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. Purified CMCase exhibited highest activity of 3.24 U/mL at pH 7.0, which was stable at pH 5-7 and thermally stable up to 60C. Agricultural waste such as wheat and rice straw and leaf litter from Mango, Poplar, Ashoka and Eucalyptus were collected, processed and subjected to different pre-treatments. Four different pre-treatment strategies such as fungal alkaline fractionation, fungal solvolysis, dilute acid alkali and microwave-acid-alkali treatment were employed for all six biomass samples to enhance saccharification. Microwave-acid-alkali pre-treatment was found to be the best and most promising technique among all pre-treatments. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Thermal gravimetric analysis (TGA), Fourier transform infrared (FTIR) spectroscopy analysis and Solid state 13C CP/MAS NMR spectroscopy of native and microwave-alkali-acid pre-treated rice straw was done to investigate physical and chemical changes after pre-treatment. Solid state 13C CP/MAS NMR spectroscopy was most reliable technique to determine biochemical changes before and after pre-treatment of biomass. Microwave-acid-alkali pre-treated rice straw was selected as feedstock for ethanol production with maximum reducing sugar (246 mg/g of biomass) yield. The process parameters were optimized using Taguchi design to obtain greater yield of ethanol using purified cellulase from Bacillus subtilis NA15 and Saccharomyces cerevisiae via simultaneous saccharification and fermentation (SSF). Maximum ethanol concentration (18.9 g/L) was found with optimized parameters of substrate loading: 11% (w/v), pH: 4.5, temperature: 30C, enzyme loading: 0.5% (v/v), inoculum size: 8% (v/v), after 24 h of incubation. The optimized conditions were further used for scale up of ethanol production in 3-L bioreactor using microwave-acid-alkali pre-treated rice straw and maximum ethanol concentration (25.2 g/L) corresponding to 59% yield was obtained after 24 h of SSF process.