CoER : Biofuels Department of Microbiology • Faculty of Natural Sciences UNIVERSITEIT • STELLENBOSCH • UNIVERSITY jou kennisvennoot • your knowledge partner Cellulosic ethanol: From revolutionary consolidated bioprocessing idea to proof of concept Emile van Zyl Department Microbiology University of Stellenbosch
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CoER : Biofuels Department of Microbiology • Faculty of Natural Sciences
UNIVERSITEIT • STELLENBOSCH • UNIVERSITY
jou kennisvennoot • your knowledge partner
Cellulosic ethanol: From revolutionary
consolidated bioprocessing idea to
proof of concept
Emile van Zyl
Department MicrobiologyUniversity of Stellenbosch
Western Cape
Map of Western Cape
2
Cape of Good Hope
Cape Town -
arial view of the city and Tafelberg
Arial view of Stellenbosch
Water Front 3
Panoramic view of Stellenbosch
Stellenbosch- Jewel of the Cape
4“Wine country”
Stellenbosch University
Central campus – “The Red Plane”
5
Dept. Microbiology
6
Content
1. Next generation technologies for cellulose conversion
2. What is Consolidated Bioprocessing?
3. Recent advances towards realizing CBP
4. Rolling out cellulosic ethanol in southern Africa
7
Next generation technologies for cellulose conversion
8
Ethanol production from sugar
Spent
yeast
Crashing
Sugar
extraction
Sugarcane\
Sugarbeet
Sweet
sorghum
Fuel
blending
Alcoholrecovery
Distillation & dehydration
Storage
tank
Yeast
Fermentation
Technologies for Ethanol Production
Sugar
Storage
tank
9
Ethanol production from cellulosics
Spent
material
Pre-treatment
Chipping
Grinding
Agric Res
Woody
Material
Grasses
Water
mixing
tank
Steam explosion
~200ºC
Cellulases
Fuel
blending
Saccharification
Alcoholrecovery
Distillation & dehydration
Storage
tank
Yeast
Fermentation
Technologies for Ethanol Production
10
Pretreatment for Ethanol Production
CelluloseLignin
Hemicellulose
Amorphous Region
Crystalline Region
Pretreatment
Pretreatment - produces an enzyme accessible substrate
Ladisch, 2006
11
Enzymes required for CBP
Component
Glucan 41.6
Xylan 15.9
Galactan 0.7
Mannan 2.2
Arabinan 0.8
Acetic acid 5
Extractives 1.4
Lignin 25.6
Ash 0.5
Total 93.7
Hemicellulose
Partially hydrolyzed
during feedstock
pretreatment
Cellulose
Cellobiohydrolases
Endo-glucanase
β-glucosidase
Endo-xylanase
α-glucuronidase
β-xylosidase
Acetyl xylan esterase
α-arabinofuransidase
Endo-mannanaseβ-manosidaseetc.
Glucose
XyloseManoseArabinoseGalactose
12
Enzyme system development
T. reesei secretome
• CBHs are the major constituent of the T. reesei cellulase system
• Second most important species are the EGs
• Broad diversity of enzymes contributes to highly active system
Largest Component of Recalcitrance Barrier:Cost of Cellulase
a) Hinman et al. 1991. Appl. Biotechnol. Bioeng. 34/35:639-657.
b) Hettenhaus & Glassner, 1997 (http://www.ceassist.com/assessment.htm).
c) NREL, 1998. Bioethanol from the corn industry. DOE/GO-1009-577.
d) Schell, 2004. ASM Natl Meeting; McMillan, 2004. DOE/NASULGS Biomass & Solar Energy Workshops.
Microbiology and Molecular Biology Reviews 66: 506-577 (2002)
Fundamentals of Microbial Cellulose Utilization
Consolidated BioProcessing (CBP)
17
Advances in Biochemical Engineering/Biotechnology 108: 205 -235 (2007)
Consolidated bioprocessing : update (2)
Consolidated BioProcessing (CBP)
18
Consolidated bioprocessing : update (3)
Consolidated BioProcessing (CBP)
Applied Microbiology and Biotechnology 87: 1195–1208 (2010)
19
Consolidated BioProcessing (CBP)
OO
O
O
O
Glu Man Gal
Xyl Ara
Ethanol + CO2
P TYFG
Glycosyl
Hydrolases
Technologies for Cellulose Conversion
20
Ethanol production from cellulosics
Spent
material
Pre-treatment
Cooling &
conditioning
Chipping
Grinding
Agric Res
Woody
Material
Grasses
Water
mixing
tank
Steam explosion
~200ºC
Cellulases
Fuel
blending
Saccharification
Alcoholrecovery
Distillation & dehydration
Storage
tank
Yeast
Fermentation
Technologies for Ethanol Production
Spent
material
Pre-treatment
Cooling &
conditioning
Chipping
Grinding
Agric Res
Woody
Material
Grasses
Water
mixing
tank
Steam explosion
~200ºC
Fuel
blending
Alcoholrecovery
Distillation & dehydration
Storage
tank
Cellulolytic Yeast
Saccharification & Fermentation
Technologies for Ethanol Production
Ethanol production from cellulosics
21
22
[REF] [EG1]
[SFI] [CEL5]
Den Haan, R., S.H. Rose, L.R. Lynd, and W.H. Van Zyl. 2007. Hydrolysis and fermentation of amorphous
cellulose by recombinant Saccharomyces cerevisiae. Met. Eng. 9: 87–94.
