Organic and inorganic geochemistry and archaeal community composition of hypersaline sediments from Orca Basin, RV Atlantis Expedition AT18-02

Among the most extreme habitats on Earth, dark, deep, anoxic brines host unique microbial ecosystems that remain largely unexplored. As the terminal step of anaerobic degradation of organic matter, methanogenesis is a potentially significant but poorly constrained process in deep-sea hypersaline environments. We combined biogeochemical and phylogenetic analyses as well as incubation experiments to unravel the origin of methane in hypersaline sediments of Orca Basin in the northern Gulf of Mexico. Substantial concentrations of methane (up to 3.4 mM) coexisted with high concentrations of sulfate (16-43 mM) in two sediment cores retrieved from the northern and southern parts of Orca Basin. The strong depletion of 13C in methane (-77 to -89 per mill) pointed towards a biological source. While low concentrations of competitive substrates limited the significance of hydrogenotrophic and acetoclastic methanogenesis, the presence of non-competitive methylated substrates (methanol, trimethylamine, dimethyl sulfide, dimethylsulfoniopropionate) supported the potential for methane generation through methylotrophic methanogenesis. Thermodynamic calculations demonstrated that hydrogenotrophic and acetoclastic methanogenesis were unlikely to occur under in situ conditions, while methylotrophic methanogenesis from a variety of substrates was highly favorable. Likewise, carbon isotope relationships between methylated substrates and methane supported methylotrophic methanogenesis as the major source of methane. Stable isotope tracer and radiotracer experiments with 13C bicarbonate, acetate and methanol as well as 14C-labeled methylamine indicated that methylotrophic methanogenesis was the predominant methanogenic pathway. Based on 16S rRNA gene sequences, halophilic methylotrophic methanogens related to the genus Methanohalophilus dominated the benthic archaeal community in the northern basin but also occurred in the southern basin. High abundances of methanogen lipid biomarkers such as intact polar and polyunsaturated hydroxyarchaeols were detected in sediments from the northern basin, with lower abundances in the southern basin. Strong 13C-depletion of saturated and monounsaturated hydroxyarchaeol were consistent with methylotrophic methanogenesis as the major methanogenic pathway. Collectively, the availability of methylated substrates, thermodynamic calculations, experimentally determined methanogenic activity as well as lipid and gene biomarkers strongly suggested methylotrophic methanogenesis as predominant pathway of methane formation in the presence of sulfate in Orca Basin sediments.

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.

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.

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.

Availability of sequences of aquatic and terrestrial taxa in genetic databases (GenBank and BOLD - the Barcode of Life Database) in 2010, 2012 and 2016

Correct species identifications are of tremendous importance for invasion ecology, as mistakes could lead to misdirecting limited resources against harmless species or inaction against problematic ones. DNA barcoding is becoming a promising and reliable tool for species identifications, however the efficacy of such molecular taxonomy depends on gene region(s) that provide a unique sequence to differentiate among species and on availability of reference sequences in existing genetic databases. Here, we assembled a list of aquatic and terrestrial non-indigenous species (NIS) and checked two leading genetic databases for corresponding sequences of six genome regions used for DNA barcoding. The genetic databases were checked in 2010, 2012, and 2016. All four aquatic kingdoms (Animalia, Chromista, Plantae and Protozoa) were initially equally represented in the genetic databases, with 64, 65, 69, and 61% of NIS included, respectively. Sequences for terrestrial NIS were present at rates of 58 and 78% for Animalia and Plantae, respectively. Six years later, the number of sequences for aquatic NIS increased to 75, 75, 74, and 63% respectively, while those for terrestrial NIS increased to 74 and 88% respectively. Genetic databases are marginally better populated with sequences of terrestrial NIS of plants compared to aquatic NIS and terrestrial NIS of animals. The rate at which sequences are added to databases is not equal among taxa. Though some groups of NIS are not detectable at all based on available data - mostly aquatic ones - encouragingly, current availability of sequences of taxa with environmental and/or economic impact is relatively good and continues to increase with time.

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.

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.

