Bacterial Diversity In Oil Shale Mines Based On Uncultured-dependent Methods

Bacterial diversity in major oil shale mines in China (Fushun in Liaoning province, Huadian in Jilin province and Maoming in Guangdong province) was investigated using the clone library of the culture-independent PCR-amplified 16S ribosomal DNA (16S rDNA). The abundance and spatial distribution of bacteria and archaea in oil shale mines were determined by fluorescence in situ hybridization (FISH). This aim is to understand the composition and structure, populations, and spacial distribution of dominant bacterial communities in the major oil shale mines in China.In order to establish an appropriate method for extracting microbial total DNA from oil shales,5 methods (SDS-high-salt extraction, SDS-grinding-in-liquid-nitrogen extraction, SDS repeated freeze-thaw extraction, SDS guanidinium isothiocyanate extraction and kit extraction) were selected to extract total DNA of 6 different samples from two mines, respectively. The total DNA extraction effects of five methods varied significantly. SDS-high-salt extraction method was the best, which could obtain total DNA with about 23 kb length and 91.44?386.85 ng·g-1 dry sample yield rate significantly higher than other methods. Further the total DNAs without purification process were used as amplification templates, and 16S rDNA was carried out PCR amplification and DGGE electrophoresis, resulting that the corresponding PCR products and DGGE fingerprint were obtained and showing the improved SDS-high-salt extraction method is suitable for oil shale samples. SDS-grinding-in-liquid-nitrogen extraction and SDS repeated freeze-thaw extraction were only able to extract microbial total DNA of partial samples, and with low yield and purity. SDS guanidinium isothiocyanate extraction and kit extraction methods were not suitable for oil shale samples.Bacterial 16S rDNA clone libraries of the three oil shale mines showed that the coverage rates of libraries in Fushun, Huadian and Maoming mines were 55.05%,73.42% and 90.12%, respectively. Proteobacteria in the three libraries were the dominant bacterial communities. In addition, Bacteroidetes, Actinobacteria and Firmicutes commonly existed in the three oil shale mines. Acidobacteria and Unclassified bacteria only appeared in Fushun mine and Maoming mine. Deinococcus-Thermus only appeared in Fushun mine. The composition and structure of bacterial communities has a slight difference at the phylum level, but more obvious difference at the generus level.The determination of bacteria and archaea in the three oil shale mines by FISH showed that the propotion of bacteria in the three mines was more than 50%, but the propotion of archaea was quitely low. Except Archaea in freshly mining oil shale and weatheren oil shale of Fushun mine accounted for 4.53% and 2.25%, respectively, they were less than 1% in other oil shale samples.In general, bacterial diversity in the three oil shale mines has a big difference. The bacterial diversity in Fushun mine is the highest, followed by Huadian mine and Maoming mine. The propotion of Archaea in each oil shale sample from different mines is very low.