1. Integration of Leading Biomass Pretreatment Technologies with Enzymatic Digestion and Hydrolyzate Fermentation DOE OBP Pretreatment Core R&D Gate Review Meeting June 9-10, 2005 Charles E. Wyman, Dartmouth College Y. Y. Lee, Auburn University Mohammed Moniruzzaman, Genencor International Bruce E. Dale, Michigan State University Richard T. Elander, National Renewable Energy Laboratory Michael R. Ladisch, Purdue University Mark T. Holtzapple, Texas A&M University John N. Saddler, University of British Columbia Biomass Refining CAFI

2. Presentation Outline Project background Technical feasibility and risks Biomass Refining CAFI Competitive advantage History and accomplishments Project overview Plan/Schedule and recent results Critical issues and show stoppers Summary and caveats Plans and resources for next stage Biomass Refining CAFI

3. Project Background: Pretreatment Needs High cellulose accessibility to enzymes High sugar yields from hemicellulose Low capital cost – low pressure, inexpensive materials of construction Low energy cost Low degradation Low cost and/or recoverable chemicals Biomass Refining CAFI

4. Technical Feasibility and Risks Dilute acid pretreatment is often favored based on more extensive development Many other options have been studied, but only a few are promising Pretreatment is most expensive single operation Difficult to compare leading pretreatments based on data available Limited knowledge of pretreatment mechanisms slows commercial use of all options Biomass Refining CAFI

5. Project Background: CAFI Biomass Refining Consortium for Applied Fundamentals and Innovation organized in late 1999 Included top researchers in biomass hydrolysis from Auburn, Dartmouth, Michigan State, Purdue, NREL, Texas A&M, UBC, U. Sherbrooke Mission: Develop information and a fundamental understanding of biomass hydrolysis that will facilitate commercialization, Accelerate the development of next generation technologies that dramatically reduce the cost of sugars from cellulosic biomass Train future engineers, scientists, and managers. Biomass Refining CAFI

6. Competitive Advantage Developing data on leading pretreatments using: –Common feedstocks –Shared enzymes –Identical analytical methods –The same material and energy balance methods –The same costing methods Goal is to provide information that helps industry select technologies for their applications Also seek to understand mechanisms that influence performance and differentiate pretreatments –Provide technology base to facilitate commercial use –Identify promising paths to advance pretreatment technologies Biomass Refining CAFI

7. Hydrolysis Stages Biomass Refining CAFI Stage 2 Enzymatic hydrolysis Dissolved sugars, oligomers Solids: cellulose, hemicellulose, lignin Chemicals Biomass Stage 1 Pretreatment Dissolved sugars, oligomers, lignin Residual solids: cellulose, hemicellulose, lignin Cellulase enzyme

8. Mass Balance Approach: AFEX Example Hydrolysis Enzyme (15 FPU/g of Glucan) Residual Solids Hydrolyzate Liquid AFEX System Treated Stover Ammonia Stover 101.0 lb 100 lb (dry basis) 36.1 lb glucan 21.4 lbxylan 39.2 lb 95.9% glucan conversion to glucose, 77.6% xylan conversion to xylose 99% mass balance closure includes: (solids + glucose + xylose + arabinose ) Wash 2 lb 99.0 lb Solids washed out 38.5 lb glucose 18.9 lbxylose (Ave. of 4 runs) Very few solubles from pretreatment—about 2% of inlet stover

9. CAFI USDA IFAFS Project Overview Multi-institutional effort funded by USDA Initiative for Future Agriculture and Food Systems Program for $1.2 million to develop comparative information on cellulosic biomass pretreatment by leading pretreatment options with common source of cellulosic biomass (corn stover) and identical analytical methods –Aqueous ammonia recycle pretreatment - YY Lee, Auburn University –Water only and dilute acid hydrolysis by co-current and flowthrough systems - Charles Wyman, Dartmouth College –Ammonia fiber explosion (AFEX) - Bruce Dale, Michigan State University –Controlled pH pretreatment - Mike Ladisch, Purdue University –Lime pretreatment - Mark Holtzapple, Texas A&M University –Logistical support and economic analysis - Rick Elander/Tim Eggeman, NREL through DOE Biomass Program funding Completed in 2004 Biomass Refining CAFI

