Methane oxidation coupled to oxygenic photosynthesis in anoxic waters

Freshwater lakes represent large methane sources that, in contrast to the Ocean, significantly contribute to non-anthropogenic methane emissions to the atmosphere. Particularly mixed lakes are major methane emitters, while permanently and seasonally stratified lakes with anoxic bottom waters are often characterized by strongly reduced methane emissions. The causes for this reduced methane flux from anoxic lake waters are not fully understood. Here we identified the microorganisms and processes responsible for the near complete consumption of methane in the anoxic waters of a permanently stratified lake, Lago di Cadagno. Interestingly, known anaerobic methanotrophs could not be detected in these waters. Instead, we found abundant gamma-proteobacterial aerobic methane-oxidizing bacteria active in the anoxic waters. In vitro incubations revealed that, among all the tested potential electron acceptors, only the addition of oxygen enhanced the rates of methane oxidation. An equally pronounced stimulation was also observed when the anoxic water samples were incubated in the light. Our combined results from molecular, biogeochemical and single-cell analyses indicate that methane removal at the anoxic chemocline of Lago di Cadagno is due to true aerobic oxidation of methane fuelled by in situ oxygen production by photosynthetic algae. A similar mechanism could be active in seasonally stratified lakes and marine basins such as the Black Sea, where light penetrates to the anoxic chemocline. Given the widespread occurrence of seasonally stratified anoxic lakes, aerobic methane oxidation coupled to oxygenic photosynthesis might have an important but so far neglected role in methane emissions from lakes.     

Quantification of methane oxidation in the rhizosphere of emergent aquatic macrophytes: defining upper limits

Rates of rhizospheric methane oxidation were evaluated by aerobic incubations of subcores collected in flooded anoxic soils populated by emergent macrophytes, by greenhouse whole plant incubations, and by CH₄ stable isotopic analysis. Subcore incubations defined upper limits for rhizospheric methane oxidation on an areal basis which were equal to or greater than emission rates. These rates are considered upper limits because O₂ did not limit CH₄ uptake as is likely to occur in situ. The ratio of maximum potential methane oxidation (MO) to methane emission (ME) ranged from 0.7 to 1.9 in Louisiana rice (Oryza sativa), from 1.0 to 4.0 in a N. Florida Sagittaria lancifolia marsh, and from 5.6 to 51 in Everglades Typha domingensis and Cladium jamaicense areas. Methane oxidation/methane emission ratios determined in whole plant incubations of Sagittaria lancifolia under oxic and anoxic conditions ranged from 0.5 to 1.6. Methane oxidation activity associated with emergent aquatic macrophytes was found primarily in fine root material. A weak correlation was observed between live root biomass and CH₄ uptake in Typha. Rhizomes showed small or zero rates of methane uptake and no uptake was associated with plant stems. Methane stable isotope data from a S. lancifolia marsh were as follows: CH₄ emitted from plants: −61.6 ± 0.3%; CH₄ within stems: −42.0 ± 0.2%; CH₄ within sedimentary bubbles: −51.7 ± 0.3%). The ¹³C enrichment observed relative to emitted CH₄ could be due to preferential mobilization of CH₄ containing the lighter isotope and/or the action of methanotrophic bacteria.     

Methane‐derived carbon flow through microbial communities in arctic lake sediments

Aerobic methane (CH₄) oxidation mitigates CH₄ release and is a significant pathway for carbon and energy flow into aquatic food webs. Arctic lakes are responsible for an increasing proportion of global CH₄ emissions, but CH₄ assimilation into the aquatic food web in arctic lakes is poorly understood. Using stable isotope probing (SIP) based on phospholipid fatty acids (PLFA‐SIP) and DNA (DNA‐SIP), we tracked carbon flow quantitatively from CH₄ into sediment microorganisms from an arctic lake with an active CH₄ seepage. When 0.025 mmol CH₄ g^?1 wet sediment was oxidized, approximately 15.8–32.8% of the CH₄‐derived carbon had been incorporated into microorganisms. This CH₄‐derived carbon equated to up to 5.7% of total primary production estimates for Alaskan arctic lakes. Type I methanotrophs, including Methylomonas, Methylobacter and unclassified Methylococcaceae, were most active at CH₄ oxidation in this arctic lake. With increasing distance from the active CH₄ seepage, a greater diversity of bacteria incorporated CH₄‐derived carbon. Actinomycetes were the most quantitatively important microorganisms involved in secondary feeding on CH₄‐derived carbon. These results showed that CH₄ flows through methanotrophs into the broader microbial community and that type I methanotrophs, methylotrophs and actinomycetes are important organisms involved in using CH₄‐derived carbon in arctic freshwater ecosystems.     

