Metal-dependent anaerobic methane oxidation in marine sediment: Insights from marine settings and other systems

Anaerobic oxidation of methane(AOM) plays a crucial role in controlling global methane emission. This is a microbial process that relies on the reduction of external electron acceptors such as sulfate, nitrate/nitrite, and transient metal ions. In marine settings, the dominant electron acceptor for AOM is sulfate, while other known electron acceptors are transient metal ions such as iron and manganese oxides. Despite the AOM process coupled with sulfate reduction being relatively well characterized,researches on metal-dependent AOM process are few, and no microorganism has to date been identified as being responsible for this reaction in natural marine environments. In this review, geochemical evidences of metal-dependent AOM from sediment cores in various marine environments are summarized. Studies have showed that iron and manganese are reduced in accordance with methane oxidation in seeps or diffusive profiles below the methanogenesis zone. The potential biochemical basis and mechanisms for metal-dependent AOM processes are here presented and discussed. Future research will shed light on the microbes involved in this process and also on the molecular basis of the electron transfer between these microbes and metals in natural marine environments.     

An inorganic geochemical argument for coupled anaerobic oxidation of methane and iron reduction in marine sediments

Here, we present results from sediments collected in the Argentine Basin, a non‐steady state depositional marine system characterized by abundant oxidized iron within methane‐rich layers due to sediment reworking followed by rapid deposition. Our comprehensive inorganic data set shows that iron reduction in these sulfate and sulfide‐depleted sediments is best explained by a microbially mediated process—implicating anaerobic oxidation of methane coupled to iron reduction (Fe‐AOM) as the most likely major mechanism. Although important in many modern marine environments, iron‐driven AOM may not consume similar amounts of methane compared with sulfate‐dependent AOM. Nevertheless, it may have broad impact on the deep biosphere and dominate both iron and methane cycling in sulfate‐lean marine settings. Fe‐AOM might have been particularly relevant in the Archean ocean, >2.5 billion years ago, known for its production and accumulation of iron oxides (in iron formations) in a biosphere likely replete with methane but low in sulfate. Methane at that time was a critical greenhouse gas capable of sustaining a habitable climate under relatively low solar luminosity, and relationships to iron cycling may have impacted if not dominated methane loss from the biosphere.     

Stratified Community Responses to Methane and Sulfate Supplies in Mud Volcano Deposits: Insights from an In Vitro Experiment

Numerous studies on marine prokaryotic communities have postulated that a process of anaerobic oxidation of methane (AOM) coupled with sulfate reduction (SR) is the main methane sink in the world''s oceans. AOM has also been reported in the deep biosphere. But the responses of the primary microbial players in eliciting changes in geochemical environments, specifically in methane and sulfate supplies, have yet to be fully elucidated. Marine mud volcanoes (MVs) expel a complex fluid mixture of which methane is the primary component, forming an environment in which AOM is a common phenomenon. In this context, we attempted to identify how the prokaryotic community would respond to changes in methane and sulfate intensities, which often occur in MV environments in the form of eruptions, diffusions or seepage. We applied an integrated approach, including (i) biochemical surveys of pore water originated from MV, (ii) in vitro incubation of mud breccia, and (iii) prokaryotic community structure analysis. Two distinct AOM regions were clearly detected. One is related to the sulfate methane transition zone (SMTZ) at depth of 30–55 cm below the sea floor (bsf); the second is at 165–205 cm bsf with ten times higher rates of AOM and SR. This finding contrasts with the sulfide concentrations in pore waters and supports the suggestion that potential AOM activity below the SMTZ might be an important methane sink that is largely ignored or underestimated in oceanic methane budget calculations. Moreover, the incubation conditions below the SMTZ favor the growth of methanotrophic archaeal group ANME-2 compared to ANME-1, and promote the rapid growth and high diversity of bacterial communities. These incubation conditions also promote the increase of richness in bacterial communities. Our results provide direct evidence of the mechanisms by which deep AOM processes can affect carbon cycling in the deep biosphere and global methane biochemistry.     

