Summary: While it is clear that microbial consortia containing Archaea and sulfate‐reducing bacteria (SRB) can mediate the anaerobic oxidation of methane (AOM), the interplay between these microorganisms remains unknown. The leading explanation of the AOM metabolism is ‘reverse methanogenesis’ by which a methanogenesis substrate is produced and transferred between species. Conceptually, the reversal of methanogenesis requires low H ₂ concentrations for energetic favourability. We used ¹³ C‐labelled CH ₄ as a tracer to test the effects of elevated H ₂ pressures on incubations of active AOM sediments from both the Eel River basin and Hydrate Ridge. In the presence of H ₂ , we observed a minimal reduction in the rate of CH ₄ oxidation, and conclude H ₂ does not play an interspecies role in AOM. Based on these results, as well as previous work, we propose a new model for substrate transfer in AOM. In this model, methyl sulfides produced by the Archaea from both CH ₄ oxidation and CO ₂ reduction are transferred to the SRB. Metabolically, CH ₄ oxidation provides electrons for the energy‐yielding reduction of CO ₂ to a methyl group (‘methylogenesis’). Methylogenesis is a dominantly reductive pathway utilizing most methanogenesis enzymes in their forward direction. Incubations of seep sediments demonstrate, as would be expected from this model, that methanethiol inhibits AOM and that CO can be substituted for CH ₄ as the electron donor for methylogenesis.

Microbial community succession is a fundamental process that affects underlying functions of almost all ecosystems; yet the roles and fates of the most abundant colonizers are often poorly understood. Does early abundance spur long term persistence? How do deterministic and stochastic processes influence the ecological contribution of colonizers? We performed a succession experiment within a hypersaline ecosystem to investigate how different processes contributed to the turnover of founder species. Bacterial and eukaryotic colonizers were identified during primary succession and tracked through a defined, 79-day biofilm maturation period using 16S and 18S rRNA gene sequencing in combination with high resolution imaging that utilized stable isotope tracers to evaluate successional patterns of primary producers and nitrogen fixers. The majority of the founder species did not maintain high abundance throughout succession. Species replacement (versus loss) was the dominant process shaping community succession. We also asked if different ecological processes acted on bacteria versus Eukaryotes during succession and found deterministic and stochastic forces corresponded more with microeukaryote and bacterial colonization, respectively. Our results show that taxa and functions belonging to different kingdoms, which share habitat in the tight spatial confines of a biofilm, were influenced by different ecological processes and time scales of succession.

Methanosarcina acetivorans produces acetate, formate, and methane when cultured with CO as the growth substrate [Rother M, Metcalf WW (2004) Proc Natl Acad Sci USA 101: 16929–16934 ], which suggests novel features of CO metabolism. Here we present a genome-wide proteomic approach to identify and quantify proteins differentially abundant in response to growth on CO versus methanol or acetate. The results indicate that oxidation of CO to CO ₂ supplies electrons for reduction of CO ₂ to a methyl group by steps and enzymes of the pathway for CO ₂ reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F ₄₂₀ H ₂ :heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO ₂ to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO ₂ reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.

Phototrophic microbial mats are compact ecosystems composed of highly interactive organisms in which energy and element cycling take place over millimeter-to-centimeter-scale distances. Although microbial mats are common in hypersaline environments, they have not been extensively characterized in systems dominated by divalent ions. Hot Lake is a meromictic, epsomitic lake that occupies a small, endorheic basin in north-central Washington. The lake harbors a benthic, phototrophic mat that assembles each spring, disassembles each fall, and is subject to greater than tenfold variation in salinity (primarily Mg²⁺ and SO²⁻₄) and irradiation over the annual cycle. We examined spatiotemporal variation in the mat community at five time points throughout the annual cycle with respect to prevailing physicochemical parameters by amplicon sequencing of the V4 region of the 16S rRNA gene coupled to near-full-length 16S RNA clone sequences. The composition of these microbial communities was relatively stable over the seasonal cycle and included dominant populations of Cyanobacteria, primarily a group IV cyanobacterium (Leptolyngbya), and Alphaproteobacteria (specifically, members of Rhodobacteraceae and Geminicoccus). Members of Gammaproteobacteria (e.g., Thioalkalivibrio and Halochromatium) and Deltaproteobacteria (e.g., Desulfofustis) that are likely to be involved in sulfur cycling peaked in summer and declined significantly by mid-fall, mirroring larger trends in mat community richness and evenness. Phylogenetic turnover analysis of abundant phylotypes employing environmental metadata suggests that seasonal shifts in light variability exert a dominant influence on the composition of Hot Lake microbial mat communities. The seasonal development and organization of these structured microbial mats provide opportunities for analysis of the temporal and physical dynamics that feed back to community function.

We used ¹³C‐labeled methane to document the extent of trace methane oxidation by Archaeoglobus fulgidus, Archaeoglobus lithotrophicus, Archaeoglobus profundus, Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Methanosarcina acetivorans. The results indicate trace methane oxidation during growth varied among different species and among methanogen cultures grown on different substrates. The extent of trace methane oxidation by Mb. thermoautotrophicum (0.05 ± 0.04%, ± 2 standard deviations of the methane produced during growth) was less than that by M. barkeri (0.15 ± 0.04%), grown under similar conditions with H₂ and CO₂. Methanosarcina acetivorans oxidized more methane during growth on trimethylamine (0.36 ± 0.05%) than during growth on methanol (0.07 ± 0.03%). This may indicate that, in M. acetivorans, either a methyltransferase related to growth on trimethylamine plays a role in methane oxidation, or that methanol is an intermediate of methane oxidation. Addition of possible electron acceptors (O₂, NO₃^–, SO₂^2–, SO₃^2–) or H₂ to the headspace did not substantially enhance or diminish methane oxidation in M. acetivorans cultures. Separate growth experiments with FAD and NAD⁺ showed that inclusion of these electron carriers also did not enhance methane oxidation. Our results suggest trace methane oxidized during methanogenesis cannot be coupled to the reduction of these electron acceptors in pure cultures, and that the mechanism by which methane is oxidized in methanogens is independent of H₂ concentration. In contrast to the methanogens, species of the sulfate‐reducing genus Archaeoglobus did not significantly oxidize methane during growth (oxidizing 0.003 ± 0.01% of the methane provided to A. fulgidus, 0.002 ± 0.009% to A. lithotrophicus and 0.003 ± 0.02% to A. profundus). Lack of observable methane oxidation in the three Archaeoglobus species examined may indicate that methyl‐coenzyme M reductase, which is not present in this genus, is required for the anaerobic oxidation of methane, consistent with the “reverse methanogenesis” hypothesis.