high efficiency production of xylitol from hemicellulose ......xylitol process optimization 0 20 40...
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High Efficiency Production of Xylitol from Hemicellulose by Fermentation
David Demirjian, Ph.D. President & CEO
World Congress of Industrial Biotechnology June 17, 2013
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Corporate Overview
Biotech R&D Group
Enabling Carbohydrate Technology
Platform of Proprietary Process Technologies and Know-How
Focus:
Food Ingredients/Specialty Chemicals
Fine Chemicals/Pharma
Biotech Company
Chicago - Headquarters
Peoria - Main Research Facility
Key Collaborators
U.S. Department of Agriculture National Center for Agriculture Utilization Research (NCAUR)
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Contributors
zuChem F. Mike Racine, Ph.D.
Ryan Woodyer, Ph.D
Paul Taylor, Ph.D.
Nathan Wymer, Ph.D.
Shama Khan
Trevor Christ
USDA - NCAUR Badal Saha, Ph.D.
Yoshikiyo Sakaibura, Ph.D.
Douglas Antibus, Ph.D.
Greg Kennedy
University of Illinois
Huimin Zhao, Ph.D.
Ryan Sullivan, Ph.D.
Nick Nair, Ph.D.
Other Collaborators/Consultants
Ian Fotheringham, Ph.D. – Ingenza Ltd.
David Dodds, Dodds & Associates
Bill Dowd, Ph.D. – fmr VP R&D Dow
David Ager, Ph.D. – Competence Mgr DSM
Funding
Biotechnology Research and Development Corporation a
U.S. Department of Energy – Biomass Program
National Science Foundation
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INPUT
Inexpensive Biomass Feedstock
Ethanol
Value Added Chemicals
e.g. Xylitol >>$3000/ton
DDG, Fiber Bagasse
~$300/ton
OUTPUT
BY-PRODUCTS
Improving Economics of Ethanol Production
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Polyol Market
$80
$370
$750
$125
Sorbitol
Xylitol - 3X growth projected
Mannitol
Erythritol and Others
Specialty Sweeteners
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Xylitol Current Chemical Process
Purified Xylose expensive
limited supply
zuChem Bioprocess
Chemical Hydrogenation
Xylitol Production
Polyol contaminants &
purification cost
driven by xylose purity
Hemicellulose inexpensive
abundant supply
Mixed C5/C6 is OK
Fermentation
Xylitol
No polyol contaminants
Simple purification
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Challenges and Process Goals
• Little or no arabitol produced
• Overcome Problems with Fermentation Inhibition
– Furfurals and other inhibitors
• Abundant Feedstock Supply
• Tolerant to Feedstock Variability
• Process Economics
– Yield – 100 g/l
– Purity – essentially free of sugar and polyol contaminants
– Throughput - >3 g/L-h
– Recovery - >85%
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Hemicellulose C5/C6 Composition
D-Xylose L-Arabinose D-Glucose D-Galactose Other
Birch Wood 89.3% 1% 1.4% - 8.3%
Rice Bran 46% 44.9% 1.9% 6.1% 1.1%
Wheat
Straw 65.8% 33.5% 0.3% 0.1% 0.1%
Corn Fiber 48-54% 33-35% - 5-11% 3-6%
Sugar Cane
Bagasse 80-90% 5-10% 0-5% 0-2% 0-5%
Saha, B., J. Ind. Microbiol. Biotech. (2003) 30: 279-91,
unpublished observations
C5 C6
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Conversion of Xylose to Xylitol
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40
Hour
Conversion of Xylose to Xylitol with XI / XDH
Glucose
D-Xylose
L-Arabinose
Arabitol
Xylitol
0
1
2
3
4
5
6
7
8
0 20 40 60 80
Hour
XR in ara+ host
Xylose
Arabinose
Arabitol
Xylitol
Co
ncen
trati
on
(W
/V)
Co
nc
en
tra
tio
n (
W/V
)
Hours Hours
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Xylose Specific Selection Strain
+ xdh
+ araB
Plate on L-arabinose/D-xylose
No
growth
LETHAL
No
growth
Growth
L-arabitol
+
Xylitol
xylitol D-xylulose
XR - active on L-arabinose and D-xylose
XR’ - D-xylose specific enzyme
araB
xdh
XR
XR’
-XR
D-xylulose
L-arabitol-5-phosphate
xdh
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Xylose Specific Mutants
0
0.2
0.4
0.6
0.8
1
1.2
Pstip XR mut#1 mut#15 mut#16 mut#17
D-x
ylo
se/
L-arab
ino
se r
ate
rati
o
WT MUT1 MUT2 MUT3 MUT4
D-X
ylo
se/L
-Ara
bin
ose
Rat
e R
atio
n
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Conversion of Arabinose to Xylitol
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Testing on Hydrolysate
0
0.5
1
1.5
2
2.5
3
3.5
4
Fiber 1 Fiber 2 Fiber 3 Fiber 3 Purified
Stover Hardwood
Fermentation Test On Unremediated Hydrolysates
O.D. 600
Slow growth on
hydrolysates due
to fermentation
inhibition
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Remediation Tests
Treatment Relative growth
Absorbance 600 nm
NaOH to pH 7 1.8
Ca(OH)2 to pH 7 2.1
Ca(OH)2 to pH 9.05
H2SO4 to pH 7
3.8
Ca(OH)2 to pH 10.5
H2SO4 to pH 7
6.2
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Remediation Comparison
Fermenter results confirmed that over-liming at high pH is best; charcoal also helps
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Xylitol Process Optimization
0
20
40
60
80
100
120
0 10 20 30 40 50
g/L
Su
bstr
ate
or
Pro
du
ct
Hours
Xylitol Production from Hydrolysate
Glucose
Xylose
Arabinose
Arabitol
Xylitol
• Optimum pH (6.8) and temperature (37°C) were determined
• Optimal nutrients identified
• Fed-batch protocol developed
• Air/agitation
requirements were determined
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Met Goals of Process
• Conversion of xylose to xylitol was 98%.
• Volumetric productivity 3.1g /L-h at 32 hours
• Yield was >100 g/L
• Complete conversion of sugars
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Summary • ~8 patents on file in four patent
families - First patents now issued.
• Piloting now underway with first
Manufacturing Partner – Godavari
Biorefineries
• Sampling to Customers Expected this
Summer
• New Manufacturing facility expected
to be operational by end of 2014
• Broad Licensing Strategy
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