Anoxic Black Sea Research Articles

Dispersion of Particle‐Reactive Elements Caused by the Phase Transitions in Scavenging

AbstractA generalized model of scavenging of the reactive radionuclide 239,240Pu was developed, in which the sorption‐desorption processes of oxidized and reduced forms on multifraction suspended particulate matter are described by first‐order kinetics. One‐dimensional transport‐diffusion‐reaction equations were solved analytically and numerically. In the idealized case of instantaneous release of 239,240Pu on the ocean surface, the profile of concentrations asymptotically tends to the symmetric spreading bulge in the form of a Gaussian moving downward with constant velocity. The corresponding diffusion coefficient is the sum of the physical diffusivity and the apparent diffusivity caused by the reversible phase transitions between the dissolved and particulate states. Using the method of moments, we analytically obtained formulas for both the velocity of the center mass and apparent diffusivity. It was found that in ocean waters that have oxygen present at great depths, we can consider in the first approximation a simplified problem for a mixture of forms with a single effective distribution coefficient, as opposed to considering the complete problem. This conclusion was confirmed by the modeling results for the well‐ventilated Eastern Mediterranean. In agreement with the measurements, the calculations demonstrate the presence of a maximum that is slowly descending for all forms of concentration. The ratio of the reduced form to the oxidized form was approximately 0.22–0.24. At the same time, 239,240Pu scavenging calculations for the anoxic Black Sea deep water reproduced the transition from the oxidized to reduced form of 239,240Pu with depth in accordance with the measurement data.

Holocene Paleosecular Variations Recorded by Relict Magnetic Minerals in the Anoxic Black Sea Sediments

Continuous paleosecular variations reconstructed from sedimentary archives have remarkably deepened our insights into the dynamics of the Earth's magnetic field as well as the chronological purpose. Nevertheless, to construct reliable sedimentary paleomagnetic records in diagenetic reducing sediments is challenging generally due to the pervasive magnetic mineral diagenesis. The relict magnetic minerals are residuals after diagenesis and probably able to record a depositional remanent magnetization, thus it is worthy to explore their paleomagnetic potentials. For this study, two Black Sea sediment cores covering the past 8 ka were subjected to mineralogical and paleo- and rock magnetic analyses. Paramagnetic pyrite framboids are pervasive in the studied sediments deposited under anoxic bottom water conditions in the Black Sea. In addition, relict magnetic minerals of ferrous hemoilmenite, Fe-Mn and Fe-Cr spinels, and magnetite inclusions are also present in the studied cores. Compared to the previous published paleomagnetic results from the same cores over the last glacial period (20-30 ka) which are dominated by detrital (titano-)magnetite particles, the studied relict magnetic mineral samples exhibit a similar behavior in recording the depositional remanent magnetization. Furthermore, the obtained paleosecular variations spanning the past 8 ka reproduce the high intensity patterns observed in the regional archeomagnetic and volcanic datasets. Thus, the successful reconstruction of paleomagnetic secular variations from the anoxic Black Sea sediments greatly extend the application of paleomagnetism in sediments deposited in water with a reducing sub-surface environments from where paleomagnetic data are generally sparse.

Aeolian imprints of multiple Mediterranean invasions of the Black Sea during Pleistocene