Biogeochemical measurements of cold seep sediments inhabit by vesicomyid clams in the Japan Deep Sea Trench

Vesicomyidae clams harbor sulfide-oxidizing endosymbionts and are typical members of cold seep communities associated with tectonic faults where active venting of fluids and gases takes place. We investigated the central biogeochemical processes that supported a vesicomyid clam colony as part of a locally restricted seep community in the Japan Trench at 5346 m water depth, one of the deepest seep settings studied to date. An integrated approach of biogeochemical and molecular ecological techniques was used combining in situ and ex situ measurements. During the cruise YK06-05 in 2006 with the RV Yokosuka to the Japan Trench, we investigated a clam colony inhabited by Abyssogena phaseoliformis (former known as Calyptogena phaseoliformis) and Isorropodon fossajaponicum (former known as Calyptogena fossajaponica). The targeted sampling and precise positioning of the in situ instruments were achieved with the manned research submersible Shinkai 6500 (JAMSTEC, Nankoku, Kochi, Japan). Sampling was first performed close to the rim of the JTC colony and then at the center. Immediately after sample recovery onboard, the sediment core was sub-sampled for ex situ rate measurements or preserved for later analyses. In sediment of the clam colony, low sulfate reduction (SR) rates (max. 128 nmol ml**-1 d**-1) were coupled to the anaerobic oxidation of methane (AOM). They were observed over a depth range of 15 cm, caused by active transport of sulfate due to bioturbation of the vesicomyid clams. A distinct separation between the seep and the surrounding seafloor was shown by steep horizontal geochemical gradients and pronounced microbial community shifts. The sediment below the clam colony was dominated by anaerobic methanotrophic archaea (ANME-2c) and sulfate-reducing Desulfobulbaceae (SEEP-SRB-3, SEEP-SRB-4). Aerobic methanotrophic bacteria were not detected in the sediment and the oxidation of sulfide seemed to be carried out chemolithoautotrophically by Sulfurovum species. Thus, major redox processes were mediated by distinct subgroups of seep-related microorganisms that might have been selected by this specific abyssal seep environment. Fluid flow and microbial activity was low but sufficient to support the clam community over decades and to build up high biomasses. Hence, the clams and their microbial communities adapted successfully to a low-energy regime and may represent widespread chemosynthetic communities in the Japan Trench.

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.

Prokaryotic growth and presence of metabolic products and contamination tracers in rocks samples from ODP Leg 187 sites

The microbial population in samples of basalt drilled from the north of the Australian Antarctic Discordance (AAD) during Ocean Drilling Program Leg 187 were studied using deoxyribonucleic acid (DNA)-based methods and culturing techniques. The results showed the presence of a microbial population characteristic for the basalt environment. DNA sequence analysis revealed that microbes grouping within the Actinobacteria, green nonsulfur bacteria, the Cytophaga/Flavobacterium/Bacteroides (CFB) group, the Bacillus/Clostridium group, and the beta and gamma subclasses of the Proteobacteria were present in the basalt samples collected. The most dominant phylogenetic group, both in terms of the number of sequences retrieved and the intensities of the DNA bands obtained with the denaturing gradient gel electrophoresis analysis, was the gamma Proteobacteria. Enrichment cultures showed phylogenetic affiliation with the Actinobacteria, the CFB group, the Bacillus/Clostridium group, and the alpha, beta, gamma, and epsilon subclasses of the Proteobacteria. Comparison of native and enriched samples showed that few of the microbes found in native basalt samples grew in the enrichment cultures. Only seven clusters, two clusters within each of the CFB and Bacillus/Clostridium groups and five clusters within the gamma Proteobacteria, contained sequences from both native and enriched basalt samples with significant similarity. Results from cultivation experiments showed the presence of the physiological groups of iron reducers and methane producers. The presence of the iron/manganese-reducing bacterium Shewanella was confirmed with DNA analysis. The results indicate that iron reducers and lithotrophic methanogenic Archaea are indigenous to the ocean crust basalt and that the methanogenic Archaea may be important primary producers in this basaltic environment.