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 CO_2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH_4), and nitrous oxide (N_2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH_4 and N_2O 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 CH_4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N_2O 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 N_2O reductase, the only known enzyme capable of microbial N_2O conversion to N_2, have only been found in classical denitrifiers. Accumulation of N_2O 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 N_2O 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

Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia

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 ^(15)N incorporation, we showed that deep-sea anaerobic methane-oxidizing archaea fix N_2, 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 N_2 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 N_2 fixation and reveals a link between the global carbon, nitrogen, and sulfur cycles

Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin

Methane vents are of significant geochemical and ecological importance. Notable progress has been made towards understanding anaerobic methane oxidation in marine sediments, however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs, and distantly related to established methanotrophic clades. Water-column associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment

ASM-Clust: classifying functionally diverse protein families using alignment score matrices

Rapid advances in sequencing technology have resulted in the availability of genomes from organisms across the tree of life. Accurately interpreting the function of proteins in these genomes is a major challenge, as annotation transfer based on homology frequently results in misannotation and error propagation. This challenge is especially pressing for organisms whose genomes are directly obtained from environmental samples, as interpretation of their physiology and ecology is often based solely on the genome sequence. For complex protein (super)families containing a large number of sequences, classification can be used to determine whether annotation transfer is appropriate, or whether experimental evidence for function is lacking. Here we present a novel computational approach for de novo classification of large protein (super)families, based on clustering an alignment score matrix obtained by aligning all sequences in the family to a small subset of the data. We evaluate our approach on the enolase family in the Structure Function Linkage Database

Chemotrophic Microbial Mats and Their Potential for Preservation in the Rock Record

Putative microbialites are commonly regarded to have formed in association with photosynthetic microorganisms, such as cyanobacteria. However, many modern microbial mat ecosystems are dominated by chemotrophic bacteria and archaea. Like phototrophs, filamentous sulfur-oxidizing bacteria form large mats at the sediment/water interface that can act to stabilize sediments, and their metabolic activities may mediate the formation of marine phosphorites. Similarly, bacteria and archaea associated with the anaerobic oxidation of methane (AOM) catalyze the precipitation of seafloor authigenic carbonates. When preserved, lipid biomarkers, isotopic signatures, body fossils, and lithological indicators of the local depositional environment may be used to identify chemotrophic mats in the rock record. The recognition of chemotrophic communities in the rock record has the potential to transform our understanding of ancient microbial ecologies, evolution, and geochemical conditions. Chemotrophic microbes on Earth occupy naturally occurring interfaces between oxidized and reduced chemical species and thus may provide a new set of search criteria to target life-detection efforts on other planets

Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex

Microbial life inhabiting subseafloor sediments plays an important role in Earth’s carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (10^2 to 10^3 cells cm^(−3)) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated

The Apparent Involvement of ANMEs in Mineral Dependent Methane Oxidation, as an Analog for Possible Martian Methanotrophy

On Earth, marine anaerobic methane oxidation (AOM) can be driven by the microbial reduction of sulfate, iron, and manganese. Here, we have further characterized marine sediment incubations to determine if the mineral dependent methane oxidation involves similar microorganisms to those found for sulfate-dependent methane oxidation. Through FISH and FISH-SIMS analyses using ^(13)C and ^(15)N labeled substrates, we find that the most active cells during manganese dependent AOM are primarily mixed and mixed-cluster aggregates of archaea and bacteria. Overall, our control experiment using sulfate showed two active bacterial clusters, two active shell aggregates, one active mixed aggregate, and an active archaeal sarcina, the last of which appeared to take up methane in the absence of a closely-associated bacterial partner. A single example of a shell aggregate appeared to be active in the manganese incubation, along with three mixed aggregates and an archaeal sarcina. These results suggest that the microorganisms (e.g., ANME-2) found active in the manganese-dependent incubations are likely capable of sulfate-dependent AOM. Similar metabolic flexibility for Martian methanotrophs would mean that the same microbial groups could inhabit a diverse set of Martian mineralogical crustal environments. The recently discovered seasonal Martian plumes of methane outgassing could be coupled to the reduction of abundant surface sulfates and extensive metal oxides, providing a feasible metabolism for present and past Mars. In an optimistic scenario Martian methanotrophy consumes much of the periodic methane released supporting on the order of 10,000 microbial cells per cm2 of Martian surface. Alternatively, most of the methane released each year could be oxidized through an abiotic process requiring biological methane oxidation to be more limited. If under this scenario, 1% of this methane flux were oxidized by biology in surface soils or in subsurface aquifers (prior to release), a total of about 10^(20) microbial cells could be supported through methanotrophy with the cells concentrated in regions of methane release

Consumption of Methane and CO_2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

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 CH_4 and CO_2 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, CO_2 reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ^(13)C 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 ^(14)C from radiolabeled CH_4 or CO_2 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 ^(14)CH_4 or ^(14)CO_2 revealed that there was interconversion of CH_4 and CO_2. The level of CO_2 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

Measurement of rare isotopologues of nitrous oxide by high-resolution multi-collector mass spectrometry

Rationale: Bulk and position-specific stable isotope characterization of nitrous oxide represents one of the most powerful tools for identifying its environmental sources and sinks. Constraining ^(14)N^(15)N^(18)O and ^(15)N^(14)N^(18)O will add two new dimensions to our ability to uniquely fingerprint N_2O sources. Methods: We describe a technique to measure six singly and doubly substituted isotopic variants of N2O, constraining the values of δ^(15)N, δ^(18)O, ∆^(17)O, ^(15)N site preference, and the clumped isotopomers ^(14)N^(15)N^(18)O and ^(15)N^(14)N^(18)O. The technique uses a Thermo MAT 253 Ultra, a high-resolution multi-collector gas source isotope ratio mass spectrometer. It requires 8–10 hours per sample and ~10 micromoles or more of pure N_2O. Results: We demonstrate the precision and accuracy of these measurements by analyzing N_2O brought to equilibrium in its position-specific and clumped isotopic composition by heating in the presence of a catalyst. Finally, an illustrative analysis of biogenic N_2O from a denitrifying bacterium suggests that its clumped isotopic composition is controlled by kinetic isotope effects in N_2O production. Conclusions: We developed a method for measuring six isotopic variants of N_2O and tested it with analyses of biogenic N_2O. The added isotopic constraints provided by these measurements will enhance our ability to apportion N_2O sources