1. Comparative Data for 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 1, Genencor International John N. Saddler, University of British Columbia 1 – now with BioEnergy International, LLC AIChE Annual Meeting San Francisco, California November 16, 2006 Biomass Refining CAFI

2. Ethanol recovery Enzymatic hydrolysis Sugar fermentation Hydrolyzate conditioning Central Role and Pervasive Impact of Pretreatment for Biological Processing Hydrolyzate fermentation Enzyme production Biomass production Harvesting, storage, size reduction Residue utilization Waste treatment Pretreatment Biomass Refining CAFI

3. Key Processing Cost Elements Capital Recovery Charge Raw Materials Process Elect. Grid Electricity Total Plant Electricity Fixed Costs Biomass Feedstock Feed Handling Pretreatment / Conditioning SSCF Cellulase Distillation and Solids Recovery Wastewater Treatment Boiler/Turbogenerator Utilities Storage (0.20)(0.10)-0.100.200.300.40 33% 5% 18% 12% 9% 10% 4% Net 4% 4% 1% (after ~10x cost reduction) Biomass Refining CAFI

4. Importance of Pretreatment Although significant, feedstock costs are low relative to petroleum: $40/dry ton ~ $13/bbl oil In addition, feedstock costs are a very low fraction of final costs compared to other commodity products Pretreatment is the most costly process step: the only process step more expensive than pretreatment is no pretreatment –Low yields without pretreatment drive up all other costs more than amount saved –Conversely enhancing yields via improved pretreatment would reduce all other unit costs Need to reduce pretreatment costs to be competitive Biomass Refining CAFI

5. Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) 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 Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI)

6. DOE OBP Project: CAFI II Started in April 2004 after completion of USDA IFAFS funded CAFI I on corn stover 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 in depth information on –Enzymatic hydrolysis of cellulose and hemicellulose in solids –Conditioning and fermentation of hydrolyzate liquids –Predictive models Evaluating AFEX, ARP, controlled pH, dilute acid, lime, sulfur dioxide pretreatments Genencor supplies commercial and advanced enzymes Biomass Refining CAFI

7. DOE OBP Project: CAFI II Started in April 2004 after completion of USDA IFAFS funded CAFI I on corn stover 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 in depth information on –Enzymatic hydrolysis of cellulose and hemicellulose in solids –Conditioning and fermentation of hydrolyzate liquids –Predictive models Evaluating AFEX, ARP, controlled pH, dilute acid, lime, sulfur dioxide pretreatments Genencor supplies commercial and advanced enzymes Biomass Refining CAFI

8. Enzymes Used and Experimental Conditions oSpezyme CP (59 FPU/ml, 123 mg protein/ml) oGC-220 (89 FPU/ml, 184 mg protein/ml) oβ-glucosidase (32 mg protein/ml) oMultifect Xylanase (41 mg protein/ml) Enzymes from Genencor International Experimental conditions o1-2% glucan in 100 ml flasks in citrate buffer plus antibiotics oDigestion time – 72 hrs oCBU : FPU ~ 2.0 oEnzyme loadings – 3.0, 7.5,15,50, and 60 FPU/g glucan prior to pretreatment Biomass Refining CAFI

9. CAFI Corn Stover Biomass Refining CAFI Second 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 Performed like CAFI I material for all pretreatments

10. Overall Yields for Corn Stover at 15 FPU/g Glucan from CAFI I 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

11. CAFI II Standard Poplar

12. Enzymatic Hydrolysis of Standard Poplar Biomass Refining CAFI

13. 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 II Initial Poplar

14. 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 II Initial Poplar

15. 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

16. Pretreatment conditions Xylose yields*Glucose yields*Total sugars* Stage 1Stage 2TotalStage 1Stage 2TotalStage 1Stage 2Combined xylose glucosetotal Maximum25.8 74.2 100 possible 190°C,5min,3% SO 2 (High lignin poplar) 20.3/13.72.723/16.41.569.971.421.8/15.272.694.4/87.8 200°C,5min,3% SO 2 (High lignin poplar) 19.3/14.02.421.7/16.42.371.974.221.6/16.374.395.9/90.6 190°C,5min,3% SO 2 (Low lignin poplar) 18.4/12.93.521.9/16.41.073.274.219.4/13.976.796.1/90.6 *Cumulative soluble sugars as total/monomers. Single number = just monomers. SO 2 Overall Yields at 15 FPU/g of Glucan (148 hours hydrolysis) Biomass Refining CAFI

