BioCulturing Using Cowdung PHD Thesis India PDF

BIOREMEDIATION OF SEWAGE WATER USING
MICROORGANISMS
THESIS SUBMITTED TO
MANONMANIAM SUNDARANAR UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
MICROBIOLOGY- ZOOLOGY (INTERDISCIPLINARY)
By
V.JEYANTHI KUMARI
Regn.No. 2417
Under the guidance of

DR.B.VICTOR
MANONMANIAM SUNDARANAR UNIVERSITY

Abishekapatti, Tirunelveli-627 012,
Tamil Nadu, India.
JUNE 2011
2
Dr.B.VICTOR.,
Guide Reader,
St.Xavier’s College,
Palayamkottai-627 002,
Tamil Nadu, South India.
CERTIFICATE
Certified that the thesis entitled “BIOREMEDIATION
OF SEWAGE WATER USING MICROORGANISMS” submitted by
Mrs.V.JEYANTHI KUMARI (Reg No.2417) for the award of Degree of Doctor of
Philosophy in Microbiology – Zoology (Interdisciplinary) of Manonmaniam Sundara-
nar University is a record of bonafide research work done by her and it has not been
submitted for the award of any degree, diploma, associate ship and fellowship of any
University/Institution.
Place:
Date: Signature of the Guide
3
V.JEYANTHI KUMARI, M.Sc., M.Phil.,
Research Scholar (Reg No. 2417), St.Xavier’s College,
Palayamkottai-627 002,
Tamil Nadu, South India.
DECLARATION
I hereby declare that the thesis entitled “BIOREMEDIATION
OF SEWAGE WATER USING MICROORGANISMS” submitted by me for the
Degree of Doctor of Philosophy in Microbiology-Zoology (Interdisciplinary) is the
result of my original and independent research work carried out under the guidance
and supervision of Dr.B.VICTOR, M.Sc., Ph.D., Reader, Centre for Aquaculture
Research and Extension (CARE), St.Xavier’s College, Palayamkottai. This work has
not been submitted for the award of any degree, diploma, associate ship and fellow-
ship of any University or Institution.
Place:
Date: Signature
(V.JEYANTHI KUMARI)
4
ACKNOWLEDGEMENT
I am indebted to my guide Dr.B.Victor, M.Sc., Ph.D., Reader, St.Xavier’s
College, Palayamkottai for suggesting the problem, the gracious guidance and critical-
ly going through the several version of this dissertation. His unfailing encouragement
and technical advice were inspired me for the completion of this research work. I owe
a deep sense of gratitude to him in ways more than one.
I must express my indebtedness and deep sense of gratitude to The Manage-
ment, Dr.S.Chockalingam, M.Sc.,Ph.D.,F.A.Z.,F.R.E.S.(London), Director and
Dr.R.Gopal, M.Sc., M.Phil., Ph.D., Principal, K.R.College of Arts and Science. Ko-
vilpatti, for their constant encouragement and their word of appreciation.
I extended my hearty thanks to Dr.B.Geetha, M.Sc., Ph.D., Professor of
P.G.Department of Zoology and Research centre, Mr.Deva Manoharan M.Sc.,
M.Phil, Professor of P.G.Department of Mathematics and Dr.S.Shanmugavel,
M.Sc., Ph.D., Reader, P.G.Department of Zoology, V.O.Chidambaram College, Tuti-
corin for their timely help and I will ever be grateful to them for their pain taking ef-
fort and suggestion for the successful completion of my work.
I have great pleasure to record my thanks to Mr.M.Dharmaraj, M.Sc.,
M.Phil Senior Technician, ICAR Lab, Sri Parasakthi College for Women, Courtal-
lam, for his encouragement during the tenure of my research.
5
My sincere thanks are also extended to Dr.M.Manohar M.Sc., Ph.D., for his
kind encouragement which enabled me in finishing this work.
I sincerely acknowledge the timely help and assistance I have had from
Er.Puthia Sekaran, Associate professor, National Engineering College, Kovilpatti
for his valuable help in maintaining biogas plants in STP campus.
I have great pleasure in expressing my deep sense of gratitude to my spouse,
son, family members, students and friends for their enthusiastic encouragement and
kind blessings from almighty for completing this research work successfully.
V.JEYANTHI KUMARI
6
CONTENTS
TITLE PAGE
Preface 1
1. Chapter I 5
1.0 Review of literature 6
2.Chapter II 22
2.0 Production of biogas using sewage and animal dung
2.0.1 Introduction 23
2.0.2 Digesters 25
2.0.3 Substrates 25
2.0.4 Organisms 27
2.0.5 Conditions 27
2.0.6 Applications 28
2.1 Materials and Methods 30

2.2 Results 37
2.3 Discussion 54
3. Chapter III 65
3.0 Biodigested slurry as biofertilizer on chilly plant-A Field trial


3.2 Results 76
3.3 Discussion 86
4. Chapter IV 93
4.0 Biodigested slurry as biological carrier for Rhizobium phaseolus.

4.0.2 Biofertilizers
4.0.3 Microflora 95
4.0.4 Carriers 96
4.0.5 Rhizobium 97

4.2 Results 106
4.3 Discussion 110
7
5. Chapter V 116
5.0 Isolation and characterization of microflora from STP


5.2 Results 127
5.3 Discussion 131
6. Chapter VI 134
6.0 Cellulose biodegradation

6.0.2 Cellulase enzyme 137
6.0.3 Cellulolytic Microbes 138

6.2 Results 145
6.3 Discussion 161
7. General summary and conclusions 167
8. Suggestions for future research work 172
9. Bibiliography 174
Paper publications and presentations
List of Appendix.
8
LIST OF TABLES
TABLE TITLE PAGE

NO
1.01 Microbial diversity in anaerobic digesters. 10
1.02 Organisms associated with phosphate solubilization 13
2.01 Characteristics of sewage water. 37
Biogas production by gradual reduction of cow dung supple-

mented with sewage water.
2.02 Total Solids (TS), Total Volatile Solids (TVS) and Volatile Fatty 38
Acid (VFA) in control bioreactor.
2.03 Total Solids (TS), Total Volatile Solids (TVS) and Volatile Fatty 38
Acid (VFA) in experimental bioreactor.
2.04 Analysis of Variance for the production of biogas between 39

control and experimental bioreactor.
2.05 Student’s ‘t’ test analysis of TS,TVS & VFA for biogas production 39
Temperature and pH in control bioreactor.

2.06 41
Temperature and pH in experimental bioreactor.
2.07 41
Biogas production by gradual replacement of cow dung with
poultry droppings supplemented with sewage water.
Total Solids (TS), Total Volatile Solids (TVS) and Volatile

2.08 Fatty Acid (VFA) in control bioreactor. 47
Analysis of Variance for the production of biogas between

2.09 control and experimental bioreactor. 48
Student’s ‘t’ test analysis of TS,TVS & VFA for biogas

2.10 prduction 48
Temperature and pH in control bioreactor.

2.11 49
Temperature and pH in experimental bioreactor.
2.12 49
THBP, THFP and TPSMP in the field soil (cfu/ml).
3.01 75
Influence of biodigested manurial sources on the morphologic
3.02 cal parameters and chlorophyll content of chilly grown at preflowe
ring stage. 79
9
3.03 Influence of biodigested manurial sources on the morphologi 80
cal parameters and chlorophyll content of chilly grown at
flowering stage.
3.04 Influence of biodigested manurial sources on the morpholocal 80

parameters and chlorophyll content of chilly grown at final stage.
3.05 Least Significance Difference (LSD) in Total height of chilly 81

plant at various stages.
3.06 Least Significance Difference (LSD) in wet weight of chilly 82

3.07 Least Significance Difference (LSD) in dry weight of chilly 83

3.08 Effect of biodigested manurial sources on NPK content of 84

chilly plant at seedling stage.
3.09 Effect of biodigested manurial sources on NPK content of 84

chilly grown at different stages of growth.
4.01 Biochemical characteristics of Rhizobium phaseolus. 106
4.02 The survival of rhizobial cultures in biodigested organic manure 108

carriers at room Temperature (28±20C).
5.01 Microbiological, biochemical and physiological analysis of 129

selected isolates from sewage treatment plant.
6.01 Microbial sources for cellulose degradation. 138
6.02 Substrate utilization by Psedomonas aeruginosa, Bacillus 146

lcheniformis and mixed cultures of both (IU/ml).
6.03 Endo and exo glucanase enzyme assay of mixed cultures (IU/ml). 150
6.04 Endo and exo glucanase enzyme assay of P.aeruginosa (IU/ml). 151
6.05 Endo and exo glucanase enzyme assay of B.licheniformis (IU/ml). 152
6.06 Friedman Two-way analysis of variance Rank test at 5% level of 153

significance in Pseudomonas aeruginosa, B.licheniformis and
mixed cultures of both.
6.07 Rank Test Analysis of cultures used in cellulose biodegradation. 154
10
LIST OF FIGURES
FIGURE TITLE PAGE

NO
Biogas production by gradual reduction of cow dung

supplemented with sewage water.
2.01 Estimation of C, N, P and K in control bioreactor. 42
2.02 Estimation of C, N, P and K in experimental bioreactor. 43
2.03 Biogas production in experimental bioreactor. 44
2.04 Biogas production in control bioreactor. 44
2.05 Total Solids (TS) in experimental bioreactor. 45
2.06 Total Volatile Solids (TVS) in experimental bioreactor. 46
2.07 Volatile Fatty Acid (VFA) in experimental bioreactor. 47
Biogas production by gradual replacement of cow dung

with poultry droppings supplemented with sewage water.
2.08 Estimation of C, N, P and K in control bioreactor. 50
2.09 Estimation of C, N, P and K in experimental bioreactor. 51
2.10 Biogas production in control bioreactor. 52
2.11 Biogas production in experimental bioreactor. 52
3.01 THBP at various stages on biodigested manurial sources 76

(cfu/ml).
3.02 THFP at various stages on biodigested manurial sources 77

(cfu/ml).
3.03 TPSMP at various stages on biodigested manurial sources 77

(cfu/ml).
3.04 Soil NPK content. 78
3.05 Effcet of biodigested manurial sources on the yield of chilly 85

(Tones/hectare).
4.01 The survival of Rhizobium phaseolus in biodigested organic 109

manure carriers at refrigerated temperature (6±20C).
11
5.01 Total Heterotrophic Bacterial Population (THBP) in STP. 127
5.02 Total Heterotrophic Fungal Population (THFP) in STP. 128
6.01 Combined glucanase enzyme assay of P.aeruginosa,

B.licheniformis and mixed cultures of both on 147
CellulosePowder(CP)
6.02 Combined glucanase enzyme assay of P.aeruginosa,

B.licheniformis and mixed cultures of both on Carboxy 148
Methyl Cellulose(CMC).
6.03 Combined glucanase enzyme assay of P.aeruginosa, 148

B.licheniformis and mixed cultures of both on Crude Filter
paper (CF).
6.04 Combined glucanase enzyme assay of P.aeruginosa, 149

B.licheniformis and mixed cultures of both on COIR (CR).
6.05 Biomass estimation of P.aeruginosa, B.licheniformis and 156

mixed culture of both on Cellulose Powder (CP).

mixed cultures of both on Carboxy Methyl Cellulose(CMC).

mixed cultures of both on Crude Filterpaper (CF).

mixed cultures of both on COIR (CR).
6.09 Protein assay of P.aeruginosa, B.licheniformis and mixed 159

cultures of both on Cellulose Powder (CP).

cultures of both on Carboxy Methyl Cellulose(CMC).

cultures of both on Crude Filterpaper (CF).

cultures of both on COIR (CR).
12
LIST OF PLATES
PLATE TITLE
NO
2.01 Sewage effluent Treatment Plant (STP).
2.02 Sewage water of STP.
2.03 Loading of bioreactors.
2.04 Bioreactors used in biogas production.
2.05 Measurement of biogas by water replacement method.
2.06 Analysis of biogas production by gradual reduction of cow dung

with poultry droppings and sewage water.
3.01 Preparation of Hydroxy Apatite Medium (HAM).
3.02 Isolation of PSO from the rhizosphere region of paddy plant.
3.03 Mass cultivation of PSO.
3.04 Seedling stage of chilly plants.
3.05 The effect of SWPS+PSO+RP on flowering stage.
3.06 The effect of SWPS+PSO+RP on yielding stage.
4.01 Isolation of R.phaseolus on CRYEMA plate.
4.02 Mass cultivation of R.phaseolus.
4.03 Biodigested slurries as carriers (SWPS, SWCS and Mixed form).
4.04 Rhizobium inoculated carrier materials.
4.05 Rhizobial count at 40th day when mixed form of slurries used as
carriers at room temperature.
4.06 Rhizobial count at 80th day at refrigerated temperature when

mixed form used as carriers.
4.07 Voges-Proskaur Test.
4.08 Simmons citrate Agar Test.
5.01 Total Heterotrophic Fungal Population.
13
5.02 Total Heterotrophic Bacterial Population.
5.03 Lactobacillus sp in MRS Agar M641.
5.04 Bacillus licheniformis in Bacillus medium M1383.
5.05 Salmonella typhi in S.S Agar M108.
5.06 Shigella sp in S.S Agar M108.
5.07(a) Escherichia coli in S.S Agar M108.
5.07(b) E.coli in EMB Agar M317.
5.08 Enterobacter aerogens in EMB Agar M317.
5.09 Vibrio parahemolyticus in TCBS M189 Agar.
5.10 Pseudomonas aeruginosa in Pseudomonas isolation Agar M406.
5.11 Staphylococcus aereus in Phenolphthalein phosphate agar M652.
5.12 Vibrio cholera in TCBS Agar M189.
5.13 Streptococcus sp in KF Streptococcal Agar M248.
6.01 Halo zone formations by Bacillus licheniformis while pouring

0.1% congored.
6.02 Halo zone formations by Pseudomonas aeruginosa while pour-

ing 0.1% congored.
6.03 Clear zone formation by B.licheniformis while acidified with 5%

H2SO4.
6.04 Clear zone formation by P. aeruginosa while acidified with 5%

H2SO4.
6.05 Various substrates utilization.
6.06 Estimation of cellulase (combined, endo and exo β 1, 4 gluca-

nase.
6.07 Microscopic view of P.aeruginosa.
6.08 Microscopic view of B.licheniformis.
14
LIST OF ABBREVIATIONS
Abbreviations Expansion
AIRCS - Australian Inoculants Research and Control Services

AP - Aluminium Phosphate
BOD - Biological Oxygen Demand
BSA - Bovine Serum Albumin
BNF - Biological Nitrogen Fixation
CFU - Colony Forming Unit
CRYEMA - Congo Red Yeast Extract Mannitol salt Agar
COD - Chemical Oxygen Demand
CMC - Carboxy Methyl Cellulose
CP - Cellulose Powder
CF - Crude Filter Paper
CR - Coir
CPPM - Cellulose Powder Peptone agar Medium
DNS - Di Nitro Salicylic acid
DAP - Di Ammonium Phosphate
DS - Dry Solids
EDTA - Ethylene Diamine Tetrachloro Acetic acid
EMB - Eosine Methylene Blue
FAS - Ferrous Ammonium Sulphate
FP - Ferrous Phosphate
FYM - Farm Yard Manure
HASL - Hybrid Anaerobic Solid Liquid
HAM - Hydroxy Apatite Medium
HRT - Hydraulic Retention Time
IAA - Indole Acetic Acid
IMViC - Indole Methyl red Voges-Proskuer Citrate Test
IU - International Unit
IPNS - Integrated Plant Nutrient System
KVIC - Kadhi Village Industries Commission
LFA - Lignite Fly Ash
L or l - Litre
µg - micro gram
µl - micro litre
Mm - Milli molar
mg - milligramp
ml - milli litre
MR-VP - Methyl Red-Vogs Proskuer medium
NH - New Hostel
N - Nitrogen
NEC - National Engineering College
NA - Nutrient Agar
OWCS - Ordinary Water treated biodigested cow dung slurry
P - Phosohorus
PGA - Peptone –Glucose Agar plates
15
PSM - Phosphate Solubilizing Microorganisms
PSO - Phosphate Solubilizing Organisms
RP - Rock Phosphate
RT - Retention Time
rpm - revolution per minute
RBD - Randomized Block Design
SWPS - Sewage Water treated biodigested Poultry Slurry
SWCS - Sewage Water treated biodigested cow dung Slurry
STP - Sewage effluent Treatment Plant
TS - Total Solids
TVS - Total Volatile Solids
TSS - Total Suspended Solids
TCP - Tri Calcium Phosphate
THBP - Total Heterotrophic Bacterial Population
THFP - Total Heterotrophic Fungal Population
TPSMP - Total Phosphate Solubilizing Microbial Population
TCBS - Thiosulphate Citrate Bile Salt agar
U-DAIS - University and Department of Agricultural Laboratory
Services
VS - Volatile Solids
VFA - Volatile Fatty Acids
VSS - Volatile Suspended Solids
VAM - Vesicular Arbuscular Mycorhizae
YEM - Yeast Extract Mannitol
16
PREFACE
17
V. Jeyanthi Kumari, 2011. Bioremediation of
Sewage water using microorganisms. Ph.D thesis,
Manonmaniam Sundaranar University,Tirunelveli,
Tamil Nadu, India.
Preface
Bioremediation is the term applied to technologies that

accelerate natural processes for degrading or detoxifying harmful chemicals in soil,
ground water and waste water. The use of microorganisms mainly bacteria for biore-
mediation or transform hazardous contaminants is not a new idea because they have
been used since 600 B.C. by the Romans and others to treat their wastewater. In fact,
bioremediation has been used commercially for almost 30 years (Ram Nayar, 2005).
The first commercial use of bioremediation system was initiated in 1972 to clean up a
Sun Oil pipeline spill in Ambler, Pennsylvania (National Research Council, 1993).
Since 1972, bioremediation has become a well-developed way of cleaning up differ-
ent contaminants. A survey prepared by the Environmental Protection Agency in
1992 received information on 240 cases of bioremediation in the United States (Alex-
ander, 1999). Most of these cases involved treating contaminated soil or groundwater.
The unprecedented population increase and anthropo-

genic activities such as industrialization and urbanization of the twentieth centuary in
the name of modernization have not only increased conventional solid and liquid
waste pollutants to critical levels but also produced a wide range of previously un-
known contaminants (Ram Nayar, 2005). Majority of the contaminants entering into
the ecosystems are in the form of chemicals and they affect man, animal life, plant life
and microbes and exert serious health and ecological problems (Bower et al., 1984).
Though many technologies have been available for

clean-up process, only a few of them have been proved to be of routine application
value (Crawford and Crawford, 1998). The choice of the technology is influenced by
the social, political, and geographical conditions. Among the techniques, bioremedia-
tion has evolved as the most promising one because of its economical,
18
safety and environmental features since organic contaminants become actually trans-
formed and some of them are fully mineralized by this technique (Salvel, 2003).
Microbes the oldest inhabitants of the earth that are versatile and adaptive to
the changing environment will be the cost-effective components to combat the present
problems (Stolz, 2001). Microbes and their diverse metabolic enzymes are typically
employed for safe removal of environmental contaminants either through direct de-
struction or indirectly through a transformation of the contaminants to a safer inter-
mediate and it can be self-sustaining and inexpensive (Watanabe et al., 2002).
Bioremediation is the application of biological treatment to cleanup of ha-

zardous chemicals and organic wastes under controlled conditions to an innocuous
state established by regulatory authorities. This process involves detoxification, where
the waste is made less toxic and mineralization and thus the waste materials are con-
verted into inorganic compounds such as carbon dioxide, water and methane (Alexan-
der and Martin, 1994; Mueller et al., 1996). There is a general interest in studying the
diversity of indigenous microorganisms capable of degrading different pollutants be-
cause of their varied effects on the environment (Fliermans and Balkwill, 1989;
Greene et al., 2000).
Organisms belonging to the group Pseudomonas and Cycloclasticus were

most abundant bacterial strains with high biodegradation potential (Watanabe, 2001).
Thiobacilli are extensively used by man for mining and bioremediation purposes.
There are other bacteria such as Ralstonia metallidurans and Deinococcus radiodu-
rans that can tolerate high levels of toxic metals and radioactivity respectively.
Another versatile bacterium Rhodobacter fixes carbon and nitrogen from the air to
make biodegradable plastics. It has also been used to produce fertilizers. It has even
been used to make a yellow pigment that is fed to hens to make their eggs more ap-
pealing to children (Miltonsaier, 2005). The use of genetic engineering to create or-
ganisms specifically designed for bioremediation has great potential (Lovley, 2003).
19
Vidali, (2001) reported the advantages of bioremediation, they are as follows
•Bioremediation is a natural process and is therefore perceived by the public as an ac-

ceptable waste treatment process for contaminated material such as soil. Microbes
able to degrade the contaminants and the residues from the treatment are usually
harmless products and include carbon dioxide, water, and cell biomass.
•Theoretically, bioremediation is useful for the complete destruction of a wide variety

of contaminants. Many compounds that are legally considered to be hazardous can be
transformed to harmless products. This eliminates the chance of future liability asso-
ciated with treatment and disposal of contaminated material.
•Instead of transferring contaminants from one environmental medium to another, for

example, from land to water or air, the complete destruction of target pollutants is
possible.
•Bioremediation can often be carried out on site, without causing a major disruption
of normal activities. This also eliminates the need to transport quantities of waste off
site and the potential threats to human health and the environment that can arise dur-
ing transportation.
•Bioremediation can prove less expensive than other technologies that are used for
cleanup of hazardous waste.
Bioremediation has been used at a number of sites worldwide, including Eu-

rope, with varying degrees of success. Techniques are improving as greater know-
ledge and experience are gained, and there is no doubt that bioremediation has great
potential for dealing with contamination (King et al., 1997).
20
CHAPTER I
21
Tamil Nadu, India.
Chapter 1
1.0 REVIEW OF LITERATURE
1.0.0 Introduction
The production, distribution, use, misuse, disposal or accidental

spills of many chemicals have polluted the environment that threatens the health of
humans, livestock, wildlife and indeed whole ecosystems. However the cost of restor-
ing the contaminated ecosystem to healthy and acceptable level is virtually incalcula-
ble. As a result the government, industry and the public have acutely felt the need for
more cost effective alternatives to traditional physical and chemical methods of re-
mediation of these contaminated sites.The microbial bioremediation program includes
immobilization or to transform the contaminants to useful products no longer hazard-
ous to human health and the environment.
1.0.01 Bioremediation
Research is underway at a number of facilities using exogen-

ous, specialized microbes or genetically engineered microbes to optimize bioremedia-
tion (Hassan et al., 2003). A successful, cost effective, microbial bioremediation pro-
gram is dependent on hydro geologic conditions, the contaminant, microbial ecology
and other spatial and temporal factors that vary widely. Microbiological assays are
carried out to assess microbial growth conditions, degrader population densities and
presences of enzymes capable of destroying contaminants of concern and microsm
studies to evaluate bioremediation potential under controlled conditions (Fredrickson
et al., 1991).
During implementation of microbial bioremediation programs,

performance monitoring plays a key role in evaluating effective treatment. Properly
22
executed, microbial bioremediation can cost-effectively and expeditiously destroy or
immobilize contaminants in a manner that protect human health and the environment
(Heitzer and Sayler, 1993; Gheewala and Annachatre, 1997; Gadd, 2000).
1.0.02 Bioremediation of animal excretes and biogas production
In Asian counties, the agro-industrial residues and sewage water form a main
contribution to the pollution of soil and waterways. Large poultry and pig farms are
often a major source of pollution from agricultural activities with odour being pollu-
tant and were most noticed by the public. At the same time these pollutants constitute
a large potential for production and of bioenergy through anaerobic digestion as well
as potential substrates for other biological fermentation process.
Technologies for treating farm wastes along with sewage polluted environ-
ments have been a major concern over the last couple of decades (Barber, 1978; Cart-
er, 1978; Fisher, 1984; Jungersen et al., 1997). Research on anaerobic degradation of
cellulosic wastes of cattle dung by rumen microorganisms for enhanced production of
biogas and ethanol has shown a clear correlation between the cellulosic content of cat-
tle dung and natural materials and their degradability by rumen microorganisms
(Huub et al., 1988). More biogas production and methane yield is achieved by Rubin-
damayugi et al., (1989; 1992) when acetogenic and methanogenic biomass are immo-
bilized on polyurethane carrier.
More amount of biogas production achieved by two phase process involving

connecting the acidogenic reactor to methanogenic reactor which leads to more effi-
cient conversion of volatile fatty acids into biogas from piggery waste treatment (Ki-
vaisi et al., 1990; 1992; Kivaisi and Mtila, 1998). Several attempts have been made
to update this technology. However more attention for bioremediation of sewage wa-
ter in many ways were seriously paid from the beginning of 1990s (Blackburn and
Hafker, 1993).
1.0.03 Biomass substrates
Kannan et al., (2003) successively produced biogas by using poultry drop-

pings, parthenium and eucalyptus leaves with donkey dung combination. Preetirao
23
and Seenaya, (1994) reported the addition of FeSO4 to cow dung and poultry litter
waste increased methanogenesis. More biogas yield obtained from the mixture of
agrotanin, cattle feces and poultry droppings by El-Hadidi and Al-Turki, (2007). The
comparative analysis of biogas yield from poultry, cattle and piggery wastes at meso-
philic temperature by Itodo et al., (2001) showed that there was significant difference
in gas yield from poultry waste. Kim et al., (2006) elucidated that a major limitation
of anaerobic digestion is long hydraulic retention time. Alvarez, (2007); Padmasiri et
al., (2006) recognized the pretreatment of any waste effectively improve the biogas
production than the performance without pretreatment.
Garcia et al.,(2006) used sewage sludge as substrate whereas Vanderzee et

al.,(2006) done an experiment using water hyacinth and water chesnut grown on toxic
metal from electroplating industry effluent as substrate for biogas production. Con-
naughton et al., (2006) employed brewery waste water for methane production. Shar-
ma et al., (1999) used the mixture of dried water hyacinth, banana stem along with
sugarcane press mud yielding average biogas production. DeSutter and Ham, (2005)
conducted an experiment for biogas production using waste from head swine revealed
that flux of biogas was very seasonal. Mc Carty, (1964); Zeikus, (1977) suggested that
different methanogic bacteria responsible for the production of biogas. Chen and Pea-
son, (1970); Pfeffer, (1974) revealed that the cellulose hydrolysis was the rate limiting
step in the conversion of cellulose into biogas. Converse et al., (1971) stated that
maintenance of oxidation-reduction potential is essential to the successful biogas pro-
duction. Trevelyan, (1975) portrayed that the more complex the substrates took longer
time to be degraded to volatile acids and the higher the production of methane in the
biogas produced.
1.0.04 Environmental conditions
The temperature has a significant influence on methanogenic bacterial activity,

bioremediation and stabilization efficiency (Golueke, 1958; Horton and Hawkes,
1972; Pfeffer, 1974; Park, 1979; Scherrer et al., 2003; Zhang et al., 2006; Lopez et
al., 2006). The effect of temperature is independent of loading rate and retention time.
Munoz and Guieysee,(2006) revealed that the microorganisms and microalgae en-
hance the removal of nutrients, organic contaminants, heavy metal and pathogens
24
from domestic water and furnish an interesting raw material for the production of bio-
gas. Bolzonella et al., (2006) achieved increased biogas production by bioremediating
municipal solid waste together with waste activated sludge. Kumar et al., (2006) used
potato waste, cattle manure with heavy metals as substrates for high methane content
of biogas production.
Fdz-Polanco et al., (2005) stated that methanogenic activity, anaerobic biode-

gradability and toxicity are key parameters in the design and operation of anaerobic
bioreactors. Gomec et al., (2005) indicated that there was no significant inhibition of
biogas production in the presence of salinity. It is obvious that anaerobic microorgan-
isms (especially methanogens) could well adopt to high salinity ratios. McCarty,
(1964) reported that methane production is very well when the pH is maintained be-
tween 6.6 and 7.6. Pathak et al., (1985) indicated that the majority of Indian biogas
plants are built on the tried KVIC design bioreactors. Ozalp et al., (2003) resulted that
high salinity and ammonium nitrogen levels have an impact on biogas production.
Ilyin et al., (2004) had more biogas yield by increasing Lactobacillus sp and Clostri-
dia sp.
The comparative study by Ogino et al., (2005) on Clostridium butyricum and

Enterobacter aerogens revealed that C.butyricum showed more biogas production on
substrates like dextrain, glucose, sucrose, maltose, galactose with yeast extract. Bagge
et al., (2005) reported that the pasteurization reduced the pathogens in biogas plant
slurry. Hae et al.,(2003); Hutnan et al.,(2005) analyzed that low pH, high concentra-
tion of iron and very low concentration of dissolved phosphorus in sewage water were
the main factor limiting the rate of biogas production. Kim et al.,(2003); Gomec et
al.,(2005) portrayed that chemical oxygen demand, dry solids, volatile solids, pH,
biogas yield, biogas composition, volatile fatty acids and reactor volume occupied by
the feed material are the important parameters in the biogas production.
Bolzonella et al., (2006) stated that the anaerobic co-digestion process of

waste activated sludge together with organic municipal solid wastes increasing the
organic loading rate and showed 50% increase in biogas production. Recently Van-
derzee et al, (2006) found that introduction of a limited amount of oxygen to anaerob-
ic bioreactors lower the level of sulfide in the biogas production. Yu and Mu, (2006)
25
have described that acetate, butyrate, capotate and ethanol were the main aqueous
products during biogas production and their concentrations were depend on both the
substrate concentration and HRT.
1.0.05 Microbes in biogas production
Effective bioconversion of organic matter in anaerobic digesters depends on a

diverse microbial population. They are listed below.
Table 1.01 Microbial diversity in anaerobic digesters.
Microorganisms References
Methanobacterium ruminatum, Methanobacterium soe- Mah et al., (1977); Zeikus, (1977); Laube
hugneii, Methanosarcina barkeri, Eubacterium tortuo- and Martin, (1981).
sum.
Clostridium butyric, Enterobacter aerogens Sonakya et al., (2003).
Acetobacterium, Clostridium, Methanospirillum, Me- DeRenzo, (1977); Haug, (1979).
thanococcus, Methanosarcina, Methanothrix.
Desulfovibrio, Desulfotomaculum, Thermoanaero bium- Anon, (1971).
brocki.
Clostridium butyricum, Selenomonas ruminantium, Bac- Malina, (1969); Abubacker and Rao,
teroides ruminicola. (2004).
1.0.06 Biodigested slurry as biofertilizer
Biofertilizers has become a hope for most countries as far as economical and
environmental view points concerned. Especially in developing countries like India it
can solve the problem og high cost of chemical fertilizers and help in saving the
economy of the country (Al Masri, 2001: Masse, et al., 2004). The disposal of biodi-
gested slurry after biogas production is a major concern for the environment (Alga-
wadi and Gaur, 1988; Gaur, 1990; Gained and Gaur, 1991; Hedge et al., 1994; Rupela
et al., 2004; Dinesh kumar et al., 2008). It contains considerable amount of plant nu-
trients and helps to improve crop production, also preventing adverse environmental
impacts of waste disposal (Singal et al., 1991; Vander zee et al., 2006). Warneke
and Siregar, (1994) reported that the application of biodigested poultry manure have
increased the uptake of nutrients in cabbage. Heathman et al., (1995) applied biodi-
26
gested poultry manure to Bermuda grass increased the N, P and K content of grass.
Increased uptake of nutrients due to poultry manure was observed by Hsieh and Hsu,
(1993) in tomato.
The biogas slurry from biogas plant used as nutrient source in agriculture (Van
Velson, 1977; Dinesh Kumar et al., 2008; Ranjana Bhatia et al., 2008; Sri Rama-
Jayam, 2008). Ghosh and Pohland,(1974); Gaur et al.,(1980); Yahiya et al.,(1996);
Gaur and Ostwal,(1980) studied the influence or impact of biogas plant slurry in the
growth, leaf area index, root length density and grain yield in wheat crops compared
to unamended plots. Odeme,(1997);El Kholy and Gomaa ,(2000); Schmidst et
al.,(2000) emphasized that the application of biodigested slurry from biogas plant as
biofertilizer with microbial sources are lighter to transport and requires less labour
for application and is more environment friendly compared to chemical fertilizers.
Indu Paul and Savithri,(2003); Badole et al.,(2004) reported that biodigested

slurry as bioorganic fertilizers improves the physical properties of the soil, increasing
water holding capacity, preventing nutrient leaching and adding more mineral nu-
trients to the poor sandy soil. Kahsnitz, (1992); Gilot, (1997) stated that the organic
slurry manure increased the microbial populations and biologically active metabolites
such as plant growth regulators. Senesi,(1989); Shweta and Kiran Kumar,(2005)
found that there was significant difference with respect to vegetative growth of plant
height, number and size of fruits, yield and shelf life of fruit in applied with organic
biodigested slurry to soil than both the uninoculated control and inorganic fertilizer
inoculated soil. Yu et al.,(2006) with their field experiment of applying biogas residue
on jujube growth showed that it could enhance the disease-resistance of jujube plant
and its fruit production also improve fruit quality and soil fertility than control also
biogas residue increased the contents of jujube fruit coarse fiber, vitamin C, aminoa-
cids, Fe and P.
The incorporation of biodigested organic slurries of biogas plant in soil pro-

motes microbial community and soil enzyme activity (Balasubramaniyan et al., 1972;
Scott and Blair, 1988; Goroji et al., 2008). The same result and high yield was ob-
tained by Anonymous,(1984); Khan et al.,(1985); Abrol and Carryall,(1990) when
biodigested poultry wastes, fish meal, pig manure, farm yard manure and press mud
27
were used as organic fertilizer on cotton crop. The application of biodigested slurry
obtained from biogas plant along with rock phosphate and phosphate solubilizing or-
ganisms induced improved crop production, increased uptake of phosphorus and plant
seed weight (Jeyanthi kumari and Victor, 2008). Hedge et al., (1994) reported that
phosphorus is the second important nutrient for plants and microorganisms. Majority
of agricultural soils in India are poor to medium in available P status and the efficien-
cy of utilization of phosphatic fertilizers is very low (20-25%) due to chemical fixa-
tion in soil and continuous deposition of P is due to regular application of phosphatic
fertilizer.
1.0.07 Soil phosphate in agriculture
Native soil phosphorus is mostly unavailable to crops because of its low solu-
bility. Saxena and Tilak, (2000); Nasreen Haqua and Dave, (2002) stated that the con-
centration of soluble phosphate in soil is usually very low which leads to deficiency of
soluble phosphate and makes it a limiting factor in plant nutrient. Algawadi and Gaur,
(1988); Gaur, (1990); Gaind and Gaur, (1991) revealed that the phosphate solublizers
has been utilized as potential microbial inoculants for crop grown in Indian soil which
contains low availability of P and amended with RP or TriCalcium Phosphate
(TCP).
Varsha Narsian et al., (1994) reported that solubilization of RP by PSM de-

