1. Enzymatic Hydrolysis of Cellulose and Hemicellulose in Solids Prepared by Leading Pretreatment Technologies Charles E. Wyman, Dartmouth College Y. Y. Lee, Auburn University Mohammed Moniruzzaman, Genencor International Bruce E. Dale, Michigan State University Tim Eggeman, Neoterics International 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 Bioprocessing of Agricultural Feedstocks: Report on Pretreatment for Biomass Refining 2 nd World Congress on Industrial Biotechnology and Bioprocessing Orlando, Florida April 20, 2005 Biomass Refining CAFI

2. USDA IFAFS Project Tasks Apply leading pretreatment technologies to prepare biomass for conversion to products Characterize resulting fluid and solid streams Close material and energy balances for each pretreatment process Determine cellulose digestibility and liquid fraction fermentability Compare performance of pretreatment technologies on corn stover Biomass Refining CAFI

3. Pretreatment and Enzymatic 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

4. 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

5. Pretreatment Yield Comparisons 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

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

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

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

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

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

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

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

13. Pretreatment Yield Comparisons at 15 FPU/g Glucan

14. Observations from IFAFS Project 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

15. 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 Biomass Refining CAFI

16. Tasks for the DOE OBP Project Biomass Refining CAFI Corn stover and poplar pretreated by leading technologies to improve cellulose accessibility to enzymes Conditioning methods developed as needed to maximize fermentation yields by a recombinant yeast, the cause of inhibition determined, and fermentations modeled Cellulose and hemicellulose in pretreated biomass enzymatically hydrolyzed, as appropriate, and models developed to understand the relationship between pretreated biomass features, advanced enzyme characteristics, and enzymatic digestion results Capital and operating costs estimated for each integrated pretreatment, hydrolysis, and fermentation system and used to direct research

17. Tasks for the DOE OBP Project Biomass Refining CAFI Corn stover and poplar pretreated by leading technologies to improve cellulose accessibility to enzymes Conditioning methods developed as needed to maximize fermentation yields by a recombinant yeast, the cause of inhibition determined, and fermentations modeled Cellulose and hemicellulose in pretreated biomass enzymatically hydrolyzed, as appropriate, and models developed to understand the relationship between pretreated biomass features, advanced enzyme characteristics, and enzymatic digestion results Capital and operating costs estimated for each integrated pretreatment, hydrolysis, and fermentation system and used to direct research

18. Measure enzymatic hydrolysis of cellulose and hemicellulose as a function of cellulase and xylanase loadings and beta glucosidase and beta xylosidase supplementation Apply fractional factorial experimental design to determine key trends and interactions Characterize enzyme and substrate features for each feedstock and pretreatment Develop kinetic models to better understand key factors impacting performance Define routes to improve cellulose and hemicellulose conversion with less enzyme Enzymatic Hydrolysis Plan Biomass Refining CAFI

19. Enzymatic Hydrolysis of Cellulose from Pretreated Poplar Wood 2% glucan concentration 50 FPU/g glucan, no β-glucosidase supplementation

20. 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

21. Non-mechanistic (NM): 2 –Based on data correlation without an explicit calculation of adsorbed enzyme concentration. Kinetic Models* Functionally-based (FB): 3 –Featuring an adsorption model, multiple enzyme activities, and substrate variables. *Zhang and Lynd ( in press) Structurally-based (SB): 0 –Structural features of cellulase and interaction between substrate and enzyme. Semi-mechanistic (SM): 8 –Based on single enzyme activity and single substrate feature (concentration). Biomass Refining CAFI

22. Predictions of Effect of Lignin by Selected Models 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

23. 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

24. Questions?

25. Stop

26. Pretreatment Yield Comparisons 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