Search

Members of the bacterial genus Sulfurimonas (phylum Campylobacterota) dominate microbial communities in marine redoxclines and are important for sulfur and nitrogen cycling. Here we used metagenomics and metabolic analyses to characterize a Sulfurimonas from the Gakkel Ridge in the Central Arctic Ocean and Southwest Indian Ridge, showing that this species is ubiquitous in non-buoyant hydrothermal plumes at Mid Ocean Ridges across the global ocean. One Sulfurimonas species, USulfurimonas pluma, was found to be globally abundant and active in cold (Sulfurimonas species, US. pluma has a reduced genome (>17%) and genomic signatures of an aerobic chemolithotrophic metabolism using hydrogen as an energy source, including acquisition of A2-type oxidase and loss of nitrate and nitrite reductases. The dominance and unique niche of US. pluma in hydrothermal plumes suggest an unappreciated biogeochemical role for Sulfurimonas in the deep ocean.

Alkanes are saturated apolar hydrocarbons that range from their simplest form, methane, to high-molecular-weight compounds. Although alkanes were once considered biologically recalcitrant under anaerobic conditions, microbiological investigations have now identified several microbial taxa that can anaerobically degrade alkanes. Here we review recent discoveries in the anaerobic oxidation of alkanes with a specific focus on archaea that use specific methyl coenzyme M reductases to activate their substrates. Our understanding of the diversity of uncultured alkane-oxidizing archaea has expanded through the use of environmental metagenomics and enrichment cultures of syntrophic methane-, ethane-, propane-, and butane-oxidizing marine archaea with sulfate-reducing bacteria. A recently cultured group of archaea directly couples long-chain alkane degradation with methane formation, expanding the range of substrates used for methanogenesis. This article summarizes the rapidly growing knowledge of the diversity, physiology, and habitat distribution of alkane-degrading archaea.

The methyl-coenzyme M reductase (Mcr) enables archaea to produce and oxidize methane, critically impacting the global greenhouse gas budget. Recently cultured archaea activate short- and long-chain n-alkanes with divergent Mcr variants, termed alkyl-coenzyme M reductases (Acrs). Here, we probed the anaerobic oxidation of mid-chain petroleum alkanes at 70°C using oil-rich sediments from the Guaymas Basin. Incubations with alkanes from pentane to tetradecane produced active cultures. In these cultures, archaea of the genus Candidatus Alkanophaga activate the alkanes with Acrs and completely oxidize the alkyl groups to CO2. Ca. Alkanophaga form a deep-branching sister clade to the methanotrophs ANME-1 and are closely related to the short-chain alkane oxidizers Ca. Syntrophoarchaeum. This suggests that multi-carbon alkane metabolism preceded methane metabolism in the class Syntrophoarchaeia. Ca. Alkanophaga shuttle the electrons from alkane oxidation to sulfate-reducing Thermodesulfobacteria. The two partners form consortia that are potential key players in petroleum degradation in heated oil reservoirs.

Members of the genus Sulfurimonas (phylum Campylobacterota) dominate microbial communities of benthic and pelagic redox clines, playing a key role in the biogeochemistry of these environments. Investigating neutrally buoyant hydrothermal plumes of the Central Arctic Ocean, we found a novel type of Sulfurimonas that inhabits hydrogen-rich and oxygen-saturated environments close to the freezing point. Using omics’ approaches, we compared the diversity and distribution of Sulfurimonas from Gakkel Ridge hydrothermal plumes with those from different Mid Ocean Ridges and other Sulfurimonas-hosting environments. A single and new Sulfurimonas species, USulfurimonas pluma, was found to be globally abundant and active in cold (US. pluma features substantial genome reduction and specific genomic traits (e.g., oxygen detoxification mechanisms, acquisition of A2-type oxidase, loss of nitrate and nitrite reductases) compared to other Sulfurimonas species. These traits are indicative for adaptation to an obligate aerobic chemolithotrophic life style in hydrogen-rich plumes, US. pluma dominates microbial communities of metal-rich neutrally-buoyant hydrothermal plumes, suggesting a so far neglected roles of Sulfurimonas in ocean biogeochemistry and food webs.