pends on the concentration and nature of the medium. An application of seed inocula-
tion with phosphate solubilizing microorganisms increased the available phosphorus
in soil and increased its uptake by crop plants were published by Vidhyasekaran et al.,
(1973). Singal et al., (1991) in their field experiment explained that the PSB inoculant
enhanced grain yield. Anjana sharma et al., (1995) described that the release of extra
cellular phosphatase help the organisms to hydrolyse the organic phosphate in their
natural habitat and to use the inorganic phosphate released and used for removal of
phosphate concentration in soil.
Microorganisms and plant roots readily dissolve insoluble phosphates and

render them easily available to plants (Gaur, 1990). Broadbent, (1957) stated that one
of the way to correct the deficiency of phosphorus in plants is to inoculate seed or soil
28
with phosphate dissolving microorganisms along with phosphatic fertilizers. Scott,
(1989) stated that high quality seed obtained by increasing P concentration with phos-
phate solubilizing bacteria, seed coating, soaking with fertilizer or nutrient concentra-
tions in seed. Seed coating has been studied by many researchers (Silcock and Smith,
1982; Scott and Blair, 1988; Ascher et al., 1994).
1.0.08 Phosphate solubilizing organisms (PSO)
The occurrence and distribution of phosphate solubilizing bacteria

found 66% gram-positive bacilli, 32% Gram-negative bacilli and 25% Gram positive
cocci (Smid and Bates, 1971; Bairk, 1994). The gram-negative bacilli were
represented by the genera Bacillus and Corynebacterium, while the gram negative ba-
cilli comprised the genera Pseudomonas, Alcaligens, Vibrio and Enterobacter. The
Gram-positive cocci were represented by the genera Micrococcus and Staphylococ-
cus. Bacillus and Pseudomonas were the dominant genera constituting 64% and 23%
of the total identified bacterial isolates. Sathiya Bama et al., (2005) revealed that the
phosphatase enzyme produced by PSO play an important role in mobilizing the soil
available phosphate to plant available form. Some of the organisms are listed below.
Table 1.02 Organisms associated with phosphate solubilization
An experiment conducted by Yahiya et al., (1996) revealed that PSO inocu-
Organisms References
Escherichia coli, Pseudomonas aeruginosa, Aerobac- Ozkanca and Flint, (1996); Day and Ingram,
ter aerogens, Alteromonas sp, Acinetobacter sp. (1973); Wolfenden and Spence, (1967); Terent
Ev et al., (1994); Cotner and Wetzel, (1991).
Staphylococcus aureus Shah and Bibel, (1968).
Bacillus subtilis Takeda and Tsugita, (1967).
Micrococcus sodonensis Glew and Heath, (1970).
Micrococcus variens, Cirtobacrer koseri Sharma et al., (1997).
Selenastrum capricornutum Klotz, (1985).
Euglena graci Price, (1962).
Nannochloris sp, Nannochloropsis Lubian et al., (1992).
Daphnia magna, Sceletonema costetum, Uchida, (1992).
Prorocentrum micans
29
lated field increased the shoot dry weight, leaf-area index, leaf area duration and crop
growth rate. It also significantly increased the pods/plant seed weight, harvest index
and seed yield in pigeon pea (Cajanus cajan). Gaur et al., (1980) found that there was
a significant increase in grain yield, when wheat was inoculated with Peudomonas
striata in the presence of rock phosphate. In another experiment, P. striata was used
as the test organism for field trails to test the inoculation effects on the yield of potato
(Gaur and Ostwal, 1980).
1.0.09 Biodigested slurries as biocarriers
In some area the biodigested slurries of biogas plant used as carriers for the
preparation of carrier based inoculums acclaimed to play a vital role in modern agri-
culture (Ananthakalaiselvi and Dharmalingam, 1995; Sharma et al., 1997; Madhiaz-
hagan et al., 2001). Saber et al., (1989) reported that application of carrier based cul-
tures increased nutrient elements and protein content. Miller and Scooter, (1997) re-
ported the possibility of getting desired plant population with good establishment by
proper seed application of carrier materials with inoculums. Mukhe, (1987) stated that
seed coating materials or proper carrier based inoculum coated seeds improve ger-
miability and increased seedling emergence at the changing soil moisture especially in
the suboptimal conditions.
A field experiment was conducted by Nagarajan and Balachandar, (2001) to

study the influence of organic amendments like farmyard manure, leaf compost and
biodigested slurry from biogas plant with rhizobium on nodulation and grain yield of
black gram and green gram. The results revealed that among the different organic
amendments used, biodigested slurry incorporation along with Rhizobium inoculation
recorded maximum plant height, nodule number, nodule weight and grain yield. Finn
and Brun, (1982); Kulkarni et al., (1984); Thakur et al., (1999) found that there was
significant increase in plant height and leaf area index, nodules/plant, number of
branches/plant, number of pods/plant noted in Phaseolus vulgaris when inoculated
with organic manure containing Rhizobium phaseoli. Prabakaran, (1998); Madhiazha-
gan et al., (2001) reported that the application of organic amendments with inoculums
gave better nodulation and grain yield of pulses in the acid soils.
30
Azoon Aguilar et al., (1981) stated that inoculation of efficient strains of rhi-
zobium with carriers have increased nodulation, nitrogen fixation, dry matter accumu-
lation in plant and grain yield either through better uptake of nutrients or by the in-
creased activities of soil microorganisms in the rhizosphere region.Varsha Narsian et
al., (1994) portrayed that to achieve the potential economic benefits by rhizobial in-
oculation in legume crop ecosystem, in addition to carriers, abiotic soil factors such as
soil texture, pH, temperature, moisture content and substrate availability are very im-
portant. These abiotic factors largely determine the survival ability of the introduced
microorganisms in the soil and host rhizosphere. Li and Alexander, (1990) stated that
the coinoculation of Rhizobium strains such as Pseudomonas, Bacillus or Streptomyc-
es were found to enhance colonization and nodulation. Benoj Mathew, (2006) mixed
the rhizibium culture with sugar solution added gum arabic powder carrier, and then
coated on Phaseolus mungo seeds favoured good plant growth when compare to un-
inoculated plants.
Raman et al., (1994) found that Prosopis juliflora seedlings inoculated with
carrier based rhizobium resulted in higher biomass, nodule dry weight, and indole
acetic acid content. Biswas et al., (2006) reported that inoculation with carrier based
Rhizobium leguminosarum R.trifolii E11, Rhizobium sp IRBG74 and Bradyrhizobium
sp IRBG 271 increased rice grain and strains yields by 8 to 22 and 4 to 19% respec-
tively at different N rates.
1.0.10 Types of carriers
Legume inoculants occupy the prime position among biofertilizers because of

the agronomic importance of legume crops Sharma et al., (1997. Ananthakalaiselvi
and Dharmalingam, (1995) stated that seed pelleting and carrier based inoculums ac-
claimed to play a vital role in agriculture for precision planting and for supplementary
nutrition through which uniform and vigorous field stand will be possible. Iswaran et
al., (1969) reported that in India, peat like material available in Nilgiri valley has been
found to be a good carrier and lignite is another carrier which is widely available from
Neyveli Lignite in South India. Date,(1974) stated that the Australia milled dry peat
carrier contained the cell count from 197 to 1010 /g. Tilak and Subbarao, (1978) found
that among the different carriers, combinations of Indian peat soil, Farm Yard Manure
31
(FYM), compost or press mud with charcoal capable of increase higher rhizobial
count than individual carriers.
1.0.11 Cellulosic biodegradation
In addition to cattle dung and poultry wastes, the deposition of cellulosic ma-
terial in close association with other compounds such as hemicellulose, lignin and
other polysaccharides also make their bioconversion more difficult (Person et al.,
1990). Cellulose is a prominent carbonaceous constituent of higher plants and proba-
bly the most abundant organic compound in nature. Sewage is the main reservoir for
cellulose accumulation. The recycling of these waste residues is necessary to prevent
pollution and to conserve scarce natural resources (Caughlan, 1985; Jeyashreepauls,
1992). The sewage water constitutes a large amount of hydrolytic, proteolytic, cellulo-
lytic, hydrogen producing, acetogenic, methanogenic and sulfate reducing organisms.
The cellulolytic microbes in sewage water play an important role in the degradation of
cellulose and in the production of valuable substances.
The cellulosic materials were solubilized to a great extend by aerobic soil in-
habiting microbes Paul et al., (1986). This microflora solubilizes the undigested cellu-
losic materials to glucose and low carbon compounds. Valente et al., (1987), Camp-
bell et al., (1995) composted primary sewage sludge and reported significant cellulose
degradation. Saravanan and Indira Jeya Raj, (2004) gave a classical example for the
degradation of coir pith using cellulolytic fungus Pleutrorus sajorcaju and Tricho-
derma viride. According to Lynd et al., (2002) view, when composition of cellulosic
biomass were provide the cellulolytic organisms showed improvement in product
yield and tolerance of microorganisms able to utilize cellulose or expressed a hetero-
logous system for cellulose hydrolysis. Demain et al., (2005) portrayed that the bio-
mass conversion to ethanol as a liquid fuel by cellulolytic organisms offers a potential
partial solution to the problem of the world’s dependence on petroleum for energy.
Ramesh Chander kuhad, (1992) found that the paddy straw samples were degraded by
microbial decomposition involves both biochemical and physical changes.
Lori Robson and Glenn Chambliss, (1984) selected B. subtilis and grown on a
variety of sugars and found that the culture supernatant was found to possess cellulo-
32
lytic activity. Alan white and Malcom brown, (1981) selected Acetobacter xylinum as
a substrate for visualizing the action of cellulase enzymes from the fungus Tricho-
derma reesei. Bhatt and Ramesh Maheswari, (1987) reported that the extensive de-
gradation occurs on whatman filter paper and cellulose powder used as substrates by
Sporotrichum thermophile. Tansely, (1971) found that the cellulolytic rates of some
thermophilic fungi Chaetomium thermophile, Sporotrichum thermophile and Ther-
moascus aurantiacus were two or three times that of Trichoderma viridae one of the
most cellulolytic fungi. Emtiazi et al., (2007) found that spore forming, nitrogen fix-
ing Paenibacillus strains isolated from soil showed high cellulase activities thus es-
tablished a plant microbe interaction. Wen-jing et al., (2005) revealed that the mixed
cultures of cellulolytic organisms showed high enzyme activity compare to individual
isolates of Bacillus pasteurii and Bacillus cereus.
The cellulolytic isolates from the effluents of gelatin factory showed higher
cellulolytic activity (Anjana Sharma et al., 1995). The isolates mostly belong to the
genus Micrococcus, Pseudomonas and Bacillus. Tulasi Raman, (1986) reported that
the microorganisms isolated from soil namely Pestolotiopsis glandicola and Myrothe-
cium indicum able to produce cellulose when cellulose membrane discs were provided
as cellulosic substrates. Thajuddin, (2004) revealed that cellulase enzymes are pro-
duced by several organisms including fungi and bacteria. In general, bacterial cellu-
lases are continuously produced, whereas fungal cellulases are produced only in the
presence of cellulose. Vijay Antony et al., (2004) their studies on microbial cellulase
production via solid state fermentation revealed that maximum enzyme production on
a mixture of wheat-bran and rice straw.
Lee and Koo, (2001) reported that the cellulolytic enzyme, endoglucanase
from Cellulomonas, Bacillus and Micrococcus sp isolated from estuarine in the pH
range of 4.0 to 9.0 with maximum activity at pH 7.0. Cooney et al., (1978) reported
maximum CMCase activity at pH 7.5 by Aspergillus nigar among the tested pH range
between 4.0 -9.0. Akiba et al., (1995) stated that a number of fungi and bacteria are
capable of producing multiple groups of enzymes, which are collectively known as
cellulases, that act in a synergistic manner to hydrolyze the β -1.4-D-glycosidic bonds
within the cellulose materials. Shah et al., (2008) done a work with cellulase produc-
tion using Bacillus subtilis and Cellulomonas individually and in mixed culture form
33
on the bioconversion of agro wastes revealed that the mixed combination of cultures
showed maximum ability to produce cellulase enzyme against agricultural residual
substrates.
1.0.12 Sewage water
The sewage water played a major role as the supplement nutrient in the anae-
robic digestion of cattle dung and poultry wastes in the biogas production and the iso-
lation of cellulolytic organisms for cellulose degradation. Also the spent slurry of bio-
gas plants can be a new source of organic manure. Johnson et al., (2003) reported that
the anaerobic digestion is an important sewage treatment process enabling stabiliza-
tion of the organic fraction of sewage prior to land application. Sommers, (1977); Mi-
sra et al., (1991); Murtaza et al.,(2003); Krishnah et al., (2004); Javid et al., (2006)
established that sewage water consist of multi-element organic wastes that are used as
a beneficial amendment for soil and nutrient source for irrigation. Nair, (1944) has
been experimentally observed that effluents from activated sludge plans increased the
rate of fish production.
Connell and Miller, (1984) viewed that domestic waste water contains metals
from metabolic wastes, corrosion of water pipes and consumer products. Murtaza et
al., (2003) found that leafy vegetables like cauliflower, cabbage and spinach grow
quite well in the presence of sewage water. Bakhsh et al., (2005) portrayed that vege-
tables such as radish are sensitive to sewage water. Like that Qadir et al., (1999) and
Murtaza et al., (2003) stated that vegetables grown by the use of sewage water contain
many heavy metals causing serious health hazards to the community and animals.
Long term and indiscriminate application of raw sewage effluent results in the accu-
mulation of heavy metals in surface and sub surface soil (Datta et al., 2000). Irrigation
with sewage effluent had enriched the soil mainly with respect to N, P, K and en-
hances the crop yields considerably (Nan and Chung, 2001). Lourenco et al., (2007)
reported that the outbreaks of diseases transmitted by water can be caused by Entero-
bacteriaceae, which proliferate in polluted waters. However, industrial pollutants to-
gether with the residues and sewage from cities have led to a severe environmental
problems significantly reflecting in the social and public health areas.
34
The application of sewage water directly to soil is not advisable because it
contains many heavy metals and pathogens that may cause serious health hazards to
the community and animals. To avoid this, the sewage water from any source should
be pre treated especially by anaerobic digestion process so that the pathogens are get
destroyed and the hardness of the heavy metals also get reduced before their usage in
agriculture field.
Having the above research background in mind the present work was carried
out with the following objectives.
To produce biogas from cow dung and poultry droppings using se-
wage water as nutrient supplement obtained from Sewage Treatment
Plant (STP).
To study the fertilizing effect of enriched and nonenriched biodigested

slurries with Rock Phosphate (RP) and Phosphate Solubilizing Organ-
isms (PSO) of biogas plant on chilly plant growth –a field trial.
To prepare the biological carriers using biodigested slurries of biogas

plant at different temperatures to analyse the lifespan and propagative
potential of Rhizobium phaseolus from Phaseolus vulgaris.
Isolation and characterization of microflora from Sewage Treatment

Plant (STP).
To biologically degrade the cellulosic substrates by selected sewage

cellulolytic bacteria isolated from STP and enzyme analysis.
35
36
37
CHAPTER II
38
Tamil Nadu, India.
Chapter II
2.0 PRODUCTION OF BIOGAS USING SEWAGE AND

ANIMAL DUNG
2.0.1 Introduction
Energy is one of the prerequisites for the economic growth
which are met mainly through fossil fuel sources like coal, natural gas and oil (Gallert
and Winter, 2002). In recent years, the prices of these commercial energy sources
have increased sharply and there is a continuous depletion of these scarce resources.
(Kannan et al., 2003). The energy crisis is influencing the economic productivity of
almost all the nations (Oblisamy et al., 1992). The growing global awareness of the
energy crisis has resulted in the commencement of the ‘Recycle revolution’ (Alich
and Inman, 1975). Increasing concern over environmental degradation and the danger
of non-sustainable development as well as the need for alternative renewable energy
resources has enhanced the role of environmental management of ecosystems (Les
Gornall, 1996; Castro, 1995).
The rising cost and shortage of conventional fuel have gen-

erated renewed interest in producing methane from organic matter through anaerobic
digestion (Hashimoto and Chen, 1978). The production of energy from alternative
sources becomes not only more desirable but economically more feasible (Hobson et
al., 1975). Biogas is one of the promising sources of alternate energy. It is the fourth
largest source of energy in the world supplying about 13% (55KJ/yr), which is
equivalent to 25 million barrels of primary energy (Mittal, 1997). The biogas technol-
ogy of modern plant produces clean renewable with nutrient rich digested slurry. Bio-
digested slurry of the biogas plant is rich in N, P, K and several other micronutrients
that can be recycled to agriculture as a secondary fertilizer (Weiland, 2000; Sehgal
39
and Sehgal, 2002). Methane is cheap and plentiful not only as natural gas, but also as
biogas (Wendlandt et al., 2001).
Whenever waste materials recycled it must be reintroduced into production

process and the non-recyclable fractions should be used as a fuel for energy recovery
(Carter, 1978). Anaerobic digestion has been considered as a means of greatly reduc-
ing the polluting power of wet organic wastes from agriculture and industry as well as
from the organic household wastes (Jewell et al., 1979; Oblisamy et al., 1992; Weil-
and, 2000). There is always a great scope for the modern anaerobic digesters for treat-
ing various types of biological wastes. The cost of such treatment facilities is lesser
when compared with conventional time consuming aerobic treatment facilities (Chin-
nanchetty et al., 2005). Other waste management options, such as land filling and in-
cineration of organic waste have become less desirable (Angelidaki et al., 2003).
India is a land of many resources. It has the largest population of livestock of

over 300million and it could generate nearly 70.22 KJ of energy annually (Govil and
Gaur, 2000). The biological treatment method is based upon the biodegradation of
organic substances by various microorganisms which lead to biogas production. The
advantage of the biodegradation in waste management allows diminishing the volume
of organic wastes thereby the biological hazard of the waste is controlled (Ilyin et al.,
2004). Economically feasible methane fermenters could lead to the production of a
significant amount of natural gas which satisfies the existing demand for this fuel
(Jewell, 1978; Hayes, 1979). It is also a form of clean energy that has become more
important due to the large in place multi-billion dollar distribution system in the
U.S.A and other countries like Bangladesh (Chan, 1973; Solly and Yarrow, 1975; Is-
lam, 1977), India ( Prasad et al., 1974; Sathainathan, 1975; Subramaniam, 1978),
China (Subramanian, 1978; Van Brush, 1979), Korea (Yoshida, 1978), Nepal (Finely,
1978; Pang, 1978), Taiwan (Chung et al., 1974), Philippines (Obias,
1978;Subramanian, 1978) and Kenya (King, 1978).
2.0.2 Digesters
Batch type anaerobic digesters were used in experiments at mesophilic tem-
perature to investigate the possibility of utilization of poultry waste as an organic fer-
40
tilizer and alternative energy source (El- Hadidi and Al-Turki, 2007). The most com-
mon types of anaerobic digesters that have been used to treat animal manure and to
produce biogas on farm are the mixed tank, plug flow and covered lagoon digester.
Janata, Deenbadhu, KVIC and pragati model biogas plants being constructed in rural
areas are suitable for cattle dung as feed material (Chastain and Linvill, 1999). Al-
though sewage sludge digesters are usually built above ground level, an earth em-
bankment to provide additional insulation surrounds many of the smaller ones
(Swanwick et al., 1969). The majority of India’s biogas gobar plants are built on the
well-tried KVICs design (Subramaniam, 1978).
Cattle manure and night soil are mainly used as methanogenic substrates
(Chirnyivi, 1978). A major limitation of anaerobic sludge digestion is the long Hy-
draulic Retention Time (HRT) required for satisfactory stabilization that results in
large digester size (Mao and Show, 2006). The greater proportion of the gas is pro-
duced from breakdown of substrates in the suspended solids and this requires intimate
contact of bacteria and substrate and bacterial colonization of the substrate (Summers
and Bousfield, 1976). Pathogens removal rate is apparently more efficient in the di-
gestion process (Zahranska et al., 2002).
2.0.3 Substrates
The main digestible components of solid wastes are carbohydrates (Cellulose

and hemicellulose), proteins and fats (Van velsen, 1977). In India, the biogas technol-
ogy is based on cattle dung as the main feed stock (Mah et al., 1977). Animal waste
is a significant source of substrate for biomethanation in biogas digesters. It is a po-
tential source for carbon, nitrogen, and other nutrients that are required for the suc-
cessful production of biogas (Kannan et al., 2003). Biogas has traditionally been pro-
duced from cattle dung but the reports about the availability of cattle dung has been
decreased in years because of the reduced number of cattle (Masse et al., 2004). Near-
ly 70% of Indian farmers own 2 animals to 3 animals per house hold. If this category
of farmers intends to own a biogas plant, the required quantity of dung transfer cannot
be met on their own farmsteads. The alternate substrate like excreta of sheep, goat,
pig and other animal wastes (Almasri, 2001) both in combination with cattle dung and
alone are possible to produces biogas. In order to widen the scope of biogas adapta-
41
bility in rural areas it is required to use supplement feed material like agricultural and
crop wastes, urban and rural organic wastes, aquatic weeds and agro industrial by
products (Edwards and Daniel, 1992).
A large amount of waste is generated from the poultry farms and if it is not
managed properly it may lead to many problems like foul odour and fly nuisance
apart from causing serious problems such as eutrophication. By carrying out anaerob-
ic digestion of poultry waste these problems could be prevented and the effluent of
such biogas plant can be new sources of organic manure (Leelawati et al., 2008). In
addition to cattle dung, human, poultry and piggery wastes have found its usage as
substrates for biogas digesters (Oblisamy et al., 1992), the other organic rich industri-
al wastes can also be used for biomethanation (Yang and Guo, 1990). Vegetable and
fruit markets produce large quantities of organic waste, which are easily biodegrada-
ble in nature; its improper management can create problems of air and water pollution
whereas its appropriate handling through anaerobic digestion can produce biogas and
manure (Rajeswari et al., 2003).
Ayurvedic pharmaceutical waste of medicinal plants (ie, root, shoot, leaf etc)
also used for biogas production (Saha et al., 2000). The wastes/ residues from most of
the agro based industries can be usefully utilized through anaerobic fermentation
process to produce biogas energy and compost or irrigation water after treatment
(Morgan, 1954). A wide variety of manures and other agricultural residues can be an
aerobically digested and converted to biogas with a reasonably high efficiency (Hob-
son et al., 1978). Also, biogas can be produced from water hyacinth (Eichhornia cras-
sipes) and channel grass (Vallisneria spiralis) (Singhal and Rai, 2003). At present, the
organic solid wastes from industrial and agricultural activities are considered to be
promising substrates for biogas production via anaerobic digestion (Lopez et al.,
2006).
A slurry manure digester on an egg producing poultry farm appears to have

the greatest potential for biogas production (John, 1970). The poultry excreta can be
digested with reduction in odour and pollutants. A gas of high methane content is
produced with lower TS content of the slurry (Hobson et al., 1978; Bousfield et al.,
1979).
42
2.0.4 Organisms
The methanogens are the key organisms in the production of methane from
waste materials (Scherer et al., 2003). Effective digestion of organic matter into bio-
gas with high methane content requires the combined and co-ordinate metabolisms of
different fermentative bacteria (Veriroglu, 1987). They are the only organisms able to
break down acetate to hydrogen to gaseous end products. Without these groups of mi-
croorganisms, effective breakdown of the total organic materials would stop due to
accumulation of the products of fermentative microbes namely fatty acids and alcohol
(McInverney et al., 1979). Methanogenic bacteria appear to be extremely sensitive to
certain environmental factors. Being obligate anaerobes, presence of small amount of
oxygen is inhibitory to these bacteria (Pfeffer, 1974).
Methanogens are a unique group of bacteria. They are obligate anaerobe and
have slow growth rate. In morphology they have different types such as cocci, bacilli,
spirilli and sarcinea. Some of them are Methanobacterium formicicum, M.Bryantii,
M.Thermoautotrophicum, Methanobrevibacter ruminantium, M.arboriphilus, Metha-
nococcus vanielii, M.voltae, Methanobacterium mobile, Methanobacterium cariaci,
M. marisnirri, Methanospirillum hungalei, Methanosarcina barkeri (Itodo et al.,
2001). Apart from microbes, earthworms were preferred for efficient composting
potential and biomass growth in a mixed bedding material composed biogas slurry,
cow dung, wheat straw, leaf litter, sawdust and kitchen waste (Schlegal and Schneid-
er, 1985; Maheswari et al .,1993).
2.0.5 Conditions
The microbial population involved in anaerobic digestion requires sufficient

nutrients to grow and multiply. Each species requires both a source of carbon and ni-
trogen. If there is too little nitrogen present the bacteria will be unable to produce the
enzymes that are needed to utilize carbon. If there is too much nitrogen particularly in
the form of ammonia, it can inhibit the growth of the bacteria (Sanders and Blood-
good, 1965). It is often suggested that an optimum ratio of carbon and nitrogen is be-
43
tween 20:1. A minimum amount of ammonia is always required in order to achieve
favorable conditions for growth (McCarty and McKinney, 1961).
To set a fermenter many the parameters like anaerobiosis, temperature, reten-

tion time, chemical oxygen demand, dry solids, volitile solids, pH, biogas yield, bio-
gas composition and volatile fatty acids are very important (Anon, 1971). A nutrition-
ally balanced substrate, biodegradability of substrate and absence of toxins are other
factors that require special consideration for application of biogas production system
(Chan and Pearson, 1970.)
One of the most important inhibitory substances in anaerobic treatment of or-

ganic compounds and methane formation is high salinity (Ozalp et al., 2003). Also the
process of anaerobic digestion is highly influenced by the environmental and opera-
tional factors like organic acid concentration and the reactor volume occupied by the
feed material. The optimum level of organic acid is commonly assumed to be in the
range between 2500 and 3500mg/l for the anaerobic digestion process (Sonakya et al.,
2003).
2.0.6 Applications
The value of biogas produced will depend on the actual amount available for
on-farm use and the value of fuel(s) displaced by biogas usage (Rauschen and Schu-
phan, 2006). The bioenergy used in U.S farms include predominantly for electricity,
heating oil and propane (Mccarty, 1964). In many countries the high quality biogas,
may be used to burn in simple gas equipment like lamps, burners, internal combustion
engines. The residues extracted regularly from the digester constitute a fertilizer in
agriculture (Weiland, 2000) and it is well established fuel for cooking and lightning
(Rosillo Calle and Hall, 1992).
The use of biogas causes the direct replacement of common fuels such as gas-
oline, diesel, oil and indirect replacement of electricity (Meynell, 1976). Energy is
needed in agriculture for irrigation and water supply, to provide fertilizer and nu-
trients, for seed bed preparation and for transportation (Revelle, 1978). Digested slur-
44
ry can also be converted to a slow release fertilizer. The process being carried out in
Ireland, combines digested slurry with urea and formaldehyde (Van velsen, 1977).
Biogenic waste anaerobic treatment shows to be advantageous when compare

to composting, incineration or combination of digestion and composting, mainly be-
cause of a better energy balance (Geethanjali et al., 2009). Therefore an alternate ap-
proach was undertaken by using sewage water as supplement for the production of
biogas using cow dung and poultry droppings as substrates.
The objectives of the work was as follows,
• To analyse the parameters of sewage water.
• To produce biogas by mixing sewage water and cow dung at equal proportion
and loaded in KVIC (Kadhi Village Industries Commission) model anaerobic
bioreactor along with control which contains equal proportion of ordinary wa-
ter and cow dung.
• To produce biogas by gradually reducing the cow dung concentration supple-

mented with sewage water (20%, 40%, 60%, 80% and100%).
• To produce biogas by gradual replacement of cow dung with poultry drop-

pings supplemented with sewage water in the order of 20%, 40%, 60% 80%
and 100% respectively.
45
2.1 MATERIALS AND MEHTODS
2.1.1 Study area
The present work was carried out using sewage water of Sewage efflu-
ent Treatment Plant (STP) of National Engineering College (NEC) Campus, Kovilpat-
ti. It is located in a 150-acre campus on NH7 at K.R. Nagar, Kovilpatti, Thoothukudi
District, Tamil Nadu, India. Around 1,850 students are studying in this college.
Among them 800 students are staying in hostels. The hostels are situated in the same
college campus. There are five deep wells, supplying necessary water for both the
college and the hostels. The total quantity of water, supply to the hostel is more than
2, 50,000 liters per day. This results in the generation of wastewater of nearly
1,00,000 liters per day. The management of this institute has installed a STP to treat
the wastewater and release the water for useful purpose.
The sewage water was directly collected from the college hostel septic
tanks, old hostel, new hostel and mess. The wastewater from all the tanks were fil-
tered through screen chamber and collected in the central collecting tank. For every
10 mt, inspecting manholes were provided to remove sludge, sedimentation particles
and for proper maintenance.
2.1.2 Sample Collection
Sewage water sample used as supplement for the production of biogas

was collected from sewage water treatment plant of NEC hostel (Plate 2.01&2.02).
2.1.3 Characteristics of sewage water
The following parameters were analysed in sewage water used both in
control and experimental bioreactors.
46
(i) Total Solids (TS) and Total Volatile Solids (TVS)
The TS and TVS were estimated as per the procedures given in MACs ma-
nual (1988) by using the following formula
TS = C-A X100
B-A
where
Weight of dish = A
Weight of dish + Sample = B
Weight of dish+Sample
After drying at 1030C-1050C = C
TVS = B-C X 100

B-A
where
Weight of silica crucible = A
Weight of drymatter+crucible
Before ignited = B
Weight of crucible+dryweight after
Ignited at 550°C (6hrs) = C
(ii) Total organic carbon
Organic carbon was estimated by the method of Walkey and Black, (1965) by
using the following formula
C (%) = (X-Y) X (Normality of Ferrous ammonium sulphate) x0.03 x100

A
where
Weight of sample = A
Volume of Ferrous ammonium sulphate required
in blank = X
47
Volume of Ferrous ammonium sulphate required
in sample =Y
1 ml of 1N K2Cr2O7 = 0.003 g carbon
(iii) Estimation of total Nitrogen
Total nitrogen in the sample was estimated by Microkjeldhal method of Jack-
son, (1958).
N (%) = V X 0.0014
W X100
where
Weight of sample = W.
Volume of 0.1N H2SO4 used for
neutralization of NH3 in boric acid = V ml
1 ml of 0.1N H2So4 = 0.0014 g nitrogen.
(iv) Estimation of Phosphorus
Phosphorus was estimated following the method of Fiske and Subba rao
(1925).
Mg/l = mg ‘p’ X 100

ml sample
(v) Estimation of Potassium
Estimation of potassium was carried outin flame photometry (Jackson, 1967).
2.1.4 Bioreactors and Loading Mode
In this experiment KVIC (Kadhi Village Industries Commission) mod-

el anaerobic bioreactors were used for biomethanation process (Plate 2.04). Semi con-
48
tinuous process was followed for biogas production. Two sets of anaerobic bioreac-
tors were used. The total capacity of a bioreactor is 46 liters. Cow dung and poultry
droppings were used as substrates. For experimental purpose these substrates were
supplemented with sewage water. The digesters were daily charged by these sub-
strates after their stabilization. The experiment was carried out in three stages. Totally
the work was carried out for the period of nearly 250 days continuously.
In the first stage of work, cow dung was used as substrate in one set of bio-
reactors (Plate 2.03). One reactor was loaded initially with 22kg of cow dung which
was mixed thoroughly with 22 liters of ordinary water and was loaded along with 2kg
of inoculum from another biogas plant situated in the same NEC campus as starter
culture. This was considered as control. About 22kg of cow dung and 22litres of se-
wage water (Plate 2.02) was mixed thoroughly and then it was loaded in another bio-
reactor which was considered as the experimental bioreactor. To that 2kg of inoculum
was added as starter culture. The RT period was maintained for 30 days. After the
completion of this period the biogas output was checked and measured by water re-
placement method. This was followed by daily charging of 750g cow dung mixed
with 750ml of ordinary water in control bioreactor and 750g of cow dung mixed with
750 ml of sewage water in experimental bioreactor. The gas production was measured
daily by water replacement method in two bioreactors (Plate 2.05).
After stabilization, in the second stage of work the feeding of cow dung
amount was gradually reduced in the order of 20%, 40% 60% 80% and100% in the
interval of 15days and the sewage water was added to maintain around 10% TS level.
During 100% reduction, the cow dung substrate was completely replaced with sewage
water and the parameters were measured. The biogas production was analysed in both
reactors. This sort of work was carried out for about 90 days.
In the third stage of work, another set of experiment was carried out in the
same manner by having one control and an experimental setup with cow dung and
sewage water (Plate 2.03). After reaching the RT period of 30days the cow dung was
replaced in 20%, 40%, 60%, 80% and 100% level with poultry droppings in the inter-
val of 15days. The sewage water level was maintained constantly so that to maintain
49
the 10% TS concentration. This sort of work was carried out for about another 90
days.
2.1.5 Methodology of preparing poultry slurry
The poultry droppings were mixed with ordinary water and soaked overnight.
In the next day the soaked poultry droppings were mixed with cow dung and sewage
water followed by charging in experimental bioreactor.
*For 20% replacement with poultry droppings slurry:
150gm of poultry droppings + 450ml ordinary water
Wetting over night
600gm of cow dung + 750ml of sewage water
Experimental set up
The biogas production in this experimental bioreactor was measured and com-
pared with the biogas production of control bioreactor. In the same manner the other
concentrations of 40%, 60%, 80% and 100% were prepared and loaded in the experi-
mental bioreactor.
2.1.6 Measurement of total biogas production
The total gas production in both sets of the bioreactors was measured by the
water displacement method. In this method, the volume of water displaced in the con-
tainer is equal to the volume of gas produced in bioreactors. The gas outlet of the bio-
reactor was connected to a rubber tube, the other end of the tube was connected to the
water filled measuring jar, which was placed in an inverted position in a tray contain-
ing water. The biogas produced was allowed to pass towards the inverted measuring
jar through rubber tube. The gas replaced the water in the measuring jar. Thus the
quantity of gas produced was equal to the volume of water replaced in the measuring
jar (Plate 2.05&2.06).
50
2.1.7 Characteristics of outlet slurry
The parameters analysed in the sewage water were also followed here. In addi-
tion to that volatile fatty acid, pH and temperature were analysed routinely both in
control and experimental bioreactor.
(i) Estimation of volatile fatty acids
Volatile fatty acid was estimated by coloumn chromatography method Swa-

minathan et al., (1982).
VFA = V X N X 60
X x1000
where
Volume of NaOH = V
Normality of NaOH = N
Volume of sample taken = X
(ii) pH
It was measured by using digital pH meter (Model No: pH scan WP 1,2).
(iii) Temperature
The temperature was ranged between 36oC to 38oC throughout the experimen-
tal period.
2.1.8 Statistical Analysis
The average (mean) and standard deviation of all the experimental data were
calculated and given in the table as mean±SD by using Microsoft Excel. Then the
values were tested by ANOVA (Analysis of varience) which revealed that there is a
significant difference between the control and experimental bioreactor both in gradual
replacement of cow dung with sewage water (Table 2.04) and gradual replacement of
51
cow dung with poultry droppings and sewage water (Table 2.09). Further the data
were subjected to Student’s‘t’ test to explained the impact and individual analysis of
TS, TVS &VFA for the biogas production in the case of gradual cow dung replace-
ment with sewage water in the first part of work (Table 2.05) and the gradual cow
dung replacement with poultry droppings and sewage water in the next part of work
(Table 2.10) which revealed that they have a very significant effect on producing TS,
TVS and VFA. The Coefficient of Varience analysis was also used to find out a better
biogas producer and the analysis report revealed that experimental bioreactor is pro-
ducing consistant biogas in the both experimental trials (Fig 2.05 &2.04).
52
2.2 RESULTS
2.2.1 Characteristic features of sewage water

The sewage effluent collected from STP was analysed and its characteristic
are presented in table 2.01
Table 2.01 Characteristics of Sewage water.

