Progress on Enzyme Technology for Bioethanol Production

1. Progress on Enzyme Technology for Bioethanol Production International Symposium Delhi Gang Duan, PhD Surendra Bade & Jay K. Shetty, PhD Mar 20-21, 2008

2. Agenda • Trends in bioethanol production • New enzymes from Genencor • Conclusion

3. Ethanol and DDGS 129 Plants are in operation 77 Plants are under construction

5. CORN TO ETHANOL 129 Plants are in operation and 77 Plants are under construction • Currently 6.0 billion gal of ethanol is produced in the US. • Estimates indicate that ethanol production in the US could be 12.0 billion gals/yr by 2009. • Most of the increase in the ethanol capacity will come from new dry grind ethanol plants • Supply of distillers dried grains with solubles (DDGS) will increase proportionately 1/3 + 1/3 + 1/3 *1lb=0.45kg

6. Starch To Ethanol -Reaction Mechanism- Enzyme+Energy SUBSTRATE + H2O ---------------------------> PRODUCT (Starch)Alpha Amylase (SPEZYME Fred,SPEZYME Xtra)(Dextrins) Enzyme SUBSTRATE + H2O ---------------------------> PRODUCT (Dextrins)Glucoamylase (Optidex L400, Fermenzyme)(Glucose) Yeast SUBSTRATE ----------------------------> PRODUCT (Glucose) Protease (FERMGEN) (EtOH,CO2,Biomass)

7. pH adjustment steps are not shown * Conventional Ethanol Production Process Thermo-Stable Alpha Amylase Alcohol Recovery Glucoamylase Yeast Milo Corn Wheat Rye Barley Tapioca Distillation & Dehydration Liquefaction Saccharification Fermentation Water JET COOKER >100° C 5–8 MIN * STORAGE TANK 60° C 8–10 HRS (optional) SLURRY TANK GRINDING SECONDARY LIQUEFACTION 95° C ~90 MIN * DDGS

8. Issues for the conventional processes • EnergyEnergy • EfficiencyEfficiency • EnvironmentalEnvironmental Enzyme ENZYME SOLUTIONS

9. Phytic acid removal and NSP modification Reduced Energy Consumption E N Z Y M E E N Z Y M E E N Z Y M E Improved Fermentation efficiency Corn to Ethanol: Manufacturing Cost 2005 (Dry Milling Plant): We make enzymes work for you Depreciation Labor Enzymes, yeast etc Utilities Net corn DDGS Ref: Gopal Chotani,2005 , Internal Communication ,Genencor-Danisco.

10. How Do we Create value using enzymes ? Process Development based on existing unit-operations never results in radical process innovation -Enzymes to reduce number of Unit operations (saving on the capital cost) -Enzymes’ Kinetic energy replacing the thermal energy in the process ( reduction in energy cost-STARGEN Platform ) -Enzymes’ Kinetic energy replacing mechanical energy ( saving on capital cost and energy cost-Macerating and viscosity reducing Enzymes,Protease , STARGEN) -Enzymes for enhancing de-watering effect-saving on drying -Enzymes resulting in value added co-products (manufacturing cost reduction,For example Phytase for value added DDGS)

11. FACTORS THAT AFFECT ENZYME ACTIVITY • Time • Temperature • pH • Substrate • Metal ions required for some enzymes • Inhibitors • Enzyme concentration

12. Factors affecting yeast performance • Microbial • Lactic acid (0.8% or 8g/L inhibitory. Overcome by higher inoculum) • Acetic acid • Ethanol • pH • … • Non microbial • Sulfite • Sodium • Temp.(Heat stress) • Nutrient stress • …

