Charles E. Wyman, Dartmouth College Y. Y. Lee, Auburn University

1. Integration of Leading Biomass Pretreatment Technologies with Enzymatic Digestion and Hydrolyzate FermentationDOE OBP Pretreatment Core R&D Gate Review MeetingJune 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. Cellulase enzyme Stage 1 Pretreatment Stage 2 Enzymatic hydrolysis Residual solids: cellulose, hemicellulose, lignin Biomass Solids: cellulose, hemicellulose, lignin Chemicals Dissolved sugars, oligomers, lignin Dissolved sugars, oligomers Hydrolysis Stages Biomass Refining CAFI

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

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 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. Increasing pH Pretreatment Yields at 15 FPU/g Glucan *Cumulative soluble sugars as total/monomers. Single number = just monomers. Biomass Refining CAFI

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

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

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

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

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

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

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

20. Pretreatment Yields at 15 FPU/g Glucan

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

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. Quang Nguyen, Abengoa Bioenergy Mat Peabody, formerly Applied CarboChemicals Gary Welch, Aventinerei Greg Luli, BC International Paris Tsobanakis, Cargill Robert Wooley, Cargill Dow James Hettenhaus, CEA Lyman Young, ChevronTexaco Kevin Gray, Diversa Paul Roessler, Dow Susan M. Hennessey, DuPont Michael Knauf, Genencor Don Johnson, GPC (Retired) Dale Monceaux, Katzen Engineers Kendall Pye, Lignol Farzaneh Teymouri, MBI Richard Glass, National Corn Growers Association Bill Cruickshank, Natural Resources Canada Joel Cherry, Novozymes Ron Reinsfelder, Shell Carl Miller, Syngenta Carmela Bailey, USDA Don Riemenschneider, USDA CAFI Project Advisory Board Serve as extension agents for technology transferProvide feedback on approach and resultsMeet with team every 6 months

26. Tasks for the DOE OBP Project • 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 Biomass Refining CAFI

27. CAFI 2 Stover • 2nd 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 Biomass Refining CAFI

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

29. Pretreated Substrate Schedule Biomass Refining CAFI

30. Pretreated Substrate Schedule Biomass Refining CAFI

31. SO2 Pretreatment of Corn Stover Pretreatment Hydrolysis 15 FPU Hydrolysis 60 FPU

32. AFEX Pretreated Poplar

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

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

35. 0.006g of protein/g of cellulose 0.03g of protein/g of cellulose 0.06g of protein/g of cellulose 31% 21% 12% Xylanase Supplementation of SO2 Treated Stover 0.9% (w/v) consistency, corn stover-190oC, 5min, 3% S02, 0.0417g Spezyme SP, 0.0073g cocktail BG-X-001 Method: High Throughput Microassay

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

37. Initial Fermentation Results after 144 hours 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 Technology96(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