Molecular Identification of Cellulose Degraders Bacillus licheniformis and Bacillus tequilensis Isolated from Soil and the Biogas Slurry

Molecular identification of bacteria is the gold standard technique for identification of particular microorganism. In the current study, 16S rDNA (genotypic) identification technique was used for the credentials of isolates by using molecular techniques and bioinformatics. Cellulase producing bacteria Bacillus licheniformis was isolated from soil and Bacillus tequilensis was isolated from biogas slurry, using serial dilution method and growing them on nutrient agar and Saubdroud Dextrose agar media. Isolates were screened by using Carboxymethyl cellulose (CMC) and Whatman filter paper degradation assay. The highly cellulolytic isolates (Bacillus sp. I and Bacillus sp. II) were identified on the basis of Gram staining, morphological cultural characteristics and biochemical tests. The two universal primers were design Set-I primers, Forward primer (5'-AACGGCTTACCAAGGGGAC-3') and Reverse primer (5'-AGGTTCTTCGCGTTGCTTC-3') Set-II primers, Forward primer (5'-AGATGGGATTAGCTAGTAGGTG-3') and Reverse primer (5'-AAGGCCCGGGAACGTATTC-3') and the organisms were identified with 16S rDNA analysis. Molecular characterization was done by phylogenetic analysis of the 16S rDNA sequence by bioinformatic tool ClustalW. The phylogenetic analysis of their 16S rDNA sequence data showed that Bacillus sp. I have 99.43% similarity with Bacillus tequilensis and Bacillus sp. II has 98.91% similarity with Bacillus licheniformis. In conclusion, Bacillus tequilensis and Bacillus licheniformis are highly cellulolytic bacterial strains were isolated and identified. These strains could be used for converting vegetable waste to compost to reduce greenhouse gas emissions and also as antifungal agent.