Bioconversion of agricultural waste biomass for ethanol production

Abstract

Conversion of lignocellulosic biomass to ethanol remains a key bottleneck at industrial scale to enhance the world energy security. Identification of promising feedstock, it s pre-treatment with process integration for enhanced saccharification by microbial conversion of substrate to ethanol is important in development sustainable energy system. Cellulose degrading bacteria from different sources such as compost, paper pulp and sugarcane bagasse were isolated, screened and characterized. Out of 32, eight bacterial isolates (designated as NA8, NA9, NA11, NA14, NA15, DGA, DGB and DGC) were found to be cellulose degraders. Biolog GEN III MicroPlateTM identification and 16S rRNA gene sequence analysis revealed that NA8, NA9, NA11, NA14, DGB and DGC showed maximum similarity with Bacillus licheniformis, isolate NA15 had maximum similarity with Bacillus subtilis whereas, DGA shared maximum similarity with Lysinibacillus fusiformis. Bacillus subtilis NA15 was found to be the best cellulose degrader among all the eight screened isolates with maximum carboxymethyl cellulase (CMCase) activity of 0.06 U/mL in cell free supernatant. Screening of 11 media ingredients using Plackett-Burman design was done to explicate the parameters that significantly influence the CMCase production. Maximum CMCase activity of 0.47 U/mL was obtained using Response surface methodology (RSM) with carboxy methyl cellulose (CMC): 18 g/L, peptone: 5 g/L, yeast extract: 5 g/L and MnCl2: 0.5 g/L. The model predicted the maximum CMCase activity (0.45U/mL) which was in good agreement with the experimental value of 0.47 U/mL showing 7-fold increase as compared to unoptimized medium. CMCase from Bacillus subtilis NA15 was purified using DEAE-Sepharose ion exchange chromatography with increase in CMCase activity from 2.65 to 30.42 U/mg of protein (11.5 fold) with a yield of 43.7%. The approximate molecular mass of the purified cellulase was 45 kDa.