Relative abundance of anaerobic methanotrophic archaea in bacterial mat Cascadia margin off Oregon

The microbially mediated anaerobic oxidation of methane (AOM) is the major biological sink of the greenhouse gas methane in marine sediments (doi:10.1007/978-94-009-0213-8_44) and serves as an important control for emission of methane into the hydrosphere. The AOM metabolic process is assumed to be a reversal of methanogenesis coupled to the reduction of sulfate to sulfide involving methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) as syntrophic partners which were describes amongst others in Boetius et al. (2000; doi:10.1038/35036572). In this study, 16S rRNA-based methods were used to investigate the distribution and biomass of archaea in samples from sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). Sediment samples from Hydrate Ridge were obtained during R/V SONNE cruises SO143-2 in August 1999 and SO148-1 in August 2000 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. The second study area is located in the Black Sea and represents a field in which there is active seepage of free gas on the slope of the northwestern Crimea area. Here, a field of conspicuous microbial reefs forming chimney-like structures was discovered at a water depth of 230 m in anoxic waters. The microbial mats were sampled by using the manned submersible JAGO during the R/V Prof. LOGACHEV cruise in July 2001. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats.

Niche differentiation among mat-forming, sulfide-oxidizing bacteria in chemosynthetic ecosystems of the Nile Deep Sea Fan (Eastern Mediterranean Sea)

Sulfidic muds of cold seeps on the Nile Deep Sea Fan are populated by different types of mat-forming sulfide-oxidizing bacteria. The predominant sulfide oxidizers of three different mats were identified by microscopic and phylogenetic analyses as (i) Arcobacter species producing cotton-ball-like sulfur precipitates, (ii) large filamentous sulfur bacteria including Beggiatoa species, or (iii) single, spherical cells resembling Thiomargarita species. High resolution in situ microprofiles revealed different geochemical settings selecting for different mat types. Arcobacter mats occurred where oxygen and sulfide overlapped at the bottom water interface. Filamentous sulfide oxidizers were associated with non-overlapping, steep gradients of oxygen and sulfide. A dense population of Thiomargarita was favored by temporarily changing supplies of oxygen and sulfide. These results indicate that the decisive factors in selecting for different mat-forming bacteria within one deep-sea province are spatial or temporal variations in energy supply. Furthermore, the occurrence of Arcobacter spp.-related 16S rRNA genes in the sediments below all three types of mats, as well as on top of brine lakes of the Nile Deep Sea Fan, indicates that this group of sulfide oxidizers can switch between different life modes depending on the geobiochemical habitat setting.

Environmental characteristics and gas composition of Lost Hammer, Canadian Arctic

We report the first microbiological characterization of a terrestrial methane seep in a cryo-environment in the form of an Arctic hypersaline (~24% salinity), subzero (-5 C), perennial spring, arising through thick permafrost in an area with an average annual air temperature of -15 C. Bacterial and archaeal 16S rRNA gene clone libraries indicated a relatively low diversity of phylotypes within the spring sediment (Shannon index values of 1.65 and 1.39, respectively). Bacterial phylotypes were related to microorganisms such as Loktanella, Gillisia, Halomonas and Marinobacter spp. previously recovered from cold, saline habitats. A proportion of the bacterial phylotypes were cultured, including Marinobacter and Halomonas, with all isolates capable of growth at the in situ temperature (-5 C). Archaeal phylotypes were related to signatures from hypersaline deep-sea methane-seep sediments and were dominated by the anaerobic methane group 1a (ANME-1a) clade of anaerobic methane oxidizing archaea. CARD-FISH analyses indicated that cells within the spring sediment consisted of ~84.0% bacterial and 3.8% archaeal cells with ANME-1 cells accounting for most of the archaeal cells. The major gas discharging from the spring was methane (~50%) with the low CH4/C2 + ratio and hydrogen and carbon isotope signatures consistent with a thermogenic origin of the methane. Overall, this hypersaline, subzero environment supports a viable microbial community capable of activity at in situ temperature and where methane may behave as an energy and carbon source for sustaining anaerobic oxidation of methane-based microbial metabolism. This site also provides a model of how a methane seep can form in a cryo-environment as well as a mechanism for the hypothesized Martian methane plumes.

Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. Samples for total cell counts were stored in formalin at 4°C until analysis. For sandy samples, the total cell count/CARD-FISH protocol was optimized to separate sand particles from the cells. Cells were dislodged from sediment grains and brought into solution with the supernatant by sonicating each sample onice for 2 minutes at 50W. This procedure was repeated four times and supernatants were combined. The sediment samples were brought to a final dilution of 1:2000 to 1:4000 and filtered onto 0.2µm GTTP filters (Millipore, Eschbonn, Germany).

Anaerobic methanotrophic archaea-2 and Desulfosarcina/Desulfococcus group in sediments at different stations

The anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). In sediment samples from Hydrate Ridge, the Isis Mud Volcano and the Gulf of Mexico, DSS cells accounted for 3-6% of all DAPI-stained single cells. Out of these, 8-17% were labelled with probe SEEP1a-1441. This translated into relative abundances of single SEEP-SRB1a cells of 0.3% to 0.7%. Contrastingly, in a sediment sample from the Gullfaks oil field, DSS cells accounted for 18% and SEEP-SRB1a for 9% of all single cells. This sediment sample also featured an unusually high abundance of single ANME-2 cells and only very few ANME-2/DSS aggregates in comparison with other AOM habitats. Considering also the nature of the sample, it is likely that the high number of single ANME-2 and SEEP-SRB1a cells were an artifact of sample preparation. Here, harsher sonication was required to remove the microorganisms from coarse sand prior to CARD-FISH analysis.

Isotopic signature of specific biomarkers derived from anaerobic methanotrophic archaea (ANME groups) and sulphate-reducing bacteria (SRB) at different cold seep provinces

Anaerobic methane-oxidizing microbial communities in sediments at cold methane seeps are important factors in controlling methane emission to the ocean and atmosphere. Here, we investigated the distribution and carbon isotopic signature of specific biomarkers derived from anaerobic methanotrophic archaea (ANME groups) and sulphate-reducing bacteria (SRB) responsible for the anaerobic oxidation of methane (AOM) at different cold seep provinces of Hydrate Ridge, Cascadia margin. The special focus was on their relation to in situ cell abundances and methane turnover. In general, maxima in biomarker abundances and minima in carbon isotope signatures correlated with maxima in AOM and sulphate reduction as well as with consortium biomass. We found ANME-2a/DSS aggregates associated with high abundances of sn-2,3-di-O-isoprenoidal glycerol ethers (archaeol, sn-2-hydroxyarchaeol) and specific bacterial fatty acids (C16:1omega5c, cyC17:0omega5,6) as well as with high methane fluxes (Beggiatoa site). The low to medium flux site (Calyptogena field) was dominated by ANME-2c/DSS aggregates and contained less of both compound classes but more of AOM-related glycerol dialkyl glycerol tetraethers (GDGTs). ANME-1 archaea dominated deeper sediment horizons at the Calyptogena field where sn-1,2-di-O-alkyl glycerol ethers (DAGEs), archaeol, methyl-branched fatty acids (ai-C15:0, i-C16:0, ai-C17:0), and diagnostic GDGTs were prevailing. AOM-specific bacterial and archaeal biomarkers in these sediment strata generally revealed very similar d13C-values of around -100 per mill. In ANME-2-dominated sediment sections, archaeal biomarkers were even more 13C-depleted (down to -120 per mill), whereas bacterial biomarkers were found to be likewise 13C-depleted as in ANME-1-dominated sediment layers (d13C: -100 per mill). The zero flux site (Acharax field), containing only a few numbers of ANME-2/DSS aggregates, however, provided no specific biomarker pattern. Deeper sediment sections (below 20 cm sediment depth) from Beggiatoa covered areas which included solid layers of methane gas hydrates contained ANME-2/DSS typical biomarkers showing subsurface peaks combined with negative shifts in carbon isotopic compositions. The maxima were detected just above the hydrate layers, indicating that methane stored in the hydrates may be available for the microbial community. The observed variations in biomarker abundances and 13C-depletions are indicative of multiple environmental and physiological factors selecting for different AOM consortia (ANME-2a/DSS, ANME-2c/DSS, ANME-1) along horizontal and vertical gradients of cold seep settings.

