesters are fitted with safety valve to prevent over and under gas
pressureinside the gas capturing system
Biogas generated is continuously sucked by a blower and supplied to gas cleaning
system. In addition to the SREL biogas about 12000 cum of biogas from Warna sugar limited
is also processed by the gas cleaning system for making BIO-CNG. Biogas produced [Fig.2]
for 30 continuous days is considered in this article.

221

Figure 1. CSTR Digester
Table-2. Performance of the digesters
S.No Parameter Data
1 Daily Loading 100 tons
2 Total solids in feed 10 to 12%
3 Volatile solids in feed 70% of TS
4 pH of the feed slurry 5.5 0.1
5 Biogas production(24 hrs) 8 000 to 9000 m
3

6 Biogas production/ton of Press mud/day 80 to100 m
3

7 Methane in biogas produced 62 2 %
8 pH of the effluent slurry 7.1 0.1
9 Digesting slurry temp 36 2
0
C
10 TS degradation 42.8%
11 Biogas production /kg TS degraded/day 1.168 m
3

12 TVS degradation 76.1%
13 Biogas production/kg VS degraded/day 0.939 m
3


222

Figure 2. Biogas production from Spectrum and WSL biogas plants
20.4 Biogas cleaning process
Raw Biogas is taken to the gas header and its composition
Biogas pressure 2500 mm WC
Methane (CH
4
) 60 -62%
Carbon Dioxide(CO
2
) 35 % (Max)
Hydrogen Sulphide(H
2
S) 3 % (Max)
Biogas from Raw gas header is taken to H
2
S removing plant through a gas flow meter.
The Bioskrubber
TM
treats H
2
S containing gases, where sulphide is biologically converted
to elemental sulfur. Biogas from bioskrubber is then passed through an adsorbent bed where
the H
2
S concentration is reduced to about 5 ppm. Online H
2
S analyzers are provided to
measure the H
2
S conc. H
2
S scrubbed biogas is stored to the clean gas holder. Biogas is
compressed to 7.5 kg/cm
2
before sending it to the CO
2
scrubber.
CO
2
removal is by absorption process Soft water as solvent at high pressure is used in
a packed column. CO
2
concentration is brought down from 35% to less than 5%. After
purification the biogas composition is comparable [Fig. 3] to that of commercial CNG and
hence it is named as BIO-CNG.
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930
Spectrum gas
WSL gas
B
i
o
g
a
s

i
n


223

20.5 BIO-CNG production & composition
After removing CO
2
biogas is dried for moisture removal and stored in a buffer vessel,
where the gas pressure is 6 to 7 kg/cm
2
. From buffer vessel gas is taken for compression and
compressed to 200 bars and filled into the cylinders [Fig. 3].
Biogas composition after purification
Biogas pressure 6-7 kg/cm2
Methane (CH
4
) minimum 95 to 95.5 %
Carbon Dioxide(CO
2
) maximum 4.0 to 3.5 %
Hydrogen Sulphide(H
2
S) less than 5 ppm
Water vapor Nil
Oxygen Nil
Hydrogen 0.2 to 0.5%
Methanol/Glycerol Absent

Recent Advances in Bioenergy Research Vol. III

Online analyzers and flow meters are provided to measure the above
Apart from on line analyzers periodically BIO
Mumbai and analyzed.
20.6 Biogas utilization / storage devices cascades
The upgraded and compressed BIOCNG storage cylinders (cascades) are placed on
specially designed trucks and shipped to the customer site, where it will be used for their
application in industrial furnaces in place of fossil fuels.
20.7 Biogas based power
A 340 kW capacity generator is installed for power production. It operates fully on
H
2
S removed Biogas. The generated power is used for captive consumption. The biogas
generator generates around 2.0 units per cum
extracts exhaust heat from the generator. Heat recovered from these sources is transferred to
the digester through heating manifold and maintained optimum digesters temperature.
20.8 Digestate (Organic Manure)
Importance of organic manure application in agriculture is gaining ground due to
increasing costs and non availability of chemical fertilizers. Farmyard manure is the oldest
organic manure used by the man. It
mixed with animal dung and urine and allowed to degrade biologically. Similarly
manure developed by SREL from Press mud
anaerobic digestion Press mud is
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
1
B
i
o
g
a
s

i
n

c
u
m

&
B
i
o
C
N

i
n

!
g
"
#
a
$
224
Figure 3. BIO-CNG produced
Online analyzers and flow meters are provided to measure the above
Apart from on line analyzers periodically BIO-CNG samples are sent to ONGC lab at
iogas utilization / storage devices cascades
application in industrial furnaces in place of fossil fuels.
Biogas based power
generator generates around 2.0 units per cum [Fig.4]. A heat recovery system is placed
(Organic Manure)
organic manure used by the man. It consists of litter; waste products from agricul
mixed with animal dung and urine and allowed to degrade biologically. Similarly
manure developed by SREL from Press mud (Table-3), is also of biological origin. During
anaerobic digestion Press mud is subjected to intense microbial enzymatic degradation. The
3 5 7 9 11 13 15 17 19 21 23 25
%ota& 'iogas puri(ie# in cum"#a$ B)*CN generate# in +g "#a$

Online analyzers and flow meters are provided to measure the above parameters.
CNG samples are sent to ONGC lab at
A heat recovery system is placed to
of litter; waste products from agricultural crops
mixed with animal dung and urine and allowed to degrade biologically. Similarly, organic
is also of biological origin. During
ymatic degradation. The
27 29
B)*CN generate# in +g "#a$

digestate is well stabilized, nutrient rich, ready to use soil conditioner
as spectrum soil conditioner. When this manure is applied it
action in soil. Humus helps in improvi
Table-3. SREL organic manure analysis
Parameter
pH
Conductivity
Moisture
Dry matter
Organic carbon
C/N
Water holding
capacity
Nitrogen
Phosporous
Potash

Note: Spectrum Soil conditioner is directly applied to the soil.Spectrum digestate is diluted
with water in 1:3 ratios and applied to the plants.
Figure 4. Biogas to Electricity generation per day in January2013
0
1000
2000
3000
4000
5000
1 3 5 7
Biogas consume# ,cum-
B
i
o
g
a
s

i
n

c
u
m

&

.
&
e
c
t
r
i
c
i
t
$

i
n

+
W
/
"
#
a
$
225
digestate is well stabilized, nutrient rich, ready to use soil conditioner (Table
as spectrum soil conditioner. When this manure is applied it helps in activating the microbial
action in soil. Humus helps in improving soil fertility and water holding capacity.
3. SREL organic manure analysis
Unit Spectrum- soil
conditioner
Spectrum digestate
7.92 7.35
Mmhos/cm 7.5 27.4
% 23.7
% 76.29
Organic carbon % 16.27 3.37
14.52 7.15
Water holding 162.32
% 1.12 0.47
% 1.14 o.45
% 0.76 0.94
and applied to the plants.
7 9 11 13 15 17 19 21 23 25 27
Biogas consume# ,cum- .&ectricit$generate# ,+W0-

(Table-3), and marketed
activating the microbial
ng soil fertility and water holding capacity.
Spectrum digestate
7.35
27.4
90
10
3.37
7.15
-
0.47
o.45
0.94

27 29 31
.&ectricit$generate# ,+W0-

226

Being tropical country, Indian soils are often deficient in humus. Humus is an
extremely important material formed after decomposition of organic manure. The application
of Spectrums Pressmud based organic manure offers one of the best ways for the
simultaneous production of humus in soil. Humus imparts grey brown or slightly black color
to soil and thereby helps the soil to absorb greater amount of radiation. Humus also resists too
much fluctuation in soil temperature.
Spectrums Digestate contains micro and macro nutrient required for the plant growth
hence like other organic material it effect the physical, chemical and biological properties of
the soil (Tandon, 1995). Results show that with Spectrums organic manure increased yield
ranging from 15-20 per cent are observed. Still better results can be obtained when it is used
in combination with other cultural practices such as improved seeds, proper crop rotation,
good soil management and supplementary dose of chemical fertilizers.
20.9 Conclusions
In the current energy scenario of growing energy demand and dwindling fossil fuel
resources, shift to renewable energy source is an obvious option. Since most sugar mills are
situated in the rural areas producing press mud year after year in large quantities.
Development of Press mud based BIOCNG plants in association with sugar mills will help in
bringing decentralized renewable energy and organic manure producing units. If exploited
properly these units minimize the dependence on fossil fuels and helps in reducing the
environmental pollution. Continuous availability of organic manure helps farmers to plan and
adopt for seasonal organic farming. This will lead to less reliance on inorganic fertilizers and
boost the self confidence of the people in agriculture. Hence, setting up SREL type of biogas
plants helps in employment generation and in the economic development of small towns and
rural areas.

References
1. http://www.economywatch.com/energy-economy/scenario.html
2. Price policy for sugarcane the 2013 sugar season.PP-39.Commission for agricultural costs and
prices, Department of agriculture & cooperation, Ministry of agriculture, Govt of India, New
Delhi.

227

3. Yaduvanshi, N.P.S. and D.V.Yadav: Effects of sulphitation pressmud and nitrogen fertilizer on
biomass, nitrogen economy, plant composition in sugarcane and on soil chemical properties.
J.Agric. Sci., 1990,114, 259-263.
4. Ranganathan, L.S. and K.Parthasarathi: Precocious development of Lampito mauritii (Kinberg)
and Eudrilus eugeniae (Kinberg) reared in pressmud. Pedobiologia, 1999, 43, 904-908.
5. Parthasarathi, K. and L.S. Ranganathan : Pressmud vermicasts are the hot spots of fungi and
bacteria, Ecol. Environ. Conser., 1998, 4,81-86.
6. Parthasarathi, K. and L.S.Ranganathan: Longevity of microbial and enzyme activity and their
influence on NPK content in pressmud vermicasts, Eur. J. Soil. Biol., 1999, 35(3), 107-113.
7. Parthasarathi, K. and L.S.Ranganathan: Aging effect on enzyme activities in the pressmud
vermicasts of Lampito mauritii (Kinberg) and Eudrilus eugeniae (Kinberg). Biol. Fertil. Soils,
2000, 30, 347-350.
8. Karan M. Agrawal, B. R. Barve and Shareena S. Khan: Biogas From Press mud, IOSR journal
of mechanical and civil engineering (IOSR-JMCE) ISSN:2278-1684, pp 37-41.
9. Tandon (1995). In: Waaste Recyling on Agriculture.Fertilizer Development Consultation
Organization, New Delhi.


228

CHAPTER 21
FEASIBILITY OF FILLING BIOGAS IN CYLINDERS
S.S. Sooch, Jasdeep Singh Saini

Abstract
A case study has been done for Ludhiana in Punjab State regarding feasibility of filling of
biogas in cylinders for cooking as well as industrial use. Biogas produced from Cattle dung in
the Corporation area can produce 30,000 m
3
/day

of biogas which can cater the needs for
cooking of 85,000 people and in addition to solve the Waste Disposal Problem and also
provide High Quality Fertilizer.
Key words: Waste disposal, Biogas, Bottling of biogas
21.1 Introduction
There is acute shortage of conventional type of fuel like petrol, diesel, kerosene and
fire wood. It is, therefore, very essential that some alternate fuel may be located. One such
fuel which can be made available in rural as well as urban areas is methane gas produced from
anaerobic digestion of organic waste. As the population of the country is increasing perhaps
the largest single problem resulting from the recent increase in confinement rearing of
livestock and poultry involve waste handling and disposal. The odour and fly nuisance of the
waste, the decline of manure as a competitive fertilizer and encroachment of urban areas close
to production units complicate the problem of waste treating and handling. Anaerobic
digestion process of well known acid is often practiced by various municipal corporations in
the country for stabilizing organic material and for production of gas.
A case study has been done for Ludhiana in Punjab. It is observed that in addition to
seven lakhs inhabitants, there are more than fifty thousand milch cattle in the city. The result
is that Ludhiana city is facing lot of difficulty in disposal of solid wastes and one finds heaps
of rubbish in every vacant plot of land or on a wide corner of a road. Although municipal
corporation has made some arrangements for disposal of such waste even then the position is
no satisfactory from health point of view. The municipal corporation has made separate
arrangement of construction of dairy sheds out side the city. But no dairy owner has so far
shifted to the dairy area.


229

The question again arises, if cattle are shifted to the new area what will be the
arrangement for the disposal of waste produced from the animals. Simple answer for this
would be to dump at one open space or sell it directly as a raw fertilizer to the farmer. This is
not possible because such a customer will not be readily available. The authors have
suggested a suitable possible method in this paper by which the whole waste of dairy sheds
can be directly handled in a safe manner.
The methods recommended for safe handling is by anaerobic digestion of organic
waste by biological means. The end products would be biogas and very good organic manure
free from any odours and flies etc. The detailed plan of the scheme is as under:

The dung from the dairies be purchased by a contractor who would run the biogas
plant and should resell the end product to the farmer at a suitable rate. Such a method is being
practised in some of the States of India.
The quantity of biogas available is calculated as under:
Number of cattle in the city = 50,000
Average dung produced = 10 to 20 kg per day (say 15 kg)
Total dung = 50,000 x 15 = 750000 kg
Biogas produced per kg of dung = 0.04 m
3

Total quantity of biogas produced/day = 750,000 x 0.04 = 30,000 m
3

DUNG FROM
CATTLE
ADDITION OF
EQUAL AMOUNT
OF WATER
ANAEROBIC
DIGESTION
DRYING
BEDS
GAS FOR
COOKING
BIOGAS
FERTILIZER
FIELDS
SEEPAGE
WATER
POWER
GENERATION
GAS
CYLINDERS

230

The gas produced may be used for cooking purposes at homes by laying distribution
pipe network in the nearby locality. The gas is sufficient to meet the demand of about 84,000
people. But the cost of network may be very high because of the cost of pipes. The other
alternative would be to generate power with the gas at the plant site to avoid losses. The
power may be supplied to the dairy sheds for light or for operation of tubewells. The power
expected from the gas is equal to 45,000 Kilo-watts per day.
The third use can be the filling of gas in cylinders which may be sold in the market at
usual rates like LP gas. This method seems to be more attractive as the increasing cost of
LPG. Although it looks very easy to fill gas in cylinders but there are many difficulties in
actual procedure such as:
1. Biogas is a mixture of methane, carbon dioxide and other gases in trace amounts like
hydrogen sulphide, carbon monoxide and ammonia. This is the reason that calorific value of
biogas is very low (4700 Kcal/ m
3
). Therefore, it is suggested that gas be purified from carbon
dioxide, H
2
S and moisture, at least, to increase its efficiency by some method to change it to
pure form of methane.
2. The other difficulty is that the gas cannot be liquefied at low pressure. It is reported that
methane gas can be liquefied at low pressure. It is reported that methane gas can be liquefied
at 162C and 20,000 psi which is not practicable. Below is a chart showing the details
regarding volume of the cylinder to store 28 m
3
at different temperatures and pressure.

From the table above it could be seen that 28 m
3
gas can be stored in 5.593 ft
3
cylinder
and such cylinders are available in the market for filling gas like oxygen, carbon dioxide etc.
The same cylinder may be used for filling methane gas. The cylinder when filled with purified
biogas free from carbon dioxide and other impurities, with high calorific value, will be
sufficient for an average family of live persons for a period of 30 days.
Sr. No. Pressure (psi) Temperature (C) Volume of cylinder (ft
3
.)
1. 1200 -70 3.195
2. 1500 -50 4.088
3. 2100 -25 4.144
4. 2400 0.0 4.641
5. 2400 25 5.593

231

21.3 Discussion
The filling of gas in cylinders as mentioned above can be tried in our country to meet
the fuel shortage Although as mentioned above to fill 28 m
3
gas a thick cylinder of about 3 cm
thickness is required. This can be managed easily as is done in the industries for the carriage
of oxygen cylinders etc. The cost of methane gas cylinders would be less and can be easily
available in the town as compared to LPG. Only draw back would be its weight because
atleast two men are required for lifting such cylinders. The cost of the cylinders is comparable
with LPG cylinder.
From the filling of gas a highly sophisticated arrangement is necessary to avoid
accidents. This would be quite costly and skilled labour is necessary to run the compressor.
From the Ludhiana town itself 1000 cylinders can be filled daily and supplied in the market.
To regulate the pressure of gas a regular similar in use for oxygen and carbon-dioxide is
necessary. Ordinary regulator used with LPG will not be suitable.
21.4 Conclusion
The method suggested in this paper would not only solve the waste disposal problem
of the Ludhiana city but also provide valuable fuel for 85,000 people. In addition high quality
fertilizer free form weeds, insects and odour would be available to the farmers for growing
crops.

References
1. Mittal, K.M. 1996 Biogas Systems: Principles and Applications, New Age International (P)
limited Publishers, New Delhi.
2. Biogas A Rural Energy Source. (1985), Ministry of Non-Conventional Energy Sources
Publication, New Delhi.
3. Grewal N.S., Sooch S.S., Ahluwalia S and Brar G.S. 2000. Hand Book Biogas Tech, PAU,
Ludhiana.
4. Sooch S.S. 2010. Biogas Plants for Rural Masses. School of Energy Studies for Agriculture,
PAU, Ludhiana.
5. Akshyay Urja 2012. Energing the re way, Ministry of Renewable Energy Government of India,
5 (6). www.mnre.gov.in.


232

CHAPTER 22
EFFECT OF PRETREATMENT ON BIOCONVERSION OF
WHEAT STRAW FOR THE PRODUCTION OF BIOGAS
Nishshesh Singh, Vivek Saini, Pranshu Gupta, Rajan Sharma, G Sanjay Kumar, Dr. Avanish
K. Tiwari

Abstract
Lignocelluloses are often a major or sometimes the sole components of different waste
streams from various industries, forestry, agriculture and municipalities. Hydrolysis of these
materials is the first step for either digestion to biogas (methane) or fermentation to ethanol.
However, enzymatic hydrolysis of lignocelluloses with no pretreatment is usually not so
effective because of high stability of the materials to enzymatic or bacterial attacks.
Pretreatment helps to improve the process of hydrolysis .In this work different methods of
pretreatment was studied.
The present work illustrates about the effect of acid, alkaline pretreatment on different
sizes of wheat straw and anaerobic digestion of treated biomass for the production of biogas
in batch stirred tank bioreactor at particular parameters. The quality of biogas formed was
analyzed by gas chromatography and quantity measured by water displacement method. The
untreated wheat straw gave a biogas yield of 104 ml/g and methane content of 64%. Acid
treated wheat straw gave biogas yield of 130, 140, 134 ml/g and methane content of 68%,
72%, 75% for respective 1%, 2%, 5% acid concentration. Similarly, for alkali treatment gave
biogas yield of 124, 128, 126ml/g and methane content of 66%, 69%, 71% for respective 1%,
2%, 5% NaoH concentration.
Key words: Pretreatment, Lignocelluloses, Biogas, Anaerobic Digestion, Batch Stirred Tank
Bioreactor.
22.1 Introduction
Biofuels are rapidly becoming a significant partner in our future energy needs. It can
provide means to mitigate deleterious impacts of greenhouse gas emissions. Production of
waste materials is an undeniable part of human society. For effective waste management


233

utilization of waste is necessary. Anaerobic digestion is the process by which the waste
treatment can be done easily and side by side it can be useful for the production of valuable
products such as Biogas and organic manure [Tsavkelova and Netrusov, 2012] .There are
different types of wastes such as industrial, forestry, agricultural and municipal waste which
can act as substrate for the Biogas production. Wheat straw is one of the most abundant
agricultural wastes produced in world [Wang, 2009]. However, due to its complex structure
and high lignin content, its degradability and gas yield are low. The degradabitity can be
improved by pre-treatment, making the material more accessible to microbial degradation
thus increasing the biogas yield.
The composition of wheat straw is - Cellulose-30%, Hemicellulose-50%, and Lignin-
15%. Lignin is responsible for the integrity, structural rigidity and resistance to swelling of
lignocelluloses. Therefore a delignification processes can improve the rate and extent of
enzymatic hydrolysis from the matrix polymers. The reason for improved rate of hydrolysis
by removal of lignin might be related to a better surface accessibility for enzymes by
increasing the population of pores after removal of lignin.
Li Sun et al. [2013] investigated the microbial response to straw as a feed stock for
biogas production. The addition of straw, pre-treatment of straw and operating temperature all
affects the cellulose degrading community in biogas digesters, but there were no major
differences in the digester performance and gas yield.[Nekema et al. 2013] evaluated biogas
production in batch and Up-flow anaerobic sludge bed (UASB) reactors from pilot-scale acid
catalyzed steam pretreated and enzymatic hydrolyzed wheat straw. The results showed that
the pretreatment was efficient and, a sugar yield of 95% was obtained. The pretreatment
improved the methane yield compared to untreated straw. [R.Chandra et al.] studied
experimental methane fermentation on untreated, NaOH and hydrothermal pretreated
substrates of wheat straw. NaOH pretreated substrate produced 87.5% higher biogas
production and 111.6% higher methane production compared to the untreated wheat straw
substrate.
Pia-Maria Bondesson [2013] investigated wheat straw in two different process
alternatives with simultaneous saccharification and fermentation (SSF) to ethanol and
anaerobic digestion (AD) to biogas. In her study three types of pretreatment were done steam
pretreatment with water, acetic acid or phosphoric acid. The overall best yield was obtained
when using phosphoric acid as impregnation material. The highest methane yield, 754 ml

234

CH
4
/g fed was achieved. [Prasad Kaparaju, 2009] investigated wheat straw hydro lysate for
biogas production in continuous stirred tank reactor (CSTR) and (UASB) reactors. In his
study methane yields increased with increase in hydro lysate concentration. However, he
concluded that, biogas process was affected by the reactor type and operating conditions.
None of above work has illustrated the effect of acid and alkali treatment on a particular
sieved size wheat straw particle.
The present work studies pretreatment of wheat straw with acid and alkali (1 vol%,
2vol %, and 5 vol% ) on mechanically grinded wheat straw (<75m, 75m, 150m, 212m
425 m). It was shown that highest lignin content was extracted in 425 m. The objective to
use 212 m particle size wheat straw was due to its abundant production during grinding.
Hence, process was economical.
Standards Chemicals used (conc. sulphuric acid, sodium hydroxide and distilled
water) were from Rankem. A Standard lignin used was from Merck. Standard biogas sample
were from Centurion scientific pvt ltd.
22.2.1 Preparation of standard lignin solution
Standard lignin samples of 1 and 10 ppm were prepared by dissolving weighed
quantity of pure lignin in appropriate volume of distilled water. The substrate wheat straw was
collected from Dehradun.
22.2.2 Experimental Method
22.2.2.1 Mechanical Treatment of wheat straw
Raw wheat straw procured from Bidholi, Dehradun was first ground. The powdered
sample was sieved. Five different sample sizes were collected: <75 m, 75 m, 150m 212
m, and 425m.
22.2.2.2 Estimation of acid soluble lignin content
0.3 g of different particle size samples were taken mixed with 3ml of 72% conc.
H
2
SO
4
[8] and kept in a water bath for 2hrs for estimation of acid soluble lignin. The solution
obtained was mixed with 84 ml water and autoclaved for 30 min at 121C and 15 psi.
Autoclaved solution was then filtered to separate residues and filtrate. Filtrate solutions were

235

diluted to 100 ppm and the same were studied under UV spectrophotometer at 205 nm for
lignin content. The process is shown in figure 1.
22.2.3 Pretreatment of Wheat Straw
Wheat Straw pretreatment was done by both acid and alkaline methods.
22.2.3.1 Acid pretreatment of wheat straw
50g of mechanically treated wheat straw of 212 m was treated with 1% H
2
SO
4
. The
sample was mixed with 500 ml of 1% H
2
SO
4
solution and kept in orbital shaker at 70C,
150rpm for 2 hrs. The solution was filtered and the residual biomass was washed twice with
water and then dried. Similar treatment was done with 2% and 5% H
2
SO
4
also. Figure 2
shows color variation of the sample after treatment with 1%, 5% H
2
SO
4.

Figure 1. Schematic representation of estimation of soluble lignin from wheat straw

Figure 2. Acid(1%.5%) pretreated wheat straw

22.2.3.2 Alkali pretreatment of wheat straw
Wheat straw was treated in presence of 500 ml of 1%, 2%, 5% NaoH in an orbital
shaker at 150 rpm for 2 hours at 70C. Reaction mixture was filtered, washed twice with
water and then dried. The residual biomass was used for anaerobic digestion in batch reactor
for biogas production.

236

22.2.4 Anaerobic digestion of pretreated wheat straw
Wheat straw after pretreatment was digested in a bio-reactor. The pH of the slurry was
maintained neutral. The cow dung was used as inoculums. The set-up is shown in Figure 3.
Gas was collected in glass collecting port via gas nozzle of digester. The quantity of biogas
produced was determined by the water displacement method. The reaction conditions
maintained in bioreactor are given in Table 1.
Table 1. Reaction specification
Reactor Batch
Working volume 500ml
Total volume 1 lt.
pH 6.8
Temperature 38
HRT 4 days

22.2.5 Gas analysis
Biogas was analyzed by GC (Nucon 5700). GC conditions are given below:
Injection volume 100 l
Mobile phase - Argon
Column Make Stainless Steel
Column ID - (HEYSEP. Q)
Run time - 10 minute

Figure 3. Batch type stirred tank reactor setup

237

22.3.1 Delignification of Wheat Straw
The absorbance of lignin standard was determined at 1 ppm and 10 ppm as 0.791 and
3.261.
22.3.2 Estimation of acid soluble lignin content
The observations obtained from acid pre treatment of wheat straw are summarized in
Table 2 and Figure 4.
Table 2. Effect of particle size on lignin removal

Figure 4 Comparative graphical representation of estimated lignin
Particle size Absorbance of soluble
lignin (1 ppm)
Absorbance of soluble
lignin (10 ppm)
%lignin
(1 ppm)
%lignin
(10 ppm)
425nm

1.363 1.643 0.8 10.5
212nm 1.370 1.671 0.9 10.7
150nm

1.376 1.732 1.0 11.1
75

1.379 1.759 1.1 11.3
<75 1.387 1.836 1.2 11.8

238

Absorbance is increased as the particle size of the wheat straw is decreased. It was
found that mechanical treatment had good impact on delignification. Particle (<75m) yield
more lignin because particle has large surface contact with acid. 212m yield 10.7% lignin.
22.3.3 Acid pretreatment of wheat straw
Above observation indicate that the acid soluble lignin removal was found more in 5%
H
2
SO
4
reaction condition as shown in Table 3. But 2% H
2
SO
4
was feasible to use for acid
hydrolysis and moreover lignin removal was found nearby to 5% H
2
SO
4
.
Table 3. Acid Treatment
% H
2
SO
4
Absorbance of 100 ppm solution Lignin removal (g/lit)
1 3.205 2.836
2 3.312 2.930
5 3.506 3.102

22.3.4 Alkaline pretreatment of wheat straw
The observations obtained from alkaline treatment of wheat straw are summarized in Table-4.
Results of alkaline pretreatment show that 2% NaOH stage is beneficial for the removal of
lignin, but more lignin was extracted out in the presence of 5% NaOH as shown in Figure 5.
Table 4. Alkali Treatment
%NaOH Absorbance 100 ppm solution Lignin removal(g/lit)
1 2.945 2.606
2 3.102 2.745
5 3.314 2.932
22.3.5 Biogas Production from Pretreated wheat straw:
A standard Biogas sample was run on GC. The composition was (CH
4
-62%), (CO
2
-36%).
This is used to calibrate the GC.
The chromatogram indicates percentage of methane content increases from 64% to
75% with increase in the conc. of sulphuric acid from 1% to 5%.As it already seen in fig 4
that 5% acid has good impact on lignin removal. Acid treatment also disrupts crystalline

239

structure of cellulose. Hence accessibility of microorganism to biomass surface increases the
2% acid pretreatment give better biogas methane yield comparable 5% sulphuric acid. Hence
preferred because it is found economical than 5% acid pretreated as require less alkaline
solution to maintain pH of pretreated slurry.

Figure 5. Comparative effect of alkali, acid treatments on lignin removal (g/lit)
Above figure indicates that acid treatment has more impact on lignin removal than alkali.

Figure 6. Comparative chromatogram of biogas after (1%, 2%, 5%) acid treatment.

Figure 7. Comparative chromatogram of biogas obtained after (1%, 2%, 5%) alkaline
treatment.

240

Above chromatograph indicates percentage methane content was found more after 5%
alkali treated wheat straw as compared to 2% NaoH. 5% alkali pretreatment extracts more
soluble lignin as compare to other.

Figure 8. Yield of biogas, methane/gm Figure 9. Yield of biogas, methane/g from
pretreated wheat straw. acid from alkali pretreated wheat straw.

The figures (8&9) indicates biogas yield/g was found more after 2%acid pretreated
and wheat straw while the rest of treatment also yield good volume of biogas per gm of
biomass. Methane yield was found more after 2%acid, 5% alkaline pretreated wheat straw.
22.4 Conclusions
Different pretreatment methods for delignification of wheat straw were tried. 5%
conc. H2SO4 yields more acid soluble lignin as compared to 2% conc. H
2
SO
4
, but 2% yield
more biogas. Alkaline pretreatment is also good method for delignification of wheat straw.
However, the methane content of biogas obtained from acid treated biomass was 10% more as
compared alkaline treated biomass.

References
1. Tsavkelova EA, Netrusov Al, (2012), Biogas production from cellulose-containing
substrates: a review. Appl Biochem Microbial 48:421-433.
2. Wang G, Gavala HN, Skiadas IV, Ahring BK, (2009), Wet explosion of wheat straw
and codigestion with swine manure: Effect on the methane productivity. Waste
Management 29:2830-2835
3. Li Sun, Bettina Mller and Anna Schnrer (2013), Biogas production from wheat
straw: community structure of cellulose-degrading bacteria, Sustainability and
Society, springer journal, 3:15.

241

4. Nkemka VN, Murto M (2013) Jan Biogas production from wheat straw in batch and
UASB reactors: the roles of pretreatment and seaweed hydro lysate as a co-substrate.
Bioresour Technol. 128:164-72.
5. R. Chandra, H. Takeuchi, T. Hasegawa, R. Kumar ,(2012) , Improving
biodegradability and biogas production of wheat straw substrates using sodium
hydroxide and hydrothermal pretreatments , Energy 43 (2012) 273-282, 2012 Elsevier
Ltd.
6. Pia-Maria Bondesson, (2010), combined production of bioethanol and biogas from
wheat straw, Lund University.
7. Prasad Kaparaju, Mara Serrano, Irini Angelidaki, (2009), Effect of reactor
configuration on biogas production from wheat straw hydro lysate. Bio resource
technology ,100(24):6317-23.
8. Sluiter A., Hames B., Ruiz R., Scarlata C., Sluiter J., Templeton D., Crocker D.,
(2012), Determination of Structural Carbohydrates and Lignin in Biomass, National
Renewable Energy Laboratory.


242

CHAPTER 23
POTENTIAL OF WHEAT STRAW FOR BIOGAS
PRODUCTION USING THERMOPHILES
Richa Singh, Shuvashish Behera, Yogendra Kumar Yadav and Sachin Kumar

Abstract
Lignocellulosic raw materials like waste biomass are considered for the production of biogas
to mitigate the increasing demand for energy. Waste Biomass includes agricultural residues,
forestry waste, municipal solid wastes, etc. Wheat straw is one of the most important
agricultural residues as a lignocellulosic source. India is the second largest producer of wheat
in the world, with production hovering around 68 to 94 million metric tonnes (MMT) and
produced around 157 MMT of wheat straw in the past few years. Therefore, the present study
was based on the biogas production using wheat straw as raw material. Wheat straw was
analyzed for proximate as well as ultimate analysis. For anaerobic digestion, a thermophilic
consortium was isolated at 50C. The optimum biogas production depends on factors such as
C/N ratio, pH, temperature and liquid content of the substrate. The C/N ratio was maintained
as 20:1 with 15% solid content. The biogas yield was obtained as 0.178 m
3
Kg
-1
of volatile
solid at a temperature of 50C in 30 days incubation period. Average methane and carbon-
dioxide composition observed in biogas was 62.77% and 37.23%, respectively. Further, after
digestion volatile solids was reduced by 37.82%, which showed the potential of wheat straw
as substrate for further digestion as well as for the production of biogas.
Key words: Biogas, Lignocellulose, Wheat straw, Thermophillic consortium
23.1 Introduction
Energy security and continuous increase in green house gases i.e. methane, CO
2

emission and their accumulation in our atmosphere has strengthened the interest in
development and utilization of alternative, non-petroleum based renewable sources of energy
(Hamelinck et al., 2005; Wendy et al., 2013). Methane production through anaerobic
digestion from the variety of biological wastes through methane fermentation technology is
growing worldwide and is considered viable solution in current scenario. It is economical and


243

environment friendly technology. In term of energy output/input ratio, this technology is the
most efficient as compared to all other technologies of energy production through biological
or thermo-chemical routes of energy conversion processes (Deublein and Steinhauser, 2008;
Chandra et al., 2012).
Various kinds of organic materials can be used as substrates for biogas production,
such as sewage sludge, manure, organic municipal waste and agricultural wastes (Appels et
al., 2011). Among the agricultural wastes, straw is an interesting feedstock for biogas
production (Tsavkelova and Netrusov, 2011). Wheat straw is a major potential source of
waste lignocellulosic biomass produced in the world, making it highly interesting as a
substrate for biogas production. India is the second largest producer of wheat in the world,
with production hovering around 68 to 94 million metric tonnes (MMT) and produced around
157 MMT of wheat straw in the past few years (Richard et al., 2005; Wang et al., 2009; Ewa
et al., 2010).
Compared to mesophillic digestion, thermophilic anaerobic digestion has additional
benefits including a high degree of waste stabilization, more thorough destruction of viral and
bacterial pathogens, and improved post-treatment sludge dewatering (Ye et al., 2008). The use
of straw for biogas production is restricted by its high C/N ratio and low levels of trace
elements, limiting microbial growth and activity. The fermentable products, called volatile
solids (VS; or volatile matter), can be fermented by various anaerobic microorganisms, either
to volatile fatty acids (VFA; or short-chain fatty acids) with C2C6 carbon chain, resulting in
acetic acid (acido- or acetogenesis), or to a mixture of carbon dioxide and hydrogen. Acetic
acid and hydrogen can be converted into a mixture of methane and carbon dioxide using
acetotrophic and hydrogenotrophic methanogenic bacteria (Angelidaki et al. 2011). Biogas
from the Anaerobic Digestion of agricultural residues is generally composed of 4865%
methane, 3641% carbon dioxide, up to 17% nitrogen, <1% oxygen, 32169 ppm hydrogen
sulphide, and traces of other gases (Pavel, 2011).
Therefore, the aim of the work presented here was to investigate the potential of
biogas productivity from wheat straw under thermophillic conditions (i.e. 50C) using the
isolated thermophillic consortia. The research was conducted to (i) determine the
characterization of raw wheat straw by its proximate and ultimate analysis; (ii) estimate the
methane and carbon dioxide composition and biogas yield; and (iii) estimate the reduction of

244

volatile solids in biogas production after digestion in the given Hydraulic Retention Time
(HRT) of 30 days.
23.2.1 Seed preparation
The seed consists of a thermophilic consortium which was prepared from a soil
samples and then stored in a sterilized glass bottle in the refrigerator at 4
o
C.
23.2.2 Feedstock preparation for the biogas digester
The dry wheat straw was used as the biomass for feedstock preparation having 5%
moisture in itself. The dry wheat straw was soaked in water for overnight to maintain 85%
(w/w) moisture holding capacity. The C/N ratio of the biomass was maintained to 20:1 using
urea as the nitrogen source. About 10% (w/w) seed containing thermophilic consortium was
added to the wet biomass and filled in the digester bottle (1L). The Table-1 shows the set
parameters used for feedstock preparation of the digester.
Table 1. Set parameters for feedstock preparation of biogas digester
Parameters Values
Dry Biomass (Wheat Straw) 113 g
Water 84.67%
Wet Biomass 670 g
Seed (inoculum) 10% of wet biomass (67 g)
C:N ratio 20:1
Urea (as nitrogen source) 3 g
Incubation Temperature 50C
Incubation 30 days

23.2.3 Experimental Set-up
Biogas production experiment was conducted in a set of three screw cap bottles i.e.
Digester bottle (1L), Gas-collecting bottle (2L) and Displaced-water collecting bottle (2L) in a
batch mode as shown in fig 1. The digester bottle was filled with about 740 g of feedstock,
which contains 670 g of wet biomass (84.67% water present), 10% w/w (on wet biomass
basis) microbial seed culture and 3 g urea as nitrogen source for the growth of microbes. The
bottles were sealed with rubber corks and wax to maintain the anaerobic condition and to

245

prevent gas leakage. The experiment was carried out at 50C for an incubation period of 30
days.
The quantity of biogas produced in anaerobic digestion was measured from time to
time by water displacement method. The methane and carbon dioxide content were calculated
by gas chromatograph (GC, NUCON 5765 series). GC was equipped with a thermal
conductivity detector (TCD) and stainless column packed with Porapak T (80/100 mesh) and
a molecular sieve (2 m). The injection port, column oven and detector were operated at 60
o
C,
70
o
C, and 70
o
C, respectively. Argon was used as the carrier gas at a flow rate of 30 ml/min.

Figure 1. Schematic layout of laboratory based biogas plant.
The Elemental analysis of wheat straw for C, H, N and S elements was done by
CHNS-analyzer (Elementar Vario Micro, Model 2400). The lignocellulosic compositional
analysis (i.e. cellulose, hemicellulose and lignin) of wheat straw was performed by
FiberCap
TM
Analysis System (Model 2100/2023), involved NDF, ADF and ADL methods.

Ash as well as total solid (TS) and volatile solids (VS) contents was calculated using muffle
furnace.

Digester
bottle

Gas
collecting
flask
BBOB
Displaced
water
collecting
bottle
Gas Sample collection
Water
Gas
collecting
bottle
T-connector

246

23.3.1 Proximate and Ultimate Analysis
Elemental analysis based on carbon (C), hydrogen (H), nitrogen (N) and sulphur (S)
content was carried out on raw wheat straw in duplicates and got the values of 42, 5.9, 0.8 and
0.04%, respectively (Table 2). Qing et al. (2009) calculated the C, H, N, and S content of
wheat straw by Vario-elemental analyzer and got 42.50, 7.26, 0.06 and 0.14%, respectively.
The elemental contents especially carbon and hydrogen contents partially determine energetic
properties of agro-residues (Demirbas, 1997). As observed in analysis, the wheat straw
biomass contains a higher proportion of C and H contents, which increases the energy value
(i.e. calorific value).
Table 2. CHNS Analysis of raw wheat straw

The results for NDF, ADF and ADL methods of FiberCap
TM
Analysis System of raw
wheat straw were observed very similar to Chandra et al., (2012) who used standard method
P.J. Van Soest [Proc. Nutr. Soc., 32, 123 (1973)] for cellulose, hemicelluloses, lignin and
others (minerals, crude fats, & proteins) and got values of 35.10, 25.60, 7.80 and 31.80%,
respectively on dry weight basis (Table 3). Also Singh et al. (2011) used TAPPI Standard test
methods and got values of cellulose, 43.24%; hemicelluloses, 28.95% and lignin, 21.12%,
respectively on dry weight basis.
There was 89.27 g of initial volatile solids before the digestion which accounts 79% of
the raw wheat straw. However, the comparative proximate analysis of wheat straw after
digestion shows the reduction of 37.82% volatile solids (33.77g) during 30 days of incubation
period. Also the ash content of the wheat straw increased from 6 to 15.35% after the digestion
(Table 4). Similarly, Chandra et al. (2012) reported the reduction of volatile solids from
88.90% to 25.20% on dry weight basis in wheat straw on 30
th
day of HRT.
23.3.2 Estimation of Biogas Yield
The production of biogas started from the first day and reached maximum on 13
th
day
during the overall incubation period. The production reduced from 14
th
day onwards in
comparison to the previous period. However, the average biogas produced was about 490
Carbon (%) Nitrogen (%) Hydrogen (%) Sulfur (%)
42 0.8 5.9 0.04

247

ml/day. Further, the methane concentration was increased from 4
th
day onwards till 13
th
day.
Finally, the average amount of biogas produced from 14
th
day onwards was 321.79 ml/day.
Table 3. Summary of lignocellulosic compositional analysis of raw wheat straw using
FiberCap
TM
Analysis System.
Fiber Components Fibre compositions (%)
Cellulose 37.6
Hemicellulose 28.8
Lignin 14.5
Residues 19.1

Table 4. Comparative proximate analysis of raw and digested wheat straw

Parameters
Values
Raw wheat straw
(Before Digestion)
Digested-wheat straw
(After Digestion)
Moisture content 5 % 85 %
Total solids 113.4 g 76.5 g
Volatile Solids 79 % 74 %
Ash content 6 15.35

The average concentration of methane and carbon-dioxide produced from the biomass
feedstock was 62.77% and 37.23%, respectively and the total biogas production yield
obtained was 0.178 m
3
Kg
-1
of VS during the incubation period of 30 days at 50C (Fig. 2).
Similarly, Ivet et al., (2008) had shown the effect of thermophilic condition on biogas
production which increased up to 30% both in batch tests and in semi-continuous experiments
at lab-scale reactor. However, Chandra et al., (2012) used wheat straw in the experiment and
reported total biogas yield of 0.188 m
3
Kg
-1
of VS in 60 days HRT at 37C.
23.4 Conclusion
From the above study it was concluded that the isolated thermophilic consortium
shows the feasibility to produce biogas from wheat straw at 50C. The properties analysis of
wheat straw after 30 days incubation period also represents the potential of wheat straw for
further biogas production on the basis of left volatile solids. The reduction of 37.82% volatile

248

solids resulted in 0.178 m
3
Kg
-1
of VS total biogas yield. The average amount of methane and
carbon dioxide during the anaerobic digestion was 62.77% and 37.23%. However, further
experimentation and analysis are required for the isolation and identification of the
microorganisms present in the thermophilic consortium.

Figure 2. Production of biogas, methane and carbon dioxide during anaerobic digestion

References
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6. Deublein D., Steinhauser A. Biogas from waste and renewable sources: an
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i
o
g
a
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P
r
o
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u
c
e
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(
m
l
/
d
a
y
)
Time (days)
Biogas 1ro#uce#2 m&"#a$
3et/ane2 m&"#a$
Car'on4#io5i#e2 m&"#a$

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7. Ewa K., Tomasz P., Wojciech B., Bogdan D. (2010) Theoretical and observed biogas
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thermal (70 C) sludge pre-treatment prior to thermophilic anaerobic digestion.
Biochemical Engineering Journal 42:186192.
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and biogas production of wheat straw substrate using sodiuym hydroxide and
hydrothermal pretreatments. Energy 43:273-282.
13. Richard E., Dan L., Gregg C., and Rosie W. (2005) Estimating straw production of
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250

CHAPTER 24
ULTRASONIC PRETREATMENT TO ENHANCE
BIOHYDROGEN PRODUCTION FROM FOOD WASTE
Abhijit Gadhe, Shriram Sonawane, Mahesh Varma

Abstract
Present study deals with the multiple-response optimization for ultrasonication pretreatment
to enhance biohydrogen production and outstanding approach to overcome the drawbacks of
conventional response surface methodology (RSM). Complex food waste was used as source
in batch fermentation was followed for this study. Response surface methodology (RSM),
based on full factorial design, was employed to obtain the best possible combination of
ultrasonication time and total solids concentration for maximum H
2
yield (HY) and hydrogen
production rate (HPR). Experimental data were evaluated by applying RSM integrating a
desirability function approach. The optimum H
2
yield and HPR conditions were:
ultrasonication time 15 min and total solids concentration 8 % with maximum overall
desirability D of 0.94. The confirmation experiment under these optimal conditions showed a
HY and HPR of 126 ml H
2
/ g-VS and 4.9 ml H
2
/ hr, respectively. This was only 0.7 % and 2
%, respectively, different from the predicted values, suggesting that the desirability function
approach with RSM was a useful technique to get the maximum H
2
yield and HPR
simultaneously.
Keywords: Optimization; Food waste; Biohydrogen production; Response surface
methodology.
24.1 Introduction
Research on biohydrogen production from waste has gained renewed interest, due to
an increased awareness of global warming effects and rapid depletion of fuel resources
(Aman, 1996). However, the rate and efficiency of biological hydrogen production is low and
the technology needs further development (Boopathy and Daniels, 1991). Among the different
biological processes, Dark fermentation process is well thought out as a highly potential
process for hydrogen production due to its inherent advantages over photo-fermentation


251

(Chiesa at al., 1985). The potentials of dark fermentation have been greatly evaluated by
different researchers for hydrogen production from different organic wastes and wastewaters
(Vazquez et al., 2005; Liu et al., 2003; Fountoulakis and Manios, 2009; Kapdan and Kargi,
2006). Among the different organic wastes, food waste has high biohydrogen production
potential, as being composed of degradable carbohydrate and proteins. However, the complex
structure of food waste limits the rate and yield of hydrogen production. Therefore, evaluation
of efficient pretreatment process for substantial mineralization of complex molecules is of
prime importance.
Ultrasonication has been increasingly used recently as a pre-treatment method for
anaerobic digestion due to its ability to enhance solubilisation of organic matter. Although,
the flair of ultrasonication has been widely evaluated to enhance methane production, few
studies addressed its applicability for enhancement of hydrogen production. An extensive
search shows that sporadic work has been reported on the application of ultrasonication for
biohydrogen production from waste activated sludge (WAS). Wang et al., 2003a evaluated the
effect ultrasonication on hydrogen production from wastewater sludge in batch mode using a
clostridium strain along with four different pre-treatments viz. acidification, sterilization,
freezing/thawing and adding methanogenic inhibitor, and found that ultrasonication
marginally improved the hydrogen yield by 16%. Guo et al. 2008 studied the effect of
ultrasonication pretreatment of WAS for biohydrogen production in a batch reactor, and
observed a lag phase of only 3 hr and a hydrogen yield of 4.68 mL/g TCOD. The effects of
ultrasonication were further evaluated by Xiao and Liu, 2009, and found that the hydrogen
yield increased from 1.21to 3.83 mL H
2
/g VS. On the other hand, some other studies applied
the ultrasonication on the seed to eliminate the methanogenesis and enrich the hydrogen
producers (More and Ghangrekar, 2010; Elbeshbishy et al., 2010).
As apparent from the aforementioned literature, all the previous studies applied the
ultrasonication on WAS. There is no previous study addressing the impact of sonication on
anaerobic digestion of food waste despite its potential solubilisation of carbohydrates and
proteins, which are conducive for hydrogen production. Moreover, no study has been reported
the various interaction effects of ultrasonication time and total solids content on yield and rate
of hydrogen production from food waste. Thus, the present work has two-fold objectives as:
(1) to study the effect of ultrasonication on food waste solubilisation and therefore
enhancement of hydrogen production, (2) application of response surface methodology

252

(RSM) integrated with desirability function approach for simultaneous optimization of yield
and rate of hydrogen production (Hu et al., 2008; Gadhe et al., 2013).
24.2.1 Food waste
Food waste was collected from a cafeteria on the Visvesvaraya National Institute of
Technology campus, Nagpur, India. The waste was made up of rice, vegetables, fruit peels
and other residues. The food waste was mixed with tap water at the volumetric ratio of 1:3
and ground in a food blender. The pH of the resulting food waste slurry was 7.2. The desired
total solids concentration was achieved by dilution or concentrating food waste.
24.2.2 Seed sludge
The anaerobic seed sludge with volatile suspended solids (VSS) of 7-8 g/L was
obtained from a full-scale anaerobic digester of Upflow Anaerobic Sludge Blanket (UASB)
reactor of the dairy industry in Nagpur, India and stored at 4
0
C. The UASB is used to produce
methane from the wastewater of dairy and food product production process. Prior to use, the
anaerobic sludge was boiled at 90
o
C for 20 min to harvest hydrogen-producing spore-forming
anaerobes and inhibit hydrogen-consuming anaerobes.
24.2.3 Ultrasonication pretreatment
The ultrasonication pretreatment of food waste was performed using an ultrasonic
processor (ChromTech, UP-1200, Taiwan), which has a converter tip of and provides a
maximum power output of 1200 W and a constant frequency of 20 kHz. 200 ml food waste
with different TS content (2%, 5%, and 8%) was sonicated for different sonication times (5,
10 and 15 min) with sonication pulses set to 2 s on and 2 s off, corresponding to different
specific energy inputs.
24.2.4 Biohydrogen production potential
Batch fermentation experiments were carried out in 125 ml serum bottles. Each serum
bottle had 100 ml mixture comprising the seed sludge and sonicated food waste slurry. The
volumes of substrate and seed calculated based on F/M ratio of 4 (Elbeshbishy et al., 2010).
The pH of the mixed liquor in each serum bottle was adjusted to 5.5 using 1 mol/L HCl or 1
mol/L NaOH. Prior to operation, each bottle was flushed with nitrogen for 5 min to ensure
anaerobic condition, sealed with butyl rubber stoppers and incubated in a water bath shaker at
120 rpm at 37
0
C. All the data obtained were the average of triplicate.

253

The amount of biogas production was determined daily using water-displacement
equipment containing 5% NaOH solution. The concentration of hydrogen was measured by a
gas chromatograph (GC-2014, Shimadzu) equipped with a thermal conductivity detector
(TCD) and a Molecular sieve 5Acolumn (30mm X 0.32mm X 30m). The operational
temperatures of the injection port, the column oven and the detector were 150, 145 and 150
0
C, respectively. Nitrogen was used as the carrier gas at a flow rate of 25 mL/min. COD,
BOD, TDS, VSS and total solid TS and Total VFA were measured according to the
procedures described in standard methods (APHA, 1995). Hydrogen gas production was
calculated from the headspace measurement of gas composition and the total volume of
hydrogen produced, at each time interval, using the mass balance Eq. (1):
I
H,
= I
H,-1
+ C
H,
(I
u,
I
u,-1
) + I
H
(C
H,
C
H,-1
) (1)
Where, V
H,i
andV
H,i-1
are the cumulative hydrogen gas volumes at the current (i) and previous
time interval (i1), respectively; V
G,i
andV
G,i-1
are total biogas volume at the current and
previous time interval; C
H,i
and C
H,i-1
are the fraction of hydrogen gas in the headspace at the
current and previous time interval; V
H
is the volume of headspace of serum bottles.
24.2.6 Kinetic modelling
A modified Gompertz Eq.(2) was used to fit the cumulative hydrogen production
curves to obtain the hydrogen production potential (P ), the hydrogen production rate (R) and
lag phase ().
E = Pcxp ]
expj
R
m
c
P
( t) + 1[
(2)
Where, H represents the cumulative volume of hydrogen produced (ml/L), P the hydrogen
production potential (ml/L), R
m
the maximum production rate (ml/h), the lag-phase time (h),
t the incubation time (h), and e is 2.718. Parameters (P, R
m
and ) were estimated using the
non-linear curve fitting function in OriginPro 8. In this study R
m
expressed as ml H
2
/ (L of
medium h
-1
), the specific hydrogen production potential P is defined as ml H
2
/L medium. The
hydrogen yield (ml H
2
/g VS) was calculated by dividing P by VS added.
24.2.7 Experimental design and Statistical analysis
In present study, %TS [X1] and ultrasonication time [X2] were chosen by borrowing
methodology as two independent factors and HY and the HPR were selected response
variables. A two-factor with three levels experimental design of full factorial method at

254

random order was used to find the optimum H
2
production condition and was given in Table
1 along with experimental data and predicted responses. The sequential model fitting test and
regression analysis were carried out in order to choose a suitable model. A second-order
polynomial model has been used to identify all possible interactions of selected factors with
response function as below:
= [
0
+ _ [
2
=1
+ _ [
2
=1
X
2
+ __ [
]
X
2
=1
X
]

Where Y is the response,
0
is the constant coefficient,
i
,
ii
, and
ij
are the coefficients
estimated by the regression for liner, quadratic and cross-product effects of X1 and X2
respectively on the response. The parameters of the response equation and analysis of
variance (ANOVA) were evaluated using MINITAB 16.
The optimization of multi-response was achieved using desirability function approach.
The general methodology is to first convert the response into an individual desirability
function (di) that varies from 0 to 1 (lowest desirability to highest desirability). The individual
desirability scores for the predicted values for each dependent variable are then combined into
overall desirability function, D, by univariate techniques as (Hu et al., 2008):
= (J
1
w1
J
2
w2
J
3
w3
J
4
w4
J
n
wn
)
1 _w
(4)
Where D is the overall desirability, di is individual response desirability and wi is a response
weight. wi is a weighted composite desirability, and the concepts and function of D and di are
identical to the desirability approach. One-sided transform of di is used in the study and given
by:
(5)
Where d
i
is individual response desirability, Y
i
is the response values, Y
i-min
is the minimum
acceptable value for response i, Y
i-max
is the maximum acceptable value for response i, and r is
a weight used to determine the scale of desirability. In present work, the value of i was chosen
to 1, implying that the d
i
increases linearly as Y
i
increases (Gadhe et al., 2013).
24.3.1 Effects of TS content and ultrasonication time on H
2
yield
The level of experimental parameters and results of all runs along with controlled
experiment are presented in Table 1.