13. Anaerobic yeast process Ethanol production C6H12O6 2C2H6O + 2CO2 + 2ATP + Heat (32 kcal) Cell growth C6H12O6+ 1.2*0.63NH3 + xATP  0.63C6H10.8O3N1.2 + 2.22CO2 + 4 NAD(P)H Glycerol production C6H12O6+ 2NAD(P)H + 2ATP  2C3H8O3 • From the above, if GA limits glucose concentration, then, it also limits ethanol production • Ethanol determines ATP production; cell growth and glycerol determine ATP consumption • However, anaerobic cell growth produces reducing power (NAD(P)H) • Nitrogen source: ammonia means more reducing power production • Nitrogen source: amino acids (AFP) mean less reducing power production • And, glycerol production recycles NAD(P)H, i.e., glycerol is proportional to cell growth • Hence net Ethanol yield on glucose is determined by growth and glycerol

14. Yeast Growth & Alcohol Production 12 enzymes involved synthesis of ethanol

15. THEORETICAL ETHANOL YIELD • Goal: Gallons of Ethanol per bushel of corn • Starch content in corn: • 56 Lbs Corn/Bu X 86% Ds (1) X 73% Starch (2) = 35.2 Lbs Starch/Bushel • Glucose content in starch • 35.2 Lbs Starch/Bu X 1.11 (3) = 39.1 Lbs Glucose/Bushel • Ethanol content based on glucose • 39.1 Lbs Glucose/Bu X .51 Lbs Ethanol/lb Glucose (4) = 19.9 Lbs Ethanol/Bu ÷ 6.54 (5) Lbs Ethanol/Gal = 3.1 Gal Ethanol/Bushel • (1) 14% Moisture In Corn • (2) Starch Content Of Corn • (3) Chemical Gain Of Starch To Glucose • (4) Lbs Ethanol Per Lb Glucose • (5) Density Of Ethanol *1lb=0.45kg 1bushel=56 lbs=25.2kg

16. THEORETICAL YIELD/Corn as an example 1 gal. 0.1 gal. 1 gal. 1 gal. Bushel = 56 lbs. corn 1000kg corn at 14% moisture and 73% starch results in 455L Alcohal Ethanol = 19.90 lbs. = 35.5% CO2 = 19.20 lb. = 34.3% DDGS = 16.9 lbs. = 30.2% *1lb=0.45kg 1bushel=56 lbs=25.2kg

17. ACTUAL ETHANOL YIELD • Theoretical ETHANOL yield = 3.1 GAL/BU (455 L/MT) • Actual ETHANOL yield • Typically 2.6-2.8 Gal/Bu (90% +/- Efficiency) (~410 L/MT) • Factors reducing yield • Grain quality, moisture content, starch content • Starch conversion rate to glucose (bound) • Glucose conversion rate to ethanol (yeast) • Plant process control *1lb=0.45kg 1bushel=56 lbs=25.2kg

18. Full Product Line for ethanol production • Liquefying enzymes • SPEZYME XTRA: High performance, thermostable, fast viscosity-reducing • Maxaliq-one: A new liquefying enzyme and system with additional phytase • Spezyme Fred: Thermostable, low calcium, alpha-amylases. • Saccharifying enzymes • Distillase L-400, Optidex L-400, Optidex L-300; • Fermenzyme L-400, Glucoamylase with acid fungal protease; • Acid Fungal Proteases(Enhancing the yeast performance, shorten the fermentation time, increase the alcohol yield) • FERMGEN • GC 106 • Viscosity reducing enzymes (Higher DS, easy to handle; potential more fermentable sugars) • OPTMASH BG, OPTIMASH TBG • OPTIMASH VR • Enzymes for no-cook process: STARGEN

19. Liquefying enzyme Liquefaction • Solubilizes starch • Hydrolyzes starch to dextrins with enzymes • Reduces viscosity >SPEZYME XTRA • Thermostable • Fast viscosity reducing >MAXALIQ • Superior viscosity reducing • more DDGs values

