1. Comparison of Selected Results for Application of Leading Pretreatment Technologies to Corn Stover Charles E. Wyman, Dartmouth College Y. Y. Lee, Auburn University 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 2003 AIChE Annual Meeting San Francisco, California November 20, 2003

2. Outline of Talk Pretreatment technologies studied General research approach Material balance data Comparison of performance Needs for the future

3. USDA IFAFS Project Overview Multi-institutional effort funded by USDA Initiative for Future Agriculture and Food Systems Program for $1.2 million to develop comparative information on cellulosic biomass pretreatment by leading pretreatment options with common source of cellulosic biomass (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 - Mike Ladisch, Purdue University –Lime pretreatment - Mark Holtzapple, Texas A&M University –Logistical support and economic analysis - Rick Elander/Tim Eggeman, NREL through DOE Biomass Program funding Emphasis on quality not quantity

4. USDA IFAFS Project Tasks 1.Apply leading pretreatment technologies to prepare biomass for conversion to products 2.Characterize resulting fluid and solid streams 3.Close material and energy balances for each pretreatment process 4.Determine cellulose digestibility and liquid fraction fermentability/toxicity 5.Compare performance of pretreatment technologies on corn stover on a consistent basis Project period: 2000-2003

5. One Source of 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 Glucan36.1 % Xylan21.4 % Arabinan3.5 % Mannan1.8 % Galactan2.5 % Lignin17.2 % Protein4.0 % Acetyl3.2 % Ash7.1 % Uronic Acid3.6 % Non-structural Sugars1.2 %

6. Dilute Acid Pretreatment Mineral acid gives good hemicellulose sugar yields and high cellulose digestibility Sulfuric acid usual choice because of low cost Requires downstream neutralization and conditioning Typical conditions: 100-200 o C, 50 to 85% moisture, 0-1% H 2 SO 4 Some degradation of liberated hemicellulose sugars Mineral acid Hemicellulose sugars solid cellulose and lignin Biomass Reactor

7. Reactor Systems Employed 5 inch long reactors Sandbaths NREL steam gun

8. Solubilized Xylan Partition vs. LogM o – Dilute Sulfuric Acid Mo = t A n exp{( T - 100)/14.75} – for acid

9. Fate of Xylan for Dilute Acid Hydrolysis

10. Schematic Diagram of Flowthrough Pretreatment Sample

11. Effect of Flow Rate and Acid on Residual Xylose at 180 o C

12. Solubilization of Xylan and Lignin for Water Only Hydrolysis

13. Controlled pH Pretreatment pH control through buffer capacity of liquid No fermentation inhibitors, no wash stream Minimize hydrolysis to monosaccharides thereby minimizing degradation

14. Solubilization of Corn Stover by Controlled pH Pretreatment

15. Cellulose Digestibility vs Hemicellulose Solubilization Controlled pH Hot Water Pretreatment (non flow- through) improves digestibility primarily by removal of hemicellulose without degrading the monosaccharides  improved porosity / enzyme accessibility Glucose YieldsXylose Yields

16. Fermentation of Pretreated, Saccharified Corn Stover Xylose Fermenting Recombinant Yeast 426A (LNH-87). Data provided by Dr. Nancy Ho.

17. What is AFEX/FIBEX? Liquid “anhydrous” ammonia treats and explodes biomass Ammonia is recovered and reused Ammonia can serve as N source downstream Batch process is AFEX; FIBEX is continuous version Conditions: 60-110 o C, moisture 20-80%, ammonia:biomass ratio 0.5-1.3 to 1.0 (dry basis) No fermentation inhibitors, no wash stream required, no overliming Only sugar oligomers formed, no detectable sugar monomers Few visible physical effects ReactorExplosion Ammonia Recovery Biomass Treated Biomass Liquid Ammonia Gaseous Ammonia ReactorExplosion Ammonia Recovery Biomass Treated Biomass Liquid Ammonia Gaseous Ammonia Moderate temperatures, pH prevent/minimize sugar & protein loss

18. Experimental Ammonia Fiber Explosion (AFEX) System

20. 60 FPU/g glucan

21. Features of Ammonia Recycle Pretreatment (ARP) Aqueous ammonia is used as the pretreatment reagent:  Efficient delignification  Volatile nature of ammonia makes it easy to recover Flowthrough column reactor is used Value-added byproducts are obtained  Low-lignin cellulose (“filler fiber” grade)  Uncontaminated lignin

22. Composition and Digestibility for ARP Pretreatment [mL/min] Lignin Glucan Xylan Liquid 1 Solid 1 Flow rate Digestibility 2 60FPU15FPU Note.; 1. All data based on original feed. 2. Enzymatic hydrolysis conditions; 60 or 15 FPU/g of glucan, pH 4.8, 50  C, 150 rpm, at 72h Glucan Xylan 2.5 5.135.610.3 6.435.910.7 5.0 7.5 5.635.710.1 0.510.1 0.510.6 0.810.5 91.386.9 91.583.2 93.486.9 Untreated17.236.121.4--21.215.7 Pretreatment conditions Liquid throughput: 3.3 mL of 15 wt% NH 3 per g of corn stover at 170  C Air dried corn stover is used without presoaking.

