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Fermentation of Sugars from HotWater Wood Extracts to Ethanol
Jian Xu and Shijie Liu
Biorefinery Research Institute
Department of Paper and Bioprocess Engineering
SUNY College of Environmental Science and Forestry
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The rest of the team
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Energy
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Thomas E. Amidon
Jennifer Putnam
Raymond Appleby
Alan Shupe
Yang Wang
Ruofei Hu
Gangesh Mishra
￭ Gary M. Scott
￭ Kathryn Gratien
￭ Christopher D. Wood
￭ Mitchell Graves
￭ Tingjun Liu
￭ Lin Lu
￭ Nourredine Abdoulmine
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Outline
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Introduction
Xylose Fermentation
Hot-Water Wood Extracts
Hydrolysis and Hydrolysate
Fermentative Microorganism Adaptation
Conclusion
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CO2
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Introduction
Energy
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Chemical components of wood
wood
21% hardwoods
Lignin
extractives
2-8%
25% softwoods
carbohydrates
35% hardwoods
hemicellulose
25% softwoods
cellulose
45%
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Lignin
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Energy
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Three precursors
a. trans-Coniferyl
alcohol
Guaiacyl
b. trans-Sinapyl
alcohol
Syringyl
c. trans-p-Coumaryl
alcohol
p-Hydroxyphenyl
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Hemicellulose
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H2O
Energy
(CH2O)n
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Common among angiosperm woody biomass:
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Hemicellulose
Type
1
2
3
CO2
H2O
Energy
(CH2O)n
Soft
wood
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Hard
wood
5 ~ 8%
0
10~15%
0
0
2~5
7 ~ 10%
Trace
Trace
15~30%
4
5
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Cellulose
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Ethanol Production
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Beverages production
Well established industrial process
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80% by fermentation
Sugar cost constitutes upto 70% production
cost
Cane, corn, wheat, rice, …
Hexoses or 6-carbon sugars
Long history
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Ethanol Production
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Fermentative organism:
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S. cerevisiae commonly used yeast
Zymomonas mobilis a fast fermenter
r E. coli very efficient ethanol producer
Stoichiometry:
C6H12O6 Æ2C2H5OH + 2 CO2
Yield:
Ethanol, “biomas”, by-products
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Energy
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Ethanol
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Fuel – energy value ~65.5% of gasoline
Fuel additive – oxygenate, octane No. 106
Ethanol fermentation
Batch: 30-40 hours for 95% conversion
Continuous: as low as 10 hours for 95%
conversion
Feed: ~ 100 g/L glucose
Ethanol concentration: ~ 5%
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Ethanol fermentation
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Energy
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Nutrients
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NH3, or NH4Cl
KH2PO4
MgSO4
CaCl2
Yeast extract
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Energy
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Ethanol Production Cost
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Brazil: Ethanol Production Costs, 2004
70.00
60.00
2.00
50.00
40.00
1.50
Cents per Gallon
30.00
1.00
20.00
10.00
0.50
Net Corn
Other Variable
Depreciation
Return
Natural Gas
Fixed w/o Depr
Income Tax
Based on: USDA’s 2002 Ethanol Cost-of-Production Survey,
Agricultural Economic Report Number 841, July 2005
Cane production
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Cane processing
Total
Storage
Others costs
Maintenance
Transport
Management
Electricity
Inputs
Labor
Total
Other costs
Rent
Management
Dry Mill
Chemicals
0.00
Harvesting
0.00
Planting
Production Costs, $/gal
US: Ethanol Production Costs, 2007
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Energy
Total
costs
Brazilian Mills are:
„ Integrated with Plantation
„ Co-produce Sugar
Brazil is Current Low Cost
Producer
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Xylose fermentation
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Energy
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5-carbon sugars resources are only second
to 6-carbon sugars, from biomass
Upto 1/3 as abundant as 6-carbon sugars
Generic fermenters not as efficient as for
glucose
Recombinant fermenters:
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rEscherichia Coli
rSaccharomyces cerevisae
rZymomonas mobilis
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Xylose fermentation
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Present state
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Efficient fermentation
Obstacle: inhibitors
Development continues
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More efficient fermenter
Toxin tolerance
Feed stock purification
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ESF Biorefinery:
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Hot-Water Extraction
Hydrolysis
Fractionation
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Acetic Acid
Methanol
Reducing Sugars
Aromatics, Furfurals
Xylan
Fermentation to Ethanol, Plastics, …
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Experimental Set-up: Extraction
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Energy
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Maple Wood Extract
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50
100
150
200
Acetyl
Aromatics
Furfurals
10
1
0.1
40
Starting conditions:
369.20 g OD Maple Woodchips
3024.04 g water, 28°C
20
HAc
30
20
MeOH
10
10
Sugars
0
0
50
100
time, min.
150
200
0
Acetic Acid, mM
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100
Concentration, mM
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Acetic Acid,
Methanol
Acetyl,
Polysaccharides
Aromatics,
Furfurals
Monomeric
Sugars
(CH2O)n
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time, min.