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing

Arctic lakes are a significant source of the greenhouse gas methane (CH₄), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH₄ flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0–1 cm) from an arctic lake in northern Alaska sampled during ice‐free summer conditions. The highest CH₄ oxidation potential was observed in the upper sediment (0–1 cm depth) with 1.59 µmol g wet weight^?1 day^?1 compared with the deeper sediment samples (1–3 cm, 3–5 cm and 5–10 cm), which exhibited CH₄ oxidation potentials below 0.4 µmol g wet weight^?1 day^?1. Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the ¹³C‐DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH₄. Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the ¹³C‐DNA. Our results show that a diverse microbial consortium acquired carbon from CH₄ in the sediments of this arctic lake.     

内陆湿地与水体甲烷厌氧氧化功能微生物研究进展

内陆湿地与水体(如湖泊、河流、水库等)是温室气体甲烷的重要排放源。微生物介导的甲烷厌氧氧化(anaerobic oxidation of methane,AOM)反应在控制内陆湿地与水体甲烷排放中起着不可忽视的作用,对缓解全球温室效应具有重要意义。内陆湿地与水体易形成缺氧环境,且电子受体的种类和数量繁多,是发生AOM反应的理想生境。近年来,不断有研究表明,内陆湿地与水体中存在多种电子受体(NO₂^-、NO₃^-、SO₄^2-、Fe (III)等)驱动的AOM途径。NC10门细菌和甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)的一新分支ANME-2d主导了湿地和水体环境中的AOM反应,其中ANME-2d具有根据环境条件选择不同电子受体的潜力。研究系统综述了内陆湿地与水体中不同电子受体驱动的AOM途径及其参与的主要功能微生物类群;分析了AOM反应在控制温室气体甲烷排放中的作用及其环境影响因素;总结了相关功能微生物的分子生物学检测方法及甲烷厌氧氧化活性测定的同位素示踪技术。最后,对未来相关研究方向进行了展望。     

Revealing the community and metabolic potential of active methanotrophs by targeted metagenomics in the Zoige wetland of the Tibetan Plateau

The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy-making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using ¹³C-labelled methane to label the genomes of active methanotrophs, examine the methane oxidation potential and recover metagenome-assembled genomes (MAGs) of active methanotrophs. We found that gammaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase coding genes, and participate in methane oxidation via H₄MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA-SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming.     

Activity and species composition of aerobic methanotrophic communities in tundra soils

The low-temperature, methane-oxidizing activities and species composition of methanotrophic communities in various tundra bog soils were investigated by radioisotopic and immunofluorescent methods. Methanotrophic bacteria carried out the methane oxidation process through all horizons of seasonally thawed layers down to permafrost. The highest activity of the process has been observed in the water surface layer of overmoistured soils and in water-logged moss covers. Up to 40% of¹⁴CH₄ added was converted into¹⁴CO₂, bacterial biomass, and organic exometabolites. By immunofluoresecent analysis it was demonstrated that the representatives of I+X (Methylomonas, Methylobacter, andMethylococcus) and II (Methylosinus, Methylocystis) methanotrophic groups occurred simultaneously in all samples at 61.6% and 38.4%, respectively. The number of methane-oxidizing bacteria in the ecosystems studied was 0.1–22.9×10⁶ cells per gram of soil. Methanotrophic organisms ranged from 1% to 23% of the total bacterial number.     

Active Methanotrophs in Two Contrasting North American Peatland Ecosystems Revealed Using DNA-SIP

The active methanotroph community was investigated in two contrasting North American peatlands, a nutrient-rich sedge fen and nutrient-poor Sphagnum bog using in vitro incubations and ¹³C-DNA stable-isotope probing (SIP) to measure methane (CH₄) oxidation rates and label active microbes followed by fingerprinting and sequencing of bacterial and archaeal 16S rDNA and methane monooxygenase (pmoA and mmoX) genes. Rates of CH₄ oxidation were slightly, but significantly, faster in the bog and methanotrophs belonged to the class Alphaproteobacteria and were similar to other methanotrophs of the genera Methylocystis, Methylosinus, and Methylocapsa or Methylocella detected in, or isolated from, European bogs. The fen had a greater phylogenetic diversity of organisms that had assimilated ¹³C, including methanotrophs from both the Alpha- and Gammaproteobacteria classes and other potentially non-methanotrophic organisms that were similar to bacteria detected in a UK and Finnish fen. Based on similarities between bacteria in our sites and those in Europe, including Russia, we conclude that site physicochemical characteristics rather than biogeography controlled the phylogenetic diversity of active methanotrophs and that differences in phylogenetic diversity between the bog and fen did not relate to measured CH₄ oxidation rates. A single crenarchaeon in the bog site appeared to be assimilating ¹³C in 16S rDNA; however, its phylogenetic similarity to other CO₂-utilizing archaea probably indicates that this organism is not directly involved in CH₄ oxidation in peat.     