Development of bacterial and archaeal communities in erupted subsurface muds at the Håkon Mosby mud volcano

The microbial oxidation of methane controls the emission of the greenhouse gas methane from the ocean floor. However, some seabed structures such as mud volcanoes have leaky microbial methane filters and can be important sources of methane. We investigated the disturbance and recovery of a methanotrophic mud volcano microbiome (Håkon Mosby mud volcano, 1250 m water depth), to assess time scales of community succession and function in the natural deep-sea environment. We analyzed 10 surface and 5 subsurface sediment samples across HMMV mud flows from most recently discharged subsurface muds towards old consolidated muds as well as one reference site (REF) located approximately 0.5 km outside of the HMMV. Surface samples were obtained in 2003, 2009 and 2010. The surface of the new mud flows at the geographical center was sampled in 2009 and 2010. Around 100 m south of the center, we sampled more consolidated aged muds in 2003 and 2010. Old mud flows were sampled around 300 m southeast and 100 m north of the geographical center in 2003, 2009 and 2010. Surface sediment samples (0-20 cm) were recovered either by TV-guided Multicorer or by push cores using the remotely operated vehicle Quest (Marum, University Bremen). Subsurface sediments of all zones (>2 m below sea floor) were obtained in 2003 by gravity corer. After recovery, sediments were immediately subsampled in a refrigerated container (0°C) and further processed for biogeochemical analyses or preserved at -20°C for later DNA analyses. Our study show that freshly erupted muds hosted heterotrophic deep subsurface communities, which were replaced by surface communities within a few years of exposure. Aerobic methanotrophy was established at the top surface layer within less than a year, followed by anaerobic methanotrophy, sulfate reduction and finally thiotrophy. Our data indicate that it takes decades in cold environments before efficient methanotrophic communities establish to control methane emission. The observed succession provides insights to the response time of complex deep-sea communities to seafloor disturbances.

Relative abundance of prokaryotes in sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

CARD-FISH was performed as previously described in Ruff et al., (2013; doi:10.1371/journal.pone.0072627) with the following modifications. 4-6 µl of 25-fold diluted sediment were used for filtration. Archaeal cell walls were permeabilized with 0.1M HCl for 2 min to detect ANME-3 cells, or Proteinase K solution (15 µg ml-1 (Merck, Darmstadt, Germany) in 0.05 M EDTA (pH 8), 0.1 M Tris-HCl (pH 8), 0.5 M NaCl) for 2-4 min at room temperature for all other archaea. Bacterial cell walls were permeabilized with lysozyme solution (1000kU/ml) for 60 min at 37°. Cells were stained with DAPI (1µg/ml), embedded in mounting medium and counted in 40-60 independent microscopic fields using an Axiophot II epifluorescence microscope (Carl Zeiss, Jena, Germany).

Microbial community composition in sediments of the Hydrate Ridge (Cascadian Margin) collected during RV SONNE cruises SO143 (1999) and SO148-1 (2000)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH). Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6×10**10cells/cm**3). Sediment samples were obtained during RV SONNE cruises SO143-2 and SO148-1 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. Sediment cores of 20 - 40 cm length were obtained using a video-guided multiple corer from gas hydrate bearing sediments and from reference sites not enriched in methane in the surface sediments. Samples for total cell counts were obtained from 1 cm core slices, fixed with 2% formaldehyde and stored cold (4°C) and the quantification of aggregates was done via epifluorescence microscopy after staining the sediments with Acridine Orange Direct Counts (AODC) according to the method of Meyer- Reil (1983, doi:10.1007/BF00395813). Total cell counts were defined as the sum of single cells plus the aggregated cells in the syntrophic consortia. DAPI staining was used to measure ANME2/DSS aggregate sizes via epifluorescence microscopy of FISH-treated samples. For FISH, subsamples of sediment cores were sliced into 1 cm intervals and fixed for 2-3 h with 3% formaldehyde (final concentration), washed twice with 1×PBS (10 mM sodium phosphate; 130 mM NaCl), and finally stored in 1×PBS/EtOH (1:1) at -20°C.