Description, Anaerobic methanotrophs and methanogens synthesize amorphous carbon., Elemental carbon exists in different structural forms including graphite, diamond, fullerenes, and amorphous carbon. In nature, these materials are produced through abiotic chemical processes under high temperature and pressure but are considered generally inaccessible to biochemical synthesis or breakdown. Here, we identified and characterized elemental carbon isolated from consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which together carry out the anaerobic oxidation of methane (AOM). Two different AOM consortia, ANME-1a/HotSeep-1 and ANME-2a/c/Seep-SRB, produce a black material with similar characteristics to disordered graphite and amorphous carbon. Stable isotope probing studies revealed that the carbon is microbially generated during AOM. In addition, we found that select methanogens also produce amorphous carbon with similar characteristics to the carbon from AOM consortia. Biogenic amorphous carbon may serve as a conductive element to facilitate electron transfer, or redox active functional groups associated with the carbon could act as electron donors and acceptors.

The anaerobic oxidation of methane (AOM) mitigates the flux of methane from marine sediments into the water column. AOM is performed by anaerobic methanotrophic archaea (ANME) that reverse the methanogenesis pathway and partner bacteria that utilize the released reducing equivalents for sulfate reduction. Here, we investigated small-molecule extracts from sediment-free thermophilic enrichment cultures of ANME-1 and sulfate-reducing bacteria using ultraperformance liquid chromatography with high-resolution mass spectrometry. During the analysis, we discovered a novel thioquinoxalinol-containing redox molecule as a major component of the chemically derivatized small-molecule pool. This compound contains both a redox active quinoxaline heterocyclic ring and a thiol group. Additionally, the same core structure was identified that contains a sulfate ester on the hydroxyl group, which likely makes the molecule more water soluble. Hydrated versions of both structures were also observed as major compounds in the extracts. On the basis of reactions of model compounds such as quinoxalin-6-ol, the hydrated version appears to be formed from the addition of water to the dehydropyrazine ring followed by an oxidation. These thioquinoxalinol compounds, which represent completely new structures in biochemistry, may be involved in electron transport processes within and/or between ANME-1 and sulfate-reducing bacteria, may serve protective roles by reacting with toxic compounds such as hydrogen sulfide, or may transport sulfate as a sulfate ester into the sulfate-reducing bacteria.

Methanogenesis and anaerobic methane oxidation through methyl-coenzyme M reductase (MCR) as a key enzyme have been suggested to be basal pathways of archaea1. How widespread MCR-based alkane metabolism is among archaea, where it occurs and how it evolved remain elusive. Here, we performed a global survey of MCR-encoding genomes based on metagenomic data from various environments. Eleven high-quality mcr-containing metagenomic-assembled genomes were obtained belonging to the Archaeoglobi in the Euryarchaeota, Hadesarchaeota and different TACK superphylum archaea, including the Nezhaarchaeota, Korarchaeota and Verstraetearchaeota. Archaeoglobi WYZ-LMO1 and WYZ-LMO3 and Korarchaeota WYZ-LMO9 encode both the (reverse) methanogenesis and the dissimilatory sulfate reduction pathway, suggesting that they have the genomic potential to couple both pathways in individual organisms. The Hadesarchaeota WYZ-LMO4–6 and Archaeoglobi JdFR-42 encode highly divergent MCRs, enzymes that may enable them to thrive on non-methane alkanes. The occurrence of mcr genes in different archaeal phyla indicates that MCR-based alkane metabolism is common in the domain of Archaea. A metagenome-based survey of archaeal genomes encoding methyl-coenzyme M reductase (MCR)—a key enzyme for methanogenesis and anaerobic methane oxidation—reveals that MCR-based metabolism is common and diverse in archaea, and may be coupled to dissimilatory sulfate reduction in single organisms.

In our Research Article, “A methylotrophic origin of methanogenesis and early divergence of …

Methanogens are considered as one of the earliest life forms on Earth, and together with anaerobic methane-oxidizing archaea, they have crucial effects on climate stability. However, the origin and evolution of anaerobic alkane metabolism in the domain Archaea remain controversial. Here, we present evidence that methylotrophic methanogenesis was the ancestral form of this metabolism. Carbon dioxide-reducing methanogenesis developed later through the evolution of tetrahydromethanopterin S-methyltransferase, which linked methanogenesis to the Wood-Ljungdahl pathway for energy conservation. Anaerobic multicarbon alkane metabolisms in Archaea also originated early, with genes coding for the activation of short-chain or even long-chain alkanes likely evolving from an ethane-metabolizing ancestor. These genes were likely horizontally transferred to multiple archaeal clades including Candidatus (Ca.) Bathyarchaeia, Ca. Lokiarchaeia, Ca. Hadarchaeia, and the methanogenic Ca. Methanoliparia.

