Improvements in Enzymes for Cellulosic Ethanol

Improvements in Enzymes for
Cellulosic Ethanol
Mian Li
Biomass Applications
Group
Danisco USA –
Genencor Division
Mian.Li@danisco.com
2009 AACC International Annual Meeting, Sep 13-16, 2009, Baltimore, Maryland
Pre-Meeting Workshops

Today’s Presentation
Background on Genencor Division
Accellerase ® Enzyme for Cellulosic Ethanol
• Accellerase ® 1500
• Accellerase ® XY, Accellerase ® XC, Accellerase ®
BG
• Enzyme Cost Projections
Summary
2

Unfolding the Potential of
Biotechnology
Core
technology/products
Agriculture Sugars
Emerging
opportunities for
innovation
Industrial
Enzymes
Food
Enzymes
Feed
Enzymes
Performance
Proteins &
Peptides
Biochemicals
Biofuels
Biorefineries

Genencor’s longstanding leadership
in cellulosic biofuels
25+ years of investment and R&D in biofuels
Pre 2000 – Genencor builds the leading production
systems for cellulase enzymes and patents key genes
required for economic biomass degradation
2004 - Genencor completes 4-year contract with NREL,
achieves 30-fold reduction in cost to convert biomass to
fermentable sugars
2007 - Launched first commercially available enzyme for
cellulosic ethanol - Accellerase ® 1000
February 2008 – DOE awarded Genencor 2nd round of
funding for cellulosic ethanol enzyme improvement
May 2008 – Genencor and DuPont enter 50/50 joint
venture: DuPont Danisco Cellulosic Ethanol LLC (DDCE)
February 2009 – Launched Accellerase ® 1500
April 2009 – Launched three Accessory Accellerase ®
Products, Accellerase ® XY, Accellerase ® XC and
Accellerase ® BG
4

Extensive feedstock / pretreatment
experience
Genencor is engaged in leading
academic / industrial consortia
pushing the boundaries of developing
cellulosic technology
CAFI 1 / 2 / 3
Corn-stover / Poplar / Switchgrass
pretreatment comparison by
leading academics
AFEX, Dilute Acid, Hot Water, Lime,
Sulfur Dioxide, Aqueous
Ammonia, etc…
Midwest Consortium
DDGS/Wet Cake, Grain Fiber
conversion
French Pulp & Paper consortium
Recycled fibers, softwood and
hardwood pulp
Wheat Straw, Sugarcane Bagasse
and More!
DDGS
Wheat Straw
Corn stover
Poplar
5

Genencor’s Biorefinery Strategy
1. Advanced Enzymes for Ethanol, Biochemicals or Biofuels from Biomass
2. DDCE Joint Venture:
Integrated Solution for
Cellulosic Ethanol
+
Dupont
Dupont Danisco
Cellulosic Ethanol
®
3. BioIsoprene ® : Biochemical
and Biofuel Production Platform
Synthetic rubber, specialty
elastomers, adhesives,
C10 Gasoline, Jet Fuel

Required operations for cellulosic
ethanol process
Agricultural
Inputs
Plant
Feedstocks
Conversion
to Sugars
Fermentation
Processes
Milling Pretreatment Saccharification Fermentation Separation
Biofuels
Biochemicals
7

Accellerase ® 1500
Accellerase ® 1500 designed to replace
Accellerase ® 1000
Strengthens Genencor’s Leadership in
Biomass Hydrolysis Enzymes
• Complex of many important activities
necessary for biomass hydrolysis.
• Provides nutrients to the yeast or other
ethanologen to support ethanol production
• Lower cost in operation than Accellerase ®
1000.
• Even higher β-glucosidase activity drives more
complete conversion to glucose.
• Improved formulation
Delivers Significantly Enhanced
Performance
8