255

Table 1 Full factorial design matrix along with predicted and experimental values of
Hydrogen yield and HPR.
Run %TS Ultrasonication
time (min)
HY
(ml H
2
/g VS)
HPR
(ml H
2
/ hr)
[X1] [X2] Observed Predicted Observed Predicted
1 2 5 38 36 0.67 0.55
2 2 10 45 47 0.8 0.85
3 2 15 48 48 0.84 0.90
4 5 5 78 80 2.8 3.01
5 5 10 93 92 3.4 3.35
6 5 15 95 94 3.6 3.43
7 8 5 109 109 4.6 4.51
8 8 10 124 123 4.9 4.89
9 8 15 126 127 4.9 4.99

In order to evaluate the effects of independent variables on HY (Y
1
) from food waste
experiment data in Table 1 were subjected to regression analysis, and the equation was
obtained as:
1
= 19.611 + 19.2778X
1
+ 4.88SX
2
u.778X
1
2
u.2uuX
2
2
+ u.117X
1
X
2

(6)
The suitability of quadratic model to evaluate the HY from food waste is fairly
represented in Table 2. Furthermore, the adequacy of the model was further justified through
analysis of variance ANOVA and several indicators viz. F-value, correlation coefficient (R
2
),
adequate precision etc. (Gadhe et al., 2013), as depicted in Table 2.
The regression model F-value implies that the model is highly significant (P =<0.001).
It is also observed that the coefficients for main, quadratic and interactive effect are highly
significant at 95% confidence interval (CI). The correlation coefficient, R
2
, of 99.86%
indicates the suitability of second order polynomial to predict the HY in terms of independent
variables, and the predicted values were found in close agreement with that of experimental
results as shown in Table 1. Moreover, the fit-value, R
2
also indicates that only 1.4% of the
total variation is not explained by the model. Furthermore, the high and reasonable agreement
of Adjusted-R
2
(98.38%) with Predicted R
2
(99.62%) indicates a negligible block effect

256

and model and/or data are best fit for optimization (Gadhe et al., 2013). Furthermore, the
Adequate Precision, a measure of the signal to noise ratio, of 148.81 indicates an adequate
signal (Gadhe et al., 2013). The small value of standard deviation (S), 2, indicates model
shows great compliance with predicted responses.
Table 2 ANOVA and result of regression analysis of full factorial design for hydrogen yield
Source Model
Term
DF Coefficient F-value P-Value
Regression
-- 5 -- 419.48 0.000
Constant
-- --
-19.6111
--
0.072
Linear
-- 2 --
60.94 0.004
TS X
1

1
19.2778 114.09 0.002
Time X
2

1
4.8833 15.43 0.029
Square -- 2 --
16.97 0.023
TS*TS X
1
*X
1
1
-0.7778 22.47 0.018
Time*Time X
2
*X
2
1
-0.2 11.46 0.043
Interaction -- 1 --
2.81 0.042
TS*Time X
1
*X
2
1
0.1167 2.81 0.042
S = 2.088; Adequate Precision = 148.810; R Sq = 99.86%; R-Sq(pred) =
98.38%; R-Sq(adj) = 99.62%

From the Table 2 it has been observed that X
1
and X
2
, and quadratic terms X
1
2
and X
2
2
had significant positive and negative effect on HY respectively. The significant main and
quadratic effects of factors predominate over interaction effects of variables. ANOVA results
of Eq. (6) shows TS X
1
has the highest positive significant effect on Y
1
followed by
ultrasonication time X
2
. The significant positive effect of the factors implies that HY
increases with increase in levels of the variables. From the full quadratic analysis for
interaction effects of variables it was confirmed that X
1
X
2
had significant positive effect on
HY. Negative quadratic effect of process variables, X
1
2
, X
2
2
, means that at high level, they
tend to decrease the yield markedly. Therefore, the marginal increase in levels of TS and
ultrasonication time are expected to increase the HY for the present situation and in the
experimental ranges used.

257

The significant plots of the response surface and contour of H
2
yield in Figs.1and 2
depicts the maximum HY evaluated using Eq. (6).

Fig. 1- 3D surface plot for the effects of TS (%) and ultrasonic time (min) on the yield of
hydrogen production
The H
2
yield was considerably low at low value of TS (2%) and ultrasonication time
(5min) but increasing TS at 8% and ultrasonication time 15 min led to increase in the HY.
This is because at a longer sonication time, there was ample opportunity for solids to come
under perpetual attack of collapsing cavitation bubbles. In additions, at higher TS content, the
ultrasonication system becomes self-viable to carry out efficient disintegration which leads to
increased yield of hydrogen production (Elbeshbishy et al., 2010).
It is evident from Figs.2 that the hydrogen yield increases up to 10min after which
there was no significant effect of ultrasonication. Specific energy [SE] calculations at
different TS content and ultrasonication time shows that as a TS content increased from 5% to
8% the specific energy input decreased by 37% and HY increased by 33% at ultrasonication
time of 15 min. This confirms that higher level of TS and ultrasonication time significantly
decreases required SE input and increases HY.
24.3.2 Effects of TS content and ultrasonication time on HPR
The TS content [X1] and ultrasonication time was found to be important parameter
that affects the rate of hydrogen production. The interaction effect of these process parameters
were evaluated by executing experimental run given by general full factorial. The

258

experimental and predicted values of SHPR are presented in Table 1. The final predicted
process model in terms of actual significant factors for SHPR (Y
2
) is given below:
1
= 19.611 + 19.2778X
1
+ 4.88SX
2
u.778X
1
2
u.2uuX
2
2
+ u.117X
1
X
2
(7)
The sufficiency of the model was evaluated through ANOVA for HPR depicted in Table 3.

Fig. 2- Contour plot for the effects of TS (%) and ultrasonic time (min) on the yield of
hydrogen production
The quadratic regression model of Y
2
was highly significant with a low probability
(P<0.05). The Predicted R
2
(94.55%) were found in close agreement with Adjested-R
2
value
(98.79%) followed by high adequate Precision value of 137.68 implying the model equation
adequately described the response surface of HPR. The correlation coefficient (R
2
) was
99.55% indicates reasonable agreement of experimental values with predicted results. From
the above statistical results, it may be inferred that the full factorial design was adequate to
predict the HPR within the range of variables studied.
It is evident from the ANOVA results that all the quadratic effects of parameters
significantly influence HPR. The positive coefficients for X1 and X2 indicate a linear effect to
increase rate of hydrogen production, while negative coefficient for square effect shows a
strong effect to decrease hydrogen production rate at very high levels of parameters. 3D
surface plot and contour plot shown in Fig. 3 and Fig. 4 clearly indicate that HPR increases
significantly with an increase in the TS content and time from 2% to 8% and from 5 min to 15
120
100
80
60
TS %
T
i
m
e

(
m
i
n
)
8 7 6 5 4 3 2
15.0
12.5
10.0
7.5
5.0

259

min, respectively. Conversely, the HPR may decrease with the further increase of TS content,
which is clearly visible from the decreased slope of response.
Table 3 ANOVA and result of regression analysis of full factorial design for HPR
Source Model
Term
DF Coefficient F-value P-Value
Regression
-- 5 -- 131.41 0.001
Constant
-- --
-2.18352
--
0.047
Linear
-- 2 --
24.67 0.014
TS X
1

1
1.18519 49.17 0.006
Time X
2

1
0.13683 1.38 0.025
Square -- 2 --
6.52 0.081
TS*TS X
1
*X
1
1
-0.05352 12.13 0.04
Time*Time X
2
*X
2
1
-0.00527 0.91 0.011
Interaction -- 1 --
0.11 0.021
TS*Time X
1
*X
2
1
0.00217 0.11 0.021
S = 0.195578; Adequate Precision = 137.680; R-Sq = 99.55%; R-Sq(pred) = 94.55%
R-Sq(adj) = 98.79%

Fig. 3- 3D surface plot for the effects of TS (%) and ultrasonic time (min) on the HPR


260

Fig. 4- Contour plot for the effects of TS (%) and ultrasonic time (min) on the HPR
The ultrasonic irradiation has a prime effect on rate of production than the extent of
production which leads to increased HPR and decreased Lag phase of the process
(Elbeshbishy et al., 2010; Guo et al., 2008). As the TS content and time increased from 2% to
8% and from 5 min to 15 min, respectively, HPR increased from 0.89 to 4.9 ml H
2
/hr.
Ultrasonication consists of the propagation of ultrasound waves (>15-20 kHz) in liquid media
that produces cavitation and acoustic streaming; while the former generates powerful shear
forces, the latter increases convection of aqueous slurries (Khanal et al., 2007). The combined
effects of these phenomenons at higher TS content and prolonged time were found to enhance
HPR by increasing surface area concomitant to disintegration and release of substrate in the
system under investigation.
24.3.3 Optimization using desirability function and Confirmation experiments
The multi-response optimization for HY and HPR was done by desirability function
approach. A detailed description of desirability functions has been discussed by Shi et al,
2010. The individual desirability of d
1
for Y
1
and d
2
for Y
2
were calculated by one side
transformation (Shi et al., 2010). The individual desirabilitys were then used to calculate
overall desirability (D) of the optimization. The best optimized conditions for new response,
D, were found to be TS content 8% and ultrasonication time 12min. The overall desirability
of optimization was found to be very high, i.e. D=0.94, and the HY and HPR were 126 ml H
2
/
g-VS and 4.9 ml H
2
/ hr, respectively.
According to the optimization results acquired by RSM and the desirability function, a
verification test with triplicate was performed under the set of optimized parameters. The
4
3
2
1
TS (%)
T
i
m
e

(
m
i
n
)
8 7 6 5 4 3 2
15.0
12.5
10.0
7.5
5.0

261

average H
2
yield and HPR were 125.12 ml H
2
/ g-VS and 4.8 ml H
2
/ hr, respectively, at
predicted optimum conditions of the variables. Verification experiment revealed only 0.7%
difference between estimated and actual hydrogen yield, while only 2% difference between
estimated and actual HPR. Results suggested that the optimal conditions attained had the least
error and can be practically applied to produce hydrogen from complex food waste.
24.4 Conclusions
In batch experiments, food waste was utilized as the substrate for H
2
production by
anaerobic sludge. The H
2
yield and SHPR were evaluated at various levels of TS content and
ultrasonication time by applying RSM. The operational condition of H
2
production from
complex food waste was also optimized with RSM and a desirability function approach. The
maximum overall desirability D of 0.94 was achieved under TS 8% and ultrasonication time
12 min. Correspondingly the H
2
yield and HPR were 125.12 ml H
2
/ g-VS and 4.8 ml H
2
/ hr,
respectively. The verification test confirms that the optimum H
2
yield and HPR measured
were in good agreement with the predicted values. The experimental results indicate that the
desirability function approach with RSM was a useful technique to get the maximum H
2
yield
and HPR simultaneously.

References
1. Aman C.A. (1996) Alternative fuels and power systems in the long term. Int J Vehicle
Design., 17:510517.
2. American Public Health Association. (1995).Standard methods for the examination of
water and wastewater. 19th ed., Washington DC USA.
3. Boopathy R. and Daniels L. (1991) Effect of pH on anaerobic mild steel corrosion by
methanogenic bacteria. Appl. Environ. Microbiol., 57:21042108.
4. Chiesa S.C., Irvine R.L. and Manning J.F. (1985) Feast/Famine growth environment
and activated sludge population selection. Biotechnol. Bioeng., 27:562569.
5. Elbeshbishy E., Hafez H. and Nakhla G. (2010) Enhancement of biohydrogen
producing using ultrasonication. Int. J. of Hydrogen Energy., 35:6184-6193.
6. Fountoulakis M.S., and Manios T. (2009) Enhanced methane and hydrogen production
from municipal solid waste and agro-industrial by-products co-digested with crude
glycerol. Bioresour. Technol., 100:30433047.

262

7. Gadhe A, Sonawane S.S. and Varma M.N. (2013) Optimization of conditions for
hydrogen production from complex dairy wastewater by anaerobic sludge using
desirability function approach. Int. J. Hydrogen Energy., 38:6607-6617.
8. Guo L., Li X.M., Bo X., Yang Q., Zeng G.M., Liao D. and Liu J.J. (2008) Impacts of
sterilization,microwave and ultrasonication pretreatment on hydrogen producing using
waste sludge. Biores. Technol., 99:36513658.
9. Hu Z.Y., Cai M. and Liang H.H. (2008) Desirability function approach for the
optimization of microwave-assisted extraction of saikosaponins from Radix Bupluri.
Sep. Purif. Technol., 61:266275.
10. Kapdan I.K. and Kargi F. (2006) Bio-hydrogen production from waste materials.
Enzym. Microb. Technol., 38:56982.
11. Khanal S.K., Grewell D., Sung S.H. and Leeuwen J.V. (2007) Ultrasound applications
in wastewater sludge pretreatment: a review. Critl. Rev. Environ. Sci. Tech., 37:277-
313.
12. Liu H., Zhang T. and Fang H.P.P. (2003) Thermophilic H
2
production from cellulose
containing wastewater. Biotechnol. Lett., 25:365369.
13. More T.T. and Ghangrekar M.M. (2010) Improving performance of microbial fuel cell
with ultrasonication pre-treatment of mixed anaerobic inoculum sludge, Bioresour.
Technol. 101: 562567.
14. Shi X., Jin D., Sun Q. and Li W. (2010) Optimization of conditions for hydrogen
production from brewery wastewater by anaerobic sludge using desirability function
approach. Renewable Energy., 35:14931498.
15. Vazquez I.V., Sparling R., Risbey D., Seijas N.R. and Varaldo H.M.P. (2005)
Hydrogen generation via anaerobic fermentation of paper mill wastes. Bioresour.
Technol., 96:190713.
16. Wang C.C., Chang C.W., Chu C.P., Lee D.J., Chang B.V. and Liao C.S. ((2003)
Producing hydrogen from wastewater sludge by Clostridium bifermentans. J.
Biotechnol., 102,8392.
17. Xiao B.Y.and Liu J.X. (2009) Effect of various pretreatments on biohydrogen production
from sewage sludge. Chinese Sci. Bull. 54: 2038-2044.

263

CHAPTER 25
Biological hydrogen production by facultative anaerobic bacteria
Enterobacter aerogens (MTCC 8100)
Virendra Kumar, Richa Kothari

and S.K.Tyagi

Abstract
Biological hydrogen is an efficient way for the degradation of organic matter to the useful
products with the help of microorganism. It basically deals with the organic matter present in
the solid or liquid substrates. Some bacteria like Clostridium, Enterobactor, and Escherichia
etc. are found to be potential to degrade the organic matter in the useful product such as
hydrogen and ethanol. Among the various process of biohydrogen production, dark
fermentation seems to be feasible in this aspect. A study of biological hydrogen production by
anaerobic facultative bacteria Enterobactor aerogens was investigated by using glucose as the
feedstock for finding the feasibility at experimental part. The growth period of the bacteria,
gas production, pH and glucose consumption was investigated during the study. In salt
medium with glucose the gas produced was comprises of 35 % hydrogen. The process shows
40% glucose consumption in 18 hours at 30C with pH of 6.5. The study reveals the selected
species is suitable for biohydrogen production from carbon source.
Key words: Biological hydrogen production, Enterobacter aerogens
25.1 Introduction
Hydrogen is a promising alternative for carbon based fuel compared to others because
it is a clean renewable source of energy having calorific value 122 Kj/g and produces water as
only by product after burning [Chang et al., 2002]. Biological routes of energy production are
taking more attention nowadays because it generates energy as well as treat the waste without
harming the environment. Biological hydrogen production is a microbial mediated process.
The generation of hydrogen by biological means is not energy intensive compared with the
conventional thermo-chemical techniques, since the operating temperature and pressure are
not very high. The method of the dark fermentation has certain advantages compared with the
other biological processes. In contrast to bio-photolysis and photo fermentation, the process


264

needs no solar radiation, but the required energy is supplied by the organic substrates and
hence the process is not interrupted during the night. Moreover, the production rate of the H
2

of the fermentative bacteria in comparison with the other biological processes is greater
(Kumar et al., 2000; Nath et al., 2005). Fermentative hydrogen production also has good
properties for the biotechnological applications, due to the capability to exploit variety of
substrates (Nath and Das, 2003).
Dark fermentation of organic substrate is manifested by the majority of diverse group
of bacteria obligate as well as facultative anaerobes that perform several biochemical
reactions. Optimization of the performance of these bacteria increases the conversion
efficiency of organic matter to hydrogen. Facultative anaerobic bacteria are gram-negative,
rods shaped, with relatively simple nutrient requirements (Schmauder, 1992). Among the
species that can produce H
2
, are Escherichia (E. coli), Proteus (P. vulgaris), Enterobacter (E.
aerogenes). These bacteria ferment sugars to a variety of end products such as acetate,
formate, lactate, succinate, ethanol, CO
2
and H
2
. The degradation of organic matter in
anaerobic environments by microbial consortia involves the cooperation of a population of
microorganisms that generate a stable, self-regulating fermentation [Das, 2001]. First,
hydrolytic bacteria hydrolyze polymeric proteins and sugars. Then, fermentative bacteria form
organic acids, H
2
and CO
2
from monomeric molecules (Fig. 1). At that point, H
2
and acetate
can be utilized and/or produced by several microbial groups. Thus, acetate is generated during
acetogenesis from CO
2
reduction and H
2
by autotrophic acetogens via the Wood Ljungdahl
pathway, a process named homoacetogenesis [Kpkea et al., 2012].
The process of hydrogen production by facultative anaerobes is given in figure 1[Jenni
et al. 2011].The organic substrate such as glucose, is degraded to pyruvate by the glycolysis.
Pyruate was further converted to acetyle coenzyme A (Co A) and formate by the enzyme
pyruate formate lyase (PFL). Formate is again converted to hydrogen and carbon dioxide by
the enzyme formate hydrogen lyase (FHL) complex. In this study the facultative anaerobic
bacteria Enterobacter aerogens, MTCC no 8100 is selected for biological hydrogen
production through anaerobic consortia.


265

Figure 1: Biohydrogen production process by facultative anaerobic bacteria
25.2.1 Microbial culture development
A bacterial culture of Enterobacter aerogens (MTCC no.8100) was brought from
Microbial Technology Culture Centre (MTCC), Chandigarh and grown on prescribed media.
One litre of growth medium contains 1gm of beef extract, 2gm of yeast extract, 5gm of
peptone and 5gm of NaCl with distilled water. The culture was grown in media at 30
o
C
temperature in an incubator shaker (New Brunswick Scintific Innova 43) for 24 hours
incubation period.

(a) (b)
Figure 2. A microscopic view of Enterobactor aerogens (a) & (b)
25.2.2 Preparation of fermentation medium
Pyruvate
Acetyl-CoA
Formate
H
2

CO
2

Ethanol Acetate
Glucose

266

Anaerobic liquid media used for experimental study contained, 10.0 g/l of Glucose ,
4.0 g/l of (NH4)
2
SO
4
, 4.0 g/l of KH
2
PO
4
, 4.0 g/l of Na
2
HPO
4
, 1.0 g/l of yeast extract, 0.20
g/l of MgSO
4
and Trace elements solution 2.0 ml/l having composition of 1.0 ml/l of HCl,
100 mg/l of MnCl
2
.4H2O, 70mg/l of ZnCl
2
, 60mg/l of H
3
BO
3
, 200mg/l of CoCl
2
.6H
2
O, 10
mg/l of CuCl
2
.2H2O, 20mg/l of NiCl
2
, 30 mg/l of Na
2
MoO
4
.2H2O.
25.2.3 Experimental set-up
Experiments were done in anaerobic jars with batch set-up plan (Walker et al., 2009).
Total volume of each jar 1000 ml was made for the study having working volume 600 ml.
The fermentation medium sample with 10% v/v microbial seed culture was kept for study
with adjusting the initial pH at 6.5.

Figure 3. Experimental Batch set-up for gas measurement.
Hydrogen content in the gas was determined with Gas Chromatograph (GC 5765
Nucon India Make ) equipped with Thermal Conductivity Detector (TCD) a stainless steel
column packed with porapak Q(80/100 mesh) . The operational temperature of the oven
injector port and detector were 70, 120 and 120
0
C respectively. Nitrogen was used as the
carrier gas at a flow rate of 20 ml/min. The sugar content was determined by DNS method
(Miller, 1959). Biomass was measured by turbidimetricaly at 600 nm using UV-Vis
Spectrophotometer (Perkin Elmer Lambda 35) also 1 ml of the sample was centrifuged and
dried to measure dry cell weight.


267

25.3.1 Growth of Bacteria
Most facultative anaerobes produce hydrogen through breakdown of glucose to
pyruate forwarded by volatile fatty acids formations during fermentation (Elsharnouby et al.,
2013). The growth pattern of the bacteria in growth medium was analyzed after each hour for
OD
600
as well as for dry cell weight and it was found that in both cases the stationary phase
was achieved after 24 period of the growth.
25.3.2 Biochemical assay for the carbohydrate fermentation
The biochemical analysis of bacteria for the fermentation of Carbohydrate (Glucose
and Sucrose) was done in lab [Aneja, 2010]. The results obtained were given in Table 1. All
test tubes in duplicate for assays observation.

Figure 4. Growth curve of bacteria Growth medium (a) OD
600
(b) Dry cell weight basis.
Table 1: Fermentation of sugar assay by selected microbial species

0
065
1
165
2
1 4 7 10 13 16 19 22 25 28
O
D
6
0
0
Time (h)
0
50
100
150
200
1 3 5 7 9 11131517192123252729
B
i
o
m
a
s
s

(
m
g
/
L
)
Time (h)
Composition Results Observation
Media + Phenol red _ No colour change
Media + Bacteria + Phenol red _ No color change
Media + Bacteria + Phenol red
Glucose
+ Yellow color appeared +
Gas bubbles seen
Media + Bacteria + Phenol red
Sucrose
+ Yellow color appeared +
bubbles seen

268

25.3.3 Hydrogen Production
The maximum yield of facultative anaerobic bacteria by dark fermentation is 2 mol
H
2
/mol glucose or 0.248L H
2
/g glucose (2 mol H
2
22.4 L/mol)/180g glucose) at standard
temperature and pressure [Hallenbeck, 2004]. In our study maximum 2g of glucose was
consumed and total water displacement was approximate 600ml. The concentration of H
2
in
the gas was 35 % so H
2
was 210 ml or 105 ml H
2
/g glucose or 0 .105 L H
2
/g glucose. This
low yield was due to the decrease in the pH of the medium during fermentation (figure 8) and
formation of volatile fatty acids that could not determined during course of experiment
however it was supported by literature available. [Byung et al., 1985; Debrock et al., 1992;
Khanal et al., 2004]. While the optimum yield was found on controlled pH 5.5 to 6.5 [Kumar
and Das, 2000; Khanna et al., 2011]

(a) (b)

(c)
Figure 5. Biochemical assay of selected microorganism (a) initial phase (b) fermentation
phase (c) gas production phase.
25.3.4 Glucose Consumption
Initial concentration of Glucose 1% (10g/l) was taken as the feedstock for study.
During the fermentation, bacteria utilized only 40% of the glucose and rest was remained as
Volatile fatty acid. The facultative anaerobic bacteria generate acetyl coenzyme A and
formate by utilizing glucose. Formate is then converted to hydrogen and carbon dioxide. An

269

acidic condition exists due to the formation of other fermentation products such as lactate,
acetate formate, succinate, ethanol and butandiol [Hallenbeck and Benemann 2002].

Figure 6. Cumulative hydrogen production.

Figure 7. Glucose consumption and cumulative gas production of the bacteria during
hydrogen production.
25.3.5 Effect of pH
The pH effect on biohydrogen production was investigated that fall during the process
from 6.5 to 4.3. As resultant in the initial stage of the fermentation the production of gas was
high as at the pH 5 to 6.5 till the 7.5
th
hours and it was decreased to the 4.3 in the 14
th
hours so
as the gas production. However in case of batch with initial pH above 6.5 the yield decreased
0
50
100
150
200
250
0 165 3 465 6 765 9 1065 12 1365 15 1665 18
H
y
d
r
o
g
e
n

P
r
o
d
u
c
t
i
o
n

(
m
l
)
Time (h)
0
1
2
3
4
5
6
0
100
200
300
400
500
600
700
0
1
6
53
4
6
56
7
6
59
1
0
6
5
1
2
1
3
6
5
1
5
1
6
6
5
1
8
&
u
c
o
s
e

,
g
"
L
-
a
s

1
r
o
#
u
c
t
i
o
n

,
m
&
-
%ime ,/-
as
1ro#uction
,m&-
&ucose
,g"L-

270

drastically. Whereas some workers found the initial pH 6.5 was optimum for hydrogen
production (Wei et al., 2010 and Lin et al., 2010).

Figure 8. pH pattern and gas production in fermentation medium
25.4 Conclusions
From the above study it was concluded that the selected species Enterobactor
aerogens shows the feasibility to produce hydrogen from glucose. During the study it was
also observed that consumption of glucose was only 40% that causes low production of
hydrogen. The low production of hydrogen was due to the lowering of pH during the process.
These experiments showed that the measurement of metabolic gases i.e. the useful method of
monitoring bacterial fermentation, which could not only measure the productivity of H
2
but
also reveal process unknown so far. Experiment also showed that the Enterobacter aerogens
utilizes glucose and convert it in to H
2
and organic acids strongly dependent on the bacterial
isolate. Thus there is a possibility to use glucose as a substrate for H
2
production, although
probably optimization of bacterial strains or their genetic modification will be desired.
Acknowledgement
The authors wish to thank Sardar Swarn Singh- National Institute of Renewable
Energy, Kapurthala Punjab. An autonomous institute of Ministry of New and Renewable
Energy, New Delhi., Dr. Sachin Kumar, Scientist B and Dr. Subhashish Behra, Post Doc.
Fellow of the institute to provide us the laboratory and guidance during the course of study.

0
1
2
3
4
5
6
7
0
20
40
60
80
100
120
0 165 3 465 6 765 9 1065 12 1365 15 1665 18
p
0
a
s

1
r
o
#
u
c
t
i
o
n

,
m
&
-
%ime ,/-
as 1ro#uction
,m&-
p0

271

References
1. Chang, J. S., Lee, K. S. and Lin, P. J. (2002) Biohydrogen production with fixed-bed
bioreactors. International Journal of Hydrogen Energy, 27:1167-1174.
2. Das, D., Veziroglu, T.N. (2001) Hydrogen production by biological processes: a survey
of literature. International Journal of Hydrogen Energy, 26:1328.
3. Hallenbeck, P.C. and Benemann J. R. (2002) Biological hydrogen production
fundamental and limiting process. International Journal of Hydrogen Energy 27:1185-
1193
4. Hallenbeck, P. C. (2004) Fundamental of the fermentative production of hydrogen.
Water Science and Technology 52: 21-29.
5. Jenni, J., Seppa, la., Jaakko A, Puhakka., Olli, Yli-Harja., Matti T, Karp., and Ville,
Santala,. (2011) Fermentative hydrogen production by Clostridium butyricum and
Escherichia coli in pure and co-cultures. International journal of Hydrogen Energy
36:10701 -08.
6. K. R. Aneja (2010) Experiments in Microbiology Plant Pathology and Biotechnology.
New age International (p) Limited Publishers, New Delhi.
7. Nath, K.., Muthukumarb, M., Kumarb, A. and Das D. (2008) Kinetics of two-stage
fermentation process for the production of hydrogen. International Journal of Hydrogen
Energy 33:11951203.
8. Lin, Y.H., Juan, M.L., and Hsien, H. J. (2010) Effects of temperature and initial pH on
biohydrogen production from food processing and wastewater using anaerobic mixed
cultures. Earth Environ. Sci., DOI: 10.1007/s10532-010-9427-2.
9. Walker, M.., Zhang,Y., Heaven, S. and Charles Banks.(2009) Potential errors in the
quantitative evaluation of biogas production in anaerobic digestion processes.
Bioresource Technology, 100: 63396346.
10. Kpke, M., Claudia, H., Hujer, S., Liesegang, H., Wiezer, A., Wollherr, A.,
Ehrenreich, A., Liebl, W., Gottschalk, G. and Drre, P.(2012) Clostridium ljungdahlii
represents a microbial production platform based on syngas. Proceeding of National
Academy of Science of United State of America. 107: 1308713092.
11. Khanna, N., Kotay, S.M., Gilbert, J.J. and Das D. (2011) Improvement of biohydrogen
production by Enterobacter cloacae IIT-BT 08 under regulated pH. Journal of
Biotechnology 152:915.

272

12. Kumar, N. and Das, D.(2000) Enhancement of hydrogen production by Enterobacter
cloacae IIT-BT 08,. Process Biochemistry 35:589593.
13. Nath, K., Das, D. (2003) Hydrogen from biomass. Current Science.85:265-271.
14. Elsharnouby, O., Hafez
,
H., Nakhla, G., Naggar, M. H. E. (2013) A critical literature
review on biohydrogen production by pure cultures. International Journal of Hydrogen
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15. Schmauder, H.-P. (1992). Hans G. Schlegel, Allgemeine Mikrobiologie. StuttgartNew
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16. Wei, J., Liu, Z.T., Zhang, X., (2010) Biohydrogen production from starch waste water
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273

CHAPTER 26
ENHANCED BIOHYDROGEN PRODUCTION FROM
GLYCEROL USING PRETREATED MIXED CULTURE
Anbalagan Krishnasamy, Mohanraj Sundaresan, Kodhaiyolii Shanmugam, Pugalenthi velan

Abstract
The anaerobic batch fermentation of glycerol was developed using pretreated dairy sludge as
mixed culture to enhance the biohydrogen production. The effect of pH and initial substrate
concentration on fermentative hydrogen production were also investigated using heat treated
mixed culture. The experimental results showed that the hydrogen yield from heat, acid, base
pretreated dairy sludge was 300, 250 and 120 mL of H
2
, respectively, and its corresponding
hydrogen content was 43 %, 38 % and 29 %. The results of the study demonstrate that the
heat treated dairy sludge enhanced the biohydrogen production when compared to other
pretreatment methods. The optimum values of pH and glycerol concentration for efficient
biohydrogen production were 6.0 and 30 g/L respectively. The highest biohydrogen
production and hydrogen content were 510 mL and 42 % respectively, under optimum
conditions.
Key words: Biohydrogen, Mixed culture, Glycerol, Pretreatment.
26.1 Introduction
Hydrogen is an ideal future energy carrier for alternative of fossil fuels because it releases a
huge amount of energy, and generates no air pollutants. Hydrogen is mainly used for many
applications such as fuel for automobile and electricity generation (Foglia et al., 2010; Wu et
al., 2011). 95% of the hydrogen produced by conventional methods is expensive and energy
intensive process (Chen et al., 2008; Foglia et al., 2010). Fermentative hydrogen process is
one of the alternative processes for conventional methods (Elsharnouby et al., 2013).
Biological methods for the biohydrogen production such as dark fermentation, photo
fermentation, and integrated system using both dark fermentation and photo fermentation
have been extensively studied (Prakasham et al., 2009; Pott et al., 2013; Chen et al 2008).



274

Among these processes, dark fermentative hydrogen production was faster than the other
processes and simple operation (Sinha and Pandey, 2013).
Biohydrogen is produced by dark fermentation from carbohydrates and other
renewable organic substrates (Li et al., 2008). Researchers have focused to develop the
hydrogen production from various organic wastes including agricultural, lingocellulosic,
industrial waste and wastewater (Elsharnouby et al., 2013). However, the biohydrogen
production from pure and waste glycerol received the great attention in recent years.
Worldwide, the development in biodiesel production sector glycerol has noticeably increased
quantity of waste (Kumar and Lin, 2013). Theoretically 3 mole of hydrogen can be produced
per mole of glycerol in suitable fermentative process. Few researchers carried out
biohydrogen production from pure glycerol and waste glycerol using pure cultures such as
Enterobactor aerogens (Markove et al., 2010), Klebsiella pneumoniae TR17 (Chookaewa et
al., 2012), Thermotoga neapolitana (Nago and Sim, 2011), and mixed cultures (Selembo et
al., 2009; Seifert et al., 2009).
Fermentative microorganisms exist enormously in natural habitat such as soil, sludge
and wastewater. These resources are used as possible source of inoculum for fermentative H
2

production (Sinha and Pandey, 2011; Selembo et al., 2009). Microbial mixed cultures are
important alternatives to pure microbial cultures in fermentation processes because it can be
easily operated under unsterile conditions (Rafra et al., 2013). The main challenges in mixed
microbial for fermentative process are; microbial growth rate is increased when dark
fermentation using open environmental source (without pretreatment) resulted low hydrogen
yield and the pathway is shifted from acidogenesis to methanogenis. These environmental
mixed cultures consume H
2
in two forms first one is consumption of NADH
2
and another one
is consumption of molecular hydrogen (Saady, 2013). Therefore, pretreatment of sludge is
one of the important features for selecting the efficient hydrogen producing microflora. The
pretreatment inoculum can control the methanogenesis and improve the activity of hydrogen
producers (Wang et al., 2011).
Pretreatment methods including heat, acid, alkali and ultrasonic treatment are widely
used for efficient fermentative hydrogen production (Cai et al., 2004; Ren et al., 2008; Wang
et al., 2011). Among these pretreatment methods, heat treatment process has been used to
create the constant inoculums for hydrogen production (Logan et al., 2002). The most
important issues for development of a fermentative hydrogen production process using mixed

275

culture require the large quantities of stable inoculums. Enrichment of mixed microbial
cultures gets considerable attention for stable inoculums in efficient hydrogen production
because they can get better reaction stability and efficiency of H
2
production (Hawkes et al.,
2002)
Hence, the efficient hydrogen production from glycerol by dairy sludge as mixed
cultures was investigated and different pretreatment methods including heat, acid and alkali
treatment methods were also studied to improve the fermentative hydrogen production. In
addition, effects of glycerol concentration and initial pH on hydrogen production were also
examined.
26.2.1 Pretreatment and enrichment of H
2
producing mixed culture
The sludge inoculum was obtained from local dairy industry, Tiruchirappalli, Tamil
Nadu, India. Prior to use the sludge as a inoculums was pretreated by three methods including
heat treatment (105
o
C for 30 min), acid treatment (pH 3, 1 N HCl, 24 h) and alkali treatment
(pH 11, 1 N NaOH, 24 h) to inactivate the hydrogen consuming bacteria and facilitating the
growth of mixed culture. Further the pH was adjusted to 6.5 by the addition of NaOH and
HCl for acid and alkali treated cultures.
The enrichment of mixed culture was carried out by following medium compositions
per liter; 10 g of glycerol 3.4 g of K
2
HPO
4
, 1.3 g of KH
2
PO
4
, 4,2g of (NH
4
) 2SO
4
, 0.2 g of
MgSO
4
, 20 mg of CaCl
2
and 5 mg FeSO
4
, according to Barbirato et al., (1995) The anaerobic
conditions were created by ushing the bottles with pure N
2
for 10 min. 48 hours grown
mixed culture was transferred to fresh growth medium and enriched mixed cultures were
maintained and preserved at 4
o
C for further use.
26.2.2 Experimental setup
Biohydrogen production experiments were conducted in a 250 mL screw cap bottle in
a batch mode. 200 ml of fermentation medium containing 6 g (30 g/L) of glycerol was
inoculated with 2 mL of the pretreated mixed culture. The pH of the growth medium was
adjusted in the range from pH 6.0 to pH 6.5. Bottles were ushed with nitrogen for 10 min to
generate anaerobic condition and placed in a magnetic stirrer at 150 rpm. All the fermentative
experiments were carried out in duplicates, and control experiments were conducted in
parallel using the dairy sludge without pretreatment. The optimization experiments were

276

carried out for heat treated mixed culture with different initial substrate concentration from 10
to 50g/L and pH values from 4.0 to 8.0.The pH was adjusted on addition of 1N NaOH or HCl.
The quantity of biogas produced in dark fermentation was measured from time to time
by water displacement method. The hydrogen, methane and carbon dioxide content were
calculated by gas chromatograph (GC, SHIMADZU GC-2014). GC was equipped with a
thermal conductivity detector (TCD) and stainless column packed with Porapak Q (80/100
mesh). The injection port, column oven and detector were operated at 40
o
C, 40
o
C, and 80
0
C
respectively. Nitrogen was used as carrier gas at a flow rate of 20 mL/min. The pH values of
the fermentation media were analyzed by a pH meter (Elico Model LI 615.)
26.3.1 Comparison of pretreatment methods
The batch experiments with dairy sludge pretreated by heat, acid and alkali were
investigated and the experiment without pretreated sludge was done as control for
comparison. As seen in the Figure 1, the cumulative hydrogen production of 300, 250 and 120
mL were heat, acid and alkali treated cultures, respectively and its corresponding hydrogen
content was 43, 38 and 29 %. On the other hand, untreated culture showed that the cumulative
hydrogen production and hydrogen content were 270 mL and 22% respectively. These
findings demonstrate that the relatively higher hydrogen production was obtained at heat
treated inoculums when compared to other pretreated cultures and control. Moreover, this
experiment indicated that the methanogenic activity in fermentative process was completely
inhibited for all pretreatment methods. This similar observation was also found by other
researchers (Wang et al., 2011). Recently, Rossi et al. (2011) reported that the heat
pretreatment was more efficient for biohydrogen production using mixed culture from
glycerol and also maximal substrate degradation were obtained than the other pretreatment
methods.
26.3.2 E ect of glycerol concentration on hydrogen production using heat treated mixed
culture
Biohydrogen production from glycerol by heat treated mixed culture was evaluated in
this study to find out the enhancement efficiency. To evaluate the fermentative hydrogen
production, glycerol concentration was varied in the range from 10 to 50 g/L (Fig. 2). The

277

experimental results show that the hydrogen production was increased when glycerol
concentration was differed from 10 to 30g/L. Further the hydrogen production was decreased
at above 30 g/L. The possible reason for low hydrogen production may be due to the
inhibition of higher substrate concentration or fermentative end product inhibition of this
process (O-Thong et al., 2008). The maximum cumulative H
2
production of 510 ml and
hydrogen content of 42% were obtained at 30 g/L of glycerol concentration. However our
data showed that the optimum concentration of glycerol was important for efficient
fermentative hydrogen production. Similar result was observed by Sittijunda and Reungsang,
(2012). They reported that the maximum hydrogen production was found to be in the range of
25-50 g/L of glycerol concentration. Seifert et al. (2009) also reported that the hydrogen
production increased from 0.345 to 0.715 l H
2
/L when the glycerol concentration was
increased from 5 to 30 g/L.

Fig. 1- Effect of Pretreatment methods on hydrogen production

Fig 2- Effect of glycerol concentration on hydrogen production

278

26.3.3 Effect of pH on hydrogen production using heat treated mixed culture
The eect of initial pH (4.0 to 8.0) on fermentative hydrogen production from glycerol
by heat pretreated mixed culture was studied. pH is an key factor for fermentative H
2

production, Kapdan and Kargi, (2006) reported that the pH in production medium influenced
the hydrogen production yield, composition of biogas content and specific hydrogen
production rate. Also, pH affected the hydrogenase activity as well as the fermentative
hydrogen producing pathway (Sinha and Pandey, 2011). From Figure 3, the experimental
result indicated that the low hydrogen production was obtained at initial pH 4.0 and hence the
lower pH was not suitable for this fermentative process. The possible reasons for lower H
2
production in fermentation were reported by (Mohan et al., 2007). They reported that the pH
values below 5.0 inhibited the acidogenic metabolism and shifting the metabolic pathway to
solventogenesis which led to inhibition of H
2
production. The cumulative hydrogen
production was increased at the initial pH from 4.0 to 6.0. Further, the hydrogen production
was decreased as the initial pH was increased from 6.0 to 8.0. The same effect was found in
hydrogen content at all the initial pH. The present study confirmed that the optimum initial
pH for efficient fermentative H
2
production was 6.0. Similar result was found by other
researchers. Venkata Mohan et al. (2007) studied effect of pH and substrate concentration
using heat-treated mixed culture and they found that the optimal pH 6.0 was suitable for
fermentative hydrogen production. Yossan et al. (2012) also reported that the maximum
hydrogen production by heat treated palm oil mill lagoon sludge at pH 6.0.

Fig 3- Effect of pH on heat treated mixed culture


279

26.4 Conclusions
The efficient hydrogen production from glycerol using heat treated inoculums under
optimum condition was investigated. The experimental results indicated that the pretreatment
methods were effectively suppressed methanogenic activity and only the hydrogen and carbon
dioxide was present in evolved gas. Among the pretreatment methods, heat pretreatment was
the most effective method for hydrogen production from glycerol. The maximum hydrogen
production of 300 mL and hydrogen content of 42% were achieved by heat treated inoculums
experiment. In addition, the fermentative H
2
production using heat treated dairy sludge
showed that the optimal condition was 30 g/L of glycerol at initial pH 6.0 for efficient
hydrogen production.

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Part IV
Chemical Conversion


283

CHAPTER 27
ISOLATION AND CHARACTERIZATION OF FRESHWATER
MICROALGAE SCENEDESMUS FROM CONTAMINATED
FIELD SAMPLES FOR BIOENERGY GENERATION
Mayur M. Phukan and B. K. Konwar

Abstract
A green alga (chlorophyceae), representative of the microalgal genera Scenedesmus was
isolated from a eutrophic water body (2640'10"N 9247'52"E) near Tezpur University,
Assam-784028, India. A mixture of antibiotics (ampicillin, kanamycin and chloramphenicol)
and antifungals (bavistin and indofil) were used to obtain a unialgal culture from field
samples with substantial bacterial and fungal contamination. The purified microalga was
cultured in liquid media and the respective microalgal biomass was examined for its physical
and chemical characteristics using SEM, CHN, TGDTA and FTIR spectroscopy. The
microalgal biomass was also studied by FTIR spectroscopy, when grown under nitrogen
limited conditions. Scenedesmus biomass showed appreciable energy (15.18 MJ/Kg) and
carbohydrate (29%) content whereas low ash (7.3%) and a shorter thermal degradation
profile. The study high lightens the use of chemical agents of control (chemotherapeutic
agents) for obtaining unialgal cultures from contaminated field samples and also additionally
suggests the importance of microalgae Scenedesmus as a second generation bioenergy
feedstock.
Keywords: Scenedesmus, Algae, Biomass, Feedstock, FTIR.
27.1 Introduction
Escalating petroleum prices, phenomenal upsurge in global energy demands and
greater concerns for environmental protection has high lightened the importance of renewable
energy resources for the energy sector. In addition, to the sustainable favorability of
renewable energy sources, they are in general more evenly distributed over the surface of the
Earth than fossil fuels or uranium and may be exploited using less capital intensive
technologies (Naik et al., 2010). Bioenergy today lies in the forefront of renewable energy


284

research and can assist in anthropogenic endeavors for curbing down reliance on fossil fuels
which are at the brink of quick depletion.
Bio-energy (energy generated from biomass) is a feasible alternative in offering
potentially attractive solutions for addressing issues related to energy security, energy crisis
and sustainable development. The total availability of biomass in the world is 220 billion
oven-dry ton (odt) per year or 4500 EJ (10
18
J) (Naik et al., 2010). Biomass has the potential
to become one of the most important global primary energy sources during the next century,
and moreover modern bioenergy systems are suggested to be important contributors to future
sustainable energy systems and sustainable development in industrialized and developing
nations (Berndesa et al., 2005). Bioenergy should play a crucial role in achieving targets to
replace petro-fuels with a viable alternative, and in reducing long-term carbon dioxide
emissions, provided environmental and economic sustainability are considered carefully
(Yuan et al., 2008). With a view to sincerely realize the importance of bioenergy and its future
implications in issues related to energy and environment, research in microalgae, cannot be
overruled.
Microalgae are sunlight driven biochemical factories which convert sunlight into food,
feed and high value bioactives (Akkerman et al., 2002; Chisti, 2007; Metzer and Largeau,
2005). They are a potential source of renewable energy, and can be converted into energy
such as biofuel oil and gas (Amin, 2009). Oleaginous microalgae are a source of renewable,
lipid-rich biomass that serves as feedstock for an emerging biofuel industry (Holguin and
Schaub, 2013). The concept of fuel production from microalgae is not new (Nagle and
Lemke, 1990; Sawayama et al., 1998), but it is now being taken seriously owing to
considerable hikes in price of petroleum and, more importantly, the emerging concern about
global warming associated with burning of fossil fuels (Gavrilescu and Chisti, 2005).
Additionally, microalgal research has also gained significant impetus as an elixir for the long
identified conundrum of Food Vs Fuel debate. They can serve as vessels for photosynthetic
biorefineries, which are believed to be the future solution for sustainable production of
various bioproducts (Xie et al., 2013). Microalgae usually have higher photosynthetic
efficiency, higher biomass production and faster growth rate, compared to lignocellulosic
biomass (Zou et al., 2009). In addition, microalgae can grow in both fresh water and saline
environments, can be cultivated on a large scale and do not require the use of agriculturally
productive or environmentally sensitive land (Ross et al., 2010). Microalgae in possession of

285

all these promising attributes have global prospects for the bioenergy sector and are likely to
foster sustainable economic development.
The present investigation aimed at isolation and purification of microalgae from
contaminated field samples via the agency of chemical agents, and subsequent investigation
of the respective microalgal biomass as a bioenergy feedstock.
27.2.1 Micro-organism and growth medium
Replicate water samples (50mL) were collected from a eutrophic water body
(2640'10"N 9247'52"E) near Tezpur University, Assam-784028, India. Compound
microscopic analysis of the water samples revealed the presence of representative genera of
the microalgal species Scenedesmus and Spirogyra. But however, the later was not taken into
consideration for the present study due to loss of sample purity. The microalgae
(Scenedesmus) were subjected to purification by serial dilution followed by plating and
quadrant streaking. The individual colonies were isolated and inoculated into liquid medium.
The purity of the culture was established by repeated streaking and routine microscopic
examination. Modified BBM media with the following compositions (per liter): NaNO
3
(400
mg), K
2
HPO
4
(80 mg), KH
2
PO
4
(30 mg), CaCl
2
.2H
2
0 (20 mg), MgSO
4
.7H
2
O (80 mg), NaCl
(25 mg), FeSO
4
(1 mg) and EDTA (45 mg) was used as the growth media (both solid and
liquid culture) for the microalgae.
27.2.2 Antibiotics and Antifungal
Three antibiotics viz. kanamycin, ampicillin (50 mg/ml, stock) and chloramphenicol
(10mg/ml, stock) was added at a volume of 1l/ml of the culture media to ward of bacterial
contamination. Prior to serial dilution the field sample of microalgae was subjected to gravity
precipitation. The upper water layer was decanted and the microalgal biomass was subjected
to three washings with distilled water via centrifugation at 3000 rpm for 15 min. The resultant
microalgal pellet was given three different treatments viz. incubation at ambient temperature
with 0.1% HgCl
2
(w/v, 10 min), 1% AgNO
3
(w/v, 10 min) and 1% bavistin (w/v, 30 min).
Additionally, Indofil M-45 (contact fungicide, Indofil Chemicals Company, Mumbai, India)
was added to the culture media of microalgae (0.5 l/ml of the culture media) from a stock
concentration of 1 mg/l. The antibiotics and antifungal (Indofil M-45) was added to the
autoclaved culture media at (~40C) inside a laminar flow cabinet.
27.2.3 Culture conditions

286

The micro algal cultures were carried out in 500ml conical flasks (200ml media) with
shaking at 100 rpm; at 282C, light intensity 1200 lux, and (16:8) light and dark cycle. A six
day old culture (exponential growth phase) was used as the inoculum at 10% (v/v) for all
subsequent experiments.
27.2.4 Growth evaluation
The growth of Scenedesmus sp. was monitored spectrophotometrically (CECIL 7400)
by reading the culture absorbance at 680nm.
27.2.5 Determination of Gross calorific value (GCV)
The Gross calorific value (GCV) of the biomass sample was calculated according to
Dulong formula (Huang et al., 2011):
Gross calorific value (MJ/kg) = 0.3383C + 1.442 (H- (O/8))
27.2.6 Determination of Net calorific value (NCV)
The NCV was calculated from the following equation as follows (Koppejan et al., 2008)
NCv = uCv [1
w
1uu
2.444 [
w
1uu
2.444 _
E
1uu
] 8.9S6
[1
w
1uu
; _
H[
Kg
, w. b. _
Where 2.444=Enthalpy difference between gaseous and liquid water at 25
0
C.
8.9S6 =
H
H
2
0
H
H
2
; i. e. the moleculai mass ielation between B
2
0 anu B
2

Where,
NCV= Net calorific value
GCV= Gross calorific value
h= Concentration of hydrogen in weight%
w= Moisture content of the fuel in weight%
27.2.7 CHN and Proximate analysis
The content of major elements viz. carbon, hydrogen and nitrogen was analyzed in a
CHN analyzer (Perkin Elmer, 2400 Series-II) and finally the oxygen content was calculated
by difference. The moisture, volatile matter and ash content of the dry algal biomass were

287

determined according to ASTM D 3173, ASTM D 3175 and ASTM D 3174 protocols. The
fixed carbon content was calculated by difference
27.2.8 Determination of cell contents
The anthrone method was used for determination of total carbohydrates (Vyas and
Kohli, 2002). Protein content was determined by the Folin Lowry method (Lowry et al.,
1951) whereas the total lipid content was determined by the method of Bligh & Dyer (Bligh
and Dyer, 1959)
27.2.9 Scanning electron microscopy (SEM)
The microalgal cells were harvested by centrifugation at 10,000 rpm for 15 min,
washed with PBS (Phosphate buffered saline) and fixed with 0.5% glutaraldehyde in 0.1M
cacodylate buffer. Dehydration was carried out in an acetone series with 30 min changes (30,
50, 70, 90 and 100%). The scanning electron micrographs were acquired using a JEOL JSM-
6390LV (Oxford Instrumentation Ltd.) model Scanning electron microscope. For SEM
analysis, the dehydrated microalgal biomass was sprinkled on the carbon tape and then coated
with 30 nm platinum coat using JOEL auto fine coater (model no. JFC-1600). The SEM was
operated at 15KV and under 1 Pascal pressure with the spot size fixed at 36.
27.2.10 FTIR analysis
For FTIR spectroscopy, briefly known weight of the dried algal biomass (1mg) was
taken in a mortar and mixed thoroughly with 2.5 mg of dry potassium bromide (KBr) using a
pestle. The IR spectra was recorded at room temperature (28C2C) in the mid infrared
range (4000-400 cm
-1
) using a PERKIN ELMER Spectrum 100 spectrometer.
27.2.11 Thermal analysis
For thermal analysis, microalgae were harvested by centrifugation at 10,000 rpm for
15 minutes. The pellet was washed twice with distilled water and then dried at 90C for 24
hours. The dried microalgal biomass was pulverized in a mortar to fine particles, and then
finally stored in a desiccator. Thermogravimetric analysis (TGA) was done to study the
degradation profile of the biomass sample. The algal biomass was subjected to
thermogravimetric analysis in nitrogen atmosphere at heating rate of 10C/min.
Approximately, 10 mg sample was heated at the preselected heating rate from ambient
temperature to 900C in a Pyris diamond TG/DT analyzer (PERKIN ELMER). High purity
nitrogen gas (99.99%) was fed at a constant flow rate of 50ml/min as an inert gas. The

288

continuous on-line records of weight loss and temperature were obtained to plot the TGA
curve and the derivative thermogravimetric analysis (DTG) curves.
27.3 Discussion
A microalga was isolated from a eutrophic water body (2640'10"N 9247'52"E) near
Tezpur University, Assam-784028, India. Compound microscopic analysis of the water
samples revealed the presence of representative genera two microalgal species viz.
Scenedesmus and Spirogyra, but however the second species was not taken into consideration
for the present investigation due to loss of sample purity. The microalga was purified using
standard microbiological techniques (serial dilution followed by plating and quadrant
streaking). Obtaining unialgal culture (a viable single species culture of algae, free from
undesirable contaminants) from contaminated field samples is the first major achievable
milestone in phycological research. Field samples might contain substantial bacterial and
fungal load. Bacteria generally produce small, limited colonies, and unialgal cultures can be
obtained if the plate has been properly streaked (Andersen and Kawachi, 2005). Fungus have
faster growth rates, they produce sporangia and spores that may hamper the microalgal
isolation processes, and moreover fungal contamination is generally worse than bacterial
contamination because fungi are harder to eliminate by physico-chemical methods, and the
fungus may also exhibit overgrowth over algae on prolonged incubation under suboptimal
conditions (Lorenz et al., 2005). Table 1 shows the treatment regimes for antibacterials and
antifungals to obtain unialgal culture of Scenedesmus sp.
Table 1- Treatment regimes for antibacterials and antifungals to obtain unialgal culture of
Scenedesmus sp

AB
1% AgNO
3
0.1%
HgCl
2
Bav Ind Bav +Ind
Amp # - BF HBMF +
Kan # - BF HBMF +
Cmp # - BF HBMF +
Amp + Kan # - MBHF MBMF +
Amp + Cmp # - MBHF MBMF +
Kan + Cmp # - MBHF MBMF +
Amp + Kan + Cmp # - HF MF U

AF

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Where, AB= Antibacterial, AF= Antifungal, Amp= Ampicillin, Kan= Kanamycin, Cmp=
Chloramphenicol Bav= Bavistin, Ind= Indofil ,-= No growth, += Mild bacterial growth, U=
Unialgal growth, BF= Heavy bacterial and mild fungal growth, #= No algal viability with
fungal growth, MBHF= Mild bacterial and heavy fungal growth, HF=Heavy fungal growth,
MF = Mild fungal growth, HBMF =Heavy bacterial and mild fungal growth, MBMF= Mild
bacterial and mild fungal growth.
As evident from the Table 1 its quiet clear that AgNO
3
and HgCl
2
are highly toxic for
the microalgae. The microalga was not resistant to incubation with 1% AgNO
3
and 0.1%
HgCl
2
at minimal exposure duration of 10 min, leading to the death of the inoculum itself.
Both the antifungals viz. bavistin (1% w/v, incubation for 30 min) and indofil (added at a
volume of 0.5l/ml of the culture media, from a stock concentration of 1mg/ l) when used
individually were only supportive in reducing the fungal load but fungal free culture was
obtained only with a combination of the two. The experimental results also indicate that
individual application of either of the antibiotics were not efficient enough in warding off
bacterial contamination. But however, as expected there was a progressive decline in the
intensity of contamination (visually obvious from plating results) with a combination strategy
of two antibiotics. A combination of three antibiotics (ampicillin, kanamycin and
chloramphenicol) and two antifungals (bavistin and indofil) was necessary to obtain unialgal
culture of the species under investigation.
Scanning electron micrographs were acquired to study the surface morphology and
size of the Scenedesmus cells. Generally Scenedesmus colonies have 4 to 8 cells. Fig 1 shows
the scanning electron micrograph of Scenedesmus spp at 2000x. Harsh processing steps
during sample preparation resulted in elimination of certain cells from the Scenedesmus
colony. The micrographs reveal that Scenedesmus cells are arranged in a flat plate. The cells
are around 2-2.5 m in diameter, with a thin cell wall. Smooth and "rough" cell walls were
observed in the Scenedesmus colony covered by an irregular network, but no spines were
visible. Boat shaped cavities were also observed on the upper side of the cells and apparently
they were of similar size.
The study aims to highlight fresh water Scenedesmus biomass as a potential bioenergy
feedstock. Significant physico-chemical properties of biomass must be taken into
consideration prior to selection of the biomass conversion technology. Biomass fuels have
significant differences with respect to chemical (volatile matter, ash content, fixed carbon)
and physical (moisture content) characteristics. These fuel characteristics are necessary for

290

choosing the proper biomass conversion technologies. Table 2 shows the biomass properties
of Scenedesmus sp. with regard to their average characteristic composition. The moisture
content of biomass may differ significantly, depending on the biomass type and its storage.
Moisture content is of great importance with regard to selection of biomass conversion
technology. Low moisture content in biomass is preferable since biomass fuels with elevated
moisture levels burns less readily and also generates less heat energy per unit mass of the
biomass being burnt. Generally biomass fuels with low moisture content are more suited for
thermal conversion technology while those with high moisture content are more suited for
biochemical processes such as fermentation conversion (Mckendry, 2002). On this basis,
Scenedesmus sp with a moisture content of 6.4% seems to be a potential candidate for
thermochemical conversion. Scenedesmus biomass had an appreciable quantum of
carbohydrate (29%). A high percentage of carbohydrate in the biomass is suggestive of its
prospective candidature for biochemical conversion via fermentation for bio-alcohol
production (a part of planned future research activity). Scenedesmus biomass had a lipid
content of 15% which is appreciable from biodiesel point of view. The coupling of bio-
alcohol production with biodiesel production may be suggestive in reducing the economics of
feedstock utility.