TS (%) TVS (%) N (%) P (%) K (%) C (%)
9.360±3.207 60.290±2.472 1.12±2.039 0.32±1.074 0.28±1.937 28.2±3.008
Values in Mean±Standard Deviation
2.2.2 Experimental biogas production by gradual reduction of cow dung supple-

mented with sewage water
a. Analysis of Total Solids (TS), Total Volatile Solids (TVS) and Volatile Fatty
Acid (VFA)
The experiment was designed in such a way so as to have the total solids
should be at 10% level. The reduction in total solids in control as well as in the expe-
rimental bioreactor which contains cow dung and sewage water was noted during
fermentation.
The total solid in control at the initial day of inoculation was at 10%. At every
15 days interval the total solids were checked and it was noted that they get decreased
from the initial level. The highest solid degradation was noted at 90th day of observa-
tion and it was 6.4% from 9.8% (Table 2.02). In experimental bioreactor the total sol-
ids ranged from 10% to 6.1%. There was a significant reduction of solids observed
when compare to the control (Table 2.03).
The percentage of total volatile solids both in control and experimental bio-
reactors were analysed. In control bioreactor, it was ranged between 62.2% and 66.2%
and the maximum volatilization of solids occurred on the 30days of observation. In
experimental bioreactor the solid volatilization occurred between 72.8% and 64.9%.
The maximum volatilization occurred during 80% reduction of cow dung with sewage
water at 60th day observation (Table 2.02). Although volatile solids destruction de-
53
creased between 62.2% and 72.8% and the high destruction of solid matters occurred
during 80% reduction of cow dung with sewage water from 61.9% to 72.8% (Table
2.03).
Table 2.02 Total Solids (TS), Total Volatile Solids (TVS) and Volatile Fatty Acids (VFA) in
control bioreactor
No of days TS (%) TVS (%) VFA (%)

Initial Every 15 Initial Every 15
days interval days interval
0day 10.0±2.516 7.6±1.835 60.1±1.311 62.2±1.997 420±0.057
th
15 day 10.0±1.732 7.9±1.646 60.3±1.352 64.2±0.900 440±0.132
th
30 day 9.8±1.250 7.6±2.657 61.7±0.702 66.2±0.9 50 400±0.374
th
45 day 9.4±1.692 7.6±1.662 60.7±0.680 64.8±1.193 430±0.497
60th day 9.6±1.587 7.8±1.743 59.5±1.789 62.4±0.680 400±1.647
th
75 day 9.4±2.285 6.6±0.208 60.3±1.200 63.8±2.066 400±0.923
th
90 day 9.8±0.513 6.4±0.351 61.4±0.808 64.2±1.153 420±0.947
Experimental bioreactor
Amount of TS (%) TVS (%) VFA (%)

No of days cow dung Initial Every 15 Initial Every 15
replacement days inter- days inter-
with sewage val val
water
0day 0% 10.0±0.100 6.3±1.053 57.4±0.585 65.8±1.504 520±1.230
15th day 20% 9.8±0.305 6.6±0.608 58.9±1.792 66.2±1.150 500±0.870
th
30 day 40% 9.4±0.493 6.9±0.550 57.3±0.642 66.8±2.954 580±0.043
th
45 day 60% 9.0±0.110 6.1±0.150 56.9±2.668 64.9±1.616 480±0.032
60th day 80% 8.8±0.152 6.8±0.519 61.9±2.193 72.8±1.101 480±0.743
th
75 day 100% 8.4±0.757 7.2±1.106 59.6±1.817 68.2±5.466 460±1.687
th
90 day 100% 8.1±1.156 7.1±0.251 59.5±2.569 68.8±6.086 520±1.1043
54
Table 2.04 Analysis of Varience (ANOVA) for the production of biogas between con-
trol and the experimental bioreactor.
Sources Degrees Sum of Sum of Mean sum Mean Table value of F

of Varia- of free- squares(x) squares(y) of quares(x) sum of
tion dom squres(y)
Between 2 17016.28 19269.21 8508.14 9634.61
Means 3.55
Between 18 13.55 42.96 0.75 2.39
products
Total 20
Calculated value of F for x and y are greater than that of the table value of F for (2, 18)
degrees of freedom. There is a significant difference between the control and the experi-
ment. Based on that the further analysis was worked out for the same separately by using
student’s t-test.
Table 2.05 Students‘t’ test analysis of TS, TVS & VFA for biogas production by gra-
dual reduction of cow dung supplemented with sewage water
Ho: There is no significant difference between the production of TS, TVS and VFA
in the control and in the experiment.
*Total Solids(TS) *Total Volatile Sol- *Volatile Fatty Ac-

No of days ids(TVS) ids(VFA)
Control Experimental Control Experimental Control Experimental
0 7.6 6.3 62.2 65.8 420 520
15 7.9 6.6 64.2 66.2 440 500
30 7.6 6.9 66.2 66.8 400 580
45 7.6 6.1 64.8 64.9 430 480
60 7.8 6.8 62.4 72.4 400 480
75 6.6 7.2 63.8 68.2 400 460
90 6.4 7.1 64.2 68.8 420 520
Total 51.5 47 447.8 473.5 2910 3540
Mean 7.357 6.714 63.971 67.643 415.714 505.714
Varience 0.2622 4.4459 911.905
Standard 0.512 2.109 30.198
deviation
T t=2.347 t=3.258 t=5.576
Table value of t at 5% level of significance; Degrees of freedom = 12; t= 2.179
* There is a significant difference between the control and experimental bioreac-

tor (p<0.05).Therefore the amount of cow dung replacement with sewage water has a
very significant effect on producing total solids, total volatile solids and volatile fatty ac-
ids.
55
The volatile fatty acid (VFA) was estimated both in control and experimental
bioreactors as mg of acetic acid per litre of the centrifuged sample. In control the
maximum level of fatty acid was observed on 15th day observation (440mg/litre). The
minimum fatty acid accumulation was noted on 30th day followed by 60th day and 75th
day. The VFA content ranged between 400 and 440mg/litre (Table 2.02).
In experimental bioreactor, the VFA content was ranged between 460 and
580mg/litre. The maximum VFA content was noted at 40% reduction of cow dung
supplemented with sewage water on 30th day. The minimum accumulation occurred
during 100% reduction of cow dung with sewage water on 75th day observation (Ta-
ble 2.03).
b. Temperature and pH
The temperature changes both in control and experimental bioreactor ranged

between 360C and 380C respectively. The results indicated that in control bioreactor
the digestion process was stable and more gas production occurred at 380C (Table
2.06) but in experimental bioreactor at 370C the digestion process was stable through-
out the retention time (Table 2.07).
The initial pH recorded was 7.2 and 7.0 in control fermentor and experimental
fermentor respectively at 0day. During the fermentation time also, there was no sig-
nificant variation in pH. In control reactor it ranged from 7.1 to 7.3 through out the
working period. In experimental reactor it ranged between 7.0 and 7.2. In control the
reduction of pH occurred from 7.3 to 7.1 and in the experimental reactor pH reduced
from 7.2 to 7.0 (Table 2.07).
56
Table 2.06 Temperature and pH in control bioreactor
Days of Control bioreactor

sampling
Temperature(0C) pH
0 37 7.2
15 38 7.2
30 37 7.3
45 37 7.1
60 37 7.3
75 36 7.2
90 37 7.2
Mean±SD 36±2.10C for temperature and 7.2±0.2 for pH
Table 2.07 Temperature and pH in Experimental bioreactor
No of days Amount of cow Experimental bioreactor

dung replacement
Temperature(0C) pH
with sewage water
0day 0% 37 7.0
15th day 20% 38 7.2
th
30 day 40% 37 7.0
th
45 day 60% 37 7.2
th
60 day 80% 37 7.2
th
75 day 100% 36 7.2
90th day 100% 37 7.0
c. Analysis of C, N, P and K
The experimental and control reactors showed variations in carbon and nitro-
gen utilization. In control reactor, the maximum carbon accumulation seen in 15th and
90th day, whereas minimum carbon value observed in 45th day (Fig 2.01). In experi-
mental bioreactor the maximum carbon value was observed at 90days of analysis
57
(100% reduction of cow dung supplemented with sewage water), and the minimum
was observed at 60% reduction work (Fig 2.02).
In control bioreactor the nitrogen content ranged between 1.2% and 1.8%
whereas in experimental bioreactor it was ranged between 1.2% and 1.52%. Phospho-
rus and potassium in the control and sewage water added sample were analysed and
the amount of phosphorus in the control ranged between 1.0% and 0.8%. In the expe-
rimental bioreactor the percentage of phosphorus ranged between 1.8% and 1.0% re-
spectively.The potassium concentration in control was ranged between 1.44% and
1.00%, whereas in experimental reactor was between 1.3% and 0.8% respectively
(Fig 2.02.).
35
30
25
20
% of N,P & K
15
10
0
0day 15th day 30th day 45th day 60th day 75th day 90th day
No of days
C(%) N(%) P(%) K(%)
Fig 2.01 Estimation of C, N, P & K in control bioreactor
58
40
35
30
% of N,P & K
25
20
15
10
5
0
0% 20% 40% 60% 80% 100% 100%
Amount of cowdung relpaced with sewage water
C(%) N(%) P(%) K(%)
Fig 2.02 Estimation of C, N, P & K in Experimental bioreactor
d. Biogas production
The gas output was calculated based on the replacement of water by gas pro-
duction per day. The gas production for every 15days was analysed. In the case of
cow dung and sewage water combination the cumulative gas production was 1421
l/Kg of dry matter) (Fig 2.03), whereas in control the total gas production was 1057
l/Kg of dry weight) (Fig 2.04). Comparatively the results indicated that the experi-
mental bioreactor revealed more biogas production than control. In control the maxi-
mum gas production occurred in 15-30days interval (200 l/Kg of dry weight/day) and
minimum gas production occurred in 0-15 day’s interval (160 l/kg of dry weight/day)
(Fig 2.04).
In experimental bioreactor the gas production was more during 80% reduction
of cow dung at 60-75 days interval (257 l/Kg of dry weight/day). The minimum gas
production was seen in initial time loading (0-15days interval- 120 l/kg of dry
weight/day). During the analysis of 100% replacement of cow dung the biogas pro-
duction was noted as 245 l/Kg of dry weight/ day in 75-90 days interval and this in
turn indicated the decreased digestion process due to very low amount of cow dung
level during this time (Fig 2.03).
59
Fig 2.033 Biogas production in Experimental bioreactor
Xe = 45.87
σe = 25.54
C.Ve= σe X100 =55.67
Xe
Fig 2.044 Biogas production in control bioreactor
XC = 44.94
σc = 25.23
C.Vc= σC X100 =56.14
XC
The Coefficient of Varience analysis revealed that Xe > Xc, which

ich means that experimental
bioreactor is a better biogas producer
producer.Also C.Ve < C.VC, which means that experimental bi
bio-
reactor is producing consistent biogas.
60
2.2.3 Analysis of biogas production by gradual replacement of cow dung with
poultry droppings
a.. Analysis of Total Solids (TS), Total Volatile Solids (TVS) and Volatile Fatty
Acid (VFA)
The experiment was designed to contain total solids approximately 10% level.
The reduction of total solids in control as well as experimental bioreactor was noted
during
uring digestion. The solid reduction in control ranged between 7.5% and 7.9% (Ta-
ble 2.06). In experimental bioreactor the total solids ranged from 7.2% and 6.7% rre-
spectively. There was a significant gradual solid reduction in experimental reaction
was notedd when compared to control ((Fig 2.05).
Fig 2.055 Total Solids in Experimental bioreactor.
The percentage of total volatile solids in experimental bioreactor and in con-

co
trol bioreactor samples was analyzed. The percentage of total volatile solids in control
contr
61
ranged between 64% and 67.4%, the maximum value being observed on the 90 days
(Table 2.06). In experimental bioreactor the total volatile solids ranged from 73.7%
to75.24%. The maximum level of total volatile solids was noted on 75days at 100%
replacement
nt of cow dung with poultry droppings ((Fig 2.06).
Fig 2.066 Total Volatile Solids in Experimental bioreactor.
The Volatile fatty acid (VFA) was estimated in control and experinmental bi
bio-
reactor as mg of acetic acid per liter of the centrifuged sample. IIn
n control, the VFA
content ranged between 426 and 464 mg/litre.The maximum level of volatile fatty aac-
id was estimated in control on the 30 days ((Table 2.05). In experimental bioreactor
VFA content ranged from 522 and 540 mg/ litre. Maximum VFA level was observed
ob
80% replacement of cow dung with poultry droppings on the 60 days observation (Fig
2.07). In both cases, the fluctuations in the level of volatile fatty acids did not present
any regular pattern.
62
Fig 2.077 Volatile Fatty Acids in Experimental bior
bioreactor.
control bioreactor
No of days TS (%) TVS (%) VFA (%)
Initial Every 15 Initial Every 15 days
days interval interval
0day 10.08±1.615 7.9±1.012 60.76±2.421 64.8±2.678 426±0.020
15th day 10.06±1.207 7.66±1.076 61.50±2.010 65.20±2.431 460±0.197
30th day 10.18±1.812 7.66±1.470 61.52±1.752 65.10±1.839 464±0.138
45th day 10.14±0.976 7.78±1.382 62.00±1.298 65.60±1.202 432±1.428
60th day 10.02±0.460 7.70±0.974 62.32±1.030 64.76±1.637 440±1.031
75th day 10.10±0.615 7.60±0.651 62.78±0.798 67.00±1.246 434±0.739
90th day 9.96±0.592 7.52±0.789 62.84±0.723 67.44±1.008 438±0.378
Values in Mean±Standard
Standard Deviation
63
Table 2.09 Analysis of Varience (ANOVA) for the production of biogas between con-
trol and the experimental bioreactor
Sources Degrees Sum of Sum of Mean Mean Table

of Varia- of free- squares(x) squares(y) sum of sum of value
tion dom quares(x) squres(y) of F
Between 2 16639.57 21823.65 8319.79 10911.83
Means 3.55
Between 18 7.25 3.04 0.403 0.1689
products
Total 20
Calculated value of F for x and y are greater than that of the table value of F for (2,18)
degrees of freedom.There is a significant difference between the control and the experi-
ment. Based on that the further analysis was worked out for the same separately by using
student,s t-test.
Table 2.10. Students‘t’ test analysis of TS, TVS & VFA for biogas production by
gradual reduction of cow dung supplemented with poultry droppings and sewage
water
Ho:There is no significant difference between the production of TS,TVS and VFA in

the control and in the experiment.
No of *Total Solids(TS) *Total Volatile Sol- *Volatile Fatty Ac-

days ids(TVS) ids(VFA)
Control Experimental Control Experimental Control Experimental
0 7.90 7.22 64.8 74.10 426 526
15 7.66 7.26 65.20 74.30 460 538
30 7.66 6.94 65.10 73.70 464 528
45 7.78 6.92 65.60 73.80 432 522
60 7.70 6.72 64.76 75.12 440 540
75 7.60 6.78 67.00 75.24 434 534
90 7.52 6.78 67.44 75.18 438 520
Total 53.82 48.62 459.9 521.44 3094 3708
Mean 7.689 6.946 65.7 74.49 442 529.71
Varience 0.0310 0.8129 134.2857
Standard 0.1760 0.9016 11.5882
deviation
T t=7.8959 t=18.2402 t=14.16
Table value of t at 5% level of significance; Degrees of freedom = 12; t= 2.179
*There is a significant difference between the control and experimental bioreactor

(p<0.05). Therefore the amount of cow dung replacement with poultry droppings has a
very significant effect on producing total solids, total volatile solids and volatile fatty ac-
ids.
64
b. Temperature and pH
The temperature of both control and experimental bioreactor was ranged from
36 C to 380C. The range was fall in mesophilic temperature condition. The initial pH
0
recorded was 7.2 and 7.1 both in control (Table 2.11) and experimental bioreactor re-
spectively at 0day. During the fermentation process, there was a significant increase
in pH that was noted in experimental bioreactor. The increase of pH in control ranged
from 7.1 to 7.4. In experimental bioreactor, pH changes were observed around neutral
with ranges being from 7.1 to 7.7 respectively (Table 2.12).
Table 2.11. Temperature and pH in control bioreactor
Days of Control bioreactor

interval
Temperature(0C) pH
0 36 7.2
15 37 7.1
30 37 7.3
45 38 7.1
60 37 7.2
75 38 7.3
90 38 7.4
Table 2.12. Temperature and pH in Experimental bioreactor
No of Amount of cow dung Experimental bioreactor

days replacement with
pH Temperature(0C)
poultry droppings
0day 0% 7.1 36
15th day 20% 7.4 37
30th day 40% 7.3 37
45th day 60% 7.5 38
60th day 80% 7.4 37
75th day 100% 7.6 38
90th day 100% 7.7 38
65
c. Analysis
is of C, N, P and K
The amount of nitrogen in control and experimental bioreactor was gradually

increased during the fermentation process. In control it was ranged from 1.04%
to1.24% (Fig 2.08).
). While in experimental bioreactor the percentage of nitrogen was
from 1.5% to 1.72% respectively ((Fig 2.09).
The amount of carbon in control bioreactor ranged between 29.2% to 30.8%.

In experimental bioreactor the carbon amount ranged between 31.0% and 32.2%.The
amount of phosphorus and potassium in the control aand
nd experimental bioreactor were
analysed and are represented in figure (2.08 & 2.09). The amount of phosphorus in
control bioreactor ranged between 0.9% and 1.10%. In experimental bioreactor the
percentage of phosphorus ranged from 0.97% to 1.20% respectively.
respectively. The amount of
potassium in the control ranged between 0.814% at 0day and 0.944% at 45th day (Fig
2.08). In experimental bioreactor the amount of potassium ranged from 0.876% to
0.964% respectively (Fig
Fig 2.09).
Fig 2.088 Estimation of C, N, P&K in control

co bioreactor
66
Fig 2.099 Estimation of C, N, P&K in Experimental bioreactor
d. Biogas production
The gas output was noted in control which ranged from 124 to 193 l/Kg of dry
matter/day. In experimental bioreactor the gas yield ranged from 226 to 41
4122 lit/Kg of
dry matter/day (Plate 2.04).
). The maximum yield was observed in 100% replacement
of cow dung with poultry droppings supplemented with sewage water during 75-
75
90days interval (Fig 2.11).
Total biogas production in control after 90th day was est

estimated
imated as 1016 lit/Kg
of dry matter/ day, and in experimental bioreactor it was observed as 1952 lit/Kg of
dry matter/day respectively (Fig 2.10).
67
250
lit/Kg of dry matter/day

200
150
100
50
0
0-15 15--30 30-45 45-60 60-75 75-90
90
Days of interval
Biogas production
Fig 2.10
.10 Biogas production in control bioreactor.
XC = 47.26
σc = 24.38
C.Vc= σc X100 =51.58
XC
Fig 2.11
.11 Biogas production in experimental bioreactor
Xe = 49.66
σe = 25.07
C.Ve= σe X100 =50.48
Xe
The Coefficient of Varience analysis revealed that Xe > Xc, which means that experimental
bioreactor is a better biogas producer
producer. Also C.Ve < C.VC, which means that experimental
bioreactor is producing consistent biogas
68
69
2.3 DISCUSSION
2.3.1 Biogas production by gradual reduction of cow dung supplemented with

sewage water
The experimental bioreactor produced more biogas than the control. The gra-
dual replacement of cow dung with sewage water in experimental bioreactor gave
maximum gas production at 80% replacement. In control bioreactor, the high solid
degradation was observed on 90day and it was 6.4% from 9.8%. The total volatile sol-
id was ranged between 62.2% and 66.2%. The maximum level of volatile fatty acid
was observed on 15th day. In experimental bioreactor the total solids ranged from 10%
to 6.1%. The maximum volatilization occurred during 80% reduction of cow dung.
The maximum VFA was noted at 40% reduction of cow dung with sewage water on
30th day. The temperature changes both in control and experimental bioreactor was
ranged between 36oC and 38oC respectively. Regarding the pH there is no significant
variation was found in both reactors and the variations were found in the C, N, P&K
utilization of both experimental and control bioreactors.
There are a large number of parameters which affect the net energy output
from a digester. Retention time will have an effect on the final gas production as well
as the total and volatile solids in the feed materials. The degree of insulation and am-
bient temperature will also have a direct bearing on the net energy produced (Hobson
and Shaw, 1976; Kaul et al., 1990; Demirer et al., 2000; Singh et al., 2001). In the
present work the total solids ranged between 6.4% and 7.9% in control and in experi-
mental bioreactor the TS ranged between 6.1% and 7.2%. The total solids reduced
from 8.8% to 6.8% level in 80% replacement of cow dung in the experimental bio-
reactor (Table 2.02). The gas production also high at this 80% reduction level. This
was supported by Wong-Chong, (1975) who stated that the total solids value would be
about 4% and do not exceed about 9%.
Hobson and Shaw, (1974) stated that volatile solids do not exceed above 90%
or drop below 40% and 73% could be chosen as a reseasonable value. In the present
work the maximum volatile solids was 72.8%. This was observed in 80% replacement
70
of cow dung with sewage water which gave more gas production than other reduction
concentration. In control the maximum volatilization occurred during 15-30 days in-
terval (66.2%). There is a definite relationship between gas yield and loading rate
(Horton and Hawkes, (1979). Also loading rate is a function of both total solid per-
centage and retention time. This was coinciding with the present work. Semi conti-
nuous fermentation process was selected for this experiment and daily charging was
done by using cow dung and ordinary water in control, cow dung and sewage water in
experimental bioreactor and gas yield was measured regularly by water displacement
method.
Zhang et al., (2006) observed the influence of temperature on the methanogen-

ic bacterial activity, which inhibit the biodegradation and stabilization efficiency of
substrates in bioreactor. Also the fluctuations of temperatures resulted in the varia-
tions of biogas production. The data obtained in the present experiment indicated that
temperature had a significant influence on anaerobic process. So it is very important
to maintain the temperature during biogas production. The effect of temperature on
anaerobic digestion of solid wastes gave conflicting results (Varsal et al., 1977).This
was true regarding this work. The mesophilic temperature was provided throughout
the experimental period and optimum gas yield was also obtained from this tempera-
ture.
Jackson, (1967) found higher gas production at 450C from sewage sludge un-
der mesophilic temperature in batch experiments. Continuously fed experiments at
different temperatures were conducted with municipal sewage sludge revealed maxi-
mum gas production between 200C to 400C by Fair and Moore, (1937). Malina (1971)
obtained lower gas production at thermophilic compared with mesophilic conditions.
In the present work, comparable high gas production occurred in mesophilic tempera-
ture provided.
The temperature fluctuation showed that the biogas production almost stop
and total VFA, such as acetate and propionate are rapidly accumulated, accompanied
by the fall in pH. Temperature fall not only affected the methanogenesis but also the
hydrolysis and acidification. The longer the duration in low temperature, the more the
decay of methanogenic archea (Wu and Sun, 2006).
71
Malina, (1962) in a series of experiments on the digestion of activated sludge
concluded that temperature has a significant influence on digester performance after
experimenting with various temperatures from 300-55oC and that the effect of temper-
ature is independent of loading rate and retention time. The investigator further re-
vealed that the total gas production was greatest at about 300C and 380C. In the
present study also the temperature was not exceeded beyond 380C both in control and
in experimental bioreactor. The present data was also supported by McCarty, (1964);
Pfeffer, (1973).
According to McCarty and Mekinney, (1961); McCarty and Stainforth, (1978)

nitrogen concentration in the range of 200-1500mg/litre has no adverse effect on the
methane formation. In this work, also the nitrogen concentration in control ranges be-
tween 1.2% and 1.8%where as in experimental set up, it was between 1.2% and 1.5%.
These threshold levels were confirmed by Hobson and Shaw, (1974).
In the present study the reduction of total solid was up to 6.4% in the case of
control, but in the experimental bioreactor the solid degradation was up to 6.1%. The
gas production was found to be maximum in 80% replacement of cow dung with se-
wage water which was also in accordance the results of Leelawathi et al., (2008). The
80% reduction of cow dung yielded good gas production, but 100% removal of cow
dung with sewage water yielded gas production less than 80% reduction of cow dung
but more than 60% reduction of cow dung level. This clearly indicated that the se-
wage alone itself without any organic solid support and methanogenic organisms
could not induces more biogas production. This clearly indicated that the sewage wa-
ter needs some solid supportiveness at least in low level as a catalyst for its excellent
biogas production. This may due to the presence of methanogenic organisms which
played a major role for biogas production. These organisms adopted themselves by
having sewage nutrients as their food and decayed the sewage material by producing
biogas.
Pfeffer and Quindry, (1978) also estimated that the maximum gas production
occurred in the study of cattle waste when the total solids are completely degraded. It
72
was true because in the present study, the maximum volatilization of total solid bio-
degradation occurred during 80% of cow dung replacement with sewage water.
Yu and Fang, (2002) stated that if there was too little nitrogen present the bac-
teria would be unable to produce the enzymes which were needed to utilize the car-
bon. If there was too much nitrogen, particularly in the form of ammonia, it inhibited
the growth of bacteria. It was often suggested that an optimum ratio of carbon: nitro-
gen is between 20:1 and 30:1. In the present study the carbon concentration in control
ranged between 26% to 30% and nitrogen concentration ranged between 1.8% and
1.2%. Where as in experimental reactor the carbon level was from 30% to 36% and
the nitrogen concentration ranged between 1.2% and 1.5%. In 80% replacement work,
the concentration of carbon was 31% and nitrogen was 1.4%. This was in limit rec-
ommended by Sanders and Blood good, (1965).
De Renzo, (1977) in his experiment with paper pulp and sewage mixtures
showed that digestion was feasible up to a point at which the C:N ratio was 45:1, and
digester failure occurred when the ratio reached 52:1%. This level was not crossed in
the present work. The maximum carbon concentration in experimental bioreactor was
seen during 75-90days interval (36%) at 100% replacement of cow dung with sewage
water and maximum nitrogen concentration was observed in 40% replacement at 15-
30 days duration (1.5%). In control the maximum carbon accumulation was in 75-90
days duration (39%) the minimum accumulation was seen 0-15 days interval (1.2%).
The pH, alkalinity, and free ammonia nitrogen concentration were the parame-
ters used to evaluate the digesters stability Hae et al., (2003). The most likely reason
for the more biogas production by sewage sludge digesters was that, there was already
enough particulate material as feed present in the digestor to support the bacteria
(Morgan, 1954). In this work also we obtained comparatively more gas production in
the experimental bioreactor than control. Because in experimental bioreactor the cow
dung was supplement with the sewage water initially and after getting stabilization,
gradually the cow dung was replaced by sewage water. In the both circumstances, the
biogas production was more in the experimental bioreactor but not in the control
where it is loaded with cow dung and ordinary tap water. Similar report was obtained
73
from Spencei, (1978) where fly ash supplemented with sewage and McConville and
Maier, (1978) work with addition of powdered activated carbon with sewage.
Park, (1979) investigated that the optimum retention time for cattle manure
was found to be 30 days at 350C. In this work also the RT provided for biogas produc-
tion in both control and experimental bioreactor was about 30 days. Chicken manure
gave a higher gas yield at a shorter retention time.Many organic substances were
used for biogas production by Borzacconi et al., (2006). They produced biogas from
malting plant waste water inoculated with sewage sledge and wastewater of slaughter
house. Tinajero and Noyola, (2006) used raw sludge plus metallic micronutrients and
a bacilli additive for biogas production maintaining raw sludge as control revealed
high gas production in micronutrients supplemented bioreactor than control reactor.
Connaughton et al., (2006) used cattle wastes supplemented with brewery ef-
fluent for biogas production. Lopez et al., (2006) revealed that organic solid wastes
from industries and agricultural activities are considered to be promising substrates
for biogas production. Boubaker and Cheikh Ridha, (2007) carried out anaerobic co-
digestion of olive mill wastewater with olive mill solid waste using semi continuous
feeding tubular digesters operated at mesophilic temperature leads to the high amount
of biogas production. In this experiment also semi continuous feeding method used
for biogas production.
Gomec and Speece, (2003); Hartmann and Abring, (2006) stated that higher
reduction in organic matter induces the highest biogas production. This was observed
in the present work. Here when compare to control, in experimental bioreactor the
total organic solids were well utilized by the microbes resulted decrease percentage of
TS than its initial percentage or concentration. Connaughton et al., (2006), from their
experiment they revealed that efficiency of biogas yield was mainly depend on the
organic loading rates. Similarly in this work the cow dung was gradually replaced by
poultry droppings with sewage water. Daily it was charged to enhance the microbial
activity.
Many factors influenced the inhibition of biogas production during biometha-

nation (Yu and Mu, 2006). One of the important inhibitory factors is high salinity and
74
ammonium nitrogen levels on mesophilic anaerobic treatment (Ozalp et al., 2003).
The process of anaerobic digestion is also influenced by organic acid concentration
and the reactor volume occupied by the feed material. The optimum level of organic
acid is commonly assumed to be in the range between 2500 and 3500 mg/l for the
anaerobic digestion process (Sonakya et al., 2003). High inorganic silt/clay and salini-
ty might have decreased the biodegradability of all sludge and methanogenesis could
be operating in early periods of digestion (Atilla et al., 2003).
Semi continuous fermentation was used in the present experiment for the bio-
gas production following the method of Wang et al., (2003) where they performed a
Hybrid Anaerobic Solid Liquid (HASL); bioreactor under a semi continuous opera-
tion system. The present results showed that the use of semi continuous bioreactor
system enhanced the methanogenesis process and increase the methane content in
biogas production.
The effect of pH on anaerobic solubilization of domestic primary sludge and

activated sludge was investigated by Gomec and Speece, (2003) in continuously
stirred anaerobic reactors at mesophilic temperature (350C) and pH was fixed at 6.5.
Total Suspended Solids (TSS) and Volatile Suspended Solids (VSS) were better in pH
controlled reactors (PH 6.5). This correlates the results of the present work where pH
provided range in two experimental conditions were between 7.0 and 7.7. The de-
struction and utilization of total solids and total volatile solids occurred from their ini-
tial readings to final one.
Centralized biogas plants in Denmark codigest mainly manure together with

other organic waste such as industrial organic waste; source sorted household waste
and sewage sludge (Angelideki and Ellegaard, 2003). In this work poultry droppings
and cow dung was supplemented with sewage water flourished with organic matter.
Biological treatment of waste waster basically reduces the pollutant concentration
through microbial degradation and removal of non settable organic colloidal solids.
Organic matter is biologically stabilized so that further oxygen demand is exerted by
it (Bal and Dhagat, 2001; Gallert and Winter, 2002).
75
Kataoka et al., (2002) did an experiment using swine wastes for biogas pro-
duction using cow dung as control revealed that swine waste produced more biogas
than the biogas production from cow dung. Krzysek et al., (2001) stated that house-
hold derived biowastes were degraded by biological methods for the recovery of
energy (biogas) and the production of the fine humus like material which can be used
as a soil amender or a substrate further thermal treatment. Like that in this work, after
biogas production the spent slurry was used for the carriers for agricultural organisms
as biofertilizers which enhance the growth of chilly plant (as field application).
2.3.2 Biogas production by gradual replacement of cow dung with poultry drop-
pings supplemented with sewage water
Comparatively the experimental bioreactor gave more biogas production than

control at 100% replacement of cow dung with poultry droppings and sewage water.
In control bioreactor the TS reduction ranged between 7.5% and 7.9%. The high per-
centage of total volatile solids observed on 90th day and the maximum level of VFA
was found in 30th day. In experimental bioreactor the TS ranged between 7.2% and
6.7% respectively. The maximum VFA was observed on 80% replacement of cow
dung with poultry droppings. The temperature of both control and the experimental
bioreactor was ranged between 36oC and 38oC. The pH changes were observed
around neutral with ranges being from 7.1 to 7.7 respectively. Variations found in C,
N P&K utilization of both experimental and control bioreactors.
In order to provide a reliable information on digester activity a monitoring sys-

tem for anaerobes is quite necessary (Ramasamy et al., 1992). The present study
was designed with the specific objectives of using the poultry droppings supple-
mented with sewage waster and also gradual replacement of cow dung by the above
said mixture content, for biomethanation process.
The total solid content was found reduced from its initial concentration during
the fermented process. Laube and Marlin, (1981) reported that among the various le-
vels of solid concentration, total solid at 8% registered a higher biogas production or
productivity followed by total solid at 6%. In the present study the reduction of solid
was from 10.06% to 7.52% in the case of control. But in the experimental bioreactor
76
the solid degradation was up to 6.72%. The gas production was found to be maximum
in 100% replacement of cow dung with sewage water treated poultry droppings sam-
ple which was also in accordance the results of Bonmati et al., (2001).
Summers and Bousfield, (1980) stated that TS of poultry slurry contained

more VFA compared with the TS pig slurry like that in this work 6.7% TS of poultry
droppings at 100% replacement contain 534mg/litre of VFA in experimental bioreac-
tor (Fig 2.07), whereas in control, the maximum total solid utilization was observed at
60-75 days duration (7.6%) which contains 434 mg/litre of VFA.
McCarty, (1964) stated that TS at 14% input slower the detoriation in digester
were noted so the very high concentration of TS would not be advised for biogas pro-
duction. This TS limit was applied in the current work. In experimental bioreactor the
maximum input TS was 9.92% at 100% replacement of cow dung with poultry drop-
pings (Fig 2.05). Whereas in control, the maximum TS found was 7.9% during 0-15
day’s interval (Table 2.08).
Semi continuous digestion of a mixture of different amounts of poultry waste

