Microbial Diversity In Subsurface Sediments From Mohe Basin, China

Mohe Basin is the only permafrost region at high latitudes in China. With global warming, permafrost regions are decreasing. Thus, research on the permafrost of Mohe Basin is extremely urgent. Mohe Basin also hold accumulation potential of gas hydrate and research on its environmental microorganism would offer support to the microbial prospecting technology. In addition, Mohe permafrost might reserve abundant and unique microbial resources due to its unique environment. This study focused on the microbial community structure and diversity in the subsurface sediments of20-1700m depth in Mohe Basin. Using a methodological combination of culture-dependent,454pyrosequencing and quantitative PCR, the following results were obtained:1. Total322bacterial strains were isolated from the sediment samples. These strains were identified to belong to121species,61genera and5phyla (i.e. Actinobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria and Bacteroidetes), among which the genera Bacillus, Acinetobacter and Brevundimonas were preponderant. Total117strains were identified to be psychrophilic or psychrotolerant, of which40strains produced amylase,73produced cellulose,53produced protease,70produced lipase and51produced β-galactosidase by preliminary screening. Eight strains with higher enzyme activities were selected to test the characteristics of amylase and cellulose under different temperature, of which at4℃two strains produced amylase that maintained53%and45%, respectively, of their highest activities and cellulose that maintained40%and70%, respectively, of their highest activities.2. Based on16S rRNA gene, bacterial and archaeal diversities in subsurface sediments of Mohe Basin were analyzed using454pyrosequencing. 20948bacterial reads were obtained that were clustered to1190OTUs and assigned to358phylotypes. The phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chlorobi, Deinococcus-Thermus, Fibrobacteres, Fusobacteria, Gemmatimonadetes, Proteobacteria, Verrucomicrobia, Firmicutes and unclassified Bacteria occurred in the samples, among which Proteobacteria and Acidobacteria were dominant followed with Firmicutes, Bacteroidetes and Deinococcus-Thermus.90948archaeal reads were obtained that were clustered to737OTUs and assigned to12phylotypes. The phyla Euryarchaeota, Crenarchaeota and unclassified Archaea were detected and differed greatly in different stratums.Analyses based on OTU showed that the bacterial richness (Chaol estimated) in samples was150-230, the Shannon index was4.3-4.7and the Simpson index was35-66, a lower level compared with the other environments. The dissimilarities of community structure between samples were high. The archaeal richness (Chaol estimated) was60-440, the Shannon index0.0-1.3and the Simpson index1.0-2.3. The archaeal distribution in different stratums was uneven and the archaeal diversity was much low. The dissimilarities of OTU membership between samples were high but some samples shared similar OTU structure.Analyses based on phylotype showed that the bacterial genera Thermopolyspora and Sphingobium were dominated with average relative abundance higher than5%in the subsurface sediments. The proportion of thermophilic bacteria (14-26%) was considerable, most of which the optimize growth temperature was45-55℃. Methanogenic archaea were detected in some stratums and thermophilic archaea were detected in the other. Combined with the abundant thermophilic bacteria, we inferred that in the subsurface, there had been a methanogenic period when biogenic methane was produced and afterward a geothermal period when thermogenic methane was produced. And thermophilic bacteria and archaea subsequently survived to be dominated.3. Microbial abundance was analyzed using quantitative PCR. The results indicated that the copy number of total Bacteria was106-107copies.gdw-1, that of Actinobacteria was104copies.gdw-1, of α-Proteobacteria was103-104copies.gdw-1, ofβ-Proteobacteria was105-106copies.gdw-1, of Bacteroidetes was104-107copies-gdw-1, of Firmicutes was105-106copies-gdw-1and of Fungi was103copies.gdw-1. Bacteroidetes, β-Proteobacteria and Firmicutes were abundant.4. A Gram-reaction-negative, yellow-pigmented, strictly aerobic bacterium, designated M0116T, was isolated from the sediment of the Mohe Basin in northeast China. Flexirubin-type pigments were produced. Cells were catalase-and oxidase-positive and non-gliding rods. Phylogenetic analysis based on16S rRNA gene sequences revealed that strain M0116T was a member of the family Flavobacteriaceae and was most closely related to members of the genera Empedobacter, Wautersiella and Weeksella with90.5-91.0%sequence similarities. The major cellular fatty acids were iso-C15:0and iso-C17:03-OH. The major respiratory quinone was MK-6and the major polar lipid was phosphatidylethanolamine. The DNA G+C content was38.2mol%. Based on the phenotypic, phylogenetic and genotypic data, strain M0116T is considered to represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Moheibacter sediminis gen. nov., sp. nov. is proposed. The type strain is Mo116T (=CGMCC1.127081JCM19634T). Emended descriptions of Empedobacter brevis, Wautersiella falsenii and Weeksella virosa are also proposed.