17. Why Is Variability Important? Impacts yields for all but SO 2 pretreatment Assumption is that all hardwoods of same type behave similarly Woody crops proponents plan to use standing forest to “store” wood, but harvest season may affect yields Important to understand what causes this and whether it impacts other feedstocks such as herbaceous crops or agricultural residues Biomass Refining CAFI

18. 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

19. Getting to the Root Cause of Factors That Impact Digestion of Different Substrates with Different Pretreatments Degree of polymerization – DP Available reducing ends Βeta-glucosidic bond accessibility Crystallinity index Other? Biomass Refining CAFI

20. Getting to the Root Cause of Factors That Impact Digestion of Different Substrates with Different Pretreatments Degree of polymerization – DP Available reducing ends Βeta-glucosidic bond accessibility Crystallinity index Other? Biomass Refining CAFI Initial data!

21. Measuring DP for Pretreated Biomass Difficult to directly measure DP for complex substrates Delignification improves measurements but could alter cellulose DP, leading to wrong conclusions ASTM capillary viscometer method modified to estimate DPv for cellulose by correcting for hemicellulose*: (DPv) = {(1.65 [η] – 116 y)/x} 1.111 η = intrinsic viscosity y = hemicellulose fraction x = glucan fraction However, no information is available on DP of corn stover for comparison *Heiningen et al.(2004) Journal of Pulp and Paper Science 30(8):211-21 Biomass Refining CAFI

22. Impact of Different Pretreatments on Cellulose DP for Corn Stover Using copper diethylenamine solution as a cellulose solvent Biomass Refining CAFI

23. Estimation of Reducing Ends for Corn Stover Copper number is often used to reflect number of reducing ends for cellulose in pulp Applied Braidy’s method Copper Number = (0.064*t) /w for which t = ceric ammonium sulfate solution (ml) consumed w = dry weight of wood/biomass In general higher DP = lower copper number and vice versa Roy L. Whistler et al.(1963). Methods in carbohydrate chemistry-III. Holtzapple, M. T. (1981) Ph.D Thesis, Deptt. of Chemical Engineering. University of Penn. Biomass Refining CAFI

24. Estimating β-Glucosidic Bond Accessibility Measured protein adsorption: At 40 o C in 50 ml tubes with 0.5- 1 % glucan content With SP-CP protein from 40mg to 800 mg/g glucan Nitrogen content was determined after decanting unabsorbed protein and drying the substrate Converted into mg protein adsorbed/gm cellulose substrate using established nitrogen factor Then determined Langmuir isotherm parameters using nonlinear regression in Polymath per Lynd, et al.(2002) Microbiol. and Mol. Biol. Reviews. 66:506-577 Biomass Refining CAFI

25. Initial Digestion Data for 7.5 FPU/g Enzyme Loading vs Substrate Features Biomass Refining CAFI

26. Closing Thoughts The resistance of one biological system (cellulosic biomass) to the other (biological conversion) requires a pretreatment interface All of the pretreatments studied were very effective in enhancing cellulose digestion for corn stover Poplar wood presented a more challenging substrate for several pretreatments, particularly those at higher pH The pretreatabilty of poplar changed with source The cause of variations in poplar performance among pretreatments and biomass sources must be understood to identify paths to 1) better pretreatments and 2) improved biomass production 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 Numerous collaborators including the CAFI Team members, students, and others who have been so cooperative Ford Motor Company and University of California for sponsoring this trip Biomass Refining CAFI

28. Questions??? Biomass Refining CAFI