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 NMMO Brett RobbinsMaster’s ThesisFAMU-FSU College of EngineeringDepartment of Chemical and Biomedical Engineering
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
22. Goals of this work Objectives: * Perform in situ enzymatic hydrolysis of high cellulose loading in NMMO Modify the Extruder so that it is capable of making high loading cellulose solutions in NMMO/H2O Perform the Enzymatic Hydrolysis of cellulose using the twin screw extruder as a reactor Analyze 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. Approach Modify extruder in which higher cellulose loading solutions could be prepared Prepare 5, 10 and 13 wt% cellulose solutions Perform cellulose hydrolysis in extruder Does Reactive Extrusion show potential? Modify reaction conditions to maximize reducing sugar yields
28. Higher Cellulose Loadings Using the developed recycle system, we were able to produce 5, 10 and 13 wt% cellulose in NMMO
29. Solution Preparation NMMO near monohydrate Preparation Starting with 50/50 NMMO/H2O, evaporate water using rotovap for 5-8 hrs Cellulose/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 (85C for Zones 1-3) Add Acetic acid into feed port 4 (50C for Zones 4-10) Collect solution at exit and manually re-feed into extruder (repeat 5 – 8 times) For Shaker bath and STR Separate sample vials containing 10.7 g Cellulose/NMMO/AA Enzyme addition Add enzyme solution to sample following NREL FPU/g loading requirements In E + DI dilution samples, DI added directly into enzyme solution before adding to sample In E + AA and E + AA + 1 dilution samples, add AA and then enzyme solution to sample vial Take 0.5 mL samples at pre-designated time Boil samples to denature enzyme and stop reaction Determine reducing sugar concentration using DNS assay technique
30. Method of Assay: DNS 3,5-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, 1959 Increasing sugar concentration
32. Approach Modify extruder in which higher cellulose loading solutions could be prepared Prepare 5, 10 and 13 wt% cellulose solutions Perform cellulose hydrolysis in extruder Does Reactive Extrusion show potential? Modify reaction conditions to maximize reducing sugar yields
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34. Reducing Sugar yield does not accurately scale with initial cellulose concentrationIs there another way to increase conversion?
37. Higher cellulose loading causes foaming when air is taken up by the extruderCellulose NMMO 10% Acetic Acid Enzymes Low Density Low Viscosity Extruder Shaker Bath Higher cellulose loading solutions can be reacted in Shaker Bath
46. Sometime between 12 and 24 hours, high pH may cause inactivation of the enzymes
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48. Samples with additional 10% acetic acid resulted in higher reducing sugar yields~2.5 mg/mL for 5 cellulose wt% ~4.5 mg/mL for 10 cellulose wt% Account for deactivation of enzymes, but high cellulose loading of 122 FPU/g may hide any substrate inhibition
62. Comparison with Literature Open Symbols are from Literature Closed Symbols are experiments Comparable to current work Literature works use ILs with regeneration, experiments combine pretreatment and hydrolysis steps
63. Conclusions ** In situ hydrolysis of high cellulose loading in NMMO/H2O solutions can be performed utilizing a twin screw extruder Modified Extruder is capable of making high cellulose loading solutions in NMMO/H2O Enzymatic Hydrolysis of cellulose can occur using a twin screw extruder as a reaction vessel Provides path to “consolidated bioprocess” Cellulose loading, enzyme loading, pH and agitation method are important factors in the initial reaction rate, reducing sugar yield, as well as overall conversion of cellulose to glucose 10wt% cellulose, 122 FPU/g, E + AA pH scheme in the STR provided the highest yield of reducing sugars (39.85 mg/mL) as well as cellulose conversion (95.55% assuming all glucose).
64. Recommendations In running the experiment in the extruder, make sure to maintain over flooded feed. This will prevent the extruder from pulling air into the extrusion system producing bubbles and reduce the pressure building capacity to push the solution through the recycle system. The hydrolysis may be run at 40 degrees Celsius, because it has been shown that higher pH is tolerated by the enzyme at low temperature. This can reduce the acid addition necessary for higher conversion. HPLC would be useful at identifying the final reducing sugar composition in solution for use in future fermentation reactions. The initial stirred tank reactor has shown to have promising results and should be investigated further.
65. Acknowledgements My Advisors, Dr Ramkrishnan and Dr. Collier for their guidance and insight in my research, without which I would not have been able to take this research the extent was able. Dr Paravastu for letting me use his laboratory and equipment to analyze my results. The FSU-FAMU College of Engineering Department of Chemical Engineering, for accepting me into the Master’s program and their financial support Bush Brothers and Sun Grant for their support of my research. Gary, Kimberly, Elizabeth and all members of the department who help with suggestions or assistance in this work.
