Bacterial communities and syntrophic associations involved in anaerobic oxidation of methane process of the Sonora Margin cold seeps, Guaymas Basin.

SUMMARY: The Sonora Margin cold seeps present on the seafloor a patchiness pattern of white microbial mats surrounded by polychaete and gastropod beds. These surface assemblages are fuelled by abundant organic inputs sedimenting from the water column and upward-flowing seep fluids. Elevated microbial density was observed in the underlying sediments. A previous study on the same samples identified anaerobic oxidation of methane (AOM) as the potential dominant archaeal process in these Sonora Margin sediments, probably catalysed by three clades of archaeal anaerobic methanotrophs (ANME-1, ANME-2 and ANME-3) associated with bacterial syntrophs. In this study, molecular surveys and microscopic observations investigating the diversity of Bacteria involved in AOM process, as well as the environmental parameters affecting the composition and the morphologies of AOM consortia in the Sonora Margin sediments were carried out. Two groups of Bacteria were identified within the AOM consortia, the Desulfosarcina/Desulfococcus SEEP SRB-1a group and a Desulfobulbus-related group. These bacteria showed different niche distributions, association specificities and consortia architectures, depending on sediment surface communities, geochemical parameters and ANME-associated phylogeny. Therefore, the syntrophic AOM process appears to depend on sulphate-reducing bacteria with different ecological niches and/or metabolisms, in a biofilm-like organic matrix.

A GC-SSIM-CRDS system: Coupling a gas chromatograph with a Cavity Ring-Down Spectrometer for onboard Twofold analysis of molecular and isotopic compositions of natural gases during ocean-going research expeditions.

Carbon dioxide (CO2) and methane (CH4) are two climate-sensitive components of gases migrating within sediments and emitted into the water column on continental margins. They are involved in several key biogeochemical processes entering into the global carbon cycle. In order to perform onboard measurements of both the molecular and stable carbon isotope ratios (δ13C) of CH4 and CO2 of natural gases during oceanic cruises, we have developed a novel approach coupling gas chromatography (GC) with cavity ring-down spectroscopy (CRDS). The coupled devices are connected to a small sample isotope module (SSIM) to form a system called GC-SSIM-CRDS. Small volumes of natural gas samples (<1 mL) are injected into the GC using a headspace autosampler or a gas-tight syringe to separate the chemical components using a Shincarbon ST packed column and for molecular quantification by thermal conductivity detection (TCD). Subsequently, CO2 from the sample is trapped in a 7 mL loop at 32 °C before being transferred to the CRDS analyzer for sequential determination of the stable carbon isotope ratios of CH4 and CO2 in 24 min. The loop is an open column (without stationary phase). This approach does not require the use of adsorbents or cooling for the trapping step. Optimization of the separation step prior to analysis was focused on the influence of two key separation factors 1) the flow of the carrier gas and 2) the temperature of the oven. Our analytical system and the measurement protocol were validated on samples collected from gas seeps in the Sea of Marmara (Turkey). Our results show that the GC-SSIM-CRDS system provides a reliable determination of the molecular identification of CH4 and CO2 in complex natural gases, followed by the stable carbon isotope ratios of methane and carbon dioxide.

Dissolved and particulate metals (Fe, Zn, Cu, Cd, Pb) in two habitats from an active hydrothermal field on the EPR at 13 degrees N.

