1. 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 Biomass Refining CAFI

2. 2 Outline of Talk Pretreatment technologies studied Research approach Material balances Performance comparison Summary of trends Future plans Biomass Refining CAFI

3. 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 Biomass Refining CAFI

4. 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 Biomass Refining CAFI

5. 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 % Biomass Refining CAFI

6. 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: 120-200 o C, 50 to 85% moisture, 0-1% H 2 SO 4 Some degradation of liberated hemicellulose sugars Biomass Refining CAFI Mineral acid Biomass Stage 1. Pretreatment Stage 2. Enzymatic hydrolysis Glucose and lignin Cellulose and lignin Hemicellulose sugars and oligomers

7. 7 Biomass Refining CAFI Combined Stage 1 and Stage 2 Yields for 0.49% H 2 SO 4 Addition in Batch Tubes

8. 8 Effect of Minerals and Temperature on Pretreatment pH Biomass Refining CAFI

9. 9 Modified Depolymerization Model 140 o C, 0.49% Added Acid Biomass Refining CAFI

10. 10 Schematic of Flowthrough and SFS System Sample Biomass Refining CAFI

11. 11 Total Xylose Fate for Batch, FT, and SFS Runs with Water at 200 o C Biomass Refining CAFI

12. 12 Concentration of Xylose in Hydrolyzate for Pretreatment with Compressed Hot Water at 200 o C Note: 60, 50, and 40mL of wash water were used for batch, SFS1, and SFS2, respectively. Biomass Refining CAFI

13. 13 Cellulose Digestibility for Batch, FT, and SFS Pretreatment ( 15 FPU/ gram cellulose, 72 h ) Water, 200 o C0.05wt% acid, 190 o C Biomass Refining CAFI

14. 14 Controlled pH Pretreatment pH control through buffer capacity of liquid No fermentation inhibitors, no wash stream Minimize hydrolysis to monosaccharides thereby minimizing degradation Biomass Refining CAFI

15. 15 Hemicellulose Solubilization from Pretreated Corn Stover (after enzyme hydrolysis followed by NREL LAP 14 to determine Xylan oligomers in solution) Biomass Refining CAFI

16. 16 Cellulose Digestibility of Pretreated Corn Stover 6.42 FPU/g glucan, cellobiase 94.4 IU/g glucan, 48 hrs., 50°C Biomass Refining CAFI

17. 17 Initial Rates of Hydrolysis at 50 o C for Different Pretreatment Temperatures Per gram glucan; 6.9 FPU cellulase, 94 IU cellobiase 190 C 200 180 170 Biomass Refining CAFI

18. 18 What is AFEX/FIBEX? ReactorExplosion Ammonia Recovery Biomass Treated Biomass Liquid Ammonia Gaseous Ammonia ReactorExplosion Ammonia Recovery Biomass Treated Biomass Liquid Ammonia Gaseous Ammonia Liquid “anhydrous” ammonia treats, explodes biomass, 99% of ammonia is recovered, remainder serves as N source downstream? No fermentation inhibitors formed, no wash stream, no overliming Conditions: 60-110 o C (pressure up to 250 psig), sample moisture 20-60%, ammonia:biomass ratio 0.5-1.3:1.0 (dry basis), enzyme loadings of 15 FPU/gm glucan give theoretical yields of glucose and ~80% of theoretical for xylose Few visible effects of pretreatment, material is left dry and can be fed at very high solids loadings, stable for long periods of time Biomass Refining CAFI

19. 19 Conversion of Glucan and Xylan vs. AFEX Treatment Temperature Biomass Refining CAFI

20. 20 Some Distinctive Features of AFEX NOT MUCH CHANGE IN APPEARANCE- DRY PROCESS Stover hydrolysis- 15 FPU @ zero hrs. NOT MUCH EXTRACTED FROM TREATED STOVER HPLC at 168 hours Glucose, xylose, arabinose, Not much else CLEAN SUGAR STREAMS FOR FERMENTATION AFEX treatedUntreated

21. 21 Fed Batch SSF of AFEX Treated and Untreated Corn Stover (15 IU/g glucan) AFEX treated stover in SSF 24% solids loading Untreated stover in SSF 24% solids loading Flows very easily Doesn’t flow worth a dang Dark appearance is deceptive- caused by autoclaving and media Biomass Refining CAFI

