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
1
Outline
 Background: fuel requirement & bioethanol
 Goal: direct microbial conversion
 Materials and methods: gene sources & expression
 Expected results: cellulose  ethanol
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The problems of petroleum utilization
Exhaustion
Petrolic resources
Global warming
fossil fuels
CO2+H2O
3
The cycle of biomass energy
Photosynthesis
Sugar
Starch
Lignocellulose
Lipid
 Renewable
CO2+H2O
 Net emission(CO2)=0
 Ethanol-gasoline
Biofuels
eg. E3
eg. bioethanol
4
Bioethanol: first generation
Unauthorized
cultivation
Food: sugar
starch
bean
Food price
5
Bioethanol: second generation
Unauthorized
cultivation
Food: sugar
starch
bean
Food price
6
Bioethanol: second generation
Unauthorized
cultivation
Lignocellulose
Food price
7
The contents of lignocellulose
Agricultural residue
Hardwood stem
Softwood stem
Corn cobs
Rice straw
Paper
Newspaper
Cellulose(%)
40–50
45–50
45
40
85–99
40–55
Hemicellulose(%)
24–40
25–35
35
18
0
25–40
Lignin(%)
18–25
25–35
15
5.5
0–15
18–30
2 M ton * 40%
=800 K ton
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(Prasad et al., 2007)
(Murphy and McCarthy, 2005)
Lignocellulose  ethanol
 Pretreatment: removed lignin & hemicellulose
 Hydrolysis: cellulose  glucose
 Fermentation: glucose  ethanol
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Pretreatment
Physical methods
Chemical methods
Biological methods
Ethanol
Cellulose
Hemicellulose
Lignin
10
(Lu and Mosier, 2008)
Cellulose  glucose
Acidic hydrolysis
Enzymatic hydrolysis
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Conversion of cellulose to glucose
Table 1. Comparison of hydrolysis methods for cellulose hydrolysis
Cellulose
Acid hydrolysis
Enzymatic hydrolysis？
Glucose
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(Bungay, 2004)
Lignocellulose  ethanol(SHF;SSF;DMC)
 Pretreatment: removed lignin & hemicellulose
 Hydrolysis: cellulose  glucose
 Fermentation: glucose  ethanol
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Lignocellulose  ethanol(SHF)
 Pretreatment
 Hydrolysis: cellulase supplementation
 Fermentation: microorganism
Cellulose  ethanol
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Lignocellulose  ethanol(SSF)
 Pretreatment
 Hydrolysis: cellulase supplementation
 Fermentation: microorganism
Cellulose  ethanol
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Lignocellulose  ethanol(DMC)
 Pretreatment
 Hydrolysis: cellulase
Cellulose
 Fermentation: microorganism
Glucose
ethanol
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The analysis of enzyme hydrosylate
and fermentation products
2D Graph 1
1.6
Ethanol and sugar concenrtion(g/L)
1.4
1.2
1.0
0.8
0.6
Cellobiose
Glucose
Ethanol
0.4
(Chen et al., 2003)
0.2
0.0
-0.2
0
20
40
60
80
Cellulase complex
Time(hours)
(劉，2010)
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Glucose  ethanol
 Saccharomyces cerevisiae
ethanol producer
ethanol tolerant
GRAS
 Zymomonas mobilis
 Escherichia coli
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Goal
Lignocellulose  ethanol(DMC)
 Pretreatment
 Hydrolysis
Cellulose
DMC
 Fermentation: S. cerevisiae
ethanol
Glucose
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Goal
Lignocellulose  ethanol(DMC)
 Pretreatment
Cellulase complex gene from rumen fungi
 Hydrolysis: cellulase
Cellulose
DMC
 Fermentation: S. cerevisiae
ethanol
Glucose
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Rumen fungi
Protocols
cellulase complex genes
E. coli
expression system
S. cerevisiae
expression system
Biochemical characterization
Promoter
Cell surface
display system
Direct microbial conversion
Bioethanol
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Expected results
Lignocellulose  ethanol(DMC)
 Pretreatment
Cellulase complex gene from rumen fungi
 Hydrolysis: cellulase
Cellulose
DMC
 Fermentation: S. cerevisiae
ethanol
Glucose
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Thanks for
your attention
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