Abstract
Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.
Highlights
MATERIALS AND METHODSThe deep subseafloor biosphere is unique in that microorganisms long persist in the high-pressure environment over geologic time
Our results show that enrichment cultures from the very deep sedimentary microbial communities were dominated by gram-positive endospore-forming piezophilic bacteria
Taxonomic classification analysis of HP enrichment cultures clearly indicated that cultivated piezophilic communities were dominated by members of the phylum Firmicutes with >80% of the reads, followed by members of Gamma-proteobacteria, which sequence frequency ranged between 1.2 and 11.6% (Figure 1)
Summary
MATERIALS AND METHODSThe deep subseafloor biosphere is unique in that microorganisms long persist in the high-pressure environment over geologic time. The primary goal of this study was to investigate microbial communities of three sediment core samples from the depths of ∼1.5 to 2.4 km below the ocean floor and, by isolating piezophilic bacterial strains, to study metabolism of the piezophilic isolates in the deep subseafloor biosphere.
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