1. CAFI 2 Project Update NREL and Neoterics Int’l. Rick Elander and Tim Eggeman March 16, 2006

2. 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

3. 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% SO 2 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.

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

5. Screening Digests w/ New GCI Hemicellulase Preps 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: C onversions are slightly lower than usual since dried material was used for these screening digestions

6. Digest of AARP Solids w/ GCI BG 1000 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 50 o C

7. 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

8. 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

11. Pretreatment Options Ideal None Dilute Acid SO 2 Hot Water ARP AFEX Lime

12. Current vs. Goal Current: Laboratory Performance –Pretreatment –Conditioning –Enzymatic Hydrolysis – Fermentation Goal: Likely Performance of Above Steps at Commercial Scale

13. Expected MESP Ranges StoverPoplar Current$2.00-4.00 Goal$1.00-1.50