Amsterdam urban canals contain novel niches for methane-cycling microorganisms

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol g_DW⁻¹ d⁻¹ that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle.     

Anaerobic methane oxidation in a coastal oxygen minimum zone: spatial and temporal dynamics

Coastal waters are a major source of marine methane to the atmosphere. Particularly high concentrations of this potent greenhouse gas are found in anoxic waters, but it remains unclear if and to what extent anaerobic methanotrophs mitigate the methane flux. Here we investigate the long-term dynamics in methanotrophic activity and the methanotroph community in the coastal oxygen minimum zone (OMZ) of Golfo Dulce, Costa Rica, combining biogeochemical analyses, experimental incubations and 16S rRNA gene sequencing over 3 consecutive years. Our results demonstrate a stable redox zonation across the years with high concentrations of methane (up to 1.7 μmol L⁻¹) in anoxic bottom waters. However, we also measured high activities of anaerobic methane oxidation in the OMZ core (rate constant, k, averaging 30 yr⁻¹ in 2018 and 8 yr⁻¹ in 2019–2020). The OPU3 and Deep Sea-1 clades of the Methylococcales were implicated as conveyors of the activity, peaking in relative abundance 5–25 m below the oxic–anoxic interface and in the deep anoxic water respectively. Although their genetic capacity for anaerobic methane oxidation remains unexplored, their sustained high relative abundance indicates an adaptation of these clades to the anoxic, methane-rich OMZ environment, allowing them to play major roles in mitigating methane fluxes.     

An obligate methylotrophic, methane-oxidizing Methylomicrobium species from a highly alkaline environment

A new, obligately methylotrophic, methane-oxidizing bacterium, strain AMO 1, was isolated from a mixed sample of sediments from five highly alkaline soda lakes (Kenya). Based on its cell ultrastructure and high activity of the hexulose-6-phosphate synthase, the new isolate belongs to the type I methanotrophs. It differed, however, from the known neutrophilic methanotrophs by the ability to grow and oxidize methane at high pH values. The bacterium grew optimally with methane at pH 9–10. The oxidation of methane, methanol, and formaldehyde was optimal at pH 10, and cells were still active up to pH 11. AMO 1 was able to oxidize ammonia to nitrite at high pH. A maximal production of nitrite from ammonia in batch cultures at pH 10 was observed with 10% of CH₄ in the gas phase when nitrate was present as nitrogen source. Washed cells of AMO 1 oxidized ammonia most actively at pH 10–10.5 in the presence of limiting amounts of methanol or CH₄. The bacterium was also capable of oxidizing organic sulfur compounds at high pH. Washed cells grown with methane exhibited high activity of CS₂ oxidation and low, but detectable, levels of DMS and DMDS oxidation. The GC content of AMO 1 was 50.9 mol%. It showed only weak DNA homology with the previously described alkaliphilic methanotroph "Methylobacter alcaliphilus" strain 20 Z and with the neutrophilic species of the genera Methylobacter and Methylomonas. According to the 16S rRNA gene sequence analysis, strain AMO 1 was most closely related to a neutrophilic methanotroph, Methylomicrobium pelagicum (98.2% sequence similarity), within the gamma-Proteobacteria. Received: July 26, 1999 / Accepted: January 4, 2000     

Intensity of the Microbiological Processes of the Methane Cycle in Different Types of Baltic Lakes

The intensity of the microbiological processes of methane formation (MF) and methane oxidation (MO) was determined in the sediments and water of different types of Baltic lakes. The emission of methane from the lake sediments and methane distribution in the water column of the lakes were studied as functions of the lake productivity and hydrologic conditions. During summers, the intensity of MF in the lake sediments and waters varied from 0.001 to 106 ml CH₄/(dm³ day) and from 0 to 3.2 ml CH₄/(l day), respectively, and the intensity of MO in the sediments and water varied from 0 to 11.2 ml CH₄/(dm³ day) and from 0 to 1.1 ml CH₄/(l day), respectively. The total methane production (MP) in the lakes varied from 15 to 5000 ml CH₄/(m² day). In anoxic waters, the MP comprised 9–18% of the total PM in the lakes. The consumption of organic carbon for methanogenesis varied from 0.03 to 9.7 g/(m² day). The role of the methane cycle in the degradation of organic matter in the lakes increased with their productivity.     