Fig 1: Scanning electron micrograph of Scenedesmus spp at 2000x.
In this investigation, ash value was determined using ASTM D 3174 protocol. The ash
content was found to be 7.3% and volatile matter content was 72.14%. The high amount of
volatile matter in Scenedesmus sp biomass will strongly influence its combustion behavior
and thermal decomposition profile. The fixed carbon content was 14.16%. The content of
major elements viz. carbon, hydrogen, oxygen and nitrogen in the microalgal biomass was
41.75, 6.36, 44.98 and 6.91% respectively. The empirical formula of the algal biomass is

291

C
7.08
H
12.97
NO
5.73.
The H/C and O/C molar ratios (on an ash free dry basis) were calculated
from elemental composition as 1.83 and 0.8 respectively. The high nitrogen content in the
biomass sample is not good from environmental point of view, and moreover high nitrogen
content in addition to high ash content (7.3%) are expected to reduce the hydrocarbon yields
during thermochemical conversion (Sanderson, 1996). The Gross calorific value (GCV) and
Net calorific value (NCV) for Scenedesmus sp biomass was 15.18 MJ/Kg and 12.69 MJ/Kg
respectively. The higher proportion of hydrogen and oxygen compared with carbon reduces
the energy value of biomass due to lower energy contained in carbonoxygen and carbon-
hydrogen bonds than in carbon-carbon bonds (Mckendry, 2002). The appreciable GCV
content in the microalgal biomass indicates that it can be a prospective biomass feedstock for
bioenergy generation.
Table 2: Properties of Scenedesmus biomass
Properties of microalgal biomass Properties Scenedesmus spp

Gross calorific value
Net calorific value
15.18 MJ/Kg
12.69 MJ/kg
Empirical formulae C
7.08
H
12.97
NO
5.71

H/C molar ratio 1.83
O/C molar ratio 0.8
Elemental analysis (wt%)
Carbon 41.75
Hydrogen 6.36
Nitrogen 6.91
Oxygen 44.98
Proximate analysis (wt%)
Moisture content 6.4
Ash content 7.3
Volatile matter 72.14
Fixed carbon 14.16
Biochemical analysis
Total carbohydrates 290.28
Protein content 410.4
Lipid content 150.69

The FTIR spectrum of the algal biomass and its nitrogen deficient analogue is shown
in Fig 2. The prominent spectral assignments in Scenedesmus biomass are 3544 cm
-1
(O-H
stretching in carboxyl functions), 2380 cm
-1
(-C-N stretch), 1643 cm
-1
(C=O stretching
vibrations of peptide bond) and 1431 cm
-1
[CH
3
(bend) or -C-O-H bending]. CH
2
stretching
vibrations in the region of 3100 - 2800 cm
-1
implies the presence of lipid. Three bands were of
particular interest in the nitrogen deficient biomass viz. 2929 and 2856 and 1740 cm
-1


292

respectively. The band at 1740 cm
-1
is associated with C=O of ester groups (mainly from fatty
acids and lipids). The most significant vibrations in the spectra of lipid are CH
2
stretching
vibrations which give rise to bands in the region of 3100-2800 cm
-1
(Stuart, 2004). This
increase in the intensity of absorption in the range of 3100-2800 cm
-1
is indicative of increase
in lipid quantum.

Fig 2: FTIR spectra of Scenedesmus biomass and its nitrogen deficient analogue
Thermogravimetric analysis was done in order to study the degradation profile of the
microalgal biomass. The thermal degradation products of the algal biomass consist of
moisture, volatiles and char.
As shown in Fig. 2 the TG-DTG profile of Scenedesmus sp biomass reveals an initial
weight loss between ambient temperature and about 130C for 10C/min .This could possibly
be explained by the elimination of physically absorbed water in the sample and/or
elimination of external or superficial water bounded by surface tension. This was followed by
continuous decrease in sample weight (region of main degradation) which ended by
approximately 460-470C. The phase is characterized by devolatization (main pyrolysis
reaction) where most of the volatile matter evolved. This zone (130-470C) has been referred
to as the zone of active pyrolysis. A very slow loss of weight occurred until 900C which

293

indicates that there was further reaction involving char. The TG-DTG profile reveals that the
initial weight loss temperature, weight loss climax point, and final end temperature for
microalgal biomass pyrolysis for 10C/min are 130, 312 and 470C respectively.

27.4 Conclusion
With scientific momentum in bioenergy research gearing up for reasons connected
with energy security, energy crisis and environmental deterioration numerous biomass sources
are being investigated as potential source for biofuel production. Research endeavors in
microalgae is no exception and as evident from the present study, it can be concluded that the
characterization of Scenedesmus biomass under the aegis of bioenergy research ensures that it
can be used as a renewable feedstock for both biochemical and thermo-chemical conversion
and may serve the exaggerating need for second generation biofuels. Scenedesmus offers
potential candidature for bioenergy generation with inexpensive nutrient regime for culture,
low ash content (5.93%), short thermal degradation profile, high energy (15.18 MJ/Kg) and
carbohydrate (29%). Scenedesmus can be efficiently harnessed as a fresh water bio-based
feedstock for biomass conversion thereby warranting future research endeavors. However, the
investigation of Scenedesmus biomass for liquid fuel (biodiesel) production is in progress.

294

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CHAPTER 28
PROSPECTS OF BIODIESEL PRODUCTION FROM NON-
EDIBLE OIL SEEDS OF NORTH EAST INDIA: A REVIEW
Debashis Sut, Rupam Kataki

Abstract
Biodiesel, an alternative diesel fuel derived from vegetable oils, animal fats or used frying
oils, is gaining increased attention due to the twin crisis of fossil fuel depletion and
environmental degradation. India, one of the fastest growing economies in the world is
becoming increasingly dependent on fossil fuel for its developmental needs. However, it is
necessary to supplement petro-based fuels with resources that are available locally for
production of liquid bio-fuel from the energy security point of view. NE region of India,
particularly Assam (24.30N and 28.10N latitude and 89.50E and 96.10E longitude) and
Arunachal Pradesh (26.28N and 29.30N latitude and 91.20E and 97.30E longitude) falls
in one of the richest biodiversity zones of the country. In the forests of Assam and Arunachal
Pradesh, a large variety of non-edible oil seed bearing trees and shrub species are available,
which can be utilised for production of biodiesel. In this paper, an attempt has been made to
review some of the non-edible oils found in Assam and Arunachal Pradesh viz. Nahar (Mesua
ferrea), Yellow oleander (Thevetia peruviana), Jatropha (Jatropha carcus), Koroch
(Pongamia glabra) etc. as feedstocks for biodiesel production. Further, some promising non-
edible oil bearing tree species available in the forests of NE India are discussed for their
potential as a source of feedstock for biodiesel production.
Keywords: Non-edible oil, Biodiesel, Methyl ester, NE region of India.
28.1 Introduction
The declining fossil fuel reserves and the growing environmental concerns regarding
the green house gas emission and global warming have made renewable energy as an
alternative energy source for the future. During the past few decades due to the growing
human population and industrialization, worldwide petroleum consumption has increased,
resulting in depletion of fossil fuel reserves and increase in petroleum price. On the other


297

hand, combustion of fossil fuels contributes to emissions of greenhouse gases, which
lead to pollution and global warming. About 98% of carbon emissions result from fossil
fuel combustion [Balat, 2011]. The climate change and energy security issues have gained
much higher priorities in recent times and the search for sustainable source of energy resulted
in biodiesel production as an alternative source of energy. Biodiesel, defined as the mono-
alkyl esters of long chain fatty acids derived from vegetable oils or animal fats and alcohol
with or without a catalyst, is one of the promising alternative fuel for diesel engine. It is
renewable, biodegradable, readily available, portable, non-toxic and ecofriendly fuel [Singh
and Singh, 2010]. The potential feed stocks for biodiesel production can be classified into
different categories. They are first generation feedstocks or edible vegetable oils , second
generation feedstocks or non-edible vegetable oils, third generation feedstock or microalgae
and others which include waste cooking oils[Atabani et al.,2013]. The price of biodiesel
depends mainly on the cost of feedstocks, which makes 7095% of the total biodiesel
cost [Karmakar et al.,2010]. At present edible oils are the main resources for biodiesel
production (more than 95%) [Xue, 2013]. However, there are many difficulties in using
edible oils as feedstocks for biodiesel production. Recently the use of edible vegetable
oils has been of great concern as it results in food versus fuel crisis that might cause food
shortage in the developing countries and also environmental problems by utilizing much of
the available land for cultivation. It can create serious ecological imbalances as the countries
are cutting down forests for plantation purposes resulting in deforestation and damage to the
wildlife. Therefore, focus is on non-edible resources, which are not suitable for human
nutrition and could grow in barren land. Microalgae have become the latest potential
feedstock for biodiesel production. They are very economical in comparison to edible oils. It
was reported that microalgae has the highest oil yield compared to other oil crops [Mata et al.,
2010]. Microalgae with high oil content have the potential to produce 25 times higher oil
yield than the traditional biodiesel crops, such as palm oil [Ahmad et al., 2011].Other
feedstocks for biodiesel production include waste cooking oil, which can be considered as a
promising option with relatively lower price than fresh vegetable oil. [Demirbas, 2009] The
feedstocks for biodiesel production should be as diversified as possible, because relying on
certain sources could bring harmful influence in the long run depending on geographical
locations in the world. Non-edible vegetable oils have gain more attention as a promising
feedstock for the sustainable production of biodiesel. The use of non-edible oils can
improve the economy of biodiesel production and its commercialisation at the industrial
scale as they are cheaper than the edible oil. Non-edible oils are unsuitable for human

298

consumption due to the presence of toxic compounds [Ahmad et al., 2011]. There are
numerous oil bearing plants in nature all over the world. Besides, non-edible oil plants
can be easily cultivated in unproductive lands with much lower costs than the edible
oil crops cultivation [Gui et al., 2008]. Moreover, growing of these plants reduces CO2
concentration in the atmosphere [Karmakar et al., 2010]. However, the main drawback is
high free fatty acids (FFAs) content which increases the biodiesel production cost
[Bankovic-Ilic , 2012].
India is one of the fastest growing economies in the world. The Indian economy has
experienced unprecedented economic growth over the last decade. India with a population of
more than 1210 million is growing at an annual rate of 1.76% and has resulted in more energy
use. India is the third largest consumer of energy in the world after USA and China
[WEO,2012]. India like many other developing countries is a net importer of energy. The
consumption as well as import of crude oil has increased year by year. High dependence on
imported energy is expensive with the ever increasing energy prices; it also impinges
adversely on energy security. To reduce the countrys dependency on conventional fuel, the
government of India has taken initiative for the growth and development of renewable energy.
Government of India has announced its national biofuel policy in 2008 to meet 20% blending
of bio-diesel and bio-ethanol by 2017 [Anon, 2008]. Due to insufficient production of edible
vegetable oils in India, the country has emphasized on non-edible feedstocks for production of
biodiesel. According to a report of Greenpeace, renewable energy can successfully meet over
35% of power demand in India by 2030 [Kumar and Sharma, 2011].
In India where fossil fuel resources are limited, the production and use of biodiesel
from vegetable oils and fats may be a viable solution to supplement the growing demand of
fuel for diesel engine in the country. However while selecting the feedstocks for biodiesel
production, special consideration should be given to utilize the local non-edible oil bearing
tree species. Various oil bearing tree and shrub species are found in the forests of India
especially north-east India. This region is famous for its high ethnic and biological
biodiversity. After the Andaman and Nicobar Islands and the Western Ghats, it forms a range
of tropical forests mainly the species-rich tropical rain forests. North eastern region is also
considered as a significant part of the Indo-Myanmar biodiversity hotspot and presently
accepted as one of the 25 global biodiversity hotspots [Chakraborty et al.,2012]. Among the
north eastern states, a large variety of oil bearing trees and shrub species are found in the
forests of Assam (24.30N and 28.10N latitude and 89.50E and 96.10E longitude) and

299

Arunachal Pradesh (26.28N and 29.30N latitude and 91.20E and 97.30E longitude). The
seeds of some of these trees are generally not used for any useful purposes. In this present
paper, some of these non-edible oils found particularly in Assam and Arunachal Pradesh are
presented as potential feedstocks for biodiesel production that can replace the current
dependence on the edible oil resources. Various aspects such as overview of non-edible oil
resources, fatty acid composition and properties and characteristics of biodiesel associated
with these feedstocks have been reviewed from recent publications.
28.2 Non-edible vegetable oils resources
Non-edible vegetable oils are not appropriate for human consumption due to the
existence of some toxic elements in the oils. The selection of non-edible vegetable oils as
feedstocks for biodiesel production requires reviewing the existing works. From the reviews
of biodiesel production from various feedstocks, it is found that non-edible oils bear certain
advantages over edible oils. Biodiesel production from non-edible oils can overcome the
problems of food shortage and also economic as well as environmental and issues related to
edible oils [Gui et al., 2008]. In addition, non-edible oil crops can be grown in barren lands,
degraded forests, fallow lands and also along railways, roads and irrigation canals. Apart from
providing energy security, biodiesel production from non-edible oils could be converted into a
major poverty mitigation program for the rural poor and improvement of the rural non-farm
sector. Non-edible feedstocks for biodiesel production can be considered as sustainable and
alternative fuels [Sastry, 2009]. In the following section, a brief account of different types of
non-edible plant oils is provided.
28.2.1 Jatropha curcas L. [Family: Euphorbiaceae]
J. Curcas popularly known as Ratanjayot is considered as one of the most promising
oil source for biodiesel production in Central and South America, Africa, India and South
East Asia. Jatropha can grow under a wide variety of climatic conditions like arid and semi-
arid conditions. J. Curcas is a multipurpose, deciduous, drought resistant plant of 57 m
height, which belongs to the family Euphorbiaceae [Pant et al. 2006]. J. Curcas can be used
in a variety of purposes including fuel. Various parts of the plant have medicinal values. J.
curcas oil can be used as lubricant, in cosmetics industry, for making soap and lighting, apart
from biodiesel production. The oil content is about 4060% in seeds and 4658% in kernels
and varies depending on the types of species, climatic conditions and the altitude where it is
grown[Kumar and Sharma,2011]. The yield of Jatropha seed ranges from 0.1 to 15 tonnes ha
-1


300

yr
-1
in different regions [Daey et al., 2007]. J. curcas plant can thrive under adverse
conditions. It requires very little irrigation and grows in all types of soils (from coastline to
hill slopes) with a healthy life cycle of 3050 years [Agarwal and Agarwal, 2007]. The plant
has abundant vegetative growth but the number of seeds produced per plant is very low
[Debnath and Verma, 2008]. In India, with an annual production of about 15,000 t, Jatropha is
one of the major feedstock for biodiesel production [Jain and Sharma, 2010]. J. curcas has
numerous advantages as a feedstock for biodiesel. It can be considered as a potential
sustainable biofuel as it is non-edible and can be grown on marginal lands and will not
compete with food crops.
28.2.2 Pongamia glabra [Family: Fabaceae]
Pongamia glabra is a medium-sized tree of 1215 m height, with a short curved trunk
and spreading or drooping branches and native to the Asian subcontinent [Karmakar et
al.,2010]. The natural distribution of koroch is along coasts and river banks in India and
Myanmar. Pongamia can grow in humid as well as subtropical environments and thrives in
areas having an annual rainfall ranging from 500 to 2500 mm [Sharma et al., 2008]. The tree
can produce seeds with a significant oil content of 35% by weight [Ahmad et al., 2009]. The
toxic substances present in the oil prevent it from using as cooking oil. Annual production of
pongamia oil in India is 55,000 t [Jain and Sharma,2010], of which only 6% is
currently used [Naik et al., 2008]. It can be cultivated to improve the soil quality, and
the exhausted land can be reused for the agricultural purpose. Koroch is a nitrogen-
fixing tree producing seeds with a significant oil content. It is reported that a single tree
can yield 990 kg seeds, which gives a yield potential of 9009000 kg seed/ha (with the
assumption of 100 trees/ha)[Karmee and Chadha, 2005] The seed oil content ranges between
30 and 40 wt% [Atabani et al.,2013]. The different parts of the plants like leaves, roots and
flowers are reported to possess medicinal properties (Kumar and Sharma, 2011).
28.2.3 Sapindus mukorossi [Family: Sapindaceae]
Sapindus mukorossi (Soapnut) is normally found in tropical and subtropical climate
areas including Asia, America and Europe. The plant grows very well in deep loamy soils and
leached soils so cultivation of it in such soils prevents the soil from erosion. The soapnut tree
can be used in numerous applications, for example in oil and sugar presses, agricultural
equipment and construction of rural building etc. Soapnut has a lot of uses from medicinal
treatments to soap and surfactant. The fruit shells can be used as natural laundry detergents
for washing fabrics, bathing and also as traditional medicines. It has a great potential as a

301

natural surfactant for washing the soils contaminant with organic compounds [Kumar and
Sharma, 2011]. Oil content of soapnut seeds is 23% of which 92% is triglycerides [Atabani et
al, 2013]. The oil from soapnut can be considered as promising feedstock for biodiesel
production.
28.2.4 Melia Azedarach [Family: Meliaceae]
M. azedarach is a deciduous tree with a height of 7- 12 m. It is native to India,
southern China, and Australia and belongs to the family Meliaceae. The oil content of dried
kernel is about 10 wt % [Sharma and Singh, 2009]. The fatty acid profile shows that the oil
contains a high percentage of unsaturated fatty acids such as oleic (21.8 wt.%) and linoleic
(64.1 wt.%) acids. The saturated fatty acids present are palmitic (10.1 wt.%) and stearic (3.5
wt.%) acids [Stavarache et al.,2008].
28.2.5 Thevetia peruviana [Family: Apocynaceae]
T. peruviana is an evergreen perennial shrub with a height of 4.56 m. It is native to
tropical America, particularly Mexico, Brazil and West Indies and naturalized in tropical
regions of the world.The leaves are deep green with linear sword shape and flowers are
funnel shaped (yellow, white or pinkish yellow in colour). The tree belongs to Apocynaceae
family and has various common names like yellow oleander, gum bush, bush milk, exile tree
in India, cabalonga in Puerto Rico, ahana in Guyana, olomi ojo by Yorubas in Nigeria
[Atabani et al, 2013]. The plant starts flowering after one and a half year and blooms thrice a
year [Balusamy and Manrappan, 2007]. Depending on the rainfall and plant age it produces
about 400800 fruits per year [Ibiyemi et al., 1995]. Almost all parts of the plant are
poisonous and bear white latex. The kernel has very high oil content (67%) [Azam et al.,
2005] and the de-oil cake has protein content of 37% [Ibiyemi et al., 2002]. The plant is
generally used as an ornamental or fencing or wasteland plant. It has annual seed yield of
52.5 t h
-1
and around 1750 L of oil can be obtained from a hectare of waste land [Balusamy
and Manrappan, 2007]. The oil has a very good thermal stability and thus has a potential for
various uses like biodiesel production [Ibiyemi et al., 2002]
28.2.6 Michelia champaca [Family: Magnoliaceae]
M. champaca belongs to Magnoliaceae plant family. It is a tall handsome evergreen
tree with straight stem and smooth brown bark. It is found in Eastern Himalayas, Burma,
China, Assam, Western Ghats and throughout India. The fruits are dark brown. Its seeds have
a oil content of 45% [Atabani et al, 2013]. The perfumed oil obtained from M. champaca

302

flowers has useful application in pharmaceutical industries as well as in perfumery industries
[Hosamani et al., 2009].
28.2.7 Terminalia belerica [Family: Combretaceae]
Terminalia (Terminalia belerica Roxb.) is a large deciduous tree that grows up to a
height of 40 m and belongs to the family of Combretaceae. It is found in the northeastern
region of India. The plant can tolerate moderate draught and heavy rainfall. The tree bears
flowers that are greenish-white or greenish-yellow in colour. The plant starts bearing fruits
from about 10 years of age and survives for more than 80 years. Fruits become mature in
November and harvesting could be continued up to January. The fruit consists of 9% kernel,
30% mesocarp and 61% endocarp. The oil content of the kernel is about 43% [Chakraborty et
al., 2009]. The mesocarp portion of the fruit has medicinal value and used for commercial
purposes. It is regularly used as traditional medicine in India in the treatment of various
diseases like dyspepsia, chronic diarrhea, dysentery etc. Dried fruit pulp is an ingredient of
Triphala (Indian Ayurvedic medicine) which is used for the treatment of intestinal disorders,
liver disorders, pancreatic cancer and many other diseases. The oil could be a potential
feedstock for biodiesel production and terminalia planting would help to curb the
deforestation in the northeastern region of India [Basumatary, 2012].
28.2.8 Mesua ferrea [Family: Clusiaceae]
Nahar (Mesua ferrea L.) is native to Sri Lanka but also grown in Assam,
southern Nepal, Indochina, and the Malay Peninsula [Sarma, 2006]. It is a moderate sized
tree known as Indian ironwood or Indian rose chestnut and belongs to Clusiaceae family.
Nahar trees can be planted on the wasteland, roadside and in the forests. The tree starts
flowering between April and July and fruits between October and November. The leaves are
linear with a length of 3-6.5 inches and white flowers from the uppermost leaf axils. Diameter
of stem at the base is about one foot only and height about 40 feet. The timber from the Nahar
tree is one of the hardest and heaviest. The oil content of the seeds is very high (70-75%) and
is non-edible [Konwer et al. 1989]. Thus it is a promising feedstock for biodiesel production
and has immense scope in NE India [Basumatary, 2012].
28.3 Fatty acid profiles of the biodiesel
The fatty acid composition of the oil is a significant property to determine its
suitability as a raw material for biodiesel production. Generally the type and percentage
of fatty acid composition depends on the plant species and their growth conditions. Biodiesel

303

properties are determined by the fatty acid composition of the oil [Gui et al., 2008]. The most
common fatty acids present in the feedstocks are C16 and C18 acids. Table 1 shows the fatty
acid composition of some of the potential non-edible oils that can be used for production of
biodiesel. Table shows that palmitic (C16:0), stearic (C18:0), oleic (C18:1) and Linoleic acid
(C18:2) are the major fatty acids present in the feedstocks.
Table 1 Fatty acid profiles of the biodiesel

28.4 Properties of the biodiesel
The properties of biodiesel depend on the fatty acid composition of the feedstock
which is used for biodiesel production [Gui et al., 2008]. The quality of biodiesel is most
important for engine part of view and various standards have been specified to check the
quality. The ASTM D6751and EN 14214 are the two most commonly used standards for
determining the quality of biodiesel. In general, biodiesel standards help in identifying the
parameters that a pure biodiesel must have, before being used as a fuel for diesel engines.
Some main properties of biodiesel produced from non-edible oils are listed in Table 2. Most
of the properties of biodiesel meet the biodiesel standards ASTM D6751 and EN 14214.

Non-edible oil
Feedstock
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 Reference
Mesua ferrea 15.9 - 9.5 52.3 22.3 - - -
[Konwer et al.,
1989]
Jatropha curcas 15.6 - 9.7 40.8 32.1 - 0.4 -
[Kumar and
Sharma, 2011]
Pongamia
glabra
11.30 - 9.80 45.25 24.75 2.90 1.75 3.20
[Basumatary,20
12]
Thavetia
peruviana
14.1 0.14 3.19 58.10 19.49 .088 1.58 0.1
[Oseni et
al.,2012]
Terminalia
belerica
32.8 0.5 6.4 31.3 28.8 - 0.3 -
[Chakraborty et
al.,2009]
Sapindus
mukorossi
4.67 0.37 1.45 52.63 4.73 1.94 7.02 1.45
[Jadon et al.,
2012]
Melia
azedarach
8.1 1.5 1.2 20.8 67.7 - - -
[ Kumar and
Sharma, 2011]
Atabani et al,
2013
Michelia
champaca
20.7 6.9 2.5 - - 42.5 - -
[Singh and
Singh,2010]
Atabani et al,
2013

304

Table 2 Properties of the biodiesel

28.5 Conclusion
North East India is considered as one of the richest biodiversity zones in India. In this
region particularly in Assam and Arunachal Pradesh, various oil seed bearing tree and shrub
species are available which can be used for biodiesel production. Considering availability,
abundance, oil content and fuel properties, these feedstocks possess a huge potential for
biodiesel production. These species will be a sustainable alternative to conventional diesel
without affecting the world food crisis.

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-1
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308

CHAPTER 29
A CRITICAL REVIEW OF ENZYMATIC
TRANSESTERIFICATION: A SUSTAINABLE TECHNOLOGY
FOR BIODIESEL PRODUCTION
Neetu Singh, M.K. Jha, A.K. Sarma

Abstract
Fossil fuels are exhaustible. Therefore transition to an economy that runs on sustainable
energy sources is both necessary and inevitable. An approach that focuses on high penetration
of renewables will provide a more effective path towards sustainable energy future. For that
liquid fuels mainly biodiesel is considered as the best sustainable energy source which is
renewable, ecofriendly. It can be produced by chemically or enzymatically via
transesterification reaction. Researchers are trying more efforts to make enzymatically
catalyzed biodiesel production process more feasible as it has certain advantages over
chemically catalyzed process. Like this process is less energy intensive, allows easy and pure
recovery of glycerol and major advantage include the transesterification of glycerides
containing high FFA contents. Although, it has some disadvantages also like high cost of
enzyme but it can be easily solvent by protein or genetic engineering and recombinant DNA
technology methods. These methods help in reducing the high cost of enzymes and also help
in making biodiesel production process more feasible for industrial purpose.
29.1 Introduction
World energy forum predicted that the fossil oil will be exhausted in less than ten
decades. And the main reason for the reduction of the energy resources is rapid population
increase and industrialization. According to the EIAs report, the world's energy demands are
set to soar in the next 21 years, with developing countries leading the way. The report says
that energy demand will rise 44% by 2030 with 70% of demand increase coming from
developing countries; oil will go to $110 per barrel in 2015 and $130 per barrel in 2030.
World renewable energy use for electricity goes from 19% in 2015 to 21% in 2030; CO
2

emissions will rise 39% unless new policies like cap and trade are implemented; world net
electricity generation increases by 77% from 18 trillion kWh (kilowatt hours) in 2006 to 23.2



309

trillion KWh in 2015 and 31.8 kWh in 2030; world coal consumption is projected to increase
from 127 quadrillion Btu in 2006 to 190 quadrillion Btu in 2030, an average annual rate of
1.7%. Electricity generation from nuclear power is projected to increase from about 2.7
trillion kWh in 2006 to 3.8 trillion kWh in 2030, as concerns about rising fossil fuel prices,
energy security, and greenhouse gas emissions support the development of new nuclear
generation capacity. Major urban areas in the non-OECD nations are expected to address
transportation congestion and strains on infrastructure with a variety of solutions, including
development of mass transit (bus and/or rail) and urban design that reduces vehicle-miles
traveled, among other improvements in transportation network [1].
So it is predicted that the renewable energy from combustible energy sources such as
biodiesel will enter the energy market in the near future to diversify the natural energy source.
29.2 Biodiesel
Biodiesel is the best alternative energy fuels which can be produced form the
vegetable oil and animal fats. Triglycerides are the main component of vegetable oil and
animal fat and are known as ester of fatty acids attached to glycerol moiety. They have
hydrophobic properties which mean that they are insoluble in water. Normally, the vegetable
oil and animal fats are obtained in crude form through solvent extracting or mechanical
pressing, containing a lot of impurities such as free fatty acids, sterols and water. Also they
posses high viscosity and low volatility which posed serious problems like deposition, ring
sticking etc. so they must be subjected to chemical reaction such as transesterification to
reduce the viscosity of oils. In this process, the triglyceride molecules of oils are converted
into fatty acid methyl ester or ethyl ester (based on alcohol source) in the presence of catalyst
with glycerol as byproduct [2].
Currently, biodiesel is mainly produced form soybean oil in U.S, rapeseed, sunflower
or soybean oil in EU, and palm oil in Southeast Asia. But the production of biodiesel from
human nutrition sources can cause a food crisis. So, the fuel versus food problem has led the
exploration of non-edible oil feedstocks to be used as source for biodiesel production.
The majority of researchers have focused on non-edible oils or waste cooking oil as
feed stock for the biodiesel production such as algal oil [3-5], microalgae [6-10], jatropha oil
[11] and grease oil [12] etc. Different feed stocks which can be used for biodiesel production
is shown in table 1.


310

Table 1: Different edible and non-edible feedstocks used for biodiesel production [13]
Conventional feedstock Non-conventional feedstock
Mahua Olive
Nile tilapia Jojoba oil
Palm Rapeseed
Poultry Canola
Sesame Copra
Sunflower Soybean
Barley Babassu
Coconut Brassica napus
Tobacco seed Brassica carinata
Rubber plant Groundnut
Rice bran Cynara cardunculus
Corn Cottonseed
Used cooking oil Pumpkin
Linseed Camelina
Mustard Peanut
Okra
Jatropha curcas
Poultry fat
Fish oil
Bacteria
Algae
Fungi
Micro-algae
Latexes
Sea mango
Palanga
Ponjamia pinnata
Tarpenes
Tallow
Lard

Biodiesel can be produced by many processes but the conventional production
technique is transesterification. It can be carried out by chemical and enzymatic approach.
Chemical technology is a well developed technology that has been commercialized
worldwide. Homogeneous alkali catalyzed process is extensively used for the large scale
synthesis of alkyl esters, due to the low cost of catalysts and their efficiency at low cost
concentrations [14]. Though great efforts have been placed in the improvement of this
process, it still suffers from high production costs and environmental concern like waste
water, chemical disposal and low quality of the glycerol co-product. This process is also
energy-intensive, requires several separation and purification steps and generates lots of waste
water which is to be treated. Additionally alkali catalyzed process accelerates the oil oxidation
which is a major drawback when dealing with the feedstock rich in high FFA content [15]. It
cannot handle conversion of free fatty acid (FFA) effectively and often leads to soap
formation. On the other hand, acid catalyzed process is limited to only esterification reaction.
Thus an oil source which contains a large amount of FFA, only a combination of acid and
alkaline process can fully utilize the feedstock oil. Also, the capital investment is doubled as
well as the operating cost, leading biodiesel to cost higher than the petroleum diesel. Common
cost allocation for biodiesel production through the chemical approach shows that the 70-90%
is allocated for the feedstock and 20-25% for operating cost while 5% for other fees.

311

Thus to obtain a product (biodiesel) with same properties as diesel, an alternative method is to
apply simple reformulation or re-synthesis that results to clearly defined pure products such as
is the case with traditional or enzymatic biodiesel production [16].
Table 2: Comparison of enzyme v/s chemical technology for biodiesel production [17]
S.No.
Parameter

Enzymatic
process

Chemical process
Alkaline process Acid process
1
FFA content in the
raw material
FFA are converted
to biodiesel
Soap formation
FFA converted to
biodiesel
2
Water content in
the raw material
It is not deleterious
for lipase
Soap formation,
Oil hydrolysis
resulting more
soaps
Catalysts
deactivation
3 Biodiesel yield
High, usually
around 99%
High, usually
>96%
High yields(>90%)
only for high
alcohol to oil molar
ratio, high catalyst
concentration and
high temperature
4 Reaction rate Low High
Slower than for
alkaline process
5 Glycerol recovery
Easy, high grade
glycerol
Complex, low
grade glycerol
Complex, low grade
glycerol
6 Catalyst recovery Easy
Difficult,
neutralized by an
acid
Difficult, catalyst
ends up in the by-
products
7 Reusability
Reusability proved
but not sufficiently
studied
Partially lost in
post-processing
steps
No reusable catalyst
8 Energy costs Low Medium High
9 Temperature 20-50C 60-80C >100C
10 Catalyst cost High Low Low
11
Environment
impact
Low: waste water
treatment not
needed
High: waste water
treatment needed
High: waste water
treatment needed

Enzymatic process is known to be clean and environment friendly technique for
biodiesel production as it can simultaneously convert both FFA and triglyceride into

312

biodiesel. Or it can be said that it is insensitive to water and FFA content. No by-product, easy
product removal, reusability without any separation step and mild reaction conditions like low
temperature and high purity products are the key advantages of this process [18]. Enzymatic
transesterification especially those using lipase has drawn researchers attention in last ten
years. There are different types of lipases that can be used as catalysts such as: Rhizaopus
oryzae, Candida rugosa, Psuedomonas fluorescens, Burkholderia, Cepacia, Aspergillusniger,
Thermomyces lanuginose and Rhizomucormiehei etc [2]. However, the use of lipase as
catalysts for biodiesel production is facing a lot of obstacles as their cost is very high which
makes the enzymatic process very expensive for industrial purpose and hence increases the
production cost of biodiesel. Moreover, their low stability is also an important drawback.
For these reasons, much effort has been directed for reducing the enzyme cost thereby
allowing the development of competitive enzymatic processes with potential for industrial
application. Production of lipases from new sources [19, 20], the development of techniques
for lipase immobilization [21] as well as to perform the enzyme-catalyzed reactions in
compressed or supercritical fluids such as propane, n-butane or carbon dioxide have appeared
as attractive alternatives for reaching these goals [22, 23]. Repeated use of the enzymes is
essential from the economic point of view, which can be achieved by using them in
immobilized form. In a continuous process using immobilized enzyme, the operational
stability, the exhaustion of enzyme activity, and inhibition by reactants and/or products play
vital roles. The use of membrane bioreactors for the enzymatic processing is increasingly
becoming more attractive, as such systems allow continuous separation of products and
prevent enzyme inhibition. Research attention is also focused on genetic engineering in
enzymes production. Recently, genes of various enzymes have successfully been cloned, and
more genes are promised to be cloned rapidly in the coming years. The use of recombinant
DNA technology to produce large quantities of recombinant enzymes will help lower the
enzymes costs. In addition, protein engineering will help to create novel enzyme proteins that
are more resistant and highly thermo-stable. The introduction of a new generation of cheap
enzymes, with enhanced activities and resilience, should change the economic balance in
favor of enzyme use [24].
In light of the increasing interest in the development of alterative energy sources, the
aim of this article is to make an overview of enzymatic biodiesel production, focusing on a
sustainable process.


313

29.3 Lipases as biocatalysts in biodiesel synthesis
Lipases (triacylglycerol hydrolase, EC 3.1.1.3.) are the enzymes that catalyze the
hydrolysis of ester link in the triglyceride molecule. They have the catalytic activity not only
in the aqueous solution but also in nonaqueous solvents [25]. Their natural function is to
catalyze the hydrolysis of ester links but they can also catalyze the esterification (link between
alcohol hydroxyl groups and carboxyl groups of carboxylic acids. They have biotechnological
applications as they can catalyze hydrolysis, esterification, alcolysis and transesterification.
Lipases are highly specific as chemo-, region- and enantioselective catalysts. Among
lipases of plant, animal and microbial origins, most commonly used are microbial lipases.
Using microorganisms it is possible to achieve a higher yield of enzymes and to genetically
manipulate the producing strain in obtaining a low-cost lipase with desired properties for the
conversion of fats into biodiesel [26].
A large number of lipases from different sources have been utilized for biodiesel
synthesis shown in table 3.
29.4 Lipase immobilization
High cost of biocatalysts is the major obstacle for industrial application. Therefore,
immobilization of lipases allows their reusability and makes them more attractive for
industrial biodiesel processes. The aim of immobilization is to enhance lipases properties like
thermo stability and activity in non-aqueous media, and to improve handling, recovery and
recycling of biocatalyst. Recycling greatly reduces the cost of the production and makes the
enzymatic biodiesel production more competitive to chemical processes.
Immobilization is defined localization or confinement of an enzyme on to a solid
support or on a carrier matrix. Supports which can be used for immobilization should be
thermally stable, chemically durable and also then its properties depends upon the mechanical
strength, lipase type, type of the reaction system, ease of regeneration, loading capacity and
cost [40]. Methods for enzyme immobilization can be classified as physical adsorption,
entrapment, covalent bonding and encapsulation, each with its advantages and disadvantages
shown in table 4.


314

Table 3: Different lipase used for enzymatic biodiesel production
S.
No.
Oil
Enzyme
catalyst
Alcohol
Reaction
conditions
Solvent
Yield
(%)
Refe
rence
1
Waste cooking
palm oil
Candida
Antarctica B
methanol
4h, 200
rpm
Tert-butanol 79.1% [27]
2 Soybean oil
Thermomyces
lanuginosa
ethanol
10h, 200
rpm
n-hexane/solvent
free
70-
100%
[28]
3 Jatropha oil
Candida
rugosa,
pseudomonas
fluorescens
ethanol
8h, 200
rpm
Solvent free 98% [29]
4 Cotton seed oil
Candida
Antarctica
methanol
24h, 200
rpm
Tert-butanol 97% [30]
5 Soybean oil
Rhizomucor
miehei,
penicillium
cyclopium
methanol
12h, 200
rpm
Solvent-free
68-
95%
[31]
6 Soybean oil
Candida
Antarctica B
Methyl
acetate
14h,200
rpm
Solvent-free 92% [32]
7
Sunflower,
soybean &
waste cooking
oil
Thermomyces
lanuginosa
Methanol
24h, 200
rpm
Solvent-free
90-
97%
[33]
8 Rapeseed oil
Thermomyces
lanuginose,
Candida
Antarctica
Methanol
12h, 200
rpm
Tert-butanol 95% [34]
9 Sunflower oil
Candida
Antarctica
Methyl
acetate
12h, 200
rpm
Solvent-free >95% [35]
10 Cotton seed oil
Candida
Antarctica
Methanol,
propanol,
butanol,amyl
alcohol
7h, 200
rpm
Solvent-free 91.5% [36]
11 Rapeseed oil
Candida
Antarctica
Methanol
24h, 200
rpm
Solvent-free 91.1% [37]
12
Jatropha,
karanja,
sunflower oil
Candida
Antarctica
Ethyl acetate
12h, 200
rpm
13 Sunflower oil
Rhizomucor
miehei,
Methanol
24h, 200
rpm
n-hexane >80% [39]
14 Sunflower oil
Thermomyces
lanuginose,
Pseudomonas
fluorescens
Methanol
24h, 200
rpm


315

Table 4: Different immobilization methods with their advantages and disadvantages

29.5 Variables affecting the enzymatic transesterification
Factors which influence the enzymatic biodiesel synthesis are lipid source, reaction
temperature, choice of acyl acceptors, alcohol to oil molar ratio, amount of water in the
system or water activity, and presence of organic solvent in the mixture. Optimal parameters
for enzymatic transesterification vary depending on the origin and type of lipase, type of oil
source, and reactor type.
29.5.1 Reaction Temperature
S.NO.
Immobilization
method
Examples of used
support
Advantages disadvantages
Refere
nce
1 Adsorption
Textile membrane,
alumina, ceramics,
sepharose, sepadex,
cellulose,
hydrotalcite, zeolites,
celite, silica gel,
polyethylene,
polypropylene etc.
Procedure is
easy, mild
conditions,
cheap method,
involve no
toxic chemical,
support can be
regenerated.
No major
activity loss
Enzyme deactivation
as enzyme are
attached to support by
weak forces (Vander
wall, hydrophobic
interactions and
hydrogen bond), not
best for industrial
application
[41-43]
2
Entrapment
(capture of
lipase within a
matrix of
polymer)
Natural and organic
support, alginate,
agarose, gelatin,
phyllosilicate sol-gel
matrix
more stable
than
adsorption,
enable
substrate and
product
diffusion
Poor diffusion and
enzyme leakage, Low
conversion
[44,
45]
3
Encapsulation
(confinement of
enzyme within a
porous
membrane
forming a
bilayer)
Natural polymers like
alginate and
carrageenan, synthetic
(resins) and acrylic
polymers, hydrogels,
microemulsion based
gels
Prevents
leaching and
provide highly
reusable
biocatalyst,,
stability of
enzyme is high
Diffusion occurs with
increase in substrate
concentration, mass
transfer problems,
[46.
47]
4
Covalent
bonding
(irreversible
bonding of the
lipase to support
matrix)
Agarose beads
Prevents
leaching of
enzyme, stable,
high reaction
activity &
better catalytic
properties
Complex process,
requires an activating
agent
[48,
49]

316

Reaction temperature may vary from 23 to 50C. In general, increasing the
temperature leads to an increase of the reaction rate of biodiesel production. And when the
optimum temperature is reached, further increase in temperature leads to decrease in the
catalytic activity due to denaturation and inactivation. The researches have shown that
immobilization of enzymes shift temperature optimum to higher values in comparison to free
enzymes. It seems that immobilization provides a more rigid external backbone for lipase
molecule, leading to the increase of the temperature optima and higher reaction rates [50].
29.5.2 Water content
Another major factor for enzymatic ester synthesis is water content in the system.
Lipases need an optimal small amount of water to maintain the activity in the organic media.
Nevertheless, increased water concentration has an 316nfavourable effect on the equilibrium
conversion, since it promotes reverse reaction of hydrolysis. The amount of water in the
system should be a compromise between minimizing hydrolysis and maximizing lipase
activity for the transesterification reaction and it should be determined for a particular reaction
system [51, 52]. Many studies have shown that immobilized enzymes show highest activity in
low water system.
Also, the content of free fatty acids (FFA) in waste oils is more as compared to refined
oils. The esterification of FFA releases water, which can shift the reaction equilibrium
towards ester hydrolysis. In these cases, molecular sieves are used for the control of water
activity and increase ester yields by removing water produced by esterification. However,
when lipases are immobilized on hydrophilic support, the molecular sieves are not need since,
in that case, they have a negligible impact on methyl esters yield [44].
29.5.3 Oil source
Generally, the main feedstock for biodiesel production can be divided in: 1) Vegetable
oils such as sunflower oil [33, 38], soybean oil [28, 31], rapeseed oil [34, 37], jatropha oil
[29], cotton seed oil [30]; 2) Animal fats such as tallow, lard; 3) Waste cooking oils and
industrial waste oils [27].
However, edible oils are not in surplus supply and the cost of oil sources accounts for
a large part in biodiesel production. In order to make the biodiesel production viable, the
solution is to develop a production based on waste cooking oils where no competition with
food production takes place. But the amount of waste oils alone is not sufficient to meet
demands. The optimal solution is to use non-edible oils which can not be used for human

317

consumption because of the presence of some anti nutritional factors, or toxic components.
The most suitable oils are those from crops with the highest productivity pre hectare, low
production cost and that can grow on waste land, such as Jatropha oil or rapeseed oil [29].
29.5.4 Acyl acceptors
The majority of enzymatic syntheses of biodiesel are performed in organic solvents.
The yield of biodiesel is greatly influenced by the type of organic solvent present in the
reaction system. Immobilized lipases showed high degree of efficiency in the presence of
non-polar solvent [39]. The polar, less hydrophobic solvents are not suitable for biocatalytic
processes since they can distort water microlayer around the enzyme influencing its native
structure, thereby, leading to denaturation. It has been shown that the highest biodiesel yield
with lipase from C. antarctica was achieved with n-hexane as solvent. The lowest yields were
obtained with polar solvents such as acetone [42].
29.6 Conclusion
Lipase catalyzed biodiesel production has been an active are of research and shows
great potential to generate environmental friendly and economic fuel in future. Researchers
have tried to standardize the parameters and optimizing the reaction conditions to produce
biodiesel from large variety of non-edible triglyceride substrates and hence eliminating the
main issue of the traditional chemical transesterification. Further improvements can make
industrial enzymatic biodiesel production a viable option for the future.

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323

CHAPTER 30
SINGLE STEP REACTION FOR BIODIESEL PRODUCTION
OF JATROPHA CURCUS SEEDS
Sanjaykumar N. Dalvi and Swati R. Sonawane

Abstract
In India, biodiesel prepared from non-edible oil seeds of Jatropha Curcus appeared as a
promising alternative energy source. The oil extraction from these seeds is a primary step for
biodiesel production. This oil is then transesterified to prepare the biodiesel. To escape the
step of oil extraction, a single step method could be used in which the Jatropha seeds crush is
directly converted into biodiesel which is fatty acid methyl & ethyl ester composition. The
objective of this study was to investigate the transesterification allowing direct production
biodiesel from Jatropha seed. This work show that the results obtained from characterization
of the product of single step reaction from non-edible oil seeds of Jatropha Curcus is
biodiesel.
Keywords Biodiesel; Jatropha Curcus; Gas chromatography-mass spectroscopy (GC-MS)
30.1 Introduction
Biodiesel is an alternative fuel made from renewable biological sources such as
vegetable oils both (edible and non-edible oil) and animal fats. Vegetable oils are usually
esters of glycol with different chain length and degree of saturation. It may be seen that
vegetable contains a substantial amount of oxygen in their molecules (Raja et al., 2011).
200 districts in 19 potential states have been identified on the basis of availability of
wasteland, rural poverty ratio, below poverty line (BPL) census and agro-climatic conditions
suitable for Jatropha cultivation. Each district will be treated as a block and under each block
15000 ha Jatropha plantation will be undertaken through farmers (BPL). Proposed to provide
green coverage to about 3 Million ha of wasteland through plantation of Jatropha in 200
identified districts over a period of 3 years (Raja et al., 2011).
Jatropha is a genus of over 170 plants from the Euphorbiaceae family, native to the
Central America but commonly found and utilized across most of the tropical and subtropical


324

regions of the world. It has a yield per hectare of more than four times that of soybean and ten
times that of corn (Nobrega. et al., 2007). Among the different species of Jatropha, Jatropha
curcas has a wide range of uses and promises various significant benefits to human and
industry. Extracts from this species have been shown to have anti-tumor activity (Juan et
al.,2003),the leaves can be used as a remedy for malaria and high fever, (Gubitz et al.,1999),(
Henning R. 1997 ), the seeds can be used in treatment of constipation and the sap was found
effective in accelerating wound healing procedure (Gubitz et al.,1999). Moreover, this plant
can be used as an ornamental plant, raw material for dye, potential feed stock, pesticide, soil
enrichment manure and more importantly as an alternative for biodiesel production(
Vasudevan et al., 2007), (Tiwari et al., 2007).
It appears very difficult to estimate unequivocally the yield of a Jatropha plant that is
able to grow in very different conditions. The yield is a function of water, nutrients, heat and
the age of the plant and other factors. Many different methods of establishment, farming and
harvesting are possible. The yield can be enhanced with right balance of cost, yield, labour
and finally cost per Mt Seed production ranges from about 2 tons
-1
hectare
-1
year to over
12.5t
-1
ha
-1
year, after five years of growth. Although not clearly specified, this range in
production may be attributable to low and high rainfall areas.
The seed reaches maturity 90 days after flowering when the capsules changes from
green to yellow and are harvested at this stage to ensure a high oil yield (Pimenta et al., 2009).
The seeds contain around 20% saturated fatty acids and 80% unsaturated fatty acids, and they
yield 25%40% oil by weight. Estimates of Jatropha seed yield vary widely, due to a
perennial life cycle. Seed yields under cultivation can range from 1,500 to 2,000 kilograms-1
hectare, corresponding to extractable oil yields of 540 to 680 litres -1 hectare. In addition, the
seeds contain other chemical compounds, such assaccarose, raffinose, stachyose, glucose,
fructose, galactose, and protein. The oil is largely made up of oleic and linoleic acids
(Bangboye and Hansen, 2008).
One of the most promising processes to convert vegetable oil into methyl ester is the
transesterification, in which alcohol reacts with triglycerides of fatty acids (vegetable oil) in
the presence of catalyst. Jatropha vegetable oil is one of the prime non edible sources
available in India. The vegetable oil used for biodiesel production might contain free fatty
acids which will enhance saponification reaction as side reaction during the transesterification
process (Raja et al., 2011).


Figure 1: Jatropha Curcus seed bearing plant and Seed powder
Jatropha can also be intercropped with other cash crops such as coffee, sugar, fruits
and vegetables. Jatropha Curcus plant oil seeds are the highest yielding feedstock for
biodiesel and also use in production of fertilizer, soaps & cosmetics.
The study of in-situ
single step of biodiesel preparation. Characterization the obtained product by GC
techniques to detect the presence of various components of transesterified product which
should give structural information of fatty acid alkyl ester of prepared biodiesel.
The In-situ transesterification carried out in a reactor which consisted of an oil bath
with 500 ml three necked round bottom flask with digital controlled
platinum resistance thermometer detector (RTD) temperature sensor with an accuracy of 1C
connected to a digital indicator and a condenser. The product was collected through a
separating funnel with valve which placed at bottom.
The Jatropha seeds were collected, dried, cleaned and separate the seed coating. By
using electric mixer with high rpm seeds grind into fine powder form. Twenty grams of seed
powder mixed with potassium hydroxide in methanol. The reaction mixture was continuou
stirring by adjusting 500 rpm oscillations. The reaction carried out at 60C for 60 minutes
(Dalvi et al., 2012). At room temperature, the solid cake & mother liquor were separated by
vacuum filtration. Solvent was separated by a rotary evaporator. The
and purify separate the water soluble impurities
used for further analysis. The product is analysed by GC
325

Jatropha Curcus seed bearing plant and Seed powder
can also be intercropped with other cash crops such as coffee, sugar, fruits
Jatropha Curcus plant oil seeds are the highest yielding feedstock for
biodiesel and also use in production of fertilizer, soaps & cosmetics.
situ transesterification reaction of Jatropha Curcus seed crush for
single step of biodiesel preparation. Characterization the obtained product by GC
ural information of fatty acid alkyl ester of prepared biodiesel.
Material and Methods
situ transesterification carried out in a reactor which consisted of an oil bath
with 500 ml three necked round bottom flask with digital controlled
separating funnel with valve which placed at bottom.
Jatropha seeds were collected, dried, cleaned and separate the seed coating. By
powder mixed with potassium hydroxide in methanol. The reaction mixture was continuou
. At room temperature, the solid cake & mother liquor were separated by
vacuum filtration. Solvent was separated by a rotary evaporator. The product was separated
and purify separate the water soluble impurities. It was preserved in airtight container and
used for further analysis. The product is analysed by GC-MS techniques.
Jatropha Curcus seed bearing plant and Seed powder
can also be intercropped with other cash crops such as coffee, sugar, fruits
Jatropha Curcus plant oil seeds are the highest yielding feedstock for
transesterification reaction of Jatropha Curcus seed crush for
single step of biodiesel preparation. Characterization the obtained product by GC-MS
ural information of fatty acid alkyl ester of prepared biodiesel.
situ transesterification carried out in a reactor which consisted of an oil bath
with 500 ml three necked round bottom flask with digital controlled mechanical stirrer, a
Jatropha seeds were collected, dried, cleaned and separate the seed coating. By
powder mixed with potassium hydroxide in methanol. The reaction mixture was continuously
. At room temperature, the solid cake & mother liquor were separated by
product was separated
. It was preserved in airtight container and
MS techniques.

326

The Jatropha curcas is better feedstock for preparation of biodiesel. This seed contain
27-40% oil. The in-situ transesterification reaction is carried out at 60C with
KOH(Potassium hydroxide). The reaction mixture continuously stirring at 500 rpm for 60
minutes.The product is anlysed by Infrared spectroscopy to determine the functional group of
product(FAME) figure 2. The final product fatty acid methyl ester gives the carbonyl
vibrational bond frequency at 1741.60 cm
- 1
, carbon oxygen alkoxy bond of ester is at
1461.94 cm
1
while finger print region hows the long chain methylene carbon hydrogen
bond frequency at 729.04 cm
1
& 694.33 cm
- 1
.
The product is characterized by Gas chromatography-mass spectroscopy technique.It
used to separate component of mixture of product (FAME) and to study molecular structure
of compound.There are 4 components were shown their presence in the GC-MS analysis. The
qualitative peaks are shown the figure 3. Following ester were found in the product which is
given with the percentage and Retention time (RT) in table 1.
The result shows that the in-situ transesterification of the jatropha curcus seeds can be
done successfully without extraction of oil from seeds. The methanol might be worked as
solvent in this reaction. There is no need to use the solvent like hexane for oil extraction ,
which are not eco-friendly.
The method to prepare the biodiesel is triple stage reaction but in-situ
transesterification single step eco-friendly reaction.

Figure 2: IR Jatropha fatty acid alkyl ester

327

Figure 3: The qualitative peak of GC-MS of Jatropha Curcus in-situ transesterified product
Table1. Jatropha Curcas Biodiesel Components with the RT, Percentage and Name of the
Ester
Sr.No. R.T. %Ester Name of Ester
1 24.289 5.29 Tridecanoic acid methyl ester
2 26.283 28.96 9,12-Octadicadienoic acid methyl ester(E,E)
3 26.356 63.72 11-Octadecenoic acid methyl ester
4 26.496 2.03 Tridecanoic acid methyl ester

30.4 Conclusions
With the in-situ transesterification which is single step reaction one can produce
biodiesel. The biodiesel content obtained from crushed seed powder of Jatropha seeds without
extraction of oil. The fatty acid methyl ester fuel characterization was done with GC-MS
techniques and results are tabulated. The non-edible oil seeds of Jatropha Curcus are better oil
yielding seeds for the preparation of biodiesel with in-situ transesterification.


