Hydrothermal plumes as hotspots for deep-ocean heterotrophic microbial biomass production.

Carbon budgets of hydrothermal plumes result from the balance between carbon sinks through plume chemoautotrophic processes and carbon release via microbial respiration. However, the lack of comprehensive analysis of the metabolic processes and biomass production rates hinders an accurate estimate of their contribution to the deep ocean carbon cycle. Here, we use a biogeochemical model to estimate the autotrophic and heterotrophic production rates of microbial communities in hydrothermal plumes and validate it with in situ data. We show how substrate limitation might prevent net chemolithoautotrophic production in hydrothermal plumes. Elevated prokaryotic heterotrophic production rates (up to 0.9 gCm-2y-1) compared to the surrounding seawater could lead to 0.05 GtCy-1 of C-biomass produced through chemoorganotrophy within hydrothermal plumes, similar to the Particulate Organic Carbon (POC) export fluxes reported in the deep ocean. We conclude that hydrothermal plumes must be accounted for as significant deep sources of POC in ocean carbon budgets.

H2-dependent formate production by hyperthermophilic Thermococcales: an alternative to sulfur reduction for reducing-equivalents disposal.

Removal of reducing equivalents is an essential catabolic process for all microorganisms to maintain their internal redox balance. The electron disposal by chemoorganotrophic Thermococcales generates H2 by proton reduction or H2S in presence of S0. Although in the absence of S0 growth of these (hyper)thermopiles was previously described to be H2-limited, it remains unclear how Thermococcales could be present in H2-rich S0-depleted habitats. Here, we report that 12 of the 47 strains tested, distributed among all three orders of Thermococcales, could grow without S0 at 0.8 mM dissolved H2 and that tolerance to H2 was always associated with formate production. Two conserved gene clusters coding for a formate hydrogenlyase (FHL) and a putative formate dehydrogenase-NAD(P)H-oxidoreductase were only present in H2-dependent formate producers, and were both systematically associated with a formate dehydrogenase and a formate transporter. As the reaction involved in this alternative pathway for disposal of reducing equivalents was close to thermodynamic equilibrium, it was strongly controlled by the substrates-products concentration ratio even in the presence of S0. Moreover, experimental data and thermodynamic modelling also demonstrated that H2-dependent CO2 reduction to formate could occur within a large temperature range in contrasted hydrothermal systems, suggesting it could also provide an adaptive advantage.

Persephonella atlantica sp. nov.: How to adapt to physico-chemical gradients in high temperature hydrothermal habitats.

A novel thermophilic, microaerophilic and anaerobic, hydrogen- sulphur- and thiosulphate-oxidising bacterium, designated MO1340T, was isolated from a deep-sea hydrothermal chimney collected from the Lucky Strike hydrothermal vent field on the Mid-Atlantic Ridge. Cells were short, motile rods of 1.4-2.2µm length and 0.5-0.8µm width. Optimal growth was observed for a NaCl concentration of 2.5 % (w/v) at pH 6.5. As for other members of the genus Persephonella, strain MO1340T was strictly chemolithoautotrophic and could oxidise hydrogen, elemental sulphur or thiosulphate using oxygen as electron acceptor. Anaerobic nitrate reduction using hydrogen could also be performed. Each catabolic reaction had a different optimal growth temperature (65 to 75°C) and an optimal dissolved oxygen concentration (11.4 to 119.7 µM at 70°C for aerobic reactions) that varied according to the electron donors utilised. These experimental results are consistent with the distribution of these catabolic substrates along the temperature gradient observed in active hydrothermal systems. They strongly suggest that this adaptive strategy could confer a selective advantage for strain MO1340T in the dynamic part of the ecosystem where hot, reduced hydrothermal fluid mixes with cold, oxygenated seawater. Phylogenetic analysis indicated that strain MO1340T was a member of the genus Persephonella within the order Hydrogenothermales as it shared a 16S rRNA gene sequence similarity <95.5 % and ANI respectively 75.66 % with closest described Persephonella (P. hydrogeniphila 29WT). On the basis of the physiological and genomic properties of the new isolate, the name Persephonella atlantica sp. nov. is proposed. The type strain is MO1340T (=UBOCC-M-3359T =JCM 34026T).

