Extremophiles (2015) 19:597–617 DOI 10.1007/s00792-015-0742-5

ORIGINAL PAPER

Biogeochemical insights into microbe–mineral–fluid interactions in hydrothermal chimneys using enrichment culture

Nolwenn Callac1,2,3,4,8 · Olivier Rouxel5 · Françoise Lesongeur1,2,3 · Céline Liorzou4 · Claire Bollinger6 · Patricia Pignet1,2,3 · Sandrine Chéron5 · Yves Fouquet5 · Céline Rommevaux‑Jestin7 · Anne Godfroy1,2,3

Received: 12 December 2014 / Accepted: 1 March 2015 / Published online: 17 March 2015 © Springer Japan 2015

Abstract Active hydrothermal chimneys host diverse and electron donors between sulfate and iron reducers at microbial communities exhibiting various metabolisms high temperature. This approach allowed the cultivation of including those involved in various biogeochemical cycles. microbial populations that were under-represented in the To investigate microbe–mineral–fluid interactions in hydro- initial environmental sample. The microbial communities thermal chimney and the driver of microbial diversity, are heterogeneously distributed within the gas-lift bioreac- a cultural approach using a gas-lift bioreactor was cho- tor; it is unlikely that bulk mineralogy or fluid chemistry sen. An enrichment culture was performed using crushed is the drivers of microbial community structure. Instead, active chimney sample as inoculum and diluted hydro- we propose that micro-environmental niche characteristics, thermal fluid from the same vent as culture medium. Daily created by the interaction between the mineral grains and sampling provided time-series access to active microbial the fluid chemistry, are the main drivers of microbial diver- diversity and medium composition. Active archaeal and sity in natural systems. bacterial communities consisted mainly of sulfur, sulfate and iron reducers and hydrogen oxidizers with the detec- Keywords Active microbial diversity · Continuous tion of Thermococcus, Archaeoglobus, Geoglobus, Sul- enrichment culture · Deep-sea hydrothermal vent · furimonas and Thermotoga sequences. The simultaneous Hydrothermal fluid · Iron cycle · Sulfur cycle presence of active Geoglobus sp. and Archaeoglobus sp. argues against competition for available carbon sources Introduction Communicated by H. Atomi. Deep-sea hydrothermal chimneys are porous mineral struc-

Electronic supplementary material The online version of this tures that form as a result of mixing of H2S and metal-rich article (doi:10.1007/s00792-015-0742-5) contains supplementary high-temperature hydrothermal fluid with oxygenated cold material, which is available to authorized users.

* Nolwenn Callac 5 Ifremer, Centre de Brest, 29280 Plouzané, France [email protected] 6 Université de Bretagne Occidentale, IUEM, UMS3113, Place Nicolas Copernic, 29280 Plouzané, France 1 Laboratoire de Microbiologie des Environnements Extrêmes, Université de Bretagne Occidentale, UEB, IUEM, UMR 7 Laboratoire Géobiosphère Actuelle et Primitive, IPGP, 6197, Place Nicolas Copernic, 29280 Plouzané, France UMR7154, 75238 Paris, France 2 Laboratoire de Microbiologie des Environnements Extrêmes, 8 Present Address: Department of Geological Sciences, Ifremer, UMR6197, 29280 Plouzané, France Stockholm University, Svante Arrhenius väg 8, SE‑106 91 Stockholm, Sweden 3 Laboratoire de Microbiologie des Environnements Extrêmes, CNRS, UMR 6197, 29280 Plouzané, France 4 Université de Bretagne Occidentale, Domaines Océaniques IUEM, UMR6538, Place Nicolas Copernic, 29280 Plouzané, France

1 3 598 Extremophiles (2015) 19:597–617 seawater. It has been well documented that the steep tem- of microorganisms (Page et al. 2008; Wang et al. 2009; perature and geochemical gradient within the chimney Kormas et al. 2006; Takai et al. 2001). wall generate specific micro-habitats that can host large In this study, we aim to better determine which param- and diverse microbial populations (Nakagawa et al. 2005; eters drive and control the structure and activity of the Page et al. 2008; Takai et al. 2008; Karl 1995). The physi- microbial populations in hydrothermal ecosystems. To ological and phylogenetical diversities of these prokaryotes gain a better control of the physico-chemical conditions have been investigated using both cultural and molecular encountered within hydrothermal habitats, we performed a approaches to determine their ecological role and impor- continuous enrichment culture using diluted hydrothermal tance for biogeochemical cycles of carbon, sulfur, nitrogen fluid as medium and an active vent flange sample as inocu- and iron (Yamamoto and Takai 2011; Byrne et al. 2009b; lum with the ultimate goal to observe steady-state condi- Slobodkin et al. 2001; Reysenbach and Cady 2001; Kashefi tions. The chimney sample was crushed to allow vigorous et al. 2002). interactions between mineral substrate and fluid medium, Although previous studies have demonstrated that thereby minimizing the range of environmental parameters microorganisms may interact with their environment by such as fluid and mineral composition changes. promoting mineral dissolution or precipitation (Houghton In addition, the cultivation system allowed maintaining et al. 2007; Page et al. 2008; Holden and Adams 2003; constant N2/CO2/H2 atmosphere through continuous gas Houghton and Seyfried 2010), their effects on vent fluid sparging (Postec et al. 2005, 2007; Byrne et al. 2009a). composition and chimney mineralogy remain poorly Here, we choose to maintain the enrichment culture at known. According to their frequency in clone libraries 85 °C to cultivate hyperthermophilic organisms. Although and isolation in pure cultures, Epsilonproteobacteria and this temperature is lower than the maximum temperature Thermococcales are considered to be key players in sul- measured in the chimney vent fluid (i.e., up to 200 °C for fur metabolism in hydrothermal chimneys (Godfroy et al. the vent fluid trapped under the flange; Fig. 1), it is closer 1997; Teske et al. 2009; Campbell et al. 2006). Sulfate to the expected temperature of the fluid diffusing through reduction has been shown to be mediated by some Del- the flange structure during mixing with seawater. Samples taproteobacteria, Thermodesulfobacterium (Jeanthon et al. composed of the growth medium and suspended materials, 2002) and Thermodesulfatator (Moussard et al. 2004; i.e., including both free cells and particle-associated cells, Alain et al. 2010). Within Archaea, sulfate reduction is the were analyzed every day for active microbial diversity prerogative of only members of the genus Archaeoglobus based on 16S rRNA (sequencing of 16S rRNA transcripts (Burggraf et al. 1990). and Q-PCR). Filtered growth medium was also analyzed Iron metabolism is likely performed by thermophilic dis- for element (major and trace) concentrations as well as sul- similatory iron reducers such as Geothermobacter ehrlichii fate, hydrogen sulfide and organic acids concentrations. (Kashefi et al. 2003), Deferribacter (Miroshnichenko et al. This experimental approach is expected to provide new 2003; Slobodkina et al. 2009a), Thermotoga (Vargas et al. insights into thermophilic microbial community, structure 1998), Geoglobus (Kashefi et al. 2002; Slobodkina et al. and dynamic in seafloor hydrothermal systems that may 2009b) and some Thermococcus isolates (Slobodkin et al. have been overlooked in previous studies using solely in 2001). Iron oxidizers belonging to the class of Zetaproteo- situ samples. bacteria were only retrieved from the outer wall of hydro- thermal chimneys and iron-rich mat. This described class of mesophilic Proteobacteria is widespread across several Materials and methods low temperature hydrothermal environments (Kato et al. 2009, 2012; Emerson et al. 2007, 2010). To date, only one Sampling and sample description thermophilic iron oxidizer, Ferroglobus placidus, known to oxidize iron with nitrate as electron acceptor (Hafenbradl In the Southern Trough of the Guaymas Basin (Gulf of et al. 1996), was isolated from a shallow vent and repre- California, Mexico), organic-rich seafloor sediments are sentatives of this genus were detected in deep hydrothermal affected by circulation of hydrothermal fluids that result in samples (Reysenbach and Cady 2001). the formation of sulfide and carbonate-rich chimneys at the It has been recently demonstrated that mineralogy is seafloor (Von Damm et al. 1985; Simoneit and Lonsdale an important driver of the bacterial community composi- 1982; De la Lanza-Espino and Soto 1999). tion (Toner et al. 2013) in inactive hydrothermal chimneys. An active hydrothermal flange sample (Sample In active chimney, at high temperature, it seems that both 1747-BIG03E01) was collected during the oceanographic micro-niches resulting from the steep and dynamic geo- cruise BIG (R/V l’Atalante June 2010) on “Rebecca’s chemical and physical gradients between vent fluid and sea- Roost” site (27°00.634′ N 111°24.405′ W; 1997 m depth) water, and chimney mineralogy may affect the distribution in the Southern Trough using the manned submersible