土壤中甲烷厌氧氧化菌多样性的分子检测

甲烷厌氧氧化作用是减少海洋底泥甲烷释放的重要生物地球化学过程,然而在陆地生态系统中甲烷厌氧氧化作用及其功能菌群的生态功能仍然不确定。对甲烷厌氧氧化菌多样性的研究可为减少甲烷排放提供重要科学依据。与传统的分离培养方法比较,分子检测方法是一种更为快速和高效的研究手段,可直接和全面的反映参与甲烷厌氧氧化作用的功能微生物。以DNA分子标记物为研究对象,重点探讨三类主要的分子标记基因,即16S rRNA,mcr A和pmo A,所采用的相关探针和引物信息,同时从定性和定量两个角度综述土壤甲烷厌氧氧化菌的多样性研究的主要进展,最后提出厌氧甲烷氧化菌多样性研究中存在的一些问题和相应的解决思路。     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.     

Gene expression and ultrastructure of meso‐ and thermophilic methanotrophic consortia

The sulfate‐dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon‐rich environments under different temperature regimes: ANME‐1a/HotSeep‐1 (60°C), ANME‐1a/Seep‐SRB2 (37°C) and ANME‐2c/Seep‐SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME‐2c encodes all enzymes, while ANME‐1a lacks the gene for N5,N10‐methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c‐type cytochromes, some predicted to be extracellular. ANME‐2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME‐1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire‐like structures and heme‐rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate‐dependent AOM, and that both partners synthesize molecules to enable it.     

Anaerobic oxidation of methane in sediments of Lake Constance, an oligotrophic freshwater lake

Anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor has been reported for various environments, including freshwater habitats, and also, nitrate and nitrite were recently shown to act as electron acceptors for methane oxidation in eutrophic freshwater habitats. Radiotracer experiments with sediment material of Lake Constance, an oligotrophic freshwater lake, were performed to follow 14CO2 formation from 14CH4 in sediment incubations in the presence of different electron acceptors, namely, nitrate, nitrite, sulfate, or oxygen. Whereas 14CO2 formation without and with sulfate addition was negligible, addition of nitrate increased 14CO2 formation significantly, suggesting that AOM could be coupled to denitrification. Nonetheless, denitrification-dependent AOM rates remained at least 1 order of magnitude lower than rates of aerobic methane oxidation. Using molecular techniques, putative denitrifying methanotrophs belonging to the NC10 phylum were detected on the basis of the pmoA and 16S rRNA gene sequences. These findings show that sulfate-dependent AOM was insignificant in Lake constant sediments. However, AOM can also be coupled to denitrification in this oligotrophic freshwater habitat, providing first indications that this might be a widespread process that plays an important role in mitigating methane emissions.     

Influence of electron acceptor availability and microbial community structure on sedimentary methane oxidation in a boreal estuary

Methane is produced microbially in vast quantities in sediments throughout the world’s oceans. However, anaerobic oxidation of methane (AOM) provides a near-quantitative sink for the produced methane and is primarily responsible for preventing methane emissions from the oceans to the atmosphere. AOM is a complex microbial process that involves several different microbial groups and metabolic pathways. The role of different electron acceptors in AOM has been studied for decades, yet large uncertainties remain, especially in terms of understanding the processes in natural settings. This study reports whole-core incubation methane oxidation rates along an estuarine gradient ranging from near fresh water to brackish conditions, and investigates the potential role of different electron acceptors in AOM. Microbial community structure involved in different methane processes is also studied in the same estuarine system using high throughput sequencing tools. Methane oxidation in the sediments was active in three distinct depth layers throughout the studied transect, with total oxidation rates increasing seawards. We find extensive evidence of non-sulphate AOM throughout the transect. The highest absolute AOM rates were observed below the sulphate-methane transition zone (SMTZ), strongly implicating the role of alternative electron acceptors (most likely iron and manganese oxides). However, oxidation rates were ultimately limited by methane availability. ANME-2a/b were the most abundant microbial phyla associated with AOM throughout the study sites, followed by ANME-2d in much lower abundances. Similarly to oxidation rates, highest abundances of microbial groups commonly associated with AOM were found well below the SMTZ, further reinforcing the importance of non-sulphate AOM in this system.