10. Feedstock: Corn Stover NREL supplied corn stover to all project participants (source: BioMass AgriProducts, Harlan IA) Stover washed and dried in small commercial operation, knife milled to pass ¼ inch round screen Glucan36.1 % Xylan21.4 % Arabinan3.5 % Mannan1.8 % Galactan2.5 % Lignin17.2 % Protein4.0 % Acetyl3.2 % Ash7.1 % Uronic Acid3.6 % Non-structural Sugars1.2 % Biomass Refining CAFI

11. Calculation of Sugar Yields Comparing the amount of each sugar monomer or oligomer released to the maximum potential amount for that sugar would give yield of each However, most cellulosic biomass is richer in glucose than xylose Consequently, glucose yields have a greater impact than for xylose Sugar yields in this project were defined by dividing the amount of xylose or glucose or the sum of the two recovered in each stage by the maximum potential amount of both sugars –The maximum xylose yield is 24.3/64.4 or 37.7% –The maximum glucose yield is 40.1/64.4 or 62.3% –The maximum amount of total xylose and glucose is 100%. Biomass Refining CAFI

12. Pretreatment Yields at 15 FPU/g Glucan Pretreatment system Xylose yields*Glucose yields*Total sugars* Stage 1Stage 2Total xylose Stage 1 Stage 2Total glucose Stage 1Stage 2Combined total Maximum possible 37.7 62.3 100.0 Dilute acid32.1/31.23.235.3/34.43.953.257.136.0/35.156.492.4/91.5 Flowthrough36.3/1.70.6/0.536.9/2.24.5/4.455.259.7/59.640.8/6.155.8/55.796.6/61.8 Controlled pH 21.8/0.99.030.8/9.93.5/0.252.956.4/53.125.3/1.161.987.2/63.0 AFEX34.6/29.3 59.8 94.4/89.1 ARP17.8/015.533.3/15.556.1 17.8/071.689.4/71.6 Lime9.2/0.319.628.8/19.91.0/0.357.058.0/57.310.2/0.676.686.8/77.2 *Cumulative soluble sugars as total/monomers. Single number = just monomers. Increasing pH Biomass Refining CAFI

13. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

14. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

15. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

16. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

17. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

18. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

19. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Pretreatment Yields at 15 FPU/g Glucan

21. General PFD for Cost Estimates Biomass Refining CAFI Boiler + Generator Different Pretreatments Hydrolysis + Fermentation Feed Handling Recovery Different Pretreatments Stover Syrup + Solids Chemicals Water Enzymes CO 2 Water EtOH Steam Power

22. Minimum Ethanol Selling Price (MESP) Biomass Refining CAFI

23. Effect of Oligomer Conversion on MESP Biomass Refining CAFI

24. DOE OBP Project: April 2004 Start Funded by DOE Office of the Biomass Program for $1.88 million through a joint competitive solicitation with USDA Using identical analytical methods and feedstock sources to develop comparative data for corn stover and poplar Determining more depth information on –Enzymatic hydrolysis of cellulose and hemicellulose in solids –Conditioning and fermentation of pretreatment hydrolyzate liquids –Predictive models Added University of British Columbia to team through funding from Natural Resources Canada to –Capitalize on their expertise with xylanases for better hemicellulose utilization –Evaluate sulfur dioxide pretreatment along with those previously examined: dilute acid, controlled pH, AFEX, ARP, lime Augmented by Genencor to supply commercial and advanced enzymes Biomass Refining CAFI