The distribution of Fe, Cu, Zn, Pb, Cd between the dissolved (<2 microm) and the particulate (>2 microm) fractions was measured after in-situ filtration in two hydrothermal habitats. The total metal concentration ranges exhibit a clear enrichment compared with the seawater concentration, accounting for the hydrothermal input for all the metals considered. Iron is the predominant metal (5-50 microM) followed by Zn and Cu. Cd and Pb are present at the nM level. At the scale studied, the behavior of temperature, pH and dissolved iron is semi-conservative whereas the other dissolved and particulate metals are characterized by non-conservative patterns. The metal enrichment of the >2 microm fraction results from the settlement and accumulation of particulate matter close to the organisms, acting as a secondary metal source. The enrichment observed in the dissolved fraction can be related to the dissolution or oxidation of particles (mainly polymetallic sulfide) or to the presence of small particles and large colloids not retained on the 2 microm frit. SEM observations indicate that the bulk particulate observed is characteristic of crystalline particles settling rapidly from the high temperature smoker (sphalerite, wurtzite and pyrite), amorphous structures and eroded particles formed in the external zone of the chimney. Precipitation of Zn, Cu, Cd and Pb with Fe as wurtzite, sphalerite and pyrite is the main process taking place within the area studied and is semi-quantitative. The distribution of the dominant observed fauna has been related to the gradient resulting from the dilution process, with the alvinellids worms colonizing the hotter and more variable part of the mixing zone, but also to the metallic load of the mixing zone. Dissolved and particulate metal concentrations are therefore necessary abiotic factors to be studied in a multiparametric approach to understand the faunal distribution in hydrothermal ecosystems.

Evidence for chemoautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes.

Symbioses between lucinid clams (Bivalvia: Lucinidae) and autotrophic sulphide-oxidizing bacteria have mainly been studied in shallow coastal species, and information regarding deep-sea species is scarce. Here we study the symbiosis of a clam, resembling Lucinoma kazani, which was recently collected in sediment cores from new cold-seep sites in the vicinity of the Nile deep-sea fan, eastern Mediterranean, at depths ranging from 507 to 1691 m. A dominant bacterial phylotype, related to the sulphide-oxidizing symbiont of Lucinoma aequizonata, was identified in gill tissue by comparative 16S rRNA gene sequence analysis. A second phylotype, related to spirochete sequences, was identified twice in a library of 94 clones. Comparative analyses of gene sequences encoding the APS reductase alpha subunit and ribulose-1,5-bisphosphate carboxylase oxygenase support the hypothesis that the dominant symbiont can perform sulphide oxidation and autotrophy. Transmission electron micrographs of gills confirmed the dominance of sulphide-oxidizing bacteria, which display typical vacuoles, and delta(13)C values measured in gill and foot tissue further support the hypothesis for a chemoautotrophic-sourced host carbon nutrition.

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.

Archaeal and anaerobic methane oxidizer communities in the Sonora Margin cold seeps, Guaymas Basin (Gulf of California).

Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the 'BIG' cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel 'ANME-2c Sonora' lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support alternative metabolic pathways than syntrophic anaerobic oxidation of methane.

Presence and diversity of anammox bacteria in cold hydrocarbon-rich seeps and hydrothermal vent sediments of the Guaymas Basin.

Hydrothermally active sediments are highly productive, chemosynthetic areas which are characterized by the rapid turnover of particulate organic matter under extreme conditions in which ammonia is liberated. These systems might be suitable habitats for anaerobic ammonium oxidizing (anammox) bacteria but this has not been investigated in detail. Here we report the diversity and abundance of anammox bacteria in sediments that seep cold hydrocarbon-rich fluids and hydrothermal vent areas of the Guaymas Basin in the Cortés Sea using the unique functional anammox marker gene, hydrazine synthase (hzsA). All clones retrieved were closely associated to the "Candidatus Scalindua" genus. Phylogenetic analysis revealed two distinct clusters of hzsA sequences (Ca. Scalindua hzsA cluster I and II). Comparison of individual sequences from both clusters showed that several of these sequences had a similarity as low as 76% on nucleotide level. Based on the analysis of this phylomarker, a very high interspecies diversity within the marine anammox group is apparent. Absolute numbers of anammox bacteria in the sediments samples were determined by amplification of a 257 bp fragment of the hszA gene in a qPCR assay. The results indicate that numbers of anammox bacteria are generally higher in cold hydrocarbon-rich sediments compared to the vent areas and the reference zone. Ladderanes, lipids unique to anammox bacteria were also detected in several of the sediment samples corroborating the hzsA analysis. Due to the high concentrations of reduced sulfur compounds and its potential impact on the cycling of nitrogen we aimed to get an indication about the key players in the oxidation of sulfide in the Guaymas Basin sediments using the alpha subunit of the adenosine-5'-phosphosulfate (APS) reductase (aprA). Amplification of the aprA gene revealed a high number of gammaproteobacterial aprA genes covering the two sulfur-oxidizing bacteria aprA lineages as well as sulfate-reducers.

