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Ethane is the second most abundant component of natural gas in addition to methane, and—similar to methane—is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps 1 – 3 , and through ethane-dependent sulfate reduction in slurries 4 – 7 . Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown 8 . Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name ‘ Candidatus Argoarchaeum ethanivorans’; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca . Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography–tandem mass spectrometry. This indicated that Ca . Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by ‘ Candidatus Syntrophoarchaeum’ 9 . Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO 2 occurs through the oxidative Wood–Ljungdahl pathway. The identification of an archaeon that uses ethane (C 2 H 6 ) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (C n H 2 n +2 ) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca . Argoarchaeum at deep-sea gas seeps 10 – 12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps. An archaeon, ‘ Candidatus Argoarchaeum ethanivorans’, which is involved in the oxidation of ethane observed in anoxic marine habitats, is identified and metabolically characterized.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C 1 -compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C 4 hydrocarbon butane. The archaea, proposed genus ‘ Candidatus Syntrophoarchaeum’, show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca . Syntrophoarchaeum expresses the genes encoding β-oxidation enzymes, carbon monoxide dehydrogenase and reversible C 1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca . Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons. Anaerobic archaea enriched in thermophilic microbial consortia completely degrade butane by modifying mechanisms which were hitherto thought to be specific to methane metabolism. Environmental oxidation of non-methane hydrocarbons Research on the anaerobic oxidation of natural gas has largely been focused on methane as the most abundant constituent. It is less clear how short-chain alkanes—including ethane, propane, n -butane and iso -butane, which together make up about 20% of natural gas—are anaerobically metabolized. Sulfate-reducing bacteria are the only organisms known to date to anaerobically oxidize short-chain hydrocarbons. Gunter Wegener and colleagues identify an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria that uses a pathway similar to anaerobic methane oxidation, which was previously thought to be specific for C 1 -compounds, to oxidize butane. Archaea activate butane, and reducing equivalents are channelled to sulfate-reducing partner bacteria. Similar consortia are detected in marine subsurface sediments, suggesting that this pathway may be widespread in nature.

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives ( Desulfurella spp.; Hippea maritima ) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.

Microbial degradation of substrates to terminal products is commonly understood as a unidirectional process. In individual enzymatic reactions, however, reversibility (reverse reaction and product back flux) is common. Hence, it is possible that entire pathways of microbial degradation are associated with back flux from the accumulating product pool through intracellular intermediates into the substrate pool. We investigated carbon and sulfur back flux during the anaerobic oxidation of methane (AOM) with sulfate, one of the least exergonic microbial catabolic processes known. The involved enzymes must operate not far from the thermodynamic equilibrium. Such an energetic situation is likely to favor product back flux. Indeed, cultures of highly enriched archaeal–bacterial consortia, performing net AOM with unlabeled methane and sulfate, converted label from 14C-bicarbonate and 35S-sulfide to 14C-methane and 35S-sulfate, respectively. Back fluxes reached 5% and 13%, respectively, of the net AOM rate. The existence of catabolic back fluxes in the reverse direction of net reactions has implications for biogeochemical isotope studies. In environments where biochemical processes are close to thermodynamic equilibrium, measured fluxes of labeled substrates to products are not equal to microbial net rates. Detection of a reaction in situ by labeling may not even indicate a net reaction occurring in the direction of label conversion but may reflect the reverse component of a so far unrecognized net reaction. Furthermore, the natural isotopic composition of the substrate and product pool will be determined by both the forward and back flux. This finding may have to be considered in the interpretation of stable isotope records.

Corrosion of iron presents a serious economic problem. Whereas aerobic corrosion is a chemical process 1 , anaerobic corrosion is frequently linked to the activity of sulphate-reducing bacteria (SRB) 2 , 3 , 4 , 5 , 6 . SRB are supposed to act upon iron primarily by produced hydrogen sulphide as a corrosive agent 3 , 5 , 7 and by consumption of ‘cathodic hydrogen’ formed on iron in contact with water 2 , 3 , 4 , 5 , 6 , 8 . Among SRB, Desulfovibrio species—with their capacity to consume hydrogen effectively—are conventionally regarded as the main culprits of anaerobic corrosion 2 , 3 , 4 , 5 , 6 , 8 , 9 , 10 ; however, the underlying mechanisms are complex and insufficiently understood. Here we describe novel marine, corrosive types of SRB obtained via an isolation approach with metallic iron as the only electron donor. In particular, a Desulfobacterium -like isolate reduced sulphate with metallic iron much faster than conventional hydrogen-scavenging Desulfovibrio species, suggesting that the novel surface-attached cell type obtained electrons from metallic iron in a more direct manner than via free hydrogen. Similarly, a newly isolated Methanobacterium -like archaeon produced methane with iron faster than do known hydrogen-using methanogens, again suggesting a more direct access to electrons from iron than via hydrogen consumption.