25. CAFI Project Advisory Board 1.Quang Nguyen, Abengoa Bioenergy 2.Mat Peabody, formerly Applied CarboChemicals 3.Gary Welch, Aventinerei 4.Greg Luli, BC International 5.Paris Tsobanakis, Cargill 6.Robert Wooley, Cargill Dow 7.James Hettenhaus, CEA 8.Lyman Young, ChevronTexaco 9.Kevin Gray, Diversa 10.Paul Roessler, Dow 11.Susan M. Hennessey, DuPont 12.Michael Knauf, Genencor 13. Don Johnson, GPC (Retired) 14. Dale Monceaux, Katzen Engineers 15. Kendall Pye, Lignol 16. Farzaneh Teymouri, MBI 17. Richard Glass, National Corn Growers Association 18. Bill Cruickshank, Natural Resources Canada 19. Joel Cherry, Novozymes 20. Ron Reinsfelder, Shell 21. Carl Miller, Syngenta 22. Carmela Bailey, USDA 23. Don Riemenschneider, USDA Serve as extension agents for technology transfer Provide feedback on approach and results Meet with team every 6 months

26. Tasks for the DOE OBP Project Biomass Refining CAFI Pretreat corn stover and poplar by leading technologies to improve cellulose accessibility to enzymes Develop conditioning methods as needed to maximize fermentation yields by a recombinant yeast, determine the cause of inhibition, and model fermentations Enzymatically hydrolyze cellulose and hemicellulose in pretreated biomass, as appropriate, and develop models to understand the relationship between pretreated biomass features, advanced enzyme characteristics, and enzymatic digestion results Estimate capital and operating costs for each integrated pretreatment, hydrolysis, and fermentation system and use to guide research

27. CAFI 2 Stover Biomass Refining CAFI 2 nd pass harvested corn stover from Kramer farm (Wray, CO) –Collected using high rake setting to avoid soil pick-up –No washing –Milled to pass ¼ inch round screen

28. Feedstock: USDA-supplied hybrid poplar (Alexandria, MN) –Debarked, chipped, and milled to pass ¼ inch round screen Biomass Refining CAFI CAFI 2 Poplar

29. Pretreated Substrate Schedule Pretreatment/SubstrateExpected Date Dilute Acid/Corn StoverSeptember 2004 Dilute Acid/Poplar (Bench Scale)October 2004 Dilute Acid/Poplar (Pilot Plant)December 2004 SO 2 /Corn StoverMarch 2005 Controlled pH/PoplarMay 2005 SO 2 /PoplarAugust 2005 Ammonia Fiber Explosion/PoplarSeptember 2005 Ammonia Recycled Percolation/PoplarOctober 2005 Flowthrough/PoplarMarch 2006 Lime/PoplarApril 2006 Biomass Refining CAFI

30. Pretreated Substrate Schedule Pretreatment/SubstrateExpected Date Dilute Acid/Corn StoverSeptember 2004 Dilute Acid/Poplar (Bench Scale)October 2004 Dilute Acid/Poplar (Pilot Plant)December 2004 SO 2 /Corn StoverMarch 2005 Controlled pH/PoplarMay 2005 SO 2 /PoplarAugust 2005 Ammonia Fiber Explosion/PoplarSeptember 2005 Ammonia Recycled Percolation/PoplarOctober 2005 Flowthrough/PoplarMarch 2006 Lime/PoplarApril 2006 Biomass Refining CAFI

31. Log (Ro)Glucose (%)Xylose (%) 3.029983 3.3510064 4.038845 3.025862 3.358860 4.038845 3.027370 3.359564 4.038845 Pretreatment Hydrolysis 15 FPU Hydrolysis 60 FPU SO 2 Pretreatment of Corn Stover

32. AFEX Pretreated Poplar

33. Biomass Refining CAFI Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar 2% glucan concentration 50 FPU/g glucan, no β-glucosidase supplementation

34. Model Predictions of Effect of Lignin South et al. Phillipidis et al. Holtzapple et al. 100 g substrate/L, 50% cellulose, 10 FPU cellulase/g cellulose, 2 CBU/FPU NM, 5 FPU/gm Biomass Refining CAFI