Climate changes determined the repeated connections between the Black Sea, Caspian Sea and Mediterranean Sea. The landlocked anoxic Black Sea basin was exposed to several transgressions throughout Quaternary by the Mediterranean Sea through the Straits of Istanbul (Bosphorus) and by the Caspian Sea through the Manych-Kerch spillway. Sedimentological records of these connections are limited mostly to the marine terrace deposits of Marine Isotope Stage (MIS) 5e while the pre-MIS 5e period remains uncertain due to a lack of robust facies and chronological data from deep-sea sedimentary sequences. Here we discuss the imprints of multiple Mediterranean transgressions during Middle Pleistocene in the Black Sea based on facies analysis and the optical age of coastal carbonate aeolianites. Contrary to today's hydro-climate of the Black Sea, the aeolianites bear witness to the transformation of the Black Sea into a warm inland sea during successive Mediterranean invasions. Prior to the onset of aeolian deposition, paleosols were formed on the Eocene-aged hardened sandy silts, suggesting strongly washed soil. This is evidenced by no calcium carbonate and a high Rb/Sr ratio, with quartz amounting to of 99.8%. According to our OSL ages, carbonates deposited on the shelf plain under higher temperature and increased evaporation conditions in MIS 15 and the later interglacial phases were transported to the coastal sand dunes during the transitional phases of MIS 15–14, MIS 13–12, MIS 11–10 and MIS 9–8. We suggest that the carbonate-rich and ooid-containing aeolianites were repeatedly formed in the multiple Mediterranean transgression stages, beginning with an increasingly severe dry phase following the Brunhes-Matuyama magnetic reversal. • The aeolianite is a robust record for Mediterranean transgressions of the Black Sea. • The ooid-rich aeolianites was repeatedly formed in multiple transgression stages. • The aeolianites bear witness to the transformation of the Black Sea into a warmer sea.

The physiology and metabolic properties of a novel, low-abundance Psychrilyobacter species isolated from the anoxic Black Sea shed light on its ecological role.

Members of the Psychrilyobacter spp. of the phylum Fusobacteria have been recently suggested to be amongst the most significant primary degraders of the detrital organic matter in sulfidic marine habitats, despite representing only a small proportion (<0.1%) of the microbial community. In this study, we have isolated a previously uncultured Psychrilyobacter species (strains SD5T and BL5; Psychrilyobacter piezotolerans sp. nov.) from the sulfidic waters (i.e., 2000 m depth) of the Black Sea and investigated its physiology and genomic capability in order to better understand potential ecological adaptation strategies. P. piezotolerans utilized a broad range of organic substituents (carbohydrates and proteins) and, remarkably, grew at sulfide concentrations up to 32 mM. These flexible physiological properties were supported by the presence of the respective metabolic pathways in the genomes of both strains. Growth at varying hydrostatic pressure (0.1-50 MPa) was sustained by modifying its membrane lipid composition. Thus, we have isolated a novel member of the 'rare biosphere', which endures the extreme conditions and may play a significant role in the degradation of detrital organic matter sinking into the sulfidic waters of the Black Sea.

Assessing the Effect of Humic Substances and Fe(III) as Potential Electron Acceptors for Anaerobic Methane Oxidation in a Marine Anoxic System.

Marine anaerobic methane oxidation (AOM) is generally assumed to be coupled to sulfate reduction, via a consortium of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). ANME-1 are, however, often found as single cells, or only loosely aggregated with SRB, suggesting they perform a form of AOM independent of sulfate reduction. Oxidized metals and humic substances have been suggested as potential electron acceptors for ANME, but up to now, AOM linked to reduction of these compounds has only been shown for the ANME-2 and ANME-3 clades. Here, the effect of the electron acceptors anthraquinone-disulfonate (AQDS), a humic acids analog, and Fe3+ on anaerobic methane oxidation were assessed by incubation experiments with anoxic Black Sea water containing ANME-1b. Incubation experiments with 13C-methane and AQDS showed a stimulating effect of AQDS on methane oxidation. Fe3+ enhanced the ANME-1b abundance but did not substantially increase methane oxidation. Sodium molybdate, which was added as an inhibitor of sulfate reduction, surprisingly enhanced methane oxidation, possibly related to the dominant abundance of Sulfurospirillum in those incubations. The presented data suggest the potential involvement of ANME-1b in AQDS-enhanced anaerobic methane oxidation, possibly via electron shuttling to AQDS or via interaction with other members of the microbial community.

Holocene paleodepositional changes reflected in the sedimentary microbiome of the Black Sea.