Growth on amorphous cellulose (PASC)
Technologies for Cellulose Conversion
23
Y294[CBH1] ~20X conc.
Y294[CBH1] ~150X conc.
Y294[CBH1] ~150X conc. treated with
Pngase
+ control CBH1 ~250 ng,
treated with Pngase
206,675115,75898,003
54,604
37,390
29,559
20,366
7,036
MW (Da)
CBH1 cellobiohydrolase production by yeast
Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl. 2007. Functional
expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of
cellulose to ethanol. Enzyme Microb. Technol. 40: 1291–1299.
Expression of cellobiohydrolases in yeast
24
CBH expressed in yeast
mU
nits
/g d
ry c
ell w
eigh
t
1
10
100
1000
10000
CBH1 CBH1-4 CBHB CBH2 CBH1Aerobic
CBH1Anaerobic
•CBH1 requirements calculated based on ratio of CBH1 to other cellulase components in T. reesei cellulase mixtures to allow growth rate of 0.02 hr-1
2.6% of t.c.p.
Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl. 2007. Functional expression of
cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme
Microb. Technol. 40: 1291–1299.
Cellobiohydrolase production by yeast
Expression of cellobiohydrolases in yeast
25
Mascoma CorporationTechnical facilities, Lebanon, NH, USA
(www.mascoma.com)
Leading Investment, Unprecedented Focus on CBP
Technical Focus: Overcoming the biomass recalcitrance barrier and enablingthe emergence of a cellulosic biofuels industry via pioneering CBP technology integrated with advanced pretreatment
Three Platforms
1. T. saccharolyticum, thermophilic bacterium able to use non-glucose sugars2. C. thermocellum, thermophilic cellulolytic bacterium3. Yeast engineered to utilize cellulose and ferment glucose and xylose
Partners in Mascoma’s CBP Organism Development Effort
• Dartmouth College, USA
• University of Stellenbosch, ZA
• VTT, Finland • BioEnergy Science Center, USA
• Department of Energy, USA
Multiple chances to succeed near-term & long-term26
27
Screen CBH1 for high level expression
Enzyme activity:• 48 hour Avicel
hydrolysis • Best enzyme
x13 greater than starting point
Enzyme Production:
• 94 mg/L CBH1• ± 2.5% of Total
cell protein in minimal medium
+ - + - + -
+ - +57 94 29 37 mg/L secreted CBHI
97
66
45
30 EndoH
0
1
2
3
4
5
6
7
8
% A
vic
el
Hyd
roly
sis
28
Screen CBH1 for high level expression
Cellulose binding domain
Attachment of CBD
• Best CBM ± 2x improvement in activity
• ± 2x decrease in protein level
EndoH
- + - + - +97
66
45
30
0
1
2
3
4
5
6
7
8
9
10
% A
vic
el H
yd
roly
sis
29
Screen CBH2 for high level expression
Enzyme activity:• 48 hour Avicel
hydrolysis • Best enzyme x2
greater than starting point
Enzyme Production:• 140 mg/L CBH2• ± 5% of Total cell
protein in minimal medium
• x3 higher than starting point
97
66
45
30
+ - + - + - + - +
EndoH
0
1
2
3
4
5
6
7
8
% A
vic
el
Hyd
roly
sis
30
Combinations of CBHs
Best combination x2.5 greater than single enzyme
0
2
4
6
8
10
12
14
16
18
20
% A
vic
el H
ydro
lysis
0
10
20
30
40
50
0
10
20
30
40
50
Prot
ein Expr
ess
ion
(mg/
g DCW
)
CBH2
expressed
(Reinikainen
et al., 1992)
March
2007
March
2008
Oct.
2008
Mascoma Cellulolytic Yeast
31
Cellulase expression Time-line
Dec.
2008
Cellulase expression in Mascoma Yeast(robust C5/C6 fermenting)
Improved CBH1;
~400X ↑
Improved CBH2;
~1500X ↑
Improved host and culture conditions; ~2500X ↑
32
Background strain
Appearance at 120 hrs.
CBP strain
0
10
20
30
40
50
60
0 50 100
Eth
ano
l co
ncent
ration
, g/
L
Time (hours)
CBP + 1 mg Xylanase
Background +BGL + Xylanase
Background + Cellulase + BGL + Xylanase
Mascoma CBP technology on 18% w/w paper sludge
Enzyme Reduction on Paper Sludge
Enzyme Reduction on Hardwood
Equivalent performance with 2.5-fold less added enzyme