Mian LiBiomass Applications GroupGenencorPalo Alto, CAMian.li@danisco.com

Development of Improved Enzyme for Lignocellulosic Biomass Conversion

4th European Bioethanol Technology MeetingDetmold, Germany, April 15-16, 2008

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Today’s Presentation

Current Enzyme Technology for Fuel Ethanol from StarchThe Challenges of Cellulosic Ethanol and Genencor’s RoleFirst Biomass Enzyme-Accellerase™1000 and its applicationConclusions

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CellFactory

Fuels

Chemicals

Polymers

Specialties

Enzym

esGlucose

MixedSugars

Sucrose

Sugar CaneBeet

Starch Grains

Lignocellulose

The BioRefinery

Co-Products

Upgraded Co-Products

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Major Feedstocks in Starch Ethanol Production

Rice Triticale Wheat Rye Barley Corn Sorghum

USEurope

Asia Pacific

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Enzymes for Fuel Ethanol Production from Starch

EnzymeLiquefying enzymes

Saccharifying enzymes

Protein hydrolyzing enzymes

Viscosity reduction enzymes

Granular starch hydrolyzing enzymes

PurposeThermostable alpha-amylases for liquefaction of starch at high temperatures

Glucoamylases and blends for hydrolysis of starch to glucose

Proteases for improved fermentation efficiency

Multi-component enzymes for wheat, barley & rye

Alpha-amylase and glucoamylase blend for processing of uncooked starch

Product nameSPEZYME® XTRASPEZYME® FREDMAXALIQ® ONE

G-ZYME ® 480 FERMENZYME ® L-400

FERMGEN ™

OPTIMASH ™ (wheat, rye and barley)

STARGEN ™ 001STARGEN ™ 002

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BG

PY

gas

olin

eBiofuel Opportunity in US Gasoline Replacement

12-15 BGPY limitfor starch ethanol

Bush SOTU target: 30% (45BGPY)

% Gasoline Replacement.% Gasoline Replacement. (All USA information)

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Sources of BiomassCourtesy of Rod Bothast

Wood chips

Switchgrass Corn stover

Sugarcane

Cottonwoods Paper

Wheat Straw

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Cellulosic ethanol : Enzyme Cellulosic ethanol : Enzyme challengeschallenges

Biomass much more recalcitrant to hydrolysisComplex, synergistic mix of cellulases & hemicellulases neededEnd product inhibition, enzyme stability, non-productive binding…..Enzyme dosing is much higher than for starch

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Hydrolyzing the β-1,4 linkage

Cellulose digestion requires at least 3 types of activities working synergistically:

• Exo-1,4-β-glucanase (cellobiohydrolase): processive, attacks reducing (CBH I) or non-reducing (CBH II) end of cellulose chain, releasing cellobiose units.

• Endo-1,4-β-glucanase: non-processive, attacks along chain; reduce dp and viscosity, creating more chain ends for the exo’s.

• Beta-glucosidase (β-glu): converts soluble oligosaccharides to glucose, relieving product inhibition.

Cellobiohydrolase

Endoglucanase

β-Glucosidase

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2nd Generation Biorefinery Concept and Challenge

Cost of EnzymesStarting ~$5/gal EtOHNeeded < 10c/gal EtOH

Pretreatment:AcidAlkali

MechanicalEtc.,

Fermentation

Product Recovery

LigninUtilization EthanolEthanol

C5 C6 C5/C6Organism Fermenting

EnzymaticCelluloseHydrolysis

Biomass

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Advanced Cellulase Development Programs

Project Structure and Funding• U.S. Department of Energy (DOE) funded >$15 million over

a 4-year period beginning in June 2000• Sub-contract through the National Renewable Energy

Laboratory (NREL)• Genencor contributed ~$5 million

Goal: Improved Low Cost Cellulase for Biomass Conversion to Ethanol

• Original program targeted a 10-fold cellulase cost reduction• Program achieved a 30-fold reduction per gallon on NREL

model substrate

Awards• R&D Magazine, Top 100 Innovations, 2004

2008: Genencor proposal chosen for new DOE Enzyme Improvement subcontract

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How Will the Market Emerge?

2008 to 2010 – pilot plants at 0.5 to ~ 2 MGPY (1,500 MT~3,000 MT)

2009 to 2012 – demonstration plants at 2 to 10 MGPY (6,000~30,000 MT)

2009 to 2014 – first commercial plants deployed @ 20 to 50 MPGY (60,000~ 150,000 MT)

2007 Unmet Need:Process developers & engineers need an enzyme solution that is

representative of final, optimal enzyme preparations so they can design their systems.