Massive microbial mats covering up to 4-meter-high carbonate buildups prosper at methane seeps in anoxic waters of the northwestern Black Sea shelf. Strong13C depletions indicate an incorporation of methane carbon into carbonates, bulk biomass, and specific lipids. The mats mainly consist of densely aggregated archaea (phylogenetic ANME-1 cluster) and sulfate-reducing bacteria (Desulfosarcina/Desulfococcus group). If incubated in vitro, these mats perform anaerobic oxidation of methane coupled to sulfate reduction. Obviously, anaerobic microbial consortia can generate both carbonate precipitation and substantial biomass accumulation, which has implications for our understanding of carbon cycling during earlier periods of Earth's history.

Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page http://www.micro-genomes.mpg.de/ebn1/.

Biological formation of methane is the terminal process of biomass degradation in aquatic habitats where oxygen, nitrate, ferric iron and sulphate have been depleted as electron acceptors. The pathway leading from dead biomass to methane through the metabolism of anaerobic bacteria and archaea is well understood for easily degradable biomolecules such as carbohydrates, proteins and lipids 1 , 2 . However, little is known about the organic compounds that lead to methane in old anoxic sediments where easily degradable biomolecules are no longer available. One class of naturally formed long-lived compounds in such sediments is the saturated hydrocarbons (alkanes) 3 , 4 , 5 . Alkanes are usually considered to be inert in the absence of oxygen, nitrate or sulphate 6 , and the analysis of alkane patterns is often used for biogeochemical characterization of sediments 7 , 8 . However, alkanes might be consumed in anoxic sediments below the zone of sulphate reduction 9 , 10 , but the underlying process has not been elucidated. Here we used enrichment cultures to show that the biological conversion of long-chain alkanes to the simplest hydrocarbon, methane, is possible under strictly anoxic conditions.

Abstract The capacity of some bacteria to metabolize hydrocarbons in the absence of molecular oxygen was first recognized only about ten years ago. Since then, the number of hydrocarbon compounds shown to be catabolized anaerobically by pure bacterial cultures has been steadily increasing. This review summarises the current knowledge of the bacterial isolates capable of anaerobic mineralization of hydrocarbons, and of the biochemistry and molecular biology of enzymes involved in the catabolic pathways of some of these substrates. Several alkylbenzenes, alkanes or alkenes are anaerobically utilized as substrates by several species of denitrifying, ferric iron-reducing and sulfate-reducing bacteria. Another group of anaerobic hydrocarbon degrading bacteria are ‘proton reducers’ that depend on syntrophic associations with methanogens. For two alkylbenzenes, toluene and ethylbenzene, details of the biochemical pathways involved in anaerobic mineralization are known. These hydrocarbons are initially attacked by novel, formerly unknown reactions and oxidized further to benzoyl-CoA, a common intermediate in anaerobic catabolism of many aromatic compounds. Toluene degradation is initiated by an unusual addition reaction of the toluene methyl group to the double bond of fumarate to form benzylsuccinate. The enzyme catalyzing this first step has been characterized at both the biochemical and molecular level. It is a unique type of glycyl-radical enzyme, an enzyme family previously represented only by pyruvate-formate lyases and anaerobic ribonucleotide reductases. Based on the nature of benzylsuccinate synthase as a radical enzyme, a hypothetical reaction mechanism for the addition of toluene to fumarate is proposed. The further catabolism of benzylsuccinate to benzoyl-CoA and succinyl-CoA appears to occur via reactions of a modified β-oxidation pathway. Ethylbenzene is first oxidized at the methylene carbon to 1-phenylethanol and subsequently to acetophenone, which is then carboxylated to 3-oxophenylpropionate and converted to benzoyl-CoA and acetyl-CoA. Anaerobic mineralization of alkanes involves an oxygen-independent oxidation to fatty acids, followed by β-oxidation. In one strain of an alkane-mineralizing sulfate-reducing bacterium, the activation appears to proceed via a chain-elongation, possibly by addition of a C1-group at the terminal methyl group of the alkane. Finally, aspects concerned with the regulation and ecological significance of anaerobic hydrocarbon catabolic pathways are discussed.

The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3- d1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional beta-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.