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

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 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. 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

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: Conversions 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 50oC

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 SO2 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