Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains

The composition of thermophilic (60 °C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 °C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had Km values of 3.11 and 1.08 mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 °C for 1 day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.