328

References
1. Dalvi S., Sonawane S. and Pokharkar R. (2012) Preparation of Biodiesel of Undi seed
with In-situ Transesterification. Leonardo Electronic Journal of Practices and
Technologies, 20:75-182
2. Gubitz G., Mittelbach M. and Trabi M. (1999) Exploitation of the tropical oil seed plant
Jatropha curcas L. Bioresour. Technol., 67: 73-82.
3. Henning R. (1997) Fuel Production Improves Food Production: The Jatropha Project in
Mali. Proceedings from the symposium Jatropha, Managua, Nicaragua.
http://www.jatrophaworld.org
4. Juan L., Fang Y., Lin T. and Fang C. (2003) Antitumor effects of curcin from seeds of
Jatropha curcas. Acta Pharmacol. Sin. 24: 241-246.
5. Nahar K. and Ozores-Hampton M. (2011). Jatropha: An Alternative Substitute to Fossil
Fuel.(IFAS Publication Number HS1193). Gainesville: University of Florida, Institute
of Food and Agricultural Sciences.
6. Nobrega W. and Sinha A. (2007) Riding the Indian Tiger: Understanding India-the
World's Fastest Growing Market. John Wiley and Sons, pp: 272.
7. Raja S. A., Robinson D.S., and Lee C. L. (2011) Biodiesel production from jatropha oil
and its characterization. Research Journal of Chemical Sciences, 1: 81-87.
8. Sharma Y. C., Singh B. and Upadhyay S. N. (2008) Advancements in development
Characterization of biodiesel: A review. Fuel, 87: 2355-2373
9. Tiwari A.K., Kumara A. and Raheman H. (2007) Biodiesel production from jatropha oil
(Jatropha curcas) with high free fatty acids: An optimized process. Biomass Bioenergy,
31: 569-575.
10. Vasudevan P.T. and Briggs M. (2007) Biodiesel production-current state of the art and
challenges. J. Ind. Microbiol. Biotechnology, 35: 421-430.


329

CHAPTER 31
PRODUCTION OF BIODIESEL FROM EDIBLE AND NON-
EDIBLE OILS: A COMPARATIVE STUDY
Aman Deep Singh, Raman Rao, L. Bhanuprakash Reddy, Hemant Kr. Raghuvanshi,
Abdullahi Isaku Kankia, Hitesh Sharma, Soumya Srivastava, Debjani Mukherjee

Abstract
As per IEA, the use of fossil fuels will be increased 56% of worlds present population. It
is already a noted fact that the depletion of fossil fuels has reached a peak value. Based on
the examples of oil crisis, several researchers are eagerly waiting to search for the
alternative biodiesel sources in order to save the economy from the oil crisis in the near
future. Searching feedstock for biodiesel production has always been a challenging step for
the researchers, as the selected one should be non-conventional, with higher yield
content, eco-friendly, and economically feasible. Based on the potential usage of the
biodiesel, the current research provides a comparative study on production of biodiesel
from non-edible and edible oils like pongamia oil, mixed vegetable oil, coconut oil,
mustard oil, soybean oil. Also this work includes a better explanation on the trans-
esterification process and physico-chemical characterization with use of ASTM standards.
After performing the sequential experiments, we obtained the following results for
coconut, mustard, pongamia, waste vegetable and soy bean oil (percentage yield: 87.49%,
60.66%, 81.66%, 79.34%, 83.99%; pH: 6.5, 7.3, 7.2, 7.1, 7.0; carbon content:0.05gm,
0.1gm, 0.04gm, 0.08gm, 0.09gm; specific gravity:0.884, 0.872, 0.892, 0.876, 0.874; Acid
value:0.34mg KOH/gm, 0.44mg KOH/gm, 0.39mg KOH/gm, 0.44mg KOH/gm, 0.34mg
KOH/gm; Viscosity at 40oC: 75.98, 80.38, 79.45, 76,58, 78.76; Moisture content:
5.06%, 5.75%, 7.06%, 6.09%, 4.94%; Density: 0.878gm/cm3 , 0.877 gm/cm3, 0.886
gm/cm3, 0.872 gm/cm3, 0.868 gm/cm3; Flash Point: 150oC 180oC, 160oC, 170oC,
160oC; Fire point: 170oC, 205oC, 180oC, 195oC, 200oC). Owing to impact of the results,
we are looking to optimize production scale of biodiesel from non-edible sources in order
to quit from food v/s fuel problems.
Key Words: Fossil fuels, Biodiesel, Non-edible oils, Edible oils and Trans-


330

esterification.
31.1 Introduction
According to the United State Standard Specification for Biodiesel (ASTM 6751),
defines Biodiesel as a fuel comprising of mono alkyl esters of long chain fatty acids derived
from renewable biomass which can be used in diesel engines and heating systems (Mittelbach
et al., 1983; Staat and Vallet, 1994). Biodiesel is considered as neat fuel in comparison with
diesel fuel; it is a renewable fuel, non toxic, safer to handle, biodegradable, requires no engine
modifications and reduces dependency on foreign oil imports (Gerpan JV, 2006 et al., 2004).
It also has favorable combustion and emission profiles. For instance, emissions of carbon
monoxide (CO) and particulate matter decease by 45%, Hydrocarbon (HC) 70% but NOx
emissions increases by 10% with 100% biodiesel (B100) as a fuel (Anon et al., 2002). The
carbon cycle, time for fixation of co2 from biodiesel is quite small compared to mineral diesel
thus contributing more to the reduction of greenhouse gas emissions compared to fossil diesel
(Gerpan JV, 2006; Carraretto et al., 2004; Agarwal et al., 2003), found that biodiesel
provides good lubricating properties that can reduce component wear and enhance engine life.
Hence algal oil is a potential alternative for fossil to harmonize agriculture, economic
development and the environment. Biodiesel is a renewable, natural and domestic fuel made
from edible and non-edible oils. It holds no petroleum, is nontoxic and biodegradable. It is an
alternative fuel for diesel engines. In our modern life there is regular and very fast
consumption of petroleum oil but the resource of petroleum oil are limited so, there is a
necessity to invent an alternative for future which is most renewable, optimal, and easily
accessible in nature. As the crude fuel resource is limited and non-renewable so, the fuel price
at faster pace is continuously increases for regularly diminishing supply and fulfilment of
demand. By continuous consumption of fossil fuel or crude oil results, speedy decline in
reserve of fossil fuels. (Rattan et al., 2012). Biodiesel is produced by the trans esterification
(alcoholises) of vegetable oil or animal fat and alcohol to yield Fatty Acid Methyl Esters
(FAME) and glycerol (Freedman et al., 1996). Searching feedstock for biodiesel production
has always been a challenging step for the researchers, as the selected one should be
non-conventional, with higher yield content, eco-friendly, and economically feasible.
Based on the potential usage of the biodiesel, the current research provides a
comparative study on production of biodiesel from non-edible and edible oils like pongamia
oil, mixed vegetable oil, coconut oil, mustard oil, soybean oil. Test quantities of ethyl and

331

methyl esters of four renewable fuels which were processed, characterized and the
performance was tested.
31.2 Scope for the Study
Analysis of the current situation with respect to availability of bio fuel resources,
processing technology, end-use applications, government policies, markets for bio fuels. The
Potential of bio fuels, considering land availability, technology advancements, future demand-
supply scenario for transportation fuels, and infrastructure and investments requirements.
Discussion and analysis on the sustainability issues in bio fuel development, with respect to,
food security, social and economic sustainability and environmental sustainability. Biodiesel
value chain and discussion on bio fuel production and utilization models. Analysis on the
national and international implications of large-scale bio fuel development, on petroleum
imports, international trade, foreign exchange balance, global environment etc.
31.3 Research Methodology
31.3.1 Praperation of Catalyst
Weigh 0.85gm of NaOH. In a beaker, add 14.28 ml of methanol. Add NaOH in the
methanol and allow to mix the solution on magnetic stirrer to obtain methoxide, which act as
a catalyst in the transesterification reaction. During mixing the beaker must be covered with
aluminium foil, to avoid the vaporisation of methanol, as it is volatile in nature.
31.3.2 Transesterification: Material Required
Measure the 85.72ml of oil in a beaker and put the beaker in hot water bath for 45-60
min at 60C. This step is called pre-treatment of oil. After pre-treatment of oil, put the oil on
magnetic stirrer, add the methoxide to the oil and allow the mixing for 60-80 min at 55C to
60C. After the oil/methoxide mixture has reacted, pour the mixture into a separatory funnel
and allow the glycerol waste to settle down for a 24hrs. After settling of glycerol, drain out
the glycerol from the bottom of the funnel and pour the top layer (crude biodiesel) back into
the beaker. Observation of the temperature during the mixing of oil and methoxide must be
continues, to maintain the temperature constant.
31.3.3 Separation

332

After the oil and methoxide were mixed, then mixture was put into the separatory
funnel for separation of crude biodiesel from glycerol. Separation of biodiesel and glycerol
started during the mixing of oil and methoxide, but generally it took 24 hrs for separation.
31.3.4 Washing
After the removal of glycerol, warm distilled water was added to the separatory funnel
that contains crude biodiesel. Tight the cork of the seperatory funnels and gently shakes it to
mix water with crude biodiesel. Let the water settle down of the biodiesel for one day. Then
drain out the water. Repeat the washing process for 3 to 4 more times until the water was
completely become transparent. Transfer the biodiesel to a clean beaker. If the pH is too high,
washing process should be performed for 2 to 3 time more.
31.3.5 Characterization
The biodiesel obtained can be characterized by following methods: ph, percentage
yield, specific gravity, carbon content, acid value, sponification value, moisture content,
viscosity, and density, flash and fire point. The procedure for these methods can be followed
by as per ASTM (American Standard for Testing Materials).
31.4.1 Results
The results obtained by different characterization methods can be shown in following
tables:
Table 1: pH of biodiesel Obtained from different oils

Sr.no. Biodiesel sample
pH
(before washing)
pH
(after washing)
Biodiesel colour
1. Mustard 10.2 7.3 Dark yellow
2. Coconut 9.2 6.5 Pale white
3. Soybean 9.7 7 Pale white
4. Pongamia 9.6 7.2 Dark brown
5. Mix vegetable oil 9.8 7.1 Light yellow

333

Table 2: The Biodiesel obtained gives the different characterization Results

Table 3: Viscosity of the Biodiesel sample in redwood records
Sr. No
Temperature
0
C
Mustard
Oil
Coconut
Oil
Soybean
Oil
Pongamia
Oil
Mix Veg
Oil
1 40 80.38 75.98 78.76 79.45 76.58
2 50 73.54 69.05 71.82 71.65 69.34
3
60
65.09 62.87 64.23 66.87 60.05
4 70 60.76 57.97 56.76 57.34 59.72

Table 4: Flash and Fire Point of Biodiesel
Sr. No. Biodiesel Sample
Flash Point
Observed at
Fire Point Observed
at
1 Mustard Oil 180 205
2 Coconut Oil 150 170
3 Soybean Oil 160 200
4 Pongamia Oil 160 180
5 Mix Vegetable Oil 170 195
Sr.
No.
Biodiesel
Sample
Percentage
yield (%)
Carbon
content
(g)
Specific
Gravity
Acid value
(mgKOH/g
oil)
Moisture
Content
(%)
Density
(M/V)
1
Mustard
Oil
60.66 0.1 0.872 0.44 5.75 0.877
2
Coconut
Oil
87.49 0.05 0.884 0.34 5.06 0.878
3
Soybean
Oil
83.99 0.09 0.874 0.34 4.94 0.868
4
Pongamia
Oil
81.66 0.04 0.892 0.39 7.06 0.886
5
Mix Veg
Oil
79.34 0.08 0.876 0.44 6.09 0.872

334

31.4.2 Graphs

Graph 1: Yield of biodiesel Graph 2: pH of biodiesel

Graph 3: Specific Gravity of biodiesel Graph 4: Acid Value of biodiesel

Graph 5: Moisture content of the biodiesel Graph 6: Density of biodiesel

0
20
40
60
80
100
Yield
Oil
sample(ml)
Yield(ml)
Percentage
yield(%)
0
2
4
6
8
10
12
pH
pH befre
!as"ing
pH after
!as"ing
0#86
0#8$
0#88
0#8%
0#%
Specific gravity
&pecific
gra'ity
0
0#1
0#2
0#(
0#4
0#)
Acid value
*cid 'al+e
0
1
2
(
4
)
6
$
8
,nitial !eig"t
f
bidesel(gm
)
-inal !eig"t
f
bidiesel(gm)
0#8))
0#86
0#86)
0#8$
0#8$)
0#88
0#88)
0#8%
Density
.ensity

335

31.4.3 Discussion
In this project, various tests were performed for the production and characterization of
the biodiesel from coconut oil, mustard oil, pongamia oil and mix vegetable oil. The
percentage yield of the biodiesel was 60.66% from mustard oil, 87.49% from coconut oil,
83.99% from soybean oil, 81.66% from pongamia and 79.34% from mix vegetable oil. The
percentage yield of biodiesel from coconut oil comes out to be maximum amongst. The pH of
the biodiesel from mustard oil is 7.3, pH of the coconut oil is 6.5, pH of the soybean oil is 7,
pH of the pongamia oil is 7.2 and the pH of the mix vegetable oil is 7.1 after washing. The
carbon content is 0.1g for mustard oil, 0.05g for coconut oil, 0.09g for soybean, 0.04g for
pongamia oil and 0.08g for mix vegetable oil. Specific gravity for the biodiesel from mustard
oil is 0.872, form coconut oil is 0.884, from soybean oil is 0.874, from pongamia oil is 0.892,
and from mix vegetable oil is 0.876. Acid value of standard biodiesel must be 0.5-0.7mg
KOH/gm oil. The acid value for the biodiesel from mustard oil is 044mg KOH/gm, from
coconut oil is 0.34 mg KOH/gm, from soybean oil is 0.34 mg KOH/gm, from pongamia is
0.39 mg KOH/gm and from mix vegetable oil is 0.44 mg KOH/gm. Viscosity of the samples
calculated at 40
0
C and the unit is redwood seconds, viscosity of biodiesel from mustard oil is
80.38, from coconut is 75.98, from soybean is 78.76, pongamia is 79.45 and mix vegetable oil
76.58. Moisture content of the samples were 5.75% for mustard B100, 5.06% for coconut
B100, 4.94% for soybean B100, 7.06% for pongamia B100 and 6.09% for mix vegetable
B100. Standard biodiesel fuel have density ranging 0.86-0.90gm/cm
3
, and the density of the
samples were 0.877, 0.878, 0.868, 0.886 and 0.872 for mustard B100, coconut B100, soybean
B100, pongamia B100 and mix vegetable B100 respectively. Flash point of standard biodiesel
must be between 130-210C. The flash point for the samples were calculated to be 180
0
C,
150
0
C, 160
0
C, 160
0
C, 170
0
C for mustard B100, coconut B100, soybean B100, pongamia
B100 and mix vegetable B100 respectively. Fire point for the samples were calculated to be
205
0
C, 170
0
C, 200
0
C, 180
0
C and 195
0
C for mustard B100, coconut B100, soybean B100,
pongamia B100 and mix vegetable B100 respectively.

References
1. Bannikov M.G., Combustion and emission characteristics of mustard biodiesel.
2. 6th International Advanced Technologies Symposium. (IATS 11), 132-136.

336

3. Bari1 M.A.A., Ali H., Rahman M., and Hossain R. Prospect of Bio-diesel Production
from soybean oil: An Alternative and Renewable Fuel for Diesel Engines. International
Journal of Mechanical Engineering, ISSN: 2277-7059, Volume 2.
4. Bobade S.N. and Khyade V.B (2012). Detail study on the Properties of Pongamia
Pinnata (Karanja) for the Production of Biofuel. Research Journal of Chemical
Sciences, Vol. 2(7), 16-20.
5. Bunyakiat K., Makmee S., Sawangkeaw R., Ngamprasertsith S., Cao W., Han H., and
Zhang J (2005). Continuous Production of Biodiesel via Transesterification from
Vegetable Oils in Supercritical Methanol. Fuel, 84: 347-351.Chandrasekhar L.A.,
Mahesh N.S., Gowda B., and Hall W (2012). Life cycle assessment of biodiesel
production from Pongamia oil in rural Karnataka. CIGR Journal, Vol. 14, No.3.
6. C.L. Peterson., D.L. Reece., B.L. Hammond., J. Thompson., and S.M. Beck (1997).
Processing, Characterization, and Performance of Eight Fuels from Lipids. Applied
Engineering in Agriculture, 13(1): 71-79.
7. Cloin J., Courty P., Deamer T., and Vaitilingom G (2004). Coconut oil as a biofuel in
Pacific Island, South Pacific Applied Geosciences Commission, SOPAC, 1-5.
8. Hasib Z.M., Hossain J., Biswas S., Islam A (2011). Bio-Diesel from Mustard Oil: A
Renewable Alternative Fuel for Small Diesel Engines. Modern Mechanical
Engineering, (1): 77-83.
9. Husain A. B. M. S., Nasrulhaq B.A., Salleh A., and Chandra S (2010). Biodiesel
production from waste soybean oil biomass as renewable energy and environmental
recycled process. African Journal of Biotechnology, Vol. 9(27): 4233-4240.
10. Issariyakul T., Dalai A.K., and Desai P (2011). Evaluating ester derived from mustard
oil (sinapis Alba) as potential diesel additives. Journal of America oil chemist, Society
88: 391-402.
11. Rattan R. Kumar M (2012). Biodiesel (a renewable alternative fuel) production from
mustard oil and its performance on domestic small diesel engines. International
Referred Research Journal, ISSN- 0975-3486, VoL. III: 45-51.
12. Scott P.T., Pregelj L., Chen N., Hadler J.S., Djordjevic M.A., Gresshoff P.M (2008).
Pongamia pinnata: An Untapped Resource for the Biofuels Industry of the
Future.Bioenergy. Res, Vol. 1, 211.
13. Sharma S.K., Kalra K.L., and Grewal H.S (2002). Fermentation of enzymatically
saccharified sunflower stalks for ethanol production and its scale up. Bioresource
Technology, 85: 3133.

337

CHAPTER 32
PRODUCTION OF BIODIESEL FROM NEEM OIL USING
SYNTHESIZED IRON NANOCATALYST
Mookan Rengasamy, Sundaresan Mohanraj, Krishnasamy Anbalagan, Shanmugam
Kodhaiyolii, Velan Pugalenthi

Abstract
The predicted shortage of fossil fuels encourages the search for substitutes of petroleum
derivatives. The production of biodiesel has been greatly increasing due to its environmental
benefits. In this present study, the production of biodiesel by transesterification of neem oil
was carried out using synthesized iron nanoparticle as a catalyst. Iron nanoparticles were
synthesized from ferric chloride solution to enhance the transestrification process for the
production of biodiesel. Ferric ions were rapidly reduced by the aqueous sodium borohydride,
leading to the formation of iron nanoparticle. Synthesized iron nanoparticles were analysed by
UVVisible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and
transmission electron microscopy (TEM). XRD results showed that the peaks were indexed
to the face-centered cubic (fcc) phase (1 1 0) of iron nanoparticles. TEM image confirms that
the formations of iron nanoparticles were predominantly cubical in shape and the size of an
iron nanoparticle was found to be within the range of 40 to 70 nm. The reaction conditions
were 60
o
C of reaction temperature, 3:1 molar ratio of methanol to oil, and 1wt % of
synthesized iron nanoparticles for the production of biodiesel. The determined values of
specific gravity, viscosity, flash point, cloud point, water content, carbon residue and copper
corrosion for biodiesel were 0.875, 4.9 cp at 40
o
C, 145
o
C, 3
o
C, 0.01 vol %, 0.03 wt %
and 1b respectively. These findings confirm that the obtained values of physical and chemical
properties are in accordance with the specifications of biodiesel as per standard ASTM
D6751. The emission characteristics were also carried out for the blend B20 of neem oil
methyl ester and the results were compared with the hydrocarbon diesel. The results of the
study conclude that the emission characteristics of produced biodiesel were found to be less
when compared to hydrocarbon diesel and hence the produced biodiesel was found to be
superior.



338

Key Word: Iron nanoparticle, Biodiesel, Neem oil methyl ester, Emission characteristics of
biodiesel.
32.1 Introduction
The rapid depletion of fossil fuel resources and increasing environmental concerns,
biodiesel has great attention in recent years as the renewable and eco-friendly fuel. Biodiesel
is a clean and reliable alternative fuel and it has been considered as the fuel substitute for
hydrocarbon diesel (Leung et al., 2010). Biodiesel is generally produced from vegetable oils
and animal fats. Commonly edible vegetable oils are used for the production of biodiesel.
However, increasing the cost of vegetable oil is the main challenges to produce the cost
effective biodiesel. In order to reduce the production cost, low cost feed stocks such as non-
edible oil, waste cooking oil and fats have been used as the raw materials (Bhatti et al., 2008).
Conventionally, biodiesel manufacturing processes employ strong acids or bases as catalysts.
But, separation of the catalyst and the by-product glycerol from the product ester is too
expensive to justify the product use as an automobile fuel (Deng et al., 2011). Hence,
heterogeneous catalysts are used since they offer exciting possibilities for improving the
economics of biodiesel synthesis. However, the preparation of heterogeneous catalysts are
more complicated and quiet expensive, which limits there industrial application (Qiu et al.,
2011). Therefore, it is necessity to find cheap and efficient catalysts to carry out the
commercial biodiesel production from non-edible oil.
Recently, nanomaterial has been considerable attention as a catalyst for biodiesel
production, owing to its large specific surface area and high catalytic activity. In addition, it
exhibits high resistance to saponification and good rigidity (Hu et al., 2011). Especially,
metallic nanoparticles have various characteristics including catalytic, magnetic, and optical
properties (Neh et al., 2006; Woo et al., 2004; Zhou et al., 2006). The metal nanoparticles are
used for various applications such as sensors, environmental remediation, catalysts and energy
storage (Lopeza et al., 2004; Akagia et al., 2012; Noubactep et al 2012; Zhao et al., 2012).
Recently, Qiu et al., (2011) reported that the transesterification from soyabean oil and
methanol was catalyzed by zirconia nanoparticles loaded with potassium bitartrate for the
production of biodiesel. Hence nanoparticles are gaining significant interest to use as a
catalyst for transesterification process to produce efficient biodiesel.
Several methods including physical, chemical and biological were available for the
synthesis of metallic nanoparticles. The main drawbacks of physical methods are inferior

339

quality of the products and also this method requires costly vacuum systems to prepare the
nanoparticles (Umer et al., 2012). Recently, biological methods such as plant and
microorganisms have been extensively studied for the synthesis of iron nanoparticle (Prasad
et al., 2007; Ahamed et al., 2011). However, many of the biological routes to produce metal
nanoparticles are time consuming process and agglomerations of the nanoparticles are the
main challenges. In order to reduce the time, chemical methods are preferred to synthesize
nanoparticles. Chemical synthesis is relatively faster than the physical and biological
methods. The main advantages of this method are to control over the size and shape of the
nanoparticles. Hence, the chemical synthesis was used in the present investigation to synthesis
iron nanoparticles.
In this present study, iron nanoparticles were synthesized using a single step, instant
chemical approach by reducing ferric chloride solution with aqueous sodium borohydride as
reducing agent. The synthesized iron nanoparticles were characterized by UV-Visible spectra,
FTIR, XRD, SEM-EDX and TEM. In addition, synthesized iron nanoparticles were used as
catalyst to investigate the transesterification of neem oil with methanol to produce efficient
biodiesel.
32.2.1 Materials
Analytical grade Ferric chloride (FeCl
3
) salt, sodium borohydride and methanol were
purchased from Merck Specialties Private Ltd, India. Neem oil was purchased from local
supplier in Tiruchirappalli, Tamil Nadu, India.
32.2.2 Synthesis of iron nanoparticles
Iron nanoparticles were synthesized using sodium borohydride as a reducing agent
with some modifications as reported by Uzum et al., (2008). A 100 mL of 2.5 mM ferric
chloride solution was prepared using sterile double distilled water and stirred at 400 rpm in a
500 mL beaker. To reduce ferric ion into iron nanoparticle, 100 mL of 2.5 mM aqueous
sodium borohydride solution was added at a rate of 0.625 mL/s. The black color was formed
after completing the reaction. The reaction mixture was centrifuged at 11000 rpm for 15 min
and then washed with distilled water for several times to remove impurities. To avoid the
oxidation of iron nanoparticles, it was stored in methanol until further use.
32.2.3 Production of biodiesel

340

The batch experiments were conducted in 250 mL three necked flask connected with
agitator, reflex condenser and thermometer for biodiesel production. 50 g of neem oil was
added to the flask and it was allowed to heat in the water bath at 60 C under stirred
conditions. Then, 1 wt % of iron nanoparticle (relative to neem oil weight) was added to the
flask as a catalyst. 3:1 molar ratio of methanol: oil was taken for this study. The reaction was
carried out for a period of 2 hr at 300 rpm. After the completion of the reaction, the mixture
was cooled to the room temperature and transferred into a separating funnel. The glycerol was
allowed to separate from the mixture. The crude fatty acid methyl esters (FAME) were
washed several times with 1:1 volume of warm distilled water about 50C in a separating
funnel until the neutral pH was obtained.
32.2.4 Characterization of iron nanoparticles
The reduction process of the iron nanoparticles was monitor using Shimadzu UV-2450
UVVisible spectroscopy. The dried iron powders were subjected to X-ray diffraction
analysis using PAN analytical- XPERTPRO diffractometer system, Netherlands.
Morphological studies of iron nanoparticles were performed using scanning electron
microscopy (SEM) equipped with an EDX detector (Model No: JSM 6390LV) at a
magnification of 5000X. The size of iron nanoparticle was analyzed by transmission electron
microcopy (TEM) made by Philips, Netherland (Model: Tecnai 10).
32.2.5 Characterization of biodiesel
The quality of biodiesel was expressed in terms of the physicochemical properties
including specific gravity, viscosity, flash point, cloud point, water content, carbon residue
and copper corrosion. These properties of the biodiesel were determined as per the methods of
American Standards for Testing Material (ASTM) and compared with the specification as per
ASTM D6751. A single cylinder, air-cooled, four-stroke, direct injection Kirloskar, diesel
engine was used to measure gaseous emissions including CO, CO
2
and NOx for biodiesel
blend B20 (20 % biodiesel and 80 % regular diesel by volume) and regular diesel.
32.3.1 UV- Visible spectrum of iron nanoparticle
The synthesis of iron nanoparticles were monitored with the color changes and UV-
Visible spectroscopy. It was observed that, the reaction mixture was turned into brownish
black within 15 mins from its original pale yellow colour, when the slow addition of 2.5 mM
of aqueous sodium borohydride into 2.5 mM aqueous FeCl
3
solution was done. As seen in the

341

Fig.1, the appearance of brownish black color indicated the reduction of Fe
3+
ions into the
formation of iron nanoparticles. Similar results on visual observations have been reported for
the formation of iron nanoparticle using tea extract by Hoag et al., (2009). In addition, the
UV-Visible spectra were recorded for the reduction of Fe
3+
ion and the results are presented in
Fig. 2. The UV-visible spectra of FeCl
3
without reducing agent did not show a distinct peak,
whereas the appearance of peak at 260 nm after the addition of reducing agent into aqueous
FeCl
3
revealed that the complete reduction of the Fe
3+
ion was confirmed and led to the
formation of iron nanoparticles.

Fig 1. Visual observation of Ferric Chloride
Reduction in stages
200 400 600 800
0.00
0.25
0.50
0.75
1.00
1.25
A
b
s
o
r
b
a
n
c
e
Wave length(nm)
Fe1
Fe2
Fe3
Fe6
Fe7
Fe8
Fe10
Fe12

Fig 2. UVVisible absorption spectra of
FeCl
3
before and after reduction

32.3.2 X-Ray Diffraction
The crystalline nature of synthesised iron nanoparticles were confirmed
using X-ray diffraction analysis (Fig.3). The result indicates that the iron nanoparticles were
cubic and observed the characteristic diffraction peak of plane at (110). The obtained peak
value of iron nanoparticles were confirmed with JCPDS 85 -1410. The similar results were
reported by Lee et al., (2004) and Sixin et al. (2006). The average particle size of the iron
nanoparticles were determined using Scherer equation and the size of the nanoparticle was in
the range of 40 to 70 nm.
32.3.3 Scanning electron microscopy and EDX
The iron nanoparticles synthesized using chemical method was analyzed in Scanning
Electron Microscope and shown in Fig.4. Typical SEM image of the synthesized iron

342

nanoparticles clearly shows that the morphology of particles was observed to be a non-
uniform shape under the tested conditions.

Fig.3. XRD Pattern of iron nanoparticle
Energy dispersive X-ray (EDX) analysis was used for identifying the elemental
composition of the iron nanoparticle. The quantitative elements present in the synthesized iron
nanoparticle are shown in Fig 5. The obtained quantitative percentage of iron nanoparticles
was found to be 43.75.

Fig. 4. SEM images of iron nanoparticles (A)
Magnification: 5,000, inset bar: 1 m.

Fig. 5. EDX image of iron nanoparticle

Position [2Theta]
10 20 30 40 50 60 70 80
Counts
0
10
20
Fe -chem

343

32.3.4 Transmission electron microscopy
Transmission electron microscopy (TEM) was used to determine the core size and
shape of the iron nanoparticles. TEM image of the synthesised iron nanoparticles is shown in
Fig 6. It was observed that the iron nanoparticles were polydisperse with different shapes
including spherical, cubical triangular and hexagonal. Average particle size was found to be
the range from 40 to 70 nm. The size obtained using Scherer equation is consistent with the
size observed using TEM.

Fig.6.a. TEM images of iron nanoparticles
at 200 nm scale

Fig.6.b. TEM images of iron nanoparticles
at 100 nm scale

32.3.5 Physical and Chemical properties of biodiesel
Transestrification from neem oil and methanol was catalyzed by iron nanoparticles for
the production of biodiesel. The fuel properties of produced biodiesel were analyzed using
ASTM test method and the obtained results are given in Table 1. The experimental results
were compared with ASTM D6751 standard and the values were found to be within the
specification range.
The important fuel properties of biodiesel including specific gravity, viscosity, flash
point, cloud point, water content, carbon residue content and copper corrosion test were analyzed
using ASTM method. The results showed that the properties of produced biodiesel were
relatively closer to that of regular diesel. Similar findings were reported by Hanna (1999);
Antolin et al., (2002). Since the viscosity of the biodiesel is closer to that of diesel, no hardware
modifications are required in the existing engine for handling this biodiesel. The cloud point is
the temperature at which the material ceases to flow when the fuel is cooled under prescribed
conditions. The cloud point of the produced biodiesel was determined to be 3
o
C. The result
suggests that the higher value of cloud point could work in cold conditions when compared to
that of regular diesel.

344

Water content in fuels imposes engine corrosion problems or reacts with glycerides to
produce soaps and glycerin. In our experimental results, water content was about 0.01 vol%.
This finding indicates the better performance of the corrosion resistance of diesel engine.
Carbon residue of the biodiesel is an indication of carbon deposition tendencies on diesel
engine. Presence of impurities like free fatty acids, glycerides, soaps, polymers and inorganic
contents are indicators of higher carbon residue (Meher et al., 2006). The result showed that
the biodiesel obtained in this study was less impurities. Copper corrosion test results indicated
that the biodiesel was superior quality when compare to normal diesel. The value of copper
corrosion for the produced biodiesel was found to be 1b. This result indicates that the
biodiesel has good resistance to copper corrosion.
Table 1. Fuel properties produced biodiesel from neem oil using iron nanoparticle
Physiochemical
Properties
ASTM Test
Method

Biodiesel
Specification
as per
ASTM D6751
Values of produced
Biodiesel
Specific gravity D4052 0.81-0.90 0.875
Kinematic Viscosity,
40 C (mm
2
/s)
D445 1.9-6.0 4.9
Flash point (C) D93 130 145
Cloud point (C) D2500 -3 to 12 3
Water content
(% vol)
D2709 0.05 0.01
Carbon residue
(% mass)
D4530 0.05 0.03
Copper strip
corrosion
D130 No.3 max. 1b

32.3.6. Emission Characteristics
The CO emission characeristics of B20 biodiesel and normal diesel was studied by
varying the load (0, 2.5, 5.0, 7.5 and 10 Kgs) of diesel engine and the results are shown in
Fig. 7. It was observed that 30 to 40 % of CO emission for B20 biodiesel was lower than the
normal diesel. The possible reason may be due to the presence of high oxygen content in B20
biodiesel, which enhanced the combustion process. This result confirms that CO emission was
inhibited by reducing the incomplete combustion process of diesel engine. Similar results
were obtained by Park et al., (2009).


345

Figure 7: CO Emission of B20 biodiesel blend and regular diesel
The emission levels of CO
2
for B20 biodiesel blend and normal diesel at various loads
of diesel engine are shown in Figure 8. The experimental result revealed that the CO
2

emission for B20 biodiesel were less as compared to normal diesel at all loads due to presence
of lower carbon content. The CO
2
emission was increased with increasing the load, because of
the higher fuel entered into the diesel engine. Similar observations were made by Ekrem
Buyukkaya. (2010).

Figure 8: CO
2
Emission of B20 biodiesel blend and regular diesel
The emission of NOx for B20 biodiesel and normal diesel is shown in Fig.9. NOx are
generally formed at high temperature since the exhaust gas temperature is higher. In the
present study, the NOx emission of B20 biodiesel was slightly higher than the regular diesel
for all the loads. Similar results were made by Lin et al.,(2007).

0
0#02
0#04
0#06
0#08
0#1
0#12
0#14
0 2 4 6 8 10 12
C
O

E
m
i
s
s
i
o
n

i
n

V
o
l
%
e
Load in Kgs
diesel
20%bidiesel
0
0#1
0#2
0#(
0#4
0#)
0#6
0#$
0#8
0 2 4 6 8 10 12
C
O
2

E
m
i
s
s
i
o
n

i
n

%
V
o
l
Load in Kgs
diesel
20%bidiesel

346

Figure 9: NOx Emission of B20 biodiesel blend and regular diesel
32.4 Conclusions
Iron Nanoparticles was synthesized by liquid phase reduction method from ferric
chloride solution using sodium borohydride as reducing agent. The size of the synthesized
iron nanoparticle was in the range of 40 70 nm. Transesterification reaction was carried out
from neem oil: methanol mixture using iron nanoparticle as a catalyst for the production of
biodiesel. The properties of produced biodiesel from neem oil were closer to normal diesel.
The results of the study conclude that B20 biodiesel had lesser emissions of CO and CO
2,
when compared to the regular diesel. The emission of oxides of nitrogen from the engine was
found to be slightly higher as compared to regular diesel. Hence the produced biodiesel can be
considered as an alternative to the regular diesel.

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10
1)
20
2)
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N
O

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m
i
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i
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20% /idiesel

347

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349

CHAPTER 33
INFLUENCE OF FREE FATTY ACIDS CONTENT IN
CATALYTIC ACTIVITY OF [BSMIM] Cl IONIC LIQUID FOR
BIODIESEL PRODUCTION FROM NON EDIBLE ACIDIC
OILS
Subrata Das, Ashim Jyoti Thakur, Dhanapati Deka

Abstract
A Brnsted acidic ionic liquid 1-(1-butylsulfonic)-3-methylimidazolium chloride
([BSMIM]Cl) was synthesised and applied as a catalyst in the pretreatment of high free fatty
acid (FFA) containing various non edible acidic oils such as: Jatropha curcas, Pongamia
pinnata, Mesua ferrea L. and Thevetia peruviana. The ionic liquid showed high esterification
activity with a FFA conversion 91-94% depending on the oil under the optimum reaction
conditions of 10wt% catalyst,1:12 oil methanol molar ratio at 70C in 6 h. Our results showed
that both the esterification and transesterification activity of the ionic liquid catalyst was
affected by the FFA content of the oil, an increased activity was observed with the increase in
FFA content. For Mesua ferrea L. oil having the highest FFA content of 20.68wt%, the
maximum methyl ester yield 52.63% was obtained out of which 19.06% and 33.56% was due
to esterification and transesterification respectively. In this particular paper a method to
determine the individual yield due to esterification and transesterification separately was
reported.
Keywords: Ionic liquid, FFA, esterification, transesterification.
33.1 Introduction
Biodiesel is obtained from the triacylglycerols of vegetable oils or animal fats and free
fatty acids (FFAs) by transesterification and esterification respectively (Borugadda and
Goudn, 2012). The edible oil feedstocks accounts up to 60-75% of the total biodiesel
production cost and hence makes the biodiesel more expensive (Ma and Hanna, 1999). The
utilization of non edible oils such as crude acidic oils(AOs) as feedstocks can be a solution to
this problem due to its low cost and availability (Atabania et al., 2013). However, the standard



350

biodiesel production by applying the conventional alkaline catalysts cannot be allowed to
these feedstocks due to the presence of FFAs which produces soaps with alkalis (Ma and
Hanna, 1999).Therefore the FFA level in the crude oils should be reduced to 2 wt% prior to
transesterification by an esterification or pretreatment step (Lam et al.,2010).Liquid inorganic
acids and solid acids are widely used in the esterification step. However, the homogenous
mineral acids such as H
2
SO
4
have serious drawbacks such as no recovery, corrosivity and
generation of waste water in washing and neutralizing the acids (Dokic et al., 2012).On the
other hand, solid acids have reduced activity, deactivated easily and adsorbed the products
into their fine powders therefore makes the separation process difficult (Zhang et al.,
2009).Therefore an alternative and environment friendly catalyst is always necessary. In this
respect the application of ionic liquids (ILs) as catalysts in this process has become a worthy
area of investigation (Zhao and Baker, 2013).
ILs is molten salts with tunable physical and chemical properties and composed of
only cations and anions (Welton, 2004). Room temperature ILs (RTILs) are applied as
catalysts because some of its unique features such as wide liquid range, large range of
solubility, variable Lewis and Brnsted acidity, high polarity, non flammability, high thermal
stability, large electrochemical window, negligible vapor pressure and potential for
recyclability and reusability (Zhao et al., 2002; Bourbigou et al., 2010). RTILs have high
catalytic activity and recyclability and thus have the potential to replace the homogenous
catalysts such as sulphuric acid, p-toluene sulphonic acids and heterogeneous solid acids in
esterification reactions (Joseph et al., 2005).
In present investigation, an acidic IL{[BSMIM]Cl} was used for the pretreatment or
esterification of crude Jatropha curcas oil(JCO), Pongamia pinnata oil(PPO),Mesua ferrea
L.oil(MFO) and Thevetia peruviana oil(TPO) and the effect of initial FFA content in the
esterification and transesterification activity of the catalyst was studied. This IL has high
catalytic performances in esterification of FFAs of the said crude AOs. Additionally the
synthetic procedure of the ionic liquid is simple, easy, requires no additional solvent and atom
efficient since the generation of byproducts does not arise.
33.2.1 Materials
Local dried seeds of Jatropha, Pongamia pinnata, Mesua ferrea L. and Thevetia
peruviana were purchased from the Kaliabor Nursery, Kaliabor, Assam, and India. 1-

351

Methylimidazole (99%), 1, 4-Butane Sultone (99%), 2-propanol (anhydrous, 99.5%) were
bought from Sigma Aldrich, India. Methanol (99%, GC grade), hexane (fraction from
petroleum), HCl (35% for analysis), KOH (Analytical grade) were procured from Merck India
Limited. All the chemicals were used as received without any further purification.
33.2.2 Instruments
1
H and
13
C NMR spectra were recorded in a 400 MHz NMR spectrophotometer
(JEOL, JNM ECS) using tetramethylsilane (TMS) as the internal standard and coupling
constants are expressed in Hertz.
33.2.3 Extraction and purification of JCO
The extraction of JCO, PPO, MFO and TPO were carried out by Soxhlet extraction
method by taking hexane (boiling point 65-70C) as solvent according to the AOAC method
2003.06. The solvent was separated from oil by rotary vacuum evaporator. The collected oils
were filtered and then heated at 100C for 10 min to remove the moisture (Jain and Sharma,
2010).
33.2.4 Preparation of IL
IL was prepared according to the method reported earlier (Kore and Srivastava, 2012).
33.2.5. Reaction procedure and calculations
The laboratory scale esterification reactions were carried out by adding 10 gm of AOs,
12:1 methanol oil molar ratio and 10wt% of [BSMIM]Cl IL catalyst in a round bottom flask
which was equipped with chilled water cooled condenser and a hot plate with magnetic
stirrer. After the reactions were performed for 6h at 60C with vigorous stirring the reaction
mixtures were cooled and centrifuged. After centrifugation of the respective cooled mixtures
for five minutes two layers were obtained. The esterified oil was in the upper layer and
separated by simple decantation while the lower layer was the mixture of ionic liquid, excess
methanol and water generated during the reaction. Pretreated oils were heated, dried over
anhydrous Na2SO4 and subjected to the determination of acid value by titration against
standard KOH solution. Conversions of FFAs due to esterification were calculated based on
acid value determination by using the following equation (Corro et al., 2013):

C
Est
(%) =

Where, A
i
is the initial acid value and A
t
is the acid value at time t.
A
i
A
t

A
i

100
(1)

352

The acid values were determined by using the following equation (Corro et al., 2013):

A=

Where, A=acid value (mg KOHgm
1
), N=strength of KOH solution (in normality),
V=volume of the KOH solution consumed, W= weight of the sample.
Acid values were measured by titration of small amounts (0.2-0.5g) of either AOs or
pretreated oils in 2-propanol with standard KOH solution. Accordingly the FFA (wt%)
content of the both acidic and pretreated oils were also determined from the equation
below(Jang et al.,2012):

Acid value= .. (3)

Following this the analysis of the pretreated oils were analysed by 1H NMR and the
total methyl ester yields were evaluated from the NMR analysis by using the following
equation (Tariq et al., 2011):

Where, A
Me
= integral of methoxy methyl ester peak at 3.6 ppm and
A
-CH2
= integral of -methylene peak at 2.3 ppm.
The yields due to transesterification were determined by subtracting the esterification
yield (in terms of methyl ester).
33.3.1 Determination of acid values of AOs
Acid values and FFA content of the acidic oils were determined according to the
equation (2) and (3) and the results are described in Table 1.

C
Tot
(%) =
2A
Me

3A
-CH2

100
(56.1NV)
W
(4)
(2)
FFA
2

353

Table 1. Acid values of the AOs
AOs Acid value(mg KOHgm
1
) FFA(wt%)
JCO 16.32 8.20
PPO 29.38 14.76
MFO 41.16 20.68
TPO 18.98 9.53

33.3.2. Influence of FFA content on catalytic activity
Different AOs with variable FFA contents (~8.20-20.68wt%) were subjected to
pretreatment or esterification reactions under the reaction conditions mentioned above and the
results are given in the table 2 and figure 1.
Table 2. Results of pretreatment reactions of AOs

a
Reaction conditions: catalyst=10wt%, methanol oil molar ratio=12:1, reaction
temperature=60C,reaction time=6h.
The FFA content of the AOs reduced below the permissible limit of 2wt% for the
alkali catalysed transesterification and hence the IL catalyst can be effectively applied in the
pretreatment of AOs (table 2).From the figure and the table 2 it was evidenced that both the
esterification and transesterification activity of the IL catalyst increases with the increase in
acid value and FFA content of the acidic oils. The most acceptable explanation of the
AOs Acid value FFA
C
Est
(%)
a

Methyl ester yield(%) due to
C
Tot
(%)
a

Initial
Fina
l
Initial
Fina
l
Esterification
Transesterificatio
n

JCO 16.32 1.01 8.20 0.50 93.8 7.69 1.84 9.53
TPO 18.98 1.10 9.53 0.55 94.2 8.97 2.29 11.26
PPO 29.38 2.57 14.76 1.29 91.2 13.46 32.51 45.97
MFO 41.16 3.22 20.68 1.61 92.1 19.04 33.59 52.63

354

observation lying on the fact that the solubility of methanol was more in high FFA content
AOs than in low (Lakhya et al.,2013). Also the increments in transesterification yields were
more as compared to esterification yields for higher FFA content AOs and vice versa. The
proper explanation of this behavior may be due to the fact that in higher FFA content AOs
methanol was more soluble and can interact with the triglycerides molecules more effectively
than at lower FFA content AOs. At lower FFA content AOs methanol due to its low solubility
remains in the FFAs and gets less interaction with the triglycerides molecules and involved in
esterification of FFAs only.

Figure 1.Variation of esterification and transesterification activities of [BSMIM] Cl catalyst
with respect to FFA content of acidic oils
33.4 Conclusion
The synthetic method of the [BSMIM] Cl IL is very easy and green since no
additional solvent is necessary and also no byproducts are generated during the synthesis. IL
catalyst is very much active in the pretreatment of AOs. Under the mild reaction conditions
(10wt% of IL catalyst, 12:1 methanol oil molar ratio for 6h at 60C) 91-94% of the FFAs of
AOs esterified.IL catalyst lowered the FFA level of AOs below 2wt% and made the oils
suitable for alkali based transesterification. Thus this catalyst makes the way to produce
biodiesel from low cost AOs and hence makes the overall biodiesel production less expensive.
The high esterification and transesterification activity of the IL catalyst for high FFA content

355

AOs paves the way for the generation new types of IL from this parent IL for the production
of biodiesel from AOs and our group is actively involved in this field.
Acknowledgements
This work was financially supported by DST Green Chemistry Project
(No.INT/FINLAND/P-02).

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16. Zhao D., Wu M., Kou Y. and Min E. (2002) Ionic liquids: applications in catalysis.
Catal. Today, 74:157189.
17. Zhao H. and Baker G.A. (2013) Ionic liquids and deep eutectic solvents for biodiesel
synthesis: a review. J. Chem.Technol. Biot. 88: 312.


357

CHAPTER 34
ANALYSIS OF PHYSICAL PROPERTIES AND BIODIESEL
PRODUCTION FROM DIFFERENT ACCESSIONS OF
JATROPHA CURCAS
Dheeraj Singh, Chiranjib Banerjee, Animesh Sinha, Diwaker Prasad Nirala, Santosh Prasad,
Rajib Bandopadhyay

Abstract
India is expected to at least double its fuel consumption in the transportation sector by 2030.
To contribute to the fuel supply, renewable energies such as Jatropha appear to be an
attractive resource for biodiesel production in India as it can be grown on waste land and does
not need intensive water supply. Hence biodiesel will act as future perspective to cope up with
the energy crisis for domestic as well as industrial purpose. Biodiesel, which is environment
friendly, non-toxic and biodegradable used in diesel engine. The jatropha oil can be used for
soap production and cosmetics production in rural areas. The oil is a strong purgative, widely
used as an antiseptic for cough, skin diseases and as a pain reliever from rheumatism. The
production of Jatropha biodiesel will be done by mainly three process- acid catalyzed, base
catalyzed, and enzyme catalyzed. Acid catalysis is generally preferred due to presence of
moisture and free fatty-acid. Although the process of biodiesel production is slow but it will
resist the production of soap during trans-esterification process and hence good quality of
biodiesel will be produced. During the trans-esterification process, generation of fatty acid
methyl ester (biodiesel) will require the methanol to react with fatty acid in the proportion of
5:1. The TLC has been performed to separate the different fatty acid including TAG
(Triacylglycerol). Different accessions of Jatropha seed were collected and their biochemical
parameters for biodiesel production were evaluated. The transesterified fatty acids were
checked through gas chromatography, leading to analysing the different fatty acid
composition. Further the major physico-chemical properties of Jatropha biodiesel will be
compared with diesel fuel. The physico-chemical properties of the produced biodiesel will be
characterized according to the ASTM D6751 standards. The physico-chemical properties
such as Cetane number, Iodine value and Saponification value, will be further analyzed. In
future 10% blending will be performed to test the engine efficiency.


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34.1 Introduction
The increasing economy of India has relatively increased its dependency on fossil
fuels where petro-based fuels account for about 95% of Indias
transportation energy demand (National Policy on Biofuels, 2009). Present analysis shows
that current growth in economy fulfils only 24% of the total oil demand so the dependency on
foreign crude oil is increasing rapidly (US EIA, 2013). According to the present scenario,
within two decades the country will nearly cross the limit of 500 million vehicles with current
of 150 million (Sharma, 2011). This transient enhancement in the fuel dependency on
imported energy causes the negative effect attributed to petro-fuel utilization such as
greenhouse gas emission.
Since biofuels can be easily mixed with fossil fuel and used in usual engines. As
biofuel seems to be challenging source of renewable energy hence, among few possible
surrogate substitutes some parts of fossil fuels are used in transportation sector. As a
legislative attempt to supplement the share of biofuels in India, the Ministry of New and
Renewable Energy set up a National Policy which defined an indicative target of 20%
blending of biofuels -whether biodiesel or bioethanol by 2017 (Ajayebi et al., 2013).
Biofuel is a renewable diesel, which is normally transformed by the transesterification
reaction of non-edible oil, vegetable oil, waste cooking oil and fats with smaller chain of
alcohols (generally methanol or ethanol). The reaction generally occurs in the presence of
acid, base or enzyme (Leduce, et al., 2009). In the absence of enzyme, high temperature and
pressure will be required while in the presence of enzyme low temperature and pressure is
required. Generally methanol is used in place of ethanol for transesterification reaction due to
its cheap cost. Usually base catalyzed reaction i.e. NaOH, KOH or corresponding alkoxides is
used for the reaction because it is faster than acid catalyzed reaction (Ataya et al., 2007).
Presence of high free fatty acid content in the seed oil results in lesser production of biodiesel.
It also causes the saponification reaction, hence sodium or potassium ions reacts with it and
form the soap which requires several washing and increase the cost of the production. So, for
commercial process people generally used cheaper feed stock (Baroi et al., 2009).
Biodiesel is a mixture of long fatty-acid chain of mono-alkyl ester which is derived
from the lipid content feed-stocks such as non-edible oil, animal fat, vegetable oil,
lignocellulosic material as an alternative fuel for internal combustion engines. Generally
biodiesel consists of long chain of fattyacids; the chain length is between C14- C22. And the
ester should be of either methanol or ethanol. The first diesel engine developed by Rudolf

359

Diesel, in 1900, was run with ground nut oil hence biodiesel is the best material for internal
combustion engines. Diesel engines got the vogue due to its higher thermal efficiency and
also have more power to weight ratio. Therefore it is largely used in the industries and
automobiles for energy generation. Hence transesterified oil is widely accepted biofuel.
34.2 Source: Jatropha Curcus
Jatropha curcus L. (commonly known as physic nut, purging nut, Barbados purging
nut, ratanjot etc.) oil is known to be the most useful biofuel production. Jatropha belongs to
the family Euphorbiaceae. The fruit of the Jatropha is generally used for the treatment of
some sexually transmitted disease, dysentery, small pox, infertility and some skin infections.
In many tropical countries, soap and cosmetics were prepared by the seed oil. Several
components derived from the leaves and latex of the Jatropha have properties of wound
healing, anti-inflammatory and also act as relief to the patients suffering from rheumatism.
Due to increase in demand of biofuel, many industries have started using vegetable oil
for the production of biofuel which caused hike in the price of vegetable oil and enhanced the
rate of malnutrition. Hence, Jatropha curcus can act as an important feed stock for biofuel
production. Jatropha curcus has wide range of adaptability in many ecological environments
and require less nutrient for development, it can also grow on non-arable land which shows
that it does not compete with food production. It can also be useful in reclamation of marsh
land. Due to its simple agricultural process, non-toxic, ecofriendly, renewable, and bio-
degradable is gaining interest of Government of India for Jatropha (Ratanjot) mission
(Chouhan & Sarma, et al., 2013).
The oil content of the Jatropha biodiesel from seed varies from 29 50%, depending
upon different geographical condition. Out of which 21% is of saturated fatty acid and 79% is
of unsaturated fatty acid. Jatropha curcus oil is rich in Nitrogen, Phosphorus and Potassium
hence used as organic manure (see Table 1 & 2).
Table 1: Content of Jatropha curcus seed
Ingredient %Composition
Moisture 06.20
Protein 18.00
Fat 38.00
Carbohydrate 17.00
Fibre 15.50
Ash 05.30

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Table 2: Detailed Fatty Acid composition of Jatropha curcus seed
Fatty acid %Composition
Palmitic acid 21.0
Stearic acid 12.2
Oleic acid 30.0
Linoleic acid 36.7
Others 01.4
* Source: Prepared from William Elliott, 2013.

34.2.1 Advantages of Jatropha
1. Irrigation of Jatropha plant on marginal land with less nutrient demand and moisture
content.
2. Jatropha plant can be cultivated on low as well as high rainfall region.
3. It can be propagated through branch pruning and seed.
4. It cannot be compete with useful agricultural land.
5. It is pest as well as disease resistant.
6. The by-product generated during chemical process can be used for other commercial
purpose.
7. After oil extraction, the seed cake generated is used as fertilizer in the field.
8. Due to presence of some toxic material it is not fit for human to use, hence Jatropha
oil eliminates tie competition with feed oil.
9. Jatropha plantation can create occupation in rural area and hence help in holding the
degraded land (Atabani et al., 2013).
34.2.2 Disadvantages of Jatropha plantation
1. It is planted in scattered location and generally mixed with the other shrubs present in
the forest region hence its collection is very much tedious job.
2. Different geographical conditions at different places causes different oil content. This
has lead to non-availability of proper quality of seed.
3. Due to improper marketing channel it has limited period of availability.
4. Improper technology for post- cultivation process and their processing.
5. There is huge fissure between actual production and competent production.
6. There is lack of Government funding and proper incentives to promote the biodiesel
production.

