Enzyme use for corn fuel ethanol production. Luis Alessandro Volpato Mereles

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Enzyme use for corn fuel ethanol production Luis Alessandro Volpato Mereles July 12 th, 2007

Agenda Global Biofuel Outlook Novozymes at a glance What are enzymes Using Enzymes to produce Fuel Ethanol from Grains Slide No. 2 7/23/2007

Growing Demand Global fuel ethanol production has doubled in the past 5 years, and will continue to grow at 20% plus annually through 2010 80 World Fuel Ethanol Production (2002-2010F) 67.1 CAGR (2006-10F) 77.5 Total 22% Others China 29% 7% Europe 53% 60 54.8 Millions of MT 40 35.5 44.5 USA 18% 20 17.4 21.4 22.5 23.4 Brazil 18% 0 2002 03 04 05 06E 07F 08F 09F 10F Source: F.O. Licht, Novozymes Analysis Slide No. 3 7/23/2007

World leader in industrial enzymes Leading-edge biotechnology expertise 600+ products in 40 different industries More than 5,000 patents granted and pending Commitment to innovation 13% of sales reinvested in R&D Products launched within the last 5 years account for 30% of turnover Slide No. 4 7/23/2007

We find the magic of nature in a handful of soil or a compost heap. Then we turn it into solutions for fuel ethanol and other applications. Slide No. 5 7/23/2007

Our commitment to the Ethanol Industry begins with our global efforts and focus Sales Offices Production Research More than 4,500 employees globally Slide No. 6 7/23/2007

Enzymes Are: Functional proteins (as opposed to structural); catalysts Primarily made up of chains of amino acids linked together by peptide bonds. Found in all living organisms Safe, however, good chemical hygiene is always recommended. Work under mild conditions Replace harsh chemicals such as strong acids Biologically degradable A clean technology Slide No. 7 7/23/2007

Why & Where Enzymes are Added Decrease Viscosity α-amylase Ammonia Slurry Jet Cooker Liquefaction Produce Dextrins α-amylase Enhance Fermentation Starter Yeast Yeast nutrients Yeast propagator Yeast nutrients Glucoamylase (Protease) Fermentation Produce glucose Slide No. 8 7/23/2007

Liquefaction The cook process Slide No. 9 7/23/2007

Liquefaction Converts large chain amylose and amylopectin to a mixture of smaller chain length dextrins DE generation (Final DE Target 10-12) Tube & Shell Heat Exchanger Cook Tubes Decreases viscosity Lower viscosity increases heat exchanger efficiency and makes the mash easier to pump Slide No. 10 7/23/2007

Smoother Operations using Liquozyme SC DS State of the art enzyme for liquefaction (protein-engineered B. stearothermophilis) Smooth your production with the highest tolerance for process variations Superior thermostability and ph tolerance maintains performance in fluxuating conditions Viscosity reducing properties excellent over a wide ph range (up to ph 6.2) The choice of over more than ¾ s of all operating ethanol plants No impact on production from calcium deficiencies or mash variances Conventional alpha-amylases suffer reduced performance due to weak thermostability and calcium dependency No band-aids required to compensate for stability issues Slide No. 11 7/23/2007

Liquefaction & Cook Liquozyme SC DS randomly cleaves large chain amylose and amylopectin to a mixture of smaller chain-length dextrins Dextrins DP6 DP4 DP3 DP2 GOALS: DE Generation with final target of 10-12 Further viscosity reduction Slide No. 12 7/23/2007

Viscosity Reduction - Demo Mixed Corn Mash + Boiling Water Note Viscosity from starch gelatinization Add 1 drop Liquozyme SC DS Ensure good agitation Note rapid viscosity break Slide No. 13 7/23/2007

Operating Conditions For Single Dose Dry Mill Liquefaction Using Liquozyme SC DS Typical Free Calcium >5ppm * Typical ph Range 5.6 6.0 Slurry Hold Temperature 83-85 C Slurry Hold Time 30-60 min Liq Hold Temperature 83-85 C Liq Hold Time 90-150 min Total Enzyme Dose 0.0155-0.031%w/w *Generally present in water and grain Slide No. 14 7/23/2007

SSF Simultaneous Saccharifaction & Fermentation Yeast Nutrition and Production of Alcohol Slide No. 15 7/23/2007

SSF Simultaneous Saccharification and Fermentation Spirizyme Fuel gluco-amylase generates fermentable sugars in the fermentor at the same time as the yeast is converting the sugar to ethanol. Slide No. 16 7/23/2007

Spirizyme Fuel ensures maximum ethanol production Higher performance than traditional glucoamylases Quicker glucose production Lower maltose levels Lower DP3 levels Concentrated formulation Reduced volume of enzyme dosage by 20 30% Less ordering and handling Greater thermostability Robust performance in temperature ranging from 32 C to 70 C Reduced rate of infection in saccharification step Proven performance The most widely used glucoamylase in the world for fuel ethanol production The leading choice for new plants since its introduction Slide No. 17 7/23/2007

Gluco-amylase: Spirizyme Fuel Application Hydrolyzes 1,4 and 1,6-alpha linkages in liquefied starch. During hydrolysis, the amylo-glucosidase activity removes glucose units in a stepwise manner from the non-reducing end of the substrate molecule. *Note: 1,4-alpha linkages are more readily hydrolyzed! α1-4 α1-6 Dextrins Glucose Slide No. 18 7/23/2007

SSF Process Flow Mash from Cook CO2 to Scrubber Mash Coolers Water Yeast Propagator Ferm. #1 Ferm. #2 Ferm. #3 Ferm. #4 Batch Fermentation Spirizyme Fuel is added to the fermenter. Spirizyme Fuel Yeast 31-32 o C ph 3.6-4.0 32-35 o C ph 3.8 4.5 To Beerwell and Distillation Slide No. 19 7/23/2007

Corn = 56 lbs (25.4 kg) Starch = 33.8 lbs (15.3 kg) Sugar = 37.5 lbs (17 kg) 1 Bushel of Corn (56 lbs) Ethanol = 17.9 lbs (2.71 gal ~ 10.27 l) Approx. a 1:1:1 ratio EtOH:CO 2 :DDGS %Efficiency = actual alcohol wt X 100% theoretical alcohol wt Max: ~93% CO 2 = 17.1 lbs (7.8 kg) DDGS = 16.2 lbs (7.3 kg) Heat 6.5 MJ Slide No. 20 7/23/2007

Simplified bio-ethanol production processes 1 st generation corn-based ethanol production Starch e.g. corn Enzyme process Fermentable sugars Fermentation process Fuel ethanol Waste biomass Pretreatment process Cellulose Enzyme process Future 2 nd generation biomass-based ethanol production Slide No. 21 7/23/2007

For more information please access www.novozymes.com www.biomass.novozymes.com Thanks for your attention! Slide No. 22 7/23/2007