Subsurface microbial communities are generally thought to be structured through in situ environmental conditions such as the availability of electron acceptors and donors and porosity, but recent studies suggest that the vertical distribution of a subset of subseafloor microbial taxa, which were present at the time of deposition, were selected by the paleodepositional environment. However, additional highly resolved temporal records of subsurface microbiomes and paired paleoenvironmental reconstructions are needed to justify this claim. Here, we performed a highly resolved shotgun metagenomics survey to study the taxonomic and functional diversity of the subsurface microbiome in Holocene sediments underlying the permanently stratified and anoxic Black Sea. Obligate aerobic bacteria made the largest contribution to the observed shifts in microbial communities associated with known Holocene climate stages and transitions. This suggests that the aerobic fraction of the subseafloor microbiome was seeded from the water column and did not undergo post-depositional selection. In contrast, obligate and facultative anaerobic bacteria showed the most significant response to the establishment of modern-day environmental conditions 5.2ka ago that led to a major shift in planktonic communities and in the type of sequestered organic matter available for microbial degradation. No significant shift in the subseafloor microbiome was observed as a result of environmental changes that occurred shortly after the marine reconnection, 9ka ago. This supports the general view that the marine reconnection was a gradual process. We conclude that a high-resolution analysis of downcore changes in the subseafloor microbiome can provide detailed insights into paleoenvironmental conditions and biogeochemical processes that occurred at the time of deposition.

The mesopelagic anoxic Black Sea as an unexpected habitat for Synechococcus challenges our understanding of global \u201cdeep red fluorescence\u201d

The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called “deep red fluorescence”, a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml−1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the “deep red fluorescence” signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.

Anaerobic Degradation of Sulfated Polysaccharides by Two Novel Kiritimatiellales Strains Isolated From Black Sea Sediment.

The marine environment contains a large diversity of sulfated polysaccharides and other glycopolymers. Saccharolytic microorganisms degrade these compounds through hydrolysis, which includes the hydrolysis of sulfate groups from sugars by sulfatases. Various marine bacteria of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum have exceptionally high numbers of sulfatase genes associated with the degradation of sulfated polysaccharides. However, thus far no sulfatase-rich marine anaerobes are known. In this study, we aimed to isolate marine anaerobes using sulfated polysaccharides as substrate. Anoxic enrichment cultures were set up with a mineral brackish marine medium, inoculated with anoxic Black Sea sediment sampled at 2,100 m water depth water and incubated at 15°C (in situ T = 8°C) for several weeks. Community analysis by 16S rRNA gene amplicon sequencing revealed the enrichment of Kiritimatiellaeota clade R76-B128 bacteria in the enrichments with the sulfated polysaccharides fucoidan and iota-carrageenan as substrate. We isolated two strains, F1 and F21, which represent a novel family within the order of the Kiritimatiellales. They were capable of growth on various mono-, di-, and polysaccharides, including fucoidan. The desulfation of iota-carrageenan by strain F21 was confirmed quantitatively by an increase in free sulfate concentration. Strains F1 and F21 represent the first marine sulfatase-rich anaerobes, encoding more sulfatases (521 and 480, 8.0 and 8.4% of all coding sequences, respectively) than any other microorganism currently known. Specific encoded sulfatase subfamilies could be involved in desulfating fucoidan (S1_15, S1_17 and S1_25) and iota-carrageenan (S1_19). Strains F1 and F21 had a sulfatase gene classification profile more similar to aerobic than anaerobic sulfatase-rich PVC bacteria, including Kiritimatiella glycovorans, the only other cultured representative within the Kiritimatiellaeota. Both strains encoded a single anaerobic sulfatase-maturating enzyme which could be responsible for post-translational modification of formylglycine-dependent sulfatases. Strains F1 and F21 are potential anaerobic platforms for future studies on sulfatases and their maturation enzymes.