The flanking regions of Guaymas Basin, a young marginal rift basin located in the Gulf of California, are covered with thick sediment layers that are hydrothermally altered due to magmatic intrusions. To explore environmental controls on microbial community structure in this complex environment, we analyzed site- and depth-related patterns of microbial community composition (bacteria, archaea, and fungi) in hydrothermally influenced sediments with different thermal conditions, geochemical regimes, and extent of microbial mats. We compared communities in hot hydrothermal sediments (75-100°C at ~40 cm depth) covered by orange-pigmented Beggiatoaceae mats in the Cathedral Hill area, temperate sediments (25-30°C at ~40 cm depth) covered by yellow sulfur precipitates and filamentous sulfur oxidizers at the Aceto Balsamico location, hot sediments (>115°C at ~40 cm depth) with orange-pigmented mats surrounded by yellow and white mats at the Marker 14 location, and background, non-hydrothermal sediments (3.8°C at ~45 cm depth) overlain with ambient seawater. Whereas bacterial and archaeal communities are clearly structured by site-specific in-situ thermal gradients and geochemical conditions, fungal communities are generally structured by sediment depth. Unexpectedly, chytrid sequence biosignatures are ubiquitous in surficial sediments whereas deeper sediments contain diverse yeasts and filamentous fungi. In correlation analyses across different sites and sediment depths, fungal phylotypes correlate to each other to a much greater degree than Bacteria and Archaea do to each other or to fungi, further substantiating that site-specific in-situ thermal gradients and geochemical conditions that control bacteria and archaea do not extend to fungi.

Methane is abundant in marine subsurface sediments, sourced from microbial or thermocatalytic products. The relative composition of its isotopologues (12CH4, 13CH4, 12CH3D and 13CH3D) is used to infer its sources and sinks. The anaerobic oxidation of methane (AOM) is an important methane sink reaction carried out by consortia ofanaerobic methanotrophic archaea (ANME) and partner bacteria in the presence of methane and sulfate. We investigated the methane isotopologue fractionations duringAOM in experiments with cultures of ANME-1 archaea and partner bacteria obtained from hydrothermally heated gas-rich sediments of the Guaymas Basin. During partial methane consumption in four sets of experiments, residual methane became enriched in 13CH4 and 12CH3D, following kinetic fractionations from 11.1 to 18.3 ‰ and from 117 to 180 ‰,respectively. Results from one set of experiments with D-depleted medium water (δD = – 200‰, whereas the control was –55‰) are inconclusive regarding the reversibility of AOM, which would lead to equilibrium as opposed to kinetic fractionations. The value of Δ13CH3D (the abundance of 13CH3D with respect to that expected from stochastic distribution) increased toward and beyond (up to 8.4‰) the value expected for isotopologue equilibrium (5.3‰ at 37 °C). The kinetic clumped isotopologue fractionation (difference between 13CH3D/12CH3D and 13CH4/12CH4 fractionations) of 4.8 to 12.8 ‰ is in contrast with our previous observation of little to no clumped isotopologue effect during aerobic methane oxidation. Our results demonstrate that AOM can contribute to nearequilibrium Δ13CH3D values observed in marine sediments and 13CH3D systematics can be used to distinguish aerobic versus anaerobic methanotrophic processes in nature.

