Optimal Conditions for Batch Tube Pretreatment Hot water only, 210 o C, 6 min -Total xylose yield is 52.1% % xylose and 106% glucose overall mass balance closure. -Cellulose digestibility at enzyme loading of 60FPU/g glucan is 57.6 % at 48 hours and 15FPU/g glucan is 19.5% at 48hours. 1wt% sulfuric acid, 140 o C, 40min -Total xylose yield is 95% (87% from pretreatment and 8% from enzymatic hydrolysis); Total glucose yield in pretreatment is 8% % xylose and 97.2 % glucose overall mass balance closure. -Cellulose digestibility at enzyme loading of 60FPU/g glucan is 94% at 48 hours and 15FPU/g glucan is 82.4% at 72 hours; -Cellulose digestibility by SSF at the 60 FPU/g glucan enzyme loading is 94.8 % after 3 days and 15FPU/g glucan enzyme loading is 86% after 7 days.

Hydrolysis 60 FPU/g of glucan, 48h Treated Solids Hydrolyzate Liquid Steam Explosion H2OH2O Corn Stover 100 lb (dry basis) 21.4 lb xylan 23.1 lb glucose (G) 2.1 lb xylose (X) 70.7 lb 57.6%Glucan to glucose conversion 83.4% mass balance closure (solids + G + GO + X + XO) 36.1 lb glucan Mass Balance for Water-Only Pretreatment (60 FPU/ g of glucan) Fermentation 2.68gal ethanol Ethanol 92% theoretical ethanol yield from glucose + xylose 0.6 lb glucose (G) 9.3 lb xylo-oligomer (XO) 1.1 lb gluco-oligomer (GO) 47.9 lb Residual Solids Washing 1.66 gal ethanol 1.03 gal ethanol 9.7 lb xylan 33.8 lb glucan

Hydrolysis 15 FPU/g of glucan, 48h Treated Solids Hydrolyzate Liquid Steam Explosion H2OH2O Corn Stover 100 lb (dry basis) 21.4 lb xylan 7.8 lb glucose (G) 2.1 lb xylose (X) 70.7 lb 19.5%Glucan to glucose conversion 83.4% mass balance closure (solids + G + GO + X + XO) 36.1 lb glucan Mass Balance for Water-Only Pretreatment (15 FPU/ g of glucan) Fermentation 1.59 gal ethanol Ethanol 92% theoretical ethanol yield from glucose + xylose 0.6 lb glucose (G) 9.3 lb xylo-oligomer (XO) 1.1 lb gluco-oligomer (GO) 47.9 lb Residual Solids Washing 0.56 gal ethanol 1.03 gal ethanol 9.7 lb xylan 33.8 lb glucan

Hydrolysis 60 FPU/g of glucan, 48h Residual Solids Hydrolyzate Liquid Batch Tube Pretreatment 1 wt% H 2 SO 4 Corn Stover 100 lb (dry basis) 21.4 lb xylan 0.2 lb gluco-oligomer (GO) 21.8lb xylose (X) 64.0 lb 95.9%Glucan to glucose % xylan to xylose conversion 89.8% mass balance closure (all solids + G + GO + X + XO) 36.1 lb glucan Mass Balance for Dilute Acid Pretreatment (60 FPU/ g of glucan) Fermentation 4.45 gal ethanol Ethanol 92% theoretical ethanol yield from glucose + xylose 2.1 lb xylose (X) 35.2 lb glucose (G) 0.1 lb xylo-oligomer (XO) 3.0 lb glucose (G) 27.4 lb Treated Solids 2.5 lb xylan 33 lb glucan 2.66 gal ethanol 1.79 gal ethanol

Hydrolysis 15 FPU/g of glucan, 48h Residual Solids Hydrolyzate Liquid Batch Tube Pretreatment 1 wt% H 2 SO 4 Corn Stover 100 lb (dry basis) 21.4 lb xylan 0.2 lb gluco-oligomer (GO) 21.8lb xylose (X) 64.0 lb 84.2%Glucan to glucose % xylan to xylose conversion 89.7% mass balance closure ( all solids + G + GO + X + XO) 36.1 lb glucan Mass Balance for Dilute Acid Pretreatment (15 FPU/ g of glucan) Fermentation 4.08 gal ethanol Ethanol 92% theoretical ethanol yield from glucose + xylose 1.3 lb xylose (X) 30.9 lb glucose (G) 0.1 lb xylo-oligomer (XO) 3.0 lb glucose (G) 32.4 lb Treated Solids 2.5 lb xylan 33 lb glucan 2.29 gal ethanol 1.79 gal ethanol

SSF Enzyme+ D 5 60 FPU/g of glucan, 3 days Batch Tube Pretreatment Corn Stover 100 lb (dry basis) 21.4 lb xylan 0.2 lb gluco-oligomer (GO) 21.8lb xylose (X) 64.0 lb 94.8%glucan to glucose conversion 36.1 lb glucan Mass Balance for Dilute Acid Pretreatment w/SSF (60 FPU/ g of glucan) 4.48 gal ethanol Ethanol 92.1% ethanol yield from glucose 0.1 lb xylo-oligomer (XO) 3.0 lb glucose (G) Treated Solids 2.5 lb xylan 33 lb glucan 2.48 gal ethanol from glucan by D 5 A 1 wt% H 2 SO 4 (1.79 gal ethanol) (0.20 gal ethanol from xylan) 92% theoretical ethanol yield from glucose + xylose

