1. Release of Sugars for Fermentation to Ethanol by Enzymatic Digestion of Corn Stover Pretreated by Leading Technologies Charles E. Wyman, Dartmouth College/University of California Bruce E. Dale, Michigan State University Richard T. Elander, National Renewable Energy Laboratory Mark T. Holtzapple, Texas A&M University Michael R. Ladisch, Purdue University Y. Y. Lee, Auburn University Mohammed Moniruzzaman, Genencor International John N. Saddler, University of British Columbia International Symposium on Alcohol Fuels San Diego, California September 26, 2005 Biomass Refining CAFI

2. Presentation Outline Project background Biomass Refining CAFI Enzymatic hydrolysis results from USDA IFAFS funded project Initial results for recently begun DOE OBP funded project Observations Biomass Refining CAFI

3. Pretreatment Needs 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

4. 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 Project Background: CAFI

5. 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 CAFI Approach

6. 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 Stage 3 Sugar fermentation

7. 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

8. 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

9. 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

10. 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

11. Pretreatment Yields at 60 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 acid32.1/31.23.335.4/34.53.953.357.236.0/35.156.692.6/91.7 Flowthrough36.3/1.70.8/0.737.1/2.44.5/4.457.061.5/61.440.8/6.157.8/57.798.6/63.8 Controlled pH 21.8/0.99.030.73.5/0.254.758.225.3/1.163.688.9 AFEXND/30.2 61.8 ND/92.0 ARP17.8/017.034.8/17.059.4 17.8/076.494.2/76.4 Lime9.2/0.320.229.4/20.51.0/0.359.560.5/59.810.2/0.679.789.9/80.3 *Cumulative soluble sugars as total/monomers. Single number = just monomers. Increasing pH Biomass Refining CAFI

12. Pretreatment Yields at 60 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 acid32.1/31.23.335.4/34.53.953.357.236.0/35.156.692.6/91.7 Flowthrough36.3/1.70.8/0.737.1/2.44.5/4.457.061.5/61.440.8/6.157.8/57.798.6/63.8 Controlled pH 21.8/0.99.030.73.5/0.254.758.225.3/1.163.688.9 AFEXND/30.2 61.8 ND/92.0 ARP17.8/017.034.8/17.059.4 17.8/076.494.2/76.4 Lime9.2/0.320.229.4/20.51.0/0.359.560.5/59.810.2/0.679.789.9/80.3 *Cumulative soluble sugars as total/monomers. Single number = just monomers. Increasing pH Biomass Refining CAFI

13. 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

14. 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

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

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

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

23. 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

24. Tasks for the DOE OBP Project Biomass Refining CAFI Pretreat corn stover and poplar by leading technologies to improve cellulose accessibility to enzymes 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 Develop conditioning methods as needed to maximize fermentation yields by a recombinant yeast, determine the cause of inhibition, and model fermentations Estimate capital and operating costs for each integrated pretreatment, hydrolysis, and fermentation system and use to guide research

25. 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

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

27. 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

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

29. 95% conversion of glucan to glucose 64% conversion of xylan to xylose 83% overall yield of sugars SO 2 Pretreatment of Corn Stover Corn stover 36.1 g glucan 21.4 g xylan Pretreatment (190 o C, 5min, 3% SO 2) Liquid phase Solid phase Sugars 1.2 g glucose 8.7 g xylose Sugars 36.9 g glucose 6.8 g xylose Hydrolysis (60FPU/g of glucan) Biomass Refining CAFI

30. 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 Biomass Refining CAFI

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

32. Initial Fermentation Results after 144 hours Control OverlimeXAD 4 Overlime + XAD4 Xylose Consumed ( %)54.142.444.541.3 Ethanol Yield (% theoretical for glucose + xylose consumed) 76.8 63.479.072.0 Biomass Refining CAFI

33. 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

34. 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 characterized Initial results are for corn stover, and performance with other feedstocks will likely be different as already found for poplar Biomass Refining CAFI

35. Impact and Opportunities 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 is important to understand reasons for performance differences Consideration should be given to taking advantage of differences among pretreatment options Biomass Refining CAFI

36. 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 Biomass Refining CAFI

37. Questions? Biomass Refining CAFI