Optimal Conditions for Batch Tube Pretreatment

1. Optimal Conditions for Batch Tube Pretreatment • Hot water only, 210oC, 6 min • Total xylose yield is 52.1%. • 49.3% 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, 140oC, 40min • Total xylose yield is 95% (87% from pretreatment and 8% from enzymatic hydrolysis); Total glucose yield in pretreatment is 8%. • 101.7 % 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.

2. Mass Balance for Water-Only Pretreatment (60 FPU/ g of glucan) H2O Enzyme @ 60 FPU/g of glucan, 48h Hydrolyzate Liquid Corn Stover Steam Hydrolysis Explosion 100 lb (dry basis) 23.1 lb glucose (G) 70.7 lb Washing Treated Solids 47.9 lb 1.66 gal ethanol 21.4 lb xylan 9.7 lb xylan 33.8 lb glucan Residual Solids 36.1 lb glucan 1.03 gal ethanol 2.1 lb xylose (X) Fermentation 9.3 lb xylo-oligomer (XO) 0.6 lb glucose (G) 1.1 lb gluco-oligomer (GO) Ethanol 2.68gal ethanol 57.6% Glucan to glucose conversion 83.4% mass balance closure (solids + G + GO + X + XO) 92% theoretical ethanol yield from glucose + xylose

3. Mass Balance for Water-Only Pretreatment (15 FPU/ g of glucan) H2O Enzyme @ 15 FPU/g of glucan, 48h Hydrolyzate Liquid Corn Stover Steam Hydrolysis Explosion 100 lb (dry basis) 7.8 lb glucose (G) 70.7 lb Washing Treated Solids 47.9 lb 0.56 gal ethanol 21.4 lb xylan 9.7 lb xylan 33.8 lb glucan Residual Solids 36.1 lb glucan 1.03 gal ethanol 2.1 lb xylose (X) Fermentation 9.3 lb xylo-oligomer (XO) 0.6 lb glucose (G) 1.1 lb gluco-oligomer (GO) Ethanol 1.59 gal ethanol 19.5% Glucan to glucose conversion 83.4% mass balance closure (solids + G + GO + X + XO) 92% theoretical ethanol yield from glucose + xylose

4. Mass Balance for Dilute Acid Pretreatment (60 FPU/ g of glucan) 1 wt% H2SO4 Enzyme @ 60 FPU/g of glucan, 48h Hydrolyzate Liquid Corn Stover Batch Tube Hydrolysis Pretreatment 100 lb (dry basis) 64.0 lb 2.1 lb xylose (X) Treated Solids 35.2 lb glucose (G) 2.66 gal ethanol 27.4 lb 21.4 lb xylan 36.1 lb glucan 2.5 lb xylan 33 lb glucan Residual Solids 1.79 gal ethanol 21.8lb xylose (X) Fermentation 0.1 lb xylo-oligomer (XO) 3.0 lb glucose (G) Ethanol 0.2 lb gluco-oligomer (GO) 4.45 gal ethanol 95.9% Glucan to glucose + 73.9% xylan to xylose conversion 89.8% mass balance closure (all solids + G + GO + X + XO) 92% theoretical ethanol yield from glucose + xylose

5. Mass Balance for Dilute Acid Pretreatment (15 FPU/ g of glucan) 1 wt% H2SO4 Enzyme @ 15 FPU/g of glucan, 48h Hydrolyzate Liquid Corn Stover Batch Tube Hydrolysis Pretreatment 100 lb (dry basis) 64.0 lb 1.3 lb xylose (X) Treated Solids 30.9 lb glucose (G) 2.29 gal ethanol 32.4 lb 21.4 lb xylan 36.1 lb glucan 2.5 lb xylan 33 lb glucan Residual Solids 1.79 gal ethanol 21.8lb xylose (X) Fermentation 0.1 lb xylo-oligomer (XO) 3.0 lb glucose (G) Ethanol 0.2 lb gluco-oligomer (GO) 4.08 gal ethanol 84.2% Glucan to glucose + 45.8% xylan to xylose conversion 89.7% mass balance closure ( all solids + G + GO + X + XO) 92% theoretical ethanol yield from glucose + xylose