     

微生物介导的甲烷厌氧氧化过程及其影响因子研究进展

温室气体甲烷减排是全球变化领域的研究热点,甲烷厌氧氧化(anaerobic methane oxidation,AOM)过程是一个以前被忽视的甲烷汇,在调控全球甲烷收支平衡及减缓温室效应等方面扮演着十分重要的角色。AOM微生物以甲烷为唯一电子供体,与硫酸盐(SO42-)、亚硝酸盐(NO2-)/硝酸盐(NO3-)、金属离子(Fe3+、Mn4+、Cr6+)等结合完成氧化还原过程,该过程是耦合碳、氮、硫循环的关键环节。本文系统整理分析了不同AOM类型、发生机理、相关功能微生物类群(ANME-1、ANME-2、ANME-3、NC10、MBG-D)及影响AOM过程的关键调控因子的最新研究进展。结果发现,目前80%以上研究都集中在对最常见电子受体类型(SO42-/NO3-/NO2-/Fe3+/Mn4+)的AOM相关过程,而忽视了潜在的新型电子受体(AQDS/HAs O42-/Cr6+/ClO4-等)的耦合作用过程和相对应的微生物类型及作用机理。对未来AOM研究方向提出展望,以期为研究甲烷厌氧氧化菌在不同生态系统中的生态分布及减缓全球温室气体排放提供新的思路。     

Contribution of quinone-reducing microorganisms to the anaerobic biodegradation of organic compounds under different redox conditions

The capacity of two anaerobic consortia to oxidize different organic compounds, including acetate, propionate, lactate, phenol and p-cresol, in the presence of nitrate, sulfate and the humic model compound, anthraquinone-2,6-disulfonate (AQDS) as terminal electron acceptors, was evaluated. Denitrification showed the highest respiratory rates in both consortia studied and occurred exclusively during the first hours of incubation for most organic substrates degraded. Reduction of AQDS and sulfate generally started after complete denitrification, or even occurred at the same time during the biodegradation of p-cresol, in anaerobic sludge incubations; whereas methanogenesis did not significantly occur during the reduction of nitrate, sulfate, and AQDS. AQDS reduction was the preferred respiratory pathway over sulfate reduction and methanogenesis during the anaerobic oxidation of most organic substrates by the anaerobic sludge studied. In contrast, sulfate reduction out-competed AQDS reduction during incubations performed with anaerobic wetland sediment, which did not achieve any methanogenic activity. Propionate was a poor electron donor to achieve AQDS reduction; however, denitrifying and sulfate-reducing activities carried out by both consortia promoted the reduction of AQDS via acetate accumulated from propionate oxidation. Our results suggest that microbial reduction of humic substances (HS) may play an important role during the anaerobic oxidation of organic pollutants in anaerobic environments despite the presence of alternative electron acceptors, such as sulfate and nitrate. Methane inhibition, imposed by the inclusion of AQDS as terminal electron acceptor, suggests that microbial reduction of HS may also have important implications on the global climate preservation, considering the green-house effects of methane.     

Thermodynamic constraints on the utility of ecological stoichiometry for explaining global biogeochemical patterns

Carbon and nitrogen cycles are coupled through both stoichiometric requirements for microbial biomass and dissimilatory metabolic processes in which microbes catalyse reduction‐oxidation reactions. Here, we integrate stoichiometric theory and thermodynamic principles to explain the commonly observed trade‐off between high nitrate and high organic carbon concentrations, and the even stronger trade‐off between high nitrate and high ammonium concentrations, across a wide range of aquatic ecosystems. Our results suggest these relationships are the emergent properties of both microbial biomass stoichiometry and the availability of terminal electron acceptors. Because elements with multiple oxidation states (i.e. nitrogen, manganese, iron and sulphur) serve as both nutrients and sources of chemical energy in reduced environments, both assimilative demand and dissimilatory uses determine their concentrations across broad spatial gradients. Conceptual and quantitative models that integrate rather than independently examine thermodynamic, stoichiometric and evolutionary controls on biogeochemical cycling are essential for understanding local to global biogeochemical patterns.     

