The Kekexili Soil Bacterial Diversity

Hoh Xil, where is situated at the Yushu Tibetan Autonomous Region in southwest Qinghai, is one of the most primitive and well-preserved natural environment in the world. The microbial diversity in this area is largely unknown till date. In the present study an attempt has been made to explore the microbial diversity using both of cultivation-dependent and independent approaches.In the culture-dependent experiment, twenty-two different media and three pretreatment methods were used to isolate general, halophilic and alkalophilic bacteria, in 25 soil samples from Hoh Xil. As a result,78 strains have been isolated, among which 48 isolates were identified to belong to 13 genera of actinobacteria, including Streptomyce, Microlunatus, Actinokineospora, Nocardia, Micromonospora, Rhodococcus, Glycomyces, Promicromonospora, Microbacterium, Gordonia, Kribbella, Williamsia and other 30 strains represent 4 genera of non-actinobacteria bacteria, such as Variovorax, Mycobacterium, Phyllobacterium, Pseudomonas, Bacillus.Based on 16S rRNA gene sequences analysis, the strains CPCC100076T showed the highest similarity with the strains of Microlunatus(95.8-98.0%), in which 98.0% with Microlunatus parietis DSM 22083T. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that the isolate CPCC100076T formed a sub-branch with the type strains Microlunatus parietis DSM 22083T. But On the basis of the results of polyphasic taxonomy, we suggested that strain CPCC100076T represents a novel species.19 isolates were obtained from the soil sample IMB08-049, which exhibited the best biodiversity among all the samples. 16S rRNA gene sequence analysis showed that these 19 strains belonged to 8 different genera, including Phyllobacterium, Variovorax, Pseudomonas, Streptomyces, Microlunatus, Kribbella, Promicromonospora and Bacillus. However, no any strain was isolated from soil sample IMB08-056.Next, Culture-independent method was also employed to investigate microbial community structure in both of soil sample. Total genomic-DNA were extracted and purified directly from the soil sample and used as template for polymerase chain reaction (PCR) amplification with universal bacterial primer sets. DGGE result shows that number of band of IMB08-049 genome 16S rRNA gene V3 fragment is 17 and that of IMB08-056 is 15.Therefore, we can roughly estimate that number of 1% of the bacterial populations of IMB08-049 is 17 and that of IMB08-056 is 15. Software Quantity one analysis showed that the biodiversity of both of samples are not good. Biodiversity was also assessed by method of 16S rRNA gene library and amplified ribosomal DNA restriction analysis (ARDRA). The result of DNA sequencing and phylogenetic analysis showed that 19 operational taxonomic units (OTUs) of the soil sample IMB08-049 were clustered in 3 the following phyla including Proteobacteria, Actinobacteria and Firmicutes, while 23 OTUs of IMB08-056 in 5 the following phyla including Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria and Firmicutes.Comparison of the results obtained from culture-dependent and -independent experiments indicates that at least 11 genera including some dominant species were not isolated using the above culture-dependent method, suggesting that new isolation method should be developed to discover more bacteria exciting.