Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment by Leading Technologies

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. CAFI Approach • 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

3. Hydrolysis Stages Cellulase enzyme Stage 1 Pretreatment Stage 2 Enzymatic hydrolysis Residual solids: cellulose, hemicellulose, lignin Biomass Solids: cellulose, hemicellulose, lignin Chemicals Dissolved sugars, oligomers Dissolved sugars, oligomers, lignin Stage 3 Sugar fermentation Biomass Refining CAFI

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 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. Increasing pH Overall Yields at 60 FPU/g Glucan *Cumulative soluble sugars as total/monomers. Single number = just monomers. Biomass Refining CAFI

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

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

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

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

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

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 • 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 Biomass Refining CAFI

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

16. Pretreated Substrate Schedule Biomass Refining CAFI

17. Effect of Enzyme Loading on Hydrolysis of SO2 Pretreated Corn Stover CBU:FPU = 2.0 Digestion time =72hr SO2 pretreated corn stover at 1% glucan concentration Biomass Refining CAFI

18. Effect of Enzyme Loading on Hydrolysis of SO2 Pretreated Corn Stover CBU:FPU = 2.0 Digestion time =72hr SO2 pretreated corn stover at 1% glucan concentration Biomass Refining CAFI

19. Increasing severity Effect of Pretreatment Severity on Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar CBU:FPU = 2.0 Digestion time =72hr 2% glucan concentration 50 FPU/ gm original glucan Biomass Refining CAFI

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

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

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

23. CAFI 2 Initial Poplar • 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

24. 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 AFEX Optimization for High/Low Lignin Poplar

25. Differences Among Poplar Species* * 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 Technology96(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 Technology96(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 Technology96(18): 1986-1993, invited. • Kim S, Holtzapple MT. 2005. “Lime Pretreatment and Enzymatic Hydrolysis of CornStover,”Bioresource Technology96(18): 1994-2006, invited. • Kim TH, Lee YY. 2005. “Pretreatment and Fractionation of Corn Stover by Ammonia Recycle Percolation Process,”Bioresource Technology96(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 Technology96(18): 2014-2018, invited. • Eggeman T, Elander RT. 2005. “Process and Economic Analysis of Pretreatment Technologies,” Bioresource Technology96(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 Technology96(18): 2026-2032, invited. Biomass Refining CAFI