Microbial community composition and bacterioplankton at time series station Helgoland Roads, North Sea

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. This study explores such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. The surface water samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' (54° 11.3' N, 7° 54.0' E) between the main island and the minor island, Düne (German for 'dune') using small research vessels (http://www.awi.de/en/expedition/ships/more-ships.html). Water depths at this site fluctuate from 6 to 10 m over the tidal cycle. Samples were processed as described previously (Teeling et al., 2012; doi:10.7554/eLife.11888.001) in the laboratory of the Biological Station Helgoland within less than two hours after sampling. Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously (Thiele et al., 2011; doi:10.1016/B978-0-444-53199-5.00056-7). To obtain total cell numbers, DNA of formaldehyde fixed cells filtered on 0.2 mm pore sized filters was stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescently labeled cells were subsequently counted on filter sections using an epifluores-cence microscope. Likewise, bacterioplankton community composition was assessed by catalyzedreporter deposition fluorescence in situ hybridization (CARD-FISH) of formaldehyde fixed cells on 0.2 mm pore sized filters.

Relative abundance of prokaryotic picoplankton populations in the Atlantic Ocean

Members of the prokaryotic picoplankton are the main drivers of the biogeochemical cycles over large areas of the world's oceans. In order to ascertain changes in picoplankton composition in the euphotic and twilight zones at an ocean basin scale we determined the distribution of 11 marine bacterial and archaeal phyla in three different water layers along a transect across the Atlantic Ocean from South Africa (32.9°S) to the UK (46.4°N) during boreal spring. Depth profiles down to 500 m at 65 stations were analysed by catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and automated epifluorescence microscopy. There was no obvious overall difference in microbial community composition between the surface water layer and the deep chlorophyll maximum (DCM) layer. There were, however, significant differences between the two photic water layers and the mesopelagic zone. SAR11 (35 ± 9%) and Prochlorococcus (12 ± 8%) together dominated the surface waters, whereas SAR11 and Crenarchaeota of the marine group I formed equal proportions of the picoplankton community below the DCM (both ~15%). However, due to their small cell sizes Crenarchaeota contributed distinctly less to total microbial biomass than SAR11 in this mesopelagic water layer. Bacteria from the uncultured Chloroflexi-related clade SAR202 occurred preferentially below the DCM (4-6%). Distinct latitudinal distribution patterns were found both in the photic zone and in the mesopelagic waters: in the photic zone, SAR11 was more abundant in the Northern Atlantic Ocean (up to 45%) than in the Southern Atlantic gyre (~25%), the biomass of Prochlorococcus peaked in the tropical Atlantic Ocean, and Bacteroidetes and Gammaproteobacteria bloomed in the nutrient-rich northern temperate waters and in the Benguela upwelling. In mesopelagic waters, higher proportions of SAR202 were present in both central gyre regions, whereas Crenarchaeota were clearly more abundant in the upwelling regions and in higher latitudes. Other phylogenetic groups such as the Planctomycetes, marine group II Euryarchaeota and the uncultured clades SAR406, SAR324 and SAR86 rarely exceeded more than 5% of relative abundance.

Composition of bacterioplankton in the North Atlantic subtropical gyre

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic. We assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions. The experimental work was performed on board the Royal Research Ship James Cook (cruise no. JC53, October-November 2010) as part of the Atlantic Meridional Transect programme, and on board the Royal Research Ship Discovery (cruise no. D369, August-September 2011). At each station, samples were collected from 20m depth with a sampling rosette of 20-l Niskin bottles mounted on aconductivity-temperature-depth profiler. Samples were collected in 1 l thermos flasks (washed with10% v/v HCl) in the dark and processed immediately. Depth of 20m was chosen because it represents the mixed layer and it was the shallowest depth unaffected by the ship's movement, including thrusting, that could artificially affect microbial metabolism in nutrient-depleted stratified surfacewaters. Molecular identification of flow-sorted cells CARD-FISH was performed on flow-sorted cells to identify the groups for which uptake rates were measured. High nucleic acid-containing bacteria, based on SYBR Green DNA staining, that had virtually undetectable chlorophyll autofluorescence, were phylogenetically affiliated with Prochlorococcus,in agreement with our previously reported results (Zubkov et al., 2007; doi:10.1111/j.1462-2920.2007.01324.x).