Isolation and identification of cellulose degrading bacteria and optimization of the cellulase production

Abstract

The cellulase producing bacteria were isolated from industrial and agricultural areas in Kerala. Potential isolates with cellulase production were identified by Grams iodine dye staining method. The isolate were tentatively identified to be Bacillus species based on cultural, morphological, biochemical analysis and labelled as CB3, CB4 and CB8. Further, the genomic DNA was isolated and amplified with universal primers 27F and 1492s specific for 16S rRNA. The amplified 16S rRNA PCR product of 1500bp was sequenced and the unknown organism was identified using the maximum aligned 16SrRNA sequences available in the GenBank of NCBI through BLAST search. The sample CB3 and CB4 showed (100% and 99% respectively) homology to Bacillus subtilis strain. The sample CB8 showed 98% homology to the Bacillus cereus strain. To test the evolutionary relationships, phylogenetic analysis was performed with the program MEGA 6.0 using the 16SrRNAsequence. The isolates were then evaluated by submerged fermentation process for maximum cellulase production. The various process parameters like pH, temperature, incubation period, substrate concentration and inoculum volume were then optimized for the maximum production of cellulase by the isolates. The optimum temperature for cellulase production for CB4, CB4 and CB8 was found to be 400C, 300C and 400C respectively. The optimum PH was found to be 7 for the three samples. The incubation period of 48 hours, 72 hours and 96 hours were found to be optimum for CB3, CB4 and CB8 respectively. An inoculum size of 6% was found to be ideal for CB3 and CB4 whereas an inoculums volume of 8% was found to be ideal for CB8 which showed a maximum activity of 4.12U/ml. The CMC concentration of 1.5% was found to be ideal for CB3 and CB4 whereas CB8 showed a maximum activity of 3.21U/ml at a CMC concentration of 1%. Among the three isolates the Bacillus cereus strain (CB8) was found to be the most active cellulase producer with maximum activity of 4.12 IU/ml in submerged fermentation.