fmicb-10-01497 July 16, 2019 Time: 16:31 # 1 ORIGINAL RESEARCH published: 16 July 2019 doi: 10.3389/fmicb.2019.01497 Hydrostatic Pressure Helps to Cultivate an Original Anaerobic Bacterium From the Atlantis Massif Subseafloor (IODP Expedition 357): Petrocella atlantisensis gen. nov. sp. nov. Marianne Quéméneur1, Gaël Erauso1, Eléonore Frouin1, Emna Zeghal1, Céline Vandecasteele2, Bernard Ollivier1, Christian Tamburini1, Marc Garel1, Bénédicte Ménez3 and Anne Postec1* Edited by: 1 Aix-Marseille Université, Université de Toulon, CNRS, IRD, MIO UM110, Marseille, France, 2 INRA, US 1426, GeT-PlaGe, Isabelle Daniel, Genotoul, Castanet-Tolosan, France, 3 Université de Paris, Institut de Physique du Globe de Paris, CNRS UMR 7154, Paris, Université Claude Bernard Lyon 1, France France Reviewed by: Rock-hosted subseafloor habitats are very challenging for life, and current knowledge Ida Helene Steen, University of Bergen, Norway about microorganisms inhabiting such lithic environments is still limited. This study Lotta Purkamo, explored the cultivable microbial diversity in anaerobic enrichment cultures from cores Geological Survey of Finland, Finland recovered during the International Ocean Discovery Program (IODP) Expedition 357 from *Correspondence: ◦ Anne Postec the Atlantis Massif (Mid-Atlantic Ridge, 30 N). 16S rRNA gene survey of enrichment ◦ [email protected] cultures grown at 10–25 C and pH 8.5 showed that Firmicutes and Proteobacteria were generally dominant. However, cultivable microbial diversity significantly differed Specialty section: This article was submitted to depending on incubation at atmospheric pressure (0.1 MPa), or hydrostatic pressures Extreme Microbiology, (HP) mimicking the in situ pressure conditions (8.2 or 14.0 MPa). An original, strictly a section of the journal anaerobic bacterium designated 70B-AT was isolated from core M0070C-3R1 (1150 Frontiers in Microbiology meter below sea level; 3.5 m below seafloor) only from cultures performed at 14.0 MPa. Received: 22 February 2019 Accepted: 14 June 2019 This strain named Petrocella atlantisensis is a novel species of a new genus within Published: 16 July 2019 the newly described family Vallitaleaceae (order Clostridiales, phylum Firmicutes). It Citation: is a mesophilic, moderately halotolerant and piezophilic chemoorganotroph, able to Quéméneur M, Erauso G, Frouin E, Zeghal E, Vandecasteele C, grow by fermentation of carbohydrates and proteinaceous compounds. Its 3.5 Mb Ollivier B, Tamburini C, Garel M, genome contains numerous genes for ABC transporters of sugars and amino acids, Ménez B and Postec A (2019) and pathways for fermentation of mono- and di-saccharides and amino acids were Hydrostatic Pressure Helps to Cultivate an Original Anaerobic identified. Genes encoding multimeric TFeFeU hydrogenases and a Rnf complex form the Bacterium From the Atlantis Massif basis to explain hydrogen and energy production in strain 70B-AT. This study outlines Subseafloor (IODP Expedition 357): Petrocella atlantisensis the importance of using hydrostatic pressure in culture experiments for isolation and gen. nov. sp. nov. characterization of autochthonous piezophilic microorganisms from subseafloor rocks. Front. Microbiol. 10:1497. doi: 10.3389/fmicb.2019.01497 Keywords: Atlantis Massif, subseafloor, oceanic crust, serpentinization, anaerobic culture, hydrostatic pressure Frontiers in Microbiology | www.frontiersin.org 1 July 2019 | Volume 10 | Article 1497 fmicb-10-01497 July 16, 2019 Time: 16:31 # 2 Quéméneur et al. Petrocella atlantisensis gen. nov. sp. nov. INTRODUCTION communities may vary depending on the age of the lithosphere, its alteration and the hydrothermal activity. The subseafloor biosphere remains largely unexplored, although The emblematic LCHF located on top of the Atlantis estimated as a huge reservoir for prokaryotic life (Whitman Massif, at 800 meters below sea level (mbsl), exhibits high et al., 1998; Orcutt et al., 2011; Kallmeyer et al., 2012). carbonate chimneys discharging alkaline hydrothermal fluids at Exploration of the rock-hosted subseafloor biosphere is especially moderate temperature (∼ pH 11 and 90◦C) and high levels very challenging and carried out through costly ocean drilling of dissolved H2 (1-15 mM) and CH4 (1-2 mM) as the most programs, which still must face several technical difficulties, obvious manifestation of underground serpentinization reactions such as poor core recovery and microbial contamination. In (Schrenk et al., 2013). The unique microbial communities addition, the very low microbial cell density, around 104 living inside the porous chimney structures are dominated cells per gram of rock at North Pond (Jørgensen and Zhao, by a single Methanosarcinales phylotype (Archaea), with 2016) or even less in the Atlantis Massif (Früh-Green et al., Proteobacteria and Firmicutes (Bacteria)(Schrenk et al., 2013). 