CAFI 2 Project Update

NREL and Neoterics Int’l.

Rick Elander and Tim Eggeman

March 16, 2006

Presentation Outline

• NREL Update

– Enzymatic digestion activities using “progressively” pretreated corn stover obtained via NREL CAFI subcontracts

• Neoterics Int’l. Update

– Process economic modeling strategy

Compositional Ranges of CAFI 2 Corn Stover Sets

Pretreatment Source Glucan Xylan Lignin_____

AFEX MSU 37.3% (avg.) 21.4% (avg.) 15.1% (avg.)

AARP Auburn 59.8 to 68.3% 22.3 to 14.2% 10.8 to 8.7%

SO2 UBC 55.5 to 60.8% 18.3 to 5.1% 19.6 to 26.3%

Dilute Acid Dartmouth ~ 64.0% (all) ~ 1.3% (all) ~ 25.0% (all)

Water/Batch Dartmouth ~ 64.0% (all) 5.6 to 3.0% 19.8 to 22.3%

Water/Flow Thr Dartmouth 72.7 to 75.8% 4.6 to 3.5% 14.4 to 12.9%

Hot Water Purdue 47.5 to 57.4% 23.3 to 6.9% 18.5 to 24.6%

Lime TA&M 38.5 to 50.8% 16.6 to 23.2% 10.8 to 15.9% ** Mass closures were poor for this set; the lime present in the biomass appears to cause problems with the 2-stage H2SO4 hydrolysis

The composition of all samples was analyzed according to the NREL LAP for Determining Structural Carbohydrates & Lignin in Biomass. Data related to 5/6 carbon repeating units, lignin and ash were documented. Ranges below are generally noted from lower to higher pretreatment severity.

Sept. 2005 D Milestone – Examples of Digestions w/ CAFI 2 Materials

Baseline Digestions of CAFI 2 Samples w/ Spzyme CP High 125mg/g cellulose Low 15mg/g cellulose Low +XynA 3mg/g cel

Source Pretreatment Conditions glucan xylan glucan xylan glucan xylan

Auburn 175C, 30min, 6.66g NH3/g stover, 19.8% Solids 97.6 83.1 94.4 76.6 94.9 78.4Auburn 170C, 30min, 3.33g NH3/g stover, 19.8% Solids 96.3 82.6 94.1 77.3 92.5 78.0Auburn 160C, 30min, 3.33g NH3/g stover, 19.8% Solids 89.7 75.9 87.6 70.8 93.6 78.8Auburn 150C, 30min, 3.33g NH3/g stover, 19.8% Solids 84.6 70.0 75.2 59.6 82.3 66.8British Columbia 190C, 5 min, 0% SO2, 20% Solids 85.9 51.8 73.0 43.3 79.7 48.7British Columbia 170 C, 9 min, 3% SO2, 20% Solids 67.4 57.8 61.1 52.2 60.5 53.4British Columbia 190 C, 5 min, 3% SO2, 20% Solids 86.4 67.2 77.8 59.9 77.9 60.3British Columbia 210 C, 7.8 minutes, 3% SO2, 20% Solids 91.3 66.2 90.4 64.5 88.8 64.3Dartmouth College 160 C, 12 minutes, 0.5% H2SO4, 5% solids (batch) 91.6 77.0 72.9 59.5 73.9 59.8Dartmouth College 160 C, 16 minutes, 0.5% H2SO4, 5% solids (batch) 98.0 83.9 76.2 53.4 75.9 56.1Dartmouth College 160 C, 20 minutes, 0.5% H2SO4, 5% solids (batch) 95.0 92.7 75.2 64.0 72.9 63.7Dartmouth College 200 C, 12 minutes, 0% H2SO4, 5% solids (batch) 97.2 75.6 93.5 71.7 94.2 73.8Dartmouth College 200 C, 16 minutes, 0% H2SO4, 5% solids (batch) 96.5 82.0 91.1 75.9 91.8 77.6Dartmouth College 200 C, 20 minutes, 0% H2SO4, 5% solids (batch) 99.2 84.4 89.2 73.0 92.8 78.0

• Baseline digestions with Spezyme CP were run on samples received prior to Sept 2005

