Statistical Optimization of Cellulase Production from Bacillus paramycoides and its Role in Saccharification of Pre-treated Brachiaria mutica (Para grass) Biomass

Abstract

The hyper-cellulase producing bacterium Bacillus paramycoides strain BTH was isolated and characterized by 16S rRNA sequencing. Its potential for saccharification of Brachiaria mutica (para grass), a lignocellulosic aquatic weed, was examined. Cellulase production from strain BTH was enhanced by optimizing various parameters in the presence of goat dung as feedstock using One factor at a time (OFAT) and Response Surface Methodology (RSM) methods. The OFAT-based non-statistical method improved cellulase activity up to 1280.32±27.3 U/g. Box-Behnken Design of RSM-based optimization exhibited 1.3-fold enhancement in cellulase activity (1725.54±32.63 U/g) as compared to OFAT technique in the presence of goat dung medium (pH 8.0), incorporated with 1.5% (w/w) CMC and incubated at 37°C. Para grass biomass was further pre-treated via hydrothermal, alkali, acid, hydrogen peroxide, and microwave heating methods and subjected to strain BTH-associated cellulase-based hydrolysis. The alkali pre-treated biomass exhibited maximum total reducing sugar production of 6.73±0.2, 9.25±0.16, 11.6±0.17, 14.11±0.16, and 11.54±0.16 mg/g in the presence of 4% (w/v) NaOH from 12 to 96 h. Likewise, 4% (w/v) NaOH pre-treated biomass showed maximum saccharification efficiency of 30.28±0.8, 41.62±0.6, 52.2±0.7, 63.49±0.6, and 51.93±0.8% from 12 to 96 h. The findings validated the role of B. paramycoides-associated cellulase in the saccharification of para grass.