Victoria J. Orphan

Manganese- and iron-dependent marine methane oxidation.

Electrons Accepted Here Methane is produced in large quantities in marine sediments during the breakdown of organic matter. Methane is a powerful greenhouse gas that plays a large role in the regulation of climate. Methane is also an energy source for the abundant anaerobic methanotrophs that consume most of it before it ever reaches the atmosphere. The anaerobic oxidation of methane in marine systems depends on the presence of sulfate, which acts as an electron acceptor and is often considered essential for the reaction to proceed. Beal et al. (p. 184) report that anaerobic methane oxidation in marine sediments can be facilitated by iron and manganese, as well as by sulfate. Thus, anaerobic methane oxidation using iron and manganese could have been an important methane sink, and energy source, for the early biosphere. Methane oxidation in marine sediments can also be driven by electron acceptors like iron or manganese, not only by sulfate. Anaerobic methanotrophs help regulate Earth’s climate and may have been an important part of the microbial ecosystem on the early Earth. The anaerobic oxidation of methane (AOM) is often thought of as a sulfate-dependent process, despite the fact that other electron acceptors are more energetically favorable. Here, we show that microorganisms from marine methane-seep sediment in the Eel River Basin in California are capable of using manganese (birnessite) and iron (ferrihydrite) to oxidize methane, revealing that marine AOM is coupled, either directly or indirectly, to a larger variety of oxidants than previously thought. Large amounts of manganese and iron are provided to oceans from rivers, indicating that manganese- and iron-dependent AOM have the potential to be globally important.

Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments.

No microorganism capable of anaerobic growth on methane as the sole carbon source has yet been cultivated. Consequently, information about these microbes has been inferred from geochemical and microbiological observations of field samples. Stable isotope analysis of lipid biomarkers and rRNA gene surveys have implicated specific microbes in the anaerobic oxidation of methane (AOM). Here we use combined fluorescent in situ hybridization and secondary ion mass spectrometry analyses, to identify anaerobic methanotrophs in marine methane-seep sediments. The results provide direct evidence for the involvement of at least two distinct archaeal groups (ANME-1 and ANME-2) in AOM at methane seeps. Although both archaeal groups often occurred in direct physical association with bacteria, they also were observed as monospecific aggregations and as single cells. The ANME-1 archaeal group more frequently existed in monospecific aggregations or as single filaments, apparently without a bacterial partner. Bacteria associated with both archaeal groups included, but were not limited to, close relatives of Desulfosarcina species. Isotopic analyses suggest that monospecific archaeal cells and cell aggregates were active in anaerobic methanotrophy, as were multispecies consortia. In total, the data indicate that the microbial species and biotic interactions mediating anaerobic methanotrophy are diverse and complex. The data also clearly show that highly structured ANME-2/Desulfosarcina consortia are not the sole entities responsible for AOM at marine methane seeps. Other microbial groups, including ANME-1 archaea, are capable of anaerobic methane consumption either as single cells, in monospecific aggregates, or in multispecies consortia.

Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics

Microorganisms play a fundamental role in the cycling of nutrients and energy on our planet. A common strategy for many microorganisms mediating biogeochemical cycles in anoxic environments is syntrophy, frequently necessitating close spatial proximity between microbial partners. We are only now beginning to fully appreciate the diversity and pervasiveness of microbial partnerships in nature, the majority of which cannot be replicated in the laboratory. One notable example of such cooperation is the interspecies association between anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. These consortia are globally distributed in the environment and provide a significant sink for methane by substantially reducing the export of this potent greenhouse gas into the atmosphere. The interdependence of these currently uncultured microbes renders them difficult to study, and our knowledge of their physiological capabilities in nature is limited. Here, we have developed a method to capture select microorganisms directly from the environment, using combined fluorescence in situ hybridization and immunomagnetic cell capture. We used this method to purify syntrophic anaerobic methane oxidizing ANME-2c archaea and physically associated microorganisms directly from deep-sea marine sediment. Metagenomics, PCR, and microscopy of these purified consortia revealed unexpected diversity of associated bacteria, including Betaproteobacteria and a second sulfate-reducing Deltaproteobacterial partner. The detection of nitrogenase genes within the metagenome and subsequent demonstration of 15N2 incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen inputs by these syntrophic assemblages.

Deep-sea archaea fix and share nitrogen in methane-consuming microbial consortia.

Balancing the Nitrogen Budget Setting the global budget for elements presents difficult challenges, such as accounting for possibly unknown sources or sinks. An unresolved imbalance in the oceanic nitrogen budget suggests that there may be additional sources of biological nitrogen fixation in the deep sea. Using high-resolution imaging techniques, Dekas et al. (p. 422; see the Perspective by Fulweiler) observed direct assimilation of isotopically labeled N2 by anaerobic methane-oxidizing archaea from deep marine sediment and the subsequent transfer of nitrogen to their sulfate-reducing bacterial symbionts. This slow and energetically costly conversion by archaea is dependent upon methane and requires physical contact with the associated bacterial partner. Such syntrophic consortia represent a potential source of nitrogen in the oceans and may help to balance the global nitrogen budget. Methane-oxidizing bacteria in marine sediments may also be a major factor in ocean nitrogen cycling. Nitrogen-fixing (diazotrophic) microorganisms regulate productivity in diverse ecosystems; however, the identities of diazotrophs are unknown in many oceanic environments. Using single-cell–resolution nanometer secondary ion mass spectrometry images of 15N incorporation, we showed that deep-sea anaerobic methane-oxidizing archaea fix N2, as well as structurally similar CN–, and share the products with sulfate-reducing bacterial symbionts. These archaeal/bacterial consortia are already recognized as the major sink of methane in benthic ecosystems, and we now identify them as a source of bioavailable nitrogen as well. The archaea maintain their methane oxidation rates while fixing N2 but reduce their growth, probably in compensation for the energetic burden of diazotrophy. This finding extends the demonstrated lower limits of respiratory energy capable of fueling N2 fixation and reveals a link between the global carbon, nitrogen, and sulfur cycles.

Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics

Methane cycling gets more diverse The production and consumption of methane by microorganisms play a major role in the global carbon cycle. Although these processes can occur in a range of environments, from animal guts to the deep ocean, these metabolisms are confined to the Archaea. Evans et al. used metagenomics to assemble two nearly complete archaeal genomes from deep groundwater methanogens (see the Perspective by Lloyd). The two reconstructed genomes are members of the recently described Bathyarchaeota and not the phylum to which all previously known methane-metabolizing archaea belonged. Science, this issue p. 434, see also p. 384 Two reconstructed archaeal genomes reveal that methane metabolism is more diverse than anticipated. [Also see Perspective by Lloyd] Methanogenic and methanotrophic archaea play important roles in the global flux of methane. Culture-independent approaches are providing deeper insight into the diversity and evolution of methane-metabolizing microorganisms, but, until now, no compelling evidence has existed for methane metabolism in archaea outside the phylum Euryarchaeota. We performed metagenomic sequencing of a deep aquifer, recovering two near-complete genomes belonging to the archaeal phylum Bathyarchaeota (formerly known as the Miscellaneous Crenarchaeotal Group). These genomes contain divergent homologs of the genes necessary for methane metabolism, including those that encode the methyl–coenzyme M reductase (MCR) complex. Additional non-euryarchaeotal MCR-encoding genes identified in a range of environments suggest that unrecognized archaeal lineages may also contribute to global methane cycling. These findings indicate that methane metabolism arose before the last common ancestor of the Euryarchaeota and Bathyarchaeota.

Single cell activity reveals direct electron transfer in methanotrophic consortia

Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

ABSTRACT The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH4 and CO2 assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO2 reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ13C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of 14C from radiolabeled CH4 or CO2 revealed one hot spot for methanotrophy and CO2 fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with 14CH4 or 14CO2 revealed that there was interconversion of CH4 and CO2. The level of CO2 reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.

Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor

ABSTRACT Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.

Variations in Archaeal and Bacterial Diversity Associated with the Sulfate-Methane Transition Zone in Continental Margin Sediments (Santa Barbara Basin, California)

ABSTRACT The sulfate-methane transition zone (SMTZ) is a widespread feature of continental margins, representing a diffusion-controlled interface where there is enhanced microbial activity. SMTZ microbial activity is commonly associated with the anaerobic oxidation of methane (AOM), which is carried out by syntrophic associations between sulfate-reducing bacteria and methane-oxidizing archaea. While our understanding of the microorganisms catalyzing AOM has advanced, the diversity and ecological role of the greater microbial assemblage associated with the SMTZ have not been well characterized. In this study, the microbial diversity above, within, and beneath the Santa Barbara Basin SMTZ was described. ANME-1-related archaeal phylotypes appear to be the primary methane oxidizers in the Santa Barbara Basin SMTZ, which was independently supported by exclusive recovery of related methyl coenzyme M reductase genes (mcrA). Sulfate-reducing Deltaproteobacteria phylotypes affiliated with the Desulfobacterales and Desulfosarcina-Desulfococcus clades were also enriched in the SMTZ, as confirmed by analysis of dissimilatory sulfite reductase (dsr) gene diversity. Statistical methods demonstrated that there was a close relationship between the microbial assemblages recovered from the two horizons associated with the geochemically defined SMTZ, which could be distinguished from microbial diversity recovered from the sulfate-replete overlying horizons and methane-rich sediment beneath the transition zone. Comparison of the Santa Barbara Basin SMTZ microbial assemblage to microbial assemblages of methane seeps and other organic matter-rich sedimentary environments suggests that bacterial groups not typically associated with AOM, such as Planctomycetes and candidate division JS1, are additionally enriched within the SMTZ and may represent a common bacterial signature of many SMTZ environments worldwide.

Trace Metal Requirements for Microbial Enzymes Involved in the Production and Consumption of Methane and Nitrous Oxide

Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological activity. Microbial enzymes involved in the production and consumption of greenhouse gases often contain metal cofactors. While extensive research has examined the influence of Fe bioavailability on microbial CO2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH4), and nitrous oxide (N2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH4 and N2O cycling. Methanogenic archaea require large amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co limits methanogenesis in pure and mixed cultures and environmental studies. Anaerobic methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse methanogenesis since ANME possess most of the enzymes in the methanogenic pathway. Aerobic CH4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N2O production via classical anaerobic denitrification is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N2O reductase, the only known enzyme capable of microbial N2O conversion to N2, have only been found in classical denitrifiers. Accumulation of N2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine systems. Future research is needed on metalloenzymes involved in the production of N2O by enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce metals, and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide-metal scavenging.