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Finally, sub-sampling normalization, alpha and beta diversity were characterized by Phyloseq, an open-source software package project for R www. In this article, we describe a field application using the pressure-retaining sampler and high-pressure systems that we developed. The prokaryotic heterotrophic production PHP measurements were performed to characterize the prokaryotic community lifestyle status with respect to hydrostatic pressure Tamburini et al.

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PHP was 3. Thus, the natural 3, m-depth prokaryotic assemblage sampled can be defined globally as piezophilic that means adapted to in situ high pressure conditions according to Tamburini et al. This global adaptation of the prokaryotic assemblage to pressure is supported by the prokaryotic respiration rate PR measurements derived from oxygen consumption rates within the two HPBs during the incubation experiment.

One set of samples was maintained at in situ hydrostatic pressure HP and the other was decompressed and incubated at atmospheric pressure DEC. Table 1 and plots presented in Figure 4 summarize the O 2 -consumption results. Results in Table 1 show that for each incubation experiment, PR was between 1.

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  • Furthermore, the sampling strategy of the incubation experiments was adjusted using real-time monitoring of oxygen concentrations. The amplicon sequencing followed by trimming and normalization processes generated 16S rDNA and 16S rRNA high quality reads average length bp per sample Table 1. The decrease of the specific richness during the experiment was indicated by the Simpson index, which increases from 0.

    Beyond the pitfalls linked to the overestimation or underestimation of OTUs abundances, this result suggests that the active community corresponded to almost the entire resident community.


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    In contrast, the shifts observed in prokaryotic community structure over time are driven by the different experimental conditions, confirming a previous study Wannicke et al. Indeed, HP vs.

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    • NMDS ordination plot Bray—Curtis distance matrix from incubation experiment performed with deep-seawater sample maintained at in situ condition HP and after decompression and incubated at atmospheric pressure DEC. The NMDS stress is equal to 0. Figure 6 shows the shifts in prokaryotic community structure from the ten most abundant families corresponding to four classes during the course of the incubation experiments. The absence of Archaea genes sequenced in the incubation experiment is confirmed by the RT-qPCR quantification showing more than 4-orders of magnitude between the number of Archaea and Bacteria gene copies per milliliter data not shown.

      The family Oceanospirillaceae is one of the rare families containing piezophilic isolates Cao et al. Bar plot of the 10 most abundant families into the four main classes during incubation experiments of deep-seawater samples maintained at in situ pressure conditions HP and after decompression and incubated at atmospheric pressure conditions DEC.

      Under DEC conditions, this decrease was in favor of Bdellovibrionaceae family Deltaproteobacteria which is mainly represented by the genus OM27, previously described as bacterial predatory Fuchs et al. This result suggests that high hydrostatic pressure could initiate growth repression of these microorganisms within this family. It is widely known that the deep-sea waters are depleted in carbon and energy sources.

      Consequently, deep-sea microorganisms harbored unique metabolic capabilities with respect to the degradation of complex organic matter and were supported by genomic and transcriptomic analysis Vezzi et al. Conserving the in situ conditions when sampling the deep ocean is becoming a major concern for the scientific community and many have tried to find solutions to sample without decompressing the samples. Likewise, McNichol et al. Peoples et al.

      Finally, Parkes et al. Based on the prototype proposed by Bianchi et al. The various improvements implemented promote the success of high-pressure sampling, sub-sampling, and transfer of samples in equi-pressure mode in replicate. In addition, it is also possible to directly measure oxygen consumption at high hydrostatic pressure and to use radiolabeled compounds in accordance with health and safety standards. Such methodology can be an outstanding tool in isolating new piezophilic strains without any pressure losses during the experiment.

      Apart from collecting samples and maintaining them under in situ conditions, it is important to note that this system and specifically HPBs are versatile. HPBs and PPGs can be used to perform laboratory experiments such as for sinking particle simulation experiments Tamburini et al.

