Prof.

Rintu Banerjee, FNAAS, FBRS, FBRIAT

Professor, Agricultural & Food Engineering Department
Indian Institute of Technology Kharagpur
India
Sector wise global CO2 emission Carbon dioxide equivalent
emissions (CO2 eq) from
transportation sector in India

Source: IPCC, 2014)

WORLD MARKET FOR BIOFUEL PRODUCTION: 2013- 2023
INDIAN SCENARIO IN BIOFUEL PRODUCTION
Bioethanol demand with blending targets (%) in India Demand for ethanol for blending and share in blending
Indian scenario in biofuel production

Government of India, is planning to set up twelve (12) 2G

Ethanol Bio-refineries across 11 States viz. Punjab, Haryana,
U.P., M.P, Bihar, Assam, Odisha, Gujarat, Maharashtra,
Karnataka and A.P.

The estimated investment for the 12 Bio-refineries is Rs 10,000

crores. These Bio-refineries shall produce around 35- 40 crore
litres of Ethanol annually, thus contributing significantly
towards the Ethanol Blended Petrol Programme.

Recently GoI has proposed a new 2G biorefinery in Paradip

(Odisha)
India to commission world’s largest green field refinery by 2022

Largest green field refinery

Signed a Joint Venture

(JV) agreement, June -
2017
Second Generation (2G)
ethanol production
Potential source of biomass for 2G Ethanol

Agricultural
Energy crops Municipal
residues (wheat Wood and paper
(switchgrass, solid waste,
straw, corn waste
poplar)
stover) organic

Low cost renewable organic resources

Global biofuels market forecast (2017-2014)
GROSS RESIDUE AVAILABILITY FROM CROP PRODUCTION IN INDIA

The above scenario illustrates the potential of some of the crop residues which can be utilized
for biofuel and biochemical generation
 Biomass Selection

Lignocellulosics

Rice straw Ricinus communis

Maize straw Lantana camara

Wheat straw Jatropha curcas

Rice husk Pineapple leaf wastes

Jute waste Bambusa bambos

Oil seed waste Datura stramonium

Grazing Non- Grazing

 2G-Ethanol production from Non-conventional and
Renewable sources

Lantana camara

Kans Grass
Ricinus communis
LIGNIN

CELLULOSE
Enzymatic
Pretreatment

Rice straw HEMICELLULOSE

-Promising Green Technology

Bambusa bambos
Cotton Stalk

Sugarcane Tops
BIOCHEMICAL CHARACTERIZATION OF LIGNOCELLULOSICS
Biochemical Cellulose Hemicellulose Lignin
composition (%, w/w) (%, w/w) (%, w/w)

Lantana camara 47.25 16.4 17.26

Ricinus communis 42.00 18.02 19.88

Bambusa bambos 45.00 17.00 19.20

Sugarcane Tops 33.00 22.76 13.45

Kans Grass 38.70 29.00 17.46

Pineapple leaf waste
42.00 25.00 13.00

Rice straw 33 16 14

Mixture 43.02 24 14.57

BIOCHEMISTRY OF LIGNOCELLULOSICS
 Steps involved in 2G ethanol generation:
soil to soil technology
Biomass
Cellulose and Hexose and
pretreatment Ethanol
Hemicellulose Pentose
biomass recovery
hydrolysis fermentation
pretreatment

Hydrolysis of Utilization of Distillation to

Enzymatic degradation of cellulose & hexose and recover
recalcitrant lignin layer hemicellulose to pentose sugar to maximum
hexose and pentose ethanol ethanol
sugar

Cyanobacterial Anaerobic Residual

Biomanure Biomethane solid
treatment Digestion
biomass
 Different Methods of Pretreatment

Acid Hydrolysis

Alkali Hydrolysis

Costly, Hazardous,
energy and labor Physical Treatment
intensive, loss of
target
biomolecules
Physico-chemical Treatment

Biological/ Enzymatic Treatment

Major Bottlenecks in Lignocellulosic
Bioethanol Production
Maximum lignin degradation with
minimum loss of cellulose and
hemicellulose

Efficient depolymerization of lignin

without the production of furfurals and
hydroxymethyl furfurals

Simultaneous utilization of Pentose and

Hexose sugars

Less tolerance of yeast towards higher

concentration of ethanol
Water requirement during biomass pretreatment

Biological pretreatment requires litre of water for per litre of ethanol production compared to more than 1 or 5 litre of water
requirement in physical, chemical and physico-chemical pretreatment methods
Leading Pretreatment technologies
• Partial hydrolysis of
hemicellulose
• Ammonia recovery during
pilot scale operation is difficult
• Lignin is deposited on the
surfaces of the material
possibly causing blockage of
cellulases to cellulose

• Expensive

• Needs improved method for

ILs recovery and reuse
Enzymes/microbes involved in second generation
bioethanol production
Lignolytic
Cellulolytic enzymes Yeast
enzymes