23. Enzymatic Hydrolysis of a Representative ARP Sample (15 wt% NH 3, 170ºC) Pretreatment conditions: 170  C, 3.3 mL of 15 wt% of ammonia per g of corn stover, 2.3 MPa; Enzymatic hydrolysis conditions : 60 or 15 FPU/g cellulose, pH 4.8, 50  C, 150 rpm ; α 93.4%@ 60FPU 86.9%@ 15FPU

24. Lime Pretreatment Biomass + Lime Gravel Air Typical Conditions: Temperature = 25 – 55 o C Time = 1 – 2 months Lime Loading = 0.1 – 0.2 g Ca(OH) 2 /g biomass

25. Reactor System for Lime Pretreatment

26. Pretreatment Time (weeks) Klason Lignin Content (wt %) 0 5 10 15 20 25 05101520 untreated Pretreatment Time (weeks) Klason Lignin Content (wt %) 25 o C 35 o C 45 o C 55 o C 0 5 10 15 20 25 05101520 untreated No Air Air Effect of Air/Temperature on Lignin

27. Enzymatic Digestibility for Lime Treated Biomass Cellulase Loading (FPU/g dry biomass) 3-d Sugar Yield (mg equiv. glucose/g dry biomass) 25 o C 55 o C 0 100 200 300 400 500 600 700 110 100 0 200 300 400 500 600 700 110 100 Air No Air untreated Cellulase Loading (FPU/g dry biomass) 0 100 200 300 400 500 600 700 110100 0 200 300 400 500 600 700 110100 Air No Air 3-d Sugar Yield (mg equiv. glucose/g dry biomass)

28. AFEX Process Yields and Material Balance Hydrolysis Enzyme (15 FPU/g of Glucan) Residual Solids Hydrolyzate Liquid AFEX System Treated Stover Ammonia Stover 100.5 lb 100 lb (dry basis) 36.1 lb glucan 21.4 lbxylan 38.7 lb 95.9% glucan conversion, 77.6% xylan conversion (up-dated on 9/10/03) 99% mass balance closure includes: (solids + glucose + xylose + arabinose ) Wash 2 lb 98.5 lb Solids washed out 38.5 lb glucose 18.9 lbxylose (Ave. of 4 runs)

29. Yield Comparisons at 60 FPU/g glucan Increasing pH ProcessXylose yields - % of total potential* Glucose yields – % of total potential* PretreatmentEnzymaticPretreatmentEnzymatic Dilute acid Flowthrough Controlled pH AFEX ARP Lime * Listed as total/monomers/oligomers in each step

30. Yield Comparisons at 15 FPU/g glucan Increasing pH ProcessXylose yields - % of total potential* Glucose yields – % of total potential* PretreatmentEnzymaticPretreatmentEnzymatic Dilute acid Flowthrough Controlled pH AFEX ARP Lime * Listed as total/monomers/oligomers in each step

31. Yield Comparisons in SSF at 15 FPU/g glucan Increasing pH ProcessXylose yields - % of total potential* Glucose yields – % of total potential* PretreatmentEnzymaticPretreatmentEnzymatic Dilute acid Flowthrough Controlled pH AFEX ARP Lime * Listed as total/monomers/oligomers in each step

32. Some Conclusions to Date Dilute acid and neutral pH pretreatments solubilize mostly hemicellulose while ammonia and lime remove mostly lignin. AFEX removes neither. –Hemicellulose hydrolysis to monomers can reduce post processing –Lignin removal can reduce cellulase use although AFEX reduces cellulase without lignin removal Greater hemicellulase activity would improve yields from higher pH and controlled pH approaches All are capital intensive for differing reasons –Costly reactor materials and waste treatment, in some cases –Pretreatment catalyst recovery, in others Some advantages in operating costs –e.g., low waste generation with AFEX

33. Future Work Evaluate performance differences in more depth –Apply hydrolyzate conditioning –Ferment hydrolyzate with recombinant organisms –Evaluate effect of pretreatment approach on cellulase effectiveness –Apply hemicellulases to improve performance –Work with Genencor as enzyme supplier –Understand effect of how pretreatment affects substrate features and digestibility –Expand research to hardwoods –Expand team to include University of British Columbia and University of Sherbrooke in Canada Funded by Office of the Biomass Program of the Department of Energy

34. Acknowledgments The United States Department of Agriculture Initiative for Future Agricultural and Food Systems Program through Contract 00-52104- 9663 for funding this CAFI research The United States Department of Energy Office of the Biomass Program and the National Renewable Energy Laboratory for NREL’s participation Our team from Dartmouth College; Auburn, Michigan State, Purdue, and Texas A&M Universities; and the National Renewable Energy Laboratory

35. IFAFS Project Participants

36. Questions?