22. 22 N2 Gas PG TG Vent Oven (Preheating Coil and Reactor) Holding Tank Pump PG : Press. Gauge TG : Temp. Gauge C.W.: Cooling Water Aqueous Ammonia Water PG C.W. ARP Experimental Set-up Temp. monitoring system (DAS) Biomass Refining CAFI

23. 23 Enzymatic Hydrolysis of Representative ARP-Treated Sample Pretreatment conditions: 170  C, 3.3 mL of 15 wt% of ammonia per g of corn stover Enzymatic hydrolysis conditions : 60 or 15 FPU/g glucan, pH 4.8, 50  C, 150 rpm 95.3%@ 60FPU 90.1%@ 15FPU Biomass Refining CAFI

24. 24 ARP Summary Representative ARP Process Conditions –Liquid throughput: 3.3 mL of 15 wt.% NH 3 per g of corn stover –Dry feed is used without presoaking. –Temperature:170  C, Residence Time: 10 minutes ARP treatment of corn stover results: –63-70% delignification –High retention of glucan –Partial solubilization of hemicellulose ARP raises the digestibility of corn stover to above 95% with 60 FPU/g of glucan and above 90% with 15 FPU/g of glucan. Biomass Refining CAFI

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

26. 26 Reactor System for Lime Pretreatment

27. 27 0 5 10 15 20 25 05101520 untreated 0 5 10 15 20 25 05101520 untreated Pretreatment Time (weeks) 25 o C 35 o C 45 o C 55 o C No Air Air Effect of Air/Temperature on Lignin Klason lignin content g Klason lignin g dry biomass treated × 100 recommended Klason lignin content g Klason lignin g dry biomass treated × 100

28. 28 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) Biomass Refining CAFI

29. 29 Mass Balance Approach: AFEX Example Hydrolysis Enzyme (15 FPU/g of Glucan) Residual Solids Hydrolyzate Liquid AFEX System Treated Stover Ammonia Stover 101.0 lb 100 lb (dry basis) 36.1 lb glucan 21.4 lbxylan 39.2 lb 95.9% glucan conversion to glucose, 77.6% xylan conversion to xylose 99% mass balance closure includes: (solids + glucose + xylose + arabinose ) Wash 2 lb 99.0 lb Solids washed out 38.5 lb glucose 18.9 lbxylose (Ave. of 4 runs) Very few solubles from pretreatment—about 2% of inlet stover

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32. 32 PretreatmentWater:Solids Ratio % Solids Solubilized Dilute acid1.536 Flowthrough~3752 Controlled pH6.237.7 AFEX0.62.0 ARP3.340.0 Lime10.023.0 Water Loadings and Stover Solids Made Soluble by Pretreatments

33. 33 Trends Dilute acid and neutral pH pretreatments solubilize mostly hemicellulose while ammonia and lime remove mostly lignin. AFEX removes neither and flowthrough removes both. All these pretreatments result in high glucose yields via enzymatic hydrolysis with somewhat better results for higher pH and flowthrough methods, possibly due to effect on lignin (cellulase adsorption and interference) –Lignin removal (ARP, lime, flowthrough) or modification (AFEX) Xylose remaining in the solids is solubilized during enzymatic hydrolysis for all methods –Enhanced hemicellulase activity could improve yields for all methods and could have particularly important benefits as pH increases and for flowthrough approach It is important to consider the sugar release pattern and solids concentrations for each pretreatment in the selection of process, enzymes, and fermentative organisms to maximize yields Biomass Refining CAFI

34. 34 Future Work Evaluate performance differences in more depth –Apply hydrolyzate conditioning –Ferment hydrolyzate with recombinant organism –Determine differences in pretreatment effects on cellulase effectiveness –Add hemicellulases to improve performance –Work with Genencor as enzyme supplier –Understand how pretreatment affects substrate features and their effect on digestibility –Broaden scope to hardwoods –Expand team to include University of British Columbia and University of Sherbrooke in Canada To be funded by Office of the Biomass Program of the Department of Energy Biomass Refining CAFI

35. 35 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 Biomass Refining CAFI

36. 36 IFAFS Project Participants Biomass Refining CAFI

37. 37 Questions? Biomass Refining CAFI