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34.3 Different criteria which effect the biodiesel production
34.3.1 Effect of free fatty acid and moisture
For transesterification reaction, the moisture content and free fatty acid should have
low values because it will cause the formation of soap and hence become difficult to separate
glycerol from it. It also increases the viscosity of the FAME.
34.3.2 Effect of molar ratio
The most important unsteady state which create problem in the ester yield is the molar
ratio of the triglycerides and alcohol. The stoichiometry shows that three moles of alcohol is
required for the proper conversion of triglyceride to three moles of fatty acid methyl ester and
one mole of glycerol. The molar ratio is generally depend upon the type of catalyst used, in
case of acid catalyzed reaction, the molar ratio should be of 9:1, because higher the reactant
more forward the reaction will proceed, according to Le- Chatlier principle (Manzetti, 2011).
34.3 Effect of catalyst
Generally there are three types of catalyst used, namely acid catalyst, base catalyst,
and enzyme catalyst. Alkali catalyzed reaction is more faster than acid catalyzed reaction but,
if the glyceride has higher content of free fatty acid and moisture content then it should be
preferred to perform the acid catalysis process because it prevents the formation of soap,
hence viscosity should be decreased.
34.4.1 Collection of Jatropha seed
Jatropha was planted during the end of the year 2010 at Nagri, Ranchi (2321.388' N;
8514.661' E and 685m above sea level) in a total area of 14 hectare. At present, plants are
three years old. According to evaluation report conducted by the funding agency (Department
of Biotechnology, Government of India, New Delhi), the performance of the trial plots is one
of the best plots in the Jatropha network project funded by DBT. For the present work, the
Jatropha seeds were procured from Institute of Forest Productivity (IFP), Lalgutwa, Ranchi,
(Jharkhand).


362

34.4.2 Climate and Soil condition of the study location
The temperature in summer is normally vary from 20 C to 42 C and in winter it is
vary from 0 C to 25 C. The average relative humidity of the experimental site is 73%. The
annual precipitation on the experimental site is 1430mm (Singh et al., 2013).
Soil at the experimental location consists of mixture of laterite and red soil having
high content of iron. Laterites are soil types rich in iron and aluminium, formed in hot and wet
tropical areas. Nearly all laterites are rusty-red because of iron oxides. They are developed by
intensive and long-lasting weathering of the underlying parent rock. Tropical weathering
(laterization) is a prolonged process of chemical weathering which produces a wide variety in
the thickness, grade, chemistry and ore mineralogy of the resulting soils. The majority of the
land area containing laterites is between the tropics of Cancer and Capricon. The texture of
Red soils varies from sand to clay, the majority being loams. Their other characteristics
include porous and friable structure, absence of lime, kankar and free carbonates, and small
quantity of soluble salts. Their chemical composition include non-soluble material 90.47%,
iron 3.61%, aluminium 2.92%, organic matter 1.01%, magnesium 0.70%, lime 0.56%,
carbon-dioxide 0.30%, potash 0.24%, soda 0.12%, phosphorus 0.09% and nitrogen 0.08%. So
the pH of the soil is generally acidic due to presence of iron in large quantity and hence it
varies between the ranges of 4.5 to 5.1.
34.4.3 Collection of Jatropha accession
IFP, Ranchi collected seeds from different parts of India through DBT, GOI. Among
them, two different accessions were selected for the preliminary study. One sample is from
Lucknow Biotech Park, UP whose accession number is IC550449, having the oil content of
40%. The other sample was collected from Ruchi, Indore, MP whose accession number is
IC569131, having the oil content of 29.64%.
34.4.4 Cultivation method and treatment
Six month old cuttings were planted at the spacing of 33 m. The normal pruning
practise is done three times in a year to make sure that more branches can grow to increase the
fruiting phenomena. Plant seedling was collected from Lucknow, having accession number
IC550449 were treated with Nitrogen, Phosphorus and Potassium fertilizer constant but the
treatment time is once in a year and twice in a year. Plant collected from Indore having
accession number IC569131 were treated with fertilizer, thrice in a year with sulphur content
and other plant were treated with vermi-compost and biofertilizers twice in a year.

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34.4.5 CHNS analysis
Different elements were analysed (Carbon, Nitrogen, Hydrogen, Sulphur) in the seed
of Jatropha by ELEMENTAL ANALYZER (Germany, VarioEL III). Which is having the
combustion temperature between 950-1200
0
C and the carrier gas used for this purpose is
Helium.
34.5 Production of Biodiesel
34.5.1 Oil extraction
For oil extraction from the Jatropha seeds, 1 g of seed was taken and decorticated and
then (Kalbande et al., 2008) crushed using Mortar pestle to make a fine powdered form to
extract oil. Solvent extraction process was used to extract oil from it. Chloroform and
Methanol was added in the proportion of 1:2 and volume makeup was done upto 20 ml for
proper extraction of oil (Bligh and Dyer, 1959). Chloroform is a non-polar solvent and
methanol is polar solvent hence both the solvent use their property in which like dissolves
like, non-polar lipid dissolves in chloroform and polar lipid dissolve in methanol. The
solution was kept for 48 hours at room temperature for maximum oil extraction. The sample
is then centrifuged at 3000 rpm for 6 minute to settle down the non-dissolved substance. The
solution was extracted and 0.9% of NaCl solution was added to separate the upper methanol
phase and lower chloroform phase. The lower chloroform phase containing non-polar lipid
was taken out. The chloroform phase was concentrated either by flushing by rotatory
evaporator (Buchi, Germany). After that the percentage of dry lipid per dry biomass (g/g) was
calculated. Finally dried lipid was again dissolving in 1 ml of chloroform for further
processing (Figure 3).
34.5.2 Transesterification
Transesterification of total lipid into Fatty Acid Methyl Ester (FAME) has been done
by acid catalyzed reaction (fig.1). To 1 ml of concentrated lipid extract, 5 ml of solvent
Methanol-Benzene-Sulphuric acid in the proportion of 8.6:1.0:0.5 respectively was added by
volume. Sample was incubated at 60 C for at least 4 hours for proper FAME conversion.
Then 7 ml of warm water was mixed well to remove the impurities and glycerol formed. After
that 5 ml of hexane was added and mixed carefully so that the transesterified lipid comes into
the hexane phase by solvent extraction method. The upper layer were properly collected and
placed in new vial. Little amount of sodium sulphate was added to absorb the water. Nitrogen

364

gas was flushed to concentrate the FAME mixture. All the chemicals were used of analytical
grade (AR).
34.5.3 Quantitative analysis
Qualitative analysis was done by Thin Layer Chromatography (TLC), it was prepared
by mixing 12 gram of Silica gel G-50 (MERCK, U.S.A) in 30 ml of distilled water and was
poured on clear TLC glass plate. After drying at room temperature the plate was finally
activated by keeping the plate in hot air oven (RIVOTEK, India) for 3 hours before use.
The transesterified mixture was run with Supelco FAME mix as standard by using
solvent system Petroleum ether: Diethyl ether: Acetic acid in the proportion of 80:20:2.

Fig.1. Transesterification of triglyceride with alcohol (adopted from Hanna et al., 1999).
Two Physical characters of the seed are shown below in table 3 and figure 2.
CHNS element analyser showed the different content of the nitrogen, carbon, sulphur and
hydrogen present in the different accessions of the Jatropha seed. The inference concluded
with this result shows that sample S4 which was grown adding of Sulphur as an extra nutrient
during manuring which may cause increase in C/N ratio and it may increase in the lipid
content of the seed.
Scanning Electron Microscopy
The below figure 2 (a) and (b) shows the SEM analysis of the seed coat which tells
about the presence of lignocellulosic fibrils inside and smooth outside coating.
After rotatory vaccum evaporator of the extracted lipid, the weight of the lipid
percentage was calculated (Table 4 & Figure 3).
The figure 3 illustrates the transesterified lipid present in the seed of the Jatropha
curcus. The figure 4 shows the TLC analysis of Fatty Acid Methyl Ester on Silica gel G-50.


365

Table 3: CHNS element analysis
S.No. Accession No. C/N ratio Content (%)
1 IC550449 18.39 N: 2.850

C: 52.40
S: 0.284
H: 9.616
2 IC569131 13.04 N: 4.124

C: 53.77
S: 0.282
H: 10.02
3 IC569131 21.35 N: 2.427

C: 51.82
S: 0.154
H: 8.935
4 IC550449 34.27 N: 1.583

C: 54.25
S: 0.126
H: 9.588
5 1C550449 25.44 N: 2.426

C: 61.72
S: 0.136
H: 11.43

Figure: 2 (a) & (b): SEM analysis of Jatropha seed.
Table 4: Lipid content of different accessions
S. No. Accession No. Lipid percentage
1
2
3
4
5
IC550449
IC569131
IC569131
IC550449
IC550449
28.85
41.27
33.20
38.08
37.58

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Fig.3: Transesterified oil of Jtaopha seeds Fig.4: TLC analysis of transesterified oil of
Jatropha seed
34.7 Conclusion
Transesterification of natural oils and fats is preferred over several methods available
for biodiesel production. This process is chosen to lower the viscosity of oil. While blending
of oil and other solvents diminishes the viscosity, engine performance, but problems, such as
carbon deposit and lubricating oil contamination, still exists. The seed coat consists of
lignocellulosic material hence biomass recovered after oil extraction will help in production
of biogas and also used as manure after pretreatment. Biodiesel can be used most efficiently
as a supplement to other energy forms, and not only as a primary source. Biodiesel is
particularly useful in mining and marine situations where maintenance of low pollution levels
is essential.
Acknowledgement
DS would like to acknowledge M. Tech. fellowship support from Centre of Excellence
(COE), TEQIP, PhaseII (Ref .No NPIU/TEQIP II/FIN/31/158; dated 16th April, 2013). AS is
highly thankful to DBT (Ref No. BT/PR 11962/PBD/26/2009 for financial support to collect
clonal materials and maintenance at IFP, Ranchi. All the authors acknowledge to HOD,
Biotechnology and Central Instrument Facilities (CIF) for providing the instrument facilities
and support during conducting this work. CB gratefully acknowledges the financial support as
Senior Research Fellow (SRF) from Council of Scientific & Industrial Research (CSIR)
[09/554/(0036)/2013 EMR-I], Government of India.

Fatty Acid Methyl
Ester

367

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I.A., Fayaj, H. (2013) Non-edible vegetable oils: A critical evaluation of oil extraction,
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oil to biodiesel under single and two phase reaction, Energy and Fuels, 21, 2450-2459.
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curcus oil using potassium carbonate as an unsupported catalyst, IJCRE,7, ISSN 1542-
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5. Bligh, E.G. and Dyer, W.J. (1959). A rapid method for total lipid extraction and
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using Lemna perpusilla Torrey ash as heterogenous catalyst, Biomass & Bioenergy, 55,
386-389.
8. Elliott, W. (2013) Biodiesel production from Jatropha oil and its characterization,
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9. Kalbande, S.R., More, G.R., Nadre, R.G. (2008) Biodiesel production from non-edible
oils of Jatropha and Karanj for utilization in electrical generator, Bioenergy Resource, 1,
170-178.
10. Leduce, S., Natarajan, K., Dotzauer, E., McCallum, I., Obersteiner, M. (2009)
Optimization of Biodiesel production in India, Applied Energy, 86, 5125-5131.
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15. Singh, B., Singh, K., Rao, G.R., Chikara, J., Kumar, D., Mishra, D.K., Saikia, S.P.,
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202.


369

CHAPTER 35
ANALYSIS OF EXHAUST EMISSION FROM A DIESEL
ENGINE FUELED WITH TRANSESERTIFIED VEGETABLE
OILS
Hemanandh.J. and Narayanan.K.V

Abstract
In this study, the emissions from Kirloskar Direct Injection 4-stroke Diesel engine, single
cylinder air-cooled, 4.4 kW, constant speed at 1500 rpm, compression ratio 17.5:1, with
different blends of diesel in comparison with Refined Corn methyl ester (CF) and Refined
sunflower methyl ester (SF) has been analyzed. Refined Corn oil and Refined Sunflower oil
was transesterified using sodium Meth oxide before blending with diesel. The main objective
of this study is to analyze and compare the CO, HC, CO
2
, NOx, and Smoke emissions by
varying the injection pressure and the load with pure diesel. The experiments were conducted
with various blends of diesel (10% CF+90% PD, 30%CF+70% PD, and 40% CF+ 60% PD,
10%SF+90% PD, 30%SF+70% PD, and 40%SF+ 60% PD) at different Injection pressures
(210 bar & 240 bar) and at different loads (0%, 25%, 50%, 75%, 100%). A 3- hole nozzle was
used to inject the fuel. The Emission results were studied using AVL gas analyzer. CF
decrease in CO and HC were noticed as 50% and 18.75% and the same for SF was measured
as 23.07% and 33.33%. On comparing diesel for CO
2
emission is insignificant for SF and 6%
decrease was observed for CF, at 240 bar with 30% blend, with the marginal increase of NO
x
.
Keywords: Kirloskar Di Diesel Engine, Injection pressure, CF Refined Corn flower
methyl ester, SF Refined Sunflower methyl ester, 3 hole Nozzle, Combustion Emission
characteristics.
35.1 Introduction
In the Present Indian scenario, an alternate fuel becomes most important due to the
continuous increase in the diesel fuel price and increasing pollution in the environment due to
diesel Engine exhaust emissions. Many types of Biodiesel can be used in Diesel Engines.


370

Biodiesel or Vegetable oil reduce the greenhouse emissions and is environmental friendly.
Biodiesel is a very good alternative for fossil fuels and is available in plenty.
The vegetable oils cannot be used directly along with diesel, since it is highly viscous.
Transesterification process is done in the presence of methanol, and added with Sodium meth
oxide as catalyst for Refined Sunflower oil and Refined Corn Oil. This improves the
performance of the engine and reduces the emissions.
35.2 Background
Similar experiments on biodiesel were conducted by many researchers. Mahin pey.N
et al., [1] explains, for transportation and safe handling low sulphur content with neutral CO
2
is essential. Jewel A. Capunitan et al. [2] conducted the experiments, one of the valuable
energy of fuels is the chemical stock produced from pyrolysis processed of corn stover. The
various studies made by, Ilknur Demiral et al.,[3] on chromatographic and spectroscopic on
bio-oil reveals that corncob stock can be classified as a renewable fuel. Significant reduction
of about 52.1% in green house gas emissions is evident [Nathan Kauffman] [4]. The literature
on production of raw material for biodiesel revealed by Xiao Huang et al., [5] that a corn
stove hydrolysate as fermentation feedstock for preparing microbial liquid reduces Nitrogen
content. N.N.A.N. Yusuf et al., [6] showed that compared with petroleum diesel reduction in
emissions of biodiesel, on CO
2
, SO
2
, particulate, CO and the HC and increase of about 10%
NO
x
are noticed. However blending biodiesel with petroleum diesel reduces NO
x
emission
with slight increase in other values but of acceptable criteria. The experiments on the DI
diesel perkinson engine were conducted by Dorado MP et, al, [7] by using reused olive oil
methyl ester to study the effect on combustion efficiency. As a result, oxygen concentration
was increased and accelerated the combustion. It was also found that the rate of combustion
efficiency in the use of reused olive oil, methyl esters, and the rate of combustion efficiency
remains almost constant as in the use of diesel oil. A lower energy rate was showed in the
palm oil combustion, done by Tashtoush G et, al, [8]. It was more efficient and higher rate of
combustion (66%) seen in burning biodiesel, when compared with the diesel combustion that
is (56%). This is because of the properties like high viscosity, less volatility and density.
Sudhir C.V. et, al, [9] conducted test on Diesel Engine, The rate of combustion temperature
and pressure was low in the operation of biodiesel, and the NOx emissions was also equal to
that of diesel. The sulphate emission was very low due to the lesser level of sulphur. The pilot
combustion caused the pre-combustion. The observation was that the blending ratio of 15%
resulted in reduced smoke opacity. The test conducted in DI stationary engine by yusuf .T. F.

371

et, al, [10] showed that as the blend increases, the brake power and CO increases in variable
speed which was less than 1800 rpm. A review was done by shareena et, al, [11] using
catalyst along with methanol in the transesterification process, which results in varying fatty
acid content of the biodiesel. This could be a good alternative fuel for diesel. Natraj.M, et, al,
[12] conducted an experiment by varying the design parameters of the DI, 4S, diesel engine
like Nozzle spray hole, Piston head clearance, Nozzle protrusion, Starting of injection timing,
Injection control pressure, Swirl level. By varying these parameters, the emissions were
reduced and analyzed by taguchi method. The method of varying the engine displacement by
Valentin Mickunaitis, et, al, [13] showed the result of mass increase by 6.5% in petrol and
7.5% in diesel. Hence, there is an increase in fuel consumption and CO
2
emission.
35.3 Methodology
The Density, Kinematic viscosity of the CF & SF is within the limits of the Biodiesel
Standards. The calorific value of the CF and SF is slightly less when compared to diesel. The
Engine requires a modification to improve better reduction in emissions. The flash point of
the CF and SF is high compared with pure diesel and is safe to store and transport.
The aim of the work is to analyze and compare the emissions, and to study the
performance of the diesel engine by using biodiesel. This has been done by varying the
injection pressure, fuelled with transesterified refined Corn oil and refined sunflower oil
combined with pure diesel at different blends.
35.3.1 Nomenclature
CF Refined Corn oil Methyl ester PD Pure Diesel
SF Refined Sunflower Methyl ester K No. of cylinders
Density, kg/m
3
L Length, mm
BIS Bureau of Indian standards A Area of the piston, mm
2

BP Brake power, kW N Engine running speed, rpm
T Torque, N- m CV Calorific Value of the fuel, kJ
R Radius of the drum, mm ASTM
American standards of Testing and
Materials


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35.3.2 Engine Specifications
Table 1: Specification of Test Engine
Type Kirloskar Vertical, 4S, Single acting, High speed, C.I. Diesel
Engine
Combustion Direct Injection
Rated Power 4.3 kW
Rated Speed 1500 rpm
Compression Ratio 17.5: 1
Injector type Single 3 hole jet injector
Fuel injection pressure 210 bar
Dynamometer Eddy current
Dynamometer arm length 200 mm
Bore 87.5 mm
Stroke 110 mm
Connecting Rod 200 mm
Cubic Capacity 661.5 cm
3

Maximum Torque 0.030 kN m (full load @1500 rpm)
Fuel tank Capacity 6.5 litres
Injection pump type Single cylinder flange mounted without camshaft
Governor type Mechanical centrifugal type

35.3.3 Details of Measuring Systems
Table 2 - Details of Measuring Systems
AVL Pressure Transducer GH 12 D
Software Version V 2.0 AVL 617 Indi meter
Data Analyzer from Engine AVL PIEZO CHARGE AMPLIFIER
To measure pressure AVL 364 Angle Encoder
Smoke meter AVL 437 C Smoke
5 Gas Analyzer ( NO
x
, HC, CO, CO
2
, O
2
) AVL DIGAS 444 Analyzer


35.3.4 Experimental Setu
A stationary kirloskar 4S, DI Diesel Engine was used in experiments. The
specification of the engine is given in Table
Electrical loading (Dynamometer). The Eddy current dynamometer for loading is coupled to
the engine for various loading ( 0%, 25%, 50%, 75%, 100% ). The exhaust gas emissions
from the engine was measured using AVL DIGAS 444 Analyser (NO
and the smoke opacity was measured using AVL 437C smoke meter. AVL 364 Angle
Encoder was used to measure the pressure and crank angle.

Fig. 1
1 - Kirloskar Vertical C.I. Diesel Engine, 2
Electrical loading device, 5

.,0&01
2*34
50602*/10
O,1 2*34
7P8 9
:O3,2O;
) 6*&
*3*1Y&0;
373
Experimental Setup
specification of the engine is given in Table 1. The load on the engine was applied using
(Dynamometer). The Eddy current dynamometer for loading is coupled to
ity was measured using AVL 437C smoke meter. AVL 364 Angle
Encoder was used to measure the pressure and crank angle.
Fig. 1 Schematic Diagram of Experimental set-up
Kirloskar Vertical C.I. Diesel Engine, 2 - Fuel Tank, 3 AVL 437 C Smoke meter, 4
Electrical loading device, 5 Engine temperature monitor
50602*/10
O,1 2*34
4,;1O&4*; .,0&01
036,30
01072;,7*1
1O*.,36 O;
.Y3*:O:020;
1. The load on the engine was applied using
(Dynamometer). The Eddy current dynamometer for loading is coupled to
x
, HC, CO, CO
2,
O
2
)
ity was measured using AVL 437C smoke meter. AVL 364 Angle

p
AVL 437 C Smoke meter, 4
Engine temperature monitor
01072;,7*1
1O*.,36 O;
.Y3*:O:020;
&:O40 :020;

374

35.3.5 Test Procedure
The experiments were conducted at different load conditions, with different pressure
at different blends viz ( 10% CF + 90% PD ), ( 30% CF + 70 % PD), (40% CF + 60% PD)
(10% SF + 90% PD ), ( 30% SF + 70 % PD) & (40% SF + 60% PD) as fuel. The test was
conducted at a constant speed of 1500 rpm. The engine was allowed to run at No load for 10
minutes, using each proportion of the blend before applying the load. The loads were
increased gradually for each blend in steps of 25 % at constant speed of 1500 rpm at different
pressures. The test was conducted to compare and analyze the emissions based on the above
conditions.
Table 3 - Comparison of properties of Diesel, Biodiesel standards, Refined palmolein oil &
Refined corn oil
S.
No.
Properties Diesel BIS
Standard
Bio Diesel
ASTM D 6751
(IS 15607:2005)
SF CF
1. Cetane Index (min) 46 51 - 38 35
2. Density at 15 C kg / m
3
820 845 860 900 (860-900 kg/m
3
) 923 923
3. Kinematic Viscosity at 40
C cst
2 4.5 2.5 6 1.9 6 mm
2
/s 4.4 5.02
4. Flash point C min 35 C 262 C 130 C min 254 282
5. Calroific Value kJ/kg 44,000 - - 39,2
84
36,824

35.4 Results & Discussions
The emissions of CF oil and SF oil (CO, HC, NO
x
, CO
2
, and smoke) and its blends are
compared with diesel for a modified diesel engine are analyzed below.
35.4.1 Carbon mono oxide (CO)
Figure 2 shows that exhaust emissions of carbon mono oxide at different injection
pressures under constant speed of 1500 rpm with various blend ratios of the SF and CF
compared with diesel. The CO emissions with CF decreases by 24% at 210 bar and 30%
blend at 75% load condition whereas emission of CO with SF is same as that of diesel at 75%
of load. At 240 bar and 30% blend there is decrement in CF and SF by 50% and 18.75%
respectively at full load condition. This might be due to the availability of excess oxygen
content that leads to better combustion of the fuel droplets that travel from jet tip to cylinder

375

wall. The literature by krahl J et al., [14] shows that the emissions of CO is decreased by 50%
with rapeseed oil containing ultra low sulphur diesel.

Fig 2 Variation of CO with respect to 30% blend of SF, CF, PD at 240 bar
35.4.2 Hydrocarbon (HC)
The fig. 3 shows the emissions of Hydrocarbon. The HC is increased at full load
condition with various blends at 210 bar for CF and SF, whereas HC decreases at 240 bar and
30% blend compared to pure diesel. It is observed that the increase in HC for CF between
20% load to 60% load, and thereafter decreases. CF and SF decrease by 23.07% and 33.33%
at full load condition. This could be due to the rich air-fuel mixture leads to the better
combustion.

Fig 3 Variation of HC with respect to 30% blends of SF, CF, PD at 240 bar
35.4.3 Carbon di oxide (CO
2
)
The CO
2
emissions of the CF and SF as shown in fig.4. There is a marginal decrease
observed at 210 bar and 30% blend. Where as at 240 bar with 30% blend emission of CO
2
is
0
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0#04
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0#08
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376

same as diesel up to 60% of load and decreases by 6% with CF and very marginal decrease
in SF at full load conditon. AL- widyan MI et al., [15] reviels that, the ethyl ester of waste
palm oil reduces the CO
2
and 50:50 blend ratio. The reduction in CF is due to more oxygen
content leads to better combustion.

Fig 4 Variation of CO2 with respect to 30% blends of SF, CF, PD at 240 bar
35.4.4 Nitrogen Oxide (NO
x
)
Fig. 5 & 6, shows the NO
x
emissions. The SF and CF is same as diesel upto 50% load
at 210 bar. The 30% blend at 210 bar and 240 bar shows the marginal increase in NO
x
for SF
and CF at full load condition,. This could be due to the reduction in flame temperature at
lower injection pressure.

Fig 5 Variation of NOx with respect to 30%
blends of SF, CF, PD at 210 bar
Fig 6 Variation of NOx with respect to 30%
blends of SF, CF, PD at 240 bar

0
1
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(
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)
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400
600
800
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1200
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7-
&-

377

35.4.5 Smoke
From fig. 7 & 8. it is noticed that, the Smoke increases at initial load condition and as
load increases the smoke decreases by 8.89 % for CF at 30% blend at both the injection
pressures when compared to pure diesel, Whereas the marginal increase in SF was observed
with the above conditons. This might be due to presence of oxygen atoms leading to better
combustion with CF and similar reason given by Kalam MA et al., [16].

Fig 7 Variation of Smoke with respect to
30% blends of SF, CF, PD at 240 bar
Fig 8 Variation of Smoke with respect to
30%blends of SF, CF, PD at 210 bar
35.5 Conclusion
The experiments conducted by varying the injection pressures on 4S diesel engine,
with the Refined Sunflower oil and Refined Corn oil and the emissions were studied are
listed below.
1. The CO decreases at 240 bar and 30% blend in CF and SF by 50% and 18.75% at
full load condition.
2. The HC decreases for CF and SF with a decrease value of 23.07% and 33.33% at full
load condition at higher injection pressure as compared with pure diesel.
3. With the NO
x
increase, the CO
2
decrease at 240 bar with 30% blend by 6% for CF and
marginal decrease for SF.
Future Scope of work
0
)
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378

The above analysis can be performed by changingthe the nozzle orifice with various
profiles and sizes like Elliptical, semi elliptical, etc., in addition to varying the number of
nozzles against different blend ratio which could yield higher efficiency and lower emissions.

References
1. Mahinpey N, Murugan P, Mani.T, Raina.R (2009) Analysis of bio oil gas and bio char
from pressurized pyrolysis of wheat straw using a tubular reactor energy, Fuel, 2736
42.
2. Jewel A. Capunitan,sergio C. Capareda (2010) Assesing the potential fr biofuel
production of corn stover pyrolysis sing a pres.surized batch reactor, Fuel, 563 -572.
3. Ilknur Demiral, Alper Eryazlci, sevgi sensoz (2012) Bio oil production from pyrolysis
of cron cob ( Zea Mays.L), Sciverse Science Direct, 43 -49.
4. Nathen Kauffman, Dermot Hayes, Robert Brown (2011), A life cycle assessemnt of
advanced biofuel production froma hectare of corn, Fuel, 3306 3314.
5. Xioa Huang, Yumei wang, Wei Liu, Jie Bao (2011) Biological removal of inhibitors
leads to the improved lipid production in the lipid fermentation of corn stover
hydrolysate by trichasporan cutaneum, Bioresources Technology, 9705 9709.
6. N.N.A.N.Yusuf, S.K. Kamarudin, Z.yaakub (2011) Overview on the current trends in
biodiesel productions, Energy Conversion and management, 2741 2751.
7. Daroda.M.P, Ballesteras E, Arnal JM, GOMEZJ, Lopez RJ (2003), Exhaust Emissions
from a Diesel Engine Fueled with Transesteified olive oil. Fuel, 1311 1315.
8. Tashtoush G, Al-widyan MI, AI shyoukh (2003) AOCombustion performance and
Emissions of methyl esters of a waste vegetable oil in water cooled furnace. Applied
Thermal Engineering, 285-93
9. Sudhir CV, Sharma NY, Mohanan .P (2007) Potential of Waste Cooking oils as
biodiesel feed stock, Emirates Journal for Engineering Research,69 -75.
10. Yusaf T.F, yosif .B.F, Elawad.M.M (2011), Crude palm oil fuel for diesel engines:
Experimental and ANN simlation approaches, Energy, 4871 - 4878.
11. Shereena,K.M, Thangaraj.T, Biodiesel (2009) An alternate fuel produced from
vegetable oils by Transesterification, European Journal of Biochemistry, 67 74.

379

12. M.Natraj, V.P. Arunachalam, & N. Dhandapani,(2005) Optimizing diesel engine
parametes for low emissions using Taguchi method variation risk analysis approach
Part I, (ijems), Indian Journal of Engineering of Material Research, 169 181.
13. Valentinas Mickunaitis, Alvydas pikunas, Ignor Mackoit, (2007) Reducing fuel
consumption and CO
2
emission in motor cars, Transport, 160-163.
14. Krahl J, Munack A, Schrder O, Stein H, Bnger J. (2003) Inuence of biodiesel and
different designed diesel fuels on the exhaust gas emissions and health effects.SAE
paper.
15. Al-WidyanMI,TashtoushG,Abu-QudaisM. (2002) Utilization of ethyl ester of waste
vegetable oil as fuel in diesel engines. Fuel Proc Technol, 91103.
16. Kalam MA, Masjuki HH. (2004) Emission and deposit characteristics of a small diesel
engine when operated on preheated crude palm oil. Biomass Bioenergy, 28997.


380

CHAPTER 36
GENETIC ENHANCEMENT OF PONGAMIA PINNATA FOR
BIO-ENERGY
M.V.R. Prasad

Abstract
Pongamia pinnata or Millettia pinnata or Derris indica (n=2x=22) is a non-edible oil-seed
yielding ever green leguminous tree adapted to be grown under conditions of marginal soils,
under rain-fed conditions. The tree is native of India and Southeast Asia and is found in
Oceania too. The Carbon sequestration potential of Pongamia pinnata during the 10 to 15
years of its growth was found to be many folds higher than that of several other tree species.
Pongamia was found to sequester around 45 to 50 kg of C per tree per annum as against 28 to
35 kg of Neem (Azadirachta indica), 23 to 26kg of Mahua (Madhuca latifolia) and 11 to 15 kg
in respect of Tendu (Diospyros melanoxylon).Pongamia produces its own nitrogen through
Symbiotic Nitrogen Fixation, thereby displacing approx $200 / ha/year of nitrates applied as
compound fertiliser. Soils under Pongamia stands of +15 years age accumulate almost 1100
to 1300 kg of Nitrogen per hectare per annum, apart from enhancing the soil organic matter
and consequent positive soil biological activity.
Considering its oil yielding potential based on the earlier work carried out at the
Directorate of Oilseeds Research under the aegis of ICAR in the decades of late nineteen
eighties and early nineteen nineties, a program of genetic enhancement of Pongamia for seed
and consequent oil yield was launched by VAYUGRID.
Based on the learning and experience gained in the genetic improvement of cashew in
Africa, the genes controlling some crucial canopy characters exhibiting consistency and
stability of expression were employed in the development of basic criteria for selection of the
elite trees of Pongamia. Around 800 elite trees of Pongamia possessing significantly higher
levels of kernel yield of the range of over 45 kg / tree were selected based on the certain stable
canopy attributes strongly associated with seed yield. The yield data collected over two
seasons (2010-2011 and 2011-2012) demonstrated consistently higher yield levels of the
selected trees. The seed oil content of the selected trees ranged from 33 to 39% as against oil


381

content of 28 to 32% in normal and unselected trees. A certain number of elite Pongamia trees
exhibited higher contents of oil and karanjin, a flurano flavanol of industrial value.
The propagation and multiplication of the selected elite trees had been carried out
adopting novel vegetative propagation and nursery technologies that ensured 90 to 100%
success rate in the recovery of the clones (VayuSaps) of the elite trees. At present
VAYUGRID possesses 8, 27,065 VayuSaps containing the elite characteristics contributing
to high yields.
VayuSap starts yielding by the end of the third year following planting with a
progressive increase in the yield every year. Normal unselected trees of Pongamia on the
other hand, start yielding poorly after the 6
th
of 7
th
year only. It is estimated that VayuSaps
planted over an area of one acre would yield around one ton of oil by the 5
th
year following
planting. VayuSap plantation technology is a low input; but highly rewarding one well
suited to wide range of agro-ecological situations including those characterized by marginal
and problem soils with low rainfall.
VayuSap plantations have been established with success at Mahindra & Mahindra
World City near Chennai, private farms near Hyderabad & Bangalore and Jindal Lignite
Mining Company (SWML) at Barmer (Rajasthan) in India and also in Ethiopia and Djibouti
in Africa.
Key words: Pongamia pinnata, genetic improvement, oil yield, karanjin
36.1 Intorduction
Pongamia pinnata or Millettia pinnata or Derris indica (n=2x=22) is a non-edible
oil-seed yielding ever green leguminous tree adapted to be grown under conditions of
marginal soils under rain-fed conditions. The tree is native of India and Southeast Asia and is
found in Oceania too. The annual seed production of Pongamia in India is estimated to be
130,000 tonne (Rattansi et al., 1997) most of which is obtained as an aggregation from avenue
and forest trees and those grown on field bunds and marginal soils without any care and
maintenance worth the name. There are no organised plantations of Pongamia in India despite
its manifold uses including as an ancient source of energy and in native medicine.
Considering its oil yield potential and suitability to be grown on marginal lands under
rain-fed conditions based on the earlier work carried out at the Directorate of Oilseeds
Research under the aegis of ICAR (Prasad, 1994) in the decades of late nineteen eighties and

382

early nineteen nineties, a program of genetic enhancement of Pongamia for seed and
consequent oil yield was launched by VAYUGRID.
36.2 Carbon Sequestration by Pongamia
The Carbon sequestration potential of Pongamia pinnata during the 10 to 15 years of
its growth was found to be many folds higher than that of several other tree species.
Pongamia was found to sequester around 45 to 50 kg of C per tree per annum as against 28 to
35 kg of Neem (Azadirachta indica), 23 to 26kg of Mahua (Madhuca latifolia) and 11 to 15
kg in respect of Tendu (Diospyros melanoxylon). (Table1) (Chaturvedi et al., 2011; Jesse
More, 2009; Gupta, 2008; Kamarkar et al., 2012; Gera and Chauhan, 2010 and Reddy et al.,
2009)
Table 1: Carbon Sequestration by Some Popular Tree Species.
Tree type
C sequestration / tree of
15yrs (kg)/ annum
C Sequestration (t)/ ha
Pongamia (Pongamia
pinnata)
45-50 23.5****
Tendu(Diospyros
melanoxylon)
11-15 6.0***
Mahua (Madhuca latifolia) 23-26 8.5**
Neem (Azadirachta indica) 28-35 13.0*
C - Sequestration by Neem is reported as 1.5 t/ha/yr from natural tree stands. He above
figure of 13.0 was arrived at based on the population density (450/ha) that would be
recommended for a regular plantation.
All the above values are for regular plantation model with appropriate tree densities per
unit area.
** Tree density of 354 / ha
*** Tree population of 460/ha
**** Tree population of 500 / ha
The current trends of global warming are of great concern. It is estimated that an
increase of 2.5% of global temperature would reduce agricultural productivity in USA by 6%;
but by 38% in India. (Sengupta 2013) It is therefore necessary to come up with appropriate
technologies involving tree species that are efficient in cutting down global warming and
bring about perceptible levels of C sequestration in addition to oil yield. Pongamia is one

383

such species that need attention in this regard. It is absolutely necessary that the tree types
grown for bio-fuels, must not occupy fertile irrigated lands to avoid competition with regular
food and commercial crops. Pongamia pinnata meets the above condition and as such forms
an excellent feed stock for bio-fuel production.
36.3 Sybiotic Nitrogen Fixation and Soil Amelioration by Pongamia
Pongamia produces its own nitrogen through Symbiotic Nitrogen Fixation, thereby
displacing approx $200 / ha/year of nitrates applied as compound fertiliser. Soils under
Pongamia stands of +15 years age accumulate almost 1100 to 1300 kg of Nitrogen per
hectare per annum, apart from enhancing the soil organic matter and consequent positive soil
biological activity (Elevitch and Wilkinson, 1999).
Table 2 - Soil Analysis Data
Parameter Under Pongamia
(6yr old trees)
Barren soil
pH 6.5 6.7
Bulk density 4.75 4.87
Oxidisable C 1.6 0.45
Organic matter 3.27 1.16
N 0.091 0.027
P 0.0093 0.0030
K 0.086 0.032

The soil under six year old trees of Pongamia exhibited marked improvements not
only soil organic matter and Nitrogen, but also in other plant nutrients viz., Phosphorous and
Potash (Table 2).
36.4 Selection of Elite Trees
Based on the learning and experience gained in the genetic improvement of cashew in
Africa (Prasad et al., 2000), genes controlling some crucial canopy characters exhibiting
consistency and stability of expression were employed in the development of basic criteria for
selection of the elite trees of Pongamia. The procedure adopted by VAYUGRID for the
identification of genetically elite trees of Pongamia pinnata for consistent higher productivity
levels is based on the selection of characters that are stable in their expression with their close

384

association with kernel yield. The selection is not based on total yield alone, which could vary
from year to year.
The characters chosen are as follows:
1. Appropriate canopy architecture with extended canopy area
2. Intensive branching pattern with higher proportion of pod bearing branches.
3. Higher number of Pod bearing clusters
4. Cluster bearing habit
5. Higher shelling percentage
6. Resistance to pod-malformation and
7. Higher kernel yield per tree
Around 800 elite trees of Pongamia possessing significantly higher levels of kernel
yield of the range of over 45 kg / tree were selected based on the certain stable canopy
attributes strongly associated with seed yield. The yield data collected over two seasons
(2010-2011 and 2011-2012) demonstrated the consistently higher yield levels of the selected
trees. (Tables 3 & 4).The seed oil content of the selected trees ranged from 33 to 39% as
against oil content of 28 to 32% in normal and unselected trees (CFTRI 2012) (Table 5). A
certain number of elite Pongamia trees exhibited higher contents of oil and karanjin, a flurano
flavanol of industrial value (CFTRI 2012). Genetically elite tree of Pongamaia
pinnaGGGEBEGta FITREE (Figure1).
36.5 Vegetative Propagation
The propagation and multiplication of the selected elite trees had been carried out
adopting novel vegetative propagation and nursery technologies that ensured 90 to 100%
success rate (Swamy 1992) in the recovery of the clones (VayuSaps) of the elite trees. At
present VAYUGRID possesses 8, 27,065 VayuSaps containing the elite characteristics
contributing to high yields.
36.6 Vayusap Plantations
VayuSaps are to be planted at the rate of 200 saplings per acre in pre-dug pits each
of 45 to 50 cubic cm at spacing of 5m X 4m, with a mixture of dug-out top-soil, 4 to 5 kg
farm yard manure and 200 g of single super phosphate per pit with the onset of rains. The
trees start yielding by the end of the third year following planting with a progressive increase

385

in the yield every year. Normal unselected trees of Pongamia on the other hand, start yielding
poorly after the 6
th
of 7
th
year only. It is estimated that VayuSaps planted over an area of
one acre would yield around one ton of oil by the 5
th
year following planting. VayuSap
plantation technology is a low input; but highly rewarding one well suited to wide range of
agro-ecological situations including those characterized by marginal and problem soils with
low rainfall.
Table 3 - Yield of Selected Elite Trees in Hiriyur of Distt.Chitradurga, Karnataka
Location Tree
Number
Total yield of the tree 2010-2012
District Village

seed kernal yield
in kg (2010-2011)
seed/kernal yield
in kg(2011/2012)
Chitradurga Mayasandra 100 31 45
Chitradurga Kyathadevaranahatti 135 35 42
Chitradurga Bhaburu 139 44 41
Chitradurga Beemanabande 142 31 69
Chitradurga Hosahalli 171 30 42
Chitradurga Hosayalandu 174 31 32
Chitradurga Hosayalandu 177 30 37
Chitradurga VV.pura 181 44 29
Chitradurga AV.kottige 189 33 52
Chitradurga Shivapura 237 53 45
Chitradurga Shivapura 238 40 44
Chitradurga Kurubarahalli 265 30 34
Chitradurga Kurubarahalli 267 32 32
Chitradurga Doddaghatta 279 46 41

unselected control

9 8


386

Table 4. Yield Of Selected Elite Trees From Tumkur District Of Karnataka
Location
Tree Number Total yield of the tree 2010-2012
District Village

seed kernal yield
in kg (2010-2011)
seed/kernal yield in
kg(2011/2012)
Thumakuru Hosahalli 312 34 44
Thumakuru Hosahalli 314 47 47
Thumakuru Chikkasarangi 316 51 46
Thumakuru Kitthaganahalli 344 49 40
Thumakuru Vaddarahalli 358 51 59
Thumakuru Honnenahalli 360 54 42
Thumakuru Yallapura 411 38 35
Thumakuru Arekere 412 49 42
Thumakuru Obalapura 413 59 63
Thumakuru Obalapura 414 48 48

Unselected
Control
9 11

Figure 2. Productive Canopy of Elite
PongamiaTree of VAYUGRID.
Figure 3. VayuSap at VAYUGRID
Pongamia Nursery


387

Table 5. Fat and Karanjin Contents Of Some Elite Pongamia Trees From Chtradurga District
For The Year 2010-2011
Elite Tree No. Fat (%) In Seed Karanjin (%) In Oil
02 38.6 5.97
05 36.2 5.79
06 37.5 5.62
08 38.0 6.10
09 37.2 5.98
11 38.7 5.84
12 36.2 5.33
17 37.4 5.45
22 36.9 4.91
35 37.1 5.54
40 33.5 6.02
55 37.6 6.00
58 38.0 5.72
61 38.6 5.17
83 33.5 6.17
125 38.4 5.98
181 36.1 5.78
202 37.0 5.38
203 36.0 4.99
211 33.4 6.04
218 32.8 6.07
236 38.2 5.21
239 37.7 5.38
Control 28.7 3.9


388

VayuSap plantations have been established with success by VAYUGRID at
Mahindra & Mahindra World City near Chennai, private farms near Hyderabad and
Bangalore and Jindal Lignite Mining Company (SWML) at Barmer (Rajasthan) in India and
also in Ethiopia and Djibouti in Africa.
Acknowledgement
The author thanks all his colleagues in VAYUGRID for their help, cooperation and
encouragement in carrying out this work.

References
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seeds for VAYUGRID, Bangalore, India. Central Food Technological Research
Institute, Mysore, India. 20.
2. Chaturvedi
,
R.K., Raghubanshi

A.S.and Singh, J.S.(2011) Carbon density and
accumulation in woody species of tropical dry forest in India, Forest Ecology and
Management, 262(8): 15761588
3. DAFF (2008) Carbon Storage in Australian Forests. ABARES Fact Sheet.
4. Elevitch, C.R. and Wilkinson, K.M. (1999) Nitrogen fixing Tree Startup Guide.
SustainableAgricultural Research. USDA. Permanent Agricultural Resources, Holualoa,
HI96726, USA.
5. Gupta, H.S. (2009) Forest as Carbon Sink: Temporal Analysis for Ranchi district.
Indian Forester, 32 (1): 7-11
6. Jesse More (2009) Carbon Sequestration Potential of the MillionTrees.NYC Initiative,
Biofuels and Bio-Based Carbon Mitigation
7. Kamarkar, A., Kamarkar,S and Mukherjee,S. (2012)Biodiesel production from neem
towards feed stock diversification, Renewable and Sustainable Energy Reviews,
16:10501060
8. Mohit Gera and Suresh Chauhan (2010) Opportunities for C sequestration benefits from
growing trees of medicinal importance on Farm lands of Haryana. Indian Forester
(2010): 287-300
9. Prasad, M.V.R. (1994) Minor Oil bearing Species of Forest Origin for Diversification of
Vegetable Oil Production. In: Prasad, M.V.R. et al(Ed). Sustainability in Oilseeds.

389

Indian Society of Oilseeds Research, Directorate of Oilseeds Research, Hyderabad.
Pp91-98.
10. Prasad, M.V.R., Langa, A. and Consolo, J.P. (2000). Selection of superior genotypes of
cashew in Nampula zone of Mozambique. The Cashew, Jan-March,2000 :8-23
11. Rattansi, R., Dikshit, M, Rattansi, R and Dikshit, M. (1997) Protease inhibitors and in
vitro digestibility of Karanja (Pongamia glabra) oil seed residue. A comparative study
of various treatments. Journal of the American Oil Chemists' Society 1997. 74 (9):
1161-1164
12. Reddy, N.S., Ramesh, G. and Suryanarayana, B. (2009) Evaluation of Tree species
under different land use systems for higher Carbon sequestration. Indian J. Dryland
Agri. Res. & Dev.24 (2):74-78.
13. Sengupta, R.P. (2013) Ecological Limits and Economic Development. Oxford
University Press, NewDelhi.
14. Swamy, K.R.M. (1992) Clonal Propagation of Elite planting materials. Soft-wood
Grafting, a novel technique. Proc. National Workshop on Cashew, Kanpur. National
Research centre for Cashew. ICAR. 76-79.

390

Part V
Electrochemical Processes


391

CHAPTER 36
EVALUATION OF ELECTRICAL PROPERTIES UNDER
DIFFERENT OPERATING CONDITIONS OF BIO-
ELECTROCHEMICAL SYSTEM TREATING THIN
STILLAGE
Ghosh Ray, S., Ghangrekar, M.M.

Abstract
Edible ethanol production industries are generating wastewater with very high concentration
of organic matter and nitrogen; hence, if not properly treated it can adversely affect the
receiving environment. Low pH, high total COD of 61 g/l and TSS of 26.4 g/l due to the
presence of dietary fiber of the thin stillage made the mesophilic conversion more challenging,
and thus leading to lesser reduction of COD. Theoretically this industrial effluent has 238
kJ/m
3
of energy present in the form of organic matter, which can be harvested in the form of
electricity by employing microbial fuel cell (MFC). In this study performance of MFC treating
thin stillage was investigated at organic loading rate of 1.5 g COD/l.d. Performance of this
Bio-electrochemical system (BES) is characterized by polarization behavior, exhibiting a
maximum power density of 22.16 mW/m
2
and current density of 699.18 mA/m
2
at 30
internal resistance. Maximum COD removal of 48% and 88% protein hydrolysis in anode
chamber was observed giving 2.91% Coulombic Efficiency (CE). Activation overpotential
was calculated to assess the kinetic properties during bio-electrochemical conversion. Charge
transfer coefficients () of 0.2 and 0.68, as well as the exchange current densities (i
0
) of 0.13
mA/cm
2
and 0.1 mA/cm
2
in the equilibrium state for cathode and anode, respectively were
observed. Further studies are required for breakdown of complex structure carbohydrate by
different physical and microbiological pretreatments to enhance the COD reduction, by
increasing the availability of acetate and to improved power generation.
Keywords: Bio-electrochemical system, Charge transfer coefficient, Exchange current
density, Mesophilic conversion, Microbial fuel cell, Thin stillage.