Net removal of dissolved organic carbon in the anoxic waters of the Black Sea

Dissolved organic carbon (DOC) concentrations in the deep Black Sea are ~2.5 times higher than found in the global ocean. The two major external sources of DOC are rivers and the Sea of Marmara, a transit point for waters from the Mediterranean Sea. In addition, expansive phytoplankton blooms contribute autochthonous carbon to the Black Sea's ~800 Tg C DOC reservoir. Here, a basin-wide zonal section of DOC is explored using data from the 2013 Dutch GEOTRACES GA04-N, cruise 64PE373. DOC distributions are interpreted with respect to well-described hydrographic and biogeochemical layers of the Black Sea. Observed DOC concentrations were >180μmolkg−1 at the surface, decreasing to ~125μmolkg−1 at the base of the oxic layer and reaching a minimum of ~113μmolkg−1 in the upper anoxic layer between ~150 and 500m. At greater depths the concentrations increased; maximum anoxic layer concentrations of 122μmolkg−1 were found in the homogeneous benthic bottom layer (>1775m). Concentrations are then predicted based on conservation with respect to salinity using linear end-member mixing models, and predictions are compared with observations to estimate net removal (i.e., deficits) and accumulation (i.e., surpluses). A maximum surplus of ~10μmolkg−1 was identified at the surface, likely due to local primary production. DOC exported to depth was non-conservative: up to ~34‐41μmolkg−1 was removed from the basin's oxic layer in <5years, and an additional 13±5μmolkg−1 was removed from the anoxic layer during its ~300 to 600‐year residence time, given steady state. These deficits represent a removal of ~19% in the oxic water and a further removal of ~10% under anoxia, for a net removal of 48μmolkg−1 (or ~29%) of allochthonous DOC, with respect to predicted concentrations. We find no evidence for DOC accumulation (i.e., net production) in the anoxic Black Sea, and suggest that concentrations are elevated relative to the ocean due to input of terrigenous DOC from rivers; we estimate that >50% of DOC in the deep Black Sea is terrigenous. The Black Sea's relatively elevated DOC pool may be analogous to a hypothesized anoxic Eocene ocean's elevated reservoir only if the Eocene ocean received a substantial amount of terrigenous DOC.

Deep-water profiling of 137Cs and 90Sr in the Black Sea: a further insight into dynamics of the post-Chernobyl radioactive contamination

The first post-Chernobyl measurements of vertical distribution of 137Cs in the abyssal Black Sea waters have been conducted in the vicinity of submarine canyon Viteaz together with profiling of 90Sr. This allowed showing an increase in 137Cs concentration and the low 90Sr/137Cs activity ratio (≈0.2) at 1,000 m water depth, suggesting the long-distance influence of the Danube River runoff through the Viteaz Canyon. Calculation of inventories of 90Sr and 137Cs in oxic and anoxic Black Sea waters and their comparison with the total input of these radionuclides to the Black Sea after the Chernobyl accident have revealed considerable accumulation of them in the deep water column.

Are iron-phosphate minerals a sink for phosphorus in anoxic Black Sea sediments?

Phosphorus (P) is a key nutrient for marine organisms. The only long-term removal pathway for P in the marine realm is burial in sediments. Iron (Fe) bound P accounts for a significant proportion of this burial at the global scale. In sediments underlying anoxic bottom waters, burial of Fe-bound P is generally assumed to be negligible because of reductive dissolution of Fe(III) (oxyhydr)oxides and release of the associated P. However, recent work suggests that Fe-bound P is an important burial phase in euxinic (i.e. anoxic and sulfidic) basin sediments in the Baltic Sea. In this study, we investigate the role of Fe-bound P as a potential sink for P in Black Sea sediments overlain by oxic and euxinic bottom waters. Sequential P extractions performed on sediments from six multicores along two shelf-to-basin transects provide evidence for the burial of Fe-bound P at all sites, including those in the euxinic deep basin. In the latter sediments, Fe-bound P accounts for more than 20% of the total sedimentary P pool. We suggest that this P is present in the form of reduced Fe-P minerals. We hypothesize that these minerals may be formed as inclusions in sulfur-disproportionating Deltaproteobacteria. Further research is required to elucidate the exact mineral form and formation mechanism of this P burial phase, as well as its role as a sink for P in sulfide-rich marine sediments.

Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition – fluorescence in situ hybridization (CARD–FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 109 to 1010 cells/mL at the sediment surface to 107–109 cells/mL below one meter depth. Based on CARD–FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Stimulation of Autotrophic Denitrification by Intrusions of the Bosporus Plume into the Anoxic Black Sea

Autotrophic denitrification was measured in the southwestern coastal Black Sea, where the Bosporus Plume injects oxidized chemical species (especially O2 and ) into the oxic, suboxic, and anoxic layers. Prominent oxygen intrusions caused an overlap of and sulfide at the same station where autotrophic denitrification activity was detected with incubation experiments. Several bacteria that have been proposed to oxidize sulfide in other low oxygen environments were found in the Black Sea including SUP05, Sulfurimonas, Arcobacter, and BS-GSO2. Comparison of TRFLP profiles from this mixing zone station and the Western Gyre (a station not affected by the Bosporus Plume) indicate the greatest relative abundance of Sulfurimonas and Arcobacter at the appropriate depths at the mixing zone station. The autotrophic gammaproteobacterium BS-GSO2 correlated with ammonium fluxes rather than with sulfide fluxes and the maximum in SUP05 peak height was shallower than the depths where autotrophic denitrification was detected. Notably, anammox activity was not detected at the mixing zone station, though low levels of DNA from the anammox bacteria Candidatus Scalindua were present. These results provide evidence for a modified ecosystem with different N2 production pathways in the southwest coastal region compared to that found in the rest of the Black Sea. Moreover, the same Sulfurimonas phylotype (BS139) was previously detected on >30 μm particles in the suboxic zone of the Western Gyre along with DNA of potential sulfate reducers, so it is possible that particle-attached autotrophic denitrification may be an overlooked N2 production pathway in the central Black Sea as well.

Exploring preserved fossil dinoflagellate and haptophyte DNA signatures to infer ecological and environmental changes during deposition of sapropel S1 in the eastern Mediterranean

In this study we used a comparative multiproxy survey (fossil DNA, calcareous nannofossils, and lipid biomarkers) to test whether preserved genetic signatures provide an accurate view of haptophyte and dinoflagellate populations during deposition of the eastern Mediterranean sapropel S1 and the organic carbon‐depleted oxidized marls flanking the S1 and to see if we could identify important environmental indicator species that did not fossilize and escaped previous microscopic identification. The marls above and below the S1 contained low concentrations of lipid biomarkers diagnostic for dinoflagellates and haptophytes (i.e., dinosterol and long‐chain alkenones), but 500 base pair long ribosomal DNA fragments of these protists were below the detection limit. In contrast, dinoflagellate and haptophyte DNA could be recovered from the organic carbon‐rich S1, but the most abundant sequences did not represent species that were part of the nannofossil (this study) or previously described dinocyst composition. The oldest section of S1 (9.8 to ∼8 14C kyr B.P.) revealed a predominance of dinoflagellate phylotypes, which were previously only detected in anoxic Black Sea sediments. In the same section of the core, the most abundant haptophyte sequence showed highest similarity with uncultivated haptophytes that were previously shown to grow mixotrophically as predators of picocyanobacteria, an adaptation that promotes growth in oligotrophic marine waters. Sequences with highest similarities to clones found in marine surface waters predominated in the S1 after ∼8 14C kyr B.P. We discuss whether the shifts in haptophyte and dinoflagellate populations inferred from the preserved DNA reflect known environmental changes that occurred during the formation of sapropel S1.

Methane and sulfide fluxes in permanent anoxia: In situ studies at the Dvurechenskii mud volcano (Sorokin Trough, Black Sea)

