Isolation and Characterization of Cellulase Producing Microbes from Ruminants for Bioremediation of Organic Wastes to Obtain Useful Byproduct

Abstract

Cellulose, the major component of lignocellulosic biomass, is enzymatically hydrolysed by cellulase, produced by different types of microbes. 71 isolates were obtained after sampling, enrichment and isolation from cow dung and rumen fluid samples and 23 isolates after screening assay (congo red and filter paper assay). The isolates were then subjected to morphological characterization followed by RAPD using OPA-11 primer which yielded 14 distinct band profiles. These were then subjected to 16S rRNA PCR, followed by sequencing and bioinformatic analysis. The sequences were used to identify the 14 microbes through BLAST and thereafter submitted to the NCBI database. The property of cellulase production of K. gyiorum and B. paralicheniformis was hitherto undescribed and is being reported for the first time. These isolates were further evaluated through various biochemical experiments (estimation of concentration of cells, glucose and protein) and statistical tools (t-test, ANOVA and correlation). B. paralicheniformis was found to have maximum glucose production and enzymatic activity. 7 most efficient isolates were then further evaluated for their bioremediation and bio-prospection properties. Office paper, leaf litter and groundnut wastes were found to be the best wastes for glucose production, cellulase production and as a medium to harvest the highest number of cells, respectively. The cell free extract of B. paralicheniformis was characterized physico-chemically and it was observed that the enzymes are thermostable (10-60ºC) with temperature optima at 40ºC, resistant to high pH and require metal ions (Mn2+, Ba2+ and Fe2+) for their activity. The kinetic parameters, KM (0.2011 g/l) and Vmax (0.007440 µmoles/min) were determined to study enzyme efficiency. Molecular characterization of the enzyme by PAGE and LC/MS analysis was followed by bioinformatic analysis. 16 peptide sequences obtained were identified as different types of cellulases- exoglucanase (1), endoglucanases (3) and endo-1-4-and#946;-mannosidase (1). The cellulose degrading bacteria obtained can be of immense commercial value as they yield enzymes, sugars, alcohols, acids or alkalis, pigments, antimicrobial agents and compost by degrading waste materials. Their production can be upscaled for industrial applications. newline newline newline