How to feed an enzyme ethane When released from ocean floor seeps, small hydrocarbons are rapidly consumed by micro-organisms. Methane is highly abundant and is both produced and consumed by microbes through well understood biochemical pathways. Less well understood is how ethane, also a major natural component of gaseous hydrocarbons, is metabolized. To understand how microbes take advantage of this energy and carbon source, Hahn et al. solved the x-ray crystal structures of an enzyme they call ethyl coenzyme-M reductase, which converts ethane into the thioether ethyl-coenzyme M as the entry point for catabolism. They found an expanded active site and, using a xenon gas derivatization experiment, a distinctive tunnel through the protein that is proposed to permit access of the gaseous substrate. Science , abg1765, this issue p. 118

Coastal sands are biocatalytic filters for dissolved and particulate organic matter of marine and terrestrial origin, thus, acting as centers of organic matter transformation. At high temporal resolution, we accessed the variability of benthic bacterial communities over two annual cycles at Helgoland (North Sea), and compared it with seasonality of communities in Isfjorden (Svalbard, 78°N) sediments, where primary production does not occur during winter. Benthic community structure remained stable in both, temperate and polar sediments on the level of cell counts and 16S rRNA-based taxonomy. Actinobacteriota of uncultured Actinomarinales and Microtrichales were a major group, with 8 ± 1% of total reads (Helgoland) and 31 ± 6% (Svalbard). Their high activity (frequency of dividing cells 28%) and in situ cell numbers of >10% of total microbes in Svalbard sediments, suggest Actinomarinales and Microtrichales as key heterotrophs for carbon mineralization. Even though Helgoland and Svalbard sampling sites showed no phytodetritus-driven changes of the benthic bacterial community structure, they harbored significantly different communities (p r = 0.963). The temporal stability of benthic bacterial communities is in stark contrast to the dynamic succession typical of coastal waters, suggesting that pelagic and benthic bacterial communities respond to phytoplankton productivity very differently.

Summary Methanogenic hydrocarbon degradation is the dominant biodegradation process in crude oil, and so far was described for syntrophic associations of hydrocarbon-degrading bacteria and methanogenic archaea. Recent culture-independent studies suggest that the archaeon ‘Candidatus Methanoliparum’ performs both reactions, involving alkyl-coenzyme M reductases for the activation of alkanes and canonical methyl coenzyme M reductases for methane formation. Here, we cultured ‘Ca. Methanoliparum’. Metatranscriptomics and isotope labelling experiments confirm the suggested pathway for the degradation of hexadecane to methane in this archaeon. Mass spectrometry identified CoM- bound intermediates for the activation of long-chain n-alkanes, n-alkylcyclohexanes and n-alkylbenzenes, showing the wide substrate range of this archaeon. The wide distribution of the ’Ca. Methanoliparum in reservoirs indicates that this alkylotrophic methanogen may play a vital role in the frequently observed transformation of reservoir oil constituents into methane

Summary Recent studies suggest an unforeseen role of archaea in the anaerobic oxidation of non-methane alkanes using enzymes of the methanogenesis pathway and requiring partnerships with bacteria that have not yet been cultured. Stable isotope probing (SIP) approaches provide an important culture-independent tool to track activity and carbon assimilation of these microorganisms in situ. However, the established stable isotope probing of lipids requires labor-intense purification of individual molecules and chemical derivatization or cleavage reactions such as ether-cleavage or saponification to produce GC-irmMS-amenable derivatives. A recently developed promising technique is isotope pattern matching (IPM) which uses isotopomer patterns of intact lipids analyzed by HPLC-MS to determine uptake of stable isotopes. We have incubated methane- and other short chain alkane-oxidizing enrichment cultures from hydrothermal sites in the Guaymas Basin with 13C-labeled bicarbonate and 13C-labeled alkane substrates at 50°C and monitored label-uptake into the lipid pool using traditional SIP and IPM methods. This presentation will show data of diagnostic lipids, including intact polar archaeal and bacterial lipids and compare IPM with traditional SIP experiments of selected biomarkers with the goal to understand carbon substrate affinity of alkane-oxidizing enrichment cultures.

The methanogenic degradation of oil hydrocarbons can proceed through syntrophic partnerships of hydrocarbon-degrading bacteria and methanogenic archaea1,2,3. However, recent culture-independent studies have suggested that the archaeon ‘Candidatus Methanoliparum’ alone can combine the degradation of long-chain alkanes with methanogenesis4,5. Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. Molecular analyses revealed that Ca. Methanoliparum contains and overexpresses genes encoding alkyl-coenzyme M reductases and methyl-coenzyme M reductases, the marker genes for archaeal multicarbon alkane and methane metabolism. Incubation experiments with different substrates and mass spectrometric detection of coenzyme-M-bound intermediates confirm that Ca. Methanoliparum thrives not only on a variety of long-chain alkanes, but also on n-alkylcyclohexanes and n-alkylbenzenes with long n-alkyl (C≥13) moieties. By contrast, short-chain alkanes (such as ethane to octane) or aromatics with short alkyl chains (C≤12) were not consumed. The wide distribution of Ca. Methanoliparum4,5,6 in oil-rich environments indicates that this alkylotrophic methanogen may have a crucial role in the transformation of hydrocarbons into methane.