The Dvurechenskii mud volcano (DMV) is located in permanently anoxic waters at 2060 m depth (Sorokin Trough, Black Sea). The DMV was studied during the RV Meteor expedition M72/2 as an example of an active mud volcano system, to investigate the significance of submarine mud volcanism for the methane and sulfide budget of the anoxic Black Sea hydrosphere. Our studies included benthic fluxes of methane and sulfide, as well as the factors controlling transport, consumption and production of both compounds within the sediment. The pie-shaped mud volcano showed temperature anomalies as well as solute and gas fluxes indicating high fluid flow at its summit north of the geographical center. The anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) was repressed in this zone due to the upward flow of sulfate-depleted fluids through recently deposited subsurface muds, apparently limiting microbial methanotrophic activity. Consequently, the emission of dissolved methane into the water column was high, with an estimated rate of 0.46 mol m −2 d −1. On the wide plateau and edge of the mud volcano surrounding the summit, fluid flow and total methane flux were lower, allowing higher SR and AOM rates correlated with an increase in sulfate penetration into the sediment. Here, between 50% and 70% of the methane flux (0.07–0.1 mol m −2 d −1) was consumed within the upper 10 cm of the sediment. The overall amount of dissolved methane released from the entire mud volcano structure into the water column was significant with a discharge of 1.3 × 10 7 mol yr −1. The DMV maintains also high areal rates of methane-fueled sulfide production and emission of on average 0.05 mol m −2 d −1. This is a difference to mud volcanoes in oxic waters, which emit similar amounts of methane, but not sulfide. However, based on a comparison of this and other mud volcanoes of the Black Sea, we conclude that sulfide and methane emission into the hydrosphere from deep-water mud volcanoes does not significantly contribute to the sulfide and methane inventory of the Black Sea.

An ENAA and PGAA comparative study of anoxic Black Sea sediments

A comparative study performed by means of both epithermal neutron activation analysis (ENAA) and prompt gamma neutron activation analysis (PGAA) concerning the distribution of 9 elements (Na, Al, Cl, K, Ca, Ti, Mn Fe and Sm) in unconsolidated sediments collected from the Black Sea anoxic region is presented. Principal component analysis (PCA) was used as a supplementary method in interpreting experimental data. The final results have shown that eight of them (Na, Al, Cl, K, Ti, Mn, Fe and Sm) have concentrations which differ by less than 30% from the corresponding concentrations of Upper Continental Crust (UCC), confirming the continental origin of sediments. At the same time, Ca concentration was about six times higher than that of UCC, this significant enrichment being due to the presence of the neighbouring oxygenated zone of considerable deposits of shell debris, characteristic of the Western Black Sea Continental Platform. Excepting Na and Mn, the concentrations of all other elements involved in this intercomparison were coincident within one standard deviation which attests the accuracy of this test. At the same time, PCA revealed the presence of five clusters, which could be put into concordance with the mineralogical composition of sediments, each of them consisting in equal proportions of elements determined by both methods.

Consumption of Methane and CO 2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

Consumption of Methane and CO ₂ by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

Consumption of Methane and CO 2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.

Subsurface Microbial Methanotrophic Mats in the Black Sea

A nodule-shaped microbial mat was found subsurface in sediments of a gas seep in the anoxic Black Sea. This mat was dominated by ANME-1 archaea and consumed methane and sulfate simultaneously. We propose that such subsurface mats represent the initial stage of previously investigated microbial reefs.

Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME‐I and ANME‐II communities

The anaerobic oxidation of methane (AOM) is one of the major sinks for methane on earth and is known to be mediated by at least two phylogenetically different groups of anaerobic methanotrophic Archaea (ANME-I and ANME-II). We present the first comparative in vitro study of the environmental regulation and physiology of these two methane-oxidizing communities, which occur naturally enriched in the anoxic Black Sea (ANME-I) and at Hydrate Ridge (ANME-II). Both types of methanotrophic communities are associated with sulfate-reducing-bacteria (SRB) and oxidize methane anaerobically in a 1:1 ratio to sulfate reduction (SR). They responded sensitively to elevated methane partial pressures with increased substrate turnover. The ANME-II-dominated community showed significantly higher cell-specific AOM rates. Besides sulfate, no other electron acceptor was used for AOM. The processes of AOM and SR could not be uncoupled by feeding the SRB with electron donors such as acetate, formate or molecular hydrogen. AOM was completely inhibited by the addition of bromoethanesulfonate in both communities, indicating the participation of methanogenic enzymes in the process. Temperature influenced the intensity of AOM, with ANME-II being more adapted to cold temperatures than ANME-I. The variation of other environmental parameters, such as sulfate concentration, pH and salinity, did not influence the activity of both communities. In conclusion, the ecological niches of methanotrophic Archaea seem to be mainly defined by the availability of methane and sulfate, but it remains open which additional factors lead to the dominance of ANME-I or -II in the environment.