20. Effect of Phytase on Viscosity ReductionUsing Maxaliq One

21. Improving the value of DDGs by using phytase Comparison of Conventional hot cook process and PALS (Phytase Amylase Liquefaction System) on the ethanol yield and phytic acid content

22. Saccharifying enzymes • Saccharification enzymes benefit fermentation • Faster fermentation times • Higher ethanol yields • Improved yeast health • Better solids separation in the centrifuge • Reduces evaporator fouling • Less recycle or backset • Higher protein in DDGS • More consistent plant operations >DISTILLASE L-400 >FERMENZYME L-400

23. Acid Fungal Protease • FERMGEN

24. Viscosity reducing enzymes Viscosity reducing enzymes to hydrolyze the non-starch polysaccharides in wheat, sorghum and tuber before/during the cooking. • OPTIMASH BG for wheat/Barley • OPTIMASH VR for sorghum/Rye and others • OPTIMASH TBG, a thermostable beta-glucanase could be effective at 80o C.

25. Enzyme for no-cook process

26. STARGEN ----Enzyme for No-cook process Alcohol Recovery STARGEN Yeast Rice/Milo + water Saccharification Liquefaction Water JET COOKER >120° C 5–8 MIN Distillation & Fermentation * STORAGE TANK SECONDARY LIQUEFACTION 95° C ~90 MIN 60° C 3–10 HRS (optional) SLURRY TANK GRINDING DDGS No cook process using STARGEN: -NO LIQUEFACTION; NO NEED for HEATING as well as for COOLING -Very low sugar concentration in the system; Relaxed and happy yeast by spooling; Less contamination. -More alcohol from raw material. Overall, a much more simplified process, and more importantly, a more EFFICIENT process .

27. The difference STARGEN could make: Comparison of the amounts of alcohol produced from conventional and STARGEN process: High temperature jet cooking: Very good process - 400 liter Good process - 390 liter Average process - 380 liter STARGEN process without any pretreatment Produces 430-460 liter or even higher!

28. Benefits of STARGEN 001 in alcohol production in No cook process • Increased Carbon Conversion Efficiency — Higher Yield • Energy Saving -Elimination of Jet Cooking • Reduction in Osmotic Stress/Reduction in By-products Formation — Glycerol, Organic Acids, etc. • Capacity Increase — High Density Fermentation - Higher Alcohol Yield • Reduction of Yeast Growth Inhibitors — High Glucose, Maillard Products, etc. • Saving on Operational Cost — Labor, Time, Chemicals • Elimination of Calcium Addition — Reduction of Calcium Salt Formation • Value Added By-product (DDGS) — Higher Protein Content • Process Simplification — Reductions in Unit Operations • Saving on Capital Cost — Capacity Increase/New Plant

29. FINLAND Jämsänkoski Hanko NETHERLANDS Leiden BELGIUM Brugge CHINA Beijing Wuxi JAPAN Tokyo USA Rochester, NY Palo Alto, CA Cedar Rapids, IA Beloit, WI SINGAPORE ARGENTINA Arroyito Buenos Aires Mfg. R&D Admin. Danisco / GenencorWuxi Application CentreDelhi Technical Service Lab India

30. Acknowledgement THANKS to all the team members in Wuxi Application Centre Lab for their hard and excellent work! Sophia Xu, Kathy Qian, John Zhou, Jessica Li, Bruce Ruan Raj Lad Roy Sim

31. Danisco (India) Pvt.Ltd. DLF Corporate Park 5thFloor,Block4B,Phase III, Gurgaon-122 002 Attn.:-- Mr.Yogesh Grover Cell No. -- +91 9999333505 Tel. No.–0124 4061510 Danisco (India) Pvt.Ltd. 302,Centre Point, J.B.Nagar,NearKohinoor Continental Hotel, Andheri-KurlaRoad,Andheri-E, Mumbai-400 059 Attn.:-- Mr.Sunil Kasat Cell No.-- +91 9833465865 Tel.No.–022 28258713 Contacts