Pressure-Retaining Sampler and High-Pressure Systems to Study Deep-Sea Microbes Under in situ Conditions

The pelagic realm of the dark ocean is characterized by high hydrostatic pressure, low temperature, high-inorganic nutrients, and low organic carbon concentrations. Measurements of metabolic activities of bathypelagic bacteria are often underestimated due to the technological limitations in recovering samples and maintaining them under in situ environmental conditions. Moreover, most of the pressure-retaining samplers, developed by a number of different labs, able to maintain seawater samples at in situ pressure during recovery have remained at the prototype stage, and therefore not available to the scientific community. In this paper, we will describe a ready-to-use pressure-retaining sampler, which can be adapted to use on a CTD-carousel sampler. As well as being able to recover samples under in situ high pressure (up to 60 MPa) we propose a sample processing in equi-pressure mode. Using a piloted pressure generator, we present how to perform sub-sampling and transfer of samples in equi-pressure mode to obtain replicates and perform hyperbaric experiments safely and efficiently (with <2% pressure variability). As proof of concept, we describe a field application (prokaryotic activity measurements and incubation experiment) with samples collected at 3,000m-depth in the Mediterranean Sea. Sampling, sub-sampling, transfer, and incubations were performed under in situ high pressure conditions and compared to those performed following decompression and incubation at atmospheric pressure. Three successive incubations were made for each condition using direct dissolved-oxygen concentration measurements to determine the incubation times. Subsamples were collected at the end of each incubation to monitor the prokaryotic diversity, using 16S-rDNA/rRNA high-throughput sequencing. Our results demonstrated that oxygen consumption by prokaryotes is always higher under in situ conditions than after decompression and incubation at atmospheric pressure. In addition, over time, the variations in the prokaryotic community composition and structure are seen to be driven by the different experimental conditions. Finally, within samples maintained under in situ high pressure conditions, the active (16S rRNA) prokaryotic community was dominated by sequences affiliated with rare families containing piezophilic isolates, such as Oceanospirillaceae or Colwelliaceae. These results demonstrate the biological importance of maintaining in situ conditions during and after sampling in deep-sea environments.