1 3 Extremophiles (2015) 19:597–617 599

Fig. 1 a Photograph of Rebec- ca’s Roost chimney and flange structure; b Bottom view of the flange showing the mirror-like interface between trapped hydrothermal fluid underneath the flange and seawater. All photos taken with the submers- ible Nautile during the BIG cruise (Dive 1745); c Schematic diagram of a flange showing the circulation of the hydrothermal fluid trapped underneath the flange and passing through the porous mineral matrix (adapted from Tivey and Delaney 1986; Tivey et al. 1999; Kerr 1997)

Nautile (dive 1747). The flange sample is similar to already in 3 sterile 5 l pouches. Each pouch was sub-sampled for described horizontal structures located near the base of shore-based chemical analysis. All pouches were degased hydrothermal spires (Tivey et al. 1999; Woods and Delaney using N2 to obtain anaerobic conditions. Pouches 2 and 3 1992; Peter and Scott 1988). As illustrated in Fig. 1, the were stored at 10 °C during, respectively, 8 and 19 days hydrothermal fluid is trapped under the horizontal flange while pouch 1 was used immediately as fluid medium. structure, forming a mirror-like contact with cold seawater. Before used as medium, the fluid of each pouch was trans- The temperature of the fluid circulating through the flange ferred aseptically into a sterile 5 l Nalgene bottle. was around 100 °C which is lower than the maximum of The first pouch served as medium until culture day 8, 201 °C measured in focused hydrothermal discharge point. the second until day 19 and the third until the end of cul- The sample was brought aseptically to the surface into ture. Hydrothermal fluid, from the same hydrothermal site, an insulated box. Immediately upon recovery, the sample was also collected near the flange outflow using titanium was crushed to grain size <1 cm and sub-sampled in an syringes (dive 1772) to determine the end-member high- anaerobic chamber onboard under N2/H2/CO2, 90:5:5 gas temperature hydrothermal fluid composition before its dilu- atmosphere. Crushed fragments were subsequently stored tion with background seawater. As explained below, chemi- 1 in sterile vials filled with sterile NaCl solution at 23 g L− , cal data indicate that the hydrothermal fluid used as growth under anaerobic condition. Sub-samples were stored at medium (i.e., PEPITO pouches) was diluted at about 65 % 1 4 °C for cultivation, frozen at 20 °C for mineralogical with ambient seawater. Before used, rezasurin (0.5 mg L− ) − analyses or frozen at 80 °C for microbial diversity molec- was added as anaerobiosis throughout the culture. Dur- − ular survey. ing all the duration of the culture, the medium remained anoxic. Enrichment culture procedure using gas‑lift bioreactor The bioreactor was inoculated with 300 mL of sus- pended slurry of crushed flange sample (1747-BIG03E01). The medium consisted of diluted hydrothermal fluid, To avoid a washout of cells before they start growing, the collected with an in situ fluid sampling system, hereaf- bioreactor was maintained in batch culture mode during the ter referred to as “PEPITO” device (Sarradin et al. 2009) first 36 h after inoculation. in the seawater/fluid mixing zone. During sampling, the minimal temperature recorded was 60 °C while maximum Culture conditions and bioreactor sampling was 190.1 °C which is close to the temperature of about 200 °C measured in the hydrothermal fluid trapped under The enrichment culture was carried out during 23 days the flange, close to mirror-like interface. (36 h in batch mode and 21 days in continuous mode cul- Care was taken to sample-diluted hydrothermal fluid ture), at 85 °C in an 850 mL glass gas-lift bioreactor as pre- from the same area where the chimney was sampled. viously described (Godfroy et al. 2006; Byrne et al. 2009a; Hydrothermal fluid samples were recovered sequentially Postec et al. 2007). Briefly this equipment allows to perform

1 3 600 Extremophiles (2015) 19:597–617 cultures in controlled temperature and pH conditions and culture samples by ion exchange chromatography (Lazar under continuous gas sweeping. The pH was monitored with et al. 2011). Dissolved hydrogen sulfide was measured an analogic control system (Infors HT™ system), associated using spectrophotometric method (Cline 1969). Concentra- with a temperature-controlled circulation bath associated tions of major elements were measured using Inductively with a PTFE-covered PT100 temperature probe (PT 100, Coupled Plasma-Atomic Emission Spectrophotometer Radley™, UK) and a gel pH electrode (Metler Toledo™). (ICP-AES, Ultima 2, Horiba JobinYvon™) and concen- The pH regulation was mediated by addition of a 0.2 M trations of trace elements were measured using High-Res- HCl or 0.2 M NaOH solutions added via the regulation sys- olution ICP Mass Spectrometer (HR-ICP-MS, Element 2, tem pumps. During the 6 first days of culture, the pH was Thermo Scientific™), both operated at the Pôle Spectromé- maintained at 6.8. Then, due to a pH probe failure at day 6, trie Ocean (PSO) in Brest (France). Prior to elemental anal- the pH decreased down to 3.8 during a few hours as a result ysis, samples were acidified at least 1 month in advance at 1 of the uncontrolled addition of HCl from the automatic 0.28 mol L− HNO3 using concentrated ultra-pure reagent titrator. The pH was then allowed to return to 6.8 within grades. Solutions for ICP-AES and ICP-MS analysis were 1 24 h by the addition of fresh medium solution. During the diluted 100-fold with 0.28 mol L− HNO3 using 2-stages last 5 days of culture time, the pH remained stable at 7.6. sub-boiled distilled acid. Three water solution standards, Consequently to the pH probe failure, the pH was measured Slew 3, Cass 4 and Nass 5, were also prepared along with every day with a portable pH meter (at room temperature). the samples. For both ICP-AES and ICP-MS analysis, two Fresh medium was continuously provided to the biore- sets of calibrating standards were used by adding multi- 1 actor with a dilution rate of 0.022 h− using Masterflex™ elemental standard solutions in either pure Milli-Q water or 1 1 peristaltic pump, corresponding to about 440 mL day− , 100-fold diluted Cass 4 acidified to 0.28 mol L− HNO3. which is about half of the culture per day. As explained Detection limits have been determined through repeated above, the fresh medium has been rendered anoxic by spar- measurements of blank solution. ging with oxygen-free N2. The effluent culture was recov- ered in sterile 1 L Nalgene bottle stored at 6 °C. Nucleic acids extractions and PCR amplifications