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      The incubation experiment, presented in this study, is an example of a field application using the pressure-retaining sampler and associated high-pressure systems. This illustrates its fully operational use during our field experiment dedicated to deep-sea microbial oceanography. Using prokaryotic diversity and community structure based on 16S rDNA- and rRNA-sequences, we have shown that the diversity decreased in both HP and DEC conditions and that the community structure evolved differently according the pressure conditions.

      Further studies will be conducted to reveal the metabolic pathways and microbial taxa involved in the biogeochemical transformation of the organic matter in the dark ocean, as many important ecological and biogeochemical processes, linked to the biological carbon pump, take place in this the largest habitat of the biosphere. MG and CT wrote the initial draft and coordinated the drafting of the paper, and participated in the experimental design.

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      SG and PB participated in molecular biological work. MG and FA performed sequencing analyses. All authors contributed to the discussion and the writing of the paper. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Gojak and K. Authors also thank D. Lefevre for discussions on oxygen measurements using oxygen planar optodes within high-pressure bottles.

      We thank F. Le Moigne for discussions and T. Bentley for English improvement. We thank the captains and crews of RV Pourquoi Pas? Guieu and K. Top end cap with optic measurement. In this experiment, we have used TA6V HPBs to follow the oxygen concentration during the experiments both at atmospheric and in situ pressure conditions. Nylon wire; 2. Stainless-steel trigger; 3. Stainless-steel frame for valve; 4. Modified handle to link trigger at spring; 6. In this view sampling valve is set in close position before sampling A.

      This mobile laboratory has been constructed in a 20 feet container. This ratio enables us to estimate a proportion of active prokaryote for each sample. The black line is bar. OTU are distributed around the line which indicates that the microbial communities identified in the sample are also active. A During the descent of the CTD-carousel. The inlet-valve is in close position; B During the sampling at the chosen depth after firing the CTD-Carousel and the inlet-valve is in open position.

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      The numbers are the same than in Figure 1: 7. HPB1 is the high-pressure bottle containing the 3, m-depth sample maintained under in situ pressure conditions. HPB2 is the high-pressure bottle containing radiolabeled compounds, such as, 3 H-Leucine to measure prokaryotic heterotrophic production PHP. National Center for Biotechnology Information , U.

      Journal List Front Microbiol v. Front Microbiol. Published online Apr 9. Marc Garel 1 Aix Marseille Univ. Patricia Bonin 1 Aix Marseille Univ. Sophie Guasco 1 Aix Marseille Univ. Marie Roumagnac 1 Aix Marseille Univ. Nagib Bhairy 1 Aix Marseille Univ. Fabrice Armougom 1 Aix Marseille Univ. Christian Tamburini 1 Aix Marseille Univ. Author information Article notes Copyright and License information Disclaimer. This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology. Received Dec 7; Accepted Feb The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

      No use, distribution or reproduction is permitted which does not comply with these terms. TIFF 4. TIFF K. TIFF 2.

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      TIFF 93K. Abstract The pelagic realm of the dark ocean is characterized by high hydrostatic pressure, low temperature, high-inorganic nutrients, and low organic carbon concentrations. Keywords: pressure-retaining sampler, deep sea, in situ sampling, prokaryotic activities, prokaryotic diversity.

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      Open in a separate window. Figure 1. Cleaning Procedure HPBs are washed with pressurized milliQ water using a pre-cleaned stainless-steel pressurized container. Figure 2. Transfer and Subsampling in Equi-Pressure Mode An important improvement was made in transferring the main sample and several sub-samples obtained with the HPSU under an equi-pressure mode maintaining at all times the high hydrostatic pressure, using a piloted pressure generator PPG. Figure 3. Figure 4. Results and Discussion In this article, we describe a field application using the pressure-retaining sampler and high-pressure systems that we developed.

      Prokaryotic Activity Measurements The prokaryotic heterotrophic production PHP measurements were performed to characterize the prokaryotic community lifestyle status with respect to hydrostatic pressure Tamburini et al. Figure 5. Figure 6. Simulation Methods in Econometrics. Tools Request permission Export citation Add to favorites Track citation.

      Share Give access Share full text access. Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article. Citing Literature. Volume 1 , Issue 1 June Pages Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure. HEAR stands for highlight, explain, apply, and respond.

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