and cow dung showed increase in plant nutrients (NPK) and decrease in TS and TVS
in the effluent slurry. The solids were degraded to varying degrees in the digesters by
the micro flora. Maximum degradation of TS (6.78%) (Fig 2.05), TVS (75.24) (Fig
2.06) and maximum biogas production was observed in 100% replacement of cow
dung with poultry droppings. This could be due to higher availability of nutrients
leading to greater degradation of solids and consequently higher biogas yield. Singh et
al., (1982) also observed greater degradation of solids and higher biogas production in
digester containing mixture of cattle dung and poultry waste as compared to cattle
dung alone.
In experimental bioreactor the volatile solid was high when compared with
control in each stage of fermentation (Fig 2.07). The increased gas production and
methanogenic population does coincide, with the occurrence of high volatile solid.
The results also collaborated with the findings of Kumar and Biswas, (1982); Hill,
(1984); Padmasiri et al., (2006) who stated that high biogas production occurred when
total volatile solid was more.
77
Park, (1979) conducted series of experiments for biogas production using cat-
tle, poultry and sewage sludge separately and combinations. The better results ob-
tained from the combinations. Like that in this work, cow dung mixed with poultry
droppings supplemented with sewage water gave comparatively maximum biogas
yield than control which was loaded with cow dung and ordinary water. Among the
various physical parameters, pH is the one which highly influences the microbial ac-
tivity (Ramasamy et al., 1992). Smith and Hungate, (1958) reported that the optimum
pH for biomethanation ranged between 6.5 and 7.7. The result of the present finding of
the experimental bioreactor also depicted the similar results with the maximum gas
yield being around neutral pH (Table 2.12).
Among the two digesters used in the present study, the maximum gas yield
was observed in experimental bioreactor on 90th day with carbon (32.2%) and nitro-
gen (1.72%). Edwards and Daniel, 1992 had also stated that in an optimum rate of di-
gestion a C: N ratio was close to 20 to30 which appear to be an ideal one as that of
our experiment.
The low amount of gas yield in control may be due to the low solid reduction,
acidic pH, high VFA and low methanogenic population. Al Masri, (2001); Kumar and
Biswass, (1982) reported that an increasing concentration of VFA decreased the spe-
cific methanogenic activity and gas output beyond a certain level. Methanogens play a
decisive role in terminal methane production in biogas digesters (Bousfield et al.,
1974).
Bousfield et al., (1979) stated that poultry excreta form a solid mass, so water
will be needed to produce a material suitable for digestion. Like that in this work be-
fore loading into the experimental bioreactor poultry droppings were soaked in ordi-
nary water over night, then next day it was mixed with cow dung and supplemented
with sewage water followed by charging of bioreactor.Leelawati et al., (2008) con-
ducted experiments by adding poultry wastes with cow dung in 10%, 20% and 30%
ratios. Maximum biogas production occurred in 30% replacement of cow dung by
poultry wastes. Like that in our experiment, the maximum biogas production was oc-
curred in 100% replacement of cow dung with poultry waste compare to control.
78
Hobson et al., (1978), their experiment with poultry and piggery waste materials for
biogas production revealed that the gas production was greater in poultry waste than
that from piggery waste. This was similar in the present work in such a way compara-
tively the poultry droppings with cow dung that 100% replacement of cow dung with
poultry droppings gave more gas production (Fig 2.11).
Bjornsson, (2006) stated that it is important of choosing substrates with a high

methane yield and high nitrogen content, and the necessity of fully utilizing both the
capacity of the equipment installed and the energy carriers produced. Many scientists
reviewed that the animal wastes and excreta are very good substrates which induces
the biogas production in large quantity. For that reasons, poultry droppings supple-
mented with sewage water and cow dung treated with sewage water were chosen for
the biogas production. In poultry droppings supplemented with sewage water work,
the concentrations of NPK were higher in poultry waste. The NPK of cow dung was
within the range reported by different workers (Cheremisinnoff and Morresei, 1976;
Pathak et al., 1985; Wohlt, 1990).
In general, the more complex the substrate, the longer it takes to be degraded
to volatile acids and the higher the proportion of methane in the gas produced (Tre-
veiyan, 1975). So in this work also, instead of suddenly using poultry droppings as
substrates for biogas production, gradually the cow dung was replaced by poultry
droppings because of its drier nature. It may take long time for degradation, in turn it
may affected the nutritional requirements of methanogenic organisms, and lower the
gas production (Burford and Varani, 1976).
The biodigested slurry from biogas plant can be a very good source of organic
manure as it is highly enriched in plant nutrients like NPK. The highest concentration
of NPK and narrowest C:N ratio were observed in digester containing 100% poultry
droppings (Fig 2.09). The results indicated that poultry droppings supplemented bio-
gas slurry has the potential of acting as novel organic manure especially in areas
where poultry waste is available.
Gunaseelan, (1987) conducted a study on the replacement of cow dung gradu-

ally with parthenium and reported that the methane content of gas varied from 60% to
79
70% for a retention period of 8 weeks, in our work also, cow dung in the experimental
bioreactor was gradually replaced by poultry droppings in turn gradually increases the
biogas production than control. Angelidaki and Ellegaard, (2003) was also reported
that the production of biogas from donkey dung supplemented with poultry droppings
was more when compare to the control reactor which contains donkey dung alone.
Kannan et al., (2003) were undertaken an investigation of three feed materials

namely, poultry droppings, parthenium and eucalyptus leaves supplemented with
donkey dung for quantitative and qualitative biogas production revealed that poultry
droppings, donkey dung combination yielded more gas production than other combi-
nations. El-Hadidi and Al-Turki, (2007) revealed that the mixture of cattle feces and
poultry droppings gave the higher production than the other combinations respective-
ly. In the present study the total gas production in experimental setup was calculated
as 1952 lit/Kg of dry matter/day. This amount was more when compare to control
reactor (1016 lit/Kg of dry matter/day).
A study was carried out by Preeti Rao and Seenayya, (1994) using cow dung
and poultry litter waste as substrates for biogas production in separate digesters sup-
plemented with iron showed faster conversion of substrates and initiate more gas pro-
duction in two digesters but comparatively more production observed in iron supple-
mented poultry droppings digester. Like that in the present study also in the experi-
mental bioreactor, the poultry droppings combination with cow dung supplemented
with sewage water gave better gas production than control bioreactor. Similar to the
present work a comparative analysis of biogas yield from poultry, cattle and piggery
wastes at the mesophilic temperatures were carried out by Itodo et al., (2001) and the
results obtained showed that there was no significant difference in biogas yield from
the piggery and cattle wastes but a significant difference was observed from poultry
waste. Anaerobic treatment of organic waste water has occupied unique place in
waste water treatment field due to the variety of reasons, which includes generation of
methane rich biogas, generation of less sludge and maximum reduction of biodegrad-
able organics (Angelideki and Ellegaard, 2003).
In the present study, the replacement of cow dung with sewage water gave
better gas production in the experimental bioreactor than the control bioreactor.
80
Moreover the 80% replacement of cow dung with sewage water in experimental bio-
reactor gave more biogas production than 100% replacement by sewage water. This
clearly indicated that the sewage water alone not enough to induce the gas production
it needs some organic supportive materials for the induction of biogas production. In
the next part of work, during the gradual replacement of cow dung with poultry drop-
pings with sewage water revealed that the experimental bioreactor gave more biogas
production (about 1952lit/kg of dry matter/day than control reactor1016 lit/kg of dry
matter/day).The gas production was step wisely increased during the gradual reduc-
tion of cow dung with poultry droppings and sewage water than control and the 100%
replacement gave better gas production which intern revealed that the poultry drop-
pings along with sewage water can be used as an alternative substrate for cow dung
in the production of biogas .
81
CHAPTER III
82
Tamil Nadu, India.
Chapter III
2.0 BIODIGESTED SLURRY AS BIOFERTILIZER ON

CHILLY PLANT-A FIELD TRIAL
3.0.1 Introduction
Phosphorus is one of the essential macronutrients necessary for proper plant

growth (Torriani, 1960; Khemka, 1998; Rogers et al., 1998; Goroji et al., 2008).
Phosphorus plays a vital role in the balanced nutrition of plants and is reported to be
the limiting plant nutrient factor in soils. A large portion of the available phosphate
present in soil is converted chemically and microbiologically to insoluble forms,
which eventually build up a pool of insoluble phosphate in soil (Vora and Shelat,
1995). Indian soils are rich in phosphate but more than two-thirds of the native
phosphates are unavailable and applied phosphate fertilizers are also rendered un-
available within a short period due to its chemical fixation in soil (Ehrilch, 1996; To-
mar et al., 1996). The phosphorus content of soil may range from one gram to several
kilo grams. Usually less than 5% of this is available to the plants and microorganisms
in a soluble form of inorganic phosphate and organic phosphorus complexes (Studp,
1982).
Phosphorus compounds in Indian soils are predominantly inorganic, chiefly

locked as tricalcium phosphate. Thus, the group of microorganisms dissolving trical-
cium phosphate appears to have implication in Indian agriculture. About 98% of In-
dian soils have inadequate supply of available phosphorus. Generally phosphorus is
supplied to the soil in the form of chemical fertilizers or organic sources such as de-
composed plants and animal materials (Vasha Narsian and Patel, 2006). Salt affected
soils (saline and sodic) occupy about 7million hectare in India. Uncultivated sodic
83
soils have large amount of insoluble phosphorus, mostly in the form of sodium phos-
phate (Thomas and Ingledew, 1990; Singh and Nigam, 1995).
Plants acquire P from soil solution as phosphate anions. However phosphate

anions are extremely reactive and may immobilized through precipitation with cat
ions such as Ca2+,Mg2+,Fe3+ and Al 3+ depending on the particular properties of soil. In
these forms P is highly insoluble and unavailable to plant (Woomer et al., 1988).
Phosphorus is especially important in the early growth of rice plants since its
deficiency depresses the root development during the early growth stages, which in-
turn leads to the decrease of grain yield (Saeki et al., 1959; Kawaguchi and Kyuma,
1974; Yoshida, 1981; Datta et al., 1982). The effect of high seed phosphate concen-
tration on early plant growth may also influence later plant growth and final grain
yield (Thomson et al., 1992). It is an essential element for plant development and
growth making up about 0.2% of plant dry weight (Shenker et al., 1992). In P defi-
cient soils, early plant growth may be depressed, forcing the plant to be strongly de-
pendent on the seed nutrient reserves to produce roots and other organs (Ascher et al.,
1994). Super phosphate is one of the common forms of phosphatic fertilizers. Rock
phosphate is one of the basic raw materials for phosphatic fertilizer production and
100million tons of rock phosphate deposits are available in India although hardly 1/6
of them is sufficiently enriched with P2O5 to be of any use for conversion into super-
phosphate (Ostwal and Bhide, 1972; Jisha and Mathur, 2005).
Phosphate transformation in the soil is influenced by several factors such as

the nature of fertilizers added, microclimate of the rhizosphere and physico-chemical
properties of the soil such as CaCO3 (Chang and Jackson, 1957). In recent years, use
of microbial inoculants as a source of biofertilizers has become a hope for most coun-
tries, as far as economical and environmental view points are concerned. Especially,
in developing countries like India, it can solve the problem of high cost of chemical
fertilizers and help in saving the economy of the country (Jeyanthikumari and Victor,
2008).
Soil micro flora plays a significant role in mobilization of phosphate for plant
growth by reduction in pH, producing chelating substances, protons, humic sub-
84
stances and CO2 which help in phosphate solublization (Lammeli, 1970; Marwaha,
1981). A considerably higher concentration of phosphate solubilizing bacteria is
commonly found in the rhizosphere soil (Suh et al., 1995; Whitelaw et al., 1999).
Several microbes including actinomycetes, cyanobacteria, fungi and yeasts are

known to solubilize phosphates (Bardiya and Gaur, 1972; Kapoor et al., 1989; Gaur,
1990; Hasan, 1999; Bhagyaraj et al., 2000). Some soil bacteria, particularly those be-
longing to the genera Pseudomonas, Bacillus Actinomycetes, Cyanobacteria and Rhi-
zobium and fungi belonging to the genera Penicillium and Aspergillus are the most
powerful phosphate solubilizers and possess the ability to bring insoluble phosphates
in soil into soluble forms by secreting organic acids such as formic, acetic, propionic,
lactic, glycolic, fumaric and succinic acids. These acids lower the pH and bring about
the dissolution of bound forms of phosphate (Garretsen, 1948; Sen and Paul, 1957;
Louto and Webley, 1959; Katznelson and Bose, 1959; Seti and Subba Rao, 1968;
Ostwal and Bhide, 1972; Gaur and Ostwal, 1972, Rudresh et al., 2004).
Phosphate which gets trapped in soil is also made available more efficiently
to crops by the action of phosphate solubilizing microorganisms in the rhizosphere of
crops and soil, which solubilize unavailable insoluble forms of phosphate such as rock
phosphate, tricalcium phosphate, iron and aluminium phosphates, by the production
of organic acids into soluble forms which are easily taken up by the plants (Vora and
Shelat, 1995). These microbial populations solubilize not only the insoluble soil
phosphates and insoluble forms of phosphatic fertilizers but also increase the efficien-
cy of soluble form of phosphatic fertilizers applied to soil (Rasal, 1996; Vora and
Shelet, 1996; Varsha Narsian and Patel, 2006).
Use of economically cheap, low grade rock phosphate and properly selected
phosphate solubilizing fungi can replace the costly currently used phosphatic fertiliz-
ers. Rock Phosphste (RP) has been reported to be solubilized by soil microorganisms
(Varshanarsian and Patel, 2006). Recently importance has been paid to the possibility
of greater utilization of indigenously available rock phosphate resources by the action
of phosphate solubilizing microorganisms. In this connection, field experiments have
been carried out in India using culture suspensions of B. polymyxa, B.circulans, Pseu-
domonas striata, P. rathonis and fungal isolates as Aspergillus awamori, A. Nigar,
85
Penicillium digitatu and Schwanniomyces occidentali, with and without superphos-
phate or rock phosphate on the yield of wheat and rice (Gaur et al., 1980). These mi-
croorganisms have shown consistently their capability to solubilize chemically fixed
soil phosphorus and rock phosphate from different sources (Bardiya and Gaur, 1974;
Arora and Gaur, 1978).
In U.S.S.R, a commercial biofertilizer under the name ‘Phosphobacterin’ was

first prepared by incorporating Bacillus megaterium var phosphobacterium are widely
used in U.S.S.R and other east European countries with yield increases of the order of
5-10% over corresponding controls (Vora and Shelet, 1996). Several field trials have
also been conducted by the Indian Agricultural Research Institute using phosphobac-
terin on wheat, berseem, maize, arhar and rice. In India, field trials were conducted in
different soil types to evaluate the efficiency of seed inoculation with phosphobacterin
(B. megaterium) (Sundara Rao, 1965) reported beneficial effect on berseem and
wheat.
The breakdown of agrowaste in rhizosphere region to simple sugars provided

energy sources for heterotrophic microorganisms such as phosphate solubilizing and
nitrogen fixing bacteria (Dhakshinamoorthy and Murugappan, 1996). Normally, the
growth and metabolic activity of soil microorganisms are limited by the availability of
nutrients. Several kinds of agro wastes and rice straw compost will be good carrier
materials for the inoculants and improve the soil conditions. The peat mass with
phosphate solubilizer showed good survival of inoculants and effects on crops (Ves-
sey Kevin, 2003).
The solubilization of phosphate by microbes is not always consistent because

the environment and plant factors influence the activity of phosphate solubilizing mi-
croorganisms (Kundu et al., 2002). Phosphatases are enzymes, which promote the de-
gradation of complex organic phosphorus compounds into orthophosphate and an or-
ganic moiety (Cembella et al., 1984). They are the extra cellular enzymes, which are
secreted and actively pass through the cytoplasmic membrane into the external envi-
ronment (Wetzel, 1991). Both alkaline and acid phosphatases have been found as ex-
ternal and internal enzymes in algae and bacteria (Suida, 1984). Also the congregation
86
of organisms producing the enzymes has an influence on the phosphatases stability
(Herbien and Neal, 1990; Tomar et al., 1996; Rengal et al., 1999).
Realizing the importance of phosphorus and Phosphate Solubilizing Organ-

isms (PSO), a field trial was conducted on chilly plant using biodigested slurry of
biogas plant enriched with RP and PSO and without enrichment of RP and PSO the
biodigested slurry alone.
The study was designed with the following objectives,
• To isolate phosphate solubilizing organisms from the rhizosphere soil of pad-

dy field using Hydroxy Apatite Medium (HAM).
• To prepare HAM medium for mass cultivation of PSO.
• Two sets of biodigested slurries were obtained from biogas plant. One set was
enriched with rock phosphate and phosphate solubilizing organisms and the
other set was used as it was without any enrichment as biofertilizers with con-
trol. They are as follows
Biodigested slurries of biogas plant

Enriched slurry Non enriched slurry
SWPS+PSO+RP SWPS
SWCS+PSO+RP SWCS
OWCS+PSO+RP OWCS
PSO+RP Inorganic manure
Control
• To apply these amendments on the chilly plant field to compare the growth
and yield.
87
3.1 MATERIALS AND METHODS
3.1.1 Isolation of Phosphate Solubilizing Organisms (PSO) from soil
One gram of rhizosphere soil from paddy field was weighed in an electronic
balance and added to 100ml of distilled water. From this it was serially diluted up to
108 dilutions. One ml of each dilution was poured into the sterile petri plates. About
15ml of HAM agar medium was dispensed into these petri plates. The plates were ro-
tated for uniform distribution and allowed to solidify. The plates were incubated at
370C for 7-14 days for the development of phosphate solubilizing zones around the
colonies. This indicated the utilization of phosphate source provided (Kannan, 1996)
(Plate 3.02).
3.1.2 Preparation of HAM for PSO
One kilogram of rhizosphere soil was mixed with two liters of distilled water.
It was steam sterilized for one hour, and then filtered using crude filter paper. The pH
of the filtrate was adjusted to 6.5 either by acid or alkali. The soil extract obtained was
taken for further manipulations (Plate 3.01).
To 100ml aliquots of soil extract, one gram of filter sterilized glucose was
added. Five ml of 10% calcium chloride and 2.5ml of 10% dipotassium hydrogen
phosphate (previously sterilized) were added under sterile environment. Now the me-
dium contained hydroxy apatite in a finely precipitated form. Then 2 grams of agar
was added to the soil extract, solid media will be obtained, otherwise the media is re-
ferred as HAM broth. The phosphate solubilizing organisms isolated from the rhizos-
phere soil was grown in HAM medium at least for 2weeks and used for phosphate
enrichment.
3.1.3 Mass Cultivation
The PSO isolated from the rhizosphere of paddy field need to be multiplied in
artificial media to harvest on a large scale so that it can be supplied to farmers. For
88
that, these cultures were grown in HAM broth (20ml) at 1% inoculum concentration.
This was incubated for 7days at 26 ± 20C. The cultures were checked for purity and
aseptically transferred to 300ml sterile medium in 500ml conical flask and incubated
at 26± 20C for 7 days on a rotary shaker. After that it was transferred to conical flasks
(2 liters capacity) which contained 1000ml of HAM. When the number of PSO in the
broth has attained the required level (not less than 108 /109 cells/ml), then it was
mixed with the finely powdered and sieved biogas slurries with Rock Phosphate (RP)
(Plate 3.03).
3.1.4 Enrichment of biodigested slurry
After biogas production, the biodigested slurries obtained from biogas plant
were prepared and applied in two ways. One set was enriched with rock phosphate
and phosphate solubilizing organisms and the other set was used as it was without any
enrichment as biofertilizers for field applications.
Enrichment of biodigested slurry using RP as phosphate source was carried

out in a pit of 175x150x45cm. About 500g of RP and one liter of PSO isolated from
rhizosphere soil, propagated in nutrient broth were added. After drying another layer
was poured in a similar manner. The content of pit was allowed to undergo bacterial
action for 21 days. This experiment was carried out with RBD (Randomized Block
Design) under field conditions in a private farm at Sankarankovil district.
3.1.5 Analytical Method
Before applying the enriched and nonenriched biodigested organic amend-
ments, the initial soil nitrogen, phosphorus and potassium were analysed by Micro-
kjeldhal method of Jackson, (1958), Fiske and Subbarow, (1925) and flame photome-
tric method given in APHA respectively. The same measurement was done at every
stage of plant growth and yield. Dry weight of the plant samples were estimated by
complete drying as per the method given in APHA, (1975). Chlorophyll content at
each stage of all field trials was identified by following the method of Aneja, (1996).
Also, THBP (Total Heterotrophic Bacterial Population, THFP (Total Heterotrophic
Fungal Population) and TPSMP (Total Phosphate Solubilizing Microbial Population)
89
were estimated by adopting serial dilution and plating on nutrient agar, rose-bengal or
cephadox dextrose agar and hydroxy apatite medium (Kannan, 2003).
3.1.6 Experimental Design
After the application of enriched and non-enriched biodigested organic

amendments in the field, 20-25days old seedlings of chilly plants were collected and
they were implanted in plots. The manurial value of RP enriched biodigested slurry
was studied on chilly plant according to the following experimental design.
SWPS+PSO+RP SWCS+PSO+RP OWCS+PSO+RP
I
N R
O P
R +
G P
A S
N O
I
C SWPS SWCS OWCS CONTROL
SWPS - Sewage Water treated biodigested Poultry Slurry

SWCS - Sewage Water treated biodigested Cow dung Slurry
OWCS - Ordinary Water treated biodigested Cow dung Slurry
PSO - Phosphate Solubilizing Organisms
RP - Rock Phosphate
In each experimental plot, the multistage sampling procedure was followed to

select 5 locations. The sample units were randomly selected using the systemic ran-
dom sampling procedure. Samples were collected at seedling (Plate3.04), preflower-
ing, flowering and final stages to study the morphological characters like root length,
shoot length, total height, wet weight, and dry weight, also biochemical constituents
which includes NPK and chlorophyll content. Chilies were harvested periodically
90
from each treatment plots, weighed individually and recorded. The cumulative yield
from each plot was expressed in tons per hectare and compared between various
treatments.
Each value is expressed as mean (±SD) of six samples. Further the data of
preflowering, flowering and final stage were subjected to Least Significant Difference
(LSD) analysis. The results revealed that there was highly significance observed dur-
ing flowering (Plate 3.05) and final stage (Plate 3.06) but in preflowering stage there
is no significance observed because at this stage they make themselves ready to ac-
cept the manurial effect and their efficacy was observed in flowering and the final
stage only. So no significant effect of manure was observed in preflowering stage and
highly significance observed in flowering and final stage.
3.1.8 Least Significance Difference (LSD)
In agricultural and biological research, pair comparision is more common. In

this experiment, a comparison is made between the control and 8 types of treatments.
Comparison is made among the treatments to find out the most effective treatment.
The most commonly used test procedure for such type of pair comparison is LSD test.
In this experiment LSD test is used because its F test is significant. This procedure
provide for a single LSD value, at a prescribed level of significance which serves as
the boundary between significant and non-significant differences between any parts of
comparison. It can be concluded that the treatment is significant if the difference ex-
ceeds the computed LSD value. Otherwise non significant (Table 3.05, 3.06 & 3.07).
LSDα = (tα) (S.Edˉ)
Where S>E is the Standard error of the mean difference and tα is the
tabulated‘t’ value at 5% or 1% of probability.
91
3.2 RESULTS
3.2.1 Analysis of THBP, THFP and TPSMP in the field soil (CFU/ml)
The rhizosphere soil of paddy field was collected, analysed and the results re-
vealed that the THBP was found to occur in the order of 106 cfu/gm and THFP in the
order of 103cfu/gm. In the case of TPSMP, the microbial density was in the order of
103cfu/gm. Before the application of the amendments, the THBP in the field soil was
16x106±2.180x106cfu/gm, THFP was 18x103±1.631x103cfu/gm and TPSMP was
51x103 ±1.078 x103cfu/gm (Table 3.01).
Table 3.01 THBP, THFP and TPSMP in the field soil (CFU/ml).
Sampling stage THBP THFP TPSMP

Initial soil sample 16±2.180 18±1.631 51±1.078
THBP - Values are expressed as counts x106 cfu/gm

THFP&TPSMP - Values are expressed as counts x103 cfu/gm
3.2.2 Analysis of THBP, THFP and TPSMP at various stages on biodigested

slurry applied field (CFU/ml)
The soil sample analysis in the seedling stage revealed that the control field
contained the lowest THBP of 21x106±0.360 x106cfu/gm followed by inorganic ma-
nure applied field which contained THBP of 27x106 ±0.264 x106cfu/gm. The highest
THBP was observed in the SWPS+PSO+RP applied field (46x106±0.264cfu/gm) fol-
lowed by SWCS+PSO+RP (42x106±1.331cfu/gm), PSO+RP (39X106±0.984 x106
cfu/gm), SWPS (39X106±700x106cfu/gm), OWCS+PSO+RP (37X106 ± 2.853x106
cfu/gm), SWCS (37X106±0.173 x106cfu/gm) and OWCS (32X106±0.400x106cfu/gm)
(Fig 3.01). The TPSMP revealed that the maximum load seen in the field which con-
tain SWPS+PSO+RP (99x103±1.637 x103cfu/gm) and the minimum load was ob-
served in the inorganic manure applied field (23x103±0.360x103cfu/gm). The PSO
92
load was decreased marginally compared with initial raw soil sample analysis
(23x103±1.005 x103cfu/gm (Fig 3.03).
The analysis of microbial load revealed that the maximum load of THBP oc-
curred in the flowering stage (112x106±2.358 x106cfu/gm) of SWPS+PSO+RP ap-
plied field and the minimum load was observed in control field of seedling stage
(21x106 ±0.360 x106 cfu/gm). The highest THFP were observed in OWCS+PSO+RP
(117x103±2.117 x103cfu/gm) (Fig 3.02) applied field on flowering stage and the low-
est THFP was seen in control field of seedling stage (22x103 ±0.655 x103cfu/gm) and
the maximum TPSMP was found in the final stage of SWPS +PSO+RP applied field
(172x103±2.707 x103cfu/gm) and the minimum TPSMP was seen in inorganic manure
applied field of seedling stage (23x103 ±0.360 x103cfu/gm) (Fig 3.03).
From the above results it was clear that there was considerable increase in the
load of THBP, THFP and TPSMP from initial stage of soil analysis to final stage. Al-
so there is a steep increase of both THBP and TPSMP was observed in
SWPS+PSO+RP applied field, where as fungal (THFP) were more in
OWCS+PSO+RP manure applied field.
120
100
Bacterial load ( x106 cfu/ml)
80
60
40
20
Biodigested manurial sources

Seedling stage Pre flowering Flowering stage Final stage
Fig 3.01 THBP at various stages on various biodigested manorial sources

(cfu/ml)
93
Fig 3.02
2 THFP at various stages on various biodigested manorial sources
(cfu/ml)
Fig 3.033 TPSMP at various stages on various bio

biodigested
digested manorial sources
(cfu/ml)
94
3.2.3 Analysis
sis of soil NPK (Initial Stage)
The NPK level of the soil was analysed before and after application of m
ma-
nures. There was marked decrease in the amount of initial NPK to final indicated the
considerable utilization
ation of NPK in the soil by plants for their growth and yield (Fig
3.04). However the soil microbial population was not affected very much under var
vari-
ous manurial sources.
Morphological and biochemical parameters of chilly plant at different stages

of growth
th were carried out and were listed. Periodically the chilies were collected and
weighed individually in each plot. The cumulative yield of each of plot was recorded
and final value was expressed as tons per hectare. There was progressive increase in
the height
eight and dry matter content of the plant which was more pronounced in plots
applied with RP and PSO enriched biodigested slurry than others.
Fig 3.04 Soil NPK content
95
3.2.4 Influence of biodigested manurial sources at pre-flowering stage
In pre flowering stage the root length, shoot length, total height, wet weight,
dry weight and chlorophyll content were more in the plants cultivated the field con-
taining SWPS+PSO+RP as biodigested organic manure compare to other biodi-
gested organic amendments (Table 3.02 ).
Table 3.02 Influence of biodigested manurial sources on the morphological parameters and
chlorophyll content of chilly grown at pre-flowering stage.
Pre-flowering stage
Manurial sources Root Shoot Total Wet Dry Chlorophyll
length(cm) length(cm) height(cm) weight(g) weight(g) content(mg/lit)
CONTROL 4.0±0.100 6.0±0.889 10.0±0.557 6.2±0.300 0.790±0.100 0.141±0.009
SWPS+PSO+RP 5.0±0.200 6.8±0.721 11.8±1.058 6.8±0.755 0.820±0.006 0.182±0.004
SWCS+PSO+RP 4.9±0.265 6.2±0.436 11.1±1.014 6.0±1.014 0.610±0.010 0.158±0.008
OWCS+PSO+RP 4.6±0.100 6.1±0.793 10.7±0.656 5.9±0.900 0.620±0.004 0.142±0.004
PSO+RP 4.7±0.173 6.3±0.458 11.0±0.917 6.1±0.917 0.630±0.005 0.162±0.009
SWPS 4.8±0.100 6.0±0.700 10.8±0.700 5.8±0.954 0.600±0.007 0.146±0.004
SWCS 4.9±0.100 6.0±0.917 10.9±0.781 5.6±0.721 0.690±0.011 0.145±0.001
OWCS 4.8±0.721 6.4±0.458 11.2±1.200 5.9±0.900 0.690±0.003 0.158±0.002
INORGANIC 4.2±0.346 6.2±0.265 10.4±0.458 5.8±0.755 0.660±0.005 0.167±0.005
Each value is expressed as Mean±Standard Deviation.
3.2.5 Influence of biodigested manurial sources at flowering stage
In flowering stage the maximum wet weight (12.6±0.079g), dry weight

(0.818±0.006g) and chlorophyll content (0.586±0.008mg/lit) were seen in
SWPS+PSO+RP applied field. The longest root length (9.8±0.700cm) and total height
(44±1.637cm) were observed in SWPS treated field and the longest shoot length
(36.9±0.700cm) was seen in SWCS+PSO+RP applied field (Table 3.03).
96
chlorophyll content of chilly grown at flowering stage.
Flowering stage
Root Shoot Total Wet weight(g) Dry Chlorophyll
Manurial sources length(cm) length(cm) height(cm) weight(g) content(mg/lit)
CONTROL 8.8±0.080 15.3±0.265 24.1±0.964 4.553±0.056 0.491±0.008 0.426±0.007

SWPS+PSO+RP 9.3±0.436 33.2±0.361 42.7±0.721 12.600±0.079 0.818±0.006 0.586±0.008
SWCS+PSO+RP 6.9±0.854 36.9±0.700 43.8±0.755 9±0.075.180 0.710±0.006 0.500±0.010
OWCS+PSO+RP 6.8±0.857 24.2±0.755 31.0±1.039 5.435±0.022 0.344±0.004 0.512±0.009
PSO+RP 6.4±0.608 36.4±0.781 42.8±0.555 9.260±0.021 0.696±0.011 0.486±0.005
SWPS 9.8±0.700 34.2±0.854 44.0±1.637 9.881±0.055 0.726±0.003 0.486±0.008
SWCS 9.1±0.265 23.6±0.265 32.7±0.656 7.381±0.016 0.714±0.005 0.526±0.006
OWCS 7.4±0.361 21.1±0.300 28.5±0.656 7.320±0.019 0.572±0.007 0.482±0.015
INORGANIC 11.2±0.200 25.4±0.872 36.6±0.654 9.433±0.014 0.618±0.002 0.448±0.002
3.2.6 Influence of biodigested manurial sources at final stage

In final stage of field plant analysis, the highest root length (16.2±1.058cm), wet
weight (20.3±0.092g) and dry weight (0.989±0.009g) of plants were seen in SWPS+PSO+RP
treated plots and the longest shoot length (42.3±0.721cm), total height(56.5±0.608cm) and
chlorophyll content (0.482 mg/lit) were more in SWCS+PSO+RP applied field (Table 3.04).
chlorophyll content of chilly grown at final stage.
Final stage
Root Shoot Total Wet Dry Chlorophyll
Manurial sources length length(cm) height(cm) weight(g) weight(g) content(mg/lit)
(cm)
CONTROL 10.4±0.458 18.6±0.557 29.0±1.039 6.21±0.046 0.52±0.006 0.397±0.009
SWPS+PSO+RP 16.2±1.058 39.9±1.136 54.1±0.954 20.30±0.092 0.989±0.009 0.473±0.007
SWCS+PSO+RP 14.2±1.311 42.3±0.721 56.5±0.608 18.37±0.101 0.823±0.008 0.482±0.012
OWCS+PSO+RP 12.1±0.361 29.2±0.872 41.3±1.153 9.610±0.019 0.473±0.007 0.394±0.005
PSO+RP 10.6±0.557 41.6±0.656 52.2±1.323 14.320±0.019 0.812±0.020 0.372±0.009
SWPS 13.4±0.608 40.1±0.625 53.5±0.755 18.012±0.024 0.813±0.012 0.353±0.012
SWCS 11.5±0.700 28.4±0.755 39.9±0.529 12.637±0.012 0.807±0.010 0.472±0.011
OWCS 10.2±1.200 27.6±0.917 37.8±0.800 14.374±1.151 0.721±0.017 0.363±0.015
INORGANIC 12.7±0.608 34.4±0.794 47.1±0.954 19.642±0.037 0.898±0.015 0.382±0.019
97
98
99
100
3.2.7 Effect of biodigested manurial sources on the NPK content of chilly grown
at different stages of growth
The NPK content of chilly plant was also increased from seedling stage (Table
3.08) to flowering stage and declined in the final stage of its growth due to the utiliza-
tion of these elements for the yield of the chilly (Table 3.09).
Table 3.08 Effect of biodigested manurial sources on the NPK content of chilly plant
at seedling stage.
STAGE N(%) P(%) K(%)

SEEDLING STAGE 1.262±2.608 0.28±0.008 0.26±0.609
Table 3.09 Effect of biodigested manurial sources on the NPK content of chilly
grown at different stages of growth.
Pre-flowering (%) Flowering (%) Final (%)