35. 0.9% (w/v) consistency, corn stover-190 o C, 5min, 3% S0 2, 0.0417g Spezyme SP, 0.0073g cocktail BG-X-001 0.006g of protein/g of cellulose0.03g of protein/g of cellulose 0.06g of protein/g of cellulose 12% 21% 31% Method: High Throughput Microassay Xylanase Supplementation of SO 2 Treated Stover

36. Dilute Acid Pretreated Corn Stover Hydrolyzate Fermentation (resin conditioned) Biomass Refining CAFI

37. Initial Fermentation Results after 144 hours Control OverlimeXAD4Overlime + XAD4 Xylose Consumed ( %)54.142.444.541.3 Ethanol Yield (% theoretical for glucose + xylose consumed) 76.8 63.479.072.0 Biomass Refining CAFI

38. CAFI Presentations/Publications Team presentations at –2004 Annual Meeting of the American Institute of Chemical Engineers, Austin, Texas, November 11 –2003 Annual Meeting of the American Institute of Chemical Engineers, San Francisco, California, November 20 –25th Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, Colorado, May 7, 2003 –2002 Annual Meeting of the American Institute of Chemical Engineers, Indianapolis, Indiana, November 4 –24th Symposium on Biotechnology for Fuels and Chemicals, Gatlinburg, Tennessee, April 28, 2002 Mosier N, Wyman CE, Dale B, Elander R, Lee YY, Holtzapple M, Ladisc1 M. 2005. “Features of Promising Technologies for Pretreatment of Lignocellulosic Biomass,” BioResource Technology 96(6): 673-686 Special issue of Bioresource Technology in progress to report USDA IFAFS findings in several papers including joint papers to introduce project and summarize results Biomass Refining CAFI

39. Critical Issues and Show Stoppers Must assure that all pretreatments realize near maximize possible yields Include both pretreatment and subsequent enzymatic hydrolysis Evaluate effect of enzymes on yields of both xylose and glucose Characterize well hydrolyzate fermentability and conditioning demands Biggest concern is unknown challenges that prove too time consuming to resolve Biomass Refining CAFI

40. Observations for Corn Stover All pretreatments were effective in making cellulose accessible to enzymes Lime, ARP, and flowthrough remove substantial amounts of lignin and achieved somewhat higher glucose yields from enzymes than dilute acid or controlled pH However, AFEX achieved slightly higher yields from enzymes even though no lignin was removed Cellulase was effective in releasing residual xylose from all pretreated solids Xylose release by cellulase was particularly important for the high-pH pretreatments by AFEX, ARP, and lime, with about half being solubilized by enzymes for ARP, two thirds for lime, and essentially all for AFEX Biomass Refining CAFI

41. Caveats The yields can be further increased for some pretreatments with enzymes a potential key Mixed sugar streams will be better used in some processes than others Oligomers may require special considerations, depending on process configuration and choice of fermentative organism The conditioning and fermentability of the sugar streams must be assessed These results are only for corn stover, and performance with other feedstocks will likely be different as initiallly shown for poplar Biomass Refining CAFI

42. Plans and Resources for Next Stage The results from this project will provide a basis for industry to select technologies to commercialize Results should also suggest new enzyme and organism strategies Further research will be important to better account for performance differences Consideration should be given to taking advantage of differences among pretreatment options Biomass Refining CAFI

43. Acknowledgments US Department of Agriculture Initiative for Future Agricultural and Food Systems Program, Contract 00-52104-9663 US Department of Energy Office of the Biomass Program, Contract DE-FG36- 04GO14017 Natural Resources Canada Our team from Dartmouth College; Auburn, Michigan State, Purdue, and Texas A&M Universities; the University of British Columbia; Genencor International; and the National Renewable Energy Laboratory Biomass Refining CAFI

44. Insanity is doing what you always have always been doing and expecting different results Biomass Refining CAFI

45. Questions? Biomass Refining CAFI