2018) (respectively 22 and 30◦N along the Mid-Atlantic Ridge), This peculiar ecosystem is assumed to picture an “open the usually low growth rate, and the lack of knowledge on window on deep serpentinizing hydrothermal system” (Lang the physiology and metabolism of the prokaryotes living in et al., 2018). However, due to the lack of core samples these extreme environments, hamper the attempt at cultivating from the LCHF basement, direct experimental evidences them. As a result, the subseafloor prokaryotic cultivability was supporting this assumption are missing. Only one study on estimated below 0.1% of total microscopically counted cells the microbiology of the Atlantis Massif (IODP Expeditions (D’Hondt et al., 2004). Moreover, the hydrostatic pressure is an 304-305, Hole U1309D) reports that the gabbroic layers host important physical parameter of these deep-sea environments a low diversity of proteobacterial lineages (Alpha-, Beta- and but was often neglected in previous subseafloor cultivation Gammaproteobacteria) hypothetically degrading hydrocarbons studies. To date, most of the enrichment tests performed on and fixing carbon and nitrogen with the potential for anaerobic rocks recovered during ocean drilling programs were made at respiration of nitrate, sulfate and metals (Mason et al., 2010). To atmospheric pressure and failed to obtain microbial growth after date no cultivated microorganism has been reported from these first incubations or subcultures (Santelli et al., 2010; Hirayama ecosystems to attest the occurrence of these metabolisms, and et al., 2015). Both hydrostatic and lithostatic pressures in deep- most of the deep-subsurface microorganisms detected so far were sea and deep subseafloor environments (increasing by about 10 refractory to cultivation. and 30 MPa km−1, respectively (Schrenk et al., 2010)) have an The primary goal of this study was to explore the diversity impact on microbial growth, metabolism and physiology, thus on of cultivable prokaryotic communities of the Atlantis Massif the cultivability of microorganisms (Bartlett et al., 2007; Lauro subseafloor from a unique collection of rock cores composed in and Bartlett, 2007; Parkes et al., 2009; Takai, 2011; Picard and various proportions of carbonate, basalt, serpentinized peridotite Daniel, 2013). Moreover, the importance of high HP for deep-sea and gabbro (Figure 1 and Supplementary Figure 1). To increase microorganisms cultivation is now well established (Tamburini our chance of success, we used a high-pressure incubation system et al., 2013). In this work, HP incubation systems were used to (Figure 1) to mimic in situ HP at the sampling sites and the cultivate and study physiology of microorganisms inhabiting the various anaerobic metabolisms likely to be present were targeted underexplored oceanic crustal biosphere. based on literature surveys (Mason et al., 2010; Schrenk et al., The Atlantis Massif, a prominent underwater oceanic core 2013). We used next-generation sequencing (NGS) of 16S rRNA complex of nearly 4 000 m high, hosts the famous Lost City gene amplicons to explore the cultivable microbial diversity Hydrothermal Field (LCHF) (Kelley et al., 2005). It is composed of Atlantis Massif core samples incubated at atmospheric or of deep crustal (gabbro) and upper mantle rocks (peridotite) in situ HP. We report novel anaerobes isolated from rock-hosted that have been exposed at the ocean floor as a result of tectonic subseafloor ecosystems and describe the phenotypic and genomic plates drifting and large active faulting (Früh-Green et al., 2018). features of strain 70B-AT, the first isolate from the Atlantis Serpentinization of ultramafic mantle rocks by deeply circulating Massif subseafloor obtained from high-pressure cultures. Finally, seawater produces heat and generates alkaline fluids enriched in we propose it to represent a novel species of a new bacterial hydrogen (H2), methane (CH4), short-chain alkanes and small genus within the newly described family Vallitaleaceae (order organic acids, representing possible carbon and energy sources Clostridiales, phylum Firmicutes). to fuel life in the absence of light and contributing to global biogeochemical cycles (Levin et al., 2016). Such environmental conditions and ecosystems may have prevailed on early Earth MATERIALS AND METHODS or other planets (Martin et al., 2008). In this context, the main objective of the IODP Expedition 357 “Atlantis Massif Rock Sample Collection Serpentinization and Life” was to explore the extent and activity A unique set of rock samples was recovered during IODP of the subseafloor biosphere in a young ultramafic substratum Expedition 357 “Atlantis Massif Serpentinization