SSF Enzyme+ D 5 15 FPU/g of glucan, 3 days Batch Tube Pretreatment Corn Stover 100 lb (dry basis) 21.4 lb xylan 0.2 lb gluco-oligomer (GO) 21.8lb xylose (X) 64.0 lb 86 %glucan to glucose conversion 36.1 lb glucan Mass Balance for Dilute Acid Pretreatment w/SSF (15 FPU/ g of glucan) 4.02 gal ethanol Ethanol 83.2 % ethanol yield from glucose 0.1 lb xylo-oligomer (XO) 3.0 lb glucose (G) Treated Solids 2.5 lb xylan 33 lb glucan 2.03 gal ethanol from glucan by D 5 A 1 wt% H 2 SO 4 (1.79 gal ethanol) (0.20 gal ethanol from xylan) 92% theoretical ethanol yield from glucose + xylose

Optimal Conditions for Flowthrough Pretreatment How water only, 200 o C, 10mL/min, 16-24min (depending on the biomass loading) -Total xylose yield is 96.2%; -98% xylose and 100% glucose overall mass balance closure. -Cellulose digestibility at enzyme loading of 60FPU/g glucan is 99.2% at 48 hours and 15FPU/g glucan is 96.5% after 72h hours; 0.05wt% sulfuric acid, 190 o C, 10mL/min, 16-20min (depending on the biomass loading) -Total xylose yield is 94.7%; -96% xylose and 100% glucose overall mass balance closure. -Cellulose digestibility at enzyme loading of 60FPU/g glucan is 99.5% at 48 hours and 15FPU/g glucan is 97.8% after 72 hours;

Mass Balance For Water-Only Flowthrough Pretreatment (60 FPU/ g of glucan) FL reactor Treated Solids Hydrolysis g of glucose, 48h Water Corn Stover Residual Solids Hydrolyzate Liquid Fermentation 43.4 lb 34.3 lb glucan 0.4 lb xylan 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 37.8 lb glucose 0.4 lb xylose 4.53 gal ethanol 7.2 lb 99.2% glucan to glucose, 88% xylan to xylose at 60 FPU/ g of glucan 97% overall glucan and xylan conversion 92% theoretical ethanol yield from glucose + xylose 97.5% overall mass balance closure (total dissolved mass+residual solids + G + X) Dissolved mass 52.1 lb 23.4 lb xylose 2.1 lb glucose 2.72 gal ethanol 1.81 gal ethanol

Mass Balance For Water-Only Flowthrough Pretreatment (15 FPU/ g of glucan) FL reactor Treated Solids Hydrolysis g of glucose, 72h Water Corn Stover Residual Solids Hydrolyzate Liquid Fermentation 43.4 lb 34.3 lb glucan 0.4 lb xylan 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 36.7 lb glucose 0.31 lb xylose 4.45 gal ethanol 7.2 lb Dissolved mass 96.5% glucan to glucose, 68.2% xylan to xylose at 15 FPU/ g of glucan 97% overall glucan and xylan conversion 92% theoretical ethanol yield from glucose + xylose 96.3% overall mass balance closure (total dissolved mass+residual solids + G + X) 52.1 lb 23.4 lb xylose 2.1 lb glucose 1.81 gal ethanol 2.64 gal ethanol

Mass Balance For Very Dilute Acid Flowthrough Pretreatment (60FPU/g of glucan) FL reactor Treated Stover Hydrolysis g of glucan, 48h 0.05wt %wt H 2 SO 4 Corn Stover Residual Solids Hydrolyzate Liquid Fermentation 46.6 lb 33.2 lb glucan 0.43 lb xylan 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 36.7 lb glucose 0.41 lb xylose 13.4 lb 99.5% glucan to glucose, 83% xylan to xylose at 60FPU/g of glucan 97.5% overall glucan and xylan conversion 92% theoretical ethanol yield from glucose + xylose 98.7% overall mass balance closure (total dissolved mass+residual solids + G + X) Dissolved mass 48.2 lb 23.0 lb xylose 3.2 lb glucose 4.55 gal ethanol 2.64 gal ethanol 1.91 gal ethanol

Mass Balance For Very Dilute Acid Flowthrough Pretreatment (15FPU/g of glucan) FL reactor Treated Solids Hydrolysis g of glucan, 72h 0.05wt %wt H 2 SO 4 Corn Stover Residual Solids Hydrolyzate Liquid Fermentation 46.6 lb 33.2 lb glucan 0.43 lb xylan 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 36.1 lb glucose 0.39 lb xylose 13.4 lb Dissolved mass 4.5 gal ethanol 97.8% glucan to glucose, 80% xylan to xylose at 15FPU/g of glucan 97.5% overall glucan and xylan conversion 92% theoretical ethanol yield from glucose + xylose 98.1% overall mass balance closure (total dissolved mass+residual solids + G + X) 48.2 lb 23.0 lb xylose 3.2 lb glucose 1.91 gal ethanol 2.59 gal ethanol

Fermentability of Hydrolyzate from Flowthrough Pretreatment of Corn Stover Pretreatment conditions Hydrolyzate (% of the medium) Time (hours) Ethanol Conc. (g/L) Fermentability (%) Water only, 200 o C, 10mL/min, 20min ~100 Water only, 220 o C, 10mL/min, 10min ~ wt% sulfuric acid, 190 o C, 10mL/min, 20min ~100 Control 0 ( Just DI water) ~100 Note: Fermentability was measured and calculated according to the attached procedure