The anaerobic oxidation of methane (AOM) is performed by methanotrophic archaea (ANME) in distinct sulfate-methane interfaces of marine sediments. In these interfaces, AOM often appears to deplete methane in the heavy isotopes toward isotopic compositions similar to methanogenesis. Here, we shed light on this effect and its physiological underpinnings using a thermophilic ANME-1-dominated culture. At high sulfate concentrations, residual methane is enriched in both

Methane is abundant in marine subsurface sediments, sourced from microbial or thermocatalytic products. The relative composition of its isotopologues (12CH4, 13CH4, 12CH3D and 13CH3D) is used to infer its sources and sinks. The anaerobic oxidation of methane (AOM) is an important methane sink reaction carried out by consortia ofanaerobic methanotrophic archaea (ANME) and partner bacteria in the presence of methane and sulfate. We investigated the methane isotopologue fractionations duringAOM in experiments with cultures of ANME-1 archaea and partner bacteria obtained from hydrothermally heated gas-rich sediments of the Guaymas Basin. During partial methane consumption in four sets of experiments, residual methane became enriched in 13CH4 and 12CH3D, following kinetic fractionations from 11.1 to 18.3 ‰ and from 117 to 180 ‰,respectively. Results from one set of experiments with D-depleted medium water (δD = – 200‰, whereas the control was –55‰) are inconclusive regarding the reversibility of AOM, which would lead to equilibrium as opposed to kinetic fractionations. The value of Δ13CH3D (the abundance of 13CH3D with respect to that expected from stochastic distribution) increased toward and beyond (up to 8.4‰) the value expected for isotopologue equilibrium (5.3‰ at 37 °C). The kinetic clumped isotopologue fractionation (difference between 13CH3D/12CH3D and 13CH4/12CH4 fractionations) of 4.8 to 12.8 ‰ is in contrast with our previous observation of little to no clumped isotopologue effect during aerobic methane oxidation. Our results demonstrate that AOM can contribute to near52equilibrium Δ13CH3D values observed in marine sediments and 13CH3D systematics can be used to distinguish aerobic versus anaerobic methanotrophic processes in nature.

Methanogens are considered as one of the earliest life forms on Earth, and together with anaerobic methane-oxidizing archaea, they have crucial effects on climate stability. Yet, the origin and evolution of anaerobic alkane metabolism in the domain Archaea remain controversial. Here, we show that methanogenesis was already present in the common ancestor of Euryarchaeota, TACK archaea, and Asgard archaea likely in the late Hadean or early Archean eon and that the ancestral methanogen was dependent on methylated compounds and hydrogen. Carbon dioxide-reducing methanogenesis developed later through the evolution of tetrahydromethanopterin S-methyltransferase, which linked methanogenesis to the Wood-Ljungdahl pathway for energy conservation. Multicarbon alkane metabolisms in Archaea also originated early, with genes coding for the activation of short- or even long-chain alkanes likely evolving from an ethane-metabolizing ancestor. These genes were likely horizontally transferred to multiple archaeal clades including Candidatus (Ca) Bathyarchaeota, Ca. Helarchaeota, Ca Hadesarchaeota, and the methanogenic Ca. Methanoliparia.

In the seabed, gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of nonmethane alkane activation by noncanonical methyl-coenzyme M reductase enzymes and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes., Cold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as the substrate, we cultured microbial consortia of a novel anaerobic ethane oxidizer, “Candidatus Ethanoperedens thermophilum” (GoM-Arc1 clade), and its partner bacterium “Candidatus Desulfofervidus auxilii,” previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieval of a closed genome of “Ca. Ethanoperedens,” a sister genus of the recently reported ethane oxidizer “Candidatus Argoarchaeum.” The metagenome-assembled genome of “Ca. Ethanoperedens” encoded a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as the sole growth substrate and production of ethyl-coenzyme M as the activation product. Stable isotope probing demonstrated that the enzymatic mechanism of ethane oxidation in “Ca. Ethanoperedens” is fully reversible; thus, its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide.