Rock-crushing derived hydrogen directly supports a methanogenic community: significance for the deep biosphere.

Microbial populations exist to great depths on Earth, but with apparently insufficient energy supply. Earthquake rock fracturing produces H2 from mechanochemical water splitting, however, microbial utilization of this widespread potential energy source has not been directly demonstrated. Here, we show experimentally that mechanochemically generated H2 from granite can be directly, long-term, utilized by a CH4 producing microbial community. This is consistent with CH4 formation in subsurface rock fracturing in the environment. Our results not only support water splitting H2 generation as a potential deep biosphere energy source, but as an oxidant must also be produced, they suggest that there is also a respiratory oxidant supply in the subsurface which is independent of photosynthesis. This may explain the widespread distribution of facultative aerobes in subsurface environments. A range of common rocks were shown to produce mechanochemical H2 , and hence, this process should be widespread in the subsurface, with the potential for considerable mineral fuelled CH4 production.

Redescription and phylogenetic analyses of Durchoniella spp. (Ciliophora, Astomatida) associated with the polychaete Cirriformia tentaculata (Montagu, 1808).

Microscopic and phylogenetic analyses were performed on endocommensal astome ciliates retrieved from the middle intestine of a marine cirratulid polychaete, Cirriformia tentaculata, collected in the bay of Roscoff (English Channel, Northwest French coast) and on the Southwest English coast. Three morphotypes of the astome genus Durchoniella were identified, two corresponding to described species (the type species Durchoniella brasili (Léger and Duboscq, 1904) De Puytorac, 1954 and Durchoniella legeriduboscqui De Puytorac, 1954) while a third morphotype remains undescribed. Their small subunit (SSU) rRNA gene sequences showed at least 97.2% identity and phylogenetic analyses grouped them at the base of the subclass Scuticociliatia (Oligohymenophorea), as a sister lineage to all astomes from terrestrial oligochaete annelids. Ultrastructural examination by transmission electron microscopy and fluorescence in situ hybridization analyses revealed the presence of endocytoplasmic cocci and rod-shaped bacteria surrounded by a very thin membrane. These endocytoplasmic bacteria may play a role in the association between endocommensal astome ciliates and cirratulid polychaetes inhabiting in anoxic coastal sediments.

Complex coupled metabolic and prokaryotic community responses to increasing temperatures in anaerobic marine sediments: critical temperatures and substrate changes.

The impact of temperature (0-80°C) on anaerobic biogeochemical processes and prokaryotic communities in marine sediments (tidal flat) was investigated in slurries for up to 100 days. Temperature had a non-linear effect on biogeochemistry and prokaryotes with rapid changes over small temperature intervals. Some activities (e.g. methanogenesis) had multiple 'windows' within a large temperature range (∼10 to 80°C). Others, including acetate oxidation, had maximum activities within a temperature zone, which varied with electron acceptor [metal oxide (up to ∼34°C) and sulphate (up to ∼50°C)]. Substrates for sulphate reduction changed from predominantly acetate below, and H2 above, a 43°C critical temperature, along with changes in activation energies and types of sulphate-reducing Bacteria. Above ∼43°C, methylamine metabolism ceased with changes in methanogen types and increased acetate concentrations (>1 mM). Abundances of uncultured Archaea, characteristic of deep marine sediments (e.g. MBGD Euryarchaeota, 'Bathyarchaeota') changed, indicating their possible metabolic activity and temperature range. Bacterial cell numbers were consistently higher than archaeal cells and both decreased above ∼15°C. Substrate addition stimulated activities, widened some activity temperature ranges (methanogenesis) and increased bacterial (×10) more than archaeal cell numbers. Hence, additional organic matter input from climate-related eutrophication may amplify the impact of temperature increases on sedimentary biogeochemistry.

Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.

Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure.

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40-80 °C) maximum in situ temperatures (∼ 23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40-60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii(T) (∼ 96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii(T)and D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼ 30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.

Phylogenetic and functional diversity of microbial communities associated with subsurface sediments of the Sonora Margin, Guaymas Basin.

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Glycine betaine as a direct substrate for methanogens (Methanococcoides spp.).

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.

Choline and N,N-dimethylethanolamine as direct substrates for methanogens.

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.

Comparison of microbial communities associated with three Atlantic ultramafic hydrothermal systems.

The distribution of Archaea and methanogenic, methanotrophic and sulfate-reducing communities in three Atlantic ultramafic-hosted hydrothermal systems (Rainbow, Ashadze, Lost City) was compared using 16S rRNA gene and functional gene (mcrA, pmoA and dsrA) clone libraries. The overall archaeal community was diverse and heterogeneously distributed between the hydrothermal sites and the types of samples analyzed (seawater, hydrothermal fluid, chimney and sediment). The Lost City hydrothermal field, characterized by high alkaline warm fluids (pH>11; T<95 °C), harbored a singular archaeal diversity mostly composed of unaffiliated Methanosarcinales. The archaeal communities associated with the recently discovered Ashadze 1 site, one of the deepest active hydrothermal fields known (4100 m depth), showed significant differences between the two different vents analyzed and were characterized by putative extreme halophiles. Sequences related to the rarely detected Nanoarchaeota phylum and Methanopyrales order were also retrieved from the Rainbow and Ashadze hydrothermal fluids. However, the methanogenic Methanococcales was the most widely distributed hyper/thermophilic archaeal group among the hot and acidic ultramafic-hosted hydrothermal system environments. Most of the lineages detected are linked to methane and hydrogen cycling, suggesting that in ultramafic-hosted hydrothermal systems, large methanogenic and methanotrophic communities could be fuelled by hydrothermal fluids highly enriched in methane and hydrogen.

Prokaryotic functional diversity in different biogeochemical depth zones in tidal sediments of the Severn Estuary, UK, revealed by stable-isotope probing.

Stable isotope probing of prokaryotic DNA was used to determine active prokaryotes using (13)C-labelled substrates (glucose, acetate, CO(2)) in sediment slurries from different biogeochemical zones of the Severn Estuary, UK. Multiple, low concentrations (5 x 100 microM) of (13)C-substrate additions and short-term incubations (7 days) were used to minimize changes in the prokaryotic community, while achieving significant (13)C-incorporation. Analysis demonstrated clear metabolic activity within all slurries, although neither the net sulphate removal nor CH(4) production occurred in the anaerobic sulphate reduction and methanogenesis zone slurries. Some similarities occurred in the prokaryotic populations that developed in different sediment slurries, particularly in the aerobic and dysaerobic zone slurries with (13)C-glucose, which were dominated by Gammaproteobacteria and Marine Group 1 Archaea, whereas both anaerobic sediment slurries incubated with (13)C-acetate showed incorporation into Epsilonproteobacteria and other bacteria, with the sulphate reduction zone slurry also showing (13)C-acetate utilization by Miscellaneous Crenarchaeotic Group Archaea. The lower potential energy methanogenesis zone slurries were the only conditions where no (13)C-incorporation into Archaea occurred, despite Bacteria being labelled; this was surprising because Archaea have been suggested to be adapted to low-energy conditions. Overall, our results highlight that uncultured prokaryotes play important ecological roles in tidal sediments of the Severn Estuary, providing new metabolic information for novel groups of Archaea and suggesting broader metabolisms for largely uncultivated Bacteria.

Microbial diversity associated with the hydrothermal shrimp Rimicaris exoculata gut and occurrence of a resident microbial community.

