Study On The Bacterial Culturability Of Water-sediment Interface

The traditional isolation and cultivation technology can only get 1% of the total natural microorganisms, and study on the microbial culturability plays an important role in improving the culturable efficiency and exploring for the microbial strain. Using the interfacial sediment from Lake Taihu as sample in this study, the original and the survival bacterial community structure were analysed by 16 S rRNA gene clone library and high throughput sequencing method respectively, and through the traditional isolation methods, the bacterial community structure which depends on cultivation were studied. Characteristics of bacterial culturability of the sediment were determined by comparing analysis.Results of the test on extraction method of genomic DNA from sediment showed that the purity of DNA by kit method was higher; the integrity of DNA extracted by the proteinase K+SDS method had the better integrity; followed by the glass beads+proteinase K+SDS method; DNA purity extracted by the chloroform/isoamyl alcohol extraction method was much lower.Genomic DNA of the sediment was extracted by kit method. PCR products of 16 S rRNA gene were obtained and connected to the pGEM-T Easy vector to construct the clone library. Result showed that 41 genotypes were identified out of 293 positive clones by amplified 16 S rRNA restrictive analysis. Based on sequencing data of the 16 S rRNA gene which represented for the genotypic clones, the main genera in the sediment included Sideroxydans, Nitrospira, Denitratisoma, Syntrophobacter and Methylobacter.Genomic DNA was extracted by the kit method after treatment of the sediment by 5 μg/mL propidium monoazide(PMA) under the light for 5 min. 16 S rRNA clone library was constructed and 22 genotypes were identified out of 191 positive clones. Based on sequencing data of the 16 S rRNA which represented for the genotypic clones, the main genera in the sediment included Candidatus Nitrotoga, Thiobacillus and Steroidobacter.The species composition of the original interface sediment and the treated one by PMA were researched by high-throughput sequencing based on the V3-V4 region of 16 S rRNA. Through high-throughput sequencing, the bacterial composition and proportion of five different classification level(phylum, class, order, family and genus) were obtained.Bacterial strains of about 25 different species were isolated from the sediment using R2 A medium at 28℃. Based on the 16 S rRNA gene sequencing and analysis, the strain mainly belonged to genera of Bacillus, Paenibacillus, Pseudomonas and Rhodococcus.By comparing analysis the bacterial community structure indentified by molecular ecology methods from the original sediment and that of PMA treatment, and also with the composition of bacterial genera indetified by cultivation, there were significant difference on the composition of bacterial genera and species between the interfacial sediment and that of PMA treatment, which indicated that part of bacteria were inactived. Some strains of genus such as Steroidobacter, Denitratisoma, Thiobacillus, Methylotenera and Candidatus Nitrotoga appeared only in the bacterial group from sediment by PMA treatment compared with the culturable method under laboratorial condition.