Speaker: Jeng-Chen Liu(劉政成) Student ID: P9921003 Graduate Institute of Bioresources, National Pingtung University of Science and Technology Proposal for Ph. D. Program Yeast expression of cellulase complex from rumen fungi for direct fermentation of cellulose to ethanol Speaker: Jeng-Chen Liu(劉政成) Student ID: P9921003

Outline Background: fuel requirement & bioethanol Goal: direct microbial conversion Materials and methods: gene sources & expression Expected results: cellulose  ethanol

The problems of petroleum utilization Exhaustion Petrolic resources Global warming fossil fuels CO2+H2O

The cycle of biomass energy Photosynthesis Sugar Starch Lignocellulose Lipid CO2+H2O Renewable Net emission(CO2)=0 Ethanol-gasoline eg. E3 Biofuels eg. bioethanol

Bioethanol: first generation Unauthorized cultivation Food: sugar starch bean Food price

Bioethanol: second generation Unauthorized cultivation Food: sugar starch bean Food price

Bioethanol: second generation Unauthorized cultivation Lignocellulose Food price

The contents of lignocellulose 2 M ton * 40% =800 K ton (Prasad et al., 2007) (Murphy and McCarthy, 2005)

Lignocellulose  ethanol Pretreatment: removed lignin & hemicellulose Hydrolysis: cellulose  glucose Fermentation: glucose  ethanol

Pretreatment Physical methods Chemical methods Biological methods Ethanol Cellulose Hemicellulose Lignin (Lu and Mosier, 2008)

Cellulose  glucose Acidic hydrolysis Enzymatic hydrolysis

Conversion of cellulose to glucose Table 1. Comparison of hydrolysis methods for cellulose hydrolysis Acid hydrolysis Enzymatic hydrolysis？ Cellulose Glucose (Bungay, 2004)

Lignocellulose  ethanol(SHF;SSF;DMC) Pretreatment: removed lignin & hemicellulose Hydrolysis: cellulose  glucose Fermentation: glucose  ethanol

Lignocellulose  ethanol(SHF) Pretreatment Hydrolysis: cellulase supplementation Fermentation: microorganism Cellulose  ethanol

Lignocellulose  ethanol(SSF) Pretreatment Hydrolysis: cellulase supplementation Fermentation: microorganism Cellulose  ethanol

Lignocellulose  ethanol(DMC) Pretreatment Hydrolysis: cellulase Fermentation: microorganism Cellulose Glucose ethanol

The analysis of enzyme hydrosylate and fermentation products (Chen et al., 2003) Cellulase complex (劉，2010)

Glucose  ethanol Saccharomyces cerevisiae ethanol producer ethanol tolerant GRAS Zymomonas mobilis Escherichia coli

Lignocellulose  ethanol(DMC) Goal Lignocellulose  ethanol(DMC) Pretreatment Hydrolysis Fermentation: S. cerevisiae Cellulose DMC Glucose ethanol

Lignocellulose  ethanol(DMC) Goal Lignocellulose  ethanol(DMC) Pretreatment Hydrolysis: cellulase Fermentation: S. cerevisiae Cellulase complex gene from rumen fungi Cellulose DMC Glucose ethanol

Protocols Rumen fungi cellulase complex genes E. coli expression system S. cerevisiae expression system Biochemical characterization Cell surface display system Promoter Direct microbial conversion Bioethanol

Lignocellulose  ethanol(DMC) Expected results Lignocellulose  ethanol(DMC) Pretreatment Hydrolysis: cellulase Fermentation: S. cerevisiae Cellulase complex gene from rumen fungi Cellulose DMC Glucose ethanol

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