Sediments within the Okinawa back-arc basin overlay a subsurface hydrothermal network, creating intense temperature gradients with sediment depth and potential limits for microbial diversity. We investigated taxonomic changes across 45 m of recovered core with a temperature gradient of 3°C/m from the dynamic Iheya North Hydrothermal System. The interval transitions sharply from low-temperature marine mud to hydrothermally altered clay at 10 meters below seafloor (mbsf). Here, we present taxonomic results from an analysis of the 16S rRNA gene that support a conceptual model in which common marine subsurface taxa persist into the subsurface, while high temperature adapted archaeal taxa show localized peaks in abundances in the hydrothermal clay horizons. Specifically, the bacterial phylum Chloroflexi accounts for a major proportion of the total microbial community within the upper 10 mbsf, whereas high temperature archaea (Terrestrial Hot Spring Crenarchaeotic Group and methanotrophic archaea) appear in varying local abundances in deeper, hydrothermal clay horizons with higher in situ temperatures (up to 55°C, 15 mbsf). In addition, geochemical evidence suggests that methanotrophy may be occurring in various horizons. There is also relict DNA (i.e., DNA preserved after cell death) that persists in horizons where the conditions suitable for microbial communities have ceased.
During the Integrated Ocean Drilling Program (IODP) Expedition 331 at the Iheya North hydrothermal system in the Mid-Okinawa Trough by the D/V Chikyu, we conducted microbiological contamination tests of the drilling and coring operations. The contamination from the drilling mud fluids was assessed using both perfluorocarbon tracers (PFT) and fluorescent microsphere beads. PFT infiltration was detected from the periphery of almost all whole round cores (WRCs). By contrast, fluorescent microspheres were not detected in hydrothermally active core samples, possibly due to thermal decomposition of the microspheres under high-temperature conditions. Microbial contamination from drilling mud fluids to the core interior subsamples was further characterized by molecular-based evaluation. The microbial 16S rRNA gene phylotype compositions in the drilling mud fluids were mainly composed of sequences of Beta- and Gammaproteobacteria, and Bacteroidetes and not archaeal sequences. The phylotypes that displayed more than 97% similarity to the sequences obtained from the drilling mud fluids were defined as possible contaminants in this study and were detected as minor components of the bacterial phylotype compositions in 13 of 37 core samples. The degree of microbiological contamination was consistent with that determined by the PFT and/or microsphere assessments. This study suggests a constructive approach for evaluation and eliminating microbial contamination during riser-less drilling and coring operations by the D/V Chikyu.
The Iheya North Hydrothermal Field in the Okinawa Backarc Basin represents an ideal environment in which to investigate the biotic temperature fringe of microbial life at depth because of its subsurface hydrothermal activity within its continental margin-type sediment profile. Geographically, the Okinawa Backarc Basin is situated along a continental margin, which is a sediment profile type commonly sampled and studied across the seafloor (e.g. Peru Margin, Costa Rica Margin, Cascadia Margin). The hydrothermal network within the subsurface here supplies an additional temperature obstacle to microbial life existing in the sediments. In particular, the sediment profile at Site C0014 exhibits a transition from hemipelagic ooze with pumiceous volcaniclastic sediments and low temperature (4°C) to a hydrothermally altered sequence of clays within the top ~10 mbsf of sediment. Temperature measurements indicate a gradient of approximately 3°C/m, which is roughly an order of magnitude greater than continental margin sites (e.g. Cascadia Margin, IODP 311 and Costa Rica Margin, IODP 344), but is more gradual than intense, centimeter-scale gradients from other hot, surface sediments. We have focused on the application of culture-independent, molecular methods to understand taxonomic and functional characteristics through this hydrothermal gradient. Confidence in DNA recovery suggests a microbial biosphere extent of approximately 15 mbsf (55°C). Results from both 16S rRNA gene surveys and metagenomics analyses suggest a temperature-dependent stratigraphy of taxonomic and functional adaptations between the shallowest and deepest sample horizons. Cosmopolitan marine subsurface bacterial and archaeal taxa are present throughout the top 10 mbsf, whereas, hyperthermophilic heterotrophic as well as thermophilic anaerobic methanotrophic archaea appear in varying local abundances in deeper, hydrothermal clay horizons.
The Okinawa Backarc Basin sediments encompass spatial diversity of physical and geochemical inputs from the subsurface hydrothermal network of the Iheya North Hydrothermal Field. Previous microbial community gene-based assessments reflect stratified and diverse communities of Bacteria and Archaea between cored IODP Expedition 331 sites. Furthermore, temperature constrains in sediments affected by hydrothermal fluid flow (Site C0014) restrict the biosphere to the upper meters of sediments. In this study, we attempt to supplement the gene-based and metagenomic studies from Sites C0014 (heated sediments) and C0017 (recharge zone sediments) with an RNA-based approach to selectively investigate the active microbial populations. The results from sequenced reverse transcribed 16S rRNA demonstrate the sensitivity of extraction and amplification to external nucleic acids and attest to the challenges in working with low biomass environmental samples, but may also reveal certain bacterial taxa (e.g. Bacteroidetes and Alphaproteobacteria) recovered from select sediment horizons. However, inconsistencies between primer sets and replicates make conclusions about the results of this RNA study extremely tenuous.