6. Mass Balance for Dilute Acid Pretreatment w/SSF (60 FPU/ g of glucan) Enzyme+ D5A @ 60 FPU/g of glucan, 3 days 1 wt% H2SO4 Corn Stover Batch Tube SSF Pretreatment 100 lb (dry basis) 64.0 lb Treated Solids 2.5 lb xylan 33 lb glucan 2.48 gal ethanol from glucan by D5A 21.4 lb xylan 36.1 lb glucan 21.8lb xylose (X) (0.20 gal ethanol from xylan) 0.1 lb xylo-oligomer (XO) (1.79 gal ethanol) 3.0 lb glucose (G) Ethanol 0.2 lb gluco-oligomer (GO) 4.48 gal ethanol 94.8% glucan to glucose conversion 92% theoretical ethanol yield from glucose + xylose 92.1% ethanol yield from glucose

7. Mass Balance for Dilute Acid Pretreatment w/SSF (15 FPU/ g of glucan) Enzyme+ D5A @ 15 FPU/g of glucan, 3 days 1 wt% H2SO4 Corn Stover Batch Tube SSF Pretreatment 100 lb (dry basis) 64.0 lb Treated Solids 2.5 lb xylan 33 lb glucan 2.03 gal ethanol from glucan by D5A 21.4 lb xylan 36.1 lb glucan 21.8lb xylose (X) (0.20 gal ethanol from xylan) 0.1 lb xylo-oligomer (XO) (1.79 gal ethanol) 3.0 lb glucose (G) Ethanol 0.2 lb gluco-oligomer (GO) 4.02 gal ethanol 86% glucan to glucose conversion 92% theoretical ethanol yield from glucose + xylose 83.2% ethanol yield from glucose

8. Optimal Conditions for Flowthrough Pretreatment • How water only, 200oC, 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, 190oC, 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;

9. Mass Balance For Water-Only Flowthrough Pretreatment (60 FPU/ g of glucan) Water Enzyme @60FPU/ g of glucose, 48h Hydrolyzate Liquid Treated Solids Corn Stover FL reactor Hydrolysis 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 37.8 lb glucose 0.4 lb xylose 43.4 lb 34.3 lb glucan 0.4 lb xylan Dissolved mass 2.72 gal ethanol Residual Solids 7.2 lb 52.1 lb 23.4 lb xylose 2.1 lb glucose 1.81 gal ethanol Fermentation 4.53 gal ethanol 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)

10. Mass Balance For Water-Only Flowthrough Pretreatment (15 FPU/ g of glucan) Water Enzyme @15FPU/ g of glucose, 72h Hydrolyzate Liquid Treated Solids Corn Stover FL reactor Hydrolysis 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 43.4 lb 34.3 lb glucan 0.4 lb xylan 36.7 lb glucose 0.31 lb xylose 2.64 gal ethanol Dissolved mass Residual Solids 7.2 lb 52.1 lb 23.4 lb xylose 2.1 lb glucose 1.81 gal ethanol Fermentation 4.45 gal ethanol 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)

11. Mass Balance For Very Dilute Acid Flowthrough Pretreatment (60FPU/g of glucan) 0.05wt %wt H2SO4 Enzyme @60FPU/ g of glucan, 48h Hydrolyzate Liquid Treated Stover Corn Stover FL reactor Hydrolysis 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 46.6 lb 33.2 lb glucan 0.43 lb xylan 36.7 lb glucose 0.41 lb xylose 2.64 gal ethanol Dissolved mass Residual Solids 13.4 lb 48.2 lb 23.0 lb xylose 3.2 lb glucose 1.91 gal ethanol Fermentation 4.55 gal ethanol 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)

12. Mass Balance For Very Dilute Acid Flowthrough Pretreatment (15FPU/g of glucan) 0.05wt %wt H2SO4 Enzyme @15FPU/ g of glucan, 72h Hydrolyzate Liquid Treated Solids Corn Stover FL reactor Hydrolysis 100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan 46.6 lb 33.2 lb glucan 0.43 lb xylan 36.1 lb glucose 0.39 lb xylose 2.59 gal ethanol Dissolved mass Residual Solids 13.4 lb 48.2 lb 23.0 lb xylose 3.2 lb glucose Fermentation 1.91 gal ethanol 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)

13. Fermentability of Hydrolyzate from Flowthrough Pretreatment of Corn Stover Note: Fermentability was measured and calculated according to the attached procedure