To maintain anaerobic condition and provide H2 as pos- sible electron donor and CO2 as possible carbon source, the Total nucleic acids (DNA and RNA) were extracted from bioreactor was continuously sparged with an N2/H2/CO2 the initial chimney sample (in duplicate from 5 g of the 1 (75:20:5) (0.1 v v min− ) gas mixture. crush raw flange), from suspended materials from the Daily samples from the liquid culture fraction were medium (recovered on filter) and from the mineral fraction directly collected from the bioreactor to determine various recovered at the end of the culture (in duplicate from 2.5 g parameters. Cell density was determined on-board by direct of remaining solid fraction). The extractions were per- cell counting using a Thoma chamber (depth 0.02 mm) formed on frozen samples (i.e., crushed samples or filters), viewed with an Olympus™ BX60 phase contrast microscope in TE-Na 1 lysis buffer (Tris–HCl pH 8, 100 mM; NaCl × (X400). For DNA and RNA analysis, the entire volume of 100 mM; EDTA pH 8, 50 mM) which Sarkosyl (1 % final), 1 medium recovered over a 24 h period was filtered through SDS (1 % final) and proteinase K (0.4 mg mL− final) were 0.22 µm (isopore™, Millipore) membrane filtered and filters added and incubated at 55 °C for 3 h. Supernatant was were stored at 80 °C until extraction. For dissolved major removed and treated once with equal volume of PCI (phe- − and trace element analysis, 5 mL of culture samples was fil- nol/chloroform/isoamyl alcohol: 25/24/1, v/v/v) and once tered through 0.45 µm (Millex®, Millipore) membrane in with an equal volume of chloroform. Then, nucleic acids LDPE acid-cleaned bottles and stored at 20 °C. For hydro- were precipitated in 0.7 volume of isopropanol and washed − gen sulfide quantification, 10 mL of culture was filtered in an equal volume of 96 % ethanol. After centrifugation at through 0.45 µm (Millex®, Millipore) membrane, precipitated 10000g during 10 min, nucleic acids were dried and finally as ZnS in 25 mL evacuated septum vials containing 0.1 g of re-suspended in 200 µL of TE 1 buffer. × zinc acetate (Sigma-Aldrich™) and stored at 4 °C. For sulfate Prior to reverse transcription, RNA was extracted from 2 ® (SO4 −) analysis, 1 mL of filtered culture was acidified with the nucleic acid pool and purified using the NucleoSpin 10 µl of 65 % HNO3 and stored at 4 °C. Filtered samples for RNA II kit (MachereyNagel) according to the manufactur- organic acid analysis (i.e., acetate, lactate, propionate and for- er’s instructions. The absence of DNA was controlled by mate) were preserved at 20 °C until analysis. PCR. The reverse transcription of the RNA in cDNA was − performed using the qScript™ cDNA SuperMix (Quanta Geochemical analysis BioSciences) reagent according to the manufacturer’s instruction. Concentrations of sulfate and organic acids—acetate, lac- After RT-PCR, samples were analyzed by DGGE to tate, propionate and formate—were quantified in the diluted select appropriate samples for the cloning sequencing. The

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DGGE were done as previously as described by Postec nuclease-free sterile deionized water were added to a final et al. (2005), using the 16S cDNA as template, and the cou- volume of 25 µL. Bacterial and archaeal standard curves ples 341F-GC/915R (Casamayor et al. 2000) and 341F-GC were calibrated using tenfold dilutions ranging from 100 to 5 (Muyzer et al. 1993)/U907R (Lane et al. 1985b) as primers. 10− of DNA from Citreicella thiooxidans (Sorokin et al. A total of seven samples were chosen according to the 2005) or DNA from Methanoculleus marisnigri (Maestro- DGGE results to assess active microbial diversity. These juan et al. 1990), respectively. All reactions were realized samples include the initial crushed flange, the 36 h batch in 96-well Q-PCR plates using Applied Biosystems StepO- culture sample, day 4, 7, 15, 21 samples and the mineral nePlus™ Instrument and its software. Q-PCR detection of fraction recovered at the end of the culture. 16S rRNA genes in sample and in tenfold dilution series The 16S rRNA transcripts were amplified using A8F/ of standard curve was run in triplicate. In Q-PCR, ampli- ARC915R primers combination (Table S1) (Kolganova fication efficiency was 96.2 %, slope was 3.42 and R2 − et al. 2002; Casamayor et al. 2000) and E8F/U907R prim- was 0.998 for Archaea and 109 % for the PCR efficiency, ers set (Table S1) (Lane 1991; Lane et al. 1985a) as spe- 3.11 for the slope and 0.998 for the R2 for Bacteria. − cific archaeal or bacterial domains’ primers in 30 cycles The abundance of microorganisms was estimated from at an annealing temperature of 58 and 52 °C, respectively. the Q-PCR data and taking into account the average of the Before cloning, PCR products were excised from agarose 16S rRNA gene per cell (1.86 for the Archaea and 4.1 for gel and gel purified using the NucleoSpin® Gel and PCR the Bacteria) (Lee et al. 2009). Clean-up kit (Macherey-Nagel) according to the manufac- The alpha diversity of each sample was calculated in turer’s instructions. Clone libraries were carried out with EstimateS (Version 9.1.0) (Colwell 2013). Thus, species pGEM®-T cloning kit (Promega) following manufacturer’s richness was estimated using the ACE (Abundance-based recommendations. Then, clones were cultured and treated coverage), and the Chao 1 (non-parametric estimator) for sequencing at GATC Biotech (Konstanz, Germany) and the species evenness was evaluated using the Shan- using M13 primers. Sequences were compared with those non index and the inverse Simpson indices [estimator non available on NCBI BLAST network service to determine affected by sampling (Campbell et al. 2013)]. As well, the their phylogenetic affiliations and aligned, edited and ana- Good’s coverage was also calculated (Good 1953). lysed using Geneious Pro version 5.6 software. Phyloge- To examine the influence of the culture time, the vari- netic trees were constructed using the MEGA 5 program. ation in the culture (e.g., pH drop, concentrations) or the The robustness of inferred topologies was tested using type of samples (e.g., raw, liquid or solid fraction) on 1000 bootstrap resampling of the trees calculated on the both archaeal and bacterial diversity, we used the UniFrac basis of neighbor-joining algorithm (Saitou and Nei 1987) computational tool (Lozupone et al. 2006). The habitats, with Kimura two-parameter correction matrix (Kimura defined by sample type and time of sampling, were clus- 1980). tered using the jackknife environment clusters analysis tool Negative controls were done during nucleic acid extrac- with 100 permutations. tions performed without sample, during RNA purification and during the reverse transcription. For each control, no PCR products were detected. Results The bacterial and archaeal sequences reported in this study have been deposited to GenBank nucleotide Geochemistry of the hydrothermal fluid medium and of sequence databases under accession number KF463052 to the flange KF463137. For all samples (i.e., chimney sample before/after cul- Geochemical characteristics of the hydrothermal fluid at ture and suspended culture samples), bacterial and archaeal Rebecca’s Roost (dives 1747 and 1772) are a circumneu- 16S rRNA gene copies’ quantification in whole DNA tral pH (6.5 at 1 atm, 25 °C) and elevated concentrations of extract was performed by Q-PCR, using SybrGreen fluo- dissolved Ca, K, Fe and Mn relative to local deep seawater rochrome system. Detection of bacterial or archaeal 16S (Table S2). End-member vent fluid composition, as deter- rRNA gene was determined using specific primers’ set mined by extrapolating element composition at Mg 0, = BACT1369f/BACT1492r (Table S1) (Suzuki et al. 2000) is generally similar to previous studies at Guaymas Basin and ARC787f/ARC1059r (Table S1) (Yu et al. 2005) in 35 (Von Damm et al. 1985). It should be noted, however, that cycles at an annealing temperature of 60 °C for the Bac- besides fluid sample 1772-B, hydrothermal fluids sampled teria or 58 °C for the Archaea. Q-PCR conditions were: with Ti syringes are largely diluted with seawater with Mg 500 nM of each archaeal primer and 600 nM for bacterial concentration >30 mM compared to about 52 mM for back- primers, 6.25 µL of PerfeCta™ SYBR® Green SuperMix, ground seawater. Maximum dissolved Fe concentration of ROX™ (Quanta Biosciences), 5 µL of DNA template and 53 µM is similar to dissolved Fe concentrations measured

1 3 602 Extremophiles (2015) 19:597–617 at other hydrothermal vents along the Guaymas Basin (Von anhydrite, ferrihydrite and sulfide minerals such as pyrrho-

