Biochemical and molecular characterization of thermo-alkali tolerant xylanase producing bacteria from thermal springs of Manikaran

One hundred ten alkalo-tolerant thermophilic bacteria were isolated from 17 samples (water and sediment) collected from Manikaran. Of 110 isolates, 70 showed the production of xylanases and were further screened for growth and production of xylanases at different temperature ranging from 40 to 75 degrees C. Eleven isolates that showed growth and xylanase production at temperatures >= 50 degrees C were selected for quantitative estimation in modified Reese mineral liquid medium containing wheat bran. Maximum xylanase activity was produced by isolate H-7 followed by H-9 and R-9 and was statistically superior to other isolates. The microscopic observation showed that the isolates possessed the typical rod with endospore, characteristic of genus Bacillus. The isolates were found to be oxidase and catalase positive. Using BIOLOG Microlog 3 software, the isolates H7, H9 and R9 were identified as Paenibacillus ehemensis, Bacillus cereus/B. thuringiensis and B. subtilis respectively, based on utilization of 95 carbon sources. PCR-RFLP analysis of 16S rDNA indicated that the isolates were genetically different from each other. DNA sequencing of the three isolates and phylogenetic analysis revealed that all the isolates obtained from Manikaran thermal springs showed 97 to 100% similarity with the sequences within the GenBank. The closest phylogenetic neighbours according to the 16S rRNA gene sequence data for the three isolates H-7, H-9, and R-9 were Paenibacillus ehemensis, Bacillus cereus and Bacillus subtilis, respectively.