Nassim NADERI MS Food Biotechnology Research Assistant Applied Biotechnology Institute, California Oct 29, 2010

Corn stover is the above ground, non-grain portion of the corn plant. It is composed of lignocellulose material which can be converted to ethanol for use as a transportation fuel. Makes up almost 80% of all agricultural residues generated in USA. Major difficulty in producing cellulosic ethanol is converting the cellulosic material into simple fermentable sugars, as it is resistant to enzymatic breakdown.

pretreatment hydrolysis Product separation Enzymatic hydrolysis Fermentation Product separation

Currently, pretreatment options involve: Enzymatic hydrolysis Fermentation Product separation Pretreatment modifies the structure of the Stover to make the cellulose more readily available for sugar conversion Currently, pretreatment options involve: Acid Hot water Ammonia Ionic solids Lime Pretreatment

Three main categories of cellulases: Enzymatic Hydrolysis of Pretreated Corn Stover Enzymatic hydrolysis of cellulose is accomplished under mild conditions ( <50°C, pH~5.0 ). Three main categories of cellulases: • Endoglucanases (E1): Produce glucans by attacking regions of low crystallinity • Exoglucanases (CBH1): Degrade the glucans into cellobiose units • Beta-Glucanases (β-Glucosidase) : Break down cellobiose into glucose

pretreatment Enzymatic hydrolysis Fermentation Product separation Fermentation converts sugar into alcohol and lastly, the product is separated and purified.

… in this study: We explore the effect of incubation conditions on the conversion of corn stover to useful sugars. Corn stover resuspended in different buffers Reaction temperature of 45oC Surface area of the reaction well

Equilibration overnight Dilution to 20% (wt/wt) ddH2O Citrate buffer pH~5 Adjusting pH to 5 Using Ammonium Hydroxide (NH4OH) Placed desired weight into 48-well plate Adding selected Enzymes Incubation 3-7 days @ 45°C Pretreated Corn Stover was provided by National Renewable Energy Laboratory (NREL)

Glucose Oxidase assay Detect and calculate glucose concentration produced during incubation by using spectrophotometer.

Prevent dehydration of sample during incubation by using parafilm. Adjusting the speed of shaker during incubation time. Extending incubation time to one week (7Days). Using different size of Multiwell plates for assay. Choosing weight of loading sample. Correcting dilution factor to 1/100 in sample preparation for Glucose oxidase assay. (To reduce the high background) Correcting dosages of added enzyme.

Selected dosage of Celluclast will be 0, 3, 10, 50 uL. Corrected dilution factor to measure glucose concentration.

Synergistic enzyme pair (E1 & CBH1) showed similar activity compared to CSE. Concentrated E1,CBH1 provided by Texas and E1,CBH1 germ extracts showed similar activity in glucose production

Do we want to use ddH2O or Citrate buffer? ddH2O was more effective in conditioning of Corn Stover and improved synergistic activity was observed. Do we want to use ddH2O or Citrate buffer?

Supplementation of Celluclast, E1, CBH1 with B-Glucosidase (Novozyme) demonstrated a significant benefit in the hydrolysis of cellulose present in pretreated Corn Stover at early time points.

Pretreated Corn Stover Hydrolyzed Corn Stover

Acid pretreated Corn Stover (Supplied from NREL) was conditioned to pH~5. Enzymatic hydrolysis was conducted with different enzyme mix assays at 45 C for 6 days. Calculation of approximate conversion was based on the amount of glucose released.

Increase enzyme loading to search for an effective synergistic activity to improve hydrolyzing cellulose in Corn Stover. Increased E1 and CBH1 crude germ extract volumes Loading purified E1, CBH1 enzymes.