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

Abstract

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.

In situ observations and modelling revealed environmental factors favouring occurrence of Vibrio in microbiome of the pelagic Sargassum responsible for strandings

“…The extractions were performed twice with an equal volume of phenol/chloroform/isoamyl alcohol pH8 (25/24/1), followed by a treatment with chloroform/isoamyl alcohol (24/1) and DNA precipitation by isopropanol. For ribosomal diversity analysis, the V4 region of the bacterial and archaeal 16S rDNA genes were amplified and analysed as described in Garel et al (2019) (see detail in Suppl Mat.). Raw sequence data are available at the NCBI Sequence Read Archive under bio project accession PRJNA597297.…”

Exploring the Deep Marine Biosphere: Challenges, Innovations, and Opportunities

“…Therefore, several pressure-retaining devices have been developed to enable sampling without decompression (Jannasch et al, 1973;Yayanos, 1977;Bianchi et al, 1999;Tamburini et al, 2013). Recently, Tamburini's team used a pressure-retaining sampler in the bathypelagic zone to measure prokaryote activity under in situ pressure conditions, and demonstrated that decompression suppresses both piezophilic metabolisms and water column community composition (Garel et al, 2019). A similar pressure-retaining sampler was developed as part of DCO's PRIME (Piezophile Research Instrumentation for Microbial Exploration) Facility.…”

“…Unfortunately, this setup requires decompression during subsampling, potentially suppressing growth rates and cell viability (Cario et al, 2016a;Rogers et al, 2016a, Oliver, 2019. Variable-volume reactors, like the PUSH and others (Seyfried et al, 1979;Bianchi et al, 1999;Cario et al, 2016a;Rogers et al, 2016b;Garel et al, 2019;Oliver, 2019; Supplementary Table S1), can overcome this limitation, and have recently been used for batch cultivation without decompression, identifying two pressure-tolerant strains previously thought to be pressure-sensitive (Cario et al, 2016a;Oliver, 2019). This technology has been extended to allow for high-pressure transfer (Oliver, 2019), a crucial step in replicating traditional liquid culture microbial cultivation and expanding the possibilities for high-pressure isolation from enrichment cultures.…”

“…Molecular techniques, which are often used to give a more robust picture of microbial diversity compared to cultivationbased approaches, have indeed revealed that subsurface microbial diversity is broader than that represented by the cultivated piezophiles (Orsi et al, 2013). However, recent work suggests that even molecular techniques can suffer from decompression bias (Garel et al, 2019). Nonetheless, continued application of next-generation -omics techniques could further elucidate the diversity and activity of the subsurface microbiome (Nunoura et al, 2010), and be used to improve culturing efforts.…”

Review and Analysis of Key Techniques in Marine Sediment Sampling

“…Sampling in a high-pressure, low-temperature water environment will also cause changes in the pressure and temperature of the sample, leading to gas phase dissolution, component loss, decomposition of organic matter, death of barophilic microorganisms, changes of chemical gradients, changes of oxidation states of variable ions, etc. [9,10]. Therefore, identifying how to provide the most primitive sediment samples for human activities and scientific research has remained the focus of research worldwide.…”