Pressure-Retaining Sampler and High-Pressure Systems to Study Deep-Sea Microbes Under In Situ Conditions

Deep ocean, poorly sampled (< 0.01%), is characterized by high hydrostatic pressure, low temperature, high inorganic nutrients and low organic carbon concentrations. Measurements of metabolic activities of bathypelagic prokaryotes are generally underestimated due to the technological limitations in recovering samples and maintaining them under in situ environmental conditions (high hydrostatic pressure, temperature,etc.). Here, we will describe a ready-to-use pressure-retaining sampler, which can be adapted to use on a CTD-carousel sampler. As sampling under high hydrostatic pressure is often not enough, we also propose a complete workflow for further incubations maintaining all the time the high hydrostatic pressure. As proof of concept, we describe a field application with samples collected at 3000 m-depth in the Mediterranean Sea. Sampling, sub-sampling, transfer and incubations were performed under in situ 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 high hydrostatic pressure 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 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.