Isolation, growth, enzyme assay and identification via 16S rRNA full sequencing of cellulolytic microbes from Nepal for biofuel production

With its rich climatic and geographical variance, Nepal harbors a habitat for a diverse set of microbes. This research aims to identify and study the potency of native cellulolytic microbes. Cellulolytic microbes are being used to produce biofuels. The microorganisms were isolated from varied sources and grown in three valid media: Tryptone Soy Broth (TSB), Nutrient Broth (NB) and Gelatin. The findings from the growth quality observations, hydrolysis capacity calculations, and total cellulase assay were used to assess the cellulase enzyme potency of the microbes. Although all three media can be used to study the growth and isolation of the cellulolytic microbes, and different media showed different application potential, Gelatin media may be preferred for screening a large sample size. Hydrolysis capacity in TSB, NB and Gelatin media ranged from 4.0-5.2, 3.8-5.4 and 4.0-5.4 for the bacterial samples and 8.8, 9.0 and 9.2 for 0.1% pure cellulase enzyme, respectively. The results from 16S rRNA full-sequence and cladogram analysis indicate the possibility of a new strain of cellulolytic bacteria. Among the isolated microorganisms, four microbes were discovered close to the Bacillus genus, and one microbe was close to Paenibacillus. The selection of media for isolation and culture of the cellulolytic microbes depends on the researcher's decision and the availability of the resources. As one of the first studies to identify the native cellulolytic strains of Nepali origin, the study pushes other national and international researchers to consider the divergent climatic conditions of Nepal for potential cellulolytic strains.