厌氧甲烷氧化微生物物质代谢与能量代谢研究进展

甲烷既是一种温室气体,也是一种潜在的能源物质,其源与汇的平衡对地球化学循环及工程应用均有重要意义。厌氧甲烷氧化(anaerobic oxidation of methane,AOM)过程是深海、湿地和农田等自然生境中重要的甲烷汇,在缓解温室气体排放方面发挥了巨大作用。AOM微生物的中枢代谢机制及其能量转化途径则是介导厌氧甲烷氧化耦合其他物质还原的关键所在。因此,本文从电子受体多样性的视角,主要分析了硫酸盐型,硝酸盐/亚硝酸盐型,金属还原型厌氧甲烷氧化微生物的生理生化过程及环境分布,并对近些年发现的新型厌氧甲烷氧化进行了梳理;重点总结了厌氧甲烷氧化微生物细胞内电子传递路径以及胞外电子传递方式;根据厌氧甲烷氧化微生物环境分布及反应特征,就其生态学意义及在污染治理与能源回收方面的潜在应用价值进行了展望。本综述以期深化对厌氧甲烷氧化过程的微生物学认知,并为其潜在的工程应用方向提供新的思路。     

Dominance of sulfur-fueled iron oxide reduction in low-sulfate freshwater sediments

A central tenant in microbial biogeochemistry is that microbial metabolisms follow a predictable sequence of terminal electron acceptors based on the energetic yield for the reaction. It is thereby oftentimes assumed that microbial respiration of ferric iron outcompetes sulfate in all but high-sulfate systems, and thus sulfide has little influence on freshwater or terrestrial iron cycling. Observations of sulfate reduction in low-sulfate environments have been attributed to the presumed presence of highly crystalline iron oxides allowing sulfate reduction to be more energetically favored. Here we identified the iron-reducing processes under low-sulfate conditions within columns containing freshwater sediments amended with structurally diverse iron oxides and fermentation products that fuel anaerobic respiration. We show that despite low sulfate concentrations and regardless of iron oxide substrate (ferrihydrite, Al-ferrihydrite, goethite, hematite), sulfidization was a dominant pathway in iron reduction. This process was mediated by (re)cycling of sulfur upon reaction of sulfide and iron oxides to support continued sulfur-based respiration—a cryptic sulfur cycle involving generation and consumption of sulfur intermediates. Although canonical iron respiration was not observed in the sediments amended with the more crystalline iron oxides, iron respiration did become dominant in the presence of ferrihydrite once sulfate was consumed. Thus, despite more favorable energetics, ferrihydrite reduction did not precede sulfate reduction and instead an inverse redox zonation was observed. These findings indicate that sulfur (re)cycling is a dominant force in iron cycling even in low-sulfate systems and in a manner difficult to predict using the classical thermodynamic ladder.     

Microbiology of flooded rice paddies

Flooded rice paddies are one of the major biogenic sources of atmospheric methane. Apart from this contribution to the 'greenhouse' effect, rice paddy soil represents a suitable model system to study fundamental aspects of microbial ecology, such as diversity, structure, and dynamics of microbial communities as well as structure-function relationships between microbial groups. Flooded rice paddy soil can be considered as a system with three compartments (oxic surface soil, anoxic bulk soil, and rhizosphere) characterized by different physio-chemical conditions. After flooding, oxygen is rapidly depleted in the bulk soil. Anaerobic microorganisms, such as fermentative bacteria and methanogenic archaea, predominate within the microbial community, and thus methane is the final product of anaerobic degradation of organic matter. In the surface soil and the rhizosphere well-defined microscale chemical gradients can be measured. The oxygen profile seems to govern gradients of other electron acceptors (e.g., nitrate, iron(III), and sulfate) and reduced compounds (e.g., ammonium, iron(II), and sulfide). These gradients provide information about the activity and spatial distribution of functional groups of microorganisms. This review presents the current knowledge about the highly complex microbiology of flooded rice paddies. In Section 2 we describe the predominant microbial groups and their function with particular regard to bacterial populations utilizing polysaccharides and simple sugars, and to the methanogenic archaea. Section 3 describes the spatial and temporal development of microscale chemical gradients measured in experimentally defined model systems, including gradients of oxygen and dissolved and solid-phase iron(III) and iron(II). In Section 4, the results of measurements of microscale gradients of oxygen, pH, nitrate-nitrite, and methane in natural rice fields and natural rice soil cores taken to the laboratory will be presented. Finally, perspectives of future research are discussed (Section 5).     

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.     

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate–methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.     

