| Summary: | Tonle Sap Lake is the largest freshwater lake in Southeast Asia, and it is regarded as one of the most biodiverse freshwater ecosystems in the world. Studies concerning aquatic cellulolytic bacteria from Tonle Sap Lake remain scarce. Cellulolytic bacteria and their cellulases play a vital role in the biogeochemical cycles of lake environments, and their application in biotechnological industries is likewise an important component of their usage. This study aimed to assess the isolation, genetic identification, bioinformatic analyses, and activity characterization of aquatic cellulolytic bacteria. The cellulolytic bacteria isolated from sedimentary water samples in the littoral zone of the lake belong to the genera <i>Aeromonas</i>, <i>Bacillus</i>, and <i>Exiguobacterium</i>. Several isolated aquatic bacteria were designated as rare cellulolytic microbes. Remarkably, <i>B. mojavensis</i> strain REP303 was initially evidenced by the aquatic cellulolytic bacterium in freshwater lake ecosystems. It was considered a highly active cellulolytic bacterium capable of creating a complete cellulase system involving endoglucanase, exoglucanase, and β-glucosidase. The encoded endoglucanase belongs to the glycosyl hydrolase family 5 (GH5), with a carboxymethylcellulase (CMCase) activity of 3.97 ± 0.05 U/mL. The optimum temperature and pH for CMCase activity were determined to be 50 °C at a pH of 7.0, with a stability range of 25–55 °C at a neutral pH of 7.0–8.0. The CMCase activity was enhanced significantly by Mn<sup>2+</sup> and was inhibited considerably by EDTA and ethyl-acetate. In conclusion, this study is the first to report data concerning aquatic cellulolytic bacteria isolated from the littoral zone of Tonle Sap Lake. A novel strain of isolated cellulolytic <i>B. mojavensis</i> could be applied in various cellulose-based industries.
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