Production of Biobutanol from Jatropha Seed Cake · Jatropha Seeds Another challenge is the cost of raw material which can be mitigated by using ligno-cellulosic biomass as the feedstock
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2 © Reliance Industries Ltd., 2013
The Reliance Group, founded by Dhirubhai H. Ambani (1932-2002), is India’s largest private
sector enterprise with businesses in the energy and materials value chain, and having annual
revenues in excess of US$ 68 billion. The flagship company, Reliance Industries Limited (“RIL”),
is a Fortune Global 500 Company and enjoys global leadership in its key product lines. RIL’s
Jamnagar refinery complex has the world’s largest refining capacity (1.3 million bpsd) at a single
location and has an impeccable track record of safe and reliable operations. RIL is continuing its
efforts to be a creator of intellectual property and is driving a “technology transformation” agenda
that promotes scientific and technical excellence by fostering an eco-system for creativity and
innovation.
RIL Overview
3
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policy or intent of Reliance Industries Ltd. (RIL).
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Disclaimer
4 © Reliance Industries Ltd., 2013
Energy Crises and Alternative Fuels
Dismal scenario of fossil fuel availability
Perils of petroleum resource exhaustion
Stringent environmental legislation
5 © Reliance Industries Ltd., 2013
Alternative Fuels
Properties Methanol Ethanol Butanol Gasoline
Molecular
formula
CH3OH C2H5OH C4H9OH Many
Energy content
(per gallon)
63k Btu 78k Btu 110 k Btu 115k Btu
Motor octane
number
91 92 94 96
Ethanol may have the early mover’s advantage, but the race is not yet over , butanol is an
emerging biofuel
6 © Reliance Industries Ltd., 2013
Butanol has higher energy content
Six times less evaporative
Non-corrosive
100% substitutable for gasoline
No engine modification
Butanol is a ‘higher grade’ fuel in many respects, the only challenge is to produce it enough
to meet the world’s insatiable demand
Butanol v/s Other Fuels
7 © Reliance Industries Ltd., 2013
Source : Ramey and Yang, DOE Report, 2004
Biological Route to n-Butanol
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Limitations of Butanol Fermentation
Low yields of butanol
Product toxicity to the bacterium
Poor recovery
Culturing and handling of anaerobes
Butanol is toxic to the bacterial system at 1.3% concentration
10 © Reliance Industries Ltd., 2013
Economic Viability
The economic viability of n-butanol fermentation is governed
mainly by three factors
High product titer
(achievable by overcoming solvent toxicity)
Raw material cost
(using lignocellulosic biomass)
Solvent recovery costs
(if titers can be raised from 12 to 19 gL-1, cost of recovery can
be cut down to half)
11 © Reliance Industries Ltd., 2013
Butanol toxicity
Butanol toxicity is the biggest
challenge in butanol fermentation
Butanol tolerance can be key to
high butanol titers
12 © Reliance Industries Ltd., 2013
Strain Improvement
Chemical mutagenesis was carried out to generate butanol tolerant
strains
Mutants were screened for solvent tolerance in medium with
varying concentrations of n-butanol
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Butanol Tolerance
Mutant strain was tolerant upto 3.0% (v/v) of butanol
Wild Type Strain Mutant Strain
14 © Reliance Industries Ltd., 2013
Mechanism of Butanol Tolerance
1. Altering the lipid composition
Myristic (C14:0) and Palmitic acid
(C16:0) were found to be present
in solvent tolerant mutant strain
Oleic acid (C18:1) conc. is high in
the solvent non-tolerant wild type
strain
Standard FAMEs
Wild type strain
Mutant strain
15 © Reliance Industries Ltd., 2013
2. Over expression of the heat shock proteins, HSP 60
Mechanism of Butanol Tolerance
Solvent tolerant mutant strain showed higher levels of GroEL
Western Blot showing GroEL expression in mutant (A) and wild-type (B)
Lanes 2–5 protein
Lane 1, standard GroEL
Lane 2, 120 h
Lane 3, 96 h
Lane 4, 72 h
Lane 5, 48 h
kDa
A B
16 © Reliance Industries Ltd., 2013
Mechanism of Butanol Tolerance
Rhodamine G accumulation in
wild-type & mutant
Rhodamine 6G
P-glycoprotein binds a large number of
lipophilic compounds, such as antibiotics,
dyes, organic solvents and mediates
the energy dependent efflux of these
toxic compounds from the bacterial cells
Rhodamine 6G (dye) is a P- glycoprotein
substrate
P-glycoprotein is an efflux pump (ABC transporter) of multidrug resistance family
17 © Reliance Industries Ltd., 2013
Lignicellulosic Biomass For Biofuels
Corn stover Switch Grass
Jatropha Seeds
Another challenge is the cost of raw material which can be mitigated
by using ligno-cellulosic biomass as the feedstock
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© Reliance Industries Ltd., 2013
Company Microbe Molecule Biomass
Butamax
(DuPont/BP)
Clostridum sp.,
E.coli
Iso-butanol,
n- butanol
Corn
Green Biologics Clostridium sp. n - butanol Lignocelluloses
Butalco Yeast Unclear Lignocelluloses
Gevo Yeast , E.coli Iso-butanol Corn
Cobalt Biofuels Clostridium sp. n- butanol Wood pulp,
sugarbeet
Tetra Vitae Clostridium sp. n- butanol variable
Lignocellulosic Biomass used for Biofuels
Source: http://www.biobutanol.com/The -Players.html
19 © Reliance Industries Ltd., 2013
Why Jatropha?