Damm et al. 1985). Maximum H2S concentration at around tite (Table 2). This is similar to previous mineral descrip- 2 mM is, however, lower than previous studies reporting tion from Guaymas Basin (Peter and Scott 1988). The main

H2S concentrations between 3.8 and 6 mM (Von Damm difference of mineral composition before and after incuba- et al. 1985). tion is the higher abundance of calcite, and lower barite at The diluted hydrothermal fluids used as medium (i.e., the end of the experiment (Table 2). Nontronite, which has PEPITO pouches) are characterized by Mg concentrations been observed at the beginning of the experiment, had also ranging from 32 to 35 mM, suggesting an overall dilution totally disappeared at the end of the culture. of the end-member hydrothermal fluid of about 65 % with ambient deep seawater (Table S2). Acetate concentrations Active microbial communities in the initial flange in the diluted hydrothermal fluid approached the detection sample and bioreactor limit (3 µM) in pouch 1 and were found at 5.2 and 5.3 µM in pouches 2 and 3, respectively. Formate concentrations A total of 624 partial 16S rRNA sequences were obtained ranged from 17 to 20 µM while lactate and propionate were and analyzed (i.e., 192 for the raw flange, 48 for each sam- not detected. H2S concentrations remained between 2.2 and ple T36, D4, D7, D15 and D21, and 192 for the solid frac- 3.6 mM (Table S2). tion collected at the end of the culture). In the raw flange In the gas-lift bioreactor, the medium that has reacted sample, the bacterial 16S rRNA transcripts detected were with the mineral substrate during the first 36 h of culture affiliated to the Epsilonproteobacteria, Deltaproteobacte- (Table 3) is characterized by near neutral pH (6.8), high ria, Thermotogales, the Guaymas bacterial group (Teske acetate concentration (288 µM), sulfate concentration up et al. 2002), Thermodesulfobacteriales, members of the to 36 mM, low formate concentration (no lactate and pro- Cytophaga–Flexibacter–Bacteroides and the Chloroflexi pionate), and dissolved H2S below detection (Table 3). divisions (Figs. 3, S1 and S2). The active archaeal com- Dissolved trace metals, such as Fe and Mn, have lower munity (from 16S rRNA transcripts) consisted of Thermo- concentrations than those measured in the initial fluid. coccus, Archaeoglobus, Methanocaldococcus, Methano- The high salinity, as reflected by Na concentration up to thermococcus and Methanopyrus (Figs. 4, 5). According to 700 mM compared to 464 mM for background seawater the quantitative real-time PCR data (Fig. 6), Bacteria were and 484 mM for end-member hydrothermal fluids (Tables dominant not only in the flange sample but also in the cul- S2, 1), suggests that evaporation occurred during the ini- ture samples. tial stage of the experiment due to heating at 85 °C and Throughout the enrichment culture, cell densities in the 6 1 efficient gas sweeping. After 36 h, when the fluid medium liquid fraction remained low and below 10 cells mL− as has been added in continuous mode, Na together with Ca, determined by direct cell counting. However, Q-PCR data 2 9 1 SO − and other elements such as Sr decreased, mainly due indicate higher cell density, with 1.13 10 cells mL− at 4 × to the dilution of the initial medium with fresh solution the beginning of the culture (i.e., T36 sample) and increas- (Table 1; Fig. 2). A correction has been, therefore, applied ing at D19. Such differences may be attributed to a large to account for evaporation and dilution effects, as explained proportion of cells being attached or stuck on mineral par- in “Discussion”. Among the organic acids, lactate and ticles and, therefore, not visualized by direct cell counting. propionate were below detection and small quantities of Also, no decrease in the cell density was observed after the formate (below 3 µM) were measured. Acetate concentra- pH failure. tions decreased progressively from 288 µM at the begin- At 36 h of culture, the active bacterial community was ning to 2 µM at the end (Table 3). As mentioned above, the made of uncultured Epsilonproteobacteria, and to two gen- pH dropped to 3.8 at day 6, and this change was associ- era of Gammaproteobacteria: Marinobacterium sp. (99 % ated with an abrupt increase in Mn, Ca and other element similarity with Marinobacterium litorale) and Idiomarina concentrations (Table 1; Fig. 2). Due to sample preserva- sp. (98 % similarity with Idiomarina baltica). The detec- tion issue, nitrate concentration was not measured but is tion of these Gammaproteobacteria at the beginning of the likely within the range of hydrothermal fluid end-member culture that are often encountered in seawater (Kim et al. which is depleted and the background seawater measured 2007; Brettar et al. 2003) or in sediments (Huo et al. 2009) at 40 µM (McHatton et al. 1996). Hence, it is possible that could be explained by the persistence of relic nucleic acid nitrate could be used as potential electron acceptor for molecules in deceased cell as previously reported (Blaze- microbial metabolisms. wicz et al. 2013). Quantitative mineralogy analysis using XRD shows During the enrichment culture, the active bacterial com- that the flange fragment used as inoculum is essentially munity was mainly composed by Sulfurimonas (98 % composed of calcite and barite, to a lesser proportion of similarity with Sulfurimonas paralvinella; 96 % similarity nontronite (possibly associated with amorphous silica), with Sulfurimonas autotrophica) at D4, 15 and 21 and by

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Table 1 Geochemical composition, organic acid concentrations and pH measured in the medium during the culture time Pepito pouch 1 Pepito pouch 2 36h D2 D3 D4 D5 D6a D7 D8 D9 D10 D11 pH 6.8 6.8 6.8 6.8 6.9 3.8 6.3 6.7 6.9 6.7 6.5 Acetate (µM) 288 181 144 84 43 nd 18 9 8 7 3 Formate (µM) 2 1 1 0 2 nd 3 1 1 2 0.2

H2S (µM) bd bd bd bd bd bd bd bd bd bd bd Na (mM) 699.45 667.46 678.93 601.58 594.08 nd 417.65 445.44 484.03 504.36 500.51 Ca (mM) 33.04 29.50 29.60 26.60 25.43 nd 40.61 31.25 28.08 25.01 22.67 K (mM) 31.09 30.21 31.44 28.64 28.45 nd 19.49 21.36 23.13 23.78 23.25 Mg (mM) 49.58 48.71 49.85 45.54 44.28 nd 31.56 34.24 37.47 39.82 39.43 S (mM) 35.08 30.59 30.39 27.09 25.53 nd 16.36 17.99 19.98 21.96 22.10 Si (mM) 1.41 1.42 1.45 1.36 1.42 nd 1.47 1.44 1.43 1.54 1.45 Ba (µM) 5.84 5.88 nd 7.45 nd nd 11.47 nd nd 7.10 nd Sr (µM) 901.17 829.67 829.21 748.03 720.73 nd 597.39 595.39 625.92 639.23 631.51 Fe (µM) 0.57 0.09 nd 0.23 nd nd 1.61 nd nd 0.77 nd Mn (µM) 30.96 30.83 30.41 30.82 30.70 nd 202.79 100.63 67.48 43.60 31.44 Al (µM) 1.37 1.31 nd 1.35 nd nd 1.50 nd nd 1.39 nd Zn (nM) 0.20 0.24 nd 0.38 nd nd 0.19 nd nd 0.33 nd Pb (nM) 4.26 18.09 nd 4.69 nd nd 12.44 nd nd 7.61 nd Sb (nM) 863.65 553.00 nd 396.81 nd nd 168.58 nd nd 279.75 nd U (nM) 0.88 1.12 nd 1.32 nd nd 3.42 nd nd 1.55 nd Pepito pouch 2 Pepito pouch 3 D12 D13 D14 D15 D16 D17 D18 D19 D20 D21 pH 6.5 6.4 6.4 6.4 7.6 7.6 7.6 Acetate (µM) 4 nd 1 1 nd 0 nd nd 3 2 Formate (µM) 1 nd 1 1 nd 2 nd nd 0.5 0.6