Manurial sources N P K N P K N P K
CONTROL 2.126±0.018 0.180±0.002 0.32±0.010 2.760±0.029 0.32±0.010 0.30±0.020 2.4±0.100 0.28±0.026 0.28±0.010
SWPS+PSO+RP 2.826±0.037 0.192±0.004 0.38±0.017 3.845±0.014 0.48±0.017 0.32±0.010 2.8±0.100 0.39±0.010 0.28±0.020
SWCS+PSO+RP 1.926±0.013 0.168±0.007 0.34±0.017 2.768±0.017 0.39±0.020 0.29±0.017 1.9±0.173 0.28±0.017 0.24±0.017
OWCS+PSO+RP 2.126±0.009 0.178±0.005 0.28±0.010 3.000±0.065 0.40±0.026 0.28±0.026 2.8±0.264 0.38±0.026 0.22±0.026
PSO+RP 1.898±0.026 0.174±0.004 0.32±0.017 2.868±0.048 0.38±0.010 0.28±0.017 1.8±0.100 0.26±0.017 0.26±0.906
SWPS 2.000±0.027 0.168±0.005 0.26±0.012 2.918±0.009 0.42±0.017 0.29±0.010 2.6±0.200 0.36±0.030 0.24±0.026
SWCS 2.128±0.008 0.168±0.006 0.30±0.026 2.926±0.015 0.41±0.020 0.31±0.017 2.4±0.115 0.38±0.017 0.26±0.602
OWCS 2.326±0.039 0.188±0.005 0.36±0.020 2.628±0.017 0.36±0.017 0.31±0.026 2.6±0.264 0.29±0.020 0.28±0.026
INORGANIC 2.300±0.035 0.168±0.004 0.28±0.010 3.257±0.010 0.38±0.017 0.32±0.010 2.8±0.100 0.32±0.017 0.29±0.017
3.2.8 Effect of biodigested manurial sources on the yield of chilly
Regarding the cumulative yield of chillies the maximum was recorded in rock
phosphate and phosphate solubilizing organisms enriched sewage water biodigested
poultry droppings slurry (6.138 tons/hectare) followed by SWCS+ PSO+RP
(5.898tons/hec), OWCS+PSO+RP(5.268ton/hec), PSO+RP (5.260tons/hec), SWPS
101
(4.928tons/hectare), SWCS(4.823tons/hectare),OWCS (4.262 tons/hectare), inorganic
fertilizer (4.100tons/hectare) and finally control (2.826 tons/hectare) ((Fig 3.005).
Therefore the biodigested slurries obtained from biogas plant enriched with
the rock
k phosphate and phosphate solubilizing organisms gave better yield than the
biodigested slurries applied without enrichment.
Manurial sources
CONTROL SWPS+PSO+RP SWCS+PSO+RP OWCS+PSO+RP PSO+RP
SWPS SWCS OWCS INORGANIC
Fig 3.05
5 Effect of biodigested manurial sources on the yield of chilly (Tones/Hectare)
102
103
3.3 DISCUSSION
In the two types of field trials on chilly plants, the biodidested slurry enriched
with PSO and RP gave more yield than the non enriched one. It clearly emphasized
the importance of phosphate solubilizing organisms in the solubilization of rocked
phosphate. In addition SWPS+PSO+RP gave maximum yield (5.268tons/hectare) fol-
lowed by SWCS+PSO+RP and others. Moreover the analysis of THBP, THFP and
TPSMP before and after the application of biodigested manure in the field revealed
that the biodigested application gave more THBP, THFP and TPSMP and found to be
increased their number from seedling stage and attained the maximum count in the
flowering stage because of the free and maximum availability of the nutrients.
Due to enormous rise in the cost of chemical fertilizers and health hazards has
made to think of alternative source of fertilizers in increasing crop production (Blatt et
al., 1980; Logakanthi et al., 2006). Thomson et al., (1992) suggested that high seed P
concentration is consistently promote early plant growth and increase final yield when
plants are exposed to environmental stresses including disease or water scarcity. In
order to fulfill the theme of action ‘Lab to land” process, the biodigested slurry pro-
duced after biogas production were applied with and without the enrichment of RP
and PSO to the chilly cultivated plots. The different biochemical and morphological
characters of plants and effect of these amendments on soil micro flora and NPK were
studied.
The possibility of greater utilization of indigenously available rock phosphate

resources by the action of phosphate solubilizing microorganisms has been empha-
sized by Blatt et al.,(1980);Gaur et al., (1980);Logakanthi et al.,(2006). In this con-
nection field experiments have been carried out in India using culture suspension of
B.polymyxa, B.circulans, P.striata with and without rock phosphate on the yield of
wheat and rice revealed that significant increase in the grain yield Dhakshinamoorthy
and Murugappan, (1996). In the present study also, the Rock Phosphate and Phos-
phate solubilizing organisms (RP+PSO) enriched biodigested slurry gave better yield
than non enriched biodigisted slurry and inorganic fertilizer applied field.
104
Phosphorus is important in the early growth of plants because its deficiency
depresses tillering and root development which inturn leads to the decrease of grain
yield (Yoshida, 1981; Ascher et al., 1994). In the present experiment this was con-
firmed by the poor growth and low yield of control chilly plants which were not sup-
plied by any other amendments.
The effect of phosphorus on plant growth has been studied in a number of spe-
cies other than chilly. De Marco, (1990) observed that germination was more rapid
from wheat seed with P concentration of 0.20% than from seed with a P concentration
of 0.14%. Thomson and Bolger, (1993) from their experiment revealed that in subter-
ranean clover, seed with P concentration of 0.75% produced faster emergence of
seedlings with a larger number of leaves and higher shoot growth than seedling grown
from seed with a concentration of 0.48%. Similarly in the present field trial also the P
enriched biodigested slurry containing plot have shown more yield when compared to
other non enriched biodigested slurries and inorganic manures applied field.
Bolland et al., (1989); De Marco, (1990); Zang et al., (1990);Thomson et al.,

(1992); Thomson and Bolger, (1993) revealed that the presence of increased amount
of P concentration plays an important role in seedling shoot growth, large number of
leaves and taller plants of crops. In this work also the highest root length, shoot
length; total height, dry matter and chlorophyll content are more in RP+PSO enriched
biodigested biogas slurry than others.
The greater root length and larger number of roots grown from high P fertiliz-
er application with phosphate solubilizing organisms were reported by Barber and Si-
berbush, (1984).These organisms may allowed the seedlings to acquire more P and
other nutrients from the soil and from the fertilizer sources (Ascher et al., 1994).
Scott, (1989) revealed that high seed P concentration, seed coating, with fertilizer or
nutrient solution are effective alternatives to improve the plant growth and yield pro-
duction.
Among 9 field trails SWPS+PSO+RP gave more yields when compared to

other amendments. This result coincided with the result of Rasal, (1996), where the
applications of phosphate solubilizers as seed inoculation with recommended doses of
105
phosphotic fertilizer increased the nodulation, dry matter, weight of plants and P up-
take in green gram significantly over control.
The seed inoculation with P solubilizing microorganisms increased the availa-

ble P in soil and increased its uptake by crop plants were published by Vidyasekaran
et al., (1973).Tomer et al., (1996) in their field experiment, judged the efficiency of
PSB with various sources and levels of phosphorus on the production of gram (Cicer
arietinum). The PSB inoculants enhanced grain yield by 2.37Q/ha (10.7%). Among
the P sources, rock phosphate+pyrite+PSO provided best result. In the present work
also SWPS+PSO+RP provided good yield when compared to other fertilizer applied
in the field.
Jisha and Mathur, (2005) revealed that higher wheat yield was obtained when
wheat crop was cultivated in the field containing RP and PSO as inoculant. Inocula-
tion effect of these isolates on yield and nutrient uptake of wheat crop was also stu-
died and found to be significant. Inoculation of P solubilizers with rock phosphate
gave higher yield and this was true in the present work, because the field enriched
with RP+PSO along with biodigested slurry gave well morphological, physiological
and yield than other fields. Similar results were found in Biennial research report;
(2004-2005) in that the increased growth of tomato and yield were obtained by the
application of rock phosphate enriched bio digested slurry. The application of bio di-
gested slurry as biofertilizer would not only increase the crop production but also di-
minish the environmental pollution.
Application of organic manure to the soil improves water holding capacity, re-
sistance to soil erosion and maintenance of soil micro flora (Bhattacharya et al., 1990;
Das et al., 2001). Such advantages are not observed with the application of inorganic
fertilizer (Singh, 1994).
In the present field trial the different morphological characters like root length,
shoot length, total height, wet and dry weight of chilly plant in different bio digested
slurry applied plots at various stages of growth were analyzed and tabulated (Table
3.02,3.03 &3.04). The height of the plant varied from 10cms to 56.5cms, the height
being maximum during final stage, the root length and shoot length in pre flowering
106
stage ranged from 4.0cm - 5.0cm and 6.0cm-6.8cm. In flowering stage both these
ranged from 6.4cm- 11.2cm and 15.3cm-36.9cm. Whereas in final stage the root
length ranged from 10.2cm to16.2cm and the shoot length ranged from 18.6cm to
42.3cm.
In the present study the wet weight of the plant ranged from 5.6g-19.6g per
plant and the dry weight from 0.6g to 0.9 g/plant. The morphological characters were
observed to be significant in the SWPS+PSO+RP treated and SWCS+PSO+RP
treated fields. Maximum growth characteristics being observed in final stage due to
the uptake of nutrients for a better yield. Similar results of increased morphological
characters were observed by Mazeen and Van Holm, (1989) in their works on bean
and maize field applied with organic manure enriched with RP and phosphate solubi-
lizing organisms.
The dry matter content of the chilly plant at various stages of growth with dif-
ferent manurial applications showed commensuration results which are similar to that
of Status report, (1984-1989). This indicated similar finding on the ground nut grown
field which had used organic and chemical fertilizer. In the present work the observa-
tions of biochemical parameters like chlorophyll, nitrogen, phosphorus and potassium
content of the plant at various stages were analyzed. The chlorophyll content in the
chilly leaves showed a increasing trend upto flowering stage from 0.141mg/lit in pre
flowering stage of control to a maximum of 0.586 mg/lit in the flowering stage of
SWPS+PSO+RP applied plot. In the final stage there was a slight reduction in the
chlorophyll content. The reduction in chlorophyll content in the final stage may be
due to the diversion of plant action towards yield and also due to the decreased photo-
synthetic activity. Similar findings were reported in Bineal Research Report (2004-
2005) in the brinjal cultivation, applied with biogas effluent and chemical fertilizer.
The NPK contents were found to be high during the flowering stage. These
contents were observed to be high in most cases in chilly plants grown in plots ap-
plied with SWPS +PSO+RP treated, SWCS+PSO+RP treated and inorganic fertilizer
treated plots. Reduction in NPK during final stage may be attributed to the growth
yield and fruiting. Research-report, (1991-1993) indicated that NPK nutrients would
have been used for food synthesis on the production of tomato, chilly and brinjal
107
when applied with organic and chemical fertilizer. Similar observations were also
made in our study also.This was again confirmed by Baqual and Das, (2006) in their
work on co-inoculation of mulberry with phosphate solubilizing microorganisms, ni-
trogen fixing bacteria has influenced its macronutrients uptake through leaf.
Results of soil NPK before and after cultivation of respective plots were tabu-
lated. In leaves, the NPK concentration considerably decreased in all treatments after
cultivation. The N2 content decreased from a maximum of 399.4kg/hectare (before the
application of manures and cultivation of crops) to 308.2kg/hectare (Fig 3.04). The
phosphorus content decreased from an initial of 22.03kg/hectare to 20.03kg/hectare.
Potassium also decreased from its initial level of 18.576kg/hectare to 16.532
kg/hectare. These results coincided with increased uptake of nitrogen by plants and
high crop yield, since the organic fertilizers are readily soluble; they are easily ab-
sorbed by the plants.
Kaur et al., (2005) compared the change of chemical and biological properties
in soil receiving FYM, poultry manure and sugarcane filter cake alone and in combi-
nation with chemical fertilizers for the cultivation of pearl millet and wheat which
changed the soil NPK level and gave the better yield. Like that in this work there is a
notable change in the NPK content before and after the application of biodigested
slurry both in enriched form and non enriched form and they gave better yield when
compare to inorganic and control field.
In chilly crop yield, the maximum were observed in organic fertilizer applied
plots. Comparatively good yields in the different biodigested slurry applied plots were
of the following order. 1.SWPS +PSO+ RP, 2.SWCS+PSO+RP, 3.OWCS+ PSO+ RP,
4.PSO+RP,5.SWPS,6.SWCS, 7.OWCS, 8.Inorganic manure and 9.Control.
The maximum chilly yield of 6.138 tons/hectare was got from the
SWPS+PSO+RP applied plot followed by SWCS+PSO+RP treated plot (5.898
tons/hectare).The minimum yield was obtained from control plot (2.826 tons/hectare)
(Fig 3.05). The quality and quantity of the product can be increased on the application
of biofertilizers especially biodigested slurries obtained from biogas plants after bio-
gas production as applied over here. Application of vermicompost and compost has
108
also been stressed as a good biofertilizer by Ranganathan and Christopher, (1996).
Since the addition of organic manure on soil has so many advantages over the inor-
ganic fertilizers. The usage of biofertilizer is thus preferable and advisable.
The quantitative data on the enumeration of bacterial (THBP), fungal (THFP),

phosphate solubilizing organisms (TPSMP) from chilly cultivated plots observed dur-
ing different stages of growth. The activity of soil organisms is very important for en-
suring sufficient nutrient supply to the plants. If the microorganisms find suitable
conditions for their growth they can be very efficient in dissolving nutrients and mak-
ing them available to plants (Sundara et al., 2002). Throughout the studies it was
noted that the microbial biomass was increased considerably from initial stage analy-
sis to final stage. This indirectly indicated the good growth of microbes which could
solubilize the nutrients in the yield very well and supplied the nutrients to the plants,
inturn they gave good yield when compared to control.
The highest count of THBP, THFP and TPSMP were noted in the flowering
stage of chilly plants, because of the free and maximum availability of nutrients.
Chhonkar and Subba rao, (1967); Illumer, (1995) also observed the increased bacteri-
al population from the organic manure applied fields. The THBP, THFP and TPSMP
were found to be less in soil treated with inorganic fertilizer and control. The results
of the present study confirmed that the soil microbial population was severely affected
on the application of inorganic fertilizer while the same increased in the biodigested
slurry applied plots. Increased micro floral activity increases the stability of the soil;
similar findings have also been outlined in Research report (1991-93) on brinjal culti-
vation plots that were applied with biogas slurry and urea. Similar phenomenon was
also documented by Bhattacharya et al., (1990); Arti Dave and Patil, (1999); El Kra-
many et al., (2007).
Recently the use of bioorganic fertilizers in agriculture has received consider-

able attention because of the environmental problems associated with alternative dis-
posal methods and the harmful affect on human health. On the other hand, bioorganic
fertilizers improves the physical properties of the soil, increasing water holding capac-
ity, preventing nutrient leaching and adding more mineral nutrients to the poor sandy
soil.
109
So based on the findings it is clearly revealed that plots applied with biodi-
gested slurry enriched with RP+PSO gave very good contributions than inorganic and
control field. This emphasized that chemical fertilizers can be replaced by the bioferti-
lizers which could be eco friendly one and the application of enriched biodigested
slurry as commercial use in agriculture is a multidisciplinary process requiring both
empirical and fundamental studies. Also this enriched biodigested slurry help in main-
taining the ecological balance, and soil micro flora and this practice can conveniently
be adopted by the farmers and will be cost effective besides being eco friendly.
110
CHAPTER IV
111
Tamil Nadu, India.
Chapter IV
4.0 BIODIGESTED SLURRY AS BIOLOGICAL CARRI-

ER FOR RHIZOBIUM PHASEOLUS.
4.0.1 Introduction
Majority of crop plants depend upon nitrate or ammonia for their ni-
trogen sources but many legumes that form root nodules can reduce atmospheric ni-
trogen in symbiosis with rhizobia (Allen and Allen, 1981). Legume crops require a
large amount of ‘N’ for seed protein synthesis. The ‘N’ is derived from symbiotic N2
fixation with soil microorganisms in addition to soil mineral nitrogen. Biological Ni-
trogen Fixation (BNF) refers to the process of microorganisms fixing atmospheric ni-
trogen mostly within subsoil plant nodules, and making it available for the assimila-
tion by plants is economically sounder and environmentally more acceptable than
chemical nitrogen fertilizer use in agriculture (Halliday, 1982). Rhizobium is the most
studied and important genera of nitrogen fixing bacteria (Hannington Odame, 1997).
Promotion of N2 fixation by inoculation of highly efficient Rhizobium

strain and improvement of soil management and cropping practice is very important
to increase seed yield of leguminous crops especially in Asia (Unkovich and Pate,
2000).
4.0.2 Bio fertilizers
Fertilizers supply essential plant nutrients, mainly nitrogen (N), phos-

phorus (P) and potassium (K). They are removed in large quantities from the soil by
each successive harvesting. Among the fertilizer elements, nitrogen and phosphorus
play key role in plant growth and development. But their application is not fulfilled a
satisfied dosage due to their alarming cost and inadequate availability (Baqual and
Das, 2006). In intensive cropping system, supplementing soil nutrients by the use of
112
chemical fertilizer is considered inevitable for obtaining optimum yield of crops. But
it has been observed that continuous use of chemical fertilizers may affect the soil
health and may lead to negative impact on soil productivity (Indu Paul and Savithri,
2003). Increasingly high inputs of chemical fertilizers for high yield agricultural dur-
ing the last 150 years has not only left our soils degraded polluted and less productive
but also posed severe health hazards (Alok adhobya and Deepak, 2003).
Higher N application may results in NO3 pollution of ground water (Mytton,

1993; Shrestha and Ladha, 1998) soil acidification (Kennedy and Tchan, 1992), dis-
ruption of nutrient balance in soil, leaching of nutrients into ground water and pollu-
tion in surface water and increased denitrification resulting in higher emission of N2O
to the atmosphere which may impact global warming (Bronson et al., 1997). In addi-
tion the burning of fossil fuels to manufacture N fertilizers is a source of hazardous
byproducts that pose a threat to human health and environment (Vitousek et al.,
1997).These problems have renewed public interest in exploring alternate or supple-
mentary nonpolluting sources of N and the need of bacterization to increase produc-
tivity in legumes for agriculture (Ladha et al., 1997; Sindhu and Daderwal, 2000).
The use of biofertilizers has been reported to be beneficial for the cultivation
of vegetable and cereals. (Mehrotra and Lehri, 1971; Dewan and Rao, 1979; Venka-
teswarlu and Rao, 1983; Hadas and Okon, 1987; Nair and Naja Chandra, 2001). There
is ample scope for increasing production through the use of organic fertilizers (Mad-
hiyazhagan et al., 2001). India is the third largest producer and consumer of fertilizer
in the world (after China and USA) accounting for 12% of world population of N and
P nutrients and 12.6% of world consumption of NPK nutrients (Alok adohobya and
Deepak, 2003). In recent years, biofertilizers have emerged as promising component
of integrated nutrient supply system in Indian agriculture.
4.0.3 Microflora
Due to excessive use of chemical fertilizers and plant protection chemicals the
rhizosphere micro flora has been affected greatly and because of it, in the place of the
beneficial associative bacteria, the harmful types pre occupy the rhizosphere (Nagina
Parmer and Dadarwal, 1997). Nitrogen fixing bacteria not only provides the nitrogen
but produce a variety of growth promoting substances like acetic acid, gibberellians,
113
B vitamins and anti fungal substances (Rao, 1986; Sindu and Lakshminarayana,
1986).
Biofertilizers are the products containing living cells of different types of mi-
croorganisms which have an ability to solubilize nutritionally important elements
from non-usable substrates. Among biofertilizers Rhizobium, Azotobacter, Azospir-
llum, blue-green algae, Azolla, P-solublizing microorganisms and mycorhizae are im-
portant (Hedge et al., 1999). Use of farmyard manure, green manure and other organ-
ic manures are fertilizers enhance the benefits from inoculation (Wani et al., 1985;
Wani et al., 1988). Application of livestock manure and inorganic fertilizer yield high
value crops (Reardon and Vosti, 1997). Compost utilization as manure is becoming
wider spread during recent years as a consequence of the rise in price of chemical fer-
tilizers (Mekki and Amal ahmed, 2005).
4.0.4 Carriers
Microbial inoculation involves the selection and multiplication of beneficial

microorganisms and applying them to plants, seed or soil (Davison, 1988). The use of
carrier based rhizobial cultures in legume has been widely recognized, especially in
the areas where indigenous nodulation has been found to be inadequate (Prabaharan
and Ravi, 1996). The money can be saved by the marginal farmers of India when they
obtained the quality tested inoculants on the farm (Subba Rao and Tilak, 1977). In-
corporation of organic amendments to the soil is very much useful (Rajput et al.,
1993).
Incorporation of microorganisms in carrier material enables easy handing,

long term storage and high effectiveness of biofertilizer Nagarajan and Balachandar,
2001). Among various types of bioferilizers, bacterial inoculant is one major group
which includes rhizobia, nitrogen fixing rhizobacteria, plant growth – promoting rhi-
zobacteria, phosphate solubilizing bacteria and so on. Biofertilizers are usually pre-
pared as carries based inoculants containing effective micro organism (FAO, 1977).
Various types of carrier materials like peat, lignite, charcoal, farmyard manure
are used for bioinoculants preparation. In addition to these coconut shell powder, cof-
114
fee husk, press mud or combination of such materials with soil and charcoal have also
been found suitable as carriers (Tilak and Subba Rao, 1978). The changes in the mi-
crobial community, composition of soil using carriers can be brought improved man-
agement practices (Baath et al., 1995; Frostegard et al., 1996, 1997; Zelles et al.,
1996). For preparation, these carrier materials are milled to fine powder (Somasegarn
and Hoben, 1994). Carrier based inoculants have almost completely replaced liquid
cultures and agar slope cultures (Abaidoo et al., 1990; Wiersma and Orf, 1992).
In USA, sedge peat is ground to fine powder and used as carrier for inoculants
(Burton, 1967). In Australia milled dry peat is packed in sealed polythene bags used
as biocarrier (Date, 1974). In India, peat like material available in the Nilagiri Vally
has been found to be a good carrier (Iswaran et al., 1969). Lignite is another carrier
which is widely used (Kandasamy and Prasad, 1971) in south India.
In India, the Indian Standards Institution has evolved methods to check the
quality of inoculants and issue ISI marks to qualified producers. The standard in New
Zealand is 100 million rhizobia (108) per gram of peat where as in Russia the standard
is range from 50 to 100 million rhizobia per gram of the carrier (Burton, 1967). The
carrier based inoculation successes at field levels have been well documented by
many scientists from time to time (Taha et al., 1967; Subba Rao, 1976; Vojinovic,
1976; Ham et al., 1976; Hera, 1976; Subba Rao and Tilak, 1977).
4.0.5 Rhizobium
Rhizobium is a common soil bacterium. Rhizobium is not toxic to humans,

plants or animals (Somasegaran and Hoben, 1994). It is one of the most beneficial
bacteria in agriculture. The native Rhizobium is probably not in sufficient numbers to
adequately nodulate the crop or may not be efficient in fixing nitrogen. Inoculation is
inexpensive and Rhizobium will spread to nearby fields after years of legume produc-
tion (Altas and Bartha, 1998). On different bacterial (microbial) fertilizers Rhizobium
inoculant, specific for different leguminous crops is the most important one (Hedge et
al., 1999). Rhizobium species have been used as legume inoculatns world wide to
supply fixed nitrogen to leguminous plans (Sindhu and Dederwal, 2000). Legume in-
oculants occupy the prime position among biofertilizers because of the agronomic
115
importance of legume crops (Sharma et al., 1997). The legume – Rhizobium symbi-
osis is the most significant source of N for agriculture (Belts and Herridge, 1987).
Rhizobia, soil bacteria have the ability to fix atmospheric nitrogen in symbiotic asso-
ciation with legumes. They normally enter the root hairs, multiply there and form no-
dules. The amount of nitrogen fixed varies with the strain of Rhizobium, the plant spe-
cies and environmental conditions (Allen and Allen, 1961). Members of the genera
Rhizobium and Bradyrhizobium inhabit the legume rhizosphere (Mahler and Wollum,
1982).
The most problematic environments for rhozobia are marginal lands with low
rain fall, extremes of temperature, acid soils of low nutrient status and poor-water
holding capacity (Brockwell et al., 1995). Rhizobium can survive at low tempera-
0
tures and tolerate temperature up to 50 C for more than few hours. It is sensitive in
plant productants, antibiotics and other agricultural chemicals (Nutman, 1965). The
bacterium can survive in soil for several years under dry storage conditions (Sen and
Sen, 1958). The development of nitrogen fixing nodules takes place in a series con-
trolled by genes of Rhizobium and host legumes (Sharma et al., 2000).
The success of biofertilizer containing rhizobia to form nodules depends on

their ability in colonize the soil and rhizosphere and then to form nodules in comple-
tion with the indigenous strains (May and Bohlool, 1983; Williams and Phillips, 1983;
Thies et al., 1991). The growth and production of Rhizobia depends on the carrier suit
abilities and soil nature (Iede Mends and Peter Bottomely, 1998). The symbiotic inte-
raction between rhizobia and leguminous plants is often very host specific (Hadri et
al., 1998). Several soil microorganisms and bacteriophages are known to inhibit the
growth of rhizobia (Chhonkar and SubbaRao, 1966; Sethi and Subba Rao, 1968; Sub-
ba Rao et al., 1972, 1974).
Therefore an effort was undertaken to study the importance of Rhizobium on

leguminous plants in the form of biodigested organic carrier based inoculums ob-
tained from biogas plant, also to find out, whether the biodigested organic carrier ma-
nures influenced the growth and propagating ability of the inoculated rhizobial culture
in it.
116
The objectives of the present work were as follows:
• To Isolate R.phaseoli from the healthy root nodules of Phaseolus vulgaris

(green gram legume nodules) using CRYEMA (Congo Red Yeast Extract
Mannitol salt Agar).
• To confirm R.phaseoli by biochemical examinations.
• To mass cultivate R.phaseoli and to prepare carrier based inoculam by using

biodigested slurries as organic carrier manure obtained from biogas plant.
• Two different environmental temperatures were provided (Refregiration tem-

perature at 6±2oC and room temperature at 28±2oC) to determine the high vi-
able load of rhizobial culture on various biodigested slurries which were used
as organic carrier manure. The multiplication of organisms in carriers were
analysed for every 10 days interval and totally for 90 days.
The following biodigested organic manure were used as carriers,
♦ Ordinary Water treated biodigested Cow dung Slurry (OWCS)
♦ Sewage Water treated biodigested Cow dung Slurry (SWCS)
♦ Sewage Water treated biodigested Poultry droppings Slurry (SWPS)
♦ Mixed form of the above mentioned organic manures (OWCS+ SWCS+SWPS).
117
4.1.1 Isolation of R.phaseolus from P.vulgaris
P.vulgaris leguminous plant was carefully uprooted from the agricultural field
Sankaran koil, Tamil Nadu, India and the root system was washed in running water to
remove the adhering soil particles. Then the shoot portion of the plant was cut off and
the roots with nodules were brought to the laboratory in polythene bags for the isola-
tion of R.phaseolus.
The method of isolation was followed by Kannan, (1996).
• Healthy, unbroken, firm pink nodules were selected and washed in tap water.
• They were immersed in 0.1% acidified HgCl2 for 4-5 minutes for surface steri-
lization.
(Nodules which were surface sterilized with HgCl2 were washed repeatedly with ste-
rile water and dipped in 70% ethyl alcohol followed by more washing with sterile wa-
ter.)
• The nodules were crushed in a mortar by adding small aliquot of sterile water
and with the help of a glass rod. This is called nodule extract.
• Serial dilutions were prepared up to 109 dilutions. The nodule extracts are de-
cimally diluted and aliquots of appropriate dilutions are spread on CRYEMA
(Vincent, 1970; Date, 1974).
• The plates were incubated at room temperature (26±20C) for 3-4 days.
• Large gummy colonies of bacteria were emerged after 5 days incubation .They
were isolated, confirmed, purified and stored. (Plate4.01).
Thus obtained colonies were picked up from the plates purified by adopting
quadrant streak method on the nutrient agar plates. After bacterial growth, they were
stored at 40C for further identification and experimental work.
118
4.1.2 Identification
Isolated rhizobial colonies were subjected to the cultural and biochemical

tests following the scheme recommended by Vincent, (1970). The pure cultures of
R.Phaseolus were transferred to nutrient broth and incubated at 37±20C for 24 hours
with agitation. These fresh broth cultures were subjected to various identification pro-
cedures.
(i) Microscopic Examination
Fresh broth cultures were checked for Gram’s reaction through Gram’s stain-
ing technique. Further, the motile or non motile nature of the isolate was tested with
the hanging drop technique using cavity slides.
(ii) Growth on Peptone-Glucose Agar
Broth cultures were streaked onto the Peptone –Glucose Agar plates (PGA).
(iii) Congo Red Test
This test is performed to differentiate rhizobia from agrobacteria. An aliquot

of 2.5 ml of 1% solution of the dye in water was added to a liter of Yeast Extract
Mannitol agar. On this medium, the strains of nodule bacteria were plated and incu-
bated at 260C for 5 days.
(iv) Hofer’s Alkaline Broth Test
This test was based on the fact that agrobacteria grow at higher pH levels,
while rhizobia are unable to do so. This medium was prepared in pH 11.0 and the
broth cultures of Rhizobium were inocubated for 24 hours.
119
(v) Lactose Agar
Plates were prepared by adding lactose at 10g/litre in agar medium and the
cultures were streaked onto the plates and incubated at 260C for 5 days, and the plates
were flooded with Benedict’s reagent.
(vi) Test for catalase
Hydrogen peroxide solution was prepared (3%) and few drops of this were placed
on a loopful of bacterial culture on a clean glass slide. Appearance of effervescence
demonstrates the cultures as catalase positive.
(vii) Hydrolysis of starch
Starch agar plates were prepared and air dried. Fresh rhizobial broth culture
was streaked on the air-dried plates and incubated at 370C for 24hours. Then Gram’s
iodine solution was flooded over the bacterial growth on the medium. Appearance of
clear zone around the bacterial growth indicates the absence of starch and in other
words the utilization of the substrates.
(viii) Hydrolysis of casein
Skim milk agar plates were prepared and air dried. Fresh rhizobial cultures
were streaked on the air-dried plates and incubated at 370C for 24-48 hours. If the or-
ganism elaborated the exo-enzyme caseinase, which hydrolysis casein, a transparent
zone exists around the bacterial out growth.
(ix) Hydrolysis of lipid
Tween-agar plates were prepared and air dried. Fresh broth cultures was
streaked on the air dried plates and incubated at 370C for 72-96 hours. Appearance of
an opaque zone around the bacterial out growth indicated lipid hydrolysis.
120
(x) Hydrolysis of gelatin
Gelatin agar plates were prepared and air dried. Fresh broth culture was
streaked on the air dried plates and incubated at 370C for 24-48 hours. After the incu-
bation period, the gelatin hydrolyzing activity of the test organism was tested using
mercuric chloride solution, which was flooded on the gelatin agar surface. Appear-
ance of clear zone around the bacterial growth is indicative of gelatin hydrolytic activ-
ity of the chosen bacterium.
(xi) Indole production
The isolated test cultures were inoculated in the peptone broth and incubated
at 37ºC for 24 hours. After incubation 0.2ml of Kovac's reagent was added and the
colour change was noted due to the production of indole. A cherry red colour ap-
peared in the alcohol layer indicated the positive results.
(xii) Methyl Red Test (MR-Test)
The tubes of MR-VP broth was inoculated with the test culture and incubated
at 370C for 48 hours. Following incubation, 5-6 drops of methyl red solution was add-
ed. A bright red colour indicated positive test.
(xiii) Voges-Proskaur test (VP-Test)
The test culture were inoculated in the MR-VP broth and incubated at 370C for
48 hours. After 48hours of incubation one ml of 40% potassium hydroxide (plus crea-
tine) and 3ml of 5% solution of alphanapthol was added in absolute alcohol. A posi-
tive reaction was indicated by the development of a pink colour with 2.5 minutes,
which become crimson red in 30 minutes. The tube was shaken at intervals to ensure
maximum reaction (Plate 4.07).
121
(xiv) Simmons citrate Test
Simmon citrate agar medium was prepared and dispensed in test tubes, steri-
lized at 1210C for 15 minutes and allowed to set as slopes. Te test organisms were in-
oculated on the agar slopes and incubated for 96 hours at 370C. Deep blue colour
change of the medium indicated the positive result (Plate 4.08).
4.1.3 Mass Cultivation of R.phaseolus:
The R.phaseolus isolated from the root nodule of P.vulgari, which is to be in-
oculated in soil as biofertilizers needed to be multiplied in artificial media to harvest
on a large scale, so that it can be supplied to farmers. Before that the stored cultures in
nutrient agar slant at 40C were periodicity checked for its purity by microscopic and
plating methods.
For mass cultivation the cultures were grown in YEM (Yeast Extract Manni-
tol) broth (20ml) at 1% inoculums concentration. This was incubated for 4 days at 26
± 2 0C. The cultures were checked for purity and aseptically transferred to 300ml ste-
rile medium in 500ml conical flask and incubated at 26±20C for 4 days on a rotary
shaker. When the number of rhizobia in the broth has attained the required standard
(not less than 108 /109 cells/ml), it should be added to the carrier materials (Plate
4.02).
4.1.4 Preparation of carrier based inoculum
The biodigested slurries of biogas plant after biogas production in the first
chapter was shadow dried to a moisture level of 5% were used as organic manure car-
riers. These carrier materials were ground into a fine powder then allowed to sieve for
removing any ungrounded soil particles in it. From the sieved the carrier materials
200gms were weighed and collected separately in 500ml beaker. It was mixed with
sterile distilled water with the final moisture content of 35 to 40%. The pH was ad-
justed to 6.8 ± 0.2 by adding CaCO3. Then these moistened carrier materials in the
container were tyndalised at 1000C for about three successive days in order to sterilize
122
completely and to eradicate if any spore formers may present. Then these were cooled
to room temperature (Plate 4.03).
4.1.5 Blending and Curing
For the preparation of carrier based inoculants, the rhizobial culture was
blended with the finely powdered and sterilized carrier. The broth was added to one
third of the carrier materials. Blending was done by thorough mixing of broth cultures
with sterilized carriers in alcohol wiped trays. After mixing the carrier with the broth
culture raw-blended carriers were kept for 24 hours for curing. During this time, the
rhizobia get acclimatized with the carriers.
4.1.6 Packing
After curing, the inoculants were ready to be packed in sterilized polythene

bags because the polythene bags are not allowed to escape moisture from the carriers.
Dispending of inoculants in each polythene bags were accomplished by sterile spatu-
la. The polythene bags were sealed leaving about two-third of space to give proper
aeration to the inoculants. A hole was made at one corner and it was cotton plugged,
so that the air can be filtered and passed through the holes and thus the anaerobic con-
ditions can be avoided (Plate 4.04).
4.1.7 Incubation and Storage
Two sets of the carrier based inoculants were prepared. One set of triplicate
carriers were stored at room temperature (28 ± 20C) (Plate 4.05) and the other set of
triplicate carriers were stored at refrigerated temperature (6±20C). Samples from both
the sets were collected at every 10 days interval and tested for its propagative ability
by adopting standard microbiological procedure of counting rhizobia by serial dilution
and plating on selective CRYEMA media plates (Plate 4.06).
4.1.8 Data Analysis

The average (mean) and standard deviation of all experimental data were cal-
culated and given in the table as mean ±S.D by using Microsoft Excel.
123
4.2 RESULTS
4.2.1 Biochemical Characteristic Features of R.Phaseolus

The cultural and biochemical characteristic features of R.phaseolus were pre-
sented in table 4.01.
Table 4.01 Biochemical characteristics of Rhizobium phaseolus
Biochemical Observations
parameters
Microscopic examination Gram negative rod
Motility Motile
Growth on PGA(Peptone Glucose Very poor growth obtained

