High-resolution temporal analysis of deep subseafloor microbial communities inhabiting basement fluids

The temporal variation in microbial communities inhabiting the anoxic, sediment-covered basaltic ocean basement is largely uncharacterized due to the inaccessible nature of the environment and difficulties associated with collection of samples from low-biomass microbial habitats. Here, a deep sea instrumented platform was employed on the Juan de Fuca Ridge in the summer of 2013 to collect 46 samples of basement fluids from the most recent generation of borehole observatories (U1362A and B), which feature multiple sampling horizons at a single location and fluid delivery lines manufactured using stainless steel or inert polytetrafluoroethylene (PTFE) parts. Included were three time-series deployments of the GEOmicrobe sled meant to resolve the fine-scale (i.e. hourly) temporal variation within in situ crustal microbial communities. Illumina technology was used to sequence small subunit ribosomal RNA (SSU rRNA) gene fragments from sediment, seawater, and subseafloor fluids. Similar to has been reported previously, basic differences in the three environments was observed. Fluid samples from depth horizons extending 30, 70, and ~200 meters sub-basement revealed differences in the observed microbial communities, indicating potential depth-specific zonation of microorganisms in the basaltic basement fluids. Extensive overlap between microorganisms collected from a single depth horizon but using two fluid delivery lines manufactured with different materials was observed, though some differences were also noted. Several archaeal (e.g. THSCG, MCG, MBGE, Archaeoglobus) and bacterial (e.g. Nitrospiraceae, OP8, KB1) lineages detected in previous years of basement fluid sampling nearby were found here, which further supports the notion that these microorganisms are stable residents of anoxic basaltic subseafloor fluids. Direct cell enumeration of samples collected from U1362A and U1362B revealed an elevated biomass compared to samples at these locations from previous years, demonstrating that high-quality borehole fluid samples may range up to ~45,000 cells x ml-1. This analysis helps to constrain subseafloor biomass estimates, reveals its variability in both short and long temporal scales, and provides a glimpse of the variability in microbial community structure that spans from hours to years.