Traditionally, the description of microorganisms starts with their isolation from an environmental sample. Many environmentally relevant anaerobic microorganisms grow very slowly, and often they rely on syntrophic interactions with other microorganisms. This impedes their isolation and characterization by classic microbiological techniques. We developed and applied an approach for the successive enrichment of syntrophic hydrocarbon-degrading microorganisms from environmental samples. We collected samples from microbial mat-covered hydrothermally heated hydrocarbon-rich sediments of the Guaymas Basin and mixed them with synthetic mineral medium to obtain sediment slurries. Supplementation with defined substrates (i.e., methane or butane), incubation at specific temperatures, and a regular maintenance procedure that included the measurement of metabolic products and stepwise dilutions enabled us to establish highly active, virtually sediment-free enrichment cultures of actively hydrocarbon-degrading communities in a 6-months to several-years' effort. Using methane as sole electron donor shifted the originally highly diverse microbial communities toward defined mixed cultures dominated by syntrophic consortia consisting of anaerobic methane-oxidizing archaea (ANME) and different sulfate-reducing bacteria. Cultivation with butane at 50 °C yielded consortia of archaea belonging to Candidatus Syntrophoarchaeum and Candidatus Desulfofervidus auxilii partner bacteria. This protocol also describes sampling for further molecular characterization of enrichment cultures by fluorescence in situ hybridization (FISH), and transcriptomics and metabolite analyses, which can provide insights into the functioning of hydrocarbon metabolism in archaea and resolve important mechanisms that enable electron transfer to their sulfate-reducing partner bacteria.

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.

At the Campeche Knolls in the southern Gulf of Mexico large-scale hydrocarbon emissions are associated with numerous salt tectonic structures. A notable feature of this area is the expulsion of highly viscous heavy oils, also referred to as asphalts, which form lava-like flows on the seafloor. These oil and asphalt seeps have been detected via satellite imaging of oil slicks at the ocean surface and by acoustically-detected gas emission sites in the water column. Here, we describe the type locality ‘Chapopote’ (Aztec word for tar) where the expelled hydrocarbons provide an energy source for microorganisms and subsequently the deep-sea fauna. In deeper asphalt layers and sediment, 16S rRNA gene sequences of archaea affiliated with Methanosaeta and bacteria of the Syntrophaceae were detected. Together with the observed light methane isotope values this indicates biogenic methanogenesis from aliphatic hydrocarbons at these depths. In shallow oil-soaked sediments gene sequences of hydrocarbon-degrading sulfate-reducing Deltaproteobacteria were found. These sediments showed high sulfate reduction but only minor methane oxidation rates. Asphalts and oil-soaked sediments freshly exposed to the surface were densely covered by microbial mats, signaling high metabolic activity. The surface microbial communities were dominated by diverse Gammaproteobacteria that likely oxidized hydrocarbons and reduced sulfur compounds. Surface asphalts were colonized by tube worms, mussels and sponges that host bacterial symbionts. In addition, grazing invertebrates such as sea cucumbers, shrimps, and crabs evidently fed on the microbial mats. This close link between the microbial and macro-benthic life demonstrates an efficient energy transfer to higher trophic levels from microbial hydrocarbon oxidation at the base of this unique asphalt-hosted ecosystem.

Dual stable isotope probing has been used to infer rates of microbial biomass production and modes of carbon fixation. In order to validate this approach for assessing archaeal production, the methanogenic archaeon Methanosarcina barkeri was grown either with H2 , acetate or methanol with D2 O and 13 C-dissolved inorganic carbon (DIC). Our results revealed unexpectedly low D incorporation into lipids, with the net fraction of water-derived hydrogen amounting to 0.357±0.042, 0.226±0.003 and 0.393±0.029 for growth on H2 /CO2 , acetate and methanol, respectively. The variability in net water H assimilation into lipids during growth of M. barkeri on different substrates is possibly attributed to different Gibbs free energy yields, such that higher energy yield promoted the exchange of hydrogen between medium water and lipids. Because NADPH likely serves as the portal for H transfer, increased NADPH production and/or turnover associated with high energy yield may explain the apparent differences in net water H assimilation into lipids. The variable DIC and water H incorporation into M. barkeri lipids imply systematic, metabolic patterns of isotope incorporation and suggest that the ratio of 13 C-DIC vs D2 O assimilation in environmental samples may serve as a proxy for microbial energetics in addition to microbial production and carbon assimilation pathways. This article is protected by copyright. All rights reserved.