392

36.1 Introduction
The growing concern in continuous depletion of natural resources has germinated the
thought in researchers to invent alternative sources of energy. India is striving hard to achieve
required per capita energy consumption and expecting increased Gross Domestic Product
(GDP) from 4% to 7.5% with annual 8% growth in energy demand. Increasing population and
a high rate of economic growth proportionately increase the environmental pollution with the
emission of Green House Gas (GHG). Gradual diminution of natural resources and limited
commencement of renewable energy recovery arise questions on sustainability of countrys
energy security. An interest has been evolved in production of bio-fuels from nonconventional
substrate, thus, promises a new horizon of better future. Till date, 13% of countrys
renewable-based electricity capacity comes from biomass.
India has only 0.5% of worlds oil reserves. So, the authority has set a target to blend
ethanol with gasoline, initially at 5% in 2006, aiming to increase it up to 20% by 2017, to
step-down the discharge of harmful gasses to protect the environment. An aspiring venture has
been taken by many industries using non-food wastes like spent wash of sugar industries,
broken and uneatable grains for the commercial ethanol production. Distillery industry waste
is a major source of pollution among the 17 industrial wastes and thus having immense
potency in becoming alternative source of energy (MNRE, 2011).
Grain based alcohol has not been much popular in India; however, this can be a better
alternative as cultivation of malt producing grain (rice, barley, wheat, maize, jowar) is much
easier in Indian climate. Even though, starchy, cellulosic sources are being proven as a better
alternative, emerging technologies are insufficient to dissolve all the ailments. Moreover,
much concern has been generated to lower per liter ethanol production presently from 20 to 23
INR. According to the CPCB report (2012), 100 grain-based distillery units are presently
running having the net capacity of 1.8 billion liters per annum. Mostly broken rice, kinki,
pearl millet and sorghum or mixed grains are being used mostly as raw material in Indian
distillery units. Increase in production of all type of rice to 12.7% from the last decade
consequently shows 9.8% increase in export quantity than the last five years. Only 5% of the
total produced rice is exported presently (AIREA, 2012). According to the statistics, around
12-24% of the annual total rice production in India is the broken rice, which is the by-product
of rice mill, also known as brewers rice. Broken rice has been used for many years for

393

commercial alcohol production. Being of almost similar biochemical composition as whole
rice, 90% of the total dry weight is composed of -glucans.
The high yield of paddy cultivation, especially in the eastern part of India generates
more interest in distillers to utilize the cellulosic feedstock as the origin of ethanol production.
Some challenges possibly can be encountered as these small grains contain high concentration
of Non-Starchy Polysaccharides (NSPs), which have high water binding capacity, leading to
increased mash viscosity. NSP covers wide variety of polysaccharides excluding -glucan
(starch). Hemicelluloses, such as arabinoxylans, xylans and xyloglucans are mostly found in
rice; the amorphous structure can be hydrolyzed easily to yield a complex coordination which
can hold water. The main cause of high viscosity mass is the presence of -glucans and
pentosans (Choct, 1997; Houston and Kohler, 1970).
During anaerobic respiration, it is difficult for micro-organisms to break down the
composite structure of NSPs, and hence Chemical Oxygen Demand (COD) reduction cannot
be achieved more than 70%. After rectification of spirit, the generated wastewater is having
COD ranging 55,000-60,000 mg/l, TSS around 13,000-15,000 mg/l and acidic pH (~3.6-3.8),
and it is 9-10 times more in volume than the total capacity of the plant. The wastewater,
typically having high concentration of yeast biomass is centrifuged and thin stillage fraction is
taken for wastewater treatment and several byproduct recoveries (Khannal, 2009). The highly
proteinaceous distillery grain or the solid fraction is used to produce cattle feed. Yeast
biomass increases the protein content of the wastewater. Therefore, pretreatment is necessary
for this wastewater to reduce suspended solids and organic matter. Fungal treatment of raw
wastewater can be done to produce Single Cell Protein (SCP), enzymes and biopolymers,
which are having high commercial value (Wilkie et al., 2000).
Minimization of organic load is been achieved by Indian industries in conventional
ways. Concentration of thin stillage followed by incineration reduces organics but this process
needs higher capital cost. Anaerobic digestion is another method where biogas generation
recoups more energy than that has been spent. As, none of the methods can suitably achieve
the distillery waste disposal standards, most recently bio-electrochemical systems (BES) are
developed that can utilize the post methanated effluent (PME) for the production of electricity
and hydrogen gas (Rabaey, 2010; Katuri and Scott, 2011; Kunusch et al., 2010). In microbial
fuel cell, microbial metabolism is utilized to convert carbohydrate to electrical energy. It is
proposed that, the utilization of remaining organics with high COD in such a manner can

394

successfully serve the purpose by producing alternative energy as well as reduction of
organics. The process is cost effective, although, it is not been established commercially and
many research is yet to be done (Rabaey and Verstraete, 2005). Energy conversion from PME,
consisting complex carbohydrates, yeast cell biomass, several metabolites of fermentation and
very less residual mono-sugar and its derivatives, into electricity is the utmost challenge that
has been faced by the researchers to make this system commercially viable. Not much work
has been pursued for complex real wastewater to sustainable energy conversion (Lu et al.,
2009; Behera et al., 2010; Huang et al., 2011). The objective of the present study is to
elucidate the prospects of producing electricity from residual sugar and soluble proteins in raw
thin stillage, as well as simultaneous reduction of carbonaceous and nitrogenous organics by
biologically active heterotrophs. Furthermore, the study contributes to the knowledge about
feasibility of secondary treatment of high organic real wastewater.
36.2.1 Wastewater characterization
Thin stillage was acquired from the distillery unit of IFB Agro Ind. Ltd., Kolkata and
kept at 4 C. Ethanol has been produced in this industry from broken rice. Enzyme treated
cellulosic mash is subjected for fermentation with specific yeast strain and ethanol is
produced. Highly viscous wastewater is generated after rectification of ethanol. Centrifuged
wastewater known as thin stillage is collected and characteristic studies are done. Estimation
of total solids (TS), total volatile solids (TVS), total suspended solids (TSS), volatile
suspended solids (VSS), COD, biological oxygen demand (BOD), NH
3
N, total protein,
lignin and phenol were done following the standard methods (APHA, 1998). Acetate, nitrate
and phosphate content are estimated by Ion Chromatograph (Albright, 2008; APHA, 1998).
36.2.2 BES operation principle and reactor fabrication
A triple chambered BES is fabricated where two chambers are assigned as anode and
cathode and the third chamber is designed as nitrification chamber. Anode contains high
organic loading of cellulose, starch and proteinaceous wastewater which is inoculated with
heat treated sludge of calculated amount for removal of organic carbon under anaerobic
condition (Jadhav and Ghangrekar, 2008; Mohanakrishna, 2010). Sludge contains mostly
mesophilic range of bacteria, having the ability to hydrolyze and ferment organic compounds
to simple sugar, amino acids and fatty acids. In the next step acidogenic microorganisms

395

convert the yields to fatty acid, acetate, CO
2
and H
2
through different fermentative pathway.
The low molecular weight carboxylic acids are utilized by H
2
producing syntrophs and acetate
is generated. Homoacetogens also produce acetate from fermented yields. The treated effluent
containing degraded organic matter and organic proteins are mostly converted to ammonium
ions by de-nitrifiers (Virdis et al., 2010). Nitrification chamber was seeded with sludge from
anode. The effluent from anode is fed to nitrification chamber where oxygen is purged to
oxidize ammonium species by facultative autotrophic bacteria converting it to nitrite and then
to nitrate by nitrifiers in that same chamber where further degradation of complex
carbohydrate is also possible. Electrons generated by anode mechanism migrates through
external metallic circuit towards cathode while, H
+
ion passes through a clay separator to
cathode and in presence of diffused oxygen it gets reduced to complete the electrochemical
cycle (Logan, 2008; Watanabe, 2008; Pant et al., 2010; Wen et al., 2010). Carbon felt is used
as the electrode material providing large effective surface area to grow the bio-film. Distance
between cathode and anode is reduced by attaching the electrodes close to separator to achieve
maximum efficiency of the cell (Rismani-Yazdi et al., 2008). The schematic diagram of C and
N mass transfer within the reactor is shown in figure 1.
R
Anode Cathode
PEM
NH
4
+
NO
3
-
Nitrification
Chamber
O
2
Final
Effluent
O
2
PEM Proton E!chan"e Membrane
#reated Effluent
Com$le!
$rotein
Pol%-&acc.
Acetate
NH
4
+
Feed
1.15 l
1.15 l
1.5 l

Figure 1: Schematic diagram of BES for simultaneous carbonaceous and nitrogenous thin
stillage treatment. Three chambers anode, cathode and nitrification chamber (NC) are labeled.
The volume of two half cells were kept similar as 1.15 l and NC as 1.5 l. The system is
maintained with 1.5 kg COD/m
3
.d

of organic loading rate in each cycle of 48 h of conversion.
Phosphate buffer (50 mM) is applied to increase the feed pH from acidic range to near neutral

396

range. Reactor is fed by gravity in an up-flow mode with 5.75*10
-4
m
3
/day flow rate. The
system is kept at 374 C to proliferate particularly mesophilic range of bacteria.
36.2.3 Treatment efficiency
The Coulombic Efficiency (CE) is directly related to degradation of complex waste
and electron migration:
CE (%) = (1)
where, M
s
is 59 gmol
-1
for acetate to transfer anodic electrons, b=8 (number of electrons
exchanged per mole of acetate), V
Anode
is the volume of anodic liquid, t is change in time over
which the measurement is taken and COD is change in COD over that time period.
Simplification of carbonaceous waste and peptide breakdown of organic nitrogen happens at
anode due to secretion of glycolytic and proteolytic enzymes by wide range of mesophilic
microorganisms. Electrons released during their metabolism are transferred to anode which
increases the flow of electron to the external circuit.
36.2.4 Calculation of thermodynamic entities
Half cell reactions:
Forward: CH
3
COO
-
+ 4H
2
O 2HCO
3
-
+ 9H
+
+ 8e
-
(E
0A
= 0.300

V) (2)
Backward: O
2
+ 4H
+
+ 4e
-
2H
2
O (E
0C
= 0.805 V) (3)
The thermodynamic units are calculated considering these half cell equation to prove
the spontaneity of the overall reaction. Free energy calculation of the fuel cell is calculated as:
-G = nFE
emf
(4)
Where n is the no. of electrons involved in the above reaction 1 and 2, F is the Faradays
constant (96,485 C/e-mol), E
emf
is the electromotive force considering the overall reaction and
E
0A,
E
0C
are equilibrium reaction potential of anode and cathode respectively. The maximum
energy efficiency and performance related to open circuit are characterized by polarization
curves where cell voltage is plotted as a function of current density to obtain kinetic data from
the steady-state current-voltage measurement.
E
0
Cell
= E
emf
+ { E
C
- E
A
} (5)
From equation 3 and 4 overall free energy of the system is calculated where E
emf
represents the difference between cathode and anode working potential (E
C
and E
A
) under no
current flow condition. From the above equation E
A
and E
C
can be derived as:

397

E
A
= E
0A
-
(6)
And, E
C
= E
0C
-
(7)
Where, represent the concentration of proton involved in forward and backward
reaction which can be calculated from the pH value of the reaction (Manohar al., 2008).
36.2.5 Consideration of overpotentials:
A non-linear model is represented in order to characterize the electrical behavior of the
BES. From the polarization behavior, overall potential losses can be considered in a more
comprehensive way by accounting kinetic overpotential, ohmic loss, mass transfer limitation
and loss due to internal current. Kinetic overpotential occurs due to loss of activation energy
to overcome the slowest step of the electrochemical reaction by showing changes in electrode
potential (Bard and Faulkner, 1980). Tafel analysis evaluates Exchange current (I
0
), passing
through the electrode surface in the equilibrium state to emphasize activation potential by
deducing from Butler-Volmer Equation:
For cathodic reaction-
(8)
For anodic reaction-
(9)
Where I stands for total current density, when reactions take place at 37C, is called as
charge transfer coefficient (A- for anode and C for cathode) which depends upon the reaction
involved and electrode material must lie between 0 to1. I
A0
and I
C0
are respective exchange
current passing through anode and cathode. and represent the overpotential observed for
anode and cathode, respectively.
36.3 Experimental results and discussion
36.3.1 Wastewater characteristics and removal of nutrients
The acidic wastewater comprises considerable level of non-fermentable organic
nutrients. Total COD and TSS range was high in between 55,000 and 60,000 mg/l and 26,000
and 28,000 mg/l, respectively (Table 1). Secondary treatment in BES is applied to reduce
COD and TSS. High level of suspended particulate matter obstructs the reaction dynamics of

398

the system by physical clogging or by increasing the viscosity of the effluent. Diluted
wastewater of pH 6.970.03 is fed to anodic chamber. Organic N is hydrolyzed resulting in
formation of organic acids and ammonium ion, which is eventually responsible for increased
alkalinity due to formation of ammonium bicarbonate. Increase in in-situ alkalinity by 30.8%
resulted due to conversion of organic matter, showing gradual increase in pH to 7.130.03
range. Catholyte pH of 7.590.05 is solely due to migration of ammonium ion through
separator. Increase in conductivity with increased TDS level of anolyte was observed, which
might have occurred due to breakdown of complex organic matter by extracellular microbial
enzymes.
Table 1 Characteristics of thin stillage
Parameter Value
pH 3.54
Conductivity (mS/cm) 2.679
TS (mg/l) 43,500
TSS (mg/l) 26,160
VSS (mg/l) 19,816
TDS (mg/l) 2704
TVS (mg/l) 40,776
Total COD (mg/l) 61,295
BOD
5
(mg/l) 28,120
NH
3
N (mg/l) 610
Acetate (mg/l) 1599
Total Protein (mg/l) 4256
Nitrate (mg/l) 0.8147
Orthophosphate (mg/l) 1670
Lignin (%) 5.586
Phenol (%) 0.532

The maximum COD removal is found to be 483 % over 12 weeks of operation. The
presence of less free acetate and because of complex nature of the waste or possibly due to
NSP, the average COD reduction in anode chamber is not more than 50%, although the higher

399

effective surface area of carbon felt encourages bio-film growth to create considerable
potential. System performance due to charge transfer is calculated by equation 1, where
coulombic efficiency is more than 3%. Nitrogenous structure of cell wall gets hydrolyzed due
to bacterial metabolism rising ammonium ion concentration in anode due to simplification by
proteolytic enzymes. Moreover, the carbonaceous waste escaping from anode goes to
nitrification chamber, where it gets digested aerobically with simultaneous nitrification of
increased ammonium ion in presence of oxygen available through air purging. Conversion of
ammonium ion to its oxidized species such as nitrate and nitrite is recorded as 856 % by
ammonia oxidizing bacteria present in nitrification chamber.
36.3.2 Analysis of thermodynamic parameters
Theoretically the collected effluent has 238 kJ/m
3
of energy present in the form of
organic matter. Thermodynamic entities are evaluated from equation 4 and 5 where theoretical
cell voltage, E
0,
at no current flow condition hence no loss condition is 1.009 V, however the
obtained open circuit potential were E
A
0.532 V and E
C
0.050 V, during no current flow
condition. From equation 6 and 7, the ratio of concentration of anodic element of real
wastewater, can be calculated which eventually assists to prove the spontaneity of
the reaction.
36.3.3 Analysis of electrical parameters
Polarization behavior i.e., voltage as a function of current and change in current
density over power density under different operating conditions (10,000-10 external
resistance) is demonstrated to understand electrical properties in Figure 2. Maximum
volumetric power density is exhibited as 22.16 mW/m
2
, when the current density is plotted as
699.18 mA/m
2
at 120 external resistance. Internal resistance under maximum power point
was 30 . Spontaneity of the overall reaction depends on reduction efficiency in cathode.
Theoretically 0.427 V more reduction potential can be produced for maximum electricity
generation by minimizing overpotential at cathode and enhancing oxygen reduction reaction.
From the polarization study we can interpret the loss in overall cell potential. Due to
voltage reduction in low current region of the graph, steep slope has been seen at the initial
stage which is solely because of activation polarization due to charge transfer. As the current
increases the slope becomes less steep as in this region loss occurs on account of the
resistivity of proton exchange membrane and cell electrolytes. Polarization of electrode

400

happens due to shift in cell potential from equilibrium potential and accordingly, this
overpotential can be measured to justify actual cell performance. Kinetic overpotential is
measured by Tafel plot shown in Figure 3.

Figure 2. Polarization behavior of BES

Figure 3. Tafel plot and charge transfer kinetics of working BES for (a) anode and (b) cathode
Exchange current density (I
0
) shows the current flow in absence of net electrolysis at
equilibrium, which is quantified as 0.13 mAcm
-2
and 0.1 mAcm
-2
considering values as 0.2
and 0.68 during reduction and oxidation for cathode and anode, respectively, using equation 8
and 9. Charge transfer efficiency is the function of organic decomposition by micro-organisms
in negative electrode. The performance analysis is based on the availability of simple organics
and presence of exoelectrogens.
36.4 Conclusion
Post fermented rice distillery wastewater shows considerable potential as a source to
produce alternative energy. Although, this process emphasized a lot of challenges due to the
complex nature of the wastewater and high level of suspended solids, hindering the normal
flow of the bio-electrochemical cell. Hence, emergence of different pretreatments to reduce

401

TSS and retrieval of different value added products followed by implementation of this
technique can be a commercially viable option to establish. Simultaneous degradation of C
and N complex matter can be suitably achieved if there is less system hindrance that
eventually can increase the electron transfer efficiency and system performance to more that
3% CE. The system shows 48 % COD removal and nearly 85 % conversion of nitrogen
species to nitrate. Activation polarization and exchange current density have also been
measured by Tafel analysis. Furthermore, it can be inferred that, the optimal behavior under
all adapted conditions could be achieved as rate of forward and backward reactions are nearly
equal considering the current exchange capacity. Although, cathode performance can be
improved by implementing pretreatment of the raw wastewater and by pursuing methods to
activate electrodes for getting higher system efficiency, the research demands more
exploration of the treatment of wastewater hereafter to develop a more efficient model.

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404

CHAPTER 37
EFFECT OF SALINITY, ACETATE ADDITION AND
ALTERATION OF SEDIMENT ON PERFORMANCE OF
BENTHIC MICROBIAL FUEL CELLS
Jadhav D.A., Ghangrekar M.M.

Abstract
Sediment/benthic microbial fuel cell (SMFC) is an attractive approach for bioelectricity
production alongwith sediment bioremediation in water bodies. This study mainly focus on
operational factors like salinity of catholyte, acetate addition and alteration of sediment
properties, which mainly affect performance of SMFC in terms of power production. The
SMFC consisting of graphite electrodes with equal surface area of 28 cm
2
for each anode and
cathode and separated by 8 cm distance between them was used. Freshwater sediment was
collected from Lower Saxony, Germany. With increase in salinity level by NaCl addition in
the water, the cell voltage increased double upto salinity of about 7 mS/cm and further
increase in salinity decreased the performance of SMFC. Conductivity of freshwater is a main
limiting parameter in SMFCs performance. The acetate can be used as the carbon source to
induce power production by electroactive bacteria. An increment was reported in cell voltage
due to addition of acetate upto 2 mM and further addition decreased the power from MFC.
While evaluating performance of SMFC with different sediments, the result shows that the
operating voltage of SMFC varies not only with locations from where sediment was collected
but also upstream to downstream of same catchment area due to variation in total organic
carbon present in the sediment and microbial activity. In future, research should be focused on
the designing of SMFCs with increasing size and scale for increased power output and
reducing operational problems.
Keywords: Sediment microbial fuel cells, Conductivity, Sediment alteration, Acetate.



405

37.1 Introduction
Extensive research toward developing reliable microbial fuel cells (MFCs) is mostly
focused on selecting suitable operating conditions, organic and inorganic substances which
can be used as sources of energy. Sediment microbial fuel cells (SMFCs) is important to
generate power for operating under sea devices like sensors, hydrophones (Bond et al., 2002).
Organic-rich sediment in freshwater and marine environments can be considered an abundant
potential source of renewable energy in SMFCs. The naturally occurred sediment water
interface serve as separator for anodic and cathodic chamber in SMFC (Rabaey et al., 2009).
The anode is placed in anaerobic sediment and connected it through an external load; the
cathode is placed in the overlying water (Fig. 1). Freshwater sediments and river water can be
used as a renewable source to generate power using sediment microbial fuel cells (Sajana et
al., 2013; Lovely, 2006). The performance of SMFC is mainly governed by the characteristics
of sediment, operating conditions like salinity, electrode spacing, electrode material and its
properties, etc. Sediment-based MFCs were first explored in 2001 by Reimer et al., and it was
demonstrated that power could be generated from the microbes and the nutrients found within
the sediment alone (De Schamphelaire et al., 2008; Nielson, 2008).

Fig.1 Mechanism of electron transfer in SMFC
Previously, Logan et al. (2006) reported that dominant ohmic losses through the
electrolyte can be reduced by increasing the electrical conductivity (EC), which results in
higher power production from SMFCs in marine environments than in freshwater
environments. Current and power densities increase with the increasing salinity of the aquatic
medium and therefore with increasing ionic strength and conductivity (De Schamphelaire et

406

al. 2010; Tender et al., 2002). Hidalgo et al. (2010) noted that the increase in NaCl
concentration resulted in an increase in both power density and Coulombic efficiency (CE) as
long as NaCl concentration remained below 2 mM. At latter value a maximum power and CE
were obtained (82.1 W/m
2
and 15.91% respectively).
Particularly in marine environments, Geobacteraceae species are involved in electron
transferring process. Acetate served as the fuel in an MFC using marine sediment as the
inoculum (Lee et al., 2003). Song et al., 2010 found that maximum power density from the
SMFC with addition of 0.2% cellulose reached to 11.2 mW/m
2
on 160 days of operation in
freshwater environment. Addition of cellulose causes increase in performance of SMFC after
few days of operation. The alteration of physico-chemical properties of sediment organic
matter (SOM) was studied by Hong et al., 2010 and reported that the physico-chemical
properties of SOM were considerably altered when electrical current was produced from
freshwater sediments in the MFC systems.
In the present study, SMFC was used to enrich electrochemically active microbes on
acetate as the sole electron donor. To date little effort has been directed towards quantifying
and predicting the effects of properties of sediment on the anodic reaction during electricity
generation from SMFC. The specic objective in this study was to evaluate the performance
of MFC under change in properties of sediments collected from different locations. The effect
of salinity or conductivity of water on performance of SMFC was also studied to find
optimum salinity limit.
37.2.1 Sediment as source of inoculum
The sediment [S
0
] was collected from downstream at Sandbach catchment area near
Hordorf and Schandelah, Lower Saxony, Germany. This site was located next to the
municipal wastewater treatment plant and not affected by any effluent contamination. The
sampling depth was restricted to 5 cm from the upper layer of the sediment in order to sample
mainly sediment under aerobic conditions (OECD, 2002). The sediment was then placed in
plastic container and covered with native water. Water/sediment samples were characterized
(Table 1) according to the OECD guideline (OECD, 2002, Mohamad, 2010).


407

Table 1 Characteristics of sediment collected for study (Mohamad, 2010)
Sediment Properties Values
Sediment type Sandy
Sand 53 %
Silt 20%
Clay 27%
TOC (Total organic carbon) 0.4% (dry substrate)
WHC (Water holding capacity) 30 %
pH 7.3
Eh (Redox potential) 220 mV
SIR (Substrate induced respiration) 1.90 mg O
2
/100 g h

37.2.2 MFC design and Construction
This study was carried out with four laboratory scale SMFCs having an anodic volume
of 5 L. The sediment was collected from downstream at the Sandbach catchment area near
Hordorf and Schandelah, Lower Saxony, Germany. The SMFC was constructed with the
cathode and anode of graphite material having a total cross sectional area of 28 cm
2
placed
parallel to each other (Fig. 2). The bottom half portion of the SMFC was filled with
homogeneously mixed sediment to work as anolyte and upper half portion with freshwater to
work as a catholyte. The spacing between electrodes was maintained at 8 cm. Both electrodes
were held in SMFC by a polyvinyl chloride (PVC) cylindrical pipe holder. The connection
between wire and electrode was made using conductive and watertight mixture of resin,
hardener and carbon powder to make it conductive. Air was introduced in the form of fine air
bubbles by using air diffuser and a air pump (ELITE 799, Hagen, Mansfield, MA) in the
freshwater on cathodic side. All connections were made with watertight connectors using
silicon and flexible parafilm. Over the period of the experimental run, the water loss occurred
due to evaporation was compensated by adding additional either freshwater from river or
distilled water daily and a constant water level in MFC was maintained. SMFC was operated
by adding wet sediment to the anodic side without giving any pre-treatment. Within a day, the
sediment re-settled at the bottom of the bucket. After stabilization, 100 external resistance
(unless stated otherwise) is connected in circuit.

408

Fig. 2 Experimental set-up of lab scale SMFC

37.2.3 Measurement and analysis
The potentials generated by the cells were measured with a digital multimeter with
data acquisition unit (model 2700 and 2701, Integra Up and Running, Keithley Instruments,
Data Acquisition, Ohio, U.S.A). The voltage difference between each electrode pair was
recorded at 5 min interval on a spreadsheet using ExceLINXTM (Keithley). Power was
calculated according to the formula: P = IV, where P = power, I = current and V =
voltage. Power density (mW/m
2
) was calculated as power production per m
2
surface area of
anode and the power output is usually normalized to the anode surface area because the anode
is where the biological reaction occurs (eq. 1, 2).
P
A
= E * j (1)
j = (2)
where, P
A
= power density, E = voltage generated, R = external resistance, Aa = area of anode,
j = current density, P = actual power generated.
The highest voltage produced in a SMFC is the open circuit voltage (OCV), which was
monitored after allowing the circuit to remain in open condition (no current flow) till the
potential value is stabilized. The anode potential, cathode potential, OCV and operating
voltage (OV) was also monitored daily during experiment under different operating
conditions. Cathode potential was measured by using Ag/AgCl reference electrode (Sat. KCl,
sensor technique, Meinsberg GmbH, Germany). The cell EMF can be calculated as eq. 3
E
emf
= E
cathode
E
anode
(3)
Conductivity of water in SMFC was measured daily by using a conductivity probe
(GLF 100, Greisinger electronics, Germany). The pH of water was measured by using pH

409

meter (GPHR 1400A, Greisinger electronic, Germany). Temperature during experiment was
maintained nearly constant about 18 0.5 C in winter and 20 0.5 C in summer season.
37.2.4 Effect of Salinity
The electrical conductivity (EC) of freshwater in SMFC [with sediment S
0,
Table 2] is
about 1030-1270 S/cm. Freshwater typically contains less than 1% sodium chloride. To
study effect of EC, salinity of water changed from 1000 to 10500 S/cm using sodium
chloride (NaCl) (added stepwise) while considering bacterial sensitivity to sodium salt.
37.2.5 Effect of Acetate addition
Acetate is the most abundant fatty acid in anaerobic ecosystems and is used as an
electron donor by anaerobic respiratory bacteria. To study the microbial response, acetate was
added and performance of SMFC with 8 cm electrode spacing (with sediment S
3,
Table 2)
performance was evaluated. Acetate was added from 0 to 15 mM at regular interval of time to
enhance activity of microorganism. The sodium acetate was added considering volume of
anodic side to maintain this concentration. However, there is possibility of diffusion of acetate
that occurs through sediment water interface. The pH of acetate solution was neutralised using
glacial acetic acid (CH
3
-COOH). In SMFC, acetate injected at different points in sediment
using syringes so that it distributed evenly in the sediment. Over the course of operational
period, bioelectrical response in association with external acetate addition was observed.
37.2.6 Alteration of sediment properties
The sediment properties changes from location to location due to different natural and
human activities so that performance of SMFC also varies. However, the relationships
between organic carbon decomposition, microbial diversity and density, and power production
remain largely unknown. In this study, aim is to find how sediment from different location
affects on cell performance. For this study, sediments were collected from Sandbach
catchment area (5216'7.16"N, 1040'51.81"E) [S
0
] from downstream, [S
3
] from upstream of
same catchment, Wiedigsteich Lake (5216'18.28"N, 1034'47.26"E) [S
1
] and Kreuzteich
Lake (5216'19.33"N, 1034'39.82"E) [S
2
] in Braunschweig, Lower Saxony, Germany. River
sediment was collected from shallow river bank. The prototype of SMFCs using these
sediments were constructed similar to SMFC as mentioned previously with maintaining 8 cm
spcing between the electrodes. The characteristics of sediments are given below in Table 2.

410

Table 2. sediment characteristics of SMFC collected at different locations in study area
Sediment Properties Values of sediment characteristics from different locations
S
0
S
1
S
2
S
3

Location in
Braunschweig
Sandbach
catchment (d/s)
Wiedigsteich
Lake
Kreuzteich
Lake
Sandbach
catchment (u/s)
pH 7.3 7.62 7.51 8.03
Conductivity (S/cm) 1060-1276 1170-1265 918-1090 910- 1575
Other Sandy type More water
content
*** Loamy type
*** Values not determined
37.3.1 Effect of Salinity
To study effect of salinity, EC was increased from 1000 to 10500 S/cm by adding
sodium chloride (NaCl) stepwise. Increase in EC of freshwater caused decrease in ohmic
losses and increase in power output. It was noted that high salinity of the water provides good
ion conductivity between the electrodes. No change in the microbes present in the sediment
was done, while changing the salinity by NaCl addition. It was reported that cell voltage
increases with increasing salinity of water (Logan et al., 2006). However, cell voltage
decreases after certain limit of salinity because of possible adverse effect on bacterial
metabolism at higher salt concentration. Bacteria can produced current only within certain
salinity limits, and thus solution conductivity cannot be increased beyond certain value.
SMFCs shows critical salinity limit at 7000 S/cm. This critical limit is helpful to determine
level of salinity of river water for feeding it to an SMFC system which also limit use of SMFC
for highly saline seafloor.
There was small apparent change in cell performance in SMFC with NaCl addition of
upto 90 mM, then cell voltage increases rapidly (Fig.3). Increasing the catholyte conductivity
decreases the ohmic resistance, and it has been shown that power density can be substantially
increased by adding NaCl upto 444 mM of to the solution. Further increase in salt
concentration reduced power production by inhibiting bacterial growth because microbes are
sensitive to certain salinity limits (Jeffrey, 2008). Also, high salt concentrations are known to
adversely affect the physiology of anaerobic microbial consortia. However, at high salt

411

concentration, cations present in the solution compete with protons in their migration towards
cathode which results into reduction in voltage produced by the cell (Wang et al., 2011).
During this experiment, cathode potential was found to be negative in some cases because of
electrical energy provided to the cell is being used to decompose organic compounds present
in sediment. The cell voltage increased from 3.81 to 7.38 mV upto maximum salinity limit
(about 7 mS/cm) when operated at 100 resistance (Fig. 3). The cell voltage decreased with
further increase in salinity. This suggests that EC (salinity) of water is main limiting factor in
SMFC performance which increased cell voltage by 93% due to reduction in ohmic losses,
when the salinity was at optimum.
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
C
e
l
l

V
o
l
t
a
g
e

(
m
V
)
Salinity (S/cm)
Cell Voltage

Fig 3 Effect of salinity on SMFC performance
37.3.2 Effect of acetate addition
To understand the effect of presence of organic matter in sediment on performance of
MFC, acetate was used as the external carbon source. Microbes like geobacter oxidizes
acetate to carbon dioxide and water while reducing compounds such as sulphur and some
metals including iron oxides, with quantitative transfer of electrons to an anode. Acetate
added get oxidised faster due to faster oxidation after some time. Small change in voltage
occurred during acetate addition initially at low concentration (Fig. 4; Fig. 5). Acetate was
used as carbon source to induce power production by electroactive bacteria (Kim et al., 2000)
because of its inertness towards alternative microbial conversions at room temperature (Zhao
et al., 2012).

412

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
2
4
6
8
10
12
14
16
C
e
l
l

v
o
l
t
a
g
e

(
m
V
)
Acetate addition (mM)
8 cm A
8cm B
8cm C

Fig 4. Effect of acetate addition on cell voltage
87 90 93 96 99 102 105 108 111
0,000
0,002
0,004
0,006
0,008
0,010
0,012
0,014
0,016
0,018
0,020
Cell voltage
Power density
Time (days)
C
e
l
l

v
o
l
t
a
g
e

(
V
)
0.3mM
0.33 mM
1mM
2mM
5mM
10mM
15mM
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4

P
o
w
e
r

d
e
n
s
i
t
y

(
m
W
/
m
2
)

Fig. 5. Voltage produced and power density vs. time at different acetate concentration
Once the current reached a stable level, acetate was consumed completely, and current
generation was dependent on the acetate supply. During consumption of acetate, cell voltage
increased and then started decreasing along with time period (Fig. 5). Addition of acetate
stimulates growth of bacteria in the MFC. Power density also increased upto 2 mM acetate
addition and then decreased. Addition of 5 mM also drastically changed electrode potential.
The results suggest that there is about ten fold increment in cell voltage due to addition
of acetate (2 mM) in sediment. There was slight increase in cathode potential after 5 mM
addition (Fig. 4); however, the potential started to decrease slowly. After 10 mM acetate
addition, cell voltage decreased further and subsequent additions of acetate typically did not

413

increase voltage output. The reduction in power production at higher acetate addition could be
attributed to higher concentration of acetate near cathode, which had adversely affected
cathodic reaction (Zhang et al., 2006) and reducing overall performance of the cell. There is a
possibility of diffusion of sodium acetate (CH
3
-COONa) through sediment water interface
during injection in sediment zone.
37.3.3 Alteration of sediment in SMFC
The sediment properties are useful for determining the microbial biomass and
microbial activities in the sediment. This gave hint for high porosity of river sediments as it
consists of mainly coarse to fine sand grains. Power generation by SMFCs was dependent on
the particulate sediments, sediment structure and texture. In general, rivers in Braunschweig
contain less organic matter. Due to this, large fish and other aquacultural animals found less in
quantities in these rivers.
In addition, assuming that temporal organic pollution has occurred in a water body
(rivers), this study examined the possibility of harvesting current by utilizing the organic
matter coexisting in water and sediment phases. Results showed that the most important
parameters that control SMFCs power output were total organic carbon in the sediment and
activity of geobacter. It was found that sediment properties had strong effect on SMFCs
performance, and Fe(III) contents in sediments were significantly related to voltage values
produced (Song et al., 2012). The environmental conditions in the sediments were suitable for
enrichment and growth of microbes. However, the activity and competitive ability of
exoelectrogenic microorganisms in sediments in different locations might show variance
(Table 1; Table 2), as the composition of sedimentary microbial communities were
significantly affected by local environmental conditions, large intra-lake variability in the
composition and the relative abundance of microbial communities in sediment was always
observed (Cordova- Kreylos et al., 2006).
During initial period of operation, SMFC with loamy sediment (S
3
) showed current
density of 4.18 mA/m
2
which is quite higher than other sediments (Fig. 6); because the
sediment S
3
belonging to this SMFC contained more mud and humic substances as compared
to the other SMFCs (with sediments S
0
, S
2
and S
1
) consisting of more fine to coarse sand as
major sediment type and some other pollutants (Table 2). The performance of SMFC utilizing
S
2
sediment was slightly lesser than SMFC with S
3
sediment. Over the course of experimental

414

period, stable OCVs were observed from SMFCs. Due to high percent of sand, SMFC with
sediment S
0
showed lower voltage (about 0.69 mV). In this MFC, due to higher porosity of
sand, there is possibility of oxygen intrusion to the anodic side. It shows that the physical
properties of sediments and the dynamics of natural environments are a special set of factors
that affect the performance of SMFCs. These results suggest that site selection for sediment
collection is important constraints before starting the experiments on SMFC. Certainly, the
values representing physical and chemical properties of sediment of the Sandbach catchment
area (upstream) has been highly humified in comparison with humus materials found in other
sediment type used in the study. Therefore, the presence of more humic substances in
sediments may boost current production in freshwater SMFC.
The sediment S
1
contains more water than others sediments used in the study. Hence,
SMFC with sediment S
1
showed lots of noise in measurement during operation over period of
time (not shown in Fig. 6). The sediment properties changes from one location to other
locations on same water bodies. So, its not correct way to consider same SMFC power
performance to all along the bank of river as noted in case of Sandbach catchment (Table 3).
The electric signal produced in SMFCs reflectes the activity and competitive ability of
exoelectrogenic microbes in sediments present at that location. Thus, these observations
support that the current production induced by microorganisms and sediment organic matter
(SOM) was dependent on the sediment characteristics: type of organic matter and diversity of
microbial populations (Hong et al., 2009).

Fig. 6 Power performance of SMFC with different sediment


415

Table 3 Summary of performance of SMFC with different sediment characteristics
SMFC
with
sediments
Operating
characteristics
Locations in
Braunschweig
Power
density
W/m
2

Current
Density
mA/m
2

Cell Voltage
at 100 ohm
(mV)
S
0
8 cm ES Sandbach
catchment (d/s)
1.7 2.46 0.69
S
1
8 cm ES Wiedigsteich 1.12 2.01 0.56
S
2
8 cm ES Kreuzteich 4.56 4.04 1.13
S
3
8 cm ES Sandbach
catchment (u/s)
4.89 4.18 1.17

37.4 Conclusions
Power performance in cell increases by 93 % with increase in the salinity of water
from 1.03 to 7 mS/cm in SMFC. Our investigation also revealed that power density varies
with sediment properties as well. Increase in concentration of acetate increases the power
density till 2 mM and further increment reduce the power production from the SMFC. The
power output for SMFC using sediment from Sandbach catchment area (upstream) is higher
than other SMFC from remaining sediments used for study. Thus, these results support that
the current production induced by microorganisms and sediment organic matter was
dependent on the sediment characteristics: type of organic matter and diversity of microbial
populations. These results can be helpful for site selection to install scale up SMFC for power
harvesting from freshwater sediment. It was noticed that cell voltage varies with locations
from where sediment is collected for the same catchment area.
37.5 Future perspectives
It seems that SMFCs are effective technique to remove organic matter from sediment
(sediment bioremediation) and also useful for energy harvesting. With few improvements, in
order to scale it up to an appropriate size, SMFC could really make a difference in the world
energy sector for future generations. The potential for energy generation from the seafloor is
large, although the accessibility will often pose a problem. Furthermore, there are many
microorganisms yet to be discovered that might be beneficial for electricity production. The
cell design in future tests should more closely reflect environmental conditions. Sediment type
fuel cells show potential for low power generation from marine contaminated and

416

uncontaminated sediment. Future research should be focused on maximizing of power output
and minimizing construction and operation cost of SMFCs.
Acknowledgement
The authors wish thanks to the German Academic Exchange Service DAAD for
providing scholarships during research stay in Braunschweig, Germany. The authors thank
Prof. Dr. Uwe Schrder, Harnisch F., Carmona-Martinez A.A. and the team of Institute
of Environmental and Sustainable Chemistry, Technical University of Braunschweig,
Germany for inspiring guidance and valuable advice throughout the period of research work.

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419

CHAPTER 38
BIOHYDROGEN PRODUCTION USING SINGLE-CHAMBER
MEMBRANE-FREE MICROBIAL ELECTROLYSIS CELL
WITH A STAINLESS STEEL CATHODE
Sundaresan Mohanraj, Krishnasamy Anbalagan, Kodhaiyolii Shanmugam, Velan Pugalenthi

Abstract
Single-chamber membrane-free microbial electrolysis cell was designed and used to
investigate the hydrogen production from glucose by mixed culture at 37C. In the present
study, the low cost anode and cathode materials were carbon graphite (1.9 cm diameter x 3.2
cm length) and stainless steel mesh (3.5 cm diameter X 4.8 cm length), respectively. In batch
experiments, the hydrogen production rate was increased from 1.42 to 1.64 ml/h when the
applied voltage was varied from 0.2 V to 0.6 V with an interval of 0.1V. Further the hydrogen
production rate was decreased with increase of applied voltage at above 0.6 V. A membrane-
less system with close electrode spacing (1.74 cm) showed that a maximum hydrogen
production rate of 1.64 ml/h was obtained at an applied voltage of E (ap) = 0.6 V. In
comparison, the control experiment (without applied voltage) indicated that the hydrogen
production rate was 1.07 ml/h. The maximum cumulative hydrogen production and hydrogen
content at 0.6 V were 149 mL and 41 %, respectively. These results suggest that the low cost
cathode was able to increase the hydrogen recoveries and hydrogen production rates. In
conclusion, the current density and volumetric hydrogen production rate of this system can be
enhanced by further modification of electrode with increasing the ratio of electrode surface
area.
Key words: Microbial Electrolysis Cell; SS cathode, mixed culture; applied voltage
38.1 Introduction
Hydrogen is an important role in a future energy fuel, owing to its high energy content
and the ability to produce from various organic substrates. Among the hydrogen production
technologies, hydrogen production from organic matter by fermentative bacteria have been



420

extensively studied (Liu et al., 2010). However, the stoichiometric potential of 12 mol H
2
/mol
glucose from fermentative process has not been achieved. Mostly, fermentative processes are
limited to a maximum yield of 4 mol H
2
/mol glucose and remaining of the energy is converted
to organic acids and alcohols. Therefore, further processes are required to convert
fermentative effluent (organic acids) to hydrogen (Lu et al., 2009). Recently, microbial
electrolysis cell (MEC) has been developed a new method for hydrogen production from
fermentative end products and other organic substrate (Liu et al., 2005; Rozendal et al., 2006;
Call and Logan, 2008; Logan et al., 2008). In microbial electrolysis cell, a substrate is
oxidized by exoelectrogens and released electrons to the anode. Further, the electrons are
transferred from anode to cathode through an external circuit and combined with protons to
produce hydrogen at cathode (Call and Logan, 2008). However, a small voltage is required to
the circuit for allowing hydrogen production at the cathode when compared to water
electrolysis. A voltage of >0.2V in practice has been required for hydrogen production from
acetate as a substrate (Cheng and Logan, 2007; Hu et al., 2008). This voltage has considerably
lower than the water electrolysis process for hydrogen production (Lu et al., 2009).
Recent years, the microbial electrolysis cell performance has been considerably
improved. However, in practice, the high cost of the reactor and low production rates are main
issues for its application (Cheng and Logan, 2007, Rozendal et al., 2007). Therefore, its recent
advances in reactor design (Call and Logan, 2008, Tartakovsky et al., 2008 and Hu et al.,
2008) and operation (Lalaurette et al., 2009 and Selembo et al., 2009) still need to be
developed for realizing the applications of this technology. Also, the high cost of cathode is
one of the most critical ones. Pt based cathodes have been widely used in MEC studies (Liu et
al., 2005) owing to its effecient catalytic properties and popularity in microbial fuel cell
(MFC) studies (Logan et al., 2008). The alternatives to Pt based cathode in MECs have been
investigated recently. Selembo et al. (2009) studied the nickel oxide catalysts through cathodic
electrodeposition of NiSO
4
and (NH
4
)
2
SO
4
on a metal sheet in MECs. Harnisch et al. (2009)
reported that the electrocatalytic behavior of synthesized tungsten carbide powder in MECs by
pasting the powder onto graphite disc with Nafion. However the main challenges associated in
an MEC for hydrogen production including low volumetric efficiency and the use of an
expensive catalyst (platinum). Hence, the researchers have led to examine non-precious
metals and alternatives for Pt based cathodes.

421

A single-chamber membrane less MEC with cost effective cathode has been attention
recently. Lack of membrane in MECs reduce the lower the internal resistance, eliminate the
pH gradient across the membrane, and enhance the hydrogen production rate (Hu et al., 2008,
Lu et al., 2009). However, in membrane less MEC, hydrogen reoxidization by exoelectrogens
and methanogens are main challenges. The present study attempts to the develop the efficient,
stable and cost-effective stainless steel cathode for hydrogen production in MECs. This
electrode performance was also examined for hydrogen production in membrane less single-
chamber MECs.
38.2.1 Reactor construction
Single-cell MEC was constructed with graphite brush (1.9cm diameter x 3.2cm length)
as anode. Stainless steel mesh (3.5 cm diameter X 4.8cm length) was used as cathode. The
distance from the middle of brush anode to the cathode was 1.75 cm. Both anode and cathode
chambers were connected to electrode and outside circuit. A power source was supplied at a
steady voltage (0.2 1.0 V) between anode and cathode electrode. A power source (DC-3002,
Beetech, India) was connected to the circuit for providing voltage, and an external resistor
(1000 ) was to calculate the current (Fig.1).

Fig.1. Photograph of MECs with stainless steel cathode
38.2.2 Start-up and operation
MEC were first operated in microbial fuel cell (MFC), with the cathode exposed to air
as mentioned previously (Lu et al., 2009). MFC was inoculated with sewage sludge, collected
from the local wastewater treatment plant, in a nutrient buffer solution (NBS) (Na
2
HPO
4
, 4.58
g/l; NaH
2
PO
4
H
2
O, 2.45 g/l; NH
4
Cl, 0.31 g/l; KCl, 0.13 g/l; trace mineral; 50:50
(v:v)wastewater and buffer) (Call and Logan, 2008) containing 1 g/l glucose. During each fed-

422

batch cycle, the solutions in the MFCs were replaced when the voltage was decreased to <
20mV (external resistance of Rex = 1000 ). Once a maximum voltage of >0.5V (Rex = 1000
) was obtained the cathodes were sealed and the reactors were switched to MEC mode.
The MEC was fed to a 200 mL of autoclaved medium containing (in 1 l of pH 7.0
phosphate buffer): NH
4
Cl, 310 mg; KCl, 130 mg; CaCl
2
, 10 mg; MgCl
2
.6H
2
O, 20 mg; NaCl,
2 mg; FeCl
2
, 5 mg; CoCl
2
.2H
2
O, 1mg; MnCl
2
.4H
2
O, 1mg; AlCl
3
, 0.5 mg; (NH
4
)6Mo
7
O
24
, 3
mg; H
3
BO
3
, 1mg; NiCl
2
.6H
2
O, 0.1mg;CuSO
4
.5H
2
O, 1mg; ZnCl
2
, 1 mg and 1.0 g/L glucose as
a carbon source. pH was adjusted by 1N NaOH solution and monitored by pH meter. A fixed
voltage (Eap) of 0.21.0 V was applied to the MEC circuit using a power supply (DC 3002,
Beetech, India) by connecting the positive pole of the power supply to the anode, and the
negative to the cathode. The voltage across a high-precision resistor (R
ex
=1000 ) in the
circuit was measured using a voltmeter at 1 h intervals to calculate current. The volume of gas
produced from MECs was measured by water displacement method. All experiments were
conducted in fed-batch mode. Fresh medium was added to the reactors and sparged with ultra
high purity nitrogen gas (99.999%) for 10min. All tests were conducted at room temperature.
Applied voltage scans were obtained by varying the voltages stepwise from 0.2 to 1.0 V at 1 h
intervals between each voltage (v) change. The current densities (cathode surface area) and the
potential of electrodes data of every applied voltage step were averaged over each time
interval.
The composition of biogas including H
2
, CO
2
and CH
4
was analyzed by Shimadzu gas
chromatograph (GC-2014) (Shimadzu Co. Singapore) equipped with a thermal conductivity
detector and a stainless column packed with Porapak Q (80/100 mesh). The operational
temperatures at the injection port, column oven and detector were 40 C, 40 C and 80 C,
respectively. Nitrogen was used as carrier gas at a flow rate of 20 mL/min. The concentration
of the volatile fatty acids (VFAs) was measured by Shimadzu gas chromatograph (GC-2014)
(Shimadzu Co. Singapore) equipped with a flame ionization detector (FID) and stabilwax
DA capillary column. The injector, column oven and detector were operated at 180, 200 and
220C, respectively. Nitrogen was used as carrier gas with a flow rate of 5 mL/min. The
concentration of glucose was determined using phenol-sulfuric acid method.


423

38.2.4 Calculations
Hydrogen yields was calculated as YH
2
= g H
2
/g glucose or mol H
2
/mol glucose and
coulombic efficiency, CE (%), was calculated as CE = C
T
/C
C
, where C
T
is the total coulombs
calculated by integrating the current over time, and C
C
is the total charge consumed. Cathodic
hydrogen recoveries (r
cat
, e to H
2
in the cathode), overall hydrogen recovery (RH
2
= CE
rcat
),
and maximum volumetric hydrogen production rates (mL/h) were calculated as previously
described assuming standard biological conditions (T=298.15K, P = 1 bar, pH 7) (Logan et
al., 2008).
38.3.1 Biohydrogen production from glucose
Hydrogen production rates were substantially increased from 1.42 (at 0.2 V) to 1.64
ml/h (0.6 V) using buffered fermentation effluent by increasing the applied voltage (Fig. 2).
Further, the hydrogen production rate was decreased from 1.64 ml/h to 0.43 ml/h, when the
Eap was varied from 0.6 to 1.0 V. The control experiment (without applied voltage) showed
that the hydrogen production rate was 1.47 ml/H. On the other hand, hydrogen content was
found to be same in Eap upto 0.5V and control experiment. The maximum hydrogen content
of 41 % was obtained at 0.6 V. Furthermore, the hydrogen content was decreased from 41 to
31 % as the Eap was varied from 0.6 to 1.0V. The maximum cumulative hydrogen volume of
149 ml was observed at 0.6V. The hydrogen production remained decreased with the further
increasing of applied voltage upto 1.0 V. These findings indicate that the hydrogen production
was considerably enhanced at an optimum applied voltage (0.6V) in single-chamber
membrane-less MEC using mixed culture when compared to the control experiment (110 mL)
(Fig.3).
38.3.2 Coulombic efficiency and current density
As seen in the Figure 4, the Coulombic efficiencies (CE) were increased from 5.4 to
33.7 % by varying the applied voltage from 0.2 to 0.7V. Further it was decreased at above 0.7
V. At low applied voltages of 0.2 and 0.4 V, CE values were found to be low due to
methanogenesis. Similar results were obtained by Tartakovsky et al., (2008). As the E
ap
was
increased from 0.7 to 1.0 V, the anode potential was increased and the CE was decreased (Fig.
4). The decrease of CE at the highest applied voltage resulted in a reduction of hydrogen
recovery (R
H2
). However, the current density was gradually increased from 0.04 to 2.32

424

mA/m
2
when the applied voltage was varied from 0.2 to 1.0 V. According to literature, a
higher resistor increased the output voltage of the power supply and shared a higher voltage
on it (Hu et al., 2012). For the MEC, the hydrogen production rate was partially dependent on
the resistor. In spite of this, a constant resistor (1000 ) was used in this study and the
maximum output current density of the external power supply was reached to 2.32 mA/m
2
.
Consequently, the high hydrogen production rate was achieved (Fig.2). Similar observation
was obtained by Chae et al. (2008).

Fig.2. Hydrogen production rate, hydrogen content and cumulative hydrogen production with
different applied voltage

Fig.3. Cumulative hydrogen production with different time interval of control and 0.6V
experiment
38.3.3 Hydrogen Recoveries

425

Factors affecting the hydrogen yield were observed to be current density (normalized
to electrodes area), coulombic efficiency, and cathodic hydrogen recovery. From Figure 4, the
cathodic hydrogen recoveries were increased from 37.5 to 79.3 % as the applied voltage was
varied from 0.2 to 1.0 V. In addition, the overall hydrogen recovery was similar to the
cathodic hydrogen recoveries. The overall hydrogen recoveries were remarkably increased
remarkably from 34.7 to 75.9 % as the applied voltage was differed from 0.2 to 1.0V (Fig. 3).
These results demonstrate that cathodic hydrogen recovery at below 0.5 V was very low. The
high hydrogen recovery was achieved at above 0.5V of applied voltage. These results show
that the low current density and coulombic efficiency reduced the hydrogen production rate.
Further study requires on optimizing the architecture to lower the internal resistance for
improving the hydrogen production.

Fig.4. Current density and Coulombic efficiency at different applied voltage

Fig.5. Hydrogen recoveries and glucose utilization at different applied voltage

426

38.4 Conclusions
The hydrogen recovery was improved in a low cost single-chamber membrane less
MEC with stainless steel cathode. In addition, small electrode spacing and lack of membrane
used in this reactor contruction reduced the pH gradients and proton diffusion resistance
which led to high hydrogen production rate. The maximum hydrogen production rate of 1.64
ml/h and overall hydrogen recovery of 69.7 % were achieved when the applied voltage was
0.6 V. In comparison, the control experiment (without applied voltage) indicated that the
hydrogen production rate was 1.47 mL/h and hydrogen recovery was 31.5%. Further,
optimizing the internal resistance and increasing the current density would improve the
hydrogen production rate in this MEC.
Acknowledgements
This research was funded by the Department of Biotechnology (Ref. No.
BT/PR12051/PBD/26/213/2009, dated 19th November 2010), New Delhi, India. Author
Mohanraj gratefully thank Ministry of New and Renewable Energy, New Delhi, India for
Senior Research Fellowship (NREFSRF).

References
1. Call D. and Logan B.E. (2008) Hydrogen production in a single chamber microbial
electrolysis cell lacking a membrane. Environ. Sci. Technol., 42:34016.
2. Chae K.J., Choi M., Ajayi F.F., Park W., Chang I.S., Kim I.S. (2008) Biohydrogen
production via biocatalyzed electrolysis in acetate-fed bioelectrochemical cells and
microbial community analysis. Int. J. Hydrogen Energy, 33:518492.
3. Cheng S. and Logan B.E. (2007) Sustainable and efficient biohydrogen production via
electrohydrogenesis. Proc. Natl. Acad. Sci., 104:188713.
4. Harnisch F., Sievers G., Schroder U. (2009) Tungsten carbide as electrocatalyst for the
hydrogen evolution reaction in pH neutral electrolyte solutions. Appl Catal B: Environ.,
89; 455458.
5. Hu H., Fan Y. and Liu H. (2008) Hydrogen production using single-chamber membrane
free microbial electrolysis cells. Water Res., 42:41724178.
6. Liu H., Grot S. and Logan B.E. (2005) Electrochemically assisted microbial production
of hydrogen from acetate. Environ. Sci. Technol, 39:431720.

427

7. Lalaurette E., Thammannagowda S., Mohagheghi A., Maness P.C. and Logan B.E.
(2009) Hydrogen production from cellulose in a two-stage process combining
fermentation and electrohydrogenesis. Int. J. Hydrogen Energy, 34:620110.
8. Liu H., Hu H., Chignell J. and Fan Y. (2010) Microbial electrolysis: novel technology
for hydrogen production from biomass. Biofuels., 1:129142.
9. Logan B.E., Call D., Cheng S., Hamelers H.V.M., Sleutels T.H.J.A., Jeremiasse A.W.
and Rozendal R.A. (2008) Microbial electrolysis cells for high yield hydrogen gas
production from organic matter. Environ. Sci. Technol., 42:86308640.
10. Lu L., Rena N., Xing D. and Logan B.E. (2009) Hydrogen production with effluent from
an ethanolH
2
-coproducing fermentation reactor using a single-chamber microbial
electrolysis cell. Biosen. Bioelect., 24:30553060.
11. Rozendal R.A., Hamelers H.V.M. and Buisman C.J.N. (2006) Effects of membrane
cation transport on pH and microbial fuel cell performance. Environ. Sci. Technol.,
40:520611.
12. Rozendal R.A., Hamelers H.V.M., Euverink G.J.W., Metz S.J. and Buisman C.J.N.
(2006) Principle and perspectives of hydrogen production through biocatalyzed
electrolysis. Int. J. Hydrogen Energy., 31:163240.
13. Rozendal R.A., Hamelers H.V.M., Molenkamp R.J. and Buisman C.J.N. (2007)
Performance of single chamber biocatalyzed electrolysis with different types of ion
exchange membranes. Water Res., 41:198494.
14. Selembo P.A., Merrill M.D. and Logan B.E. (2009) The use of stainless steel and nickel
alloys as low-cost cathodes in microbial electrolysis cells. J. Power Sources., 190, 271
278.
15. Tartakovsky B., Manuel M.F., Neburchilov V., Wang H. and Guiot S.R. (2008)
Biocatalyzed hydrogen production in a continuous flow microbial fuel cell with a gas
phase cathode. J. Power Sources., 182:2917.


428

CHAPTER 39
FEASIBILITY OF INTERLINKING TWO TECHNOLOGIES
FOR SIMULTANEOUSLY TWO BIOENERGIES
GENERATION
Prashant Pandey, Vikas Shinde, S.P. Kale, R.L. Deopurkar

Abstract
Microbial fuel cell for bioelectricity generation rapidly extending interdisciplinary area, has
attracted great research efforts to fulfill the demand of renewable energy. Here, we studied
bioelectricity generation using food waste leachate obtained from aerobic predigestion tank of
two-stage aerobic-anaerobic sequential biogas reactor (Nisargruna Biogas Technology).
Volatile fatty acids content of food waste leachate was 8000 mgl
-1
; it inhibits the methanogens
for methane production in anaerobic reactor. Hence, after electrochemical evaluation of MFC
for food waste leachate, it was observed that maximum open circuit voltage (OCV) 0.830 V
was obtained for the COD load of 1000 mgl
-1
. At optimum condition, MFC current density
was reached up to 5.897 Am
-2
and maximum power density 2.379 Wm
-2
along with substrate
removal more than 80% in terms of COD. This study infers that simultaneously bioelectricity
and biogas production can be done using food waste by linking bioelectrochemical technology
(Microbial fuel cell) to Nisargruna Biogas technology.
Key words: Microbial fuel cell (MFC), Electricity, Biogas, Wastewater treatment
39.1 Introduction
Three Es (Energy, Environment and Economy) are the prime and inescapable concern
of modern society. Society is always ultimate beneficiary of scientific research, knowledge
derived and its understanding. Biotechnology is employed for the betterment three Es in the
well being of human beings.
The first E i.e. Energy resources scenario if considered, current global energies in use
are coal, gas, oil, nuclear and renewable energies. There are limits of non-renewable
resources. Oil reserves will not appreciably run out for at least 100 years or more, demand for


429

oil is expected to exceed production within 2015 to 2025 timeframe (Rifkin, 2002). Nuclear
energy has its own limits having concern of attack on nuclear power stations, nuclear war,
accidental discharge, thermal pollution, radioactive waste disposal problem etc. This
vulnerability of conventional sources of energy bound world to think about reliable and
sustainable energy sources. Hydrogen is considered the most viable energy carrier for the
future. Hydrogen is high in energy content as it contains 120.7 KJg
-1
. This is the highest
energy content per unit mass among known fuels (Krishna, 2013). Therefore, Fuel cell and
Renewable energy resources are most promising technology.
The second E i.e. environmental scenario if considered, presently, rapid population
growth results in globalization, industrialization and rapid economic development that
produces in addition enormous amount of waste in its daily activities. 1/3 food produced lost
or wasted globally amounting to 1.3 billion tons per year. In India, Fruit and vegetables loss is
more than the UK consumes and Grain loss is more than Australia produces (Gustavsson et
al., 2011). 1,27,486 Tons per day municipal solid waste is generated in the India during 2011-
12 (CPCB, MOEF, Govt. of India, 2013). Generally, food waste accounts about 27% of total
MSW (Behera et al., 2010) which amounted to 34421.22 Tons per day. In India, Food waste is
disposed by land filling. Alternatively, food waste is utilized in biogas production. Both
utilization and disposal processes resulted in discharge of large amount of leachate, which is
troublesome for handling, storage and transportation. Acidogenic food waste leachate has
complex composition and heavy pollutant load. It causes ground and surface water
contamination, vermin attraction, offensive odour emanation and produce putrefying gases
(Han et al., 2004). For satisfying discharge quality standards of food waste, some easy
treatment options are needed. For reducing global green house gas emission a method need to
be developed that did not leak CO
2
into the atmosphere at an average rate (globally!) of more
than 1% over centuries (Lewis and Nocera, 2006). Thus, promising solution of these
environmental concerns is MFC technology.
If we consider scenario of third E i.e. Economics, need for energy in the world
increases 1.6 % in average annually (ztrk et al., 2013). Increasing world human population;
declining reserves of cheaply extracted fossil fuels; increasing price of crude oil, coal and
natural gas in international markets, affect economic stability in the world. Moreover, human
population of the world will increase by 40 % by 2050 ( Holechek, 2013) if effective steps are
not taken soon then the world will face economical consequences. Although, huge amount of

430

food waste is utilized as resource in two-stage aerobic-anaerobic sequential biogas reactor
(Nisargruna Biogas Technology) but it produces acidogenic food waste leachate in anaerobic
digestion or methane production process. High content of volatile fatty acids in acidogenic
food waste leachate has adverse effect on growth of methanogens resulting in reduced amount
of biogas production. Microbial fuel cell is promising sustainable technology for bioelectricity
generation, wastewater treatment and resource recovery.
Our aim is to evaluate the feasibility of interlinking of Nisargruna Biogas technology
(anaerobic digester) to microbial fuel cell technology (anodic compartment) so that
simultaneously both energies i.e. Methane and Bioelectricity generation can be done.
Therefore, efficacy of acidogenic food waste leachate obtained through the Autothermal
anaerobic digestion of Nisargruna Biogas plant predigestion tanks food waste is examined as
microbial fuel cell resource for bioelectricity generation.
39.2.1 Acidogenic food waste leachate : Production
Food waste was collected from the predigester tank of Nisargruna plant based on
kitchen waste from Symbiosis International University, S.B. road, Pune (India) developed by
Bhabha Atomic Research Centre (BARC), Navi Mumbai, (India). Batch mode digestion was
carried out in a simulated Autothermal thermophillic aerobic digester (ATAD). The total
volume of the conical flask was 1000 ml with effective volume of 600 ml. The temperature of
45C in the digester was maintained using water bath for 144 h. High amount of VFAs causes
reduction in Biogas production (Wang et al., 2009) in Nisargruna Biogas plant. Therefore,
Volatile fatty acids were used as indicators of process imbalance in anaerobic digesters as
suggested by Ahring et al., 1995. After analysis of VFAs at regular intervals for 144 hrs, it
was observed that with progression of time, the volume of leachate was increased with
increased in COD. pH of leachate was depleted day by day due to accumulation of VFAs. hrs
of digestion at 45C gave maximum concentrations for all VFAs under observation. (Figure1).
Then the Acidogenic food waste leachate digested at hrs at 45C and was stored at 4
o
C for
use as substrate in anodic compartment.
39.2.2 Microbial innocula:
Two types of inocula: 1) municipal wastewater 2) anaerobic sludge from a sequential
two stage aerobic-anaerobic digester, were mixed in ratio of 3:7 (100 ml). First inocula were

431

collected from Sangam Bridge, Pune, Maharashtra, India. Second inocula was collected from
sequential two stage aerobic-anaerobic digester (Nisargruna Biogas technology), Matheran,
Maharashtra, India.

