Eco-friendly Production Of Cellulose Using Different Agro-wastes By Bacillus subtilis

Plant biomass waste consists almost entirely of lignocellulose, which is formed by three types of polymers, namely cellulose, hemicellulose and lignin. Cellulose is a linear polymer composed of D-glucose linked by b-1,4 glucoside bonds. The cellulose enzyme system usually comprises three cellulolytic enzyme classes which hydrolyze the cellulose: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and cellobiases (EC 3.2.1.21). Celluloses are used in various industries, such as fuel, food, detergent, animal feed, agriculture, wine, beer, pharmaceuticals and cosmetics. The main objective of the study is to explore an easy and cost effective method to produce the cellulase using various agricultural wastes, such as sugarcane bagasse, sago waste and rice bran as substrates. Cellulase producing bacteria were isolated from forest soil. Among the isolates, three strains show the maximum activity on carboxymethyl cellulose (CMC) agar plates. Among the three strains, a strain was showed maximum enzyme activity, which was measured by dinitro salicylic acid (DNS) method. The isolates were identified as Bacillus subtilis. Optimization of the fermentation medium for the production of maximum cellulase was carried out through submerged fermentation (SmF). The highest production of cellulase was obtained as 7.9 U/mL at 3% glucose as a carbon source, 8.1 U/mL at 3% meat extract as a nitrogen source and 7.0 U/mL at 3% sugarcane bagasse at 37°C, 8.2 U/mL at pH 9, 6.9 U/mL at 3.5% inoculum level at 48 hr. Cellulase was purified to centrifugation, ammonium sulphate precipitation and diethylaminoethyl-cellulase (DEAE-cellulase) chromatography. By adopting these steps, a fold purification of 18.02 with 86% overall yield was obtained. The purified cellulase with a molecular mass of 22 kDa determined by SDS-PAGE. Bacillus subtilis are capable to produce the cellulase for industrial application.