Bioenergy-Hydrolysis Wood Chemistry PSE 406 Bioenergy-Hydrolysis

Agenda Enzymatic hydrolysis Cellulases Experimental

Bioconversion of biomass to ethanol (hydrolysis) Liquid phase Sugars Ethanol Pretreatment Fermentation Biomass Lignin Recovery Solid phase Cellulose Hydrolysis Fermentation Sugars Ethanol

Enzyme Function There are a large number of fungal enzymes responsible for the breakdown of each wood component. Each enzyme plays specific roles: Endo-beta-1,4-glucanase acts within the chain, breaking it into smaller units and providing more "ends" for CBH. Cellobiohydrolase (CBH), acts on the end of the molecule successively cleaving off the disaccharide cellobiose. Beta-glucosidase (or cellobiase) which cleaves cellobiose to two glucose units.

Trichoderma reesei Trichoderma reesei is an industrially important cellulolytic filamentous fungus. T. reesei: present in nearly all soils and other diverse habitats favored by the presence of high levels of plant roots. Trichoderma reesei

Cellobiohydrolyses (CBH) Cellulases Binding domain Catalytic domain Endoglucanases  (EG) cutting the cellulose chains randomly Cellobiohydrolyses (CBH) cutting cellobiose units of the ends of the cellulose chains

“Rapid microassay method (1)” Pretreated substrate Flasks Pretreated substrate Handsheet Microplate

“Rapid microassay method (2)” Microplate Handsheets HPLC Shaker Microplate Reader

“Rapid microassay method (3)” Advantages over the flask based method: Faster processing time (10x) Cheaper (enzyme and substrate requirements 200x) Efficient (768 versus 30 samples) Smaller lab area required (30x) Equipment and development cost (<$3,000) Berlin, A., Bura, R., Gilks, N., and Saddler, J.N., (2005)

Equipment 1mL 200 mL 4L 40L

Enzymatic hydrolysis