Document Type

Dissertation - University Access Only

Award Date

2012

Degree Name

Doctor of Philosophy (PhD)

Department / School

Health and Nutritional Sciences

First Advisor

Basil Dalaly

Abstract

Bioethanol was considered as one of the alternative fossil fuel replacements. It was produced by fermenting plant sugars and involves a multi-step production process: pretreatment, enzymatic hydrolysis and fermentation of biomass. The ability to produce ethanol from low-cost biomass will be crucial for making ethanol competitive with gasoline. The high cost of cellulase and hemicellulase enzymes were the key barrier for economic production of cellulosic ethanol and therefore, immobilization of enzymes would lower the cost permitting enzyme recycling. The main objectives of this study were to find a suitable matrix and method for immobilization of cellulase and hemicellulases and to optimize the activity of immobilized enzyme on biomass. Immobilization of cellulase and hemicellulases were done on porous siran™ beads. Enzyme immobilization methods and carriers were selected based on protein loading, activity loading and extent of recycling of the enzyme for biomass hydrolysis. In the first paper (Chapter 2), immobilization of cellulase complex ( cellulase) and enzyme complex ( cellulases and hemicellulases) were performed on porous siran TM beads using covalent and non-covalent bonding methods. Free enzyme complex was used for hydrolyzing various pretreated biomass such as com stover, distillers dried grains with solubles (DGGS), switchgrass and bluestem grass. Maximum activity of enzyme complex was found with com stover, and was used as a substrate for measuring cellulase and hemicellulases activities. Protein loading and activity loading for immobilized cellulase complex and enzyme complex on siran TM carriers were measured. In addition, activity loading for immobilized cellulase complex and enzyme complex were measured on different substrates. Recycling experiments for immobilized cellulase complex and enzyme complex were carried out using various substrates and the residual enzyme activity was measured after the third cycle. Carbodiimide bonded cellulase complex retained more residual enzyme activity with avicel ( 49 .5 % ) which, indicates it's a good cross linker for cellulase immobilization. Enzyme complex bonded to carbodiimide and glutaraldehyde cross-linkers, retained residual activity of 44.2 % with xylan, 73.2 % with com stover, respectively. Recycling studies showed greater retention in the residual enzyme activity with covalent bonded enzymes than non covalent bonded enzymes. Glutaraldehyde and carbodiimide were found to be effective cross linkers for immobilizing both cellulase and hemicellulases in the covalent bonding method and were selected for immobilizing cellulase and hemicellulases in further studies. In the second paper (Chapter 3) immobilization of cellulase ( cellulase complex and P-glucosidase) was performed on porous siran TM beads using the covalent bonding method with both cross-linkers: carbodiimide and glutaraldehyde. Free cellulase complex/enzyme complex in combination with p-glucosidase at different enzyme activity levels and ratios, were used for hydrolyzing pretreated corn stover and switchgrass. Maximum enzyme activity was found with corn stover followed by switchgrass for both the enzymes at various enzyme doses. Cellulase complex and P-glucosidase together have showed good sugar yields compared to enzyme complex and p-glucosidase combination on both the biomass. Maximum enzyme activity was found with cellulase complex (20 FPU units) and P-glucosidase (80 CBU units) combination. This ratio of cellulase (cellulase complex and p-glucosidase (1 :4)) was immobilized onto siran ™ carriers by covalent bonding method. Protein loading, activity loading and recycling of unbound enzyme ( after first immobilization cycle) were studied for seven consecutive cycles with both the cross-linkers on the two substrates. Recycling experiments with unbound cellulase ( cellulase complex and Pglucosidase) on both the substrates retained~ 70% of the residual enzyme activity even after sixth cycle for both immobilization methods. Carbodiimide bonded cellulase retained more residual enzyme activity which, indicates a good cross- linker for cellulase immobilization and therefore, carbodiimide bonded cellulase was chosen for studying cellulase kinetics. Studying kinetic parameters provides information on cellulase activity and stability under varying substrates and operating conditions. Kinetic parameters such as Km, Vmax, optimum pH and temperature, activators and inhibitors and product inhibition studies were determined for both free and carbodiimide bonded cellulase on switchgrass and corn stover. From these studies, it was suggested that carbodiimide bonded cellulase could be used in biofuel industry, but further investigation on bench scale trials needs to be conducted before its final application. Future work using bench scale bioreactor needs to be carried out for saccharification of biomass with carbodiimide bonded cellulase.

Library of Congress Subject Headings

Biomass
Cellulose
Hemicellulose
Lignocellulose
Ethanol as fuel
Cellulosic ethonal

Publisher

South Dakota State University

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In Copyright