Charles E. Wyman, Dartmouth College/University of California Rajeev Kumar, Dartmouth College

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 Moniruzzaman1, 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. Central Role and Pervasive Impact of Pretreatment for Biological Processing Enzyme production Biomass production Harvesting, storage, size reduction Pretreatment Enzymatic hydrolysis Sugar fermentation Hydrolyzate conditioning Hydrolyzate fermentation Ethanol recovery Residue utilization Waste treatment Biomass Refining CAFI

3. 33% Biomass Feedstock Capital Recovery Charge 5% Feed Handling Grid Electricity 18% Pretreatment / Conditioning Raw Materials SSCF 12% Total Plant Electricity (after ~10x cost reduction) 9% Cellulase Process Elect. Distillation and Solids 10% Recovery Fixed Costs 4% Wastewater Treatment Net 4% Boiler/Turbogenerator Utilities 4% Storage 1% (0.20) (0.10) - 0.10 0.20 0.30 0.40 Key Processing Cost Elements 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) • 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

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 • Enzymes from Genencor International • Spezyme CP (59 FPU/ml, 123 mg protein/ml) • GC-220 (89 FPU/ml, 184 mg protein/ml) • β-glucosidase (32 mg protein/ml) • Multifect Xylanase (41 mg protein/ml) • Experimental conditions • 1-2% glucan in 100 ml flasks in citrate buffer plus antibiotics • Digestion time – 72 hrs • CBU : FPU ~ 2.0 • Enzyme loadings – 3.0, 7.5,15,50, and 60 FPU/g glucan prior to pretreatment Biomass Refining CAFI

9. CAFI Corn Stover • 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 Biomass Refining CAFI

10. Increasing pH Overall Yields for Corn Stover at 15 FPU/g Glucan from CAFI I *Cumulative soluble sugars as total/monomers. Single number = just monomers. Biomass Refining CAFI

11. CAFI II Standard Poplar Biomass Refining CAFI

12. Enzymatic Hydrolysis of Standard Poplar Biomass Refining CAFI

13. CAFI II 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

14. CAFI II 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

15. 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

16. SO2 Overall Yields at 15 FPU/g of Glucan (148 hours hydrolysis) *Cumulative soluble sugars as total/monomers. Single number = just monomers. Biomass Refining CAFI

17. Why Is Variability Important? • Impacts yields for all but SO2 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* * 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? Initial data! Biomass Refining CAFI

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. Using copper diethylenamine solution as a cellulose solvent Impact of Different Pretreatments on Cellulose DP for Corn Stover 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 40oC 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