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Http://www.genencor.com/cellulosicethanol

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Accellerase™1000 Application

Hydrolysis of different pretreated lignocellulosic biomass

Process Development• Separate Hydrolysis and Fermentation (SHF)• Simultaneous Saccharification and Fermentation (SSF)• Hybrid Saccharification and Fermentation (HSF)--pre-hydrolysis substrate for a period of

time between 4-16 hours at 50 C

Effect of additional nutrient of the unclarified enzyme on fermentation

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Applications Data Across Different Biomass Substrates

Bagasse Corn Stover

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% C

ellu

lose

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estib

ility

Accellerase 1000Spezyme CP

Softwood Pulp

0 1 2 3

Day(s)

Accellerase 1000Spezyme CP

0 1 2 3

Day(s)

Accellerase 1000

Acid pretreated sugar cane bagasse

Acid pretreated corn stover

Softwood pulp (Kappa = 14)

All samples with 7% cellulose loading, 50°C, and pH 5.0

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Significantly Lower Cellobiose Levels During Hydrolysis

Sugar profiles of AccelleraseTM 1000 vs cellulase product Spezyme ® CP on acid pretreated corn stover (▲) and sugarcane bagasse (■), 7% cellulose loading, 50°C, and pH 5.0.

Cellobiose

Glucose

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Suga

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AccelleraseTM 1000Spezyme® CP

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Enhanced SSF Performance

Ethanol profiles of AccelleraseTM 1000 vs. cellulase product Spezyme ® CP in SSF on sugarcane bagasse, 7% cellulose loading, 50°C, and pH 5.0.

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ield

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AccelleraseTM 1000Spezyme® CP

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Time (hr)

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nol (

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acid-pretreated corn stover

acid-pretreated sugarcanebagasseSoftwood pulp

Theoretical Ethanol Yield

SSF of pretreated lignocellulosic biomass

Simultaneous saccharification and fermentation of acid-pretreated corn stover, acid-pretreated sugar cane bagasse, and softwood pulp using Accellerase 1000 at 0.24 mL product /g cellulose at 7% cellulose loading, 38°C, and pH 5.0.

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Effect of solids loading on SSF12.4% DS vs. 26% DS

Simultaneous saccharification and fermentation of acid pretreated bagasse using Accellerase 1000 38°C, and pH 5.0.

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Time (hr)

Etha

nol (

g/L)

7% cellulose13% cellulose

~ 7% V/V

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Comparison of SSF, Hybrid Hydrolysis and Fermentation, and Separate Hydrolysis and Fermentation—12.4% solids loading, 7% cellulose, APB

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4 hr prehydrolysis

8hr prehydrolysis

16 hr prehydrolysis

Separate Hydrolysis andFermentation

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Benefits of an Unclarified Product

Yeast Nutrients• 1 g/L Yeast Extract• 1 g/L Peptone• 1 g/L Urea

Urea supplementation used at full scale ethanol fermentation may be unnecessary!

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AccelleraseTM 1000 + yeast nutrientsAccelleraseTM 1000 - yeast nutrientsClarified AccelleraseTM - yeast nutrients

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Conclusions

Genencor has launched its first cellulase product targeted at process developers for biomass application

• Synergistic effect of multiple enzyme activities including Endo, exoglucanase, β-glucosidase and hemicellulase for broad substrate application

• Ability to minimize residual cellobiose and increase saccharification rate & glucose concentration

• Ability to operate in different process configurations.

• Unclarified product with remaining nutrients from enzyme production available to the yeast in addition to the hydrolysis sugars after saccharification

Further improvements are on-going to meet the demand for• Improved performance• Lower cost

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Genencor Biomass Team

R&D Team:Suzanne LantzEd LarenasBrad KelemenSteve KimNicole ChowMeredith FujdalaBob CaldwellLouise WallaceJasan ZimmermanBecky KongLuis Cascao-PereiraMike PepsinAlisha JarnaginSigrid PaechMegan HisJeff Pucci

Biomass Applications:Aaron Kelley Mian LiTom Huang Ryan WarnerBen Liu John McLaughlinChristina GutierrezColin Mitchinson

Business:Jack HuttnerScott PowerGeorg AnderlBjarne AdamsenLandon SteeleBrian CarterMichael Kolman

Plants:Mika RistimakiHenna LehtolaFrederik VaningelgemJing Yang

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