Methyl‐coenzyme M reductase (MCR) has been originally identified to catalyse the final step of the methanogenesis pathway. About 20 years ago an aerobic me thane‐oxidizing archaea (ANME) were discovered that use MCR enzymes to activate methane. ANME thrive at the thermodynamic limit of life, are slow‐growing, and in most cases form syntrophic consortia with sulfate‐reducing bacteria. Recently, archaea that have the ability to anaerobically oxidize non‐methane multi‐carbon alkanes such as ethane and n ‐butane were described in both enrichment cultures and environmental samples. These an aerobic multi‐carbon alka ne‐oxidizing archaea (ANKA) use enzymes homologous to MCR named alkyl‐coenzyme M reductase (ACR). Here we review the recent progresses on the diversity, distribution and functioning of both ANME and ANKA by presenting a detailed MCR/ACR‐based phylogeny, compare their metabolic pathways and discuss the gaps in our knowledge of physiology of these organisms. To improve our understanding of alkane oxidation in archaea, we identified three directions for future research: (i) expanding cultivation attempts to validate omics‐based metabolic models of yet‐uncultured organisms, (ii) performing biochemical and structural analyses of key enzymes to understand thermodynamic and steric constraints and (iii) investigating the evolution of anaerobic alkane metabolisms and their impact on biogeochemical cycles.

Analysis of cellular membrane lipids has been widely applied to describe the microbial community composition in natural systems. When combined with stable isotope probing (SIP) experiments, deuterium label (D 2 O) uptake into lipids enables assessment of microbial biomass production. We performed SIP on methane-rich, hydrothermally-heated sediments to examine the de novo production of individual archaeal intact polar lipids (IPLs) by mesophilic anaerobic methane oxidizing group-1 archaea (ANME-1). The greatest extent of label uptake was by phosphatidyl glycerol archaeol (PG-AR), reaching 50% of the medium’s label concentration in only 10 days. This indicates PG-AR as an important cell membrane lipid during the active growth phase of mesophilic ANME-1. Much less label uptake was into intact polar glycerol dibiphytanyl glycerol tetraethers (GDGTs), especially for those bound to diglycosidic head groups. The low production of these GDGTs contrasts with their predominance in ANME-1-dominated natural samples. We attribute the differential label uptake among individual IPLs to the ANME-1 tetraether biosynthetic pathway. This mechanism likely involves head-to-head condensation of two molecules of PG-AR and progressive substitution of PG by glycosidic head groups. The observation that ANME-1 invest in the synthesis of diethers during optimum growth suggests intact ARs and/or phosphate-bearing tetraethers as important biomarkers for actively growing populations in natural environments, while the diglycosidic GDGTs appear to signal stationary ANME-1 communities.

The anaerobic oxidation of methane (AOM) is mediated by consortia of anaerobic methane-oxidizing archaea (ANME) and their specific partner bacteria. In thermophilic AOM consortia enriched from Guaymas Basin, members of the ANME-1 clade are associated with bacteria of the HotSeep-1 cluster, which likely perform direct electron exchange via nanowires. The partner bacterium was enriched with hydrogen as sole electron donor and sulfate as electron acceptor. Based on phylogenetic, genomic and metabolic characteristics we propose to name this chemolithoautotrophic sulfate reducer Candidatus Desulfofervidus auxilii. Ca. D. auxilii grows on hydrogen at temperatures between 50°C and 70°C with an activity optimum at 60°C and doubling time of 4-6 days. Its genome draft encodes for canonical sulfate reduction, periplasmic and soluble hydrogenases and autotrophic carbon fixation via the reductive tricarboxylic acid cycle. The presence of genes for pili formation and cytochromes, and their similarity to genes of Geobacter spp., indicate a potential for syntrophic growth via direct interspecies electron transfer when the organism grows in consortia with ANME. This first ANME-free enrichment of an AOM partner bacterium and its characterization opens the perspective for a deeper understanding of syntrophy in anaerobic methane oxidation.

