Enzymatic Hydrolysis of Rye Straw and Bermudagrass for Ethanol Production

Abstract

Dilute sulfuric acid pretreatment of rye straw and bermudagrass was investigated under acid concentrations of 0.6% to 1.5% (w/w) and residence time 30 min to 90 min. The pretreatment effectively solubilized the hemicellulose components to monomeric sugars such as xylose, arabinose, and galactose. Cellulose in the rye straw was not hydrolyzed into glucose during the acid pretreatment, while part of the cellulose in the bermudagrass was converted into glucose by the acid pretreatment. After enzymatic hydrolysis with excessive cellulases, the glucose yields of 30% to 52% and 46% to 81% of the theoretical potentials were obtained for rye straw and bermudagrass, respectively.

The pretreated solid residues were hydrolyzed with cellulases supplemented with β-glucosidase. The addition of β-glucosidase greatly improved the glucose production rate. There was no cellobiose accumulation when the β-glucosidase loading was up to 25 CBU/g (CBU, cellobiase unit, expressed as μmol of cellobiose that is converted into glucose per minute). The conversion rate was 45% for bermudagrass hydrolyzed with cellulases of 10 FPU/g (FPU, filter paper unit, expressed as μmol of glucose produced per min with filter paper as a substrate) and &#946-glucosidase of 25 CBU/g. The further increase of enzyme loadings did not significantly improve the glucose yield. Rye straw was more resistant to enzymatic hydrolysis than bermudagrass. The conversion rate was 38% with the additions of cellulases at 15 FPU/g and &#946-glucosidase at 25 CBU/g rye straw.

The production of cellulase enzymes in transgenic plants has the potential to significantly reduce enzyme costs. Expression of cellulase enzyme, Acidothermus cellulolyticus E1 endoglucanase, in transgenic duckweed Lemna minor was examined. The recombinant E1 protein was biologically active with the expression level of 0.24% of total soluble protein. HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH = 8) extracted more proteins including E1 from duckweed fronds than sodium citrate buffer (pH = 4.8) and sodium acetate buffer (pH = 5). The E1 protein was thermotolerant and exhibited the optimal enzyme activity at temperature 80°C and pH 5. The E1 activity remained unchanged after heating at 60°C for 6 h. However, it was inactivated after 15-min heating at 90°C.