0
Methanol, mM
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CO2
H2O
Energy
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CO2
H2O
Wood
Extracts
Unbleached
Pulp
Pulping
Chemicals
Hydrolysis/
Saccharification
Separation / Hydrolysis
Xylan
Methanol
Acetic
Acid
Paper,
Board, or
Cellulose
Products
Bleached
Pulp
Black
Liquor
Separation/Co-generation
Carbohydrates
Sugars
Aromatics
Bleaching
Residual
Chips
(CH2O)n
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Alkaline Pulping
or Oxidation
Hot-Water Extraction
Woodchips
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Energy
Energy
Lignin
Fermentation
Ethanol
Butanol
Bioplastics
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Hydrolysis
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Dissolved carbohydrates and lignin in liquid
Monomeric sugars (~1/3) and polysaccharides
Hydrolyzate
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(CH2O)n
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Extract or Dissolved woody biomass solution
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CO2
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Energy
Monomeric sugars (> 80% of all carbohydrates)
Hydrolysis
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Hydrolysis
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Depolymerize macromolecules (of
carbohydrates) by inserting water
molecules between the monomeric units
Enzymatic hydrolysis
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Using a hydrolytic enzyme as catalyst
Acid Hydrolysis
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Using acid (proton) as catalyst
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Concentration and Wash
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Energy
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Wood Extract Liquor:
„ Dissolved solids - < 5%
„ Acetic acid, formic acid, methanol,…
Concentration:
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Increase dissolved solids content
Wash off small molecules: acetic acid, formic
acid, methanol, furfural, …
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Schematic Diagram
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P
Permeate
Holding
Tank
Feed and
Concentrate
Tank
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Reducing sugars as xylose:
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Energy
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200
Starting point
First pass 23.4 g/L
Second pass –
18.2 g/L
Third pass –
16.0 g/L
Reducing Sugars, g/L
1
2
150
3
100
50
0
1
2
3
4
5
6
7
8
V0/V
9
10
11
12
13
14
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Concentration of wood extracts
CO2
H2O
Energy
Time, minutes
0
60
120
180
240
300
360
Xylose
Total Monomeric Sugars
10
5
Concentrate Stream
0
0.00
0.02
Permeate Stream
0.04
0.06
0.08
0.10
0
60
120
180
Time, Minutes
240
300
360
C, g/L
C, g/L
15
(CH2O)n
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Toxins during concentration
1.6
1.4
C, g/L
1.2
Methanol
HMF
Furfural
Formic Acid
1.0
0.8
0.6
0.4
0.2
0.0
1
2
3
4
5
6
V0/V
7
8
9
10
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H2O
Energy
(CH2O)n
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Detoxification: fresh extract
CO2
H2O
Energy
(CH2O)n
active charcoal:Extract = 1:10 g/ml
0.5
Acetyl
Xylose
Acetate
0.4
0.3
0.2
HMF
0.1
0
0
6 12 18 24 30 36 42 48 54 60 66 72 78
detoxification time, min
Furfural and HMF
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Furfural
HAc and Xylose, g/L
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Detoxification
CO2
H2O
Energy
(CH2O)n
4.5
active charcoal:Extract = 1:20 g/ml
Acetyl
3.5
Xylose
3
2.5
Acetate
2
Furfural
HAc and Xylose, g/L
4
1.5
1
0.5
HMF
0
0
6 12 18 24 30 36 42 48 54 60 66 72 78
detoxification time, min
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Furfural and HMF
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Adaptation of E. Coli fbr5
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Energy
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Acetic
acid
methanol
furfural
inoculum
xylose
arabinose
HMF
10
round 1a
2.66
0.21
0.18
10
0.35
0.023
round 2b
3.73
0.29
0.26
10
0.49
0.033
round 3c
5.33
0.41
0.37
10
0.70
0.047
a
: dilution of wood extract to a concentration of 25% (v/v);
b : dilution of wood extract to a concentration of 35% (v/v);
c : dilution of wood extract to a concentration of 50% (v/v)
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Without pH control
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H2O
Energy
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Ethanol concentration, g/L
2.50
2.00
1.50
1.00
0.50
0.00
0
20
40
60
80
Fermentation time, h
100
120
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With 4mL buffer pH control
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H2O
Energy
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Ethanol concentration, g/L
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0
20
40
60
80
Fermentation time, h
100
120
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Ethanol concentration, g/L
With 8mL buffer pH control
CO2
H2O
Energy
(CH2O)n
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8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0
20
40
60
80
Fermentation time, h
100
120
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Ethanol concentration, g/L
With 12mL buffer pH control
CO2
H2O
Energy
(CH2O)n
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7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0
20
40
60
80
Fermentation time, h
100
120
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Ethanol concentration (g/L)
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60
Energy
50
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40
A
B
C
D
CO2
H2O
(CH2O)n
30
20
10
0
8
10
12
16
Inoculum (%)
Ethanol productivity of FBR5 under different operation conditions (g/L)
A: agitation with pH control
Fermentation condition: 35ºC, 150rpm,
B: agitation without pH control
Overnight seed culture (OD600=2.127
1.06x109 cells/mL), 5 days, 100 g/L xylose
C: still with pH control
D: still without pH control
Note: pH control obtained by adding 5 ml NaH2PO4-Na2HPO4 (0.2 M, pH 6.8)
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CO2
H2O
OD600
1.8
Energy
1.5
0
1.2
A
0.9
B
0.6
C
0.3
D
0
8
10
12
original inoculum quantity (%)
16
cells grow th during the fermentation process
0: original OD600 in the medium
A: OD600 after 5 days fermentation under agitation with pH control
B: OD600 after 5 days fermentation under agitation without pH control
C: OD600 after 5 days fermentation under still with pH control
D: OD600 after 5 days fermentation under still without pH control
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Pichia Stipitis
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Pichia Stiptis
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OD600
1.1
1.05
E
1
0.95
0.9
0.85
F
0.8
8
12
16
Original inoculum quantity (%)
cells concentration after 5 days fermentation
E: OD600 after 5 days still fermentation
F: OD600 after 5 days agitated fermentation
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Conclusions
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Fermentation of wood extract sugars
is ready for adoption
Further developments needed in
Adaptation of fermenters to hydrolysate
economically produced today
Inhibitor removal
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Thanks!
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