Oil-rich sediments from the Gulf of Mexico were found to contain diverse alkane-degrading groups of archaea. The symbiotic, consortium-forming “Candidatus Argoarchaeum” and “Candidatus Syntrophoarchaeum” are likely responsible for the degradation of ethane and short-chain alkanes, with the help of sulfate-reducing bacteria. “Ca. Methanoliparia” occurs as single cells associated with oil droplets. These archaea encode two phylogenetically different methyl-coenzyme M reductases that may allow this organism to thrive as a methanogen on a substrate of long-chain alkanes. Based on a library survey, we show that “Ca. Methanoliparia” is frequently detected in oil reservoirs and may be a key agent in the transformation of long-chain alkanes to methane. Our findings provide evidence for the important and diverse roles of archaea in alkane-rich marine habitats and support the notion of a significant functional versatility of the methyl coenzyme M reductase., Crude oil and gases in the seabed provide an important energy source for subsurface microorganisms. We investigated the role of archaea in the anaerobic degradation of non-methane alkanes in deep-sea oil seeps from the Gulf of Mexico. We identified microscopically the ethane and short-chain alkane oxidizers “Candidatus Argoarchaeum” and “Candidatus Syntrophoarchaeum” forming consortia with bacteria. Moreover, we found that the sediments contain large numbers of cells from the archaeal clade “Candidatus Methanoliparia,” which was previously proposed to perform methanogenic alkane degradation. “Ca. Methanoliparia” occurred abundantly as single cells attached to oil droplets in sediments without apparent bacterial or archaeal partners. Metagenome-assembled genomes of “Ca. Methanoliparia” encode a complete methanogenesis pathway including a canonical methyl-coenzyme M reductase (MCR) but also a highly divergent MCR related to those of alkane-degrading archaea and pathways for the oxidation of long-chain alkyl units. Its metabolic genomic potential and its global detection in hydrocarbon reservoirs suggest that “Ca. Methanoliparia” is an important methanogenic alkane degrader in subsurface environments, producing methane by alkane disproportionation as a single organism.

Seepage of methane (CH4) on land and in the sea may significantly affect Earth's biogeochemical cycles. However processes of CH4 generation and consumption, both abiotic and microbial, are not always clear. We provide new geochemical and isotope data to evaluate if a recently discovered CH4 seepage from the shallow seafloor close to the Island of Elba (Tuscany) and two small islands nearby are derived from abiogenic or biogenic sources and whether carbonate encrusted vents are the result of microbial or abiotic processes. Emission of gas bubbles (predominantly CH4) from unlithified sands was observed at seven spots in an area of 100 m(2) at Pomonte (Island of Elba), with a total rate of 234 ml m(-2) d(-1). The measured carbon isotope values of CH4 of around -18 parts per thousand (VPDB) in combination with the measured delta H-2 value of -120 parts per thousand (VSMOW) and the inverse correlation of delta C-13-value with carbon number of hydrocarbon gases are characteristic for sites of CH4 formation through abiogenic processes, specifically abiogenic formation of CH4 via reduction of CO2 by H-2. The H-2 for methanogenesis likely derives from ophiolitic host rock within the Ligurian accretionary prism. The lack of hydrothermal activity allows CH4 gas to become decoupled from the stagnant aqueous phase. Hence no hyperalkaline fluid is currently released at the vent sites. Within the seep area a decrease in porewater sulphate concentrations by ca. 5 mmol/l relative to seawater and a concomitant increase in sulphide and dissolved inorganic carbon (DIC) indicate substantial activity of sulphate-dependent anaerobic oxidation of methane (AOM). In absence of any other dissimilatory pathway, the delta C-13-values between -17 and -5 parts per thousand in dissolved inorganic carbon and aragonite cements suggest that the inorganic carbon is largely derived from CH4. The formation of seep carbonates is thus microbially induced via anaerobic oxidation of abiotic CH4.