• High (125mg/g) and low (15mg/g) Spezyme loadings w/ excess beta-glucosidase

• Low (15mg/g) Spezyme load coupled with a single component xylanase* (3mg/g)

*(purified from a Sigma T. lanuginosus xylanase preparation)

Screening Digests w/ New GCI Hemicellulase Preps

120hr Digestions of Selected CAFI Samples w/ New GCI Enzyme Preparations - Glucan Conversions

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AARP 160C, 30m SO2 190C, 5m 0.5% H2SO4 160C, 12m

% G

luca

n C

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ion

Spezyme CP (25)

Sp CP (15) MF XL (10)

Sp CP (15) MF A40 (10)

Sp CP(15) BG 1000 (10)

Sp CP (15) Optimash BG (10)

Sp CP (15) Optimash XL (10)

120hr Digestions of Selected CAFI Samples w/ New GCI Enzyme Preparations - Xylan Conversions

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30.00

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AARP 160C, 30m SO2 190C, 5m 0.5% H2SO4 160C, 12m

% X

ylan

Co

nve

rsio

n

Spezyme CP (25)

Sp CP (15) MF XL (10)

Sp CP (15) MF A40 (10)

Sp CP(15) BG 1000 (10)

Sp CP (15) Optimash BG (10)

Sp CP (15) Optimash XL (10)

• Selected CAFI Samples

• Washed/dried for quick digestion set-up

• Spezyme CP at 25mg/g cellulose vs. Spezyme CP at 15mg/g + 10mg/g of each GCI prep

GCI Hemicellulase Preparations:

• Multifect XL - endoxylanase

• Multifect A40 – cellulase/hemicellulase w/ low levels of xylanase & mannanse

• BG 1000 – beta-glucanase

• Optimash BG – beta-glucanase

• Optimash XL – beta-glucanase/xylanase* Note: Conversions are slightly lower than usual since dried material was used for these screening digestions

Digest of AARP Solids w/ GCI BG 1000

Digestion of AARP Pretreated Corn Stover w/ Spezyme CP & BG 1000:

Glucan Conversion After 120hrs at 50oC

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AARP#1 AARP#2 AARP#3 AARP#4

% G

luca

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Spez(25)

Spez(15) BG 1000 (10)

Digestion of AARP Pretreated Corn Stover w/ Spezyme CP & BG 1000:

Xylan Conversion After 120hrs at 50oC

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AARP#1 AARP#2 AARP#3 AARP#4

% C

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ion

Spez(25)

Spez(15) BG 1000 (10)

BG 1000:• Primary Component is an endo-1, 3(4) beta-glucanase• G. emersonii• 10 Units/mg; 34 mg/ml

Digestions:• Washed/Non-dried Solids• Numbered most severe (#1) to least severe (#4)• Load 1: 25mg of Spezyme CP per gram of cellulose• Load 2: Spezyme CP at 15mg/g & BG1000 at 10mg/g• 100mg cellulose per 10ml digestion; incubated at 50oC

Current Work with CAFI 2 Substrates• Moving towards exploring single component accessory enzymes vs. multi-component commercial enzyme preparations

• Working “in-house” to produce/purify some components

• Working with industry to obtain others

• Currently have genes for six individual components

• Have recently produced and purified small batches of two enzymes

• Over the next few months we will begin to increase production and test these enzymes on a number of the CAFI corn stover and newly received poplar substrates

Model Basis• Ethanologenic Fermentation of Enzymatic

Hydrolyzates w/ Boiler + Power Export

• 2000 Tonnes/day (dry)

• Hydrolysis & Fermentation Performance to Vary with Feedstock and Pretreatment

• 2002 NREL Design Model - Aspen GUI w/ Hierarchy

Pretreatment Options

IdealNoneDilute AcidSO2

Hot WaterARPAFEXLime

Current vs. Goal

• Current: Laboratory Performance– Pretreatment– Conditioning– Enzymatic Hydrolysis– Fermentation

• Goal: Likely Performance of Above Steps at Commercial Scale

Expected MESP Ranges

Stover Poplar

Current $2.00-4.00 $2.00-4.00

Goal $1.00-1.50 $1.00-1.50