Cellulolysis Process Overview Summary found at: en.wikipedia/wiki/Cellulosic_ethanol

1. Cellulolysis Process Overview Summary found at: http://en.wikipedia.org/wiki/Cellulosic_ethanol Harvest Expose Cellulose for Hydrolysis Break down hemicellulose Cellulose to Sugars Convert Sugars to alcohol Separate alcohol from water Chopping or Baling Chemical Hydrolysis Storage Pretreatment OR OR Fermentation Distillation Enzymatic Hydrolysis Combined Storage-Pretreatment (silos, piles, agribags, dry storage) Combined Hydrolysis & Fermentation

2. Cellulolysis Pretreatment Explanations • 1) Acid Hydrolysis • 2) Dilute Acid - Steam Explosion • -feedstock is impregnated with acid (H2S04 – sulfuric acid) H2SO4 is used because of its low cost • - feedstock is then processed in a steam explosion reactor • time in the reactor (residence time) and temperature levels of the reactor seem to determine the ethanol yield • down the line • - A good sample experiment of this using corn stover is at: http://www.nrel.gov/docs/gen/fy03/32119.pdf • 2b – How is sulfuric acid made – answer below from http://wiki.answers.com/Q/How_is_sulfuric_acid_made • There are two main ways: • 1. Step 1:- Sulfur is burned to produce sulfur dioxide. = S(solid) + O2(gas) → SO2(gas) • Step 2:- This is then oxidised to sulfur trioxide using oxygen in the presence of a vanadium(V) oxide catalyst. = 2 SO2 + O2(g) → 2 SO3(g) (in presence of V2O5) • Step 3.a:-The sulfur trioxide is treated with water (usually as 97-98% H2SO4 containing 2-3% water) to produce 98-99% sulfuric acid. =SO3(g) + H2O(i.e. water) → H2SO4(liquid) Hey Presto! • Or, way 2.The SO3, produced at step 2 above, can be processed in an alternative way. i.e. • Step 3.b. The SO3 can be absorbed into H2SO4 (concentrated sulfuric acid!) to produce oleum (H2S2O7) = H2SO4(l) + SO3 → H2S2O7(l) • Step 4:- Oleum can then be reacted with water to form more concentrated H2SO4. = H2S2O7(l) + H2O → 2 H2SO4(l)

3. Cellulolysis Pretreatment Explanations Continued 3) Ammonia Fiber Expansion – Liquid ammonia is used to pretreat and explode biomass - Ammonia is recycled - Process is run at 60-100°C, 20-80% moisture, and biomas ration is .5 to 1.3-1.0 - see http://www1.eere.energy.gov/biomass/pdfs/34861.pdf - Ammonia Fiber Expansion (AFEX) is a promising pretreatment with no inhibitory effect in resulting hydrolysate (meaning it leaves no impurities that hinder Fermentation) 4) Lime Pretreatment - see http://www1.eere.energy.gov/biomass/pdfs/34861.pdf - Takes 1 to 2 months – looks to be done in a pile or bunker 5) Flowthrough Pretreatment - see http://www1.eere.energy.gov/biomass/pdfs/34861.pdf 6) Controlled PH Pretreatment - see http://www1.eere.energy.gov/biomass/pdfs/34861.pdf 7) Ozone Pretreatment 8) Alkaline Wet Oxidation

4. Cellulolysis Chemical (Acid) Hydrolysis Explanations • Dilute Acid • - process uses more heat and pressure • - sugar degradation is a problem and can lower sugar yield and toxins can be left over that hamper • fermentation • - uses 1% sulfuric acid solution in a continuous flow reactor at 215°C • - Sugar conversion efficiency is 50% • - Two step process, since hemicellulose (5 carbon sugar) degrades faster than 6 carbon sugars (Cellulose) • a) mild conditions to recover 5 carbon sugars • b) harsher process to recover 6 carbon sugars • - both resulting hydrolyzed solutions are then fermented to alcohol. Lime is used to neutralize acids • prior to fermentation, and leftover lignin is used to as boiler fuel or to make steam to produce • electricity • 2) Concentrated Acid • - process uses lower heat and pressure • - uses concentrated sulfuric acid and then dilution with water to dissolve and hydrolyze material into sugar • - process converts cellulose to glucose, and hemicellulose to 5 carbon sugars • - acid is recycled in the process • - Two step process: • a) Hemicellulose hydrolyzation uses a 70% sulfuric acid solution and is hydrolyzed at 100°F for • 2-6 hours in a hemicellulose hydrolysis reactor. Material is then soaked with water and drained • many times to recover the sugars • b) Cellulose hydrolyzation – from step a, the solids are taken and soaked in a 30-40% sulfuric acid • solution for 1-4 hours. Material is then drained and dried, and the acid rate is increased to 70% • and placed in another container for 1-4 hours at low temperatures. The sugar and acid are then • recovered. This acid is then used in step A. • - This process has about a 90% sugar conversion efficiency.