• Lignin • Endo β- • Saccharomyces

peroxidase glucanase cerevisae
• Manganese • β-glucosidase • Saccharomyces
peroxidase • Cellobiohydrolas pastorianus
• Laccase e
• Pichia pastoris
• Versatile • Oxidative • Saccharomyces
peroxidase cellulases
fermentati
• Cellulose
phosphorylase • Saccharomyces
paradoxus
• Xylanase
• Swollenin
• Expansin
IIT Kharagpur contribution
in biofuel production
Enzymatic Delignification of Lignocellulosics
Microbial Biotechnology and
DSP Laboratory, Department of
AgFE, IIT Kharagpur

Blue laccase from a

hyperactive strain of Pleurotus Yellow laccase
djamor from Lentinus
squarrosulus
 Result of Delignification

Biomass Condition Solid loading % Delignification

(% dry
weight/vol.)
Ricinus communis Initial mixing followed by static 30 75-80
Lantana camara Initial mixing followed by static 30 75-80
Kans grass Initial mixing followed by static 30 75-80
Bamubusa bambos Initial mixing followed by static 30 75-80
Cotton stalk Initial mixing followed by static 25 77-81
Sugarcane top Initial mixing followed by static 30 75-80
Banana waste Initial mixing followed by static 20 70-72
Pineapple leaf waste Initial mixing followed by static 30 75-80
Rice straw Initial mixing followed by static 20 78-82
Mixture Initial mixing followed by static 30 75-80
SEM analysis

Control
Lantana camara Pretreated
Lantana camara

Control
Ricinus communis Pretreated
Ricinus communis

Control
Bambusa Bambos Pretreated
Bambusa Bambos

Control
Pineapple leaf Pretreated
Pineapple leaf
SEM analysis cont…

Control
Kans grass Pretreated
Kans grass

Control
Sugarcane top Pretreated
Sugarcane top

Control
Mixture Pretreated
Mixture
 Porosity Analysis by BET/BGH Analyzer

Biomass Pore size Pore volume (Before (b); % Delignification

(Before (b); After After (a)) cm3/g
(a)) Angstrom

Ricinus communis 189.20 (b); 13.01 x 10-3 (b); 75-80

297.98 (a) 20.50 x 10-3 (a)

Lantana camara 86.74 (b); 5.968 x 10-3 (b); 75-80

108.957 (a) 7.496 x 10-3 (a)

Kans grass 61.9 (b); 4.259 x 10-3 (b); 75-80

128.5 (a) 5.365 x 10-3 (a)

Pineapple leaf waste 120 (b); 4.26 x 10-3 (b); 75-80

134 (a) 6.57 x 10-3 (a)
 FTIR analysis

Ricinus communis Lantana camara Bambusa bambos Kans grass

Pineapple leaf Sugarcane top Mixture

Wavenumber (cm-1 ) Particular
3425-3300 OH Vibration
2919-2932 C–H methyl and
methylene groups
1060-1262 O–H phenolic
626-1055 lignin
XRD Analysis

Control
Lantana camara Pretreated
Lantana camara

Control
Sugarcane top Pretreated
Sugarcane top

Control
Kans grass Pretreated
Kans grass
 XRD Analysis cont…
25000

20000

15000

PSD
10000

5000

0
10 20 30 40 50 60 70

Angle

Pineapple leaf Mixture

Lignocellulosic biomass Increase in crystallinity (%)

Ricinus communis 6.82

Lantana camara 7.46

Kans grass 8.54
Bambusa bambos 4.62
Pineapple leaf waste 6.25
Sugarcane top 4.30
Mixture 3.66
 HPLC chromatogram of Lignin degradation
Products

• With time peak area of the individual peaks increased

• Lignin degraded products released over time

 GC-MS Analysis of lignin degraded compounds

Identified compounds 0h 1st h 2nd h 3rd h 4th h 5th h 6th h 7th h Retention time
Propanoic acid - + + + + + + + 4.748
Butanoic acid - + + + + + + + 5.731
Acetic acid - + + + + + + + 5.974
Chromone - + + + - - - - 7.167
Methanone - - - - - + + + 7.544
Pentacene - + + + - - - - 8.007
Butanedioic acid - + + + + + + + 8.194
2-Butenedioic acid - + + + + + + + 8.747
2(3H)-Furanone - + + + + + + + 9.186
3,4-Methylenedioxyphenylacetic acid - - - + + + + - 10.876
3,5-di-tert-Butyl-4-hydroxyphenyl propionic acid - + + + - - + + 11.282
3-Bromo-5-ethoxy-4-hydroxybenzaldehyde - - - - + + + + 12.269
2',6'-Dihydroxyacetophenone - + + + - - - - 15.882
11H-Dibenzo[b,e][1,4]diazepin-11-one - - - - - + + + 15.863
5(2-Dimethylamino-1-phenyl)-vinyl-thiadiazol - + + + - - - - 16.264
Trimethylsilyl-3-methoxy-4-(trimethylsilyloxy)cinnamate - - - + + + + + 17.023
Ferulic acid - - - + + + + + 17.013
Trimethylsilyl-3,4-bis(trimethylsiloxy)cinnamate - - + + + + + + 17.452
Ethyl-2-ethoxyquinoline-4-carboxylate - - + + - + - - 21.628
Salbutamol - + + + + - - - 23.876
2-(p(Dimethylamino)phenyl) benzimidazole - - - + + + + + 25.070
3,4-Dimethylbenzamide - - - - + + + + 25.375
Some of the detected compounds
Compounds detected during laccase mediated delignification

