Identification and whole genome sequencing analysis of Bacillus subtilis K35-1, a highly efficient cellulose in forage degrading bacterium

Abstract

This study reports the isolation and characterization of Bacillus subtilis K35-1, a novel cellulolytic strain with exceptional forage degradation capabilities. From eight B. subtilis isolates obtained from yak rumen fluid through Congo red screening (hydrolysis capacity = 2.61 ± 0.23), K35-1 demonstrated superior enzymatic performance, achieving peak cellulase (77.26 U/mL) and hemicellulase (222.85 nmol/min/mL) activities at 36 h of fermentation. The whole genome sequencing revealed a 4.06 Mb circular chromosome (GC content 43.83%) encoding 3,980 protein-coding sequences. Comprehensive CAZy annotation identified 703 carbohydrate-active enzymes, including: 87 cellulases spanning 7 GH families (GH5, GH6, GH9, GH12, GH44, GH45, GH48) and 34 hemicellulases from 4 GH families (GH10, GH11, GH26, GH30). Comparative genomic analysis showed K35-1 possesses 40% more glycoside hydrolases than reference strains (Srivastava et al., Mol Genet Genomics 298:361–74, 2023), explaining its enhanced degradation efficiency (53.2% cellulose reduction vs. 7.3% in conventional treatments). Functional annotation revealed: 275 carbohydrate metabolism genes (KEGG), 228 cell wall/membrane biogenesis genes (COG) and 53.91% reduced-virulence mutations (PHI database). The strain’s robust enzymatic profile, coupled with minimal antibiotic resistance (11 genes, including ermB), positions K35-1 as both an efficient forage degrader and safe probiotic candidate. These findings provide a genomic foundation for developing novel feed additives to improve livestock nutrition.