Cellulases are enzymes synthesized by diverse groups of microorganisms. These enzymes have demonstrated biotechnological potentials in various sectors, including food, animal feed, brewing and wine, and pulp and paper industries. In this study, 100 strains of Bacillus spp. were incubated in MT1 agar medium, and cellulolytic activity was qualitatively determined by measuring the diameters of the hydrolytic clear zones. The three most active strains (A1.2, A1.8, and B6.4) showing clear zone diameters above 24 mm were selected. Cellulase activity in a liquid medium was further quantitatively determined by the dinitrosalicylic acid (DNS) method. Cellulolytic bacteria were identified by 16S rRNA gene sequencing, and neighbor-joining phylogenetic analysis was conducted to determine the evolutionary relationships between the selected strains and reported strains from the GenBank database. The strains were identified as Bacillus cereus (A1.2 and A1.8), and Bacillus pumilus (B6.4). Cellulase produced by B. pumillus B6.4, a GRAS bacterium showing the highest cellulase production in a liquid medium, was partially purified and characterized. The enzyme was most active at 55°C and pH 6.5. Half-lives (conducted at pH 5.0) of the enzyme at 55, 65, 75, and 85°C were 180, 180, 30 and 20 min, respectively. Similarly, half-lives (conducted at 37°C) of the enzyme at pH 5.5, 6.5, 7.5, and 8.5 were 130, 135, 80 and 70 min, respectively. The broad range of working temperatures and the stability under mild acidic conditions suggest that the cellulase of B. pumilus B6.4 could be a good candidate for application in the lignocellulosic bioethanol industry.
