The document summarizes research on enzymatic hydrolysis of cellulose in the ionic liquid NMMO. Key findings include: 1) A twin screw extruder was modified to produce high cellulose loading solutions of up to 13% in NMMO/H2O. Enzymatic hydrolysis reactions were then performed in the extruder. 2) Reaction conditions like cellulose loading, enzyme loading, pH, and agitation method significantly impacted sugar yields and conversion rates. The highest yield was obtained at 10% cellulose, 122 FPU/g enzymes, and acidic pH in a stirred tank reactor. 3) A stirred tank reactor provided better mixing than a shaker bath and achieved near complete conversion of cell

1. Enzymatic Hydrolysis of Cellulose in NMMOBrett RobbinsMaster’s ThesisFAMU-FSU College of EngineeringDepartment of Chemical and Biomedical Engineering

2. Traditional Biomass to Ethanol Schematic- DOE report

3. - DOE report

4. Pretreatment of Lignocellulose- DOE report

5. Ionic LiquidsSolutions of organic cations and inorganic anions

6. Strong intermolecular interaction

7. Very low vapor pressures

8. Easy to recycle due to phase separation

9. Can be modified for specific properties by changing cation or anion combination

10. Ionic properties tend to alter reaction kinetics- R. Rogers, 2002

11. N-methylmorpholine-N-oxide (NMMO)Similar properties of ionic liquids

12. Low vapor pressure solvent: Easily contained & recovered

13. Excellent solvent for dissolution of cellulose in monohydrate form

14. Environmentally friendly UN award for green process

15. Biodegradable solventDissolves cellulose by breaking the hydrogen bonds of cellulose layers- J. Collier and co-workers (2000, 2001)Commercially used as a cellulose solvent in the Lyocell Process

16. Previous WorkProvedEnzymes are active in NMMO

17. Initial reaction rates are higher in cellulose suspended and AA buffered solutions

18. Combined pretreatment and reaction step possible LimitationsOnly 1 wt% cellulose solutions

19. Higher cellulose loadings could not be prepared in batch method mixing

20. Higher cellulose solutions produced a gelatin-like formation  Poor mixing

21. Higher reducing sugars yields desired for fermentation-S.Rama, R.Oyetunji (2009)

22. Goals of this workObjectives:* Perform in situ enzymatic hydrolysis of high cellulose loading in NMMOModify the Extruder so that it is capable of making high loading cellulose solutions in NMMO/H2OPerform the Enzymatic Hydrolysis of cellulose using the twin screw extruder as a reactorAnalyze how cellulose loading, enzyme loading, pH and agitation affect the initial reaction rate, reducing sugar yield, as well as overall conversion of cellulose to glucose

23. ApproachModify extruder in which higher cellulose loading solutions could be preparedPrepare 5, 10 and 13 wt% cellulose solutionsPerform cellulose hydrolysis in extruderDoes Reactive Extrusion show potential?Modify reaction conditions to maximize reducing sugar yields

24. MaterialsCelluloseDissolving pulp with 1160 degree of polymerizationObtained as from Buckeye TechnologiesDissolving pulp is cellulose in which most of the hemicellulose and lignin has been removedCellulaseAccelerase ™ 1000 Enzyme complex solution obtained from Genencor. Composed of a mixture of endogluconase, exoglucanase, β-glucosidase and hemicellulose enzymesCommercially availableEndoglucanase structures of CBDEGZ

25. The enzyme active site extends over 3 glucose monomer links

26. This allows for multiple breaks per a single absorption

27. Right is the enzyme overlaying a cellulose chainTwin Screw Extruder & Modifications

28. Higher Cellulose LoadingsUsing the developed recycle system, we were able to produce 5, 10 and 13 wt% cellulose in NMMO

29. Solution PreparationNMMO near monohydrate PreparationStarting with 50/50 NMMO/H2O, evaporate water using rotovap for 5-8 hrsCellulose/NMMO Solutions (5, 10 and 13 wt%)Dissolve measured pure cellulose in NMMO using extruder (make sure flow through recycle hose is established)Cellulose/NMMO Acetic acid Solution (no recycle system)Add NMMO/Cellulose solution to extruder Port 1 (85C for Zones 1-3)Add Acetic acid into feed port 4 (50C for Zones 4-10)Collect solution at exit and manually re-feed into extruder (repeat 5 – 8 times)For Shaker bath and STRSeparate sample vials containing 10.7 g Cellulose/NMMO/AA Enzyme additionAdd enzyme solution to sample following NREL FPU/g loading requirementsIn E + DI dilution samples, DI added directly into enzyme solution before adding to sampleIn E + AA and E + AA + 1 dilution samples, add AA and then enzyme solution to sample vialTake 0.5 mL samples at pre-designated timeBoil samples to denature enzyme and stop reactionDetermine reducing sugar concentration using DNS assay technique

30. Method of Assay: DNS3,5-Dinitrosalicylic acid (DNS or DNSA) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. A reducing sugar is any sugar that, in basic solution, forms some aldehydeor ketone. Reducing sugars include glucose, fructose, glyceraldehyde, lactose, arabinose and maltose. - Miller, 1959Increasing sugar concentration

31. Reactive ExtrusionIncreasing cellulose loadings yield higher reducing sugar concentrationsExtruder shows potential for enzymatic hydrolysis

32. ApproachModify extruder in which higher cellulose loading solutions could be preparedPrepare 5, 10 and 13 wt% cellulose solutionsPerform cellulose hydrolysis in extruderDoes Reactive Extrusion show potential?Modify reaction conditions to maximize reducing sugar yields

33. Reactive ExtrusionAlthough reducing sugar yield is increasing, no major change in percent conversion