In silico and in vitro Study of Two Novel Closely Related Biogas Digestate Bacilli Strains

Abstract

Biochemical, morphological and molecular variance in Bacillus subtilis and B. tequilensis is reported here. Gen Bank-submitted partial rRNA sequences of KIIT VSKC006 (K6; KF410850.1) and KIIT VSKW003 (K3; KF410851.1) isolated from biogas digestate while formulating effective microbial consortia, upon being subjected to a BLAST search, revealed that they belonged to Bacillus. These rRNA sequences with high maximum-identity-score values were selected and aligned using multiple alignment software ClustalW. Phylogenetic trees constructed using MEGA 5 (neighbour-joining) revealed K6 and K3 as B. tequilensis and B. subtilis respectively. BioEdit analyses for the similarity in the gene sequences through the constructed nucleotide composition graph revealed that, the percent GC and AT contents (K6: 55.09, 44.91; K3:55.22, 44.78, respectively) and the molecular weights (single-stranded; 3897 d) in both the strains differed. Global alignment of the sequences using Needleman-Wunsch algorithm (EMBL-EBI) showed a 98.9 % identity match. When grown on agar at 30°C for 96 hrs, K6 showed irregular, cream colonies whereas K3 exhibited circular, off-white colonies with prominent concentric circles throughout. When re-grown in nutrient broth, the growth patterns were visibly different; K6 showed white, clumped, suspended particles whereas K3 displayed uniform turbidity. These in vitro and in silico analyses infer that, the difference in the two reported isolates was obvious, to the extent of being different species though closely related.