Screening and Molecular Identification of Hypercellulase and Xylanase-Producing Microorganisms for Bioethanol Production

The present study projects the baseline work for bioconversion of pine needles to second generation biofuel, which deals mainly with screening, molecular identification and optimization of process parameters for cellulase and xylanase production. In total, 89 hydrolytic enzymes producing isolates were isolated from the soils and ten potential enzyme producers (seven for cellulase and three for xylanase) were subjected to secondary screening by inducing physical and chemical mutation. The wild and mutant strains of hypercellulase producers N₁₂ and Kd₁ were identified as Bacillus stratosphericus N₁₂ and Bacillus altitudinis Kd₁ using 16S rRNA technique. The fungal isolates RF1 and F2 were identified on the basis of 5.8 rRNA ITS technique and identified as Rhizopus oryzae, RF1 and Rhizopus delemar, F2 respectively. The mutant strains B. stratosphericus N₁₂ (M) and B. altitudinis Kd₁ (M) are highly stable till 10 generations. Cellulase activity increased from 3.230 to 5.983 IU, i.e. 85.23% increase in cellulase activity was achieved. Xylanase production increased from 51.32 to 95.25 IU with 85.60% increase in production. Solid-state fermentation was also performed by potential fungal strains, i.e. R. delemar F2 and R. oryzae RF1 using pine needles as the substrate.