Study On Microbial Diversity And Chitinolytic Bacteria Of Surface Sediments From Shantou Bay

We made an investigation of the microbial diversity in Shantou Bay surface sediments, using 16S rDNA and conservative chitinase gene fragments as the breakthrough points; and we isolated a number of chitinolytic bacteria, amplified their chitinase gene fragments and explored the optimal chitinase fermentation conditions.Used PCR-DGGE band analysis and 16S rDNA library, we got 97 16S rDNA sequences, belong to 10 bacteria phylum: Proteobacteria, Acidobacteria, Bacteroidetes, Deferribacteraceae, Spirochaetes, Chloroflexi, Verrucomicrobia, Nitrospirae, Deinococcus-Thermus and Planctomycetes. Compared these sequences to NCBI records,the max identity rates from 81% to 100%, 97% as a standard,47 sequences higher than 97 % , accounting for 48.5%, 50 sequences lower or equal to 97%, accounting for 51.5 %, and there were four sequences could not identify their classification status, because of the max identity rates were too low. It suggested that Shantou Bay sediments contain abundant of microorganism resource, and many of the microbes are still not studied.We amplified 63 chitinase coding gene fragments through PCR, compared these sequences to NCBI records, the identity rates from 41% to 97%, and the majority below 70%, only 8 sequences higher than 70%, 27 sequences have the highest identity rates with the chitinase sequence of Herpetosiphon aurantiacus ATCC 23779 in NCBI records, the highest similarity chitinase protein sequences were 18, including some uncultured bacteria chitinase sequences. It suggested that there are many different chitinase coding genes in Shantou Bay sediments, most of them are still not studied from now.Isolated 69 chitinolytic bacteria from the surface sediments, according to 16S rDNA sequences, we identified their genuses of SWCH-1, SWCH-2, SWCH-3, SWCH-5, SWCH-6 and SWCH-9, were Brevibacillus, Paenibacillus, Bacillus, Aeromonas and Stenotrophomonas, respectively. We amplified the chitinase gene fragments of the 6 strains, suggested that they all contained 18 family chitinase coding genes, and their coding protein sequences had some differents from the NCBI records, SWCH-1 and SWCH-3 had low identities; according to the morphological and physiological and biochemical characteristics of SWCH-6, we identified it belonged to Aeromonas hydrophila species. The optimal chitinase fermentation conditions of strain SWCH-6 were conformed by single-factor experiments and orthogonal experiments, they were colloidal chitin 25.0g/L, tryptone 10.0g/L, seawater 1L, pH8.5, 32℃, 150 rpm for 72h. In these conditions, its chitinase activity reached 0.39U/ml. In addition, at 40℃and pH5.0, its chitinase performed the highest catalytic activity and its chitinase activity could be enhanced by Cu2+, Fe3+ and surfactant Tween-80; weakened by Zn2+, Mn2+ and surfactants SDS, detergent powder, and there were some differences from orther chitinases. In addition, found a 90kDa extracellular chitinase in SWCH-6 supernatant, but its coding gene had not been cloned, and there was a 60kDa intracellular protein, whether it was relative to chitin degradation still unknown.