H2S (µM) bd bd bd bd nd bd nd nd bd bd Na (mM) 494.57 495.22 527.07 520.27 nd 515.63 nd nd 512.74 518.51 Ca (mM) 21.74 22.18 22.91 23.42 nd 23.07 nd nd 21.75 22.40 K (mM) 22.76 22.99 24.66 24.02 nd 24.07 nd nd 23.82 24.04 Mg (mM) 39.00 39.00 41.82 41.50 nd 40.14 nd nd 40.70 41.26 S (mM) 22.24 22.61 23.73 23.89 nd 23.42 nd nd 22.86 23.23 Si (mM) 1.41 1.39 1.57 1.46 nd 1.41 nd nd 1.98 2.01 Ba (µM) nd nd nd 5.53 nd 5.71 nd nd nd 5.71 Sr (µM) 622.05 634.14 665.36 672.52 nd 654.35 nd nd 633.76 640.44 Fe (µM) nd nd nd 0.21 nd 0.34 nd nd nd 0.22 Mn (µM) 27.86 27.55 29.43 29.01 nd 28.29 nd nd 30.08 31.66 Al (µM) nd nd nd 3.54 nd 1.40 nd nd nd 1.35 Zn (nM) nd nd nd 0.24 nd 0.24 nd nd nd 0.34 Pb (nM) nd nd nd 17.27 nd 29.31 nd nd nd 21.39 Sb (nM) nd nd nd 156.51 nd 127.29 nd nd nd 130.80 U (nM) nd nd nd 0.95 nd 0.54 nd nd nd 1.91 nd not determined, bd below the detection limit (bd is 10 µM for H2S) a The day when pH falls down to 3.8

Thermotoga (99 % similarity with Thermotoga maritima) Some Betaproteobacteria, in particular sequences in the solid fraction (Figs. 3, S1 and S2). Sequences close closely related to Leptothrix, were detected at D7 (after the to Sulfurospirillum were also detected at D15. pH drop). Leptothrix phylotypes were already detected in

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Fig. 2 Time series of calcium (Ca), sulfur (S), potassium (K), magnesium (Mg), silica (Si), manganese (Mn), barium (Ba), strontium (Sr), aluminum (Al), iron (Fe), zinc (Zn), antimony (Sb) and uranium (U) con- centrations and pH during the enrichment culture. All data were corrected for evaporation and dilution by normalization all elemental concentrations to the same Na concentration at 470 mM. Note that Si, Zn, Fe are multiplied by 10 and Na, Sr and Sb are multiplied by 0.1 for scaling purposes. Ca, K, Mg, Mn, Na, S, Si and Sr were measured by ICP-AES; Al, Ba, Fe, Sb, U and Zn were measured by HR-ICP-MS

several hydrothermal systems (Reysenbach et al. 2000) and strain isolated from deep seawater collected in the South- at 75 °C in the Steep Cone Hot Spring (Yellowstone) (Ina- West Indian Ridge (Lai et al. 2011) and 99 % with Parvi- gaki et al. 2001). baculum hydrocarboniclasticum, a mesophilic strain iso- Alphaproteobacteria were detected at the end in D21 lated from hydrothermal fluid (Rosario-Passapera et al. and in the solid fraction collected at the end of the culture, 2012). In previous enrichment cultures, it was shown that including sequences closely related to the Parvibaculum Parvibaculum lavamentivorans, formerly described as an sp., sharing 99 % of similarity with Parvibaculum indi- aerobic strain, is able to grow autotrophically in anaero- cum (strain P31T) (8 % of the total clones), a mesophilic bic condition, oxidizing Fe(II) using nitrates as electron

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Table 2 Relative abundance of Mineral Chemical formula Raw flange sample Bulk sample (end of the culture) each mineral phase expressed in percentage of total abundance Anhydrite CaSO4 <5 <5 before (in situ sample) and after Calcite CaCO 65 90 enrichment culture (ex situ 3 sample) Barite BaSO4 13 <5

Pyrrhotite Fe(1-x)S 9 <5 3 Ferrihydrite (Fe +)2O3.0.5H2O <5 <5 3 Nontronite (CaO0.5.Na)0.3Fe2 +(Si.Al)4 8 0 O (OH) H2O 10 2·n Sphalerite ZnS <5 <5

Aragonite CaCO3 <5 <5 Galena PbS <5 <5

Mineral phases having their relative abundances modified after the enrichment culture are given in bold

Fig. 3 Relative abundance of the active bacterial community based on the frequency of bacte- rial 16S rRNA gene in clone libraries at different time of the enrichment culture. “Raw flange sample” corresponds to the in situ sample before culture. “Bulk” corresponds to the solid fraction remaining at the end of the culture time (i.e., ex situ). *Involved in the sulfur cycle, †Involved in the iron cycle, α Alphaproteobacteria, β Betaproteobacteria, γ Gam- maproteobacteria, δ Deltapro- teobacteria, ε Epsilonproteo- bacteria, T Thermotogales

acceptor (Straub et al. 1996; Blöthe and Roden 2009). Thermococcales (Thermococcus sp. at 36h, D4 and in the Several studies also reported the detections of Alphapro- solid fraction and Palaeococcus sp. at D21 and in the solid teobacteria in active deep-sea hydrothermal chimneys fraction) and to uncultivated Archaeoglobales (96–97 % (Olins et al. 2013; Flores et al. 2011, 2012; Kato et al. similarity with Ferroglobus placidus) (D21 and in the solid 2010), as well as in a Tunisian geothermal spring (Sayeh fraction) were also detected (Figs. 5, 6). In addition, at the et al. 2010). end of the culture (D21 and in the solid fraction), sequences For the Archaea, Geoglobus (99 % similarity with Geo- affiliated to the Desulfurococcales and to the Thermopro- globus ahangari) were detected in all samples (36h, D4, 7, teales were found. 15 and in the solid fraction), except at D21 and Geoglo- The Good’s coverage calculated for each culture sample bus were the sole archaeal phylotype detected after the was higher for the Archaea (i.e., between 0.65 and 0.93) pH drop. Sequences affiliated to Archaeoglobus (98 % and for the Bacteria (i.e., between 0.59 and 0.82) (Table 3). similarity with Archaeoglobus profundus) at 36h, D15, 21 The richness estimates with the Chao 1 and the ACE, for and in the solid fraction, and in lesser proportion to the the Archaea and the Bacteria, were higher in the culture

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Fig. 4 Relative abundance of the active archaeal com- munity based on the frequency of archaeal 16S rRNA gene in clone libraries at different time of the enrichment culture. “Raw flange sample” corresponds to the in situ sample before culture. “Bulk” corresponds to the solid fraction remaining at the end of the culture time (i.e., ex situ)

