1. Maximum Total Time for Talk = 25 minutes

2. Comparative Sugar Recovery Data from Application of Leading Pretreatment Technologies to Corn Stover and Poplar 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 28 th Symposium on Biotechnology for Fuels and Chemicals Nashville, Tennessee May 1, 2006 Biomass Refining CAFI

3. Biomass Refining Consortium for Applied Fundamentals and Innovation organized in late 1999 and early 2000 Included top researchers in biomass hydrolysis from Auburn, Dartmouth, Michigan State, Purdue, NREL, Texas A&M, U. British Columbia, 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 CAFI Background

4. 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

5. USDA IFAFS Project Overview: CAFI 1 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

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

7. Pretreatment system Temperature, o C Reaction time, minutes Chemical agent used Percent chemical used Other notes Dilute acid 1 16020Sulfuric acid0.4925% solids concentration during run in batch tubes Flowthrough 2 20024none0Continuously flow just hot water at 10mL/min for 24minutes Partial flow pretreatment 2 20024none0Flow hot water at 10mL/min from 4-8 minutes, batch otherwise Controlled pH 3 19015none016% corn residue slurry in water AFEX 4 905Anhydrous ammonia 10062.5% solids in reactor (60% moisture dry weight basis), 5 minutes at temperature ARP 5,6 17010ammonia15Flow aqueous ammonia at 5 mL/min without presoaking Lime 7 554 weekslime0.08 g CaO/g biomass Purged with air. Key Features of CAFI Pretreatments

8. CAFI 1 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

9. Consistent Mass Balance Approach as Applied to AFEX 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 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. Overall Sugar Yields from Corn Stover 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. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

13. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

14. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

15. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

16. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

17. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

18. Dilute acid Flowthrough Controlled pH Maximum possible ARP AFEX Lime Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

19. Sugar Yields from Corn Stover at 15 FPU/g Glucan Biomass Refining CAFI

20. CAFI Economic Estimates Pretreatment Model Aspen Plus Bioethanol Plant Model 2001 NREL Design Case 2000 Metric Tons Stover (dry)/Day Stover Cost: $35/ton Enzyme Cost: ~$0.15/gal ethanol Thermodynamics Process Analogies Design Methods Chemistry CAFI Researcher Biomass Refining CAFI Updated Model Basis and Feedstock Basis in “CAFI 2” Project

21. General Process Flow Diagram Biomass Refining CAFI Boiler + Generator Hydrolysis + Fermentation Feed Handling Recovery Pretreatment Stover Syrup + Solids Chemicals Water Enzymes CO 2 Water EtOH Steam Power Poplar

22. Capital Cost Estimates Biomass Refining CAFI Pretreatment System Pretreatment Direct Fixed Capital ($MM) Pretreatment Breakdown, (% Reactor/ % Other) Total Fixed Capital ($MM) Ethanol Production (MM gal/yr) Total Fixed Capital ($/gal Annual Capacity) Dilute Acid25.064/36208.656.13.72 Controlled pH Hot Water 4.5100/0200.944.04.57 AFEX25.726/74211.556.83.72 ARP28.325/75210.946.34.56 Lime22.319/81163.648.93.35 No Pretreatment 0-200.39.022.26 Ideal Pretreatment 0-162.564.72.51 Basis: 2000 metric tons (dry basis) corn stover/day, assumes only monomers fermented

23. Minimum Ethanol Selling Price (MESP) Biomass Refining CAFI Assumptions: 2.5 years construction, 0.5 years start up, 20 year plant life, zero net present value when cash flows are discounted at 10% real after tax rate

24. Effect of Oligomer Conversion Biomass Refining CAFI

25. 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 during enzymatic hydrolysis in Stage 2 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 The projected costs were similar due to the high yields and similar capital costs for the overall processes Biomass Refining CAFI

26. Publication of Results from CAFI 1 Bruce Dale of the CAFI Team arranged for and edited a special December 2005 issue of Bioresource Technology entitled “Coordinated Development of Leading Biomass Pretreatment Technologies” to document these results: –Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. 2005. “Coordinated Development of Leading Biomass Pretreatment Technologies,” Bioresource Technology 96(18): 1959-1966, invited. –Lloyd TA, Wyman CE. 2005. “ Total Sugar Yields for Pretreatment by Hemicellulose Hydrolysis Coupled with Enzymatic Hydrolysis of the Remaining Solids, ” Bioresource Technology 96(18): 1967-1977, invited. –Liu C, Wyman CE. 2005. "Partial Flow of Compressed-Hot Water Through Corn Stover to Enhance Hemicellulose Sugar Recovery and Enzymatic Digestibility of Cellulose, ” Bioresource Technology 96(18): 1978-1985, invited. –Mosier N, Hendrickson R, Ho N, Sedlak M, Ladisch MR. 2005. “ Optimization of pH Controlled Liquid Hot Water Pretreatment of Corn Stover, ” Bioresource Technology 96(18): 1986-1993, invited. –Kim S, Holtzapple MT. 2005. “ Lime Pretreatment and Enzymatic Hydrolysis of Corn Stover, ” Bioresource Technology 96(18): 1994-2006, invited. –Kim TH, Lee YY. 2005. “ Pretreatment and Fractionation of Corn Stover by Ammonia Recycle Percolation Process, ” Bioresource Technology 96(18): 2007-2013, invited. – Teymouri F, Laureano-Perez L, Alizadeh H, Dale BE. 2005. “ Optimization of the Ammonia Fiber Explosion (AFEX) Treatment Parameters for Enzymatic Hydrolysis of Corn Stover, ” Bioresource Technology 96(18): 2014-2018, invited. – Eggeman T, Elander RT. 2005. “ Process and Economic Analysis of Pretreatment Technologies, ” Bioresource Technology 96(18): 2019-2025, invited. –Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. 2005. “ Comparative Sugar Recovery Data from Laboratory Scale Application of Leading Pretreatment Technologies to Corn Stover, ” Bioresource Technology 96(18): 2026-2032, invited. Biomass Refining CAFI