Low molecular weight compounds like

 2(3H)-Furanone (RT 9.186),
 3,4-Methylenedioxyphenylacetic acid (RT 10.876),
 Ethyl-2-ethoxyquinoline-4-carboxylate (RT 21.628)

 Aromatic compounds
 Trimethylsilyl-3-methoxy-4-(trimethylsilyloxy) cinnamate (RT 17.023),
 3-Bromo-5-ethoxy-4-hydroxybenzaldehyde (RT 12.269) and
 Ferulic acid (RT-17.013)

 Low molecular organic acids

 Propanoic acid (RT 4.748),
 Butanoic acid (RT 5.731),
 Acetic acid (RT 5.974),
 Butanedioic acid (RT 8.194)
Microscopic analysis of raw and pretreated biomass

Raw biomass Pretreated biomass

Raw biomass Pretreated biomass

Established biomass pretreatment technology
with different substrates
Blue laccase

Substrates: L. camara, R. communis, S. spontaneum, B. bambos, S.

Officinarum tops, A. comosus leaf waste, Lignocellulosics Mixture
Optimum conditions: 35-40 ºC, 6-8 h, 15-25% Solid loading, 6-7 pH, 300-500
IU/mL laccase titre, 75-86% delignification %
Ref: Gujjala et al., 2016; Mukhopadhyay et al., 2011; Rajak and Banerjee, 2015;
Kulia et al., 2011; Avanthi and Banerjee, 2016.

Yellow laccase

Substrates: L. camara, R. communis, S. spontaneum, B. bambos, S. Officinarum

tops, A. comosus leaf waste, Lignocellulosics Mixture
Optimum conditions: 35-40 ºC, 4-6 h, 15-25% Solid loading, 5-7 pH, 400-800
IU/mL laccase titre, 65-87% delignification %
Ref: Mukhopadhyay et al., 2011; Rajak and Banerjee, 2016.
ENZYMATIC SACCHARIFICATION OF PRETREATED LIGNOCELLULOSIC
BIOMASS

Delignification

A mixture of carbohydratases produced

from T. reesei RUT C30
Correlation between Delignification, Reducing sugar and Ethanol
Production from Lignocellulosic Biomass
90 900

80 800

70 700

Delignification (%)

Reducing sugar (mg/g)

60 600

Ethanol (mg/g)
50 500

40 400

30 300

20 200

10 100

0 0
Ricinus Lantana Bambusa Sugarcane top Kans grass Pineapple leaf Mixture
communis camara bambos waste
Delignification (%) Reducing sugar Ethanol
 Strategies to improve the ethanol yield from
lignocellulosic biomass
Lignocellulosic Ethanol (%, v/v)
biomass
SHF SSF CBP PCBP SSCF

2.78 3.58 6.81 7.12 6.24

Ricinus communis
2.51 3.38 6.5 6.9 6.95
Lantana camara
3.21 6.02 5.91 5.96 7.50
Sugarcane top
3.50 6.30 4.9 7.80 8.00
Kans grass
3.25 6.42 6.78 7.18 6.95
Pineapple leaf waste
3.00 5.14 7.52 7.65 7.50
Mixture
Integrated approach towards soil to soil technology

Processing Microbes / Enzymes

Processed solid Fermentation
biomass
Agricultural residue

Residual Ethanol,
fermented butanol, lactic
applied back to the biomass
soil acid

Enrichment with
cyanobacteria Left over residual solid
Biomanure Fermentation (MAC)
biomass

By-products
(Biogas)
Removal of CO2 by
water stripping
Biodiesel

CO2 as carbon source Enriched

used in algal cultivation biomethane
 Pilot Plant facility for Ethanol Production
(Capacity: 500 kg fresh biomass processing per batch)
Lignocellulosic
Biomass

Hot Air Oven

Pulverizing unit
 Pilot plant

Pre-treatment tank Saccharification tank Fermentation tank

Control panel Tincture press Gantry system

Boiler assembly Distillation assembly
Biomass collection Gantry system for lifting the immobilization tray
tank
 STUDENTS’S CONTRIBUTION

Dr. Mainak Dr. Arindam Kuila

Mukhopadhyay
Mr. Rajiv Ch. Mr. Sanjeev Kumar
Rajak

BIOENERGY GROUP AT
P.K.SINHA CENTRE FOR
Ms. Anjani BIOENERGY, Ms. Althuri Avanthi
Devi Chintagunta IIT KHARAGPUR

Ms. Knawang Ch.

Mr. G. Lohit K. Sherpa
Srinivas

Mr. Debajyoti Kundu Ms. Tania Dev Ms. Reddhy Mahle

NEWS TIME