samples than in the in situ flange sample (Table 3). Glob- Discussion ally, the richness estimates increased with the culture time for both the Archaea and the Bacteria, and maximum val- Geochemical and mineralogical evolution of the ues were obtained for the solid substrate collected at the medium end of the culture (Table 3). The same trend was observed for the diversity indi- Changes in medium chemistry could result from (1) evapo- ces estimated using the Shannon index and the inverse ration and dilution process within the bioreactor; (2) change Simpson indices. Indeed, lower values were calculated of primary composition of the fresh medium; (3) mineral for the raw flange sample while higher values were dissolution and precipitation resulting, for example, from obtained for the solid fraction recovered at the end of the temperature or pH changes; and (4) microbial activity. In enrichment culture. Also, for both Archaea and Bacteria, addition, dissolved sulfide concentration in the medium is the species evenness increased throughout the culture likely affected by H2S loss due to vigorous and continuous time (Table 3). gas sweeping in the bioreactor. Overall, the results show that both archaeal and bacte- As mentioned above, during the initial 36 h of batch rial diversity depend upon the type of sample in the bio- culture, and despite the presence of a condenser, signifi- reactor (i.e., suspended versus precipitated fraction), sam- cant evaporation occurred in the bioreactor. Evaporation pling time, and initial (i.e., in situ) versus final (i.e., end was enhanced by both increased temperature and active of culture) chimney sample (Figs. 3, 4 and 7). Indeed, gas purging, and lack of new medium filling. During the only two archaeal OTUs and none of the bacterial phylo- continuous culture period, evaporation was attenuated by 1 type were found across all types of samples. A maximum the input of fresh medium at a rate of about 400 mL day− . of 8 archaeal OTUs were common between the suspended The effect of medium evaporation and dilution was cor- and precipitated fractions and only one bacterial OTU was rected by normalizing all elemental concentrations to the found in both in situ sample and the precipitated fraction at same Na concentration taken at 470 mM which is similar the end of the culture (Fig. 7). to the measured concentration in the initial hydrothermal In addition, the cluster trees obtained with the archaeal fluid. Since Na is generally conservative during mixing and bacterial sequences (Fig. 8) using the statistical jack- and evaporation, this approach allows discussing Na-nor- knife environment clusters did not show any correlation malized values of other elements, resulting solely from between the microbial diversity, the type of samples and microbe–mineral–fluid interactions. Time-series concentra- the time of sampling, excepted for samples D4 and D15 tions reported in Fig. 2 are, therefore, all corrected in this for the Bacteria and samples D7 and D15 for the Archaea manner. Finally, the medium composition was negligibly which were clustered together. affected by differences in chemical composition between This absence of similarity among all the samples, as well the three pouches (Table S2) and did not need specific as the species richness and the diversity indices, illustrates corrections. that not only the pH drop had an effect on the microbial Using this approach, it is possible to determine the net structure and activity but also other parameters can drive effect of mineral dissolution on medium composition dur- the microbial diversity. ing the initial step of the experiment. At 36h, Ca, Sr and

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Fig. 5 Phylogenetic relation- ships of archaea based on 16S rRNA gene sequences after RNA reverse transcrip- tion obtained from the in situ hydrothermal flange sample and during the enrichment culture, determined by neighbor-joining analysis. Bootstrap values above 50 % based on 1000 replicates are displayed. Thaumarchaeota are used as outgroup

S (as sulfate), concentrations are higher than the initial and precipitation of Ca-sulfate phases (e.g., gypsum) as fluid composition. In the initial fluid medium, sulfate con- shown by the concomitant decrease in Ca concentrations centration was measured at around 20 mM. At 36 h, sul- (trend 2 in Fig. 10). Note that gypsum has not been deter- fate concentration was measured at 23 mM probably due to mined by XRD after the experiment, most likely because it anhydrite dissolution (trend 1 in Fig. 9). After D7, sulfate dissolved during cleaning of the sample for XRD analysis concentration decreased substantially due to the saturation to remove sea salt.

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Fig. 6 Total number of 16S rRNA gene copies per ng of whole DNA in the in situ sample and in the liquid fraction in the bioreactor determined by Q-PCR results. Archaea and Bacteria ratio expresses in percentages of number of 16S rRNA gene copies per ng of whole DNA determined by Q-PCR. B corresponds to crude flange sample, T36 sample just after inoculation and D at each day of culture time

Table 3 Analysis of the archaeal and bacterial diversity, from 16S rRNA gene libraries, in the raw flange sample and in the enrichment culture Sample Number of OTUs (with Good’s ACE Chao1 Chao1 lcia Chao1 hcib Shannon Inverse Simp- >97 % similarity) coverage index son indice

Archaea Raw flange sample 7 0.83 5.12 4.9 4.33 9.99 0.94 2.58 T36h 3 0.80 8.79 7.99 7.22 14.79 1.42 3.67 D4 2 0.87 10.71 9.67 8.75 17.58 1.66 4.55 D7 1 0.93 12.62 11.07 10.21 19.35 1.83 5.34 D15 2 0.87 13.91 12.17 11.34 20.75 1.95 5.93 D21 5 0.65 15.15 13.17 12.20 23.28 2.03 6.30 Solid fractionc 7 0.83 17.05 14.49 13.15 28 2.11 6.59 Bacteria Raw flange sample 31 0.25 22.98 16.5 10.72 38.01 1.44 5.14 T36h 3 0.78 43.22 34.71 22.69 73.25 2.18 8.66 D4 2 0.86 53.91 44.61 30.38 88.07 2.51 10.44 D7 5 0.59 72.71 60.72 41.26 116.17 2.79 12.26 D15 3 0.81 83.44 70.47 49.13 128.84 2.98 13.82 D21 6 0.67 94.98 80.80 56.99 144.04 3.11 14.55 Solid fractionc 7 0.82 109.13 93.82 66.36 164.74 3.24 15.31 a Low confidence interval b High confidence interval c Solid fraction collected at the end of the culture

Consecutive to medium acidification at D7, Ca and Sr Manganese also shows a marked increase at D7, sug- increased while S remained mainly constant (Fig. 2), which gesting dissolution of Mn-rich phases at lower pH. is consistent with calcite (CaCO3) dissolution (Fig. 8). Because Mn-oxyhydroxide phases were not identified by Sr is considered as a good proxy of the calcite dissolu- either optical microscopy or X-ray diffraction, we propose tion because it occurs as a trace element in this mineral. that Mn sources reside within carbonates as Mn(II) spe- Thus, an increase in both Ca and Sr concentrations in the cies. It is also unlikely that any significant Mn(V) phases medium suggests significant calcite dissolution. After D7, are formed within active hydrothermal chimneys due to when medium returns back to circumneutral pH, Ca and Sr the very slow kinetic of Mn oxidation in vent environ- decrease as a result of the addition (i.e., dilution effects) of ments. As for Ca and Sr, the decrease in Mn concentra- fresh medium rather than mineral precipitation due to the tions after D7 is due to the dilution effects rather than min- continuous renewal of half volume of the culture per day. eral precipitation.

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Fig. 7 Diagram representing the archaeal (a) and the bacte- rial (b) phylotypes partitioning according to the three types of samples: in situ sample, sample from the liquid fraction and from the solid fraction

Fig. 8 Microbial communities associated with the culture time, the Frac metric-based 16S rRNA gene sequences determined by neigh- variation in the culture (e.g., pH drop, concentrations) or the type bor-joining tree) showing the phylogenetic relationships among the of samples (e.g., raw, liquid or solid fraction). a Jackknife environ- bacterial lineages detected in each sample according to the culture ment cluster tree (made using the weighted UniFrac metric-based 16S time and the type of samples. The jackknife statistical analysis was rRNA gene sequences determined by neighbor-joining tree) showing done with one hundred replicates; the jackknife value was tagged the phylogenetic relationships among the archaeal lineages detected near their corresponding nodes (values higher 50 %). The scale bar in each sample according to the culture time and the type of samples. corresponds, in the Unifrac unit, to the distance between the different (b) Jackknife environment cluster tree (made using the weighted Uni- habitats

Sulfide minerals, in particular metal monosulfides such higher value is best explained by the release of organic as pyrrhotite, galena, and sphalerite, are also prone to dis- compounds from the chimney sample, possibly resulting solution at low pH. Due to the very low abundances of from the lyses of microbial and/or eukaryotic cells (e.g., sulfide minerals in the original flange sample (Table 2), present in the top of the flange at lower temperature, in only Fe shows significant increase upon medium acidifica- contact with seawater, Fig. 1), at high temperature (85 °C). tion (Fig. 2). Overall, trace metal concentrations remained After D10, acetate concentration was lower than its pouch below initial vent fluid concentrations. concentration (e.g., 5.2 µM in pouch 2 and 5.3 µM in PEP- Acetate concentrations in the initial vent fluids ranged ITO pouch 3) suggesting that acetate was actively used from 3.8 to 5.3 µM, which is much lower than acetate by microorganisms as energy or carbon sources. Formate concentration (288 µM) measured in the bioreactor. This concentration in the medium was always lower than those