First respiration estimates of cold-seep vesicomyid bivalves from in situ total oxygen uptake measurements.

Vesicomyid bivalves are one of the most abundant symbiont-bearing species inhabiting deep-sea reducing ecosystems. Nevertheless, except for the hydrothermal vent clam Calyptogena magnifica, their metabolic rates have not been documented, and only assessed with ex situ experiments. In this study, gathering benthic chamber measurements and biomass estimation, we give the first in situ assessment of the respiration rate of these bivalves. The giant pockmark Regab, located at 3160m depth along the Congo-Angola margin, is a cold-seep site characterised by dense assemblages of two species of vesicomyids: Christineconcha regab and Laubiericoncha chuni with high dominance of C. regab. Two sites with dense aggregates of vesicomyids were selected to measure total oxygen uptake (TOU), and methane fluxes using IFREMER's benthic chamber CALMAR deployed by the ROV Quest 4000 (MARUM). Photographs were taken and bivalves were sampled using blade corers to estimate density and biomass. Total oxygen uptake was higher at Site 2 compared to Site 1 (respectively 492 mmol.m(-2).d(-1) and 332 mmol.m(-2).d(-1)). However, given vesicomyid densities and biomass, mean oxygen consumption rates were similar at both sites (1.9 to 2.5 µmol.g total dry mass(-1).h(-1) at the Site 1 and 1.8 to 2.3 µmol.g total dry mass(-1).h(-1) at Site 2). These respiration rates are higher than published ex situ estimates for cold-seep or hydrothermal vent bivalves. Although methane fluxes at the base of sulphide production were clearly higher at Site 2 (14.6 mmol.m(-2).d(-1)) than at Site 1 (0.3 mmol.m(-2).d(-1)), they do not seem to influence the respiration rates of these bivalves associated to sulphide-oxidizing symbionts.

Temperature resistance studies on the deep-sea vent shrimp Mirocaris fortunata.

The shrimp Mirocaris fortunata is a hydrothermal vent species that is found at most vent-sites along the Mid-Atlantic Ridge. This endemic species is found across a hydrothermal gradient, with thermal conditions ranging from 2-9 degrees C in ambient seawater to fairly warm values of about 25 degrees C. We performed in vivo experiments on M. fortunata specimens originating from different sites and depths (850 m to 2300 m), both at atmospheric pressure and in pressurized aquaria, to characterise the upper thermal limits of this species. Atmospheric pressure results show that thermal physiology should be studied at each population's native pressure. At in situ pressure, shrimps from Menez Gwen (850 m depth) and Lucky Strike (1700 m depth) do not survive temperatures of 39 degrees C, and the 'loss of equilibrium' response suggests that their critical thermal maximum (Ctmax), is about 36+/-1 degrees C for both sites. This value is similar to those found for another vent shrimp, Rimicaris exoculata, which is thought to be a more temperature-resistant organism, so temperature resistance does not appear to be a crucial factor for explaining differences in distribution of shrimp species in a given vent site. Finally, the data for both vent shrimps are also comparable to those of other non-vent tropical caridean species.

Dual symbiosis in a Bathymodiolus sp. mussel from a methane seep on the Gabon continental margin (Southeast Atlantic): 16S rRNA phylogeny and distribution of the symbionts in gills.

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 microM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.