Biogeography of microbial communities in high-latitude ecosystems: Contrasting drivers for methanogens, methanotrophs and global prokaryotes.

Methane-cycling is becoming more important in high-latitude ecosystems as global warming makes permafrost organic carbon increasingly available. We explored 387 samples from three high-latitudes regions (Siberia, Alaska and Patagonia) focusing on mineral/organic soils (wetlands, peatlands, forest), lake/pond sediment and water. Physicochemical, climatic and geographic variables were integrated with 16S rDNA amplicon sequences to determine the structure of the overall microbial communities and of specific methanogenic and methanotrophic guilds. Physicochemistry (especially pH) explained the largest proportion of variation in guild composition, confirming species sorting (i.e., environmental filtering) as a key mechanism in microbial assembly. Geographic distance impacted more strongly beta diversity for (i) methanogens and methanotrophs than the overall prokaryotes and, (ii) the sediment habitat, suggesting that dispersal limitation contributed to shape the communities of methane-cycling microorganisms. Bioindicator taxa characterising different ecological niches (i.e., specific combinations of geographic, climatic and physicochemical variables) were identified, highlighting the importance of Methanoregula as generalist methanogens. Methylocystis and Methylocapsa were key methanotrophs in low pH niches while Methylobacter and Methylomonadaceae in neutral environments. This work gives insight into the present and projected distribution of methane-cycling microbes at high latitudes under climate change predictions, which is crucial for constraining their impact on greenhouse gas budgets.

Widespread soil bacterium that oxidizes atmospheric methane.

The global atmospheric level of methane (CH4), the second most important greenhouse gas, is currently increasing by ∼10 million tons per year. Microbial oxidation in unsaturated soils is the only known biological process that removes CH4 from the atmosphere, but so far, bacteria that can grow on atmospheric CH4 have eluded all cultivation efforts. In this study, we have isolated a pure culture of a bacterium, strain MG08 that grows on air at atmospheric concentrations of CH4 [1.86 parts per million volume (p.p.m.v.)]. This organism, named Methylocapsa gorgona, is globally distributed in soils and closely related to uncultured members of the upland soil cluster α. CH4 oxidation experiments and 13C-single cell isotope analyses demonstrated that it oxidizes atmospheric CH4 aerobically and assimilates carbon from both CH4 and CO2 Its estimated specific affinity for CH4 (a0s) is the highest for any cultivated methanotroph. However, growth on ambient air was also confirmed for Methylocapsa acidiphila and Methylocapsa aurea, close relatives with a lower specific affinity for CH4, suggesting that the ability to utilize atmospheric CH4 for growth is more widespread than previously believed. The closed genome of M. gorgona MG08 encodes a single particulate methane monooxygenase, the serine cycle for assimilation of carbon from CH4 and CO2, and CO2 fixation via the recently postulated reductive glycine pathway. It also fixes dinitrogen and expresses the genes for a high-affinity hydrogenase and carbon monoxide dehydrogenase, suggesting that atmospheric CH4 oxidizers harvest additional energy from oxidation of the atmospheric trace gases carbon monoxide (0.2 p.p.m.v.) and hydrogen (0.5 p.p.m.v.).

Methylocapsa palsarum sp. nov., a methanotroph isolated from a subArctic discontinuous permafrost ecosystem.

An aerobic methanotrophic bacterium was isolated from a collapsed palsa soil in northern Norway and designated strain NE2T. Cells of this strain were Gram-stain-negative, non-motile, non-pigmented, slightly curved thick rods that multiplied by normal cell division. The cells possessed a particulate methane monooxygenase enzyme (pMMO) and utilized methane and methanol. Strain NE2T grew in a wide pH range of 4.1­8.0 (optimum pH 5.2­6.5) at temperatures between 6 and 32 °C (optimum 18­25 °C), and was capable of atmospheric nitrogen fixation under reduced oxygen tension. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C16 : 1ω7c, and the DNA G+C content was 61.7 mol%. The isolate belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the facultative methanotroph Methylocapsa aurea KYGT (98.3 % 16S rRNA gene sequence similarity and 84 % PmoA sequence identity). However, strain NE2T differed from Methylocapsa aurea KYGT by cell morphology, the absence of pigmentation, inability to grow on acetate, broader pH growth range, and higher tolerance to NaCl. Therefore, strain NE2T represents a novel species of the genus Methylocapsa, for which we propose the name Methylocapsa palsarum sp. nov. The type strain is NE2T ( = LMG 28715T = VKM B-2945T).