27. 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

28. 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

29. CAFI 2 Corn 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

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

31. 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

32. CAFI 2 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 Ammonia Fiber Explosion/PoplarSeptember 2005 Ammonia Recycled Percolation/PoplarOctober 2005 Flowthrough/PoplarMarch 2006 SO 2 /PoplarApril 2006 Lime/PoplarApril 2006 Biomass Refining CAFI

33. Overall Yields for Corn Stover 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 SO 2 Steam explosion 14.7/1.020.034.7/21.02.5/0.856.759.2/57.517.2/1.876.793.9/78.5 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

34. Effect of Pretreatment Severity on Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar Biomass Refining CAFI 2% glucan concentration 50 FPU/ gm original glucan CBU:FPU = 2.0 Digestion time =72hr Increasing severity For 50 FPU, Total Protein ( mg/gm original glucan) POP1122.2 POP2122.0 POP3142.0 POP4160.3

35. Digestion time =72hr Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids Biomass Refining CAFI

36. Digestion time =72hr Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids Biomass Refining CAFI

37. Digestion time =72hr Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids Biomass Refining CAFI

38. Feedstock: USDA-supplied hybrid poplar (Arlington, WI) –Debarked, chipped, and milled to pass ¼ inch round screen –Not enough to meet needs Biomass Refining CAFI CAFI 2 Initial Poplar

39. Feedstock: USDA-supplied hybrid poplar (Arlington, WI) –Debarked, chipped, and milled to pass ¼ inch round screen –Not enough to meet needs Biomass Refining CAFI CAFI 2 Initial Poplar

40. AFEX Optimization for High/Low Lignin Poplar C - Cellulase (31.3 mg/g glucan) X - Xylanase (3.1 mg/g glucan) A - Additive (0.35g/g glucan) UT - Untreated AFEX condition 24 h water soaked 1:1 (Poplar:NH3) 10 min. res. time

41. Differences Among Poplar Species* Original Poplar - Low LigninPoplar Standard - High Lignin Arlington, WI near Madison Very rich, loamy soil Demonstrated some of best growth rates Harvested and shipped in February 17, 2004 Planted in 1995, probably in spring but possibly in fall Alexandria, Minnesota Lower growth rate than Arlington Slightly shorter growing season Harvested and shipped in August 2004 Planted in spring 1994 * Based on information provided by Adam Wiese, USDA Rheinlander, WI Biomass Refining CAFI

42. Fermentation of Dilute Acid Treated Corn Stover corn stover 175  C Fermentation 30  C Ethanol H 2 SO 4 Cells pH 6.0pH 1.2 A Fermentation at 0 hr at 48 hr Ethanol020 Glucose240 Xylose7550 Acetic Acid13 13.3 Furfural 2.00 HMF 3.00 Streamg/L Inhibitor Liquid Ca(OH) 2 Solids pH 1.2 A consumed B B S. cerevisiae 424A(LNH-ST) 80% of theoretical

43. Fermentation of Hot Water Treated Corn Stover corn stover 190  C50  C Fermentation 30  C Ethanol WaterEnzyme Cells + Solids pH 6.0pH 4.5 Fermentationat 0 hrat 48 hr Ethanol022 Glucose320 Xylose185 Acetic Acid 1.2 Furfural 0.40 HMF 0.10 Streamg/L No Xylanase A A B B consumed Ca(OH) 2 S. cerevisiae 424A(LNH-ST) 95% of theoretical below threshold

44. Fermentation of SO 2 Treated Corn Stover corn stover Fermentationat 0 hrat 48 hr Ethanol029 Glucose27 0 Xylose375 Acetic Acid 4.6 Furfural 0.20 HMF 0.30 Streamg/L below threshold A consumed 180  C50  C Fermentation 30  C Ethanol SO 2 EnzymeS. cerevisiae 424A(LNH-ST) Cells pH 6.0 ~pH 1 A pH 4.8 Solids No Xylanase B B Ca(OH) 2 96% of theoretical

45. Observations 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 Initial data on conditioning and fermentation shows mostly good yields All pretreatments gave similar results for corn stover Initial hydrolysis results for poplar are not as good, with one variety more recalcitrant than other Biomass Refining CAFI

46. Planned Work Maximize yields with standard poplar for each pretreatment Evaluate differences with initial poplar at optimal conditions for standard poplar Develop fermentation data with hydrolyzate for each material Upgrade technoeconomic model with corn stover and poplar Identify key features that distinguish performance of different pretreatments Biomass Refining CAFI

47. 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 All of the CAFI Team members, students, and others who have been so cooperative Biomass Refining CAFI

48. CAFI DOE Project A&I Advisory Board: Meetings Every 6 Months Quang Nguyen, Abengoa Bioenergy Mat Peabody, Applied CarboChemicals Gary Welch, Aventinerei Greg Luli, BC International Paris Tsobanakis, Cargill Robert Wooley, Cargill Dow James Hettenhaus, CEA Steve Thomas, CERES Lyman Young, ChevronTexaco Kevin Gray, Diversa Paul Roessler, Dow Julie Friend, DuPont Jack Huttner, 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 Robert Goldberg, NIST Joel Cherry, Novozymes Ron Reinsfelder, Shell Andrew Richard, Sunopta Carl Miller, Syngenta Carmela Bailey, USDA Don Riemenschneider, USDA

49. Questions?? Louisiana Rice Hulls Pile