Geochemical and physiological evidence for mixed aerobic and anaerobic field biodegradation of coal tar waste by subsurface microbial communities

We used geochemical analyses of groundwater and laboratory-incubated microcosms to investigate the physiological responses of naturally occurring microorganisms to coal-tar-waste constituents in a contaminated aquifer. Waters were sampled from wells along a natural hydrologic gradient extending from uncontaminated (1 well) into contaminated (3 wells) zones. Groundwater analyses determined the concentrations of carbon and energy sources (pollutants or total organic carbon), final electron acceptors (oxygen, nitrate, sulfate), and metabolic byproducts (dissolved inorganic carbon [DIC], alkalinity, methane, ferrous iron, sulfide, Mn2+). In the contaminated zone of the study site, concentrations of methane, hydrogen, alkalinity, and DIC were enhanced, while dissolved oxygen and nitrate were depleted. Field-initiated biodegradation assays using headspace-free serum bottle microcosms filled with groundwater examined metabolism of the ambient organic contaminants (naphthalene, 2-methylnaphthalene, benzothiophene, and indene) by the native microbial communities. Unamended microcosms from the contaminated zone demonstrated the simultaneous degradation of several coal-tar-waste constituents at the in situ temperature (10°C). Lag phases prior to the onset of biodegradation indicated the prevalence of both aerobic and anaerobic conditions in situ. Electron acceptor-amended microcosms from the most contaminated well waters demonstrated only aerobic naphthalene degradation. Collectively, the geochemical and microbial evidence show that biodegradation of coal-tar-waste constituents occurs via both aerobic and anaerobic terminal electron accepting processes at this site.     

Enrichment of ANME-1 from Eckernförde Bay sediment on thiosulfate, methane and short-chain fatty acids

The microorganisms involved in sulfate-dependent anaerobic oxidation of methane (AOM) have not yet been isolated. In an attempt to stimulate the growth of anaerobic methanotrophs and associated sulfate reducing bacteria (SRB), Eckernf?rde Bay sediment was incubated with different combinations of electron donors and acceptors. The organisms involved in AOM coupled to sulfate reduction (ANME-1, ANME-2, and Desulfosarcina/Desulfococcus) were monitored using specific primers and probes. With thiosulfate as sole electron acceptor and acetate, pyruvate or butyrate as the sole electron donor, ANME-1 became the dominant archaeal species. This finding suggests that ANME-1 archaea are not obligate methanotrophs and that ANME-1 can grow on acetate, pyruvate or butyrate.     

Microbial methane turnover at Marmara Sea cold seeps: a combined 16S rRNA and lipid biomarker investigation

Lipid biomarkers and their stable carbon isotopic composition, as well as 16S rRNA gene sequences, were investigated in sediment cores from active seepage zones in the Sea of Marmara (Turkey) located on the active North Anatolian Fault, to assess processes associated with methane turnover by indigenous microbial communities. Diagnostic ¹³C‐depleted archaeal lipids of anaerobic methane oxidizers were only found in one core from the South of Çinarcik Basin and consist mainly of archaeol, sn‐2 hydroxyarchaeol and various unsaturated pentamethylicosenes. Concurrently, abundant fatty acids (FAs) and a substantial amount of monoalkylglycerolethers (MAGEs), assigned to sulphate‐reducing bacteria, were detected with strong ¹³C‐depletions. Both microbial lipids and their δ¹³C values suggest that anaerobic oxidation of methane with sulphate reduction (AOM/SR) occurs, specially in the 10‐ to 12‐cm depth interval. Lipid biomarker results accompanied by 16S rRNA‐based microbial diversity analyses showed that ANME‐2 (ANME‐2a and ‐2c) archaea and Desulfosarcina/Desulfococcus and Desulfobulbus deltaproteobacterial clades are the major AOM assemblages, which indicate a shallow AOM community at high methane flux. Apart from the typical AOM lipid biomarker pattern, a ¹³C‐depleted diunsaturated hydrocarbon, identified as 7,14‐tricosadiene, occurred in the inferred maximum AOM interval at 10–12 cm depth. Its isotopic fingerprint implies that its microbial precursor occurs in close association with the AOM communities. Interestingly, the presence of 7,14‐tricosadiene coincides with the presence of the so‐far uncultured bacterial Candidate Division JS1, often detected in AOM areas. We propose the hypothesis that the JS1 bacterial group could be the potential source of ¹³C‐depleted tricosadiene. Future testing of this hypothesis is essential to fully determine the role of this bacterial group in AOM.