Rimicaris exoculata dominates the megafauna of several Mid-Atlantic Ridge hydrothermal sites. Its gut is full of sulphides and iron-oxide particles and harbours microbial communities. Although a trophic symbiosis has been suggested, their role remains unclear. In vivo starvation experiments in pressurized vessels were performed on shrimps from Rainbow and Trans-Atlantic Geotraverse sites in order to expel the transient gut contents. Microbial communities associated with the gut of starved and reference shrimps were compared using 16S rRNA gene libraries and microscopic observations (light, transmission and scanning electron microscopy and FISH analyses). We show that the gut microbiota of shrimps from both sites included mainly Deferribacteres, Mollicutes, Epsilon- and Gammaproteobacteria. For the first time, we have observed filamentous bacteria, inserted between microvilli of gut epithelial cells. They remained after starvation periods in empty guts, suggesting the occurrence of a resident microbial community. The bacterial community composition was the same regardless of the site, except for Gammaproteobacteria retrieved only in Rainbow specimens. We observed a shift in the composition of the microbiota of long-starved specimens, from the dominance of Deferribacteres to the dominance of Gammaproteobacteria. These results reinforce the hypothesis of a symbiotic relationship between R. exoculata and its gut epibionts.

Archaeal communities associated with shallow to deep subseafloor sediments of the New Caledonia Basin.

The distribution of the archaeal communities in deep subseafloor sediments [0-36 m below the seafloor (mbsf)] from the New Caledonia and Fairway Basins was investigated using DNA- and RNA-derived 16S rRNA clone libraries, functional genes and denaturing gradient gel electrophoresis (DGGE). A new method, Co-Migration DGGE (CM-DGGE), was developed to access selectively the active archaeal diversity. Prokaryotic cell abundances at the open-ocean sites were on average approximately 3.5 times lower than at a site under terrestrial influence. The sediment surface archaeal community (0-1.5 mbsf) was characterized by active Marine Group 1 (MG-1) Archaea that co-occurred with ammonia monooxygenase gene (amoA) sequences affiliated to a group of uncultured sedimentary Crenarchaeota. However, the anoxic subsurface methane-poor sediments (below 1.5 mbsf) were dominated by less active archaeal communities, such as the Thermoplasmatales, Marine Benthic Group D and other lineages probably involved in the methane cycle (Methanosarcinales, ANME-2 and DSAG/MBG-B). Moreover, the archaeal diversity of some sediment layers was restricted to only one lineage (Uncultured Euryarchaeota, DHVE6, MBG-B, MG-1 and SAGMEG). Sequences forming two clusters within the Thermococcales order were also present in these cold subseafloor sediments, suggesting that these uncultured putative thermophilic archaeal communities might have originated from a different environment. This study shows a transition between surface and subsurface sediment archaeal communities.

Extending the sub-sea-floor biosphere.

Sub-sea-floor sediments may contain two-thirds of Earth's total prokaryotic biomass. However, this has its basis in data extrapolation from ~500-meter to 4-kilometer depths, whereas the deepest documented prokaryotes are from only 842 meters. Here, we provide evidence for low concentrations of living prokaryotic cells in the deepest (1626 meters below the sea floor), oldest (111 million years old), and potentially hottest (~100 degrees C) marine sediments investigated. These Newfoundland margin sediments also have DNA sequences related to thermophilic and/or hyperthermophilic Archaea. These form two unique clusters within Pyrococcus and Thermococcus genera, suggesting unknown, uncultured groups are present in deep, hot, marine sediments (~54 degrees to 100 degrees C). Sequences of anaerobic methane-oxidizing Archaea were also present, suggesting a deep biosphere partly supported by methane. These findings demonstrate that the sub-sea-floor biosphere extends to at least 1600 meters below the sea floor and probably deeper, given an upper temperature limit for prokaryotic life of at least 113 degrees C and increasing thermogenic energy supply with depth.