Agar)
Congo red test Produce,white,translucent, glistening colonis

evaluated with entire margin.
Hofer’s Alkaline Broth test No growth observed
Lactose Agar +
Catalase test +
Starch hydrolysis -
Casein hrdrolysis -
Gelatin Hydrolysis -
Lipid hydrolysis -
Indole production -
Methyl red test -
Voges-Proskaur test +
Simmons Citrate Agar +
After the preparation of carrier based rhizobial culture it was stored at two dif-
ferent environmental conditions. They were
• Storing at 28 ± 20C (Room temperature)
• Storing at 6 ± 20C (Refrigerated temperature).
124
Every 10 days interval one gram carrier material was taken and they were
tested to determine the viability of rhizobial culture.The overall results revealed that
all carrier manures provided were able to support the growth of inoculated rhizobial
culture both in 28±20C and 6±20C temperature. But among the 4 biodigested organic
manure carriers, the mixed biodigested organic manure carrier supported the maxi-
mum growth and propagation of rhizobial culture in both climatic conditions provided
compared to other carrier manures. Next to mixed carrier manure, SWPS carrier ma-
nure provided the prolonged survival and that was followed by SWCS and OWCS.
4.2.2 Survival of organisms in biodigested carrier manures at room temperature

(28±20C)
When the carrier manures were stored at 28±20C temperature, the mixed carri-
er manure, showed the gradual increase in rhizobial load up to 40th day (Plate 4.05)
and other carrier manures (SWPS, SWCS and OWCS) showed the maximum viability
at 30th day observation.
In mixed cultures, the starting inoculum was enumerated as 11.2x109

±4.02x109cfu/ml, then there was a gradual increase in the viability of rhizobial cul-
ture, maximum survival observed at 40th day (29.3x109±4.01x109cfu/ml), (Plate 4.05).
Then there was a stepwise decrease of rhizobial load from 50th day onwards, and min-
imum organisms seen at 90th day screening (17.3x109± 4.09 x10 9cfu/ml). Next to
mixed carrier manures, SWPS manure gave high viable count compared to other two
carriers. Here the maximum viable count was seen at 30 days (25.4x109 ± 7.01 x109
cfu/ml) and the rhizobial culture started to decline from 40 days onwards
(21.6x109±6.06 x10 9cfu/ml). The minimum load was observed at 90 days (5.4x109 ±
2.01 x109cfu/ml).When SWCS was used as carrier, during the initial day observation
the microbial load was about 7.4x109 ± 3.01 x109cfu/ml, and it obtained the high
countable colonies at 30 days (16.9x109 ± 6.03 x109cfu/ml) then it was gradually de-
creased from 40 days onwards (13.2x109 ± 5.03 x109cfu/ml) and least counted colo-
nies were seen at 90 days observation (2.9 x109±1.09 x109cfu/ml).OWCS carrier ma-
nure able to show only the marginal increase in its rhizobial load. The maximum level
observed at 30 days (13.7x109 ± 4.08x109 cfu/ml) and minimum at 90 days
(1.2x109±0.05x109 cfu/ml)(Table 4.02).
125
Shortly, in the room temperature the mixed biodigested organic manure carrier
supported the growth maximum at 40 days, and other individual carriers started to de-
cline their supportiveness from 40 days onwards. OWCS organic carrier manure
showed less supportive to rhizobial cultures when compared to other carriers.
Table 4.02 Survival of rhizobial cultures in biodigested organic manure carriers

at Room Temperature (28±20C).
DAYS Mixed carrier SWPS SWCS OWCS
1 11.2x10 9 ± 4.02 x10 9 8.9x109 ± 3.01 x10 9 7.4x109 ± 3.01 x10 9 5.2 x109 ± 2.00 x10 9
10 16.3x109 ± 6.01 x10 9 14.1x109 ± 5.06 x10 9 11.9x109 ± 4.07 x10 9 8.7 x109 ± 2.09 x10 9
20 23.7x109 ± 7.07 x10 9 19.8x109 ± 7.05 x10 9 14.6x109 ± 5.01 x10 9 10.6 x109 ± 4.01 x10 9
30 27.6x109 ± 8.06 x10 9 25.4x109 ± 7.01 x10 9 16.9x109 ± 6.03 x10 9 13.7 x109 ± 4.08 x10 9
40 29.3 x109 ± 4.01 x10 9 21.6x109 ± 6.06 x10 9 13.2x109 ± 5.03 x10 9 11.5 x109 ± 4.02 x10 9
50 26.5x109 ± 7.09 x10 9 15.3x109 ± 4.07 x10 9 9.8x109 ± 3.08 x10 9 8.3 x109 ± 3.09 x10 9
60 24.5x109 ± 6.03 x10 9 11.7x109 ± 4.07 x10 9 6.6x109 ± 3.02 x10 9 5.9 x109 ± 2.05 x10 9
70 23.3x109 ± 5.05 x10 9 9.7x109 ± 4.01 x10 9 5.3x109 ± 2.02 x10 9 4.2 x109 ± 2.00 x10 9
80 20.1x109 ± 5.01 x10 9 7.8x109 ± 3.09 x10 9 3.2x109 ± 2.06 x10 9 2.4 x109 ± 1.01 x10 9
90 17.3x109 ± 4.09 x10 9 5.4x109 ± 2.01 x10 9 2.9x109 ± 1.09 x10 9 1.2 x109 ± 0.05 x10 9
4.2.3 Survival of organisms in biogested carrier manures at refrigerated temper-
ature (6±20 C)
In refrigerated conditions, all the carrier manures showed minimum number of
rhizobial load compared to incubation at room temperature. But all the carrier mate-
rials supported the rhizobial cultures approximately up to 80 days except OWCS or-
ganic manure carrier where it supported the microbial load only up to 60 days.
In mixed carriers, at this refrigerated temperature the rhizobial propagation

was gradually increased and the maximum survival was occurred at 80 days
(29.7x109±4.06 x109cfu/ml) observation (Plate 4.06). The analysis of 90th day report
revealed that, the rhizobial load was started to decrease (27.6x109±3.03 x109cfu/ml) in
its count. From this study it was clear that, a gradual increase of rhizobial load was
observed from 1st day to 80th day observation.
126
When SWPS and SWCS were analyzed at this refrigerated temperature, both
these carrier showed the maximum viability up to 70 days. There was a stepwise re-
r
duction of bacterial load
d observed in 80 and 90 days of analysis. Among these two
organic manure carrier SWPS manure contained the maximum load of 23.1x109±5.01
x109cfu/ml. This maximum rhizobial count observed in 70days and started to decline
from 80 days (21.6x109 ± 6.06 x109cfu/ml),
u/ml), where as in SWCS carrier manure the
maximum enumerated colonies in 70 days was 22.3 x109 ± 6.04 x109cfu/ml, and they
were also started to decline from 80 days (18.6x109 ± 5.08 x109cfu/ml) onwards. But
17.1x109cfu/ml),
the OWCS carrier manure started to decline from 70 days onwards (17.1x10
and the maximum propagation observed up to 60 days (18.9x109 ± 5.07 x109cfu/ml).
Among the 4 carriers analysed, OWCS showed the least supportiveness to rhizobial
cultures in refrigerated conditions (Fig 4.01).
Fig 4.01 survival of Rhizobium phaseolus in biodigested organic manure carriers

at refrigerated temperature (6±20C).
127
128
4.3 DISCUSSION
In the present investigation, the room temperature provided was the suitable
growth condition for rhizobia. Hence the load was gradually increased from the 1st
day observation to 30th day observation (Table 4.02). But a gradual decrease of load
was observed on 40th day onwards. This may due to prolonged incubation and loss of
moisture from carrier material. The above mentioned observation was coincided by
Gibson, (1967); Burton, (1964) stated that, a marked effect of room temperature on
the efficiency of rhizobia and sometimes the survival and growth was hampered at
prolonged exposure (or) incubation of room temperature.
Dev and Tilak, (1976) reported that farmyard manure as carriers provided the
better nodulation of legumes than other chemical fertilizers. Their results strongly
supported the efficiency of carrier materials along with inoculants into the soil. Inocu-
lation of rhizobia along with charcoal carrier enriched the productivity of certain
pulses (Iswaran et al., 1969; Singh and Tilak, 1977).
Gaur et al., (1980) showed that the dose of minerals, macro and micro nu-
trients influence the growth of rhizobial cultures and the symbiosis with legume
plants. In the present study also showed that the depletion of nutrients leads to a con-
siderable loss of organisms in both environmental conditions provided. Under room
temperature the viability of cultures decreased from 40days whereas in refrigerated
temperature, the decrease of load started from 70 days onwards. The reason may due
to the nutrient loss on prolonged storage.
Rhizobia are not very particular in their nutritional requirements. For commer-
cial production of cultures the utilization of cheap sources of special nutritive value is
found to be more economical (Burton, 1967). In this experimental work also, the easi-
ly available, very cheap but high nutritional factors containing biogas spent organic
slurries (SWPS, SWCS and OWCS) were used as carrier materials.
The medium in which rhizobia were allowed to multiply is an important factor

in rhizobial culture preparation. Tilak, (1998) stated that among various carrier
129
materials used in his work, peat based inoculants were found to be superior because it
offer some good advantages such as increased population and providing high organic
matter content. Finely produced charcoal, farmyard manure, coconut shell powder,
coffee husk, press-mud or combination of such materials with soil or charcoal have
been tested and found useful. Following this findings, the biogas plant spent slurries
were used as carrier for R.phaseolus.
Tilak and SubbaRao, (1978) observed that coconut shell powder, coffee husks,
press mud or combination of such materials with soil or charcoal helped to retain the
viability of rhizobia for more days when stored at room temperature. Comparable re-
sults were obtained in the present investigation, where the mixed organic carriers re-
tained the maximum concentration both in room and refrigerated conditions. Van-
Schrevan, (1970) reported that the moisture content of the carrier based culture influ-
enced the number of rhizobia. So in the present work the depletion of rhizobial load in
room temperature provided may due to the moisture loss in the carriers.
According to Thies et al., (1991) normally at room temperature (300C), the

rhizobial population decreases after 30days of storage, whereas at refrigerated tem-
perature the decline of the rhizobial population is rather slow when R.leguminosirium
was used as experimental cultures. This finding is exactly coinciding with present day
work, in which the microbial decline occurs after 30days in room temperature and af-
ter 60 days at refrigerated temperature (Fig 4.01).
The viable nature of the bioinoculant and the holding capability of this organ-
ism by carrier materials depend upon the physicial and chemical characteristics of the
individual carriers. It includes organic matter, total nitrogen, bulk density, particle
density, porosity, water holding capacity and total surface area (Tiles, 1978). Bardiya,
(1970) stated that among the different carriers, combination of Indian peat soil, and
farm yard manure, compost or press- mud with charcoal (1:1) were capable of sup-
porting more propagation and gave high viable count than individual carriers. In the
individual cells at the end of 20 days, the population of viable cells started declining
but the charcoal, FYM and press mud helped to retain the viability of cells up to 3
months. In the present work also, all the carriers started to decline in the inoculant
number from 30 days but not in the case of mixed carrier.
130
According to Burton, (1967) the U.S.A sledge peat is ground to fine powder
and neutralized with CaCO3 to pH 6.8 and blended with rhizobial culture
(109cells/ml). The resulting products have at least 300 million (3x108 cells/ml) rhizo-
bia per gram of peat. R.japanicum reaches a maximum population of more than
25x1010 cells per gram in 4 weeks, while R.meliloti reaches the same level in 2 weeks,
similar to that, in the present work during 30th day analysis, all organic carrier mate-
rials contained 109 cells per gram. At higher temperatures the number of rhizobia
falls below optimum when carrier based inoculants are stored beyond 2-3 months.
Distribution of Rhizobium to augment the nitrogen requirement of growing le-

gume is well established by Singh, (1994) in their experiment of inoculating the le-
guminous plants with VA mycorrhizal fungi, efficient strains of Rhizobium and phos-
phate solublizing organisms separately and in dual form have increased nodulation,
nitrogen fixation and accumulation in plants. Such agriculturally important Rhizobium
species was propagated using organic manures revealed high population.
The success of nodulation can be achieved by seed applied strains of carrier

based rhizobia. They colonize in the soil and rhizosphere and then to form nodules in
competition with the indigenious strains (May and Bohlool, 1983; Williams and Phil-
lips, 1983; Thies et al., 1991). Kuy Kendall et al., (1982); Woommer et al., (1988)
portrayed that the abiotic factors include nutritional availability, moisture, pH, salinity
and temperature influence the growth of bacteria. In the present study also, approx-
imately after 40days, due to steep increase of rhizobial inocula cause the mass or
heavy depletion of nutrients in carrier materials. Hencewhy the nutritional depletion
decreases the count of Rhizobium step wisely. This condition happened after 60 days
in refrigerated inoculation.When analyses the microbial load, the initial days contain-
ing more number of rhizobia.
The inoculation of rhizobia into soil in the form of seedlings and free rhizobi-
al inoculations was done by Robinson, (1969) and observed that the nodulation was
found to be more by the effective strain. Rhizobium can survive at low temperature
and tolerate temperatures up to 500C (Nutman, 1965). In the present investigation, the
refrigerated temperatures influenced the growth of rhizobial culture more than
80days, and room temperature augmented the growth up to 40 days.
131
In many countries varies types of carriers were used. In U.S.A sedge peat is
used as carrier. After inoculation with rhizobial culture, the carriers at least should
have 300million of rhizobia (3x108)per gram peat (Burton ,1967).When room temper-
ature was provided population of R.japanicum was more than 25x109cells/g in 4
weeks, but storage at 40C prolonged the life of culture up to 6 months. The propaga-
tive ability of our culture also more or less similar to this report.
In Australia, milled dry peat (15%moisture) is used as carriers. The carriers

with inoculum are packed in polythene bags and sterilized by gamma radiations. Then
the broth is introduced into polythene bags by means of a hallow needle (Date, 1974)
and the cell count is ranging from 109 to 1010g/peat up to 3months. In this work also,
the organic manure carriers containing rhizobial cultures were packed in the sterilized
polythene bags and the viability of the cultures also in the considerable amount.
In India, lignite, Indian peat soil, charcoal, FYM and press mud are used as
carrier. They helped to retain the viability of rhizobia up to 2 months at 300C and ex-
tend to more than 6months at 40C (Tilak and Subba Rao, 1978). Among these carriers,
peat soil retains the maximum viability and be extended to 12 months when stored at
40C. El Kramany et al., (2007) performed an experiment by using organic manures
with and without microbial inoculants produced the greatest number of pods, highest
seed, and straw in ground nut than chemical and organic farmyard manure with inocu-
la. This trail implemented the importance of organic manure with inoculum in yield
production by plants. Also, this type of low-organic fertilizers helps to get clean agri-
cultural product free of undesirable high doses of heavy metals and other pollutants
plus increasing yield and yield components, secondary for improving biological,
physical and chemical properties.
Benoj Mathew, (2006) studied the effect of carrier oriented Rhizobium inocu-
lation on imbibed seed and germinated seed on Phaseolus mungo which revealed that
the carrier Rhizobium inoculation on emerging radicle of Phaseous mungo was highly
effective in nodulation and biomass production when compared to inoculation on im-
bibed seeds. Nagarajan and Balachandar, (2001) experimented the influence of organ-
ic amendments along with carriers on nodulation and grain yield of black gram and
green gram (like farmyard manure, leaf compost and biodigested slurry) revealed that
132
there was a registered significant enhancement in the plant biomass. They also re-
ported that among the different organic amendments used, biodigested slurry obtained
from biogas plant with Rhizobium inoculation recorded the maximum plant height,
nodule number, nodule height and grain yield both in black gram and green gram re-
spectively. For the present work also, the organic manure obtained from biogas slurry
was used as carrier.
Barber and Siberbush, (1984) in work on French bean (Phaseolus vulgaris),

significant increase in plant height, leaf area index, nodules per plant, number of
branches per plants, number of pods per plants and green pod yield was noted when
inoculated with Rhizobium phaseoli.
Ranjana Bhatia et al., (2008) investigated the use of different available carriers
suitable for the use of bioinoculants. Kumarimanimuthu veeral, (2008) showed that
organic manures such as Farm Yard Manure(FYM), press mud and industrial wastes
such as rice husks, rice hulled ash and fly ash have been reported to be effective in
influencing the physio-chemical and biological properties of the spoils. Prabaharan,
(2008) revealed that the application of organic manure like poultry manure increased
nitrogen of leaf tissue at harvest in strawberry and application of coir pit based poultry
manure increased nutrient uptake in sorghum, cabbage, Bermuda grass and tomato.
To summarize the results, better survival and growth of R.phaseolus was rec-
orded in refrigerated temperature (6±20C) than the room temperature provided be-
cause in refrigerated temperature the growth, metabolic activities and propagation was
occurred in slow manner compared to normal room temperature. Also among the four
biodigested organic carrier manures, the mixed carrier manure showed the maximum
concentration of rhizobial load followed by SWPS and SWCS manures. OWCS was
less supportive for rhizobial growth even though it had ensured their survival. The use
of carrier based inoculum reduces the dependency of agricultural crops on cost of
chemical fertilizers, rapid depletion of non renewable energy sources, release of pol-
lutants during fertilizer production, disruption of nutrient balance in soil leaching of
nutrients into ground water and pollution in surface water. Considering the economic
point of view, the prime objective of this work is to provide highly efficient Rhizo-
bium culture. This was achieved when it was provided as carrier based inoculants.
133
CHAPTER V
134
Tamil Nadu, India.
Chapter V
5.0 ISOLATION AND CHARACTERIZATION OF

MICROFLORA FROM STP
5.0.1 Introduction
Water is a prime natural resource, a basic human need and a precious national
asset (Harish babu et al., 2004). It is indeed required in all aspects of life and health,
for producing food, industrial activities, energy generation and maintenance of envi-
ronment and substance of life and development (Tiwari, 2000). Dumping of industrial
and municipal wastes causes toxic substances to be leached and seep into the soil and
affects the ground water source (Khan and Khan, 1983). Different chemical processes
are operating simultaneously making the polluted water an extremely complex system
(Dhankar and Sushila sangwan, 2004).
The release of huge quantities of municipal and domestic waters by drains into
the canals and rivers cause the pollution of major rivers of our country (Chitra and
Jeya, 2005). Sewage discharge is a major component of pollution and the disposal of
raw sewage into water bodies deteriorates their water quality and contributing to oxy-
gen demand and nutrient loading, promoting toxic algal blooms and leading to a des-
tabilized aquatic ecosystem (Ogunfowokan et al., 2005). The sewage contains human
faces, urine, kitchen wastes, street wastes and organic substances that provide nutri-
tion for bacteria and fungi. Reckless discharge of domestic sewage and other wastes
into the water bodies such as ponds, lakes, streams accounts for 70% of water pollu-
tion (Krishna kumar et al., 1998).
Untreated domestic waste or raw sewage wastewater is usually gray brown,

odiferous. Some of the important constituents of domestic and raw sewage wastewa-
ters that are targeted for removal through treatment are total suspended solids, bio-
135
chemical oxygen demand, nutrient-nitrogen and phosphorous (Girisha et al..2006).
High nitrate levels in waste effluents could also contribute to eutrophication effects,
particularly in freshwater (Fried, 1991; OECD, 1982). Sewage treatment is subject to
use one or more process to reduce its impurity contents, promoting corrections of un-
desirable characteristics in a way that final products and sub products can be reused or
returned to environment according rules and criteria defined by environment legisla-
tions in force(Van haandel and Lettinga, 1994).
Sewage is used for agricultural purposes notably in India, Latin, Tunisia, Unit-
ed States of America and west Asia. In addition to that the waste water reuse can be
observed in several countries as Spain, Portugal, France, Mexico, USA, Israel, and
India. Practices vary from the use of raw sewage to well treated sewage. Out of vari-
ous usage of water, its application for irrigation ranks top on priority in most of the
countries including India. In India, more than 70,000 hectares of land is irrigated with
waste water (Agarwal, 2002). It must be admitted however that due to scarcity of
freshwater and 70% of Indian population being dependent upon farming the farmers
have no option other than to grow their agriculture and horticultural crops either under
rain fed conditions or waste water due to its easy availability near cities and even
smaller towns. Therefore its use not only solves the disposal problem causing the en-
vironmental pollution but also serves as a source of nutrients to plants as most of the
essential nutrients are generally present in waste water (Wendland et al., 1994; Akhtar
et al., 2006).
Practice of waste water farming is an important development in water resource

management. This practice has resulted due to the scarcity of alternative waste sup-
plies and need to increase local food and vegetable production (Asano et al., 1985).
Soil has a great capacity for decomposing wastes and pollutants of different kinds.
The nutrients may be either precipitate out by the soil microflora or absorbed by the
plants. The utility of municipal and industrial waste waters for irrigation of crops is
well documented (Stirban et al., 1984; Aziz et al., 1999). Sewage sludge consists of
multi-element organic wastes that are used commonly as manures (Hagler, 1986). It
has been experimentally observed that effluents from activated sludge plans can be
made use of to increase the rate of fish production (Nair, 1944).
136
Anaerobic treatment of organic waste water has occupied unique place in
waste water treatment field due to the variety of reasons, which includes generation of
methane rich biogas apart from reducing the COD and BOD of the waste water (Bull
et al., 1984). In this process, the organic pollutants of waste water are converted by
anaerobic bacteria into biogas and biomass (Vasder Berg, 1984). Generation of less
sludge and maximum reduction of biodegradable organics obtained by this process
(Annapurna jetty et al., 2004). Biological waste water treatment processes such as la-
goons, trickling filters and activated sludge treatment may sustainably reduce the
number of pathogens in the waste (EPA, 1989). The practice of utilization of sewage
for pisciculture has been described by Bose, (1944); Fowler, (1944); Saha, (1970) in
the Vidyadhari spill area located in a suburb of Calcutta.
The heterotrophic bacterial population constitutes an important part of sewage

water community (Radha and Seenayya, 2004; Gangotri and Malkar, 2004). Popula-
tions of 108-109 hydrolytic bacteria per ml of mesophilic sewage sludge (Tiwari,
2000) or 1010 to1011 hydrolytic bacteria per gram of volatile solids present in sewage
sludge. Enumeration studied of hydrolytic bacteria that utilize specific carbon sub-
strates demonstrated 107 proteolytic and 105 cellulolytic bacteria per ml of sewage
sludge. It should be expected that genera common to both fecal/gastrointestinal and
soil/sediment environments are present in mesophilic anaerobic digesters.
So the sewage water from STP was treated anaerobically with cow dung and
poultry droppings for biogas production in the previous chapter. In addition to that the
enumeration of total microbial population in four cycles and isolation of microbes
were also carried out by following the objects.
● To enumerate Total Heterotrophic Bacterial Population (THBP) and Total

Heterotrophic Fungal Population (THFP) by decimal dilution-agar plating
technique.
● To isolate organisms by using selective Hi-media.
● To confirm and characterize the isolated bacterial population by

microbiological, biochemical and physiological experiments.
137
5.1.1 Sample collection
Sterile 250ml conical flasks were used to collect sewage water samples from
STP of NEC, Kovilpatti, Tamil Nadu, India. They were transported to the laboratory
in an icebox. They were subjected to microbiological, biochemical and physiological
assays. Monthly samples were collected for a period of four months (June 2006-
September 2006).
5.1.2 Microbiological Analysis
The present study was subjected to find out the influence of microbial diversity in
particular to THBP and THFP in STP.
(i) Enumeration of Total Heterotrophic Bacterial Population (THBP)
One ml of sample was serially diluted and was plated on nutrient agar plates
adopting pour-plating technique. The plates were incubated for 24-48 hours at 37◦C.
Bacterial outgrowth in countable plates were selected and enumerated. The bacterial
populations were expressed as number of Colony Forming Units per ml (CFU/ml)
(Nallathambi et al., 2002) (Plate 5.02).
(ii) Enumeration of Total Heterotrophic Fungal Population (THFP)
One ml of STP sample was serially diluted and was plated on fungal selective
media called Rose Bengal agar. Then plates were incubated for about 3-7days at room
temperature. The number of colonies appeared on dilution plates were counted, aver-
aged and multiplied by the dilution factor to find the number of cells of the sample
(Theroux et al., 1999) (Plate 5.01).
CFU/ml = Mean plate count

Dilution plated x Volume of sample in plate in milliliters
138
5.1.3 Isolation of bacterial population by using selective Hi-media
The following selective media were used for the isolation of bacteria. They are
▪ Lactobacillus MRS Agar M641 for the isolation of Lactobacillus sp (Plate 5.03).
▪ Bacillus medium M1383 used for the isolation and maintenance of Bacillus
licheniformis (Plate 5.04).
▪ S.S Agar (Salmonella and Shigella Agar) M108 for the isolation of i)Salmonella
sp (ii) Shigella sp (iii) E. coli (Plate 5.05; 5.06& 5.07(a).
▪ EMB Agar M317 used for the selective isolation of E. coli (Matallic sheen colo-
nies)
Enterobacter sp (Pink colonies) (Plate 5.07 (b);5.08)
▪ KF Streptococcal Agar M248 for the isolation of Streptococcus sp (Plate5.13).

▪ Pseudomonas isolation Agar 406 for the selective isolation of Pseudomonas
aeruginosa (Plate5.10).
▪ Phenolphthalein phosphate AgarM652 isolation of Staphylococcus sp (Plate5.11).
▪ TCBS Agar M189 used for the selective isolation of Vibrio cholerae (Plate5.12)
and Vibrio parahemolyticus (Plate5.09)
Thus obtained colonies were stored in agar slants for further microbiological,
biochemical and physiological characterization. Cultures obtained were made free of
contamination at frequent intervals by streak plating and sub-culturing. The isolated
bacterial cultures by using selective media were subjected to the following tests
adopting the scheme recommended by Bergey's Manual, (1998).
5.1.4 Characterization of isolated bacterial population by microbiological, bio-

chemical and physiological experiments
(i) Simple staining
The bacterial cultures were smeared on a clean microscopic slide, air-dried

and heat fixed. The slide was flooded with methylene blue reagent for one minute.
Then the smear was washed with tap water, air-dried and examined under microscope.
139
(ii) Gram staining
The bacterial smear was prepared, air-dried and heat fixed. The slide was
flooded with crystal violet reagent for one minute. The smear was washed with tap
water for 2 seconds and then flooded with ethanol for 30seconds. Then the slide was
washed with water. Finally, the slide was flooded with counter stain safranin for 30
seconds and washed with tap water. It was air-dried and examined under the micro-
scope.
(iii) Spore staining
The bacterial smear was prepared, air-dried and heat fixed. The smear was
flooded with Melachite green. Then the slide was exposed to steam of boiling water
for about 5minutes, followed by washing and counter stained by safranin. It was ob-
served under the microscope.
(iv) Motility
Hanging drop method was used for observing motility of organisms.
(v) Test of catalase
Nutrient agar slants or broth solutions with the test cultures were inoculated
and incubated at 370C for 24 hours. Following incubation one ml of 3% hydrogen pe-
roxide was trickled down the slant (or) broth. They were examined immediately after
five minutes. Evolution of effervescence indicated a positive reaction.
(vi) Hydrolysis of gelatin
Agar medium supplemented with 0.4% was streak plated and incubated at the
optimum temperature for the growth of organisms. After incubation the plates were
flooded with gelatin precipitin reagent (15% HgCl2 in 20% (V\V) Con HCl). A clear
zone around the colony indicated the positive result.
140
(vii) Hydrolysis of starch
Starch agar plates (starch 2%) were prepared and streak of organisms crossed
the plates were inoculated. The plates were incubated at 370C for 24 hours for suffi-
cient growth. After incubation, the plates were flooded with iodine solution. Clear
zones around the line of bacterial growth indicated hydrolysis and unchanged starch
gave blue colour.
(viii) Hydrolysis of lipid
Sterile Tween agar medium plates were prepared and the test cultures were
streaked on them. Then the inoculated plates were incubated for 2-6 days at 370C.
Formation of opaque zone indicated the lipolytic activity of the microorganisms.
(ix) Hydrolysis of casein
Skim milk agar plates were prepared inoculated with isolated cultures and
were incubated for 24-48 hours at 370C. Formation of clear zone around the colonies
indicated the proteolytic activity of the microorganisms.
(x) Test for Carbohydrate fermentation
In a sterile test tube containing oxidation-fermentation basal medium incorpo-

rated with the different sugars such as dextrose, sucrose, mannitol, arabinose, xylose
and lactose with Durgam's tubes were prepared and a loopful of broth culture was in-
oculated and incubated at 370C for 24-48 hours. For anaerobic fermentation a layer of
sterile mineral oil was added on the top of the medium. Change in colouration and
production of gas was observed and recorded.
(xi) Triple Sugar Iron Agar (TSIA) Test
Test colonies were picked up and inoculated them into a TSIA slant by first
streaking the surface of the slant and then stabbing the medium in the butt region, and
incubated them at 370C for 24hours. An alkaline reaction was indicated by a red co-
141
louration, production of acid by yellowing of the medium, gas formation by the for-
mation of gas bubbles and production of H2S by a blackening of the medium. Non
fermenting ability is indicated by alkaline slant butt.
(xii) Nitrate Reduction test
Sterile nitrate free medium was prepared in test tubes and inoculated with the
test cultures and incubated at 370C for 96 hours. Following incubation 0.1ml of the
test reagent was added to the cultures. Formation of red colour in a few minutes indi-
cated the presence of nitrate reducing bacteria into nitrite.
(xiii) Indole productionTest
The isolated test cultures were inoculated in the peptone broth and incubated
at 37ºC for 24 hours. After incubation 0.2ml of Kovac's reagent was added and the
colour change was noted due to the production of indole. A cherry red colour ap-
peared in the alcohol layer indicated the positive results.
(xiv) Methyl Red Test (MR Test)
The colonies were inoculated into MR-VP broth tubes and incubated at 370C for 24-
48hours. Formation of red colour on the addition of 5-6 drops of methyl red indicator
indicated positive results.
(xv) Voges-Proskuer (VP) test
The test colonies were inoculated in the MR-VP broth and incubated at 370C
for 48hours. After incubation period one ml of potassium hydroxide (plus creatine)
and three ml of 5% solution of alphanaphthal was added in absolute alcohol. A posi-
tive reaction was indicated by the development of a pink colour within 2-5 minutes,
which become crimson red in 30 minutes. The tube was shaken at intervals to ensure
maximum reaction
142
(xvi) Citrate Utilization Test
Slants of Simmon citrate agar medium was inoculated with the test organisms
and incubated at 370C for 24 hours. Change of colour from green to blue indicated the
positive result.
(xvii) Test for Urease
A loopful of isolates was inoculated on Christensen urea agar slants incubated

at 370C for 18-24 hours. An appearing pink colouration was taken as positive and rec-
orded.
(xviii) MacConkey Agar Bile salts
Sterile MacConkey agar bile salts were prepared, streaked by the isolated cul-
tures and incubated at 370C for 24 hours. This test was mainly used for the isolation
and differentiation of lactose fermenting organisms from nonlactose fermenting or-
ganisms.
(xix) pH tolerance test
Varying pH (5.7, 6.8, and 8.0) was provided to the test cultures inoculated
with nutrient broth tubes. These tubes were incubated at 37oC for 24 hours and the
growth was indicated by the turbidity in nutrient broths.
(xx) Temperature tolerance
Varying temperatures (4◦C, 15◦C, 30◦C, 41◦C, 50◦C and 65◦C) were provided to
the test cultures inoculated with nutrient agar plates. These plates were checked for
growth and that was indicated by colony appearance on the Nutrient agar (NA) plates.
143
(xxi) NaCl tolerance
Varying concentration of NaCl (2.5%, 5%and 7%) were added to peptone

broth medium which were inoculated with test organisms and checked for growth and
was indicated by turbidity after incubation at 370C for 48hours.
144
5.2 RESULTS
5.2.1
.2.1 Total Heterotrophic Bacterial and Fungal Population (THBP&THFP)
The sewage water was collected and processed to determine the heterotrophic
bacterial and fungal population. Throughout the sampling period, the THBP was
found to be in the order of 107CFU/ml (Fig 5.01).
1). Among the four cycling periods, the
lowest count was observed in the first sampling cycle (98x107± 4.013CFU/ml) and
highest at the third sampling cycle (151x107± 3.654CFU/ml). In the case of THFP, the
population density was consistently low in the order of 103CFU/ml (Fig 5..02). Here
the highest fungal population occurred in the first sampling cycle (21x103±
3.018CFU/ml-) and the lowest in the fourth sampling cycle. In both cases, THBP and
THFP, the variations were marginal signifying relatively consistent nature of their ooc-
currence.
Fig 5.011 Total Heterotrophic Bacterial Population (THBP) in STP (x107 cfu/ml).
145
Fig 5.022 Total Heterotrophic Bacterial Population (THBP) in STP (x103 cfu/ml).
al, biochemical and physiological characterizati