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Fig. 9 Calcium versus sulfur (sulfate) behavior during the whole experiment time. The trend 1 (①) corresponds to the anhydrite dissolution during the first 36 h of batch mode; the trend 2 (②) corresponds to the gypsum precipitation during the culture performed in continu- ous mode. Note that the data are corrected for evaporation effects after normalizing to Na at 470 mM

measured in the pouches, also suggesting its consumption retrieved at 36h, D4, D21 and in the solid fraction (Campbell by microorganisms. et al. 2006; Bertoldo and Antranikian 2006). Thermotoga was also detected at the end of the culture and in the solid Microbial diversity and implication in sulfur and iron fraction. Sulfur sources in the medium may include (Fig. 10) biogeochemical cycles (1) elemental sulfur present in the original sample (but below

detection limit of XRD) which generally formed after H2S The range of detected microorganisms in the enrichment oxidation and/or pyrrhotite alteration in chimney exposed to culture should translate into a range of metabolisms includ- seawater and (2) sulfur resulting from anaerobic microbial ing those involved in sulfur or iron biogeochemical cycles. sulfide (H2S, FeS) oxidation (Jost et al. 2010). Although phylogenic affiliation may not be necessarily Sulfate reduction may be also active in the bioreac- linked to specific metabolic or physiological properties, tor and likely mediated by Archaeoglobus (98 % similar- here we cautiously inferred potential metabolic and physi- ity with Archaeoglobus profundus) that were retrieved at ological trends for clusters of microorganisms using their the beginning (T36) and at the end (D15 and D21) of the related cultivated and described species sharing similar culture and in the solid remaining fraction. Yet changes of metabolic property (e.g.: Archaeoglobus sp., Geoglobus sulfate concentration in the medium (i.e., between 17 and sp., Thermococcus sp., Sulfurimonas sp. or Thermotoga 23 mM) were principally the result of fluid dilution and sp., cluster). Similar approaches have been already applied Ca-sulfate precipitation rather than microbial sulfate reduc- and discussed in previous studies (Callac et al. 2013; Alain tion. This is consistent with (1) the low cell-specific sulfate et al. 2002). reduction rate of Archaeoglobus profundus (Callac et al., in Although it is beyond the scope of this paper to infer the prep); (2) the absence of positive amplification by PCR of potential importance of microorganisms in mineral disso- the dsrA gene encoding for a subunit of the dissimilatory lution and precipitation processes, we tentatively propose sulfite reductase involved in the sulfate reduction [using the a model for prokaryote–fluid–mineral interactions in the specific primers DSR1F and DSR4R (Wagner et al. 1998)] culture (Fig. 10) that can explain the nature, dynamics and (Figs. 2, 9). diversity of microbial communities observed in the culture. Thus, the presence of coexisting sulfur species, includ- ing elemental sulfur, sulfate, organic-sulfur compounds or The role of the microbial communities in controlling sulfur dissolved hydrogen sulfide and the availability in hydro- cycling gen, nitrate, CO2 and organic carbon, allowed establishing convenient conditions for the growth of the detected sulfur Active sulfur (S°) reduction is likely occurring in the bio- metabolizing species (Fig. 10). Our results of microbial reactor as revealed by the presence of sequences related to diversity in the bioreactor demonstrate that complete sulfur Sulfurospirillum at D15 and to the Thermococcales that were cycle can occur in anaerobiosis at hydrothermal conditions.

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Fig. 10 Schematic diagram showing the geochemical and microbiological processes related to coupled iron and sulfur cycles in the enrichment culture. See text for discussion

This suggests that similar sulfur biogeochemical cycling It is important to note that, despite the presence of dis- can occur in an active hydrothermal chimney at the same or similatory iron reducers and the abundance of sulfide min- different spatial or time scales (Fig. 10). erals, Fe(II) concentrations remained lower (<0.8 µM) than in the hydrothermal fluid (Table 1). Presumably, this low The role of microbial communities in controlling iron concentration was due to active Fe(II) precipitation with cycling H2S in the medium to form FeS precipitate (Rickard 1995; Vazquez et al. 1989) (Fig. 10). Dissimilatory iron reduction (DIR) was likely active in the The mineral fraction (i.e., from inoculum) remaining at bioreactor, as revealed by the detection of Geoglobus until the end of the culture in the bioreactor contained Thermo- day 21. Geoglobus species are known to grow on poorly toga and Geoglobus species. It is important to note that Geo- crystalline ferric iron (Fe(III)) as a sole electron acceptor. In globus species were not present in the suspended fraction at the initial medium, iron oxide minerals such as ferrihydrite the same time (Fig. 4) which is best explained by the absence and Fe(III)-rich clay such as nontronite (Table 2) are pre- of available Fe oxide substrates in the suspended fraction. sent either as primary minerals directly precipitating from Parvibaculum sequences were detected both in the the hydrothermal fluid mixed with seawater or as second- medium and in the solid fraction collected at the end of ary minerals resulting from the oxidative alteration of Fe- the culture. These species are frequently detected in habi- sulfide minerals (e.g., pyrrhotite) at the seafloor (Fig. 10). tat containing hydrocarbon (Schleheck et al. 2011) like the X-ray mineralogical analysis on the solid fraction collected Guaymas Basin hydrothermal field (Martens 1990; Welhan at the end of the culture showed a complete loss of nontron- 1988). Thus, even if little is known about the Alphaproteo- ite while ferrihydrite was below detection either before or bacteria in hydrothermal systems, we cannot exclude that after incubation. This suggests that Fe(III)-containing min- some of Parvibaculum species are able to grow at high erals may have been available for DIR by Geoglobus, using temperature in anaerobic condition and potentially as iron

H2 and CO2 or acetate and other organic compounds in the oxidizers as previously shown in mesophilic and anaerobic medium as energy and carbon sources (Fig. 10). conditions (Straub et al. 1996; Blöthe and Roden 2009).