Growth and Population Dynamics of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a Continuous-Flow Bioreactor

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (μ_ANME-2 = 0.167 · week⁻¹), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (μ_ANME-1 = 0.218 · week⁻¹). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 · week⁻¹, and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.     

Niche separation within aerobic methanotrophic bacteria across lakes and its link to methane oxidation rates

Lake methane (CH₄) emissions are largely controlled by aerobic methane-oxidizing bacteria (MOB) which mostly belong to the classes Alpha- and Gammaproteobacteria (Alpha- and Gamma-MOB). Despite the known metabolic and ecological differences between the two MOB groups, their main environmental drivers and their relative contribution to CH₄ oxidation rates across lakes remain unknown. Here, we quantified the two MOB groups through CARD-FISH along the water column of six temperate lakes and during incubations in which we measured ambient CH₄ oxidation rates. We found a clear niche separation of Alpha- and Gamma-MOB across lake water columns, which is mostly driven by oxygen concentration. Gamma-MOB appears to dominate methanotrophy throughout the water column, but Alpha-MOB may also be an important player particularly in well-oxygenated bottom waters. The inclusion of Gamma-MOB cell abundance improved environmental models of CH₄ oxidation rate, explaining part of the variation that could not be explained by environmental factors alone. Altogether, our results show that MOB composition is linked to CH₄ oxidation rates in lakes and that information on the MOB community can help predict CH₄ oxidation rates and thus emissions from lakes.     

Evaluation of methane oxidation in therhizosphere of a Carex dominated fen in northcentral Alberta, Canada

Rhizospheric methane oxidation was evaluated at a Carex (spp.) dominated fen in Alberta, Canada overthree growing seasons. Aerobic incubations of bulkpeat and live roots in the laboratory show a clearassociation between active methane oxidizing bacteriaand the rhizosphere. Aerobic incubations also show anoxidation potential that far exceeds methaneproduction potential measured in the laboratory. Quantitative estimates of how this oxidation potentialis expressed in situ depend strongly on which of twocommon approaches are used. (1) Subtracting in situmethane emission rates from methane production ratesmeasured in the laboratory with anaerobic incubationssuggest that methane oxidation may attenuate emissionsby 58 to 92%. (2) Applying the inhibitor methylfluoride (CH₃F) to whole plants in situ suggestmethane oxidation attenuates emissions by less than20% seasonally. The production minus emissiontechnique likely overestimates methane oxidationbecause methane production measured via anaerobicincubations in the laboratory are probablyoverestimates. Oxidation percentages measured byCH₃F were greatest early in the growing seasonwhen emission rates were low and fell to almostnondetectable levels as emission rates peaked in latesummer. Estimates provided by the CH₃F techniquewere generally in better agreement with estimates ofoxidation based on a stable isotope mass balance(0–34%) determined in a companion study (Popp et al. 1999).     

Biogeochemical and physical controls on methane fluxes from two ferruginous meromictic lakes

Meromictic lakes with anoxic bottom waters often have active methane cycles whereby methane is generally produced biogenically under anoxic conditions and oxidized in oxic surface waters prior to reaching the atmosphere. Lakes that contain dissolved ferrous iron in their deep waters (i.e., ferruginous) are rare, but valuable, as geochemical analogues of the conditions that dominated the Earth's oceans during the Precambrian when interactions between the iron and methane cycles could have shaped the greenhouse regulation of the planet's climate. Here, we explored controls on the methane fluxes from Brownie Lake and Canyon Lake, two ferruginous meromictic lakes that contain similar concentrations (max. >1 mM) of dissolved methane in their bottom waters. The order Methanobacteriales was the dominant methanogen detected in both lakes. At Brownie Lake, methanogen abundance, an increase in methane concentration with respect to depths closer to the sediment, and isotopic data suggest methanogenesis is an active process in the anoxic water column. At Canyon Lake, methanogenesis occurred primarily in the sediment. The most abundant aerobic methane‐oxidizing bacteria present in both water columns were associated with the Gammaproteobacteria, with little evidence of anaerobic methane oxidizing organisms being present or active. Direct measurements at the surface revealed a methane flux from Brownie Lake that was two orders of magnitude greater than the flux from Canyon Lake. Comparison of measured versus calculated turbulent diffusive fluxes indicates that most of the methane flux at Brownie Lake was non‐diffusive. Although the turbulent diffusive methane flux at Canyon Lake was attenuated by methane oxidizing bacteria, dissolved methane was detected in the epilimnion, suggestive of lateral transport of methane from littoral sediments. These results highlight the importance of direct measurements in estimating the total methane flux from water columns, and that non‐diffusive transport of methane may be important to consider from other ferruginous systems.     

