Date on Master's Thesis/Doctoral Dissertation

5-2012

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemical Engineering

Committee Chair

Berson, Robert E. (Eric)

Author's Keywords

Adsorbed cellulase inactivation; Kinetics; CBHI; Cellulose hydrolysis

Subject

Cellulose; Hydrolysis; Ethanol fuel industry--Technological innovations

Abstract

Several technical and economic obstacles currently hamper the industrial development of ethanol from biomass. One of the key bottlenecks is the slow kinetics of the enzymatic hydrolysis of cellulose, and the subsequent rate reduction as the reaction proceeds. As a result, this research focused on understanding underlying causes for the slow kinetics, rate reduction, and low yield during cellulose hydrolysis. Mechanisms traditionally thought to cause these results were investigated, such as change of substrate properties and deactivation of enzyme due to environmental mechanisms, but neither was found to contribute significantly to the slow kinetics and low yield. Inactivation due to enzyme-substrate interactions was then proposed as a key factor. Results here show that inactivation of adsorbed enzyme played the most significant role for the hydrolysis rate reduction and low yield based on the following findings: (1) a kinetic model featuring inactivation of adsorbed enzyme accurately accounted for experimental cellulose hydrolysis data for two different types of substrates; the enzyme's apparent maximum reaction rate was found to decrease with a first order exponential decay function of time due to inactivation of the adsorbed enzyme, which has historically always been considered to remain constant. (2) comparison of relative extents of enzyme activity loss due to environmental mechanisms (such as thermal and/or mechanical factors) with inactivation due to enzyme-substrate interactions revealed that enzyme- substrate interactions contributed more towards the overall activity loss than did environmental mechanisms; (3) AFM imaging visualized crowding of Cellobiohydrolase 1 (CBHl) on cellulose substrate surface and thereafter became inactivated; (4) desorption of inactive CBHl was slower compared to desorption of active CBHl, implying that once inactivated, CBH 1 cannot dissociate immediately to find another site on a substrate surface to start another digestive cycle. The overall conclusion is that inactivation of adsorbed enzyme is a primary contributor to the hydrolysis rate reduction. Near complete conversion (99%) of cellulose was predicted by the model to occur within 10-20 hours if inactivation of adsorbed cellulase can be prevented, compared to 7-10 days or more to achieve a lower yield when inactivation occurs. Finally, factors to consider when developing a cellulose hydrolysis process were proposed based on the inactivation mechanism. One important strategy proposed is to desorb inactive cellulases from the substrate, such as with the addition of GdnHCl. Additionally, a technique for scaling-up separation of CBHl was developed. The technique allows for efficient purification of active CBHl from commercial cellulose cocktails at a cost of less than 10% compared to the conventional small-scale FPLC method.

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