5.2.2 Microbiological, characterization
on of the se-
s
wage water isolates
Using selective media 11 bacterial cultures were isolated and they were su
sub-
jected to various microbiological, biochemical and physiological analysis to identify
its species level
evel and it was tabulated (Table 5.01).
146
147
148
149
150
5.3 DISCUSSION
By using selective media 11 bacterial cultures were isolated from STP and
their characteristic features were confirmed by biochemical experiments. In addition
to that monthly samples were collected for THBP and THFP analysis through four
sampling cycle.
Sewage is commonly a cloudy aqueous solution containing minerals and or-

ganic matter. The sewage water without treatment was simply discharged into the
nearest large body of water such as river, which leads to contamination of rivers and
lakes chronically affecting the flora and fauna (John Ingraham and Catherine Ingra-
ham, 2004). Bacteria are considered as serious pathogens, which cause diseases and
subsequent economic losses. Among the bacterial diseases, Gram-negative bacteria
such as Pseudomonas, and Vibrio predominantly affected the fishes and among the
three Aeromonas sp are the main pathogenic organisms for the fresh water fishes
(Munn and Trust, 1984; Manohar, 2005). These organisms were present in the sam-
ples taken from the STP for the present study.
The microorganisms present not only in the sewage water but also in the
wastewater from the distilleries which contains the microbial stains such as Entero-
bacter sp, Corynebacterium sp and Micrococcus in large amount (Vasanthy and Tha-
marai selvi, 2006). Also these organisms were used for the colour removal from car-
bon treated spent wash individually by the bioremediation technology. Similarly in
present study among 11 bacterial isolates B.licheniformis and P.aeruginosa were se-
lected for cellulosic biodegradation studies. Tharekeshwari and Shobha Jeyannath,
(2006) reported that the effluent from distillery plant was used for the germination of
the seedling growth and root meristem cells of Vigna radiata. Similarly in present
study the treated sewage water in STP was used for the irrigation purposes.
Sometimes the disposal of municipal and industrial wastes in the neighboring

streams and rivers have quite significant role in raising the pathogenic organism’s
151
level and toxic metals in coastal areas (Martin deva prasath, 2006). Sometimes ground
water is also polluted due to the discharge of industrial effluents (Subha rao and Subba
rao, 1994). The sewage water irrigation showed increased soil chemical concentrations
Girisha et al., (2006). Studies on the industrial effluent toxicity levels are essential to
develop effective measure for the conservation of the already depleted aquatic fauna
D’Cruz and Miranda, (2006).
Apart from the disposal of sewage water into the water bodies, tannery indus-
try is one of the major industries of India, which creates heavy water pollution Sanjith
Sen Gupta et al., (2006). Not only the fresh water bodies and its flora and fauna alone
affected by the disposal of sewage and other industrial waste materials, the impacts of
pollutants on marine environment is more acute and its deleterious effects on living
resources are much more evident in coastal areas (Everaats et al., 1989).
In addition to heavy metals the pathogenic organisms present in sewage and

industrial wastewater disposal into water bodies plays a vital role in disease emerging
in aquatic biota (Hazen et al., 1978). Higher concentrations of these metals in the eco
system may lead to excessive accumulation of metals, becoming toxic and possibly
endanger to human health (Stephan and Kochian, 1997).
Increasing population densities and urbanization during eighteenth and nine-

teenth centuries initially were not accompanied by adequate sanitation practices.
These situations created conditions for devoting epidemics caused enteropathogenic
microorganisms such as Vibrio cholera (cholera), Salmonella typhi (Typhoid fever),
various other Salmonella and Shigella strains (gastrointestinal infections of varying
sevearity) and Entamoeba histolytica (amoebic dysentery) (Atlas and Bartha, 1993).
In present studies the report of the work revealed that the presence of above said pa-
thogenic organisms in central collecting tank of STP.
Natural water has an inherent self-purification capacity. Hence human population

community living patterns and large scale agricultural and industrial activities typical-
ly produces liquid wastes on a scale that overwhelms the homeostasis of aquatic
152
communities and cause unacceptable deterioration of water quality (Dart and Stretton,
1977). But the STP of NEC, the treated sewage water is used for irrigation purpose.
In the present study 11 bacterial cultures were isolated from sewage water of
STP and among them B.licheniformis and P.aeruginosa were selected for further
study on cellulosic biodegradation.
153
CHAPTER VI
154
Tamil Nadu, India.
Chapter VI
6.0 CELLULOSE BIODEGRADATION
6.0.1 Introduction
A prominent carbonaceous constituent of higher plants and probably the most

abundant organic compound in nature is cellulose. Since a large part of the vegetation
added to the soil is cellolosic, the decomposition of these carbohydrates has a special
significance in the carbon cycle. Recycling of these waste matters is necessary to pre-
vent pollution and to conserve scarce natural resources (Coughlan, 1990; Jeyashree-
pauls, 1992; Kasing apun et al., 2000; Padmaja and Leena lavanya, 2006).
Cellulose is the primary product of photosynthesis in terrestrial environment

and the most renewable bioresource produced in the biosphere (Jarvis, 2003; Zhang
and Lynd, 2004). Cellulose biodegradation by cellulases and cellulosomes produced
by numerous microorganisms which represents a major carbon flow from fixed car-
bon sinks to atmospheric CO2 (Falkowski et al., 2000; Melillo et al., 2002; Berner,
2003), is very important in several agricultural and waste treatment processes
(Humphrey et al., 1977; Russell and Rychlik, 2001; Van Wyk, 2001; Hamer, 2003;
Angenent et al., 2004; Das and Singh, 2004; Haight, 2005; Schloss et al., 2005), and
could be widely used to produce sustainable biobased products and bioenergy to re-
place depleting fossil fuels(Lynd et al.,1991; 1999; Hall et al., 1993; Wyman, 1994,
1999, 2003; Lynd,1996; Mohanty et al., 2000; Mielenz,2001; Galbe and Zacchi,
2002; Hoffert et al., 2002; Angenent et al., 2004; Demain et al., 2005; Moreira, 2005;
Reddy and Yang, 2005).
Cellulose, the largest renewable carbon available (approximately 180 billion

tons of organic materials is photosynthesized annually) is frequently found in close
association with other compounds such as hemicellulose, lignin and other polysaccha-
155
rides, which make its bioconversion more difficult (Person et al., 1990). It constitutes
40-60% of woody plant cell walls, and its bioconversion to fuels and chemicals is of
great interest (Philips and Humphrey, 1983). The hydrolysis of cellulose by cellulolyt-
ic microorganisms occurs mainly through the concerted action of several hydrolytic
enzymes (Coughlan, 1990), which may be truly extracellular, located intracellularly,
or bound to the cell wall. Occasionally, additional non-hydrolytic enzymes have also
been reported to be involved in cellulose degradation, but little attention has been giv-
en to their role in the overall process (Zhou et al., 1999).
Large quantities of cellulose are locked up in manufactured products such as

textiles, paper and building materials (Bakare et al., 2005). Disposal of solid wastes
by biological methods using cellulolytic microorganisms can reduce environmental
pollution, compared to other methods of waste disposal such as incineration which
increases the atmospheric carbon dioxide concentration (Wyman et al., 1993). Annual
production of cellulose is estimated to be 4.0X10-1 tons (Singh and Hayashi, 1995).
The proportion of cellulose in plant tissue ranges from 20 to 45% of dry weight and to
almost over 90% in cotton fibre (Stephens and Heichel, 1975). Much of the cellulose
in nature exists as waste paper (Cruger and Crueger, 1990).
A number of biomass conversion methods have been proposed and employed

ranging from direct chemical methods like acid hydrolysis and pyrolysis to biological
methods such as application of cellulase enzymes (Cooney et al., 1978). There are
two methods for degrading the cellulosic materials, which are economically feasible.
They are acid hydrolysis and enzymatic hydrolysis (Saravanan and Indrajeyaraj,
2004). Cellulosic materials can be hydrolysed into soluble sugars by acid treatment,
but the process is uneconomical and yields poor products (Haper and Lynch, 1981).
Enzymic hydrolysis has advantage of being energy sparing because of relatively mild
reaction conditions, and avoids the use of toxic substances (or) corrosive acids (Xin
and Kumakura, 1992). Enzymatic hydrolysis of wastes may give a relatively pure
product with the consumption of less energy during the process (Bakare et al., 2005).
Cellulolytic microorganisms produce a wide variety of enzymes which acts

synergistically on their substrate (Bayer et al., 1998). The bacterial cellulase is be-
coming a well-understood multi-protein complex found in cellulolytic microorgan-
156
isms (Ding et al., 2003; Bayer et al., (2004). Agricultural and industrial wastes are
among the celluloses with its immense importance is being causes of environmental
pollution (Milala et al., 2005). Cellulose degradation occurred in both aerobic and
anaerobic environment (Szakacs and Tengerdy, 1997).
6.0.2 Cellulase enzyme
The enzyme cellulase is an adaptive enzyme which degrades cellulose chains

by hydrolyzing β 1 4 glucosidic bonds Mandals and Reese, (1957, 1960); Talboys,
(1958). The catalytic system required for microorganisms to convert cellulose to the
simple sugars that penetrate the cell typically includes three types of enzymes, they
are endoglucanase or C1, exo β 1,4 glucanase or Cx and β glucosidase (Parry et al.,
1983; Kang et al., 1999; Saravanan and Indra Jeyraaj, 2004).
The cellulolytic enzymes have vast industrial applications because of their

property of saccharification of cellulosic material to glucose. The cellulase is a com-
plex system comprised mainly of three-enzyme endo-β glucanase, exo-
cellobiohydralase and β glucosidase (Dhake and Patil, 2006). The enzyme β –
glucosidase has been used in the industries for production of fuel alcohol. They are
also used in synthesis of useful glucosides also used in enzyme mixture for animal
feed due to their high cellulytic activity (Xin et al., 2001).
Complete cellulose hydrolysis to glucose demands the action of exoglucanases

and β glucosidases. Exoglucanases are usually active on crystalline cellulose and are
lacking from incomplete cellulase systems. Endoglucanases are the most active
against the amorphous region of cellulose and they can also hydrolyse substituted cel-
luloses, such as Carboxy Methyl Cellulases (CMC) and hydroxymethyl-cellulose
(Han et al., 1995; Akiba et al., 1995; Siddiqui et al., 2000; Arul ray et al., 2007). Ex-
oglucanases cleave disaccharide units either from non reducing ends, whereas endog-
lucanases hydrolysis the cellulose chain internally. β -glucosidases are needed to
cleave cellobiose and other soluble oligosaccharides to glucose (Beguin, 1990).
157
6.0.3 Cellulolytic Microbes
Cellulases are produced by a variety of microbial forms included Protozoa,

fungi, bacteria and actinomycetes (Fred Stutzenberger, 1972). Organisms produce cel-
lulase enzyme in culture filtrates which can rapidly degrade solid cellulosic sub-
stances such as cotton fibers (Selby, 1963). Cellulolytic microorganisms are common
in field and forest soils, in manure and on decaying plant tissues. Cellulose- utilizing
species are aerobic, anaerobic mesophilic bacteria, filamentous fungi, basidiomycetes,
thermophilic bacteria and actinomycetes (Nevalainen et al., 1991; Ghosh and Ghosh,
1992). The organisms can be isolated from soil, water, sewage and faeces. Microbial
sources for cellulose degradation are listed below
Table 6.01 Microbial sources for cellulose degradation
Organisms References
Pseudomonas fluorescens, Actinomycetes, Brock and Madigan, (1991)

Fibrobacter succinogens.
Ruminococcus flavefacins, Ruminococcus Weimer, (1996); Parry et al., (1983)
albus
Bacillus brevis, B.firms, B.pulmis, B.subtilis, Julio et al., (2002); Hazel-wood et al., (1988)
B.licheniformis, B.polymyxa, B.cereus, B.
spharricus, B. circulans
Clostridium thermocellum, Paenibacillus Andrew Hudson et al., (1991); Kang et al.,
azotofianns,Gluconacetobacter, Azospirillum, (1999).
Sinorhizobium fradii
Aspergillus, Chaetomium ,Curvularia, Fusa- Vipul wadhwa and Sheela Srinivastava,
rium, Memnoniella, Phoma, Thielavia and (1997).
Trichoderma.
Corynebacterium,Cytophaga, Sporocytopha- Arul ray et al., (2007); Breznak, (1975).
ga and Vibrio.
Apart from microorganisms, termites are the best cellulose degrader in soil
(Shsilendra saxena et al., 1993). In the more primitive families, obligate flagellate
protozoa digest cellulose intra cellular (Honigbergh, 1970), while this function is car-
158
ried out by bacteria in higher families. However in higher termites the role of bacteria
and their population maintenance till today is an unknown mystery (Breznak, 1975).
The alternative and environmentally friendly technologists that introduce cel-

lulase enzymes at different stages of pulp and paper manufacture as a pretreatment to
pulping, bleaching or wastewater treatment has allowed considerable electrical power
savings and a reduction of pollutants in the waste water from these industries (Wy-
man, 1999; Caldeira et al., 2003; Wirth et al., 2003; Angenent et al., 2004; Demain et
al., 2005; Moreira, 2005).
So an attempt was made to degrade the cellulose substrates by using Bacillus

licheniformis, Pseudomonas aeruginosa and mixed cultures of both which were iso-
lated from the Sewage Treatment Plant (STP) of National Engineering College,
K.R.Nagar, Kovilpatti.
The study was conducted based on the following objectives,
• To prepare Bacillus licheniformis, Pseudomonas aeruginosa and mixed cul-

tures of both for experimental work and biochemical testing to confirm their
activity and characterization.
• To Prepare cellulolytic strains in large scale.
• To inoculate B.licheniformis, P.aeruginosa and mixed cultures of both by pro-

viding cellulosic substrates like Cellulose Powder (CP), Carboxy Methyl Cel-
lulose (CMC), Crude Filter paper (CF) and Coir (CR) for degradation.
• To analysis the production of combined, endo and exo β I, 4 glucanase, bio-

mass and total protein content by the selected organisms, with different above
mentioned substrates provided.
159
6.1.1 Sample Collection
Sewage water sample was collected from Sewage effluent Treated Plant (STP)
of National Engineering College, Kovilpatti, Tamilnadu, India by using sterile conical
flask. The collected sample was transported to the laboratory in an icebox. This was
used for further microbiological and biochemical assays.
6.1.2 Bacteriological analysis
By using selective Himedia, 11 bacterial cultures ware isolated in the previous

chapter. Among those organisms, the bacterial cultures taken for the experimental
work were B.licheniformis and P.aeruginosa. These two organisms were isolated us-
ing selective media namely Bacillus medium M1383 for the selective isolation and
maintanence of B.licheniformis (Plate 6.08) and Pseudomonas isolation Agar Base
M406 for selective isolation and identification of P.aeruginosa (Plate 6.07). Biochem-
ical tests were carried out for their confirmation in the previous chapter (Dhaka and
Patil, 2006).
6.1.3 Media employed
The media as described by Berg’s manual, (1994) was used for the isolation of
cellulolytic microorganisms. Cellulose Powder Peptone agar Medium (CPPM) was
used to confirm the cellulolytic ability of the selected organisms.
6.1.4 Preparation
The required quantity of water and ingredients of CPPM were taken, mixed
well and the pH 7 was checked and finally sterilized. The cultures were inoculated in
CPPM broth separately. After 24 hours incubation, the cultures were streaked onto
two sets of CPPM agar and incubated at 350C±0.050C for 24-48 hours.
160
6.1.5 Cellulolysis
After 24hours of inoculation, one set of plates were flooded with 0.1% Congo
red and kept for 10 minutes. The plates were then washed with 1M NaCl. The Congo
red stained the cellulose region and formed a red colour (Plate 6.01 & 6.02). Halo
zone appeared if bacteria utilizing the cellulose were found. This was considered as a
positive result. The plates could also be acidified by using 5% H2SO4 to arrest the cel-
lulose activity. The cellulose region would turn to blue (Plate6.03&6.04). Colonies
showing clear zones were picked up from the other set of plates and streaked onto
Berg’s media slant (Teather and Wood, 1983). After bacterial growth, they were
stored at 40C for determining their biomass, protein assay and enzyme activity.
6.1.6 Experimental design
The two isolated cellulolytic strains were subjected to the following experi-
ments.
(i) Substrate utilization
Different substrates like Carboxy Methyl Cellulose (CMC), Crude filter paper
(CF), Cellulose Powder (CP) and Coir (CR) were taken in 20ml of CPPM medium
which do not contained cellulose. Instead of cellulose the above said substrates were
added with media composition in various concentrations (mg levels) like 200,400,600
& 800 (Plate 6.05).
(ii) Initial stage

These isolated cellulolytic colonies were inoculated in nutrient broth and incu-
bated at 370C for approximately 24 hours. After obtaining the required initial bacterial
load (even for all), which was determined using a colorimeter at 580nm, the colonies
were now ready for transfer. Media with different substrates and at varied concentra-
tions were prepared and sterilized. One ml of the culture with the same optical densi-
ties were transferred into 20ml of sterile prepared medium, homogenized well and
kept for a period of 15 days at 370C. After 15days, the growth was observed using co-
lorimeter at 580nm.
161
6.1.7 Estimation of cellulase (Endo and Exo β 1,4 glucanase)
After 15 days, the inoculated media were subjected to the estimation of cellu-
lase enzyme activity (Endo and exo β 1, 4 glucanase) as per the methodology of Sad-
hasivam and Manickam, (2008) (Plate 6.06).
a. Preparation of enzyme extract

The inoculated samples were centrifuged at 10,000rpm for 20 minutes. The
cell free supernatant phase were collected and used as a stock crude enzyme solution.
b.Endo β 1, 4 glucanase assay
The scheme of endo β 1, 4 glucanase assay was shown as follows,
0.05 ml of enzyme extract + 0.45ml of 1% CMC solution 550C.
Incubation at 550C for 15 minutes in a water bath.
0.5ml DNS reagent was added.
The mixture heated in boiling water bath for 5 minutes.
1.0ml potassium sodium tartarate solution was added.
Solution was cooled to the room temperature
Distilled water added to make the volume up to 5ml.
OD measured at 540nm.
The standard was prepared using glucose by taking different concentrations ranging
from 50µg to 1000µg/ml of distilled water.
162
c.Combined assay
The methodology was as follows:
00.5ml of enzyme extract + 32mg of dry what man No1 filter paper.
Incubation for 1hour at 500C in a water bath
0.5ml DNS reagent was added.
The mixture was heated in a boiling water bath for 5 minutes.
1.0ml potassium tartarate solution was added.
Solution cooled to the room temperature.
Distilled water was added to make the volume up to 5ml
OD measured at 540nm.
The standard was prepared with glucose by taking different concentrations rang-
ing from 50µg to 1000µg/ml of distilled water.
d. Exo glucanase assay
• The production of exo β,1,4 glucanase enzyme was calculated by subtracting the
values of endo β 1, 4 glucanase enzyme values from the values of combined en-
zyme assay (Vijaya lakshmi, 1994).
163
6.1.8 Estimation of Proteins
Protein was measured in spectrophotometer by Lowery’s method using Bo-

vine Serum Albumin (BSA) as standard substrates (Lowry et al.,1951).
6.1.9 Biomass assay
As per the methodology described by Muthuvelayutham et al., (2006) the

biomass was estimated. For biomass estimation, 5ml of culture broth was centrifuged
for 20 minutes and the supernatant was discarded. The resulting pellets were dried and
dry weight was estimated.
The average (mean) and standard deviation (SD) of all the experimental data
were calculated and given in the table as mean±SD by using Microsoft Excel. Then
the values were tested by Friedman Two-way Analysis of Varience Rank Test to un-
derstand the interactions of enzyme secretion by the selected strains (Table 6.06).
Friedman Two-way Analysis of Varience Rank Test:

Formula for this method is,
χr2 = 12 ΣK j=1(Rj)2 - 3.N.(k+1)
N.K.(K+1)
N – refers number of rows

K – refers number of column
Rj – Rank total of jth column of calculated value of χr2 ≤ table value of χr2 for (K-1)
degrees of freedom. Then only there is a significant difference otherwise not (Table
6.07).
164
6.2 RESULTS
6.2.1 Substrate Utilization
Of the 11 cultures obtained from sewage water, the two organisms’ namely P.
aeruginosa and B. licheniformis were selected individually as well as in the mixed
form for cellulose degradation. Among various substrates tested for the growth of
cellulolytic microorganisms at varied concentrations, Cellulose Powder (CP) was
found to be the best utilizable substrate by the two individuals and mixed organisms
followed by Carboxy Methyl Cellulose (CMC), Crude Filter paper (CF) and Coir
(CR).
When CP was provided as substrates, the maximum growth was shown at

800mg level by mixed cultures (2.789±0.154IU/ml), P.aeruginosa,
(1.789±0.225IU/ml) and B. licheniformis (1.285±0.335 IU/ml). The minimum
growth rate was observed at 200mg level by all these three types. Utilization of
CMC at 800mg level was found to be the most optimum for mixed cultures
(1.518±0.417 IU/ml), P.aeruginosa (0.591±0.030 IU/ml). This was followed by
B.licheniformis which showed comparatively low level maximum production at
800 mg concentration (0.469 ± 0.020 IU/ml). The use of crude filter paper as sub-
strate recorded the higher growth at 800 mg level for P.aeruginosa (0.701 ± 0.018
IU/ml) and mixed cultures (0.542 ± 0.048 IU/ml). Here also B.licheniformis
showed comparatively minimum but high utilization at 800 mg level (0.392 ±
0.013 IU/ml). But when coir was used as a substrate maximum growth rate was
portrayed by mixed cultures (0.405 ± 0.024 IU/ml) followed by B. licheniformis
(0.250 ± 0.026 IU/ml) and P.aeruginosa, which showed least at the same time
maximum growth at 800 mg level (Table 6.06). On comparison, it was noted that
P.aeruginosa, B.licheniformis and mixed cultures preferred to grow cellulose
powder first followed by carboxyl methyl cellulose, crude filter paper and coir
(Table 6.02).
165
Table 6.02 Substrate utilization by Pseudomonas aeruginosa, Bacillus lichenifor-
mis and mixed cultures of both (IU/ml).
Substrate P.aeruginosa B.licheniformis Mixed cultures

Conc(mg)
Cellulose Powder (CP)
200 0.480 ± 0.038 0.266 ± 0.008 0.687 ± 0.044

400 0.695 ± 0.006 0.415 ± 0 .050 1.468 ± 0.183
600 1.547 ± 0.229 0.831 ± 0 .039 1.528 ± 0.012
800 1.789 ± 0.225 1.285 ± 0 .333 2.789 ± 0.154
Carboxy Methyl Cellulose (CMC)
200 0.387 ± 0.020 0.172 ± 0 .011 0.266 ± 0.025

400 0.466 ± 0.036 0.194 ± 0.012 0.391 ± 0.004
600 0.500 ± 0.058 0.384 ± 0 .012 0.498 ± 0.007
800 0.591 ± 0.030 0.469 ± 0 .020 1.518 ± 0.417
Crude Filterpaper (CF)
200 0.235 ± 0.022 0.079 ± 0 .009 0.254 ± 0.029

400 0.387 ± 0.040 0.249 ± 0 .021 0.319 ± 0.031
600 0.500 ± 0.031 0.275 ± 0 .013 0.399 ± 0.023
800 0.701 ± 0.018 0.392 ± 0 .013 0.542 ± 0.048
COIR(CR)
200 0.147 ± 0.018 0.197 ± 0 .011 0.251 ± 0.014

400 0.159 ± 0.034 0.207 ± 0 .003 0.348 ± 0.044
600 0.189 ± 0.020 0.230 ± 0.000 0.388 ± 0.012
800 0.200 ± 0.037 0.250 ± 0.026 0.405 ± 0.024
Standard deviation of the mean (n=6).
6.2.2 Combined glucanase enzyme assay of P.aeruginosa, B.licheniformis and

Mixed cultures
In enzymatic studies, the maximum combined glucanase activity was observed

at 800 mg concentration (2.789±0.154 IU/ml), when cellulose powder was used as
substrate by mixed cultures, followed by P.aeruginosa at 800mg concentration
(1.789±0.225 IU/ml).The minimum combined glucanase was observed by
B.licheniformis (0.266±0.008 IU/ml) at 200mg concentration (Fig 6.01). When CMC
(Carboxy Methyl Cellulose) was provided as substrates, the maximum combined glu-
canase production was given by mixed cultures at 800mg concentration (1.518±0.417
IU/ml) and the minimum production was given by B.licheniformis (0.172±.011
IU/ml) at 200mg concentration (Fig 6.02).
166
The third best utilized substrate was the crude filter paper. Here the maximum
combined enzyme production occurred at 800mg concentration by P.aeruginosa
(0.701±0.018
0.018 IU/ml) followed by mixed cultures at 800mg co
concentration
ncentration
(0.542±0.048
0.048 IU/ml) and the minimum production was observed at 200mg concentr
concentra-
tion by B.licheniformis (0.079±0.009
(0.079 IU/ml) (Fig 6.03).
When compared to all provided substrates coir was the least utilized substrates
by individual as well as mixe
mixedd cultures. Here the high enzyme concentration was oob-
served at 800mg concentration by mixed cultures, followed by B.licheniformis at
800mg concentration (0.250
(0.250±0.026
0.026 IU/ml) and the low enzyme production was ob-
o
served at 200mg concentration by P.aeruginosa (0.147± 0.018) (Fig 6.04).
Fig 6.011 Combined glucanase enzyme assay of Pseudomonas aeruginosa, Bacillus

licheniformis and Mixed cultures of both (IU/ml) – Cellulose Powder (CP).
167
Bac
licheniformis and Mixed cultures of both (IU/ml) – Carboxy Methyl Cellulose
(CMC).

licheniformis and Mixed cultures of both (IU/ml) – Crude Filterpaper
paper (CF).
168
ombined glucanase enzyme assay of Pseudomonas aeruginosa, Bacillus
Fig 6.04 Combined
licheniformis and Mixed cultures of both (IU/ml) – Coir (CR).
6.2.3
.2.3 Endo and exo glucanase enzyme assay of mixed cultures
The exo glucanase secretion was more than endo glucanase productio
production in all
four provided substrates. When mixed cultures were to degrade cellulose powder, the
maximum exo glucanase activity observed at 800mg concentration (2.575±0.152
(2.575
IU/ml) and minimum at 200mg concentration (0.550
(0.550±0.040). Like that more endo
glucanase was occurred at 400mg concentration of cellulose powder (0.588
(0.588±0.004
IU/ml) and low endo glucanase was observed at 600mg concentration (0.128
(0.128±0.005
IU/ml).
In CMC, the maximum exo glucanase production was observed at 800mg co

con-
centration by mixed cultures (0.499±0.043
0.043 IU/ml) and the maximum endo glucanase
was also observed at 800mg concentration (0.214±0.005
(0.214 0.005 IU/ml). The minimum exo
169
glucanase was observed at 200mg concentration, and the minimum endo glucanase
synthesis occurred at 600mg concentration (0.028±0.003 IU/ml).
Table 6.03 Endo and exo glucanase enzyme assay of both mixed cultures (IU/ml).
Substrate Endo glucanace Exo glucanace
Con(mg) Assay Assay
200 0.137 ± 0.044 0.550 ± 0.040
400 0.588 ± 0.004 0.880 ± 0.179
600 0.128 ± 0.005 1.400 ± 0.007
800 0.214 ± 0.002 2.575 ± 0.152
200 0.077 ± 0.003 0.183 ± 0.026
400 0.087 ± 0.003 0.184 ± 0.025
600 0.028 ± 0.003 0.354 ± 0.020
800 0.214 ± 0.005 0.499 ± 0.043
200 0.071 ± 0.003 0.186 ± 0.011
400 0.135 ± 0.006 0.205 ± 0.001
600 0.045 ± 0.003 0.252 ± 0.058
800 0.043 ± 0.005 0.343 ± 0.020
COIR(CR)
200 0.065 ± 0.003 0.186 ± 0.011
400 0.143 ± 0.045 0.205 ± 0.001
600 0.136 ± 0.007 0.252 ± 0.058
800 0.072 ± 0.003 0.343 ± 0.020
Next to CMC, in CF, the high exo glucanase production was seen at 800mg
concentration but high endo glucanase occurred at 200mg concentration (0.071±0.003
IU/ml). The least exo glucanase synthesis seen at 200mg level (0.186±0.011 IU/ml)
and the least endo glucanase observed at 800mg concentration (0.043±.005 IU/ml).
The least degradation occurred in coir substrates. Here also, the maximum exo gluca-
nase found at 800mg concentration by mixed cultures (0.314±0.020 IU/ml), and the
minimum endo glucanase was observed at 200mg level (0.065±0.003 IU/ml) (Table
6.03).
6.2.4 Endo and exo glucanase assay of P. aeruginosa

Next to mixed cultures, the P.aeruginosa gave comparatively more enzyme
production than B.licheniformis when CP was provided as substrates. It portrayed the
highest exo glucanase activity at 800mg concentration (1.525±0.220 IU/ml), and the
lowest production was seen at 200mg concentration (0.469±0.038), and the maximum
170
endo glucanase production was seen at 800mg concentration (0.264±0.006 IU/ml) and
the minimum endo glucanase production was observed at 200mg concentration
(0.011±0.002 IU/ml)
The best substrate utilization was showed by CMC next to cellulose powder.
Here the more exo glucanase activity was observed at 800 mg concentration
(0.573±0.030 IU/ml) and the low production observed at 200mg concentration
(0.208±0.007 IU/ml). Like that the maximum endo glucanase was observed at 200mg
level (0.179±0.013 IU/ml) and the minimum was observed at 800mg concentration
(0.018±0.007 IU/ml).
Table 6.04 Endo and exo glucanase enzyme assay of Pseudomonas aeruginosa (IU/ml).

Con(mg) Assay Assay

200 0.011 ± 0.002 0.469 ± 0.038
400 0.120 ± 0.020 0.575 ± 0.010
600 0.157 ± 0.002 1.390 ± 0.228
800 0.264 ± 0.006 1.525 ± 0.220
200 0.179 ± 0.013 0.208 ± 0.007
400 0.172 ± 0.007 0.294 ± 0.030
600 0.082 ± 0.004 0.418 ± 0.096
800 0.018 ± 0.007 0.573 ± 0.030
200 0.131 ± 0.005 0.104 ± 0.012
400 0.138 ± 0.004 0.249 ± 0.030
600 0.228 ± 0.004 0.272 ± 0.021
800 0.264 ± 0.025 0.377 ± 0.012
COIR(CR)
200 0.046 ± 0.040 0.101 ± 0.160
400 0.030 ± 0.014 0.129 ± 0.026
600 0.071 ± 0.005 0.118 ± 0.002
800 0.092 ± 0.005 0.108 ± 0.027
The crude filter paper utilized by P.aeruginosa showed maximum endo gluca-
nase activity at 800 and 600mg concentratios. The production of endo glucanase was
high at 800mg concentration (0.264±0.025 IU/ml) and low at 200mg concentration
(0.131±0.005 IU/ml).
171
The use of coir portrayed a highest exoglucanase production at 400 and 600mg
concentration. The maximum exo glucanase was pointed out at 600mg concentration
(0.118±0.002 IU/ml) with the minimum being at 800mg concentration. The produc-
tion of endoglucanase was more at 800mg concentration (0.092±0.005 IU/ml) fol-
lowed by 600mg concentration (0.071±0.005 IU/ml), 200mg concentration
(0.0461±0.040 IU/ml) and the least production of endo glucanase occurred at 400mg
concentration (Table 6.04).
6.2.5 Endo and exo glucanase enzyme assay of B. licheniformis:
The enzyme production by B.licheniformis was less compared to the enzyme

activity of mixed cultures and P.aeruginosa.
Table 6.05 Endo and Exo glucanase enzyme assay of Bacillus licheniformis
(IU/ml).

Con(mg) Assay Assay
200 0.046 ± 0 .005 0.220 ± 0.003
400 0.045 ± 0.047 0.370 ± 0.003
600 0.204 ± 0.007 0.627 ± 0.032
800 0.193 ± 0.009 1.092 ± 0.324
200 0.057 ± 0.006 0.155 ± 0.005
400 0.085 ± 0.008 0.109 ± 0.004
600 0.083 ± 0.011 0.301 ± 0.001
800 0.074 ± 0.015 0.395 ± 0.005
200 0.047 ± 0.007 0.032 ± 0 .002
400 0.062 ± 0.006 0.187 ± 0.016
600 0.146 ± 0.055 0.129 ± 0.042
800 0.048 ± 0.010 0.344 ± 0.003
COIR(CR)
200 0.055 ± 0.007 0.142 ± 0.004
400 0.038 ± 0.007 0.169 ± 0.004
600 0.077 ± 0.003 0.153 ± 0.001
800 0.056 ± 0.013 0.194 ± 0.013
Enzymatic activity using CP as substrate reflected the maximum combined

glucanase at 800mg concentration. The minimum value was seen at 200 and 400mg
concentration (Table 5.8.1). The maximum endo glucanase was observed at 600mg
172
concentration (0.204± 0.007 IU/ml) with the minimum at 400mg concentration
(0.045± 0.047 IU/ml). Exo glucanase production was high at 800mg concentration
(1.092±0.324 IU/ml) while low values were observed at 200,400 and 600mg concen-
tration.When CMC was provided, exo glucanase production was more at 800mg con-
centration (0.395±0.005IU/ml). The endo glucanase production was more at 400mg
concentration, and the production was low at 200mg concentration (0.057±0.006
IU/ml). On using B.licheniformis, the maximum exo glucanase production was found
at 800mg concentration (0.344±0.003 IU/ml) when CF was used and the minimum
observed at 200,600 and 400mg concentration. The maximum endo glucanase activity
was analysed at 800mg concentration, and the minimum was observed at 200mg con-
centration (0.032±0.002 IU/ml).
When coir was used, the maximum endo glucanase enzyme synthesis was ob-
served at 600mg concentration (0.077±0.003 IU/ml) followed by 800,200 and 400mg
concentration. The more exo glucanase production occurred at 800mg concentration
(0.194±0.013 IU/ml) followed by 400,600 and 200mg concentrations (Table 6.05).
Table 6.06 Friedman Two-way analysis of variance Rank test at 5% level of sig-
nificance in Pseudomonas aeruginosa, B.licheniformis and Mixed cultures.
Enzyme assay 5% level of significance
Mixed cultures P.aeruginosa B.licheniformis
Combined enzyme 7.815 10.875 7.5
Endo glucanase 7.815 1.725 3.0
Exo glucanase 7.2 12.0 6.6
173
174
6.2.6 Biomass (mg/ml)
In biomass analysis the overall highest pellets dry weight were shown by
mixed cultures followed by Pseudomonas aeruginosa and Bacillus licheniformis .The
maximum biomass were shown by these 2 organisms also in mixed cultured form at
800 mg level and minimum production was at 200 mg concentration. The highest pel-
lets dry weight was shown by mixed cultures at 800 mg level when cellulose powder
was used as a substrate (396±6.1 mg/ml) (Fig 6.05) followed by crude filter paper at
the concentration of 800 mg level by both Bacillus licheniformis and mixed cultures
(377±10.0 mg/ml and 366±13.0 mg/ml) respectively. The minimum biomass quantity
was observed at 200 mg level by P. aeruginosa when crude filter paper was used as a
substrate (93±10.4 mg/ml) (Fig 6.07).
When Pseudomonas aeruginosa was used for degrading the cellulosic sub-
strate cellulose powder, the maximum biomass production was seen at 800 mg level
(273±15.2 mg/ml) and identical minimum biomass production was observed at both
200 and 400 mg levels (113±14.7 mg/ml). In CMC cellulosic substrate the maximum
dry weight of pellets were recorder at 800 mg level (178±7.63 mg/ml) and minimum
was recorder at 200 mg level (103±15.2 mg/ml) (Fig 6.06). On using CF as substrates
the highest biomass occurrence was portrayed at 800 mg level (143±23.1 mg/ml) and
the lowest was at 200mg level (93±10.4 mg/ml). When coir was used as a substrate,
the maximum occurrence at 800 mg level and minimum at 200 mg (129±18.0 mg/ml
and 116±11.1 mg/ml) was observed (Fig 6.08).
175
300
Biomass (mg/ml) 250
200
150
100
50 Mixed form
0 B.licheniformis
200 P.aeruginosa
400
600
800
Substrate concentration (mg)
P.aeruginosa B.licheniformis Mixed form
Fig 6.055 Biomass estimation of P.aeruginosa, B.licheniformis and Mixed cultures

of both on Cellulose Powder (CP).
300
250
Biomass (mg/ml)
200
150
100
50 Mixed form
0 B.licheniformis
200 P.aeruginosa
400
600
800
Substrate concentration

of both on Carboxy Methyl Cellulose Powder (CMC).
176
300
250
Biomass(mg/ml)
200
150
100
50 Mixed form
0 B.licheniformis
200 P.aeruginosa
400
600
800

of both on Crude Filter paper (CF).
300
250
Biomass (mg/ml)
200
150
100
50 Mixed form
0 B.licheniformis
200 P.aeruginosa
400
600
800

