1. Pretreatment Fundamentals Bruce E. Dale, Richard T. Elander, Mark T. Holtzapple, Rajeev Kumar, Michael R. Ladisch, Yoon Y. Lee, Nate Mosier, Jack Saddler, Mohammed Moniruzzaman, Charles E. Wyman CAFI BIO 2006 Annual International Convention Chicago, Illinois April 12, 2006 Biomass Refining CAFI

2. Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): Who we are…. Pretreatment researchers working together in a coordinated, disciplined way to understand the fundamentals underlying lignocellulosic biomass pretreatment and hydrolysis. Organized in 1999 -2000 CAFI recognizes that pretreatment is part of a system that includes hydrolysis and fermentation—pretreatment effects on downstream processes must be understood Biomass Refining CAFI

3. USDA IFAFS Project: September 2000 to 2003 USDA Initiative for Future Agriculture and Food Systems Program for comparative information on leading cellulosic biomass pretreatments with common source of corn stover and identical analytical methods –Aqueous ammonia recycle pretreatment - YY Lee, Auburn University –Water only and dilute acid hydrolysis by co-current and flowthrough systems - Charles Wyman, Dartmouth College –Ammonia fiber explosion (AFEX) - Bruce Dale, Michigan State University –Controlled pH pretreatment - Michael Ladisch, Purdue University –Lime pretreatment - Mark Holtzapple, Texas A&M University –Logistical support and economic analysis - Rick Elander/Tim Eggeman, NREL through DOE Office of the Biomass Program funding Emphasis on quality not quantity Concluded September, 2003 Biomass Refining CAFI

4. DOE USDA Project: September 2004 to 2007 Biomass Refining CAFI Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment by Leading Technologies Water, AFEX, acid pretreatments Hydrolysis characteristics Mixed sugar fermentations using yeast Researchers: 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, Nate Mosier, Nancy Ho, Purdue University, Y. Y. Lee, Auburn University, Mohammed Moniruzzaman, Genencor International, John N. Saddler, University of British Columbia

5. CAFI 1: Corn Stover Glucan36.1 % Xylan21.4 % Arabinan3.5 % Mannan1.8 % Galactan2.5 % Lignin17.2 % Protein4.0 % Acetyl3.2 % Ash7.1 % Uronic Acid3.6 % Non-structural Sugars 1.2 %

6. Biomass Refining CAFI CAFI 2 Standard Poplar

7. Key Comparisons: Per 100 lbs PoplarCorn Stover Glucan (cellulose) 43.8 36.1 Xylan (hemicellulose) 14.9 21.4 Lignin 29.1 17.2 Ash 1.1 7.1 Energy in Lignin (est) 116,000 86,000 Ethanol (at 90% Yield) 4.9 4.8

8. Ethanol Fermentation Pretreatment is needed to get us there Feedstock Preparation Biomass Pretreatment Enzyme hydrolysis

9. Crystalline Region Amorphous Region Cellulose Lignin Hemicellulose Effect of Pretreatment Pretreatment

10. Pretreatment Modeling C C* GnGn GDegradation k1k1 k2k2 k3k3 k4k4 K k 2, k 3, >> k 1

11. Pretreatment Converts Cellulose to Reactive Form C C* GnGn GDegradation k1k1 k2k2 k3k3 k4k4 K

12. Enzymes convert cellulose to glucose and xylan to xylose (no degradation products) C H,C* GnGn X,G Degradation Products k1k1 k2k2 k3k3 k4k4 C = native cellulose H = hemicellulose (xylan) C* = hydrated cellulose G n = glucans (oligosaccharides) G = glucose (monomer) X = xylose (monomer) k 2 > k 1

13. Yeast Metabolism: pentose fermentation Glucose Glucose-6-P Fructose-6-P 3-Phosphoglycerate Phosphoenolpyruvate Pyruvate Acetaldehyde Ethanol TCA Cycle Xylose Xylitol Xylulose Xylulose-5-P Glyceraldehyde-3-P NAD(P)H NADH NAD+ NAD(P)+ NAD+ PPP Ho et al

14. Several Pentose Fermenting Microbes Yeast (Ho et al) E. coli (Lonnie Ingram et al) Zymomonas (NREL)

15. 1 Bale = 970 lbs = 2000 miles Using Hay Assuming 50 gal x 40 mpg