Accellerase ® 1500 vs Accellerase ®
1000 Saccharification
Glucan Conversion (%)
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
> 75% conversion a full day earlier
0 20 40 60 80 100 120 140
Time (hr)
APB, Accellerase 1500
APB, Accellerase 1000
% Glucan conversion of a washed acid-pretreated sugarcane bagasse (APB)
using ACCELLERASE ® 1500 and ACCELLERASE ® 1000
at 0.24 mL/g cellulose, 7% cellulose loading, 50˚C, and pH 5.0.
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Saccharification—Glucan Conversion
Effect of Enzyme Loading
Glucan Conversion (%)
120%
100%
80%
60%
40%
20%
Pretreated Softwood Pulp
0.24 mL/g celllulose
0.96 mL/g cellulose
0%
0 10 20 30 40 50
Time (hr)
60 70 80 90 100
Acid-pretreated Sugarcane Bagasse
0%
0 10 20 30 40 50
Time (hr)
60 70 80 90 100
% Glucan conversion of a pretreated softwood pulp**, and a washed
acid-pretreated sugarcane bagasse (APB) using ACCELLERASE ® 1500
at 0.24 and 0.96 mL/g cellulose, 7% cellulose loading, 50˚C, and pH 5.0.
Glucan Conversion (%)
120%
100%
80%
60%
40%
20%
0.24 mL/g celllulose
0.96 mL/g cellulose
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Accellerase ® 1500 performance in
SSF mid to high solids loading
% Theoretical Ethanol Ethanol (g/L) Yield
60 80.0%
70.0%
50
60.0%
40
50.0%
30
40.0%
20 30.0%
20.0%
10
10.0%
0
0.0%
Day 2 Day 3 Day 4
Day 2 Day 3
Time
Time
Day 4
7% cellulose
7% cellulose
13% cellulose
13% cellulose
Simultaneous saccharification and fermentation of washed acid-pretreated
sugarcane bagasse using ACCELLERASE ® 1500 at 0.24 mL/g cellulose, 7%
and 13% cellulose loading, pH 5.0, and 38°C with THERMOSACC ® DRY yeast
(Milwaukee, WI).
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Accellerase ® 1500 performance in
SSF mid to high solids loading
Concentration (g/L)
80
70
60
50
40
30
20
10
0
0 20 40 60 80 100 120 140 160
Time (hr)
Fed-batch simultaneous saccharification and fermentation of pretreated
softwood pulp** using ACCELLERASE ® 1500 at 0.24 mL/g cellulose, 16%
cellulose loading, pH 5.0, and 36°C with THERMOSACC ® DRY yeast
(Milwaukee, WI). (♦-Ethanol ■-Glucose)
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Three new Accellerase ® enzyme products for
cellulosic ethanol process development
• Accessory enzyme products for small-scale process
development that enhance biomass hydrolysis and
ethanol production
• ACCELLERASE ® XY: Accessory xylanase enzyme
complex
• ACCELLERASE ® XC: Accessory xylanase/cellulase
enzyme complex
• ACCELLERASE ® BG: Accessory beta-glucosidase
enzyme

Enzyme Activities Important in
Biomass Hydrolysis

Accellerase ® XY—Accessory
xylanase enzyme complex
% Xylan conversion for ammonia fiber-expansion (AFEX) corn
stover and steam expanded corn stover

Accellerase ® XC—Accessory
xylanase/cellulase enzyme complex
% Glucan and Xylan conversion for ammonia fiberexpansion
(AFEX) corn stover

Accellerase ® BG—Accessory betaglucosidase
enzyme
Simultaneous saccharification and fermentation (SSF) of a
pretreated softwood pulp** at 7% cellulose loading, pH 5.0
and 38°C with THERMOSACC® DRY yeast (Milwaukee, WI)

Enzyme Cost in Use – A Historical
Perspective from the Starch Ethanol
Industry
Outset - 1985 Maturity - 2009
Plant scale (MGPY) 10-20 60-110
Solids 25-28% 32-35%
Enzymes
- Liquefaction wild type B.
licheniformis
engineered proteins /
advanced hosts
- Saccharification wild type A. niger engineered proteins /
advanced hosts
Enzyme cost in use $0.08 to $0.12 per gallon
of ethanol
- Liquefaction Rel Cost 100 65
- Saccharification Rel Cost 100 25
$0.025 to $0.040 per
gallon of ethanol
Yeast Modest ethanol tolerance tolerant up to 18% v/v
Purification Azeotropic distillation molecular sieves
Heat and Water Highly integrated

$/ Gal Ethanol
Enzyme Cost in Use – Cellulosic
Ethanol
3.00
2.50
2.00
1.50
1.00
0.50
0.00
1.00
Pilot and Demo Demo +
0.50
0.35 0.30
2009 2010 2011 2012
Enzyme Cost of Capital Feedstock Production Remaining Cost

Future improvements for enzyme
efficacy driven by active RnD today!
Reduce product inhibition of enzymes
Increase ethanol tolerance of enzymes
Increase thermo tolerance of enzyme suite
Increase hemicellulase activity
Reduce ineffective binding/ interaction with substrate
Increase specific activity of enzymes
= Improved Process Economics
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Summary
ACCELLERASE ® 1500 provides a new benchmark for process
developers and plant designers.
ACCELLERASE ® XY, XC, and BG are accessory enzyme
products for small-scale process development that will enhance
biomass hydrolysis and ethanol production.
These enzymes offer a wide range of activities and synergies
that allow for greater flexibility with multiple different
feedstocks and pretreatment technologies.
Genencor is actively working on future generations of
Accellerase ® products.
Cost of enzymes has already come a long way, and is projected
to improve significantly in coming years.
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Acknowledgements:
Genencor Biomass Team
Biomass Applications Group
Biomass R&D Teams
Manufacturing Plants
Biorefinery Business Group
** Some research on pulp and paper feedstocks funded by
Agence Nationale de la Recherche, France (ANR-05-BIOE-
007)
www.accellerase.com
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