5. Cellulolysis Enzymatic Hydrolysis Explanations Enzymatic Hydrolysis In this process, enzymes are used to hydrolyze the cellulose (C6 Sugar) and hemicellulose (C5 sugars) Trichoderma reesei– produces cellulase enzymes – needed to convert cellulose and hemicellulose to sugars (glucose molecules) Enzyme Producers Iogen - http://www.iogen.ca/ Genecor - http://www.genencor.com/wps/wcm/connect/genencor/genencor Novozymes - http://www.novozymes.com/en Dyadic International Inc - http://www.dyadic.com/wt/home Verenium - http://www.verenium.com/ Direct Microbial Conversion In this process, ethanol and required enzymes are produced from the same microorganism

6. Combined Hydrolysis & Fermentation Some bacteria have been found to convert cellulose directly to ethanol. Examples of these bacteria are: Clostridium thermocellum(C. thermocellum) – this bacterium will convert cellulose directly to ethanol, but has some other byproducts that can reduce efficiency during fermentation

7. Cellulolysis Fermentation Process Explanations Do C5 and C6 sugars need to be fermented separately? Zymomonas mobilis(Z. mobilis) – bacterium that converts sugars to pyruvate, which is then fermented to ethanol and carbon dioxide. NREL has developed a version that leads to more efficient fermentation of both C5 and C6 sugars. Arkenol will be using the NREL version. Saccharomyces cerevisiae – bakers yeast used in brewery industry to produce ethanol from C6 sugars Escherichia coli– (E. coli) uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide

8. Cellulolysis Distillation Process Explanations This process of separating alcohol from water is that same as is used in making corn ethanol – in simplistic terms, the “beer” is heated to the point where the Alcohol (which boils at a lower temperature than water” evaporates up leaving water behind. The alcohol vapor is contained and cooled which turns it back to a liquid, with this liquid being mainly alcohol (some water will/may remain, so to purify the alcohol it may be run thru the distillation system repeatedly until the desired % of alcohol is achieved.

9. Cellulolysis Process Definitions Assistance with definitions found at: http://dictionary.reference.com/ Pretreatment – separation of 4 components of biomass – hemicellulose, cellulose, lignin, and extractives so that they can then be broken down into their sugar components Saccharification - the process of breaking a complex carbohydrate (as starch or cellulose) into simple sugars Hydrolyze – to subject to hydrolysis, which is the breaking down of a chemical compound into two or more simpler compounds by reacting with water. The proteins, fats, and complex carbohydrates in food are broken down in the body by hydrolysis that is catalyzed by enzymes in thedigestive tract Hemicellulose - (5 carbon sugar) - any of a group of gummy polysaccharides, intermediate in complexity between sugar and cellulose, that hydrolyze to monosaccharides more readily than cellulose Cellulose - (6 carbon sugar) - a polysaccharide(C6H10O5) of glucose units that constitutes the chief part of the cell walls of plants, occurs naturally in such fibrous products as cotton andkapok, and is the raw material of many manufactured goods (as paper, rayon, and cellophane) Lignin - A complex polymer, the chief noncarbohydrate constituent of wood, that binds to cellulose fibers and hardens and strengthens the cell walls of plants xylose (C5H10O5)– 5 carbon sugar (C5 sugar)derrived from hemicellulose arabinose – 5 carbon sugar (C5 sugar) derrived from hemicellulose hexoses – 6 carbon sugars (C6 sugars) Glucose (C6H12O6)– 6 carbon sugar (C6 sugar) derrived from cellulose Mannose (C6H12O6) – 6 carbon sugar (C6 sugar) Galactose (C6H12O6) – 6 carbon sugar (C6 sugar) that is derrived from hemicellulose

10. Gasification Process Overview

11. My Fantasy – Local Cellulosic Ethanol then Pellet Production Transport feedstock to local plant Fuel to sell at local gas station Home heat fuel to sell at local stores Central Local Processing Drying of Material Harvest Ethanol Production Pellet Pressing Clean Energy Sources Electric and pellet heat sources for distillation and drying and cooling