Characteristics of Biomass Pretreatments Studied by the CAFI

1. Characteristics of Biomass Pretreatments Studied by the CAFI Bruce E. Dale, Richard T. Elander, Mark T. Holtzapple, Michael R. Ladisch, Yoon Y. Lee and Charles E. Wyman ISAF XV International Symposia on Alcohol Fuels September 26-28, 2005 San Diego, CA Biomass Refining CAFI

2. Brief History of Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) • Pretreatment researchers working together in a coordinated, disciplined way to understand the fundamentals underlying lignocellulosic biomass pretreatment and hydrolysis • Organized in late 1999, early 2000 • CAFI recognizes that pretreatment operates as part of a system that includes hydrolysis and fermentation—pretreatment effects on downstream processes must be better understood Biomass Refining CAFI

3. 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

4. Characteristics of CAFI Pretreatments

5. Stage 2. Enzymatic hydrolysis Glucose and lignin Dilute Acid Pretreatment Stage 1. Pretreatment Biomass Cellulose and lignin Mineral acid • 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: 120-200oC, 50 to 85% moisture, 0-1% H2SO4 • Some degradation of liberated hemicellulose sugars Hemicellulose sugars and oligomers

6. Glucose and Xylose Yields in Pretreatment and Enzymatic Hydrolysis

7. Schematic of Flowthrough Pretreatment System Sample

8. Xylose Fate for Batch, FT, and SFS Runs with Water at 200oC

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

10. Stepwise Process Yields & Mass Balance for 15 FPU Spezyme (168 hrs) Water 620 lb Controlled pH Liquid Hot Water Stover 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan Treated Stover Slurry 62.8 lb undissolved solids 37.2 lb dissolved solids 620 lb water Cellulase Enzyme 2209 FPU per lb stover (30 FPU/ml) Hydrolysis Fermentation Hydrolyzate Liquid 36.32 lb glucose 19.89 lb xylose Cellobiase Enzyme 5891 IU per lb stover (309 IU/ml) Residual Solids 22.0 lb Ethanol 25.2 lb 90.5% total glucan conversion (raw stover basis) 81.8% total xylan conversion (raw stover basis) 88% of theoretical ethanol yield from glucose + xylose

11. Ammonia Ammonia Gaseous Gaseous Recovery Recovery Liquid Liquid Ammonia Ammonia Ammonia Ammonia Treated Treated Biomass Biomass Biomass Biomass Reactor Reactor Explosion Explosion The AFEX/FIBEX Process • 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: 60-110oC, moisture 20-80%, ammonia:biomass ratio 0.5-1.3 to 1.0 (dry basis) • Moderate temperatures, pH prevent/minimize sugar & protein loss • No fermentation inhibitors, no wash stream required, no overliming • Only sugar oligomers formed, no detectable sugar monomers • Few visible physical effects

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

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

14. Preliminary Mass Balance for ARP Pretreatment Ammonia recycling SSF Ammonia Biomass Treated stover 100 lb (dry basis) Hydrolysis Washing ARP Reactor G: 35.6 lb X:10.8 lb Hydrolyzate G:36.1 lb X: 21.4 lb O: 7.8 lb Liquid Glucose 34.9 lb Xylose 17.0 lb Soluble sugar G: 0.5 lb X: 10.6 lb Residual solids 18.9 lb Fermention Lignin 12.1 lb Others 20.8 lb Residual solid 19.3 lb Ethanol 23.4 lb Conversion Efficiency: 87% glucan + 70% xylan conversion at 15 FPU/g of gulcan 3.56 gallons ethanol (90% of theoretical yield, glucan+xylan) 95% mass balance closure (solids + G + X + EtOH + Others) Other sugar is excluded for Ethanol production G: Glucan X: Xylan O: Other sugar

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

16. Mass Balance for Lime Pretreatment Lime + air 43 lb carb. 4 lb lignin Enzyme Hydrolyzate Liquid Solids Liquid Lime System Stover Hydrolysis 100 lb (dry basis) 58 lb carb. 19 lb lignin 55-60 lb sugars 15 lb lignin Residual Solids 15 lb sugars 15 lb lignin 27 lb Ethanol 3.8 – 4.1 gal Fermentation To date: 80 – 90% overall carbohydrate conversion

17. Pretreatment Yields-Stage 1 and Overall* * Soluble oligomers plus monomers/monomers only

18. Characteristics of CAFI Pretreatments

19. 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 • Genencor International • Our team from Dartmouth College; Auburn, Michigan State, Purdue, and Texas A&M Universities; the University of British Columbia; and the National Renewable Energy Laboratory • First group publication: Bioresource Technology vol. 96 #18 pgs. 1959-2032 December 2005 Biomass Refining CAFI

20. Purdue University IFAFS Project Institutions

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

22. Cellulose Digestibility of Pretreated Corn Stover190C – 15 minute