1. Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment by Leading Technologies Charles E. Wyman, Dartmouth College/University of California Rajeev Kumar, Dartmouth College 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 BIO Meeting Chicago, Illinois April 12, 2006 Biomass Refining CAFI

2. 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

3. 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

4. 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

5. 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

6. Overall 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

7. Overall 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

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

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

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

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

12. Total Yields at 15 FPU/g Glucan

13. 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

14. Tasks for the DOE OBP CAFI 2 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 (corn stover and poplar), 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

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

16. 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

17. Effect of Enzyme Loading on Hydrolysis of SO 2 Pretreated Corn Stover SO2 pretreated corn stover at 1% glucan concentration Protein ( mg) FPU/gm (SP-CP) 6.43.0 16.17.5 32.215 42.920 107.450 128.960 CBU:FPU = 2.0 Digestion time =72hr Biomass Refining CAFI

18. Effect of Enzyme Loading on Hydrolysis of SO 2 Pretreated Corn Stover SO2 pretreated corn stover at 1% glucan concentration Protein ( mg) FPU/gm (SP-CP) 6.43.0 16.17.5 32.215 42.920 107.450 128.960 CBU:FPU = 2.0 Digestion time =72hr Biomass Refining CAFI

19. 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

20. Digestion time =72hr Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids

21. Digestion time =72hr Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids

22. Digestion time =72hr Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids

23. 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

24. 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

25. Differences Among Poplar Species* Original PoplarPoplar Standard 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

26. Observations 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 All pretreatments gave similar results for corn stover Initial performance for poplar is not as good, with one source more recalcitrant than other Yields can be further increased for some pretreatments with enzymes a potential key Biomass Refining CAFI

27. 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

28. Questions? Biomass Refining CAFI

29. 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