of both on Coir (CR).
177
On using Bacillus licheniformis, it showed maximum biomass production at
800 mg level in all 4 cellulosic substrates (280.3+18.5 mg/ml, 157±6.01 mg/ml,
366±13.0 mg/ml and 141±13.3 mg/ml) respectively. But in cellulose powder, the
minimum biomass occurrence reported on 100 mg level (126±9.68 mg/ml) followed
by 200 and 600 mg level and in CMC, the least sedimentation seen at 400 mg level
(127±9.8 mg/ml). Crude filter paper and coir (Fig 6.08) revealed minimum biomass
accumulation at 200 mg levels.
When mixed cultures used, the maximum dried pellets in all cellulosic sub-
strates occur at 800 mg level (396±6.1 mg/ml, 343±5.0 mg/ml, 377±10.0 mg/ml and
268±9.6 mg/ml) and the minimum was recorded at 200 mg levels (144±4.5
mg/ml, 147±7.0 mg/ml, 231 ± 9 .4 mg/ml and 100±12.5 mg/ml) respectively.
6.2.7 Protein assay (mg/ml)
At various substrate concentrations, the maximum protein concentration was

recorded when cellulose powder was used as a substrate at the concentration of 800
mg level by mixed cultures of both P. aeruginosa and B.licheniformis. It was fol-
lowed by P. aeruginosa (3.260±0.012 mg/ml and B. licheniformis at 800 mg level
(3.260±0.012 mg/ml and 2.82±0.665 mg/ml). Like that minimum protein estimation
was observed at 200 mg levels by mixed cultures of P.aeruginosa and B. lichenifor-
mis (3.665±0.226 mg/ml, 2.664±0.170 mg/ml and 2.058±.011 mg/ml)(Fig 6.09).
On using CMC (Fig 6.10), CF(Fig 6.11) and coir (Fig 6.12) the maximum pro-
tein assay revealed at 800 mg concentrations, by mixed cultures, P. aeruginosa and B.
licheniformis (3.660±0.355 mg/ml, 2.750±0.111 mg/ml and 2.500±0.083 mg/ml) re-
spectively, and the minimum was at 200 mg level in the case of mixed cultures and B.
licheniformis when CMC was used but P. aeruginosa showed minimum protein pro-
duction at 400 mg level (2.205±0.111 mg/ml).
178
Fig 6.09 Protein assay of Pseudomonas
omonas aeruginosa, Bacillus licheniformis and
mixed cultures of both oon Cellulose Powder (CP)
Fig 6.10 Protein assay of Pseudomonas aeruginosa, Bacillus licheniformis and

mixed cultures of both onn Carboxy Methyl Cellulose (CMC)
179
mixed cultures of both oon Crude Filter Paper (CF)

mixed cultures of both on
o Coir(CR)
180
181
6.3 DISCUSSION
Among various cellulosic substrates tested for the growth of cellulolytic

microorganisms at varied concentrations, cellulose powder was found to be the
best utilizable substrate by P.aerugenosa, B.licheniformis and a mixed format of
these organisms followed by carboxy methyl cellulose, crude filter paper and coir.
When CP was provided as substrates, the maximum enzyme synthesis occurred at
800mg concentration by mixed cultures of P.aerugenosa and B.licheniformis. The
minimum production was observed at 200mg concentration by all 3 types of cul-
ture. Utilization of CMC at 800mg level was found to be the most optimum for
mixed cultures followed by P.aerugenosa, and B.licheniformis. When coir was
used for the biodegradation studies, the maximum growth rate was found in
mixed cultures and was followed by B.licheniformis and P.aerugenosa (Table
6.07). In biomass assay, the overall highest dry weight of pellets was shown by
mixed cultures of P.aerugenosa and B.licheniformis. In protein assay also, at var-
ious substrate concentrations, the maximum protein concentration was recorded
when cellulose powder was used as a substrate at the concentration of 800 mg
level by mixed cultures followed by P. aeruginosa and B.licheniformis.
Cellulose bioconversion through enzymatic hydrolysis to obtain sugar which

can be fermented to produce liquid fuels has received global attention during the past
fifteen to twenty years. Cellulose produced in millions of tons annually through pho-
tosynthesis all over the world and notably in the tropical regions of the world (Srini-
vasan and Seeta laxman, 1988). Often cellulolytic microbes have been grown on these
for producing microbial biomass for animal feed supplement as single cell protein
(Krikkson and Larson, 1975).
Among the various substrates used for the cellulolytic utilization at varied
concentrations, cellulose powder was found to be the best utilized substrates by all the
organisms used. Mixed cultures showed better utilization and more secretion of en-
zymes on the substrates provided and it was followed by Pseudomonas aeruginosa
and Bacillus licheniforms. This corroborates with the investigations of Stoppok et al.,
182
(1982), who pointed out that cellulose powder was better used by Cellulomonas uda
as compared to microcrystalline cellulose, printed newspaper and some monosaccha-
ride.
Lu et al., (2005) stated that the isolation and characterization of predominant

mesophilic cellulose-utilizing bacteria from flower stalks-vegetable waste co-
composting and their cellulase activities were investigated. The result showed that a
high cellulose degradation rate can be achieved by mixed-cultures. In the present ex-
periment also the maximum substrate utilization and enzyme production was observed
in the mixed cultures of P.aeruginosa and B.licheniformis. Odom and Swall, (1983);
Lewil et al., (1988) revealed that in nature, cellulosic materials are degraded by the
co-operation of many microorganisms. It has been reported that a mixed culture com-
prising a cellulolytic bacterium and a non-cellulolytic bacterium is ideal for degrading
cellulose.
The experimental study of Horton and Osborne, (1967) on cellulase enzyme

activity using an extreme thermophilic strain Clostridium thermocellum with carboxy
methylcellulose as a substrate reported that pH exerted a greater influence on enzyme
secretion. The enzyme activity was optimum at neutral pH (6.5-7.0). Higher (80%)
activity was observed at acidic PH (5.5), and less enzyme activity was seen in an alka-
line pH. In this experimental work the neutral pH was provided for enzyme produc-
tion.
Degradation of highly ordered cellulose in native cotton fibre is almost com-

pletely dependent on the synergistic action of the three hydrolytic enzymes such as
endo glucanase, exo glucanase and β-glucosidase. None of the enzymes acting alone
can solubilize cotton to any significant extent (Erikson and Wood, 1984). Synergism
is evident between exoglucanases and endo glucanases, but for a maximum rate and
extent of hydrolysis all three hydrolytic enzymes must be present at the same time.
The induction and repression phenomena of enzymes involved in cellulose

hydrolysis have been best studied in the white-rot fungus Sporotrichum pulverulentum
and the soft rot fungus Trichoderma viride (Vijaya Antony et al., 2004). The studies
showed that, rapid induction of endoglucanase occurred in the growth media contain-
183
ing cellobiose along with CMC by Sporotrichum pulverulentum. However this cello-
biose did not induce any endoglucanase activity in Trichoderma viride in their work.
It was observed in the present study that, the strains that were used for the cellulolytic
purpose showed more exoglucanase activity than endoglucanase.
The activity of cellulose hydrolyzing enzymes in cultures is dependent not on-

ly on mechanisms regulating their biosynthesis, but also on the presence of specific
inhibitors regulating the activity of the enzyme themselves (Eriksson,1981). (Huang et
al., 2004; Lu et al., 2005) indicated that enhanced cellulose degrading ability was due
to the complementary of cellulases from different strains. Wen-jing, (2005) observed
that mesophilic organisms showed maximum enzyme activity at the temperature
range between 300 and 400C. Aguiar, (2001) stated that the effect of temperature on
the activity and the stability of an enzymatic extract is important in biological
processes.
In the present work, the production of endoglucanase was less not only in the
cellulose powder used as a substrate, but also in the case with CMC, crude filter paper
and coir substrate. This finding is similar to that of Vijay Antony et al., (2004), where
increased activity in exoglucanase than endoglucanase when T. viride AVCM4 was
used on the bran amended with rice straw substrate. The genus Bacillus comprises a
large number of spore forming, aerobic and facultative anaerobic species, some of
which posses cellulolytic activity (Greaves, 1971; Fogarty and Griffin, 1973; Thayer,
1978). Some even contain enzymes for the hydrolysis of hemi cellulose. (Indoka and
Soda, 1956; Emi and Yamamoti, 1972; Esteban et al., 1982).
The cellulolytic enzymes present in most Bacillus forms can hydrolyze car-
boxy methyl, with cellobiose as the main product. The study of Greaves, (1971) on
Bacillus polymyxa indicated that the cellulase was extra cellular and could be ex-
tracted from the mixtures of cells. The remaining cellulosic substrates could also be
removed with distilled water. The formation of the Bacillus polymyxa cellulase is de-
pendent on the carbon source present in the growth medium. Thus, the enzyme was
produced when starch, glucose, xylan, maltose and sucrose were added to a basal salt
peptone medium. It was found that the cellulase enzyme production did not occur in
184
the basal salts peptone medium alone (Fogarty and Griffin, 1973). Here in the me-
dium cellulose was provided as carbon source for enzyme synthesis.
Similar study was done by Thayer, (1978) found that glucose and cellobiose
could stimulate the enzyme formation of Bacilus cereus and Robson and Chambliss,
(1984) working with a new isolate related to Bacillus subtilis demonstrated that the
production of carboxy methyl cellulase is enhanced by the presence of sucrose, mal-
tose, glucose, cellobiose or lactose in the medium. In the present work, instead of bas-
al salts peptone medium Cellulose Powder Peptone Medium (CPPM) was used, which
enhances the growth of cellulolytic bacteria. Cellulose powder, carboxymethyl cellu-
lose, crude filter paper and coir were added at varied concentrations as carbon source,
to check their ability to produce the cellulase enzyme.
Among the cellulolytic strains used for experimentation, mixed cultures of

Pseudomonas aeruginosa and Bacillus licheniformis showed the maximum combined
and exoglucanase activity at 800 mg level followed by Pseudomonas aeruginosa at
800 mg concentrations. Han and Srinivasan, (1968) were able characterize cellulose
utilizing organisms as mainly belonging to Pseudomonas sp next to Cellulomonas sp.
Tewari and Chahal, 1977) were able to report the production of extra cellular cellulase
from Pseudomonas. Bakare et al., (2005) portrayed that the comparative study on mu-
tant strains of Pseudomonas on CMC cellulose substrate revealed that the mutant
strain produce large amount of cellulase enzyme when compare to wild type and the
cellulase activity of the mutants and the wild type increased with increase incubation
time during the growth of the mutants and wild type.
Wen-jung Lu, (2005) used Bacillus pasteurii and Bacillus cereus as cellulose
degrader by providing CMC and filter paper as substrates. The results revealed that
the degrading ability of the substrate was low when single culture used. Significant
synergistic cellulose degradation was detected in mixed cultures. In the present work
also the maximum substrate utility and enzyme secretion was observed when mixed
culture used than the individual inoculation.
The culture used to inoculate the fermentation medium must be in a healthy,

active state and of optimum size, possibly minimizing the length of log phase, thus in
185
its high rate for substrate conversion (Arul ray et al., 2007). The inoculum quantity
normally used is between 3% and 10% of the medium volume (Lincoln 1960; Mey-
rath and Suchanek, 1972; Hunt and Stieber, 1986). Large inoculum volume may be
used to generate the maximum production in a short time, thus increasing the vessel
productivity. The physiological condition of the inoculum when it is transferred to the
next culture stage can have a major effect on fermentation performance. The optimum
transfer time must be determined, so that the inoculation with an ideal culture can be
achieved (Arul ray et al., 2007).
The experimental study of Stoppok et al., (1982) on cellulose degrading en-

zymes from Cellulomonas uda by using CMC, aviel and printed newspaper found that
Cellulomonas uda could rapidly degrade not only aviel and CMC but also lignocellu-
loses materials (like printed newspaper). The obtained growth yield of cultivation
with Avicel in which the carbon source was higher than CMC, followed by printed
newspaper. They acquired cellobiose and glucose as the end product of the hydrolytic
action by Cellulomonas uda.
It has already been outlined that when the extra cellular glucose level rises
during cellulose degradation, the β 1, 4 glucosidase activities is inhibited non compe-
titively with an increase in the cellobiose concentration. High cellobiose concentra-
tions, on the other hand inhibit the extra cellular endo β1, 4-glucanase activities, thus
slowing down the hydrolysis of cellulose. In the present work, when CP, CMC, CF
and coir were used as substrates, minimum enzyme synthesis obtained at 200 mg level
and maximum obtained at 800 mg level.
Choudhary et al., (1980) were able to show that increasing concentrations of

mineral medium helped in increasing the cell density as well as their extra cellular
system. Like wise in the present work the growth of the cellulolytic microbes depen-
dent on the concentrations of the medium provided ie, minimum biomass occurrence
seen at 200 mg level where as maximum biomass reported on 800 mg level by both P.
aeruginosa and mixed cultures. The B.licheniformis showed the minimum occurrence
seen in 400 mg level compared to 200 mg when cellulose powder and Carboxy Me-
186
thyl Cellulose (CMC) were used and maximum growth was all shown at 800 mg lev-
el.
Tulasi Raman, (1986) in his experiments on the induction and elaboration of

the enzyme cellulase in soil fungi by carbohydrate supplements stated that, there was
resistance in the elaboration of cellulase, but an increase was noted with an increase in
the incubation period. Raman gave 20 days incubation for obtaining increased level of
cellulase enzyme. In present work also had 20 days incubation to the cellulolytic
strains to check their ability to produce cellulase enzymes. If the incubation time ex-
tended means there may be more cellulase enzyme production.
The study of Padmaja and Leena Lavanya, (2006) on degrading coir pith us-
ing the fungal culture Humicola grisea Eraaen revealed considerable decrease in the
cellulose content from its initial levels, (35.35% to 30.93%,27.43% and 17.45%) over
30,60,and 90 days. The present work also correlated with the above work for degrad-
ing the cellulose content in coir substrate. Protein and biomass content were more in
mixed cultures than individual cellulose degrader in present study. These results can
be positively correlated with Rajendren et al (1994) while their study with Humicola
fuscotra and Vijay Antony et al., (2004) by using Trichoderma viride and Trichoder-
ma harzianum.
In the present study, when CP and CMC were used as substrates the best de-
gradation effect was given by mixed cultures followed by P.aeruginosa and
B,licheniformis. In CF better degradation was given by P.aeruginosa, mixed cultures
and B.licheniformis. Better CR degradation was given by mixed culture followed by
B.licheniformis and P.aeruginosa. The substrate utilization was determined by esti-
mating the cellulase enzyme production. In biomass and protein assay, the overall
high pellets dry weight and maximum protein concentration were reported in CP by
mixed cultures followed by P.aeruginosa and B.licheniformis.
187
GENERAL SUMMARY
AND CONCLUSIONS
188
Tamil Nadu, India.
7.0 GENERAL SUMMARY AND CONCLUSIONS
Bioremediation is emerging as the most ideal alternative technology for re-

moving pollutants from the environment, restoring contaminated sites and preventing
further pollution. This environment friendly technology contains a range of organisms
used for bioconversion, to clean up pollution and to degrade environmental pollutants.
Therefore the present study is aimed to use the bioremediation technology to reme-
diate sewage water into beneficial ways.
From the results obtained, the following conclusions have been drawn:
7.0.1 Production of biogas using sewage and animal dung
The gradual replacement of cow dung with sewage water concluded that there was
a constant increase in biogas production in the experimental bioreactor than con-
trol upto the level of 80% replacement. The maximum biogas production also oc-
curred in that particular level.
At 100% replacement, the amount of biogas production became lower which in-
turn indicated that the importance of organic rigid supportiveness and methanoge-
nesis in the biogas production. Thus cow dung is a good source of anaerobic me-
thanogenic organisms while sewage water acted as a good nutrient supplier.
The gradual replacement of cow dung with poultry droppings and sewage water
revealed that there was an excellent biogas production in the 100% replacement
of cow dung which inturn indicated that poultry droppings contains more total
solid level which can be easily degraded by methanogenic organisms and sewage
water could be a good nutrient source for biogas production.
189
Thus the animal dung and the sewage water could be easily bioremediated for
biogas production. This technology helps us to reduce the environmental pollution
by dumping them. Furtherlly after the biogas production the spent biodigested
slurry could be used as a good fertilizer for agriltural field.
7.0.2 Biodigested slurry as biofertilizers
The impact of applied biodigested slurries in chilly cultivated field, obtained from
biogas plant revealed that the SWPS+PSO+RP applied field influenced better
yield and high chilly production than the other combinations applied.
The analysis of total height, wet weight and dry weight by using LSD method in-
dicated that all the given manurial treatments were highly significant in flowering
and final stage but non significant in pre flowering stage because at this stage
plants got ready to accept the manurial effect and their efficacy was observed in
flowering and final stage only.
The microbial population analysis revealed that the biodigested manure applied
fields enhanced more microbial growth than before its application.
7.0.3 Biodigested slurry as biological carriers for Rhizobium phaseolus
The biodigested slurries obtained from the biogas plant were used as biocarriers
for R.phaseolus by providing two different climatic conditions such as room and
refrigerated temperature. In room temperature, the mixed biodigested organic car-
rier manure showed the gradual increase in rhizobial load up to 40 days. Other
carrier manures (SWPS, SWCS and OWCS) showed the maximum viability up to
30days.
In refrigerated condition also, the mixed carriers showed maximum viability of

rhizobial culture at 80 days incubation. The better survival of R.phaseolus was
recorded in refrigerated temperature than room temperature because at this tem-
perature the growth, metabolic activities and propagation was occurred in slow
manner compared to normal room temperature.
190
7.0.4 Isolation and characterization of microflora from STP
Selective Himedia were used for the isolation of organisms from STP. At the end
of the analysis 11 bacterial cultures which were predominantly present in the se-
wage water was identified.
They were E.coli, E.aerogens, S.typhi, Shigella sp, Streptococcus sp, S.aerius,
B.licheniformis, P.aeruginosa, Lactobacillus sp, V.cholera and
V.parahemolyticus. THBP and THFP were also analysed and the results revealed
that in both cases, the variations were marginal signifying relatively consistent na-
ture of their occurrence.
7.0.5 Cellulose Degradation
Among the 11 bacterial cultures analysed P.aeruginosa, B.licheniformis and

mixed format of both these organisms were used for cellulose degradation studies
by providing cellulosic substrates.
Four celulosic substrates were used for the biodegradation. When CP and CMC
were used as substrates the best degradation effect was given by mixed cultures,
P.aeruginosa followed by B.licheniformis.
When CF was used, the better degradation effect was given by P.aeruginosa fol-
lowed by mixed cultures and B.licheniformis. During CR degradation again mixed
culture has given better performance in degradation followed by B.licheniformis
and P.aeruginosa. These substrates utility were determined by or based on the cel-
lulase secreation by these organisms.
In biomass analysis also, the overall high dry weight of pellets were shown by
mixed cultures followed by P.aeruginosa and B.licheniformis.
191
In protein assay, at various substrates provided the maximum protein concentra-
tion was reported when CP was used as a substrate at the concentration of 800mg
levels by mixed cultures followed by P.aeruginosa and B.licheniformis .
From this study it is concluded that bioremediation is the eco-friendly and

economical novel alternative to conventional treatment technologies.
*********************************************************
192
SUGGESTIONS FOR
FUTURE RESEARCH
WORK
193
Tamil Nadu, India.
9.0 SUGGESTIONS FOR FUTURE RESEARCH WORK
The probiotic value of spent biodigested slurry can be further investigated.
Comparative study could be carried out in the genome size of normal envi-
ronmental isolated B.licheniformis and P.aeruginosa with sewage isolated
B.licheniformis and P.aeruginosa.
Plasmid isolation studies in cellulose degradative organisms can be conducted

because plasmids play a vital role in the degradation of toxic materials.
Mutations studies in phosphate solubilizing organisms can be performed to in-

duce more phosphatase enzyme synthesis because it plays a vital role in the
conversion of insoluble bound phosphate to soluble form also mutant strains
produces better results compare to wild strains.
Studies can be conducted by adding organic additives to the biodigested slur-

ries sto increase their potential effect and thus they might extend their suppor-
tiveness period as carrier for agriculturally important organisms.
194
`
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LIST OF PAPER
PUBLICATIONS
&PRESENTATION
APPENDIX
APPENDIX Yeast extract - 0.1
*Congo-red(1%solution) 2.5ml
Bacillus Medium (HIMEDIA M1383) Agar - 20
Ingredients Quantity Distilled water - 1000 ml
(gms/litre) pH - 7.2 ± 0.2
L-Glutamic acid - 4.00 * Congo-red (2.5ml/ litre of 1% solution) is incorporated
Citric acid - 2.00 into yeast extract mannitol medium. Congo-red solution is
Dipotassium hydrogen sterilized separately and added to the sterilized medium.
Phosphate - 0.50 Colonies of rhizobia stand out as white, translucent, glis-
Ferric ammonium tening and elevated, with entire margins.
Citrate - 0.50
Magnesium sulphate - 0.50 Casein Agar
Agar - 20.00 Ingredients Quantity
Distilled water - 1000 ml (gms/litre)
pH - 7.4 ± 0.2 Skim milk powder - 100
Peptone - 5
Agar - 15
Cellulose Powder Peptone agar Medium (CPPM) Distilled water - 1000 ml
Ingredients Quantity pH - 7.2 ± 0.2
(gms/100 ml) EMB Agar (HIMEDIA M317-500G)
Cellulose - 2 Ingredients Quantity
Peptone - 2 (gms/litre)
K2HPO4 - 0.2 Peptone - 10.00
Mgso4.7H2o - 0.05 Dipotassium phosphate - 2.00
Yeast extract - 0.05 Lactose - 5.00
Tween 80 - 0.1 Sucrose - 5.00
Agar - 1.5 Eosin-Y - 0.40
Distilled water - 100 ml Methylene blue - 0.065
pH - 7.0 ± 0.2 Agar - 13.50
Distilled water - 1000 ml
pH - 7.2 ± 0.2
Carbohydrate consumption Broth (HIMEDIA M 124)
Ingredients Quantity Gelatin Agar
(gms/litre) Ingredients Quantity
Peptone - 10.00 (gms/litre)
Sodium chloride - 5.00 Peptone - 5
Beef extract - 1.00 Beef extract - 3
Bromocresol purple - 0.10 NaCl - 5
Sterilized carbohydrate Gelatin - 4
Solution - 10 ml Agar - 15
Distilled water - 1000 ml Distilled water - 1000ml
pH - 6.8 ± 0.2 pH - 7.0 ± 0.2
Congo Red Yeast Mannitol Agar (CRYEMA) Hofer’s Alkaline Medium

Ingredients Quantity Ingredients Quantity
(gms/litre) (gms/litre)
Mannitol - 10 *Mannitol - 10
K2HPO4 - 0.5 K2HPO4 - 0.5
MgSO4.7H2O - 0.2 MgSO4.7H2O - 0.2
NaCl - 1.0 NaCl - 1.0
*Yeast extract - 0.1 Beef extract - 10.00
Agar - 20 Yeast extract - 5.00
Distilled water - 1000 ml Dextrose - 20.00
pH - 11.0 ± 0.2 Poly sorbate 80 - 1.00
Ammonium citrate - 2.00
*The reaction of yeast extract mannitol medium is raised Sodium acetate - 5.00
to pH 11.0 with 1N NaOH. 1 ml of 1.6% thymol blue per Magnesium sulphate - 0.10
litre is added. In contrast to Rhizobium the allied genus Manganese sulphate - 0.05
Agrobacterium can grow in this medium, a characteristic Dipotassium phosphate - 2.00
feature which serves as a useful criterion to separate the Agar - 12.00
two bacteria on agar plates during routine isolation of root Distilled water - 1000ml
nodule bacteria. pH - 6.5 ± 0.2
Hydroxy Apatite Medium (HAM)

Ingredients Quantity
(gms/litre) Lactose Agar
Glucose - 10 Ingredients Quantity
CaCl2 (10%) - 50 (gms/litre)
K2HPO4(10%) - 25 Beef Extract - 3
*Soil extract - 1000 ml Peptone - 5
Agar - 15 Lactose - 5
*1000 g of sieved garden soil is mixed with 1200 ml of tap Agar - 15
water and steamed in an autoclave for 30 minutes, then Distilled water - 1000ml
filter through a double layer of Whatman No. 1 filter pa- pH - 6.9 ± 0.2
per.
pH of the medium to 7.0 ± 0.2 by adding 0.1 N NaOH MR-VP Medium (HIMEDIA M070)
solution. Ingredients Quantity
(gms/litre)
Buffered peptone - 7.00

KF Streptococcal Agar Base (HIMEDIA M248) Dextrose - 5.00
Ingredients Quantity Di potassium
(gms/litre) Phosphate - 5.00
Peptone - 10.00 Distilled water - 1000 ml
Yeast extract - 10.00 pH - 6.9 ± 0.2
Sodium chloride - 5.00
Sodium glycerol Mac Conkey Agar Bile Salts (HIMEDIA M 008-100G)
Phosphate - 10.00 Ingredients Quantity
Maltose - 20.00 (gms/litre)
Lactose - 1.00 Peptone - 17.00
Sodium azide - 0.40 Protease peptone - 3.00
Agar - 20.00 Lactose - 10.00
Distilled water - 1000 Bile salts - 1.50
PH - 7.0 ± 0.2 Sodium chloride - 5.00
Neutral red - 0.03
Agar - 15.00
Lactobacillus MRS Agar (HIMEDIA M641) Distilled water - 1000 ml
Ingredients Quantity pH - 7.1 ± 0.2
(gms/litre)
Peptone - 10.00
Ferrous sulphate - 0.0001
Nutrient Agar Agar - 15.00
Ingredients Quantity Distilled water - 1000ml
(gms/litre) pH - 7.0 ± 0.2
Peptone - 5
Beef extract - 1.5
Yeast extract - 1.5 *Phenolphthalein Phosphate Agar (HIMEDIA M652)
NaCl - 5 Ingredients Quantity
Agar - 15 (gms/litre)
pH adjusted to 7.2 ± 0.2 using 10N NaOH Peptone - 5.00
Peptone Glucose Agar (PGA) Beef extract - 3.00
Ingredients Quantity Sodium chloride - 5.00
gms/litre) Sodium Phenolphthalein
Peptone - 10 Phosphate - 0.012
Glucose - 5 Agar - 15.00
Agar - 15 Distilled water - 1000 ml
Bromo cresolpurple pH - 7.4 ± 0.2
(1% alcoholic solution) - 10 ml *76.4 grams were suspended in 1000ml. distilled water,
KH2PO4 - 1 boiled to dissolve the medium completely and steri-
Agar - 15 lized.This was cooled to50C and aseptically added 10 ml
Distilled water - 1000 ml of 1% Triphenyl Tetrazolium Chloride to sterilize me-
pH - 7.1 ± 0.2 dium.It should be mixed well before pouring into sterile
petriplates.
*Pseudomonas Isolation Agar Base Rose Bengal Agar

(HIMEDIA M406) Ingredients Quantity
Ingredients Quantity (gms/litre)
(gms/litre) Glucose - 10
Peptone - 20.00 Peptone - 5
Magnesium chloride - 1.40 K2HPO4 - 1
Potassium sulphate - 10.00 MgSO4.7H2O - 0.05
Triclosan - 0.025 *Streptomycin - 0.03
Agar - 13.60 Agar - 15
Distilled water - 1000 ml Rose-bengal - 0.035
pH - 7.0 ± 0.2 Distilled water - 1000 ml
pH - 7.2 ± 0.2
* 45.03 grams were suspended in 1000 ml distilled water *The antibiotic is sterilized separately and added to the
containing 20 ml glycerol. It was heated to boiling to sterilized medium.
dissolve the medium completely and sterilized.
Pikovskaya’s Agar (HIMEDIA M520) Simmons Citrate Agar (HIMEDIA M099)

Ingredients Quantity Ingredients Quantity
(gms/litre) (gms/litre)
Yeast extract - 0.50 Magnesium sulphate - 0.20
Dextrose - 10.00 Ammonium dihydrogen
Calcium phosphate - 5.00 Phosphate - 1.00
Ammonium sulphate - 0.50 Dipotassium phosphate - 1.00
Potassium chloride - 0.20 Sodium citrate - 2.00
Magnesium sulphate - 0.10 Sodium chloride - 5.00
Manganese sulphate - 0.0001 Bromo thymol blue - 0.08
Agar - 15.00 Lactose - 10.00
Distilled water - 1000 ml Sucrose - 10.00
pH - 6.8 ± 0.2 Dextrose - 1.00
Sodium chloride - 5.00
Ferrous sulphate - 0.20
Starch Agar Sodium thiosulphate - 0.30
Ingredients Quantity Phenol red - 0.024
(gms/litre) Agar - 12.00
Peptone - 5 Distilled water - 1000 ml
Beef Extract - 3 pH - 7.4 ± 0.
Soluble starch - 2
Agar - 15
Distilled water - 1000 ml TCBS Agar (HIMEDIA M189-100G)
pH - 7.0 ± 0.2 Ingredients Quantity
(gms/litre)
Sabouraud Dextrose Agar (SDA)(HIMEDIA M063) Peptone - 10.00
Ingredients Quantity Yeast extract - 5.00
(gms/litre) Sodium thiosulphate - 10.00
Dextrose - 40 Sodium citrate - 10.00
Peptone - 10 Oxgall - 8.00
Agar - 15 Sucrose - 20.00
Chloramphenical - 0.04 Sodium chloride - 10.00
Distilled water - 1000 ml Ferric citrate - 1.00
pH - 5.6 ± 0.2 Bromo thymol blue - 0.04
Thymol blue - 0.04
*SS Agar (HIMEDIA M 108) Agar - 15.00
Ingredients Quantity Distilled water - 1000 ml.
(gms/litre) pH - 8.6 ± 0.2
Peptone - 5.00
Beef extract - 5.00 Tween Agar
Lactose - 10.00 Ingredients Quantity
Bile salts mixture - 8.50 (gms/litre)
Sodium citrate - 10.00 Peptone - 10
Sodium thiosulphate - 8.50 CaCl2 - 0.1
Ferric citrate - 1.00 Tween 80 - 10ml
Brilliant green - 0.00033 Agar - 20
Neutral red - 0.025 Distilled water - 1000ml
Agar - 15.00 pH - 7 ± 7.4
pH - 7.0 ± 0.2 *Urea Agar Base (Christensen) (HIMEDIA M II2)
(*Do not autoclave and over heat the media). Ingredients Quantity
(gms/litre)
Triple Sugar Iron Agar Peptone - 1.00
Ingredients Quantity Dextrose - 1.00
(gms/litre) Sodium chloride - 5.00
Peptone - 10.00 Dipotassium phosphate - 1.20
Casein enzymatic Mono potassium
Hydrolysate - 10.00 Phosphate - 0.80
Yeast extract - 3.00 Phenol red - 0.012
Beef extract - 3.00 Agar - 15.00
pH - 6.8 ± 0.2
*24.09 grams of this media were suspended in 50 ml dis-
tilled water. It was boiled to dissolve the components
completely and sterilized. It was cooled to 500 C and 50ml
of 40% urea solution were added aseptically and mixed
well. This was dispensed into sterile tubes and was put in
the slanting position. The medium should not be over
heated.

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BIOREMEDIATION OF SEWAGE WATER USING

Under the guidance of

MANONMANIAM SUNDARANAR UNIVERSITY

Certified that the thesis entitled “BIOREMEDIATION

OF SEWAGE WATER USING MICROORGANISMS” submitted by

Mrs.V.JEYANTHI KUMARI (Reg No.2417) for the award of Degree of Doctor of

Philosophy in Microbiology – Zoology (Interdisciplinary) of Manonmaniam Sundara-

Date: Signature of the Guide

I hereby declare that the thesis entitled “BIOREMEDIATION

OF SEWAGE WATER USING MICROORGANISMS” submitted by me for the

Degree of Doctor of Philosophy in Microbiology-Zoology (Interdisciplinary) is the

and supervision of Dr.B.VICTOR, M.Sc., Ph.D., Reader, Centre for Aquaculture

ship of any University or Institution.

I am indebted to my guide Dr.B.Victor, M.Sc., Ph.D., Reader, St.Xavier’s

a deep sense of gratitude to him in ways more than one.

I must express my indebtedness and deep sense of gratitude to The Manage-

ment, Dr.S.Chockalingam, M.Sc.,Ph.D.,F.A.Z.,F.R.E.S.(London), Director and

vilpatti, for their constant encouragement and their word of appreciation.

I extended my hearty thanks to Dr.B.Geetha, M.Sc., Ph.D., Professor of

P.G.Department of Zoology and Research centre, Mr.Deva Manoharan M.Sc.,

M.Phil, Professor of P.G.Department of Mathematics and Dr.S.Shanmugavel,

M.Sc., Ph.D., Reader, P.G.Department of Zoology, V.O.Chidambaram College, Tuti-

fort and suggestion for the successful completion of my work.

I have great pleasure to record my thanks to Mr.M.Dharmaraj, M.Sc.,

lam, for his encouragement during the tenure of my research.

kind encouragement which enabled me in finishing this work.

Er.Puthia Sekaran, Associate professor, National Engineering College, Kovilpatti

for his valuable help in maintaining biogas plants in STP campus.

I have great pleasure in expressing my deep sense of gratitude to my spouse,

1.0 Review of literature 6

2.0 Production of biogas using sewage and animal dung

2.1 Materials and Methods 30

3.0 Biodigested slurry as biofertilizer on chilly plant-A Field trial

3.1 Materials and Methods 71

4.0 Biodigested slurry as biological carrier for Rhizobium phaseolus.

4.1 Materials and Methods 100

5.0 Isolation and characterization of microflora from STP

5.1 Materials and Methods 120

6.0 Cellulose biodegradation

6.1 Materials and Methods 140

7. General summary and conclusions 167

8. Suggestions for future research work 172

Paper publications and presentations

TABLE TITLE PAGE

1.01 Microbial diversity in anaerobic digesters. 10

1.02 Organisms associated with phosphate solubilization 13

2.01 Characteristics of sewage water. 37

Biogas production by gradual reduction of cow dung supple-

2.04 Analysis of Variance for the production of biogas between 39

Temperature and pH in control bioreactor.

Total Solids (TS), Total Volatile Solids (TVS) and Volatile

Analysis of Variance for the production of biogas between

Student’s ‘t’ test analysis of TS,TVS & VFA for biogas

Temperature and pH in control bioreactor.

3.04 Influence of biodigested manurial sources on the morpholocal 80

3.05 Least Significance Difference (LSD) in Total height of chilly 81

3.06 Least Significance Difference (LSD) in wet weight of chilly 82

3.07 Least Significance Difference (LSD) in dry weight of chilly 83

3.08 Effect of biodigested manurial sources on NPK content of 84

3.09 Effect of biodigested manurial sources on NPK content of 84

4.01 Biochemical characteristics of Rhizobium phaseolus. 106

4.02 The survival of rhizobial cultures in biodigested organic manure 108