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Due to the inferred occurrence of Fe-oxyhydroxides in notable exception of few clones detected at the Suiyo Sea- the mineral substrate and nitrate in the vent fluid, the results mount, Izu-Bonin Arc (Higashi et al. 2004) and at the Mar- of microbial diversity reveal the importance of iron redox iner field, Lau Basin (Takai et al. 2008). To date, most of cycling at high temperature and under strictly anaerobic the detected phylotypes are related to cultivated species of conditions. Our results differ from the thermodynamic cal- sulfur reducers, sulfur oxidizers and sulfate reducers. For culations previously established. Indeed, in the few models example, phylotypes related to Geoglobus sp. were never considering the iron reduction, this reaction is consider- reported in natural hydrothermal chimneys (Wang et al. ing to have low energy yield (Nakamura and Takai 2014; 2009; Byrne et al. 2009a; Kormas et al. 2006; Kato et al. McCollom and Shock 1997; Boettger et al. 2013; McCol- 2010, 2012). Here, Geoglobus were detected in almost lom 2007) and, therefore, contrary to the thermodynamic all the culture samples suggesting that thermophilic iron models, iron reduction seems to be key metabolism. reduction is probably an important process in hydrother- mal systems along the mixing zone between hydrothermal Comparison with previous enrichment cultures fluid and seawater. In addition, the simultaneous presence in hydrothermal conditions of Geoglobus sp. and Archaeoglobus sp. suggests a lack of competition between sulfate reducers and iron reducers. The detection of microorganisms potentially involved in S and Fe biogeochemical cycles differ from previous results Insights into the driver of microbial diversity obtained in other enrichment cultures performed in ther- in enrichment culture and in situ sample mophilic and anaerobic conditions using either synthetic or selective medium and active hydrothermal chimney as Using our experimental approach, we have been able to inoculum. compare the microbial diversity of three different types of Here, we provide evidences for active sulfur, sulfate and samples: (1) the initial (in situ) chimney sample; (2) the iron reduction, as well as sulfur and potentially iron oxida- final (i.e., after incubation) chimney sample; and (3) the tion, occurring simultaneously in the enrichment culture. suspended fraction composed of fluid medium and small Previous enrichment cultures done at 85–90 °C in anaer- substrate particles stirred in the bioreactor. To overcome obic conditions have shown phylotypes potentially involved the inherent molecular bias that could affect such compari- in sulfur reduction, such as Thermococcales [Thermococ- sons (Wintzingerode et al. 1997; Teske and Sorensen 2007; cus sp., or Pyrococcus sp. (Byrne et al. 2009a)] and Marini- Huber et al. 2009), we used, for all samples, the same toga sp. (Postec et al. 2005), or in the ferric iron reduction methods and protocol (nucleic acid extraction, RT-PCR, with Geoglobus sp. (Byrne et al. 2009a). However, no evi- 16S rRNA PCR pair primers, cloning vector). Hence, we dences were reported for Fe or S oxidation metabolic path- consider that the large differences in microbial diversity ways, and for the simultaneous presence of sulfur, sulfate obtained between sample types and sampling time result and iron reducers. from the sampling procedure itself rather than biases in Other experimental studies performed in anaerobic con- analytical methodology. dition, at 70 °C using a medium made for sulfate reducers However, an important question remains to be addressed and inocula containing iron-rich minerals (e.g., pyrite, pyr- in evaluating whether the suspended fraction reflects tem- rhotite, sphalerite) reported microbial phylotypes related to poral variations or heterogeneity of the microbiome in the sulfate reducer Thermodesulfatator sp., to the methanogen bioreactor (e.g., free cells vs particle-associated cells). Methanocaldococcus vulcanius and to putative iron reduc- First, it is important to consider the large differences in tion (Houghton et al. 2007). terms of number of phylotypes and phylogenetic affiliation Two other enrichments done at 60 °C also in anaero- between sample types (Fig. 7). Lower bacterial diversity bic conditions showed for one that iron reduction (with and an increase in the number of archaeal phylotypes in the detection of Geoglobus sp.) and sulfur reduction (with the suspended or solid fractions compared to initial sam- Thermococcus sp. and Deferribacter sp.) occurred at the ple have already been shown in a previous study (Byrne same time (Byrne et al. 2009a), while for the other, that et al. 2009a). Hence, the differences in microbial diversity iron reduction, sulfur reduction and sulfate reduction took between the initial mineral sample and the suspended frac- place simultaneously (Postec et al. 2007). tion may be easily explained by the drastic differences in In all these studies, sulfur and/or iron oxidizers could growth medium between in situ conditions and in the bio- not be active in the absence of available electron acceptor reactor. For example, even in the presence of both CO2 and as nitrates. H2, no methanogen was detected whereas 16S rRNA tran- The evidences for iron reducer with Geoglobus sp. scripts affiliated to several methanogens (Methanopyrus reported in our study are generally lacking in microbial sp., Methanothermococcus sp. and Methanocaldococcus diversity survey of active hydrothermal chimneys, with the sp.), were detected in the raw flange sample. This also

1 3 Extremophiles (2015) 19:597–617 613 contrasts with thermodynamic modeling which predicts hydrothermal fluid and mineral substrate (i.e., flange frag- that in hydrothermal conditions, dominating metabolisms ment) as the culture medium to compare in situ and ex situ are sulfur and sulfate reduction, as well as methanogen- diversity. The time-series analysis of the chemical composi- esis and methanotrophy (McCollom 2007; McCollom and tion of culture medium together with suspended microbial Shock 1997). Presumably, the lack of strong geochemical communities (16S rRNA) allowed the assessment of major gradients in our bioreactor compared to in situ chimney biogeochemical reactions occurring in the bioreactor as well conditions plays an important role in controlling microbial as key parameters affecting microbial diversity. Although diversity. the experimental approach in bioreactor is not designed to Secondly, our results suggest that active microbial com- reproduce the complexity of mineral–fluid–microbe inter- munities are extremely heterogeneous and/or unstable actions found in natural systems, it permits to tune different in the suspended fractions (Figs. 3, 4, 7 and 8). A statis- parameters to evaluate their first-order influences on natural tical jackknife environment analysis shows no correlation microbial communities. We proposed a model accounting for between microbial diversity, sample types and sampling the interactions between microorganisms and their medium time. This demonstrates that steady state of the culture was (hydrothermal fluid and chimney substrate), which is con- never reached, even when microbial communities were sistent with the observed microbial community structures. maintained under stable and well constrained chemical The availability of substrates (electron donors and accep- and physical conditions. With the exception of the pH drop tors, carbon sources) fueling microbial growth appeared to experienced at Day 6, the overall chemical composition of be highly dependent on the interaction that occurred between the medium and mineral substrates did not change signifi- the mineral fractions, the fluid, and culture conditions (gas cantly. Hence, neither changes of fluid chemistry nor min- sweeping, temperature, pH). All together the results dem- eralogy may directly explain the observed variation in the onstrate a possible linkage between iron and sulfur cycles at microbial community structures. We also consider that it is high temperature and under strictly anaerobic conditions. In also unlikely that minor elements or chemical compounds, particular, we provided evidences for simultaneous activity which were not analyzed in our study, could have a signifi- of Geoglobus and Archaeoglobus, suggesting no competition cant impact on the microbial activity and structure. between the sulfate and the iron reducers. Considering that the precipitated mineral fraction in the We showed that, although both precipitation and disso- bioreactor is less prone to vigorous mixing and cell wash- lution processes occurred throughout the culture, changes out than the suspended fraction, the development of biofilm in medium fluid composition are not the primary drivers of and micro-niches along mineral grains boundaries (e.g., microbial community structure and diversity. Overall, we sulfides/sulfate) may favor specific microbial populations. show that the microbial community is highly dynamic in We suggest that interaction between fluid flow dynamics the bioreactor and that steady state could not be reached and mineral may also impact microbial diversity observed even after 23 days of incubation. Microbial diversity is in natural samples of hydrothermal chimneys. highly different between the suspended and precipitated It is also important to note that several species detected mineral fraction, despite vigorous fluid stirring in the bio- in the bioreactor, such as Geoglobus sp., Palaeococcus sp., reactor and well-controlled culture parameters. This dem- Parvibaculum sp., are rarely detected in natural hydrother- onstrates that the nature and stability of micro-habitats mal chimney samples. This demonstrates that cultivation are also important drivers of microbial diversity. Further methods in bioreactor allow to access uncultured prokary- experimental studies, using the same device and hydrother- otes that are poorly represented in natural ecosystems and/ mal fluid as medium coupling, should aim to better evalu- or exhibiting a long latency phase as previously suggested ate the nature of micro-habitats and investigate the nature, (Postec et al. 2005, 2007; Byrne et al. 2009a). In addition, composition and diversity of mineral-cell aggregates. This species richness and diversity increased throughout the cul- could be achieved through the combination of fine-scale ture time, suggesting that this cultivation method allows geochemical and mineralogical studies with the use of radi- access to more diverse microbial phylotypes. otracers and metatranscriptomic analysis.

Acknowledgments We want to thank all the shipboard cruise party Conclusion for their work and support during the BIG cruise: officers, crew and technicians of the R/V L’Atalante, the DSV Nautile team and the on-board scientific team. This cruise was funded by IFREMER This study further validates the use of continuous enrich- (France) and has benefited from a work permit in Mexican waters ment cultures performed in bioreactors to access micro- (DAPA/2/281009/3803, October 28th, 2009). We also acknowledge bial diversity and dynamics and the interactions between the anonymous reviewers for their constructive suggestions and com- ments. We are grateful to Carole Decker and Jean-Claude Caprais for microorganisms and growth medium (Postec et al. 2005, their assistance with hydrogen sulfide measurement. Yoan Germain is 2007; Byrne et al. 2009a). We used for the first time in situ thanked for clean laboratory assistance and Karine Estève for her help

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