Hardy nature
Short gestation time of about 3 years
Productive life of 50 - 100 years
Not browsed by animals
Adaptability to varied agro-climatic
conditions and soil type
Drought resistance
Non competing with food crops for land and
water sources
Jatropha Is Better For Bio-fuel Production
20 © Reliance Industries Ltd., 2013
Optimization of Jatropha Seed Cake (JSC)
Concentration
Acid pretreated JSC hydrolysates (2%, 5%, 7% &10% w/v )
examined for butanol production
Mutant grown in JSC (7 %w/v) with 2 % glucose yielded 13.2 g L−1
butanol
21 © Reliance Industries Ltd., 2013
14.8 g L−1 of n-butanol in 96 h
Acetone : Butanol : Ethanol
2.6: 6.6: 0.8
Scale-up of Butanol Production using JSC (3L)
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Scale-up of Butanol Production using JSC (15L)
A butanol titer of 18.6 g L-1 obtained in 72 h with a productivity of 0.26 g L-1 h-1
23 © Reliance Industries Ltd., 2013
Butanol Titers Using Biomass
Biomass Hydrolysate Additives in
medium
Butanol titer
(gL-1)
Productivity
(gL-1h-1)
Yield
(g/g)
Wheat straw
(Pfromm et al. 2010)
Glucose, YE ,
Vitamin Sol
12.7 in 72h 0.176 0.26
Barley straw
(Qureshi et al. 2010a)
Glucose, YE ,
Vitamin Sol
18.01 in 68h 0.264 0.43
Corn Strover
(Qureshi et al. 2010b)
Glucose 10.4 in 96h 0.108 0.43
Switch grass
(Qureshi et al. 2010b)
Glucose 9.55 in 84h 0.114 0.37
Rice Straw
(Ranjan et al. 2013)
None 13.5 in 12 days 0.047 0.34
Corn Fiber
(Qureshi et al. 2008)
Glucose, YE ,
Vitamin Sol
6.4 in 88 h 0.072 0.27
Jatropha Seed cake
(This study)
Glucose, YE ,
Peptone
18.6 in 72 h 0.255 0.45
24 © Reliance Industries Ltd., 2013
Solvent-tolerant mutant strain was developed
Increasing n-butanol productivity using cheap substrate JSC, a
potential lignocellulosic substrate
The process could be successfully scaled up at 15 L
Entire fermentation process was run in a single batch mode
Conclusion
25 © Reliance Industries Ltd., 2013
Production of high yield of butanol (18.6 gL-1) in a single batch
process using jatropha seed cake as the biomass
Highly solvent tolerant mutant
No stripping of butanol produced during fermentation
Highlights
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Forward Path
Optimization Strategies
Use of Flexible feedstocks
Efficient pre treatment method
Scale up of the process up to pilot size
Developing down stream processing of
butanol
Recovery of byproducts (CO2, H2)
© Reliance Industries Ltd., 2013
27 © Reliance Industries Ltd., 2013
Acknowledgements
Dr. Vidhya Rangaswamy
Harshvardhan Joshi
Dr. Sanjeev Katti
Reliance Life Sciences
Reliance Industries Ltd.
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