Figure 1: Variation in concentration of VFAs with respect to ATAD digestion time.
39.2.3 Preparation of Substrate:
3.36 g of acidogenic food waste leachate diluted to 300 ml (COD, 1000 mgl
-1
), then
the 100 ml of mixed inocula were added, making the working volume of the reactor 400 ml.
The prepared fuel is ultrasonically treated using Ultrasonic Processor (Piezo U- Sonic brand)
having constant supplied power of 100 W, frequency of 53 kHz for 5 minutes. This substrate
was used in MFCs anodic compartment. In Cathode, 400 ml of 100 mM potassium
ferricyanide in phosphate buffer solution (pH 7.1) was inoculated.
39.2.4 Microbial fuel cell reactor configuration and operation
Laboratory scale dual chamber MFC made of transparent Plexiglas material was used
in this study. The basic concept of design was taken from Logan et al., 2006. The two
chambers were designed with about 0.500 l capacities. The working volume of each chamber
was 0.400 l. The anode compartment was maintained anaerobic condition. The anode and
cathode chamber had two openings connected through a pipe for inlet and outlet and for

432

circulation of fuel. Two Pure carbon brush electrodes were used with 16*12.5*2 mm in
dimensions in anodic compartment having 1.0 cm apart from each other. In cathode, a pure
carbon brush electrode having dimensions of 16*12.5*2 mm was used in opposite side of the
CEM in between anodic compartment electrodes. Electrodes were placed equal distance from
membrane. The internal distance of each electrode was fixed i.e. 2.0 cm from membrane. The
terminal of each electrodes were connected with ultrathin copper conceal wires. The effective
surface area of both carbon electrodes in anode compartment was 0.00102 m
2
. Both the
compartments were connected with CEM having 7 cm in diameter (Nafion T117; Dupont,
Wilmington, Delaware) by using rubber coupling arrangement. The two chambers with
membrane coupling assembly were fixed with nut- bolts. The anode chamber was washed
with nitrogen gas to remove traces of oxygen to maintain the anaerobic condition. The
systematic diagram of dual chambered MFC is given in Figure 2 (A).

Figure 2: Systematic diagram (A) and Actual figure (B) of dual chambered MFC.
MFC was operated in batch fed mode. For inoculation and start up of MFC, the
synthetic wastewater was prepared with NH
4
Cl (0.31gl
-1
), KCl (0.13 gl
-1
), NaH
2
PO
4
.H
2
O
(2.69 gl
-1
), Na
2
HPO
4
(4.33 gl
-1
), Acetate (10mM), Trace element solution (12.5 ml), Vitamin
solution (12.5 ml). At the starting condition, MFC was operated in open circuit voltage (OCV)
mode. The pH of the solution influent was 4.70 in anode and 7.01 in cathode. The MFC was
operated once with synthetic wastewater. After stable voltage generation, MFC was switched
for different food waste leachate concentration to decide the optimum organic influent rate.
The influent concentration was tested for 500 mg l
-1
, 1000 mg l
-1
and 2000 mg l
-1
of organic
matter as COD. The MFC was kept in incubator at 30
C. To prevent the sedimentation of fuel


433

a magnetic bead was kept in anode chamber and placed at magnetic stirrer (REMI brand) at
170 rpm throughout the operation. The MFC operating condition is given in the figure 2 (B).
39.2.5 Data collection, Analysis and Calculation
Voltage was verified directly by using digital multimeter (MASTECH brand Digital
Multimeter 10 A DC) for every hour. Current (I) and Power (P = IV) were recorded. The
power density was normalized to anode surface area and anode void volume. The polarization
curve was prepared for measuring stable voltage for various external resistances (39-470
ohms). The curve then used to calculate the maximum power density. The current density (I
d
=
V/RA) was calculated, where V (V) is voltage, R () is resistance and A (m
2
), the geometric
anode surface area. The power density (P
d
= V
2
/RA) was calculated. The columbic efficiency
(CE) was calculated for batch fed mode system by using the formula (Logan et al., 2006) as:

where COD is the substrate concentration for batch fed mode system by the time ( tb ) , F is
the faradays constant,
an
liquid volume in anode chamber, I, is the current in ampere. pH and
conductivity was measured by probes (HACH, USA). COD (including both soluble and
particulate) was determined using a standard dichromate oxidation (open reflux) method and
VS, SCOD was analyzed by the standard method (APHA, 2005). To quantify VFA 1ml
filtrate sample of the supernatant was inserted into glass syringe and inserted into the
Shimadzu GC -2010 with a flame ionization detector. Before the assay was started, the
machine was calibrated and 1ml of standard VFA solution was added. The sample injection
volume was 1ml. Nitrogen was the carrier gas. The injection port and detector were
maintained at 180C respectively. The determination result of VFA constituents were
respectively expressed in the form of height of the peaks in the graph and their occupied area
was used to calculate their respective concentration in mgml
-1
. Carbohydrate estimation was
done by Anthrones method (Scott and Melvin, 1953). Protein content was measured by
Biuret method (Layne, 1955).
39.3 Results
39.3.1 Acidogenic food waste leachate characterization

434

Before anaerobic digestion raw material has pH of 6.1 that after anaerobic digestion
gradually decreases with increase in VFA concentration. After 72 hrs of digestion the
characterization of Acidogenic food waste leachate is given in the table 1.
Table 1: Characterization of Acidogenic food waste leachate after 72 hrs digestion.
Parameter Unit Food waste leachate ( after 72 hrs digestion )
pH - 4.40
Soluble COD mgO
2
l
-1
12400
Conductivity mScm
-1
19.39
VS gl
-1
78.3
VFA mgl
-1
8010
Temperature C 45
Carbohydrate mgml
-1
11.80
Protein mgml
-1
7.61

39.3.2 Pretreatment of Anodic Substrate
For the selective enrichment of specific group of bacteria, pre-treatment of parent
inoculum was carried out to use in anode chamber of a MFC. Symbiotic relationship within
population and composition of the substrates (wastewater) strongly affects the composition of
the bacterial associations in the anode chamber. Inocula in present study are a mixture of
different consortia of bacteria and therefore the susceptibility of this to ultrasonication
depends on the resistance of the predominant bacterial groups. Resistant microorganisms to
ultrasonication stress have more possibility to survive and become a dominant species in the
mixed culture. Ultrasonication can suppress activity of Grampositive methanogens by
retaining Gram-negative bacteria. Major electrogens are gram-negative bacteria.
Methanogenic bacteria lacks protective spores forming capability in restrictive environment
such as high temperature, extreme acidity and alkalinity, and also have a slower growth rate
unlike other hydrogen producing and electrogenic bacteria (Zhu and Beland, 2006).
Therefore, the application of moderate duration ultrasonic pre-treatment to inoculums
might have enhanced the activity of electrogenic bacteria and somewhat change in leachate
composition, which resulted in enhanced current production. The pretreatment of ultrasonic
waves at 53 kHz at 100 W for 5 minutes ( Figure 4 ) causes reversible damage although 2, 10

435

and 15 minutes treatment produces poor results. Ultrasonication of fuel (acidogenic food
waste leachate and mixed inocula) produced initial open circuit voltage of 0.130 V with
respect to untreated 0.92 V only. Thus, with pretreatment initial OCV is increased by 41.30%.

Figure 4: Microscopic image of ultrasonically treated inocula before and after ultrasonic
treatment (53 Htz, 100W for 5 minutes) at 1000X magnification.
39.3.3 MFC performance:
39.3.3.1 Determination of Organic Loading Rate (OLR)
During start up, it was decided to operate MFC almost for 12 days to reach the stable
OCV voltage of approx 0.536 V for 0.400 l of anode solution. It means that anode and
cathode were considered fully enriched (Zhang et al., 2011). MFC was freshly re-inoculated
with food waste leachate solution of varying concentration of 500, 1000 and 2000 mgl
-1
of
COD. It was observed that, at different COD input, MFC gave different OCV. For leachate
concentration of 500, 1000, 2000 mgl
-1
as COD, OCV was 0.631 V, 0.830 V, 0.638 V
respectively.
The MFC gave maximum voltage of 0.830 V with carbon brush electrode for 1000
mgl
-1
COD. After that, voltage was reduced for further substance load. This was because of
high COD concentration may affect the microbial activity of anode chamber. It also leads to
increase in internal resistance of anode chamber and may increase the charge transfer
resistance (Nam et al., 2010). Performance of MFC during in open circuit voltage mode to

436

determine optimum organic loading rate is given in figure 5. Thus, the total substrate with
concentration of 1000 mgl
-1
was considered optimum to study the electrochemical
performance of MFC for various external loads.

Figure 5: Open circuit voltage at various substrate concentration with time.
39.3.3.2 Polarization Behavior and Power density
In closed circuit voltage mode with various external resistances (39 - 470 ) gave
maximum closed circuit voltage output of 0.730 V at 220 resistance i.e. good current,
Afterwards sudden and continuous drop in voltage was observed. Therefore, optimum
external resistance was considered 220 for MFC operations. The Closed circuit Voltage
(CCV) at a range of resistances is given in the figure 6.
Maximum power density and internal resistance of MFCs were obtained by
polarization curves. Polarization curves were plotted against potential and power density
varying resistances. Current generation showed decreasing trend with increase in the
resistance which is in concurrence with the literature reported earlier (Mohan et al., 2008). A
low resistance allows more electrons flow in the fuel cell circuit and this result in the potential
drop especially at lower resistances in spite of higher power density (Mohan et al., 2010).
According to Ohms law, at maximum power density, the internal and external resistances are

437

equal. Thus, at Maximum Power density (2.379 Wm
-2
) MFC external resistance equal to its
internal resistance i.e. 220 at 3.258 Am
-2
current density. MFC current density was reached
up to 5.897 Am
-2
. The trend of Voltage and Power density as function of current density is
given in the figure 7.

Figure 6: Closed circuit Voltage at a range of external resistances (39 to 470 ).

Figure 7: The polarization behavior of MFC and Power density.
39.3.3.3 Columbic efficiency and Substrate removal efficacy
Columbic efficiency (CE) is the ratio of the coulombs obtained in a MFC to the
theoretical coulombs if all the substrate oxidized produces current. CE percentage was
obtained 24 %. MFC demonstrated the high COD removal efficacy of 83%.

438

39.4 Discussion
The application of low-frequency ultrasonication was reported to be very effective in
decreasing the amount of bacterial population in sewage sludge. As the frequency and time
period increases, the bacterial population decreases (Kesari and Behari, 2008). However, with
the prolonged exposure time, the flaw may expand and the structure of cell wall will be
destroyed, which leads to the decrease of bacterial activity (Xie et al., 2009). Effect of
pretreated anaerobic sludge was reported by More and Ghangrekar, 2009. They reported
maximum 0.745 V OCV (40 kHz, 120 W for 5 minutes) with simple synthetic wastewater as
substrate rather in present study high molecular wastewater is used and maximum OCV of
0.830 V (53 kHz, 100 W for 5 minutes) is produced.
The effects of three different inocula (domestic wastewater, activated sludge, and
anaerobic sludge) on the treatment of acidic food waste leachate in MFCs were evaluated by
Li et al., 2013. Using 1000 mgl
-1
COD food waste leachate (pH 4.76) as the substrate highest
power (0.432 Wm
-3
) was obtained using anaerobic sludge inoculum. COD removal and CE%
was obtained >87% and 20% respectively. In another experiment, using 5000 mgl
-1
COD food
waste leachate produced maximum power density 15.14 Wm
-3
with COD removal efficacy of
90% and columbic efficacy of 66.40% (Rikame et al., 2012).
Rather in present study, although the OCV is less than Rikame et al., 2012 but it is
greater than Li et al., 2013. Volumetric power density is less than Rikame et al., 2012 and Li
et al., 2013 because large volume to anode surface area ratio. If both power density is
converted to Wm
-2
(with respect to anode surface area) then there is no any literature available
as best of my knowledge until date reporting such high power density with Acidogenic food
waste leachate. Columbic efficacy and COD removal efficacy although is lesser than all of
above because of lesser surface area of anode and respectively large anodic chamber working
volume.
39.5 Conclusion
This study infers the feasibility of interlinking of Nisargruna Biogas technology
(anaerobic digester) to microbial fuel cell technology (anodic compartment) is possible with
further research and improvement of Power density, Current density and Voltage, so that
simultaneously both energies i.e. Biogas and Bioelectricity generation can be done.


439

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process imbalance in anaerobic digestors. Applied Microbiology and Biotechnology,
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2. APHA, AWWA and WPCF (2005) Standard Methods for Examination of Water and
Wastewater. 22nd ed., APHA Washington, DC.
3. Behera S.K., Park J.M., Kim K.H. and Park H.S. (2010) Methane production from food
waste leachate in laboratory-scale simulated landfill. Waste Management,30:1502-1508.
4. Gustavsson J., Cederberg C., Sonesson U., Otterdijk R. and Meybeck A. (2011) Global
food losses and food wastes. Food and Agriculture Organization of the United Nations,
pp. 1-23.
5. Han S. K. and Shin H.S. (2004) Biohydrogen production by anaerobic fermentation of
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8. Kesari K.K. and Behari J. (2008) Ultrasonic impact on bacterial population in sewage
sample. International Journal of Environment and Waste Management, 2: 233244.
9. Krishna R. H. (2013) Review of Research on Production Methods of Hydrogen: Future
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10. Layne E. (1955) Biuret method. In: S. P. Colowick & O. Kaplan (Ed.), Enzymology,
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energy utilization. PNAS, 103:15729-15735.
12. Li X.M., Cheng K.Y., Selvam A. and Wong J.W.C. (2013) Bioelectricity production from
acidic food waste leachate using microbial fuel cells: Effect of microbial inocula. Process
Biochemistry, http://dx.doi.org/10.1016/j.procbio.2012.10.001.
13. Logan B.E., Hamelers B., Rozendal R.A., Schrorder U., Keller J., Freguia S., et al. (2006)
Microbial fuel cells: methodology and technology. Environmental Science Technology,
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14. Mohan S.V., Mohanakrishna G., Reddy B.P., Saravanan R. and Sarma P. N. (2008)
Bioelectricity generation from chemical wastewater treatment in mediatorless (anode)
microbial fuel cell (MFC) using selectively enriched hydrogen producing mixed culture
under acidophilic microenvironment. Biochemical Engineering Journal, 39:121130.
15. Mohan S.V., Mohanakrishna G., Velvizhi G., Lalit Babu V. and Sarma P. N. (2010) Bio-
catalyzed electrochemical treatment of real field dairy wastewater with simultaneous
power generation. Biochemical Engineering Journal, 51:3239.
16. More T.T. and Ghangrekar M.M. (2010) Improving performance of microbial fuel cell
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Energy, 31: 19801988.

441

CHAPTER 40
INHIBITORY EFFECTS OF FLUORIDE ON BACTERIAL
METABOLISM PRESENT IN MICROBIAL FUEL CELLS
Iti Sharma, M.M. Ghangrekar

Abstract
Microbial fuel cells (MFCs) catalyzed by the microorganism offer dual advantages by
generating power along with wastewater treatment. Mixed anaerobic sludge is the most
popular source for inoculation of bacterial population. As a result of use of various products
such as toothpastes, tea, coffee, carbonated beverages and non vegetarian food, fluoride
remains in the inoculum sludge collected from septic tank. Fluoride-ion restrains glycolysis of
bacteria present in MFC by competitive inhibition of Enolase and subsequently lessens the
power generation in MFC. An earthen pot cum separator as anode chamber, stainless steel
meshe as anode and carbon felt as cathode materials were used to fabricate the MFC.
Synthetic wastewater having acetate as carbon source was fed in MFC-1 and powder of dried
mango peels as carbon source was fed in MFC-2, both mixed in tap water. Higher
accumulation and inhibition of fluoride-ion was observed in case of MFC-2 than in MFC-1.
Fluoride concentration in MFC-1 was found initially 0.016 mg/L, which reached to 3.387
mg/L after 7 days of retention time and in MFC-2 initial concentration of 16.605 mg/L
reached to final concentration of 78.67 mg/L after 7 days. COD removal was 70% and 50%
for MFC-1 and MFC-2, respectively. Maximum power density of 37.79 mW/m
2
and 4.48
mW/m
2
, respectively, was observed in MFC-1 and MFC-2. For improving the prospects of
significant COD removal and power generation, external treatment of mango peels and/or the
use of fluoride absorbing material will be vital for improving performance of MFC.
Key-words: Inhibition, Fluoride ion, Enolase, Glycolysis, 2-PG (2 phosphoglyceric acid),
Microbial fuel cells
40.1 Introduction
Modern style of living demands a stable, reliable supply of energy. It lies at the heart
of our mobility, our success and our daily comfort. Fossil fuels currently supply most of the


442

worlds energy needs; and howsoever unacceptable their long term impacts; the supplies are
likely to remain for the next few generations (Dresselhaus M.S. and I.L. Thosmas, 2001).
Recovery of usable biochemical energy from plant biomass is one strategy that could be used
to counteract the petroleum consumption. Available methods to extract energy from
lignocellulosic waste rely on either thermo or chemical pre-treatment methods, which have
numerous environmental as well as process limitations (Gregoire, 2012). Among these
alternatives, MFCs are attractive because they produce societys most widely useful energy
form i.e. electricity directly without combustion.
The microbial fuel cell works using oxidation-reduction reaction that involves the
transfer of electrons from one molecule to another. The electron donors are organic materials
in waste that is used to feed fuel cells. The microorganisms perform half a redox reaction.
They free electrons from the waste compounds. But those electrons are sent through a circuit
reducing anode, instead of being immediately transferred to molecular oxygen. Microbes that
can transfer electron to extracellular electron acceptors are important in organic matter
degradation and nutrient cycling (Reguera G., 2005; Lovely, 2003) and to understand how
bacteria make their livings inside fuel cells, we need to study communities, not individuals
(Raghoebarshing, 2006).The current generated by these microbial fuel cells are small;
however their potential, in the non-voltage sense, may be surprisingly large (Girguis Peter,
2006).
In MFCs, substrate is regarded as one of the most important factor affecting electricity
generation. Depending upon the composition of the substrate, new enzymes are synthesized
and the synthesis of some other enzyme is repressed (Todar Kenneth, 2012). A great variety of
substrate as feed can be used in MFCs ranging from pure compounds to complex mixtures of
organic present in wastewater. The inedible parts of plants are feeding the next generation of
biofuels. But extracting the energy-containing molecules is a challenging task (Sanderson
Katharine, 2011). However, there are many potential sources of enzymes that digest wood,
from the microbes found in termite guts or from wood-decomposing fungi that thrive on tree
trunks. For lignocellulosic biofuels production, there are obviously questions about sourcing
the required materials.
In the current study, the waste part of mango was selected as substrate.
Mango (Mangifera indica L.) is the most important fruit of India. It is grown over an area of
1.23 million hectares in the country producing 10.99 million tones. It accounts for 22.1 per

443

cent of total area (5.57 million ha) and 22.9 per cent of total production of fruits (47.94
million tons) in the country. India ranks first among worlds mango producing countries
accounting for 52.63 per cent of the total worlds mango production of 19 million tons. About
30-40% of fruits production go waste due to lack of proper processing and packaging (Jadhav
P.B., 2010). However, the utilization of this fruit for processing receives less attention
internationally. It is estimated that only 0.22% of mangoes produced in the world is utilized
for processing (Nanjundasswamy, 1998). One of the major problems in mango processing is
that they produce a large amount of waste (40-50% of total fruit weight), which poses
considerable disposal problems and ultimately can lead to pollution problems. Mango
processing is no exception (Gantioque, 2011). The expense connected to the problem of
hygiene and odor of the fruit waste could possibly be turned into an advantage by
transforming fruit waste to renewable energy through MFC. All the potential for generating
electricity from waste is in a pool of waste water.
Mixed anaerobic sludge, collected from septic tank bottom, is the most popular source
for inoculation of bacterial population in wastewater. Toxic elements present in sludge can be
a challenge for microbes that may be too toxic for bacteria to deal with. There are various
elements such as fluoride, chloride and bromide which may be present in the sludge that can
cause inhibitory actions against the bacterial metabolism (Ochoa-Herrera et.al, 2009). As a
result of various products such as toothpastes, tea, coffee, carbonated beverages and non
vegetarian food (Mercola, 2012; Maria Hoven, 2011; Judy Heilman, 1999; M.C. Kiritsy,
1996; Conde Nast, 2013), fluoride remains in the inoculum sludge collected from domestic
wastewater.
Fluoride, being the most electronegative element, bonds with almost any element, both
metals and nonmetals. Fluoride affects many metabolic enzyme however Enolase is very
sensitive because, fluoride is a known competitor of Enolases substrate 2-PG. The fluoride is
part of a complex with magnesium and phosphate, which binds in the active site instead of 2-
PG (2 phosphoglyceric acid) and disrupt the bacterias glycolytic pathways (G. N. Jenkins,
1962). Inhibition of this enzyme reduces the supply of phosphoenolpyruvate for use by the
phosphotransferase system and so inhibits sugar uptake as well as glycolysis (Hamilton,
1977), leading to a diminished production of adenosine triphosphate and consequent failure of
the energy supply to the microbial cells. Fluoride also inhibits other processes such as
glycogen synthesis, peroxidase activity and the proton translocating ATPase of bacterial

444

membranes (Marquis, 2003). Low level of 0.025 mg/L fluoride is able to inhibit proton
excretion by approximately 50% in cells grown even at pH of 7.0 under anaerobic condition
(Guha-Chowdhury, 1997). Fluoride easily reacts with other elements to form toxic
compounds inside the reactor and causes bacterial death due to accumulation of these
compounds (Rao and Pal, 1978; Wilke, 1987).
Thus the specific goals of this study were (1) to analyze fluoride concentration present
in sludge collected from septic tank bottom (2) its inimical effects on microbial community of
MFCs due to inhibition and accumulation and (3) its consequences in power generation.
40.2 Materials and Method
40.2.1 Construction of MFC reactor
In this study, three earthen pots of 500 ml of capacity (as anode chamber) were used
to fabricate dual chambered MFCs. The outside wall of these pots (5 mm thick) acted as
separator to prevent intermixing of electrolytes between anode and cathode. These pot, were
made from locally available red soil (major compound present: Na
2
O - 3.95%, MgO - 0.654%,
Al
2
O
3
- 26.3%, SiO
2
- 57.5%, P
2
O
5
- 1.13%, K
2
O - 1.78%, SO
3
- 0.258%, CaO - 0.791%,
Fe
2
O
3
- 4.75%, Ti - 0.658%), (Ghadge A.N., 2013). These earthen pots were placed inside the
plastic container of 5 litre capacity, working as a cathode chamber. Tap water was used as
catholyte which was continuously aerated with aquarium pump (SOBO, Aquarium, China) in
all these MFCS. Stainless steel mesh (area 1341.83 cm
2
) was used as the anode material
while carbon felt (Panex33, Zoltek corporation) (area 2152.18 cm
2
) was used as cathode to
construct three identical MFCs. The MFCs were operated in batch mode. The electrodes were
connected externally with concealed copper wire through external resistance of 100 (Figure
1 and 2).
40.2.2 Operation of MFC
Anaerobic mixed sludge collected from septic tank bottom (100 ml) was used as the
inoculum in the anode chamber of MFCs after giving heat treatment (Jadhav and Ghangrekar,
2008). In MFC-1, synthetic wastewater containing acetate as carbon source having 2000 mg/l
of COD was used, and other ingredients of the synthetic wastewater were similar to as
described by (Jadhav and Ghangrekar, 2008) were used. Powder of dried mango peels mixed
in tap water was used as substrate in MFC-2 while in MFC-3; juice of mango peels, pretreated
with S.cerevisiae was used as carbon source. The mango waste used in this study was

445

collected from the IIT-Kharagpur campus. These MFCs were operated at room temperature
varying from 26 to 34C. The MFCs were operated under batch mode with reaction time of
168 hours.

Figure 1: Schematic diagram of earthen pot anode chamber, plastic container as cathode
chamber and electrodes position used in MFC

Figure 2: Schematic diagram working of MFC

Aerator
connected to
diffuser
O2
O2
Effluent
Influent
CO2
Anode (SS) Anode (SS) Anode (SS) Anode (SS)
e
-
Earthen pot working as PEM Earthen pot working as PEM Earthen pot working as PEM Earthen pot working as PEM
Outside Outside Outside Outside
view view view view
O2
Cathode (ar!on Cathode (ar!on Cathode (ar!on Cathode (ar!on
felt) felt) felt) felt)
O2
"esistane
e-
e-
e
-
e-
e-
e-
e-
e
-
water
Earthen pot
Anode
ha#!er
Stainless steel wire
#esh (Anode)
Car!on felt (athode)
Outside
view
Inside view
Aerator connected to diffuser
100 resistance
Anode chamber
Cathode chamber
Sludge with su!strate
$ater

446

40.2.3 Analysis and calculation
The suspended solids (SS), volatile suspended solids (VSS), and chemical oxygen
demand (COD) were monitored according to APHA standard method. Open circuit voltage
(OCV) and operating voltage (OV) across 100 resistance were measured after reaching to
stable value. The voltage and current were measured using a digital multimeter with data
acquisition unit (Agilent Technologies, Malaysia) and converted to power according to P=
I*V; where, P = power, I = current, and V = voltage (V). The Coulombic efficiency (CE) was
estimated by integrating the measured current relative to the theoretical current on the basis of
consumed COD (Logan B.E., 2006). Polarization studies were carried out at variable external
resistances (10000-10 ) using 10 K variable resistors. Internal resistance of the MFCs was
measured from the slope of line from the plot of voltage versus current.
Ion chromatography was done to check the presence of fluoride ion. A Metrohm ion
chromatography system (761 Compact IC) was used with a conductivity detector (0-100%
organic modifier). The column used for separation was Metrosep A Supp 5-250 (6.1006.530)
at 4.0 x 250 mm dimensions. Analysis was carried out with a flow rate of 0.7 ml/min till 35
min with max pressure 15 MPa. The particle size was 5.0 m, carrier material was polyvinyl
alcohol with quaternary ammonium groups, and injection loop volume was 25.0 L. Eluent
was prepared by dissolving approximately 339 mg anhydrous sodium carbonate and
approximately 84 mg sodium hydrogen carbonate in distilled water . The Solution was diluted
to 1.00 L with Millipore water in a volumetric flask. Samples were filtered first with 0.45 m
and then with 0.20 m filter paper. The standard solutions (50 ml) were prepared in the range
of 2-20 ppm to prepare calibration curve.
40.3 Results
40.3.1 Substrate degradation
Illustrated substrates, supplemented with anaerobic sludge inside the anode chamber
were consumed by anaerobic bacteria community or biofilm to continue their metabolism. The
initial retained COD of synthetic wastewater, dried mango peels, and mango juice was 2000
mg/l. Average COD removal efficiencies in the anode chambers of MFC-1, MFC-2 and MFC-
3 were 70 3 %, 50 2 % and 61 4 %, respectively in 5 days retention time (Figure 3).
Maximum COD removal was attained in MFC-1 due to synthetic wastewater being used as
feed.However, in MFC-2, dried mango peels mixed in tap water was not as easily edible

447

substrate for bacteria, as it was in MFC-1. In MFC-3, substrate was provided in less knotty
mode, compared to MFC-2. Mango peels in juicy fashion and treated with S. cerevisiae was
the substrate for MFC-3. Degraded lignin as well as carbohydrate by S. cerevisiae, contributed
more COD removal in case of MFC-3. Thus the substrate played dominant role in successive
analysis. In the presence of inhibitors which enter with the sludge, the substrate has to face
competition to bind with the enzymes, which bacteria use for its metabolic activities. This is
another reason for less COD removal efficiency inside the anode chamber.

Influent COD (mg/l) Effluent COD (mg/l)
Figure 3: COD removal / substrate reduction pattern in the three MFCs

40.3.2 Power generation
Electricity generation in MFC-1, MFC-2 and MFC-3, increased progressively.
However MFC-1, engender more power than MFC-2 and MFC-3 in the beginning. OCVs of
MFC-1, MFC-2 and MFC-3 were 852 mV, 731 mV and 802 mV, respectively (Figure 4).
However, the accumulation and inhibition of fluoride progresses slowly and caused voltage
reduction later up to 728 mV, 512 mV, and 678 mV in MFC-1, MFC-2 and MFC-3
respectively. Initially OV of MFC-1, MFC-2 and MFC-3 was 670 mV, 221 mV and 336 mV
at 100 external resistance, which reduced up to 532 mV, 67 mV and 180 mV, respectively
(Figure 5). Maximum power density of 37.79 mW/m
2
, 4.48 mW/m
2
and 2.61 mW/m
2
,
respectively, was observed in MFC-1, MFC-2 and MFC-3 (Figure 6, 7 and 8). Coulombic
efficiency of 6.8%, 4.1% and 2.3%, respectively was observed in MFC-1, MFC-2 and MFC-3.
The gradual downturn in power generation was observed in all MFCs in consecutive cycles
and an escalation in fluoride concentration was noted in coordination with power loss.
0
500
1000
1500
2000
2500
1 2 3
Initial COD
Final COD
MFC- MFC- MFC-

448

Figure 4: Power reduction in successive cycles
in three MFCs (OCV results)
Figure 5: Power reduction in successive cycles
in three MFCs (OV results)

Figure 6: Polarization curve for MFC-1 Figure 7: Polarization curve for MFC-2

40.3.3 Fluoride analysis in MFC
Possibility of fluoride inhibition in MFCs was investigated by Ion Chromatography.
The wastewater samples from all three MFCs were collected six times during operation for 6
days. The chromatograms generated from analysis corroborate the presence of fluoride anion
in all three MFCs. The accumulation of the fluoride in MFC-1 was found initially 0.016 mg/L,
which reached to 3.387 mg/l and in MFC-2 initial concentration of 16.605 mg/L and final
concentration of 78.67 mg/L was observed. While in MFC-3 initial concentration was 0.90
mg/l and final concentration 0.445 mg/l was noticed (Figure 9). The fluoride concentration in
inoculated sludge and sludge present in the bottom of anode chamber was analyzed. In the
inoculum sludge 154.96 mg/l fluoride was found. However, fluoride concentrations in sludge
analyzed after 6 days of operation in MFC-1, MFC-2 and MFC-3 were 0.62 mg/l, 16.50 mg/l
0
100
200
300
400
500
600
700
800
900
1 2 3
Initial OCV
Final OCV
MFC- MFC- MFC-
0
100
200
300
400
500
600
700
800
1 2 3
Initial OV
Final OV
MFC-
MFC-
MFC-
0
5
10
15
20
25
30
35
40
0
100
200
300
400
500
600
700
800
900
0 50 100 150 200 250
P
o
'
e
r
d
e
n
&
i
t
%

m
(
)
m
2
*
o
l
t
a
"
e

+
m
*
,
Current den&it% mA)m2
MFC-1
0
1
2
3
4
5
0
100
200
300
400
500
600
700
800
900
1000
0 10 20 30 40 50
P
o
'
e
r

d
e
n
&
i
t
%

m
(
)
m
2
*
o
l
t
a
"
e

m
*
MFC-2

449

and 0.25 mg/l , respectively (Table 1 and Table 2). The fluoride concentration in tap water
used for the preparation of synthetic wastewater, in mixing of dried mango peels and for
dilution of mango peels juice, was 0.017 mg/l.

Figure 8: Polarization curve for MFC-3
Figure 9: Fluoride concentration in all three MFCs

Table 1: Concentration of fluoride in sludge
Sludge
characteristics (for
50 ml each)
Initial conc. of
fluoride(mg/l)
Final
conc.
(mg/l)
Initial Conductivity
of sludge slurry
446(s/cm)
Finial
Conductivity100
(s/cm)-after dilution
MFC 1 154.96 0.62 245 100
MFC 2 154.96 16.50 236 100
MFC 3 154.96 0.25 233 100

Table 2: Concentration of fluoride in wastewater from all three MFCs
Wastewater samples Initial conc. (mg/l) Final conc. (mg/l)
MFC-1synthetic wastewater 0.016 3.387
MFC-2 mango peel 16.60 78.67
MFC-3 mango peels juice
+yeast
0.095 0.442

40.4 Discussion
The achieved results of COD removal, displayed that degradation of substrate in
MFCs is entirely dependent upon the type of substrate. In MFC-1, where synthetic wastewater
was used, the COD removal was highest. The reason behind, the substrate is present in form
what bacteria can easily use for its metabolic activities. Hence highest COD removal
efficiency was found in MFC-1 that is 70 3 %. While in MFC-2, dried mango peels were
impenetrable to be degraded by the bacteria, due to its real complex form and also due to
0
1
2
3
0
100
200
300
400
500
600
700
0 10 20 30 40 50
P
o
'
e
r

d
e
n
&
i
t
%

m
(
)
m
2
*
o
l
t
a
"
e

m
*
0
10
20
30
40
50
60
70
80
90
0
0.5
1
1.5
2
2.5
3
3.5
4
0 2 4 6
F
l
u
o
r
i
d
e

c
o
n
c
e
n
t
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a
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i
o
n

+
m
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)
-
,
F
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o
r
i
d
e

c
o
n
c
e
n
t
r
a
t
i
o
n

+
m
"
)
-
,
.a%&
MFC 1
MFC 3
MFC 2

450

lignin contents in this lignocellulosic waste. Thus COD removal efficiency in MFC-2 was
only 50 2 %. In MFC-3, to make the mango peels waste more accessible to bacteria, the
juice of mango peels was fed to bacteria and to avoid the restrain by lignin contents in the
same, the S.cerevisiae was used, which has enzyme to degrade lignin. Hence the COD
removal efficiency obtained in MFC-3 was better than MFC-2 (61 4 %). This whole study
concluded that the available substrate concentration which bacteria can use was more in MFC-
1 than in MFC-2 and MFC-3, which may be relevant for any kind of real waste. The binding
of fluoride, being the competitive inhibitor of enolase enzyme, depends upon the substrate
concentration in the chamber. In MFC-1, easily accessible substrate concentration was more
than other two MFCs. Enolase is the important enzyme in carbohydrate pathways for
conversion of sugars to acids, and to generate ATP, the final product of bacterial metabolism.
This directly affects the power generation of MFCs. In parallel the power generation was also
estimated for all three MFCs, and results showing the mirror image of the previous substrate
degradation and its correlation with fluoride inhibition. The maximum power was obtained in
MFC-1 than MFC-2 and MFC-3. In the steady state where max production or power can be
achieved, comes for a short time before the down time. However the continuous accumulation
of the toxic elements either present in sludge or in feed can shorten this peak time or
eventually can decrease the performance of the microbial fuel cells. Fluoride reacts with
almost every element and forms complexes. These complexes gradually accumulate and do
competition with substrate to bind with the enzyme. When more inhibitor complexes
containing fluoride ions, binds with enzymes active site in place of substrate and disrupt the
bacterias glycolytic pathway and other processes such as glycogen synthesis, peroxidase
activity and the proton translocating ATPase of bacterial membranes. These blockage in
bacterial metabolism finally reduced active bacterial population and causes power reduction as
found in MFC-2.
40.5 Conclusion
The treatment and power generation from many real wastes has been done so far.
However, the mango peels waste is not used for the treatment and energy generation in
microbial fuel cells as evident from available literature. As a result of higher fructification of
mango in India and less processing units, demand a substitute operation to convert this waste
in to energy likewise its treatment. In addition, the critical elements, which disturb the
bacterial metabolism in MFCs, should be exercised. The fluoride ion, exist in septic tank

451

sludge used as inoculum, hinder bacterial metabolism during companionship with substrate.
This study has opened the new area of research for new methods to remove fluoride from the
MFCs, to maintain the biofilm to get steady power generation. Significant feasibility can be
brought about from real waste by external treatment and/or the use of fluoride absorbing
material like bioadsorbents, carbon based sorbents, and zirconium based adsorbents etc.,
which may prove to be vital in MFC research to enhance its power.

References
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(6861), 332-337.
2. Gantioque, G. G., Lu, Z. E. N. G., & Yan-bin, X. I. A. (2011). Production of fungal
pectinase using mango peel as substrate by submerged fermentation.Journal of Hunan
Agricultural University (Natural Sciences), 5, 023.
3. Germaine, G. R., & Tellefson, L. M. (1986). Effect of endogenous phosphoenolpyruvate
potential on fluoride inhibition of glucose uptake by Streptococcus mutans. Infection
and immunity, 51(1), 119-124.
4. Ghadge, A. N., Pradhan, H., Prasad, S., & Ghangrekar, M. M. (2013). Enhancing
Activity of Electrogenic Bacteria In Microbial Fuel Cell By 2-Bromoethanesulphonate
Dosing. International Journal of Engineering, 2(5).
5. Girguis, P. R., & Lee, R. W. (2006). Thermal preference and tolerance of
alvinellids. Science, 312(5771), 231-231.
6. Gopalakrishnan, S. (2013). Marketing System of Mangoes in India. World Applied
Sciences Journal, 21(7), 1000-1007.
7. Goyal, A., Gauba, K., & Tewari, A. (1998). Bioavailability of fluoride in humans from
commonly consumed diets in India. Journal of the Indian Society of Pedodontics and
Preventive Dentistry, 16(1), 1.
8. Gregoire, K. P., & Becker, J. G. (2012). Design and characterization of a microbial fuel
cell for the conversion of a lignocellulosic crop residue to electricity. Bioresource
Technology, 119, 208-215.

452

9. Guha-Chowdhury, N., Iwami, Y., & Yamada, T. (1997a). Effect of low levels of
fluoride on proton excretion and intracellular pH in glycolysing streptococcal cells
under strictly anaerobic conditions. Caries research, 31(5), 373-378.
10. Hamilton, I. R. (1977). Effects of fluoride on enzymatic regulation of bacterial
carbohydrate metabolism. Caries research, 11(Suppl. 1), 262-291.
11. Henrique, M. A., Silvrio, H. A., Flauzino Neto, W. P., & Pasquini, D. (2013).
Valorization of an agro-industrial waste, mango seed, by the extraction and
characterization of its cellulose nanocrystals. Journal of environmental
management, 121, 202-209.
12. Iwami Y.,Guha-Chowdhury N., & Yamada T. (1997b). Effect of sodium and potassium
ions on intracellular pH and proton excretion in glycolyzing cells of Streptococcus
mutans NCTC 10449 under strictly anaerobic conditions. Oral microbiology and
immunology, 12(2), 77-81.
13. Jadhav, G. S., & Ghangrekar, M. M. (2008). Improving performance of MFC by design
alteration and adding cathodic electrolytes. Applied biochemistry and
biotechnology, 151(2-3), 319-332.
14. Jadhav, P. B., Chavan, N. D., Modi, P. K., & Suryawanshi, G. B. (2010). Production of
by-products from wastes of food processing industries.International Journal of
Processing and Post Harvest Technology, 1(2), 114-117.
15. Jana, P. S., Behera, M., & Ghangrekar, M. M. (2010). Performance comparison of up-
flow microbial fuel cells fabricated using proton exchange membrane and earthen
cylinder. International Journal of Hydrogen Energy, 35(11), 5681-5686.
16. Jenkins G. N. (1962). Biological Action of Fluorides, Nature, 193, 23-24
17. Kiritsy, M. C., Levy, S. M., Warren, J. J., Guha-Chowdhury, N., & Marshall, T. (1996).
Assessing fluoride concentrations of juices and juice-flavored drinks.The Journal of the
American Dental Association, 127(7), 895-902.
18. Logan, B. E., & Regan, J. M. (2006). Electricity-producing bacterial communities in
microbial fuel cells. TRENDS in Microbiology, 14(12), 512-518.
19. Logan, B. E., Hamelers, B., Rozendal, R., Schrder, U., Keller, J., Freguia, S., &
Rabaey, K. (2006). Microbial fuel cells: methodology and technology.Environmental
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453

20. Lovley, D. R. (2003). Cleaning up with genomics: applying molecular biology to
bioremediation. Nature Reviews Microbiology, 1(1), 35-44.
21. Maria Hoven (2011). Foods containing fluoride, LIVESTRONG.COM, 1-3
22. Marquis, R.E., Clock, S.A., & Mota-Meira, M. (2003), Fluoride and organic weak acids
as modulators of microbial physiology. FEMS microbiology reviews, 26(5), 493-510.
23. Mercola (2012). This Food Has 180 Times the Fluoride of Drinking Water. Care2 make
a difference, http://www.care2.com/greenliving/this-food-has-180-times-the-fluoride-of-
drinking-water.html, 1-5
24. Nanjundaswamy, A.M. (1991). Mango processing; present status and future outlook.
Acta Hort. (ISHS) 291:525-545
25. Ochoa-Herrera, V., Banihani, Q., Len, G., Khatri, C., Field, J. A., & Sierra-Alvarez, R.
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26. Raghoebarsing, A. A., Pol, A., Van de Pas-Schoonen, K. T., Smolders, A. J., Ettwig, K.
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methane oxidation to denitrification. Nature, 440(7086), 918-921.
27. Rao, D. N., & Pal, D. (1978). Effect of fluoride pollution on the organic matter content
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1098-1101.
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activity of mull, moder and mor soils. Biology and fertility of soils, 5(1), 49-55.


454

Part VI
Hybrid Systems


455

CHAPTER 42
CONVERSION OF PLASTIC WASTES INTO LIQUID FUELS
A REVIEW
Arun Joshi, Rambir and Rakesh Punia

Abstract
Various technologies are being developed to overcome the drawback of plastics, namely, their
non-biodegradability. Though work has been done to make futuristic biodegradable plastics,
there have not been many conclusive steps towards cleaning up the existing problem.
Recycling waste plastics into reusable plastic products is a conventional strategy followed to
address this issue for years. However this technique has not given impressive results as
cleaning and segregation of waste plastics was found difficult. Over a 100 million tones of
plastics are produced annually worldwide, and the used products have become a common
feature at overflowing bins. Plastics is placed in a landfill, it becomes a carbon sink,
Incineration, blast furnace, gasification are not much appreciated solution to the problem, as
toxic gases are produced and their cost of production is quite high. Pyrolysis of waste plastics
into fuel is one of the best means of conserving valuable petroleum resources in addition to
protect the environment. This process involves catalytic degradation of waste plastic into fuel
range hydrocarbon i.e. petrol, diesel and kerosene etc. A catalytic cracking process in which
waste plastic were cracked at very high temperature, the resulting gases were condensed to
recover liquid fuels. Type of plastics also effect the rate of conversion of into fuel and the
results of this process are found to be better than other alternate methods which are used for
the disposal of waste plastic.
Key words: waste plastics, thermal degradation, pyrolysis, catalyst degradation.
42.1 Introduction
Plastics play an important role in day- today life. It is unique material because of their
toughness, light weight, resistance to water and chemicals, resistant to heat and cold, low
electrical and thermal conductivity, ease of fabrication, remarkable color range, more design
flexibility, durability and energy efficiency. Due to above properties it is used in packaging


456

materials, agriculture, construction, insulation, automobile sector, electronic devices, textiles
and sports equipment and toys.
Plastics constitutes in two main categories. It is thermoplastics and thermoset plastics.
Thermoplastics make up 80% of the plastics and thermoset plastics make up of remaining 20
% of plastics produced today (Birley et al, 1988), etc. Thermo plastics can re-melt or re-
mould and therefore it recyclable easily but thermoset plastics cannot re-melt or reshape and
therefore it is difficult to recycling. Use of different type of some thermo plastics is given in
table1 below. Plastics are relatively cheap, easy available, easy to manufacture and their
versatility replace to conventional materials.
Plastic waste management is biggest problem now due to their non- biodegradability
nature. Now plastics manage by plastics recycling technologies.
Table 1:Uses of different types of plastics.
Type of Plastics Uses
Polyester Textile fiber
PET Carbonated drink bottles, plastics film
PE Supermarket bags, plastics bottle
HDPE Milk jugs, detergent bottles, thicker
Plastics film, pipes
LDPE Floor tiles, shower curtains, cling film
PVC Agriculture (fountain) pipe, guttering
Pipe, window frame, sheets for
building material
PS foam use for insulation of roofs and
walls, disposal cups, plates, food
Container, CD and cassette box.
PP Bottle caps, drinking straws,
Bumper, house ware, fiber carpeting and rope.

42.1.1 Plastics in environment
The quantum of solid waste is ever increasing due to increase in population,
developmental activities, changes in life style, and socio-economic conditions, Plastics waste
is a significant portion of the total municipal solid waste (MSW). In India generation of

457

plastics are increased from about 2.6 MT in 2003 to about 3.6 MT in 2007(MOEF, 2007).
Also it is estimated that approximately 10 thousand tons per day (TPD) of plastics waste is
generated i.e. 9% of 1.20 lacks TPD of MSW in the India(CPCB, 2003). 32 million of plastics
were generated in 2011 in America, representing 12.7 percent of total MSW (EPA, 2011). It is
estimated that 100 million tones of plastics are produced each year with PE, PS, PVC and PP
amounting to more than 65% of total produced. The average European throws away 36kg of
plastics each year. Discarded plastic products and packaging materials make up a growing
portion of municipal solid waste. Plastics packaging totals 42% of total consumption and very
little of this is recycled (Vogler et al, 1984), etc. Only 8 percent of the total plastic waste
generated in 2011 was recovered for recycling (EPA, 2011).
Plastics waste may grow in India in future because more and other countries like as
U.S, China and U.K will comes in Indian market. There is a much wider scope for recycling
in developing countries mainly in India due to low labor cost, plastics consumption increase
and therefore raw materials increase.
42.1.2 Environmental hazards due to mismanagement of plastics waste
Plastics are no biodegradable material. It takes time to biodegrade is 300-500 years
and therefore environmental hazards due to improper manage include following aspect:
1. Littered plastics spoils beauty of the city and choke drains and make important public
places dirty.
2. Garbage containing plastics, when burnt may cause air pollution by emitting polluting
gases.
3. Garbage mix with plastics gives problem in landfill operation.
4. Lack of recycling plant to posing unhygienic problem to environment
42.1.3 Side Effect of plastics in nature
1. Durability and chemical structure greatly influences the biodegradability of some
organic compounds therefore an increased number of functional groups (groups of
atoms) attached to the benzene ring in an organic molecule usually hinders microbial
attack.
2. Instead of biodegradation, plastics waste goes through photo-degradation and turns
into plastic dusts which can enter in the food chain and can cause complex health
issues to earth habitants.

458

3. Plastics are produced from petroleum derivatives and are composed primarily of
hydrocarbons but also contain additives such as antioxidants, colorants, and other
stabilizers.
4. However, when plastic products are used and discarded, these additives are
undesirable from an environmental point of view.
5. Burning of plastics give NO
X
,

CO
X,
SO
X
, particulate, dioxins, furans and fumes to
increase air pollution with result acid rain and increase global warming.
6. Plastics in land fill area leaching of toxins into ground water.
42.2. Target of waste plastics into liquid fuel
42.2.1 Recycling Technologies
1. Mechanical Recycling of waste plastics into reusable product is difficult and
unfeasible due to contamination of plastics, difficulty to identifying and separating
different type of plastics.
2. Uncontrolled incineration of plastics at higher temp above 850 deg Celsius to
produces polychlorinated dibenzo-p-dioxins, a carcinogen (cancer causing chemical).
Open-air burning of plastic occurs at lower temperatures, and normally releases such
toxic fumes and many oxide gases. So flue gases treatment use for protect
environment and health problems in incineration plant.
3. Chemical recycling could lead to useful raw materials via by degradation and
monomerization of plastics waste, but no method of this primary recycling currently
available. The degradation of some plastics into chemicals has been reported in
research level.
Gasification and blast furnace of plastics waste to produce gases that are carbon
dioxide, nitrogen, carbon mono oxide, hydrogen and methane at higher temp above 800 deg.
Celsius.
42.2.2 Biodegradability
Plastics are non biodegradable material that resists microbial attack. Though work has
been done to make futuristic biodegradable plastics, there have not been many conclusive
steps towards cleaning up the existing problem because prices of biodegradable plastics is
more than petrochemicals based plastics. It may be due to high cost of production and low
availability or high cost of raw materials. Some degradable plastics have been developed, but

459

none has proved compatible with the conditions required for most waste landfills. Thus, there
is an environmental problem associated with the disposal of plastics.
42.2.3 Energy Demand
Fossil fuel i.e. coal, petroleum and natural gas age is expected to span only 1000 years
of human civilization (1700 AD to 2700 AD). It is limited sources which are likely to be
exhausted in a few more decades or centuries. Increasing population and fuel consumption
rates increase in petroleum prices and due to this the energy starvation is felt by every
developing and less developed country. The Growing energy demand in table 1.2 is below.
Some developing countries like as India have to import petroleum for transportation
and chemical industry sector. The prices of petroleum are increasing due to increase prices in
international market. Conversion of waste plastics into fuel is complete the some part of
objectives in National Energy Strategy is:
1. To reduce petroleum Imports
2. To reduce the annual growth of total energy demand to 2 percent From 4 to 6% by
conservation of energy.
3. To develop alternative sources of energy.
Table 2: Growing Energy Demand.
Year World Primary Energy Demand (exajoules/year)
1972 270
1985 390
2000 590
2020 840
(S. Rao and Dr B.B. Parulekar, 2012)
42.3 Plastics Recycling Technologies
Recycling of plastics should be carried in a manner to minimize pollution during the
process and enhance efficiency and conserve the energy. There is different type of technology
include following aspect:
1. Mechanical Recycling- Recycling of plastics waste into reusable product.
2. Chemical Recycling Gasification, blast furnace
3. Incineration- Burning of waste plastics to obtain energy.
4. Pyrolysis Conversion of waste plastics into liquid fuels.


460

42.4 Process technology
42.4.1 Raw materials
Type of Plastics as raw materials and its contents in table 3 is below.
Table 3: Type of plastics and its content.
Type of plastics contents
PE (HDPE, LDPE), PP, PS hydro carbons
PET, PVA, PF hydro carbons with oxygen
PVC, PVCD hydrocarbons with chlorine
Nylon (polyamide), PU hydrocarbons with nitrogen
Polyphenylene sulfide hydrocarbons with sulfur

42.4.2 Effect of raw material as plastics in production
If PE, PS, PP with other plastics gives flue gas pollution and contaminated to reactor
by making other unexpected compound. In contamination to reactor resulting liquid may
contain alcohol, waxy hydrocarbons and inorganic substance. Type of plastics and their
product in table 4 is below.
Table 4: Effect of plastics in production.
Type of plastics Product
PET terephthalic acid and benzoic acid
PVA water and alcohol
PVC, PVDC HCL gas and carbonous compound
PU, PF, NYLON carbonous product
PE, PS, PP liquid fuels
(UNEP, 2009)
42.4.3 Pyrolysis
It is thermal degradation process in the absence of oxygen. It prevent of formation of
C0
X
, NO
X
, SO
X
due to absence of oxygen. It breaks large hydrocarbon chain into smaller
ones, but this type of pyrolysis requires higher temperature and high reaction time. Also
resulting fluid have low octane value, higher pour point of diesel and high residue content.
42.4.4 Catalytic Pryolysis

461

Pyrolysis of waste plastics in presence of catalyst lower the pyrolysis temp and
reaction time, increase conversion rate of waste plastics into fuel, increase the yield of fuel
and satisfying diesel, petrol quality of fuel by increase octane value of petrol and decrease
pour point of diesel. Catalyst use for this purpose is solid acids such as silica, alumina,
zeolite, zeoliteY, mordenite, HZSM-5, MCM-41. Acidic catalysts (HZSM-5, Zeolitey,
mordenite and so on) have greater efficiency than less acidic ones, for example amorphous
alumina silicate. The pore size and structure of catalyst determine their performance on
cracking reaction as well as production, for example mordenite size( about 7x8) larger give
large product molecules while HZSM-5 have smaller pore size(5x5) give small product
molecules.(P.A. Parikh and Y.C. Rotliwala, 2008)
42.4.5 Process of formation
Collect waste plastics and separate that clean and recyclable. Store the waste plastics
that cant separate. Shredding of waste plastics to reduce volume of its. Shredded plastics is
treated in a cylindrical reactor at temperature of 300C 350C(Pawar harshal and Lawankar
Shailendra, 2013).Plastics waste further cracked with catalyst and resulting hydrocarbons are
condensed from water cool condenser and collected in receiver. Then liquid fuel fractionates
to get diesel, kerosene, petrol etc.
Gases produced are toxic, corrosive with non toxic gases. For example hydrogen
chloride, hydrogen sulfide etc is toxic and non toxic is butanes, methane, ethane and
propylene. So all the gases are treated from this process before it discharge into atmosphere.
Therefore flue gas treated through scrubbers and water/ chemical treatment for neutralization
i.e. Solution of methanol amine is use in hydrogen sulfide absorption. Treated flue gas can
incinerate use in dual Fuel diesel-generator set for generation of electricity. After process
remove the formed carbonous substance or residue in reactor to work as insulator for
maintaining the efficiency of process. The block diagram of process is given in figure1.
42.4.6 Yield
The average percentage yield of various fuel fractions by fraction distillation
depending on composition of waste plastics are Gasoline (60% ) and Diesel (30%). The
percentage of liquid distillate is mentioned in terms of weight by volume (Antony Raja and
Advaith Murali 2011).
42.5. Advantages of process of fuel production
42.5.1 Eco-friendly

462

The fuel satisfies quality of liquid fuel with low sulfur content and low carbon residue.
The properties of waste plastic pyrolysis oil and diesel in table 5.
collection and segregation of plastic waste

storing of plastic waste

shredding of plastic waste

Figure 1- Conversion waste plastics into liquid fuel (Pawar Harshal and lawankar, 2013)
Table 5: Properties of Waste Plastic Pyrolysis Oil and Diesel.
Sr. No. Properties WPPo Diesel
1. Density(kg/m2) 793 850
2. Ash content (%) <1.01%wt 0.045
3. Calorific value(kJ/kg) 41,800 42,000
4. Kinematic viscosity @ 2.149 3.05
40C(cst)
5. Cetane number 51 55
6. Flash point oC 40 50
7. Fire point oC 45 56
8. Carbon residue (%) 0.01%wt 0.20%
9. Sulphur content (%) <0.002 <0.035
10. Pour point oC -4 3-15
(Pawar Harshal and Lawankar, 2013)
feeding into hopper
Flow of waste into heating vessel in absence of oxygen and presence of catalyst
movement of liquid-vapor into condenser vessel tarry waste
Tapping of liquid fuel
Fractionation of liquid fuel to obtain diesel, petrol, kerosene etc.

