Production of Biobutanol from Jatropha Seed Cake Jasmine Isar
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Energy Crises and Alternative Fuels Dismal scenario of fossil fuel availability Perils of petroleum resource exhaustion Stringent environmental legislation 4
Alternative Fuels Properties Methanol Ethanol Butanol Gasoline Molecular CH 3 OH C 2 H 5 OH C 4 H 9 OH Many formula Energy content (per gallon) Motor octane number 63k Btu 78k Btu 110 k Btu 115k Btu 91 92 94 96 Ethanol may have the early mover s advantage, but the race is not yet over, butanol is an emerging biofuel 5
Butanol v/s Other Fuels 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 6
Biological Route to n-butanol Source : Ramey and Yang, DOE Report, 2004 7
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 8
Global Players Source: http://www.biobutanol.com/the -Players.html 9
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) 10
Butanol toxicity Butanol toxicity is the biggest challenge in butanol fermentation Butanol tolerance can be key to high butanol titers 11
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 12
Butanol Tolerance Mutant strain was tolerant upto 3.0% (v/v) of butanol Wild Type Strain Mutant Strain 13
Mechanism of Butanol Tolerance 1. Altering the lipid composition Wild type strain Myristic (C14:0) and Palmitic acid (C16:0) were found to be present in solvent tolerant mutant strain Mutant strain Oleic acid (C18:1) conc. is high in Standard FAMEs the solvent non-tolerant wild type strain 14
Mechanism of Butanol Tolerance 2. Over expression of the heat shock proteins, HSP 60 Solvent tolerant mutant strain showed higher levels of GroEL A kda B 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 15
Mechanism of Butanol Tolerance 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 G accumulation in wild-type & mutant Rhodamine 6G (dye) is a P- glycoprotein substrate Rhodamine 6G P-glycoprotein is an efflux pump (ABC transporter) of multidrug resistance family 16
Lignicellulosic Biomass For Biofuels Another challenge is the cost of raw material which can be mitigated by using ligno-cellulosic biomass as the feedstock Corn stover Switch Grass Jatropha Seeds 17
Lignocellulosic Biomass used for Biofuels 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 Source: http://www.biobutanol.com/the -Players.html 18
Why Jatropha? Jatropha Is Better For Bio-fuel Production 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 19
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 20
Scale-up of Butanol Production using JSC (3L) 14.8 g L 1 of n-butanol in 96 h Acetone : Butanol : Ethanol 2.6: 6.6: 0.8 21
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 22
Butanol Titers Using Biomass Biomass Hydrolysate Additives in medium Butanol titer (gl -1 ) Productivity (gl -1 h -1 ) Yield (g/g) Wheat straw (Pfromm et al. 2010) Barley straw (Qureshi et al. 2010a) Corn Strover (Qureshi et al. 2010b) Switch grass (Qureshi et al. 2010b) Rice Straw (Ranjan et al. 2013) Corn Fiber (Qureshi et al. 2008) Glucose, YE, Vitamin Sol Glucose, YE, Vitamin Sol 12.7 in 72h 0.176 0.26 18.01 in 68h 0.264 0.43 Glucose 10.4 in 96h 0.108 0.43 Glucose 9.55 in 84h 0.114 0.37 None 13.5 in 12 days 0.047 0.34 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 23
Conclusion 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 24
Highlights 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 25
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 (CO 2, H 2 ) 26
Acknowledgements Dr. Vidhya Rangaswamy Harshvardhan Joshi Dr. Sanjeev Katti Reliance Life Sciences Reliance Industries Ltd. 27
Thank You! 28