Spatiotemporal variations in an assemblage of closely related planktonic aerobic methanotrophs

1. The assemblage of aerobic methane‐oxidising bacteria (MOB) was investigated in different seasons in the water column of a stratified freshwater lake. Species composition was analysed by performing denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes and cloning analysis of the pmoA gene, which encodes the α‐subunit of particulate methane monooxygenase. The relative abundance of MOB to total bacteria was deduced from the copy number of the pmoA gene and 16S rRNA gene using real‐time polymerase chain reaction. 2. The profiles of the DGGE banding patterns changed with water depth, and these changes correlated with oxygen concentration and water temperature. The sequences of the DGGE bands obtained were all associated with the genus Methylobacter. During the analysis of pmoA gene, all clones sequenced were that of the Methylobacter/Methylosarcina group. The relative abundances of pmoA gene peaked around the oxycline, and small peaks of pmoA gene were also observed near the surface when peaks of methane were observed at the corresponding depth. 3. Profiles of the DGGE banding patterns suggested that ecophysiological characteristics differ among members of the genus Methylobacter; this indicates the importance of investigating the MOB assemblage at the species level or lower. Planktonic MOB seemed to be abundant around oxycline.     

Utilization of Methane and Carbon Dioxide by Symbiotrophic Bacteria in Gills of Mytilidae (Bathymodiolus) from the Rainbow and Logachev Hydrothermal Fields on the Mid-Atlantic Ridge

Bivalve mollusks Bathymodiolus asoricus and Bathymodiolus puteoserpentis collected from the Rainbow and Logachev hydrothermal fields during dives of the Mir 1 and Mir 2 deep-sea manned submersibles were studied. Rates of methane oxidation and carbon dioxide assimilation in mussel gill tissue were determined by radiolabel analysis. During oxidation of ¹⁴CH₄, radiocarbon was detected in significant quantities not only in carbon dioxide but also in dissolved organic matter, most notably ¹⁴C-formate and ¹⁴C-acetate, occurring in a 2 : 1 ratio. Activities of hexulose-phosphate synthase, phosphoribulokinase, and ribulose 1,5-bisphosphate carboxylase were shown in the soluble fraction of gill tissue cells. At the same time, no activity of hydroxypyruvate reductase—the key enzyme of the serine pathway of C₁-assimilation—was detected. The results of PCR amplification using genetic probes for membrane-bound methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF) attest to the presence of the genes of these enzymes in the total DNA extracted from gill samples. However, no appropriate PCR responses were obtained with the mmoX primer system, which is a marker for soluble methane monooxygenase. All samples studied showed amplification with primers for the genera Methylobacter and Methylosphaera. At the same time, no genes specific to the genera Methylomonas, Methylococcus, Methylomicrobium, or MethylosinusandMethylocystis were detected. Electron microscopic examinations revealed the presence of two groups of endosymbiotic bacteria in the mussel gill tissue. The first group was represented by large cells possessing a complex system of cytoplasmic membranes, typical of methanotrophs of morphotype I. The other type of endosymbionts, having much smaller cells and lacking intracellular membrane structures, is likely to be constituted by sulfur bacteria.     

Global co-occurrence of methanogenic archaea and methanotrophic bacteria in Microcystis aggregates

Global warming and eutrophication contribute to the worldwide increase in cyanobacterial blooms, and the level of cyanobacterial biomass is strongly associated with rises in methane emissions from surface lake waters. Hence, methane-metabolizing microorganisms may be important for modulating carbon flow in cyanobacterial blooms. Here, we surveyed methanogenic and methanotrophic communities associated with floating Microcystis aggregates in 10 lakes spanning four continents, through sequencing of 16S rRNA and functional marker genes. Methanogenic archaea (mainly Methanoregula and Methanosaeta) were detectable in 5 of the 10 lakes and constituted the majority (~50%–90%) of the archaeal community in these lakes. Three of the 10 lakes contained relatively more abundant methanotrophs than the other seven lakes, with the methanotrophic genera Methyloparacoccus, Crenothrix, and an uncultured species related to Methylobacter dominating and nearly exclusively found in each of those three lakes. These three are among the five lakes in which methanogens were observed. Operational taxonomic unit (OTU) richness and abundance of methanotrophs were strongly positively correlated with those of methanogens, suggesting that their activities may be coupled. These Microcystis-aggregate-associated methanotrophs may be responsible for a hitherto overlooked sink for methane in surface freshwaters, and their co-occurrence with methanogens sheds light on the methane cycle in cyanobacterial aggregates.