463

42.5.2 Feasibility
Process of conversion of waste plastics into liquid fuels is feasible. Also the rate of
fuel does not vary widely along the period. The cost for per kg of input and related output in
table 1.6 is below.
Table 6: cost for 1 kg of input and the yield, cost of output.
Input Qty Kg Rate per Kg Amount (Rs) Output Qty (l) Rate per liter
Amount (Rs)
Plastic 1.00 12.00 12.00 Petrol 0.600 37.50 22.50
Labour 5.00 Diesel 0.300 25.50 7.65
Service
Charge 2.50 Lube oil 0.100 15.00 1.50
Total 1.00 19.50 1.00 31.65
(Antony Raja and Advaith Murali, 2011)
42.5.3 Good performance
Liquid fuels from petroleum is diesel, petrol, kerosene require to mix various additives
for improving burner and engine performance but fuel from waste plastics does not require to
add these additives for work on burner and engines. Tarry waste or residue in reactor can use
as solid fuel.
42.6 Conclusion and recommendation
Based on review papers, waste plastics liquid fuel is good alternative method for
obtaining new energy resource and eliminate greater problem of plastics waste management.
In India 3.6 million ton of plastics waste generated in 2007. Improper management of plastics
gives hazardous problem to human and environment. Mechanical recycling is not effective to
reduce to problem of plastics waste. Incineration, gasification , blast furnace is other method
does not effectively eliminate to this problem due to air pollution, economical unfeasibility
compare to waste plastics fuel method. Biodegradable plastics are not meet at same rate as
petroleum based plastics.
Growth of energy demand due to urbanization, population, industrialization and also
increased price of fuel need to reduce to this demand and increased rate of fuel. Waste plastics
fuel is eco friendly due to low content of pollutants, good performance characteristics on
engine, burner with no added any additives like as lubricants and good feasibility with earning
profit.

464

Abbreviation
PET- polyethylene terephthalate
HDPE- high density polyethylene
LDPE- low density polyethylene
PS- polystyrene
PVC- polyvinyl chloride
PP- polypropylene
PF- phenol formaldehyde
PU- poly urethane
PVA- poly vinyl alcohol
PVDC- polyvinylidene chloride

References
1. Antony Raja and Advaith Murali, 2011 Conversion of Plastic Wastes into Fuels
Journal of Materials Science and Engineering B 1 (2011) 86-89
2. Birley, A. W., Heath, R. J., and Scott, M. J. (1988) Plastics Materials. Blackie, 2nd ed.
Introductory scientific textbook.
3. Central Pollution Control Board. Study on solid waste management CPCB Delhi.
(2003).
4. Environment Protection Agency, U.S.A. Study on solid waste management (2011).
5. Ministry Of Environment and Forest. News letter on solid waste management, New
Delhi, (2007)
6. Pawar Harshal R. and Lawankar Shailendra M.(2013) Waste plastic Pyrolysis oil
Alternative Fuel for CI Engine A Review Research Journal of Engineering Sciences
ISSN 2278 9472 Vol. 2(2), 26-30, February (2013)
7. P.K Parikh PhD, Y.C Rotliwala (2008) DOI: 10.1680/warm.2008.161.2.85
ISSN : 1747-6526
8. S Rao, Dr. B.B Parulekar (2012) Energy Technology (NONCONVENTIONAL,
RENEWABLE & CONVENTIONAL), Khanna Publishers, ISBN NO. 81-7409-040-1
9. Tiwari D.C., Ejaz Ahmad, Kumar Singh K.K. Catalytic degradation of waste plastic
into fuel range hydrocarbons International Journal of Chemical Research, ISSN: 0975-
3699, Volume 1, Issue 2, 2009, pp-31-36
10. UNEP, Converting Waste Plastics into Resource, (2009).

465

11. Vogler, Jon, Small-scale recycling of plastics. Intermediate Technology Publications
1984. A book aimed at small-scale plastics recycling in developing countries


466

CHAPTER 43
KINETICS OF NOX REDUCTION IN BIODENOX PROCESS
WATER: EFFECT OF TEMPERATURE AND IRON
CHELATE
B. Chandrashekhar, Heena Tabassum, Nidhi Sahu, Padmaraj Pai, R. A. Pandey

Abstract
The aqueous solution of various iron chelates viz. Fe
II
EDTA, Fe
II
NTA has been utilized for
NOx absorption from gaseous emissions using wet-scrubbers. In order to make the spent
solution recyclable to the scrubber, the BioDeNOx process involves treatment of the spent
scrubber solution wherein the reduction of NOx adduct of Fe
II
EDTA takes place by
employing denitrifying bacteria. The influence of temperature on batch NOx reduction
process using different concentration of Fe
II
EDTA was investigated and modelled. The
specific NOx reduction rates in 0 to 30 mM Fe
II
EDTA solution were estimated in the
temperature range of 298-313 K. The values of Arrhenius factor (Ar) and activation energy
(Ea) were determined using the Arrhenius equation. The values of Ar and Ea were highest in
the absence of Fe
II
EDTA (319.54 moles gVSS
-1
L
-1
h
-1
and 88.298 J/mol respectively).
Addition of 5 mM Fe
II
EDTA to the solution decreased the values of Ar and Ea, which
however increased with further increase of Fe
II
EDTA concentration. The sensibility of NOx
reduction rates to temperature was modeled using the equation R = R
293
x 10
K(T-293)
and the
values of temperature constant (K
T
) was predicted, which was found to be highest in 30 mM
Fe
II
EDTA solution (0.0034 K
-1
). The temperature coefficient (Q
T
) was also calculated at each
Fe
II
EDTA concentration to determine the sensitivity of reduction rate to temperature.
Keywords: Nitrogen oxides, Ferrous EDTA, Denitrification, NOx reduction, Arrhenius
Equation
43.1 Introduction
The adducts of chelating agents and metals such as Ferrous (Fe
II
) EDTA (ethylene
diamine tetra acetic acid) and Ferrous NTA (nitrilotriacetic acid) have been used to enhance
the absorption rate of NOx from into aqueous phase in wet scrubbers (Gambardella et al.,



467

2006; Demmink et al., 1997). Fe
II
EDTA is the most commonly used chelates for absorption
of NOx. In order to make absorption process economical, the spent metalligand solution has
to be regenerated and recycled which is done by the biological denitrification process in
which the NO adduct of Fe
II
EDTA is reduced to molecular nitrogen. It is reported that
reduction of NO is enzymatically catalyzed by potential denitrifying bacterial strains viz.
Bacillus azotoformans (Kumaraswamy et al., 2005), Pseudomonas (Zhang et al., 2007),
Paracoccus denitrificans (Li et al., 2012) and anaerobic sludge (Van der Maas et al., 2008;
Dilmore, 2004) which use an organic electron donor as a reducing agent. The regenerated
adduct solution therefore is recycled back to the scrubber where it continuously absorbs NOx
from the emission gases. This process is also known as BioDeNOx (van der Maas, 2008;
2005) and a general schematic of the process is shown in Fig-1.

Fig. 1 General schematic of the BioDeNOx concept of NOx removal from gaseous emissions
In order to develop a full scale engineered process for biological NOx removal using
Fe
II
EDTA solution, it is necessary to evaluate the kinetic parameters associated with NOx
reduction under the operating conditions. The emission gases containing NOx have high
temperatures; therefore, scrubbing of such gases with the scrubber solution would also lead to
a rise in temperature of the solution, which consequently would affect the biological reduction
process. In the present investigation, the biomass from a wastewater denitrification process
was evaluated for NOx reduction in different concentrations of Fe
II
EDTA solutions using
ethanol as carbon source as well as electron donor. The bioreduction rates (R) were
determined and the impact of temperature on the reduction rate was investigated in the range

468

of 298-313 K. Arrhenius equation was applied for the calculation of activation energy (E
A
)
and Arrhenius factor (A
r
). Also, a kinetic model was applied to determine the value of
temperature constant at each Fe
II
EDTA constant.
43.2.1 Source of biomass for conducting NOx reduction experiments
The bacteria used for this study were obtained from the sludge obtained from a
denitrification tank of an effluent treatment plant. The biomass was cultivated for a period of
3035 days in a 1 L batch reactor with a medium containing 0.020 M Fe
II
EDTA with
nutrients and trace elements. NaNO
2
(as a source of NOx) was added to the medium along
with ethanol and trace element solution from a concentrated feed stock whenever required.
The unit was operated in anaerobic conditions to allow bacterial growth. The temperature of
unit was controlled at 37 C the help of a water bath. The inoculum was prepared by
harvesting the cultivated bacteria from the medium by centrifugation (8000 rpm, 10 min). The
pellet obtained was again washed with 0.1 N phosphate buffer saline and used as inoculum for
the batch experiments.
43.2.2 Media composition and batch experiments
All the batch experiments for NOx reduction were conducted in 250 mL Erlenmeyer
flasks containing 200 mL media. The flasks were sealed with silicon stoppers and kept in a
shaking incubator at 100 rpm. The experiments were conducted in order to find out the rate at
which Fe
II
EDTA-NO was reduced by the biomass at different Fe
II
EDTA concentration.
Hence, the assays were conducted by varying the Fe
II
EDTA (0 - 30 mM) in a medium
containing: NaNO
2
1 mM, K
2
HPO
4
3 mM, KH
2
PO4 4 mM, MgCl
2
0.002 mM,
MgSO4.7H
2
O 0.4 mM, Na
2
SO
3
0.5 mM, FeSO
4
0.06 mM, CuSO
4
.5H
2
O 0.03 mM,
Na
2
- MoO
4
0.02 mM and Ethanol- 4.5 mM. Fe
II
EDTA-NO medium was prepared by
mixing equimolar concentrations (0 - 30 mM) of FeCl
2
and Na
2
H
2
EDTA to the above
medium under anoxic conditions according to a method described by van der Maas et al
(2005). The media as well as head space was flushed with pure nitrogen gas in order to
maintain anaerobic conditions. The pH of the media was adjusted to 6.5 by adding NaOH or
HCl for all the experiments. The biomass was inoculated to the medium at initial volatile
suspended solid (VSS) concentration of about 2.0 g VSS L
-1
. A medium without the biomass
inoculation was kept as control for each experiment. The NOx concentration (N) after

469

different time intervals was monitored in the media and the specific NOx reduction rate (R)
was calculated as follows-
R = - (lnN
2
- lnN
1
)/(t
2
- t
1
) * x (1)
where, x is the initial VSS concentration in the medium.
To study the impact of temperature NOx reduction at each Fe
II
EDTA concentration,
the experiments were set up as previously described and conducted at 25, 30 and 35 and 40 +
0.5 C under anoxic conditions as most of the denitrifying bacteria are mesophiles that are
known to grow in the range of 20-45 C. The kinetics of the reduction reaction included the
estimation of activation energy (E
A
) and the temperature coefficient (K) determination that
determine the process sensibility and the change of the NOx reduction rates along with the
change in temperature. E
A
measures the change in the potential energy that is required to
begin the reaction to convert the reactants into products. The reactions with a low E
A
are less
sensitive to the change of temperature. The change of the NOx reduction rates along with the
temperature was given by the Arrhenius equation

R = A
r
e
-(Ea/Rg.T)
(2)
where R is the specific NOx reduction rate, A
r
is the Arrhenius factor (mM.g
-1
.h
-1
), E
A
is the
activation energy (J mol
-1
), Rg is the gas constant (8.314 J mol
-1
K
-1
) and T is temperature
(K). A linear form of the equation obtained after taking logarithm is shown below-
lnR = lnA
r
E
A
/Rg T ... (3)
The graphic plot of lnR versus 1/T is a straight line with a slope of - E
A
/Rg and
intercept of lnA
r
which enables the estimation of E
A
and A
r
. The sensitivity of the reaction to
increasing temperature was expressed by the following equation [18]:
R = R
293
10
K
T
(T-293)
(4)
Where, K
T
is the temperature constant. The temperature coefficient (Q
T
), which determines
the change of the reduction rate along with the change of temperature, can be calculated as the
ratio of the specific reduction rates at different temperatures as shown below-
Q
T
= R
(t+t)
/R
t
(5)
Where R
(T+T)
and R
T
are the specific NOx reduction rates at times T+T and T respectively.

470

43.3 Analytical methods
The concentration of NOx in the media was measured in the form of NO
2
-
in all the
experiments. In case of the medium without Fe
II
EDTA, NO
2
-
was measured directly by standard
protocol using sulphanilamide and NED reagent (APHA, 2005). In case of media with
Fe
II
EDTA, some amount of NOx was present in the form of NO adduct of Fe
II
EDTA,
therefore the samples were oxidized by 0.1 N NaOH (to convert NO to NO
2
-
) before
subjecting to nitrite estimation by the above method. All the analyses results reported in this
paper are average values of duplicate samples.
43.4.1 Specific NOx reduction rates under various conditions
The biomass used for the batch NOx reduction experiments were initially cultivated
for 30 days in a medium containing 20 mM Fe
II
EDTA along with NOx, using ethanol as
electron donor. The acclimatized biomass thus possessed the ability to grow in the presence of
Fe
II
EDTA using NOx as the available electron acceptor. It has been earlier reported that
Fe
II
EDTA has the potential to act as an electron donor for the chemical reduction of NOx (van
der Maas et al, 2008). The objectives of the batch experiments were to investigate the effect
of Fe
II
EDTA concentration on NOx reduction under different temperatures of incubation. The
experiments were designed to simultaneously determine the effect of Fe
II
EDTA as well as
temperature on NOx reduction rates. The batch NOx reduction experiments were carried out
in a medium containing 1 mM NOx using ethanol as the organic electron donor. The biomass
concentration in each experiment was kept constant (2 mg VSS/ml). Four sets of reduction
media with varying Fe
II
EDTA concentration were prepared; each set was incubated at
different temperature viz. 25, 30, 35 and 40 C and monitored for NOx concentration at
regular intervals.
43.4.1.1 Effect of Fe
II
EDTA concentration
The NOx reduction curves obtained at 298 K for different concentration of Fe
II
EDTA
in the media are shown in Fig (2).
Similar curves were obtained for different incubation temperature, and the specific
NOx reduction rate for each experimental condition was calculated using Eq. 1. The specific
reduction rates using various concentration of Fe
II
EDTA at different temperatures (298 313
K) are depicted in Fig (2). It was observed that highest NOx reduction rates (R) were obtained
in the absence of Fe
II
EDTA. However, addition of 5 mM Fe
II
EDTA drastically reduced the

471

specific reduction rate, which indicated that Fe
II
EDTA inhibited the biological reduction of
NOx. However, it was also observed that with further increase in Fe
II
EDTA concentration, the
specific NOx reduction rates also increased. Therefore it can be suggested that though the
biological reduction process was inhibited at higher Fe
II
EDTA concentration, the chemical
reduction of NOx by Fe
II
EDTA occurred. The chemical reduction of NOx by Fe
II
EDTA is
shown in the Eq. 6-7 (van der Maas et al., 2008). The reactions clearly show that 2 moles of
Fe
II
EDTA
2-
is required for complete reduction of each mole of NO
2
-
to N
2
. As more unbound
Fe
II
EDTA would be available at higher concentration of Fe
II
EDTA in the medium for the
chemical reaction to occur, this provides the reason for the enhanced reduction rates.
2Fe
II
EDTA
2-
-NO + 2H
+
N
2
O + H
2
O + 2Fe
III
EDTA
-
.. (6)
N
2
O + Fe
II
EDTA
2-
+ 2H
+
N
2
+ H
2
O + 2Fe
III
EDTA
-
. (7)

Fig. 2 NOx (as NO2-) reduction curves obtained at 298 K with various concentration of
Fe
II
EDTA
43.4.1.2 Effect of temperature on NOx reduction rates
The temperature range selected for the investigation was based on the fact that most
denitrifying bacteria grow under mesophilic conditions and also that the stability of the
Fe
II
EDTA-NOx complex would be lower at temperatures higher than 323 K (Gambardella et
al, 2006). Therefore, the batch experiments were carried out at 298 313 K. The percentage
0.000
0.200
0.400
0.600
0.800
1.000
0 5 10 15 20 25 30 35 40 45
N
O
2
-
(
m
M

L
-
1
)
Time (h)
0.000 M FeEDTA 0.005 M FeEDTA 0.010 M FeEDTA
0.020 M FeEDTA 0.030 M FeEDTA

472

NOx reduction at different concentration of Fe
II
EDTA incubated for 40 hours at various
temperatures is summarized in Table-1.
Table 1 NOx reduction (%) at various temperatures and Fe
II
EDTA concentrations after 40
hours of incubation
% NOx reduction
Fe
II
EDTA
concentration (mM)
Temperature (K)
298 303 313 323
0 85.92 87.65 89.38 88.25
5 67.68 67.96 68.24 66.63
10 73.81 74.79 75.76 75.38
20 80.59 81.30 82.00 81.22
30 80.82 82.58 84.33 84.41

Also, it is evident from Fig (3) that at all Fe
II
EDTA concentrations used in the present
investigation, the specific reduction rates increased with temperature.

Fig. 3 Specific NOx reduction rates at different Fe
II
EDTA concentrations and temperatures
The impact of temperature on reduction rate can be studied by applying Arrhenius equation,
which enables to calculate the Arrhenius factor (A) and activation energy (E
a
). The change of
reduction rates along with temperature is given by Eq. 3. A linear plot drawn between 1/T
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
0 5 10 15 20 25 30 35
S
p
e
c
i
f
i
c

N
O
x

r
e
d
u
c
t
i
o
n

r
a
t
e

(
R
)

m
M
o
l
e
s

g
-
1
h
-
1
e
!!
"#T$ (mMoles L
-1
)
25 degrees C 35 degrees C 40 degrees C

473

and lnR showed good degree of linearity and was used to determine the values of A and E
a
for
each concentration of Fe
II
EDTA as shown in Fig. 4. The values of E
a
and A obtained for each
concentration of Fe
II
EDTA are summarized in Table 2.

Fig. 4 Plots between log values of specific NOx reduction rate (ln R) and inverse of
temperature (1/T) to determine the value of activation energy and Arrhenius factor at each
Fe
II
EDTA concentration
Table 2 Activation energy and Arrhenius factor for NOx reduction at different Fe
II
EDTA
concentration
e
!!
"#T$
(mM)
$rrhenius factor% $
r

(mM/gV/!)
$cti&ation "nerg'% "a
("/m#$)
0 319.54 38.43
5 28.21 3.39
10 34.83 4.19
20 75.14 9.04
30 257.21 30.94

% & '89.078( ) 2.9418
*+ & 0.9938
2.64
2.64
2.65
2.65
2.65
2.66
2.66
0.0031 0.0032 0.0033 0.0034
l
n

R
1(T ()
-1
)
* mM e
!!
"#T$
% & '200.54( ) 3.5504
*+ & 0.9625
2.87
2.88
2.89
2.90
2.91
2.92
0.0031 0.0032 0.0033 0.0034
l
n

R
1(T ()
-1
)
1+ mM e
!!
"#T$
% & '377.12( ) 4.3193
*+ & 0.7549
3.04
3.06
3.08
3.10
3.12
0.0031 0.0032 0.0033 0.0034
l
n

R
1(T ()-1)
2+ mM e
!!
"#T$
% & '721.03( ) 5.5499
*+ & 0.9737
3.12
3.16
3.20
3.24
3.28
0.0031 0.0032 0.0033 0.0034
l
n

R
1(T ()
-1
)
,+ mM e
!!
"#T$

474

It was observed that similar to the specific NOx reduction rates, the values of both E
a

and A were highest in the absence of Fe
II
EDTA, and lowest in the presence of 5 mM which
gradually increased along with Fe
II
EDTA concentration. Generally, reactions with low
activation energies are less sensitive to temperature. The sensitivity of the reaction to
increasing temperature can be also expressed by the Eq. 4. The values of R obtained at
various temperatures (298-313 K) for 0 - 30 mM Fe
II
EDTA concentration was modeled using
this equation by Solver function as shown in Fig. (5), and the predicted values of temperature
constant (K
T
) are summarized in Table 3.
Table 3 Temperature constants of NOx reduction at different Fe
II
EDTA concentration
e
!!!
"#T$ (mM)
R
2-,

(mM/gV/!)
)
T

0 26.21 0.00337
5 13.98 0.00041
10 17.59 0.00094
20 20.38 0.00277
30 22.00 0.00339

Since, higher values of K
T
signifies higher sensitivity of the reaction to temperature, it
can be suggested from the results that NOx reduction in the medium without Fe
II
EDTA
(completely biological reaction) was highly sensitive to temperature.
On the other hand, lesser values of K
T
obtained in the presence of 5- 10 mM
Fe
II
EDTA indicate that the process was less sensitive to temperature. Further increase in
Fe
II
EDTA concentration increased the value of K
T
, which means increasing the concentration
of Fe
II
EDTA increases the chemical reduction of NOx and also renders the reduction reaction
to be more temperature sensitive. The temperature coefficient determines the change in
reaction rate along with change in temperature. The temperature coefficient (t = 10 K) for
each Fe
II
EDTA concentration was calculated from the predicted values of R, using Eq. 5 and
as shown in Fig. 5, value of Q also slightly increased along with Fe
II
EDTA concentration.
However the value of Q was found to be very less as it is well accepted that that a 10 K rise in
temperature results in doubling of the reaction rate.
The effect of temperature on reduction of NOx in the presence of Fe
II
EDTA has not
been reported elsewhere. The effect of temperature on biological reduction of NOx in the
absence of Fe
II
EDTA has been studied previously (Pfenning and McMohan, 1996; Rusmana,
2007; Carrera et al, 2003; Kadlec, 2001). Casey (1997) and Foglar et al (2010) have studied

475

the effect of temperature on bio-denitrification rates and reported similar values of K
T
, as
reported in the present investigation. However, higher values of temperature coefficient (2.03)
within the same temperature range has been reported by Foglar et al (2010; 2005) and
Delanghe et al (1994).

Fig. 5 Modeled NOx reduction rates as a function of temperature at various concentration of
Fe
II
EDTA
26.0
27.0
28.0
29.0
30.0
31.0
290 300 310 320
R

(
m
M
.
g
-
1
h
-
1
)
Temperature ())
+ mM e
!!
"#T$
14.00
14.05
14.10
14.15
14.20
14.25
14.30
295 300 305 310 315
R

(
m
M
.
g
-
1
h
-
1
)
Temperature ())
* mM e
!!
"#T$
17.7
17.9
18.1
18.3
18.5
290 300 310 320
R

(
m
M
.
g
-
1
h
-
1
)
Temperature ())
1+ mM e
!!
"#T$
20.5
21.0
21.5
22.0
22.5
23.0
23.5
295 300 305 310 315
R

(
m
M
.
g
-
1
h
-
1
)
Temperature ())
2+ mM e
!!
"#T$
22.5
23.0
23.5
24.0
24.5
25.0
25.5
26.0
26.5
295 300 305 310 315
R

(
m
M
.
g
-
1
h
-
1
)
Temperature ())
,+ mM e
!!
"#T$

476

43.5 Conclusion
The biological reduction of NOx in the presence of Fe
II
EDTA is a slow process, since
the growth of bacteria in Fe
II
EDTA medium is inhibited. Biological reduction of NOx is
therefore the limiting step of the two stage BioDeNOx process for NOx removal. The results
obtained from the present investigation indicate that chemical reduction of NOx by 30 mM
Fe
II
EDTA show comparable results with biological NOx reduction (in the absence of
Fe
II
EDTA) under the same conditions. Using higher concentrations of Fe
II
EDTA can make
the NOx reduction process more sensitive to temperature. It can be suggested that chemical
reduction of NOx by Fe
II
EDTA itself can be enhanced by optimizing the concentration of
Fe
II
EDTA (i.e. ratio of Fe
II
EDTA/NOx) and temperature in order to decrease the process cost
and improve NOx removal efficiency of the process.
Acknowledgements
The authors are thankful to Director, CSIR-NEERI, to give kind permission to present
this research work. The financial support extended by Department of Biotechnology and
Council of Scientific and Industrial Research, Ministry of Science & Technology,
Government of India, for execution of this project work is duly acknowledged.

References
1. American Public Health Association (APHA) (2005). Standard Methods for the
Examination of Water and Wastewater, nineteenth ed. American Public Health
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2. Demmink, J.F., Van Gils, I.C.F., Beenackers, A.A. (1997). Absorption of nitric oxide
into aqueous solutions of ferrous chelates accompanied by instantaneous reaction. Ind.
Eng. Chem. Res. 36:49144927.
3. Dilmore, R. (2004). Evaluation of the kinetics of biologically catalyzed treatment and
regeneration of NOx scrubbing process waters. Doctoral Thesis. University of
Pittsburgh, 224pp
4. Gambardella, F., Winkelman, J.G.M., Heeres, H.J. (2006) Experimental and modeling
studies on the simultaneous absorption of NO and O
2
in aqueous iron chelate solutions.
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5. Rusmana I. (2007) Effects of Temperature on Denitrifying Growth and Nitrate
Reduction End Products of Comamonas testosteroni Isolated from Estuarine Sediment,
Microbiology Indonesia, 43-47.
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Muyzer, G. (2005). Characterization of microbial communities removing nitrogen
oxides from flue gas: the BioDeNOx process. Appl. Environ. Microbiol. 71: 63456352
10. Li, N., Zhang, Y., Chen, M., Dong, X., Zhou, J. (2012). Reduction of Fe(II)EDTANO
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Microbiol. Biotechnol. 76: 11811187


478

CHAPTER 44
STATUS OF WASTE TREATMENT, UTILIZATION AND
MANAGEMENT IN AGRO PROCESSING
Yogender Singh and Y. K. Yadav

Abstract
The present situation of increase in world population brought severe problems to the
sustainable agriculture with simultaneous food scarcity and climatic change. In the near
future the management of food and agricultural systems will play an important role in the
conservation of the natural resources. Agro processing comprise of techno-economic
activities associated for conservation and handling of agricultural produce and to
make it usable for feed, food, byproduct utilization and industrial raw material.
Agricultural and food processing industrial waste contains many reusable substances of high
value. The agricultural residue occurs at the time of harvesting, handling transportation,
storage, marketing and processing resulting in waste. Depending on availability of an
adequate technology this residual matter can be converted into commercial products either as
raw material for further secondary processes, as operating supplies or as ingredients of new
products. Efficient management of these wastes can help in preserving vital nutrients of our
foods, feeds and byproducts utilization, bringing down the cost of production of processed
foods, besides minimizing pollution hazards. It causes serious pollution problem if not
utilized or disposed off appropriately. A clear, concise and consistent policy is necessary to
establish and set up waste management systems and backed by legislations for all kinds of
violations. Implementation and monitoring of the different waste management rules should
also be identified at a regular interval, to study the effects of inappropriate disposal of waste
on the environment. This is essential for guiding the management of waste in a manner that is
environmentally responsible and which minimizes danger to public health. Thus, it is
recommended that phrase of Reduce, Reuse, Recycle should be used for waste
management.
44.1 Introduction
The rapid increasing of world population brought serious food, health and


479

environmental problems such as global food availability and climatic changes. In the near
future the management of food and agricultural wastes will play an important role in the
conservation of the natural resources in many countries, including India. Agro processing is
a set of techno- economic activities carried out for conservation and handling of
agricultural produce and to make it usable for feed and food or industrial raw material.
Agro processing industries occupy an important position economically and generate large
volumes of mostly biodegradable wastes. Large amount of wastes is generated every year
from the industrial processing of agricultural raw materials. Agro-industrial wastes can be
used as solid support, carbon and/or nutrient source for the production of a variety of value-
added products. The presence of carbon sources, nutrients and moisture in these wastes
provides conditions suitable for the development of microorganisms and their possibilities of
reuse. The generation of biodegradable waste increased linearly with the growth and
development of agro and food processing industries. A huge amount of waste in solid and
liquid form is produced in agro processing industries is valuable but biodegradable natural
resources with large economic prospects. It causes serious pollution problem if not utilized or
disposed off appropriately. However, hazardous wastes are also occasionally causes
depending on situations, as contamination by pesticides/herbicides and pathogens.
Wastes are materials which are discarded after use at the end of their intended life
period. Waste also defined as materials that are not prime products for which the
generator has no further use for their own purpose of production and consumption,
which discarded or intends to discard. Wastes may be generated during the extraction
and processing of raw materials to intermediate and final products during consumption
and any other human activity. Food wastes and effluents are rich in biodegradable
components with high biological oxygen demand (BOD) and chemical oxygen
demand. If they remained untreated their uncontrolled decomposition is hazardous to
the environment due to the production of methane and toxic materials (Waldron, 2004).
Wastes derived from agro processing industries are categorized into three groups:
(a) manufacturing losses,
(b) food products thrown away as municipal solid waste (MSW), and
(c) discarded wrappers and containers.
The lack of pilot scale testing of the developed technologies, negative attitude of the
industrialists and perhaps, less helping hand from the government sector are the major
constraints in utilization of the waste.

480

Due to poor postharvest management, the post harvest losses of farm produce in
India have been assessed of a very high order. According to Indian Agricultural Research
Data Book estimated post production losses in food commodities to the tune of INR
0.75 to 1.0 lakh crore per annum. The estimated loss includes losses during handling, storage
and processing. The extent of losses could be drop down to less than 50% of the estimated
existing level on adopting proper agro processing technology. For reducing the maximum
losses new initiatives need to be searched. It would be the long term interest of the
economy to developing suitable infrastructure processing systems to avoid the losses.
44.1.1 Agro processing industrial wastes
Agro processing industrial wastes are generated during the industrial processing of
agricultural and animal products. The agricultural activities includes the waste materials such
as straw, stalk, stem, leaves, husk, peel, shell, seed, pulp/stubble from fruits, legumes and
cereals, bagasses produced from sugarcane or sorghum milling, brewers spent grains and
many others. These wastes are generated in large amounts throughout the year with the
most abundant renewable resources on the earth. They are mainly composed of fibres, sugars,
proteins and minerals which are major compounds of industrial interest.
Due to the large availability and composition rich in compounds that could be used in
other refining processes, there is a great potential on the reuse of these wastes on economical
and environmental aspects. The economical aspect is based on the fact that such wastes may be
used as low-cost raw materials for the production of other value addition of compounds, with
the expertise of reducing the production and input costs. The agro-industrial wastes may
contain phenolic compounds and other compounds of toxic potential, which may cause
deterioration of the environment when the waste is discharged to the nature.
44.1.2 Solid Wastes
Solid wastes are generated in food industries can be categories in two groups. One
group is organic residual wastes such as sludge from wastewater treatment and food wastes or
garbage accompanied with consumption. For organic food wastes, the options of feed use,
biogas then composting and heat recovery is adopted for reuse and recycling. Another type of
waste relating to food industries is the waste originating from packaging materials. These
wastes comprise of a large portion of municipal solid waste. Among these wastes, plastic
wastes in particular should be focused on from an environmental standpoint.
44.1.3 Agricultural Wastes

481

Cereals are a major source of agricultural waste in many countries. Part of plant
residues (utilized/non-utilized) are considered as agricultural wastes. The amount of wastes
from livestock and especially of liquid manure is also included in wastes. Liquid manure
contains different microorganisms that are dangerous for people as well as for animals. On
the other hand, the manure has high energy potential and it is a significant source of
renewable energy.
44.1.4 Food Wastes
Food-processing wastes are those end products of various food-processing industries
that have not been recycled or used for other purposes. Food industry produces large volumes
of solids and liquid wastes from the production, processing and consumption of product.
These wastes results increasing disposal and potentially severe pollution problems and
represent a loss of valuable biomass and nutrients. In general, wastes from the food-
processing industry have the following characteristics (Litchfield, 1987):
1. Large amounts of organic materials
2. Varying amounts of suspended solids
3. High biochemical oxygen demand or chemical oxygen demand
44.1.5 Hazardous Wastes
Food that has been accidentally contaminated by, herbicides, pesticides and fumigants
may be treated as hazardous waste. For hygienic cleaning chlorine is frequently used in food
processing and daily operations. Hence, chlorinated organic compounds should be observed
in the wastewater treatment of industries. The wastewater may also contain certain levels of
trihalomethane and other related compounds. Food products contaminated with pathogenic
microbes or food poisoning sometimes result in hazardous wastes.
44.2 Agro processing industrial wastes treatment/utilization
The utilization and treatment of product specific waste is not easy due to its
inadequate biological stability, potentially pathogenic nature, high water content, potential for
rapid autoxidation as well as its high level of enzymatic activity. The two general methods of
traditional waste utilization have been to use the waste as either animal feed (e.g., spent
grains, distillers wash) or fertilizer (filtration sludge, carbonation sludge). Disposal of this
waste can be difficult for the following reasons (Russ and Meyer-Pittroff, 2004):
- Biological stability

482

- High water content
- Rapid autoxidation
- Changes due to enzymatic activity
There are three general methods of waste disposal which not associated with
agricultural practices are incineration, anaerobic fermentation and composting, but very little
of value (e.g., energy, fertilizer) can be recovered using these processes, it is often
necessary to pay for these kinds of disposal.
44.2.1 Solid State Fermentation
Solid state fermentation consists of the microbial growth and product formation on
solid particles in almost absence of water. However, the substrate may contain sufficient
moisture to allow the microorganism growth and metabolism. This bioprocess has been subject
of several studies and it has been proved that solid state fermentation has the important
advantage of leading to higher yields and productivities or better product characteristics than
submerged fermentation, which is characterized by the cultivation of the microorganisms in a
liquid medium. Another great advantage is the lower capital and operating costs due to the
utilization of low cost agricultural and agro-industrial wastes as substrates (Nigam, 2009).
44.2.2 Ethanol production
Bioethanol production by agro-industrial wastes have been considered as an excellent
alternative for reusing these wastes with additional technological and economic advantages,
since this process is of easy operation and save energy. The waste can be subjected to solid
state fermentation for the production of ethanol for several uses. It can be used as a liquid fuel
supplement and as a solvent in many industries. Ethanol production by SSF using grape and
sugar beet pomaces (Rodriguez et al., 2010), and apple pomace (Joshi and Devrajan, 2008)
as solid substrates, has been recently evaluated.
44.2.3 Biogas Production
The constituents of the Biogas are methane (CH
4
), carbon dioxide (CO
2
) making up
approximately 90%. Other impurities such as nitrogen, hydrogen sulphide and oxygen
complete the unrefined fuel source (Zinoviev et al., 2007). Biomass consisting of agricultural
crop residues, solid as well as liquid wastes from industries and sludge can also be utilized
for production of biogas through microbial technology.
Biogas is produced by anaerobic digestion of agricultural and horticultural produce
wastes. Methanotropic bacteria can utilize CO
2
from waste materials results production of

483

methane. The process follows the complex polymers are first hydrolyzed into simple
substances by acid forming bacteria and finally these are digested anaerobically by
methanotropic bacteria and methane gas is liberated. Thus, the waste from agro processing
wastes in real sense is not a waste as everything can be potentially recycled and utililzed in
other form as food, feed or fodder. Thus, proper waste utilization will add to the wealth of the
nation and will benefit all involved in the process.
44.2.4 Byproducts Utilization
The new methods of byproduct utilization focus on certain contents of the food and
agricultural waste. The content of fibrous material (soluble and insoluble) in food is gaining
importance in human nutrition. To produce building materials, fibrous materials from spent
grains can be used as well, as filler and structural material in fiber board. The fibers of the
used up grains of brewery industry waste increase the strength of bricks before they are
kilned, which increases the bricks ability to thermal insulator as well. Pectin, a soluble
fiber, can be extracted from apples, citrus fruit, and beet waste through another extraction
process. Pomace can be used as fertilizer after it has been subjected to a special fermentation
process. Single cell proteins can be produced from dried and pectin extracted apple pomace
by using Aspergillus niger and Trichoderma viride. The grape apple pulp wastes have also
been employed as a substrate for Aspergillus niger to generate crude protein and cellulose.
The waste obtained from processing of fruits and vegetables is rich in fibre with poor quality
of protein. Fermented potato waste has been successfully used as animal feed purpose. Apple
pomace after fermentation followed by drying makes the produce enriched with proteins,
vitamins amd minerals which can be used for animal feed.
Waste from wineries, breweries and distilleries can be used for feeding livestock.
Natural colors of blue grapes skin, kokum (Garcinia indica), Phalsa (Grewia subin acqualis),
and Jamun (Syzygium cumini) have been thoroughly investigated for their nature,
concentration, extractability, stability and suitability as food colors. Fat is partially removed
from slaughterhouse waste, and is then used as a basis for many products in the chemical and
cosmetic industries.
44.3 Waste water in agriculture and food processing
Wastewater is the most serious environmental problem in the manufacturing and
processing of foods. Whenever and wherever food, in any form, is handled, processed, packed
and stored, there will always be an unavoidable generation of wastewater. Most of the volume

484

of wastewater comes from cleaning operations at almost every stage of food processing and
transportation operations. Wastewater from food processing operations is defined by the food
itself. There are common pollutants present in the majority of food and agricultural
wastewater and effluents from each stage of the typical waste water treatment processes, they
are free and emulsified oil/grease, suspended solids, organic/inorganic colloids, acidity or
alkalinity, and sludges. The effluents from fruit and vegetable processing operations consist of
mainly carbohydrates, sugars, pectins, vitamins, and other components of the cell walls
components that have been severed during processing.
44.3.1 Waste water Treatment
Different sources contribute to the generation of wastewater in agro processing
industries, including fruits and vegetable processing, cereal processing, dairy products, meat
processing, seafood and fish processing, sugar processing and alcoholic/non-alcoholic
beverages. Wastewaters released from these industries are turbid, with high concentrations
of bio-chemical oxygen demand, Fats, oils/grease and suspended solids. The main parameters
for physicochemical and biological treatment of wastewater are pH, solids content (suspended
solids and dissolved solids), temperature, odor, biochemical oxygen demand and total organic
carbon.
Usually it is desirable to group wastewater as high, medium and low concentration.
High concentration wastewater sometimes may be concentrated further, treated and
recycled/disposed as solid wastes. Medium concentration wastewater may be treated on site.
Low concentration wastewater such may be discharged without any treatment. Activated
sludge processes are generally employed in food processing industries. Sometimes various
advanced treatment systems are used such as coagulation and filtration.
44.3.2 Waste management
Waste management is a collective activity involving segregation, collection,
transportation, re-processing, recycling and disposal of various types of wastes. Sustainable
waste management involves managing waste in an environmental, socially satisfactory and a
technological economic viable manner. Waste management differs for different types of
wastes and for wastes in different geographical locations such as urban, rural and hilly areas.
While the management of non-hazardous domestic waste is the joint responsibility of the
government, industrial and hazardous waste is the responsibility of the waste generators i.e.
commercial and industries establishments.

485

44.4 Importance of waste management
Waste management allows to save money on commodities, labor, energy and disposal
costs. Waste leads to considerable carbon emission in the environment. In the case of food
industry waste, farm inputs, storage and transportation each require additional input costs and
landfill disposal leads to production of methane gas, by decreasing this, can reduce various
environmental hazard.
44.5 Challenges in Waste management
1. Segregation at source
2. Quality of waste (High moisture content, low calorific value)
3. Poor quality of landfill
4. Lack of information on substrate specific biocatalyst
5. High lag period of biomethanation process,
6. Process parameters control
Sustainable waste management can be achieved through strategic planning,
institutional building capacity, fiscal encouragement, technological and economically viable
techniques, public private partnerships programmes, community participation and many
others. There is a need for efforts to manage and safely dispose of wastes, in order to get rid
of these environmental hazards. Moreover, waste management is a significant environmental
justice issue.
44.6 Conclusion
A clear, concise and consistent policy is necessary to establish and set up waste
management systems and backed by legislations for all kinds of violations. Implementation
and monitoring of the different waste management rules should also be identified at a regular
interval, to study the effects of inappropriate disposal of waste on the environment. This is
essential for guiding the management of waste in a manner that is environmentally
responsible and which minimizes danger to public health. Thus, it is recommended that
phrase Reduce, Reuse, Recycle should be used for waste management.

References
1. Joshi, V.K. and Devrajan, A. (2008). Ethanol recovery from solid state fermented apple
pomace and evaluation of physico-chemical characteristics of the residue. Natural
Product Radiance. 7: 127-132.

486

2. Litcheld, J. H. (1987). Food Biotechnology. 1(1): 29.
3. Nigam, P. S. (2009). Production of bioactive secondary metabolites, In: Biotechnology
for Agro- Industrial Residues Utilization. Nigam, P.S., and Pandey, A. (Eds.).
Springer, Netherlands. pp. 129-145..
4. Rodriguez, L. A., Toro, M. E., Vazquez, F., Correa-Daneri, M. L., Gouiric, S. C. and
Vallejo, M. D. (2010). Bioethanol production from grape and sugar beet pomaces by
solid-state fermentation. International Journal of Hydrogen Energy. 35: 5914-5917.
5. Russ, W. and Meyer-Pittroff, R. (2004). Utilizing waste products from the food
production and processing industries. Crit. Rev. Food Sci. 44(1): 5762.
6. Waldron, K. W. (2004). Plant residues. In: Waldron K. W., Faulds C. B. and Smith A.
C. (eds.). Total Food 2004 Exploiting Co-Products, Minimising Waste. Proceedings
volume, Institute of Food Research. pp. 117131.
7. Zinoviev, S., Arumugam, S. and Miertus, S. (2007). Biofuel Production Technologies.
Dubrovik, Croatia.


487

CHAPTER 45
DEVELOPMENT OF NANO BASED THERMIC FLUID:
RHEOLOGICAL ASPECTS OF NEW ENERGY SYSTEM
Vijay Juwar, Shriram Sonawane

Abstract
Nanofluids are emerging as highly efficient thermic fluids due to its enhanced thermal
conductivity, to used nanofluids as heating or cooling media on industrial level it becomes
mandatory to study the flow related aspects of nanofluids. The viscosity of nanofluid plays
decisive role in economic viability nanofluids as thermal fluid. Variation of viscosity of
nanofluid with temperature and concentration of nanoparicle directly affects pressure drop
which in turn affects pumping cost in heat exchange equipments. Present study deals with
measurement of viscosity nanofluid, prepared by dispersing Fe
3
O
4
nanoparticles in ethylene
glycol. Viscosity is measured both as function of volume concentration and temperature. Pure
base fluid displays Newtonian behaviour in the experimental temperature range. After
addition of nanoparticles nanofluid retains Newtonian behaviour. Our study shows that
viscosity of nanofluid increases with increase in volume concentration and decreases with
increase in temperature
Key Words: Nanofluid, Thermic fluid Nanofluid viscosity
45.1 Introduction
Nanotechnology is emerging field, to which entire world is looking forward as a
solution to many issues. Amongst those, energy and environmental issues are occupying the
topmost attention. Fossil fuels are depleting and creating environmental problems like air
pollution, green house effect etc. so need of the hour is to design the system which can
efficiently utilize the conventional fuel with least environmental issues like air pollution or
search of alternative which replace the existing fuel which can be economic and
environmental friendly.
Nanofluid is one such potential alternative which can be used to reduce the
dependence on the conventional fuel and also reduce load of pollution. Nanofluid is colloidal


488

suspension of nanoparticles in base fluid. Nanofluid shows high thermal conductivities which
attracted great attention of many researchers across the globe. Due to high thermal
conductivity there are numerous potential application in microelectronics, energy supply,
transportation. Nanofluid is proposed as next generation heat transfer fluid .At constant
Nusselt number convective heat transfer coefficient is directly proportional to thermal
conductivity. In convective heat transfer process flow properties of heat transfer media such
as viscosity plays important role. Till the date very few research works are done on the
viscosity of nanofluids. Al
2
O
3
nanoparticles dispersed in propylene glycol were studied by
Prasher et al (2006). Viscosity of copper oxide nanoparticle in engine oil was studied by
Koley and Dey (2010). Viscosity of Fe
3
O
4
nanoparticles was investigated by Sundar et al
(2013). The behavior of nanofluid viscosity with changing temperature is one of the important
aspects of studies in nanofluid. Aldag et al (2012) studied effect temperature on Al
2
O
3
water
and CNT water nanofluids. Namburu et al (2007) and Koley and Dey (2010) studied effect
temperature on viscosity of copper oxide nanoparticle dispersed in ethylene glycol water
mixture and car engine coolant respectively. Duangthongsuk et al (2009) studied TiO
2
and
water nanofluid. Lee et al (2011) investigated SiC nanofluid.
Ferrous nanofluids possess distinct importance due its magnetic property. Magnetic
property of nanoparticle can particularly important for the recovery of nanoparticles from
nanofluid. Thermal conductivity acquires direct importance in convective heat transfer
process. Viscosity and its temperature dependence plays vital role in design of heat exchange
equipments. Change of properties such as shear stress, shear strain with change concentration
of nanoparticle and temperature of nanofluid are important in understanding of ferrous
nanofluid as heat transfer fluid.
In present work the effect of changing concentration and temperature on shear rate and
viscosity of Fe
3
O
4
ethylene glycol are studied.
45.2 Materials and Method
In our experiment we used Fe
3
O
4
nanoparticles with average size of 16nm with
density of 5.17gm/cc. Nanofluids with different volume concentration of 1%, 2%, 3% & 4%
were dispersed in ethylene glycol. Ethylene glycol of Fisher Scientific of SQ grade was used
in experiment. While making samples high precision mass balance of Shimadzu was used for
weighing nanoparticles. To ensure uniform dispersion of nanoparticles in the mixture ultra
sonication treatment was given to the samples for 30 minutes. A ChromTech sonicator
(Taiwan) with 40 kHz and 1200W was used for ultra sonication treatment. Measurement of

489

viscosity was done with the help of AR G2 Rheometer. Plate cone geometry with diameter 40
mm and A 1
0
was used. A gap of 32pm was maintained when geometry acts as parallel
plate.It has facility to raise temperature with help of peltier plate that offers temperature range
of -4u
0
C to 2uu
0
C with typical heating rates up to Su
0
C/min and accuracy of u.1
0
C. They
incorporate four Peltier heating elements to cover an 80mm diameter plate surface. These
Peltier elements are placed directly in contact with thin copper disc with an externally rugged,
hardened chrome surface. A platinum resistance thermocouple, PRT is placed at exact centre
ensuring accurate temperature measurement and control. The unique design provides for
rapid, precise and uniform temperature control over entire 80mm diameter surface allowing
standard geometries up to 60mm in diameter.The viscosity of nanofluid was measured with
increasing shear rate in the range of 1-500 s
-1
. Each sample of different volume concentration
was taken for measurement of viscosity in temperature range 10
0
-50
0
C.
45.2.1 Modeling of Viscosity
After experimentation it becomes necessary to correlate results with the effective
viscosity of nanofluid. In theoretical point of view to understand the properties of nanofuid is
another challenge. Since nanofluid is two phase fluid, must have common features of solid
liquid mixture. Application of relation of solid liquid mixture to nanofluid is still doubtful to
predict the properties of nanofluid.
Some of the widely used models are mentioned below:
Einsteins model is used for relatively low volume fraction ( 0.02) which is given as
p
n]
= p
b]
(1 +2.S)
Where, p
b]
is base fluid viscosity.
Brinkman et al (1952) extended Eiensteins model for the use of moderate volume fraction as

n]
b]
=(1 )
-2.5

Batchelor (1977) considered Brownian motion of particles on the bulk stress of an isotropic
suspension of spherical particles and the expression for viscosity is

n]
b]
=1+2.5+6.5
2

Maiga et al (2004) proposed another model

490

n]
b]
= 306
2
-0.19 +1
45.2.2 Temperature dependence of viscosity
Generally it is observed that fluids are having low viscosity near to their boiling point
and high viscosity near freezing point. To study the effect of temperature on viscosity of
nanofluid various models are proposed. Some widely used are given in a table1
Table 1 Models for temperature dependence of viscosity.
Authors Equations
White(2004)
ln
n]
b]
= a + b(
10
1
) + c(
10
1
)
Reid et al (1987) p
n]
=Aexp(BT)
Yaws (1977) Log(p
n]
) =A + BI
-1
+ CT +DI
2

Kulkarni et al (2006) lnp
n]
= AI
-1
- B
Namburu et al (2007)
Log(p
n]
) = Ac
-B1

The viscosity of nanofluid as function of shear rate is shown in figure 1. It may be
seen that that viscosity of nanofluid remains independent of shear rate as the shear rate is
increased; at lower shear rate viscosity sharply declines and for further increase in shear rate
remains constant under experimental temperature range of 10
o
C to 50
o
C.

This indicates the
Newtonian behavior of nanofluid in experimental temperature and shear rate range of 01/s to
500 l/s.

Figure 2 shows relationship between shear rate and shear stress of nanofluid, a linear
relationship is observed with zero intercept in experimental range, indicates Newtonian
behavior of nanofluid.
In figure 3 the effect of concentration of nanoparticles and temperature on the
viscosity of nanofluid is shown. The viscosity of nanofluid increases with increase in
concentration of nanoparticle and decreases with increase in temperature of nanofluid.


491

A

B

C

D
Figure 1 Nanofluid viscosity as a function of shear rate: (A) 1%; (B) 2%; (C) 3% (D) 4%
0
0.01
0.02
0.03
0.04
0.05
0 100 200 300 400 500 600
N
a
n
o
f
l
u
i
d

/
i
s
c
o
s
i
t
'

(
0
a
.
s
)
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283 , 293 , 303 , 313 , 323
0
0.01
0.02
0.03
0.04
0.05
0.06
0.1 100.1 200.1 300.1 400.1 500.1
N
a
n
o
f
l
u
i
d

/
i
s
c
o
s
i
t
'
%

(
0
a
.
s
)
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0
0.05
0.1
0.15
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0.25
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a
n
o
f
l
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i
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s
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s
i
t
'

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a
.
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)
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5.00E'02
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4.50E'01
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a
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i
t
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a
.
s
)
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283 , 293 , 303 , 313 , 323 ,

492

A

B

C

Figure 2 Relation between shear stress and shear strain : (A) 1% (B) 2% (C) 4%

0
2
4
6
8
10
12
14
0 200 400 600
S
1
"
$
R

S
T
R
"
S
S

(
0
a
.
)
S1"$R R$T" (1(S)
283 , 293 , 303 , 313 , 323 ,
0
2
4
6
8
10
12
14
16
0 100 200 300 400 500 600
S
h
e
a
r

S
t
r
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.
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)
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293 , 293 , 303 , 313 , 323 ,
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1.20E)01
0 100 200 300 400 500 600
S
h
e
a
r

S
t
r
e
s
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493

Figure 3 Effect of concentration and temperature on nanofluid viscosity
Figure 4 discuss model studies of Brinkman, (1952), Batchelor (1977) and Maiga
(2004). All of these models consider the effect of nanoparticle concentration on viscosity of
nanofluids. At lower temperature all models over predict the viscosity of nanofluid and higher
temperature all models under predicts the viscosity of nanofluid.
In figure 5 temperature dependence of nanofluid viscosity is shown. Models shown in
table 1 are proposed for prediction of nanofluid viscosity at different temperatures. Out of all
mentioned models only Namburus model fits well to the experimental data.
Log(p
n]
) = Ac
-B1
where A and B are constants, T is temperature in Kelvin and p
n]

is nanofluid viscosity in centipoises. Values of curve fit constants A and B are shown in table
2.

0
0.005
0.01
0.015
0.02
0.025
0.03
280 290 300 310 320 330
N
a
n
o
f
l
u
i
d

&
i
s
c
o
s
i
t
'

(
p
a
.
s
)
Temperature ())
4-
3-
2-
1-

494

A

B

C

D

E

Figure 4 Study of Models of viscosity at constant temperature: (A) 283 K (B) 293K (C)
303K (D)313K (E) 323K
0
0.5
1
1.5
0 0.002 0.004 0.006
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a
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&
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495

Table 2 Curve fit values
NP volume conc. (v/v)
2% 3% 4%
A 54.49996 54.49996 56.77083
B 0.013077 0.013372 0.012931
A

B

C

Figure 5 Effect of temperature on nanofluid viscosity by Naburus model: (A) 2%(B) 3% (C)
4%
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0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
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1
1.2
1.4
1.6
280 300 320 340
#
$
%
(
&
#
$
%
(
&
#
$
%
(
&
#
$
%
(
&
' '' '
) )) )
Temperature ())
E(.r/me012$
62m78r89s
:/0e2r (62m78r89s)

496

45.4 Conclusion
F
3
O
4
Nanoparicles in ethylene glycol shows Newtonian behavior in temperature range
of 10
o
C to 50
o
C and in volume concentration of 1% to 4%. The shear rate was increased from
1s
-1
to 500s
-1
. The viscosity of nanofluid is increased when volume concentration of
nanoparticle is increased. At temperature of 50
o
C approximately 63.1% increase in base fluid
viscosity is observed at 4% volume concentration of nanoparticle. As the temperature
increases, the viscosity of nano-fluid decreases exponentially.

References
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C. To prevent the sedimentation of fuel

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