Metatranscriptomic analysis of arctic peat soil microbiota.

Recent advances in meta-omics and particularly metatranscriptomic approaches have enabled detailed studies of the structure and function of microbial communities in many ecosystems. Molecular analyses of peat soils, ecosystems important to the global carbon balance, are still challenging due to the presence of coextracted substances that inhibit enzymes used in downstream applications. We sampled layers at different depths from two high-Arctic peat soils in Svalbard for metatranscriptome preparation. Here we show that enzyme inhibition in the preparation of metatranscriptomic libraries can be circumvented by linear amplification of diluted template RNA. A comparative analysis of mRNA-enriched and nonenriched metatranscriptomes showed that mRNA enrichment resulted in a 2-fold increase in the relative abundance of mRNA but biased the relative distribution of mRNA. The relative abundance of transcripts for cellulose degradation decreased with depth, while the transcripts for hemicellulose debranching increased, indicating that the polysaccharide composition of the peat was different in the deeper and older layers. Taxonomic annotation revealed that Actinobacteria and Bacteroidetes were the dominating polysaccharide decomposers. The relative abundances of 16S rRNA and mRNA transcripts of methanogenic Archaea increased substantially with depth. Acetoclastic methanogenesis was the dominating pathway, followed by methanogenesis from formate. The relative abundances of 16S rRNA and mRNA assigned to the methanotrophic Methylococcaceae, primarily Methylobacter, increased with depth. In conclusion, linear amplification of total RNA and deep sequencing constituted the preferred method for metatranscriptomic preparation to enable high-resolution functional and taxonomic analyses of the active microbiota in Arctic peat soil.

Physiological basis for atmospheric methane oxidation and methanotrophic growth on air.

Atmospheric methane oxidizing bacteria (atmMOB) constitute the sole biological sink for atmospheric methane. Still, the physiological basis allowing atmMOB to grow on air is not well understood. Here we assess the ability and strategies of seven methanotrophic species to grow with air as sole energy, carbon, and nitrogen source. Four species, including three outside the canonical atmMOB group USCα, enduringly oxidized atmospheric methane, carbon monoxide, and hydrogen during 12 months of growth on air. These four species exhibited distinct substrate preferences implying the existence of multiple metabolic strategies to grow on air. The estimated energy yields of the atmMOB were substantially lower than previously assumed necessary for cellular maintenance in atmMOB and other aerobic microorganisms. Moreover, the atmMOB also covered their nitrogen requirements from air. During growth on air, the atmMOB decreased investments in biosynthesis while increasing investments in trace gas oxidation. Furthermore, we confirm that a high apparent specific affinity for methane is a key characteristic of atmMOB. Our work shows that atmMOB grow on the trace concentrations of methane, carbon monoxide, and hydrogen present in air and outlines the metabolic strategies that enable atmMOB to mitigate greenhouse gases.

Environmental transcription of mmoX by methane-oxidizing Proteobacteria in a subarctic Palsa Peatland.

Methane-oxidizing bacteria (MOB) that possess the soluble form of methane monooxygenase (sMMO) are present in various environments, but unlike the prevalent particulate methane monooxygenase (pMMO), the in situ activity of sMMO has not been documented. Here we report on the environmental transcription of a gene (mmoX) for this enzyme, which was attributed mainly to MOB lacking a pMMO. Our study indicates that the sMMO is an active enzyme in acidic peat ecosystems, but its importance for the mitigation of methane releases remains unknown.

Thermal acclimation of methanotrophs from the genus Methylobacter.

Methanotrophs oxidize most of the methane (CH4) produced in natural and anthropogenic ecosystems. Often living close to soil surfaces, these microorganisms must frequently adjust to temperature change. While many environmental studies have addressed temperature effects on CH4 oxidation and methanotrophic communities, there is little knowledge about the physiological adjustments that underlie these effects. We have studied thermal acclimation in Methylobacter, a widespread, abundant, and environmentally important methanotrophic genus. Comparisons of growth and CH4 oxidation kinetics at different temperatures in three members of the genus demonstrate that temperature has a strong influence on how much CH4 is consumed to support growth at different CH4 concentrations. However, the temperature effect varies considerably between species, suggesting that how a methanotrophic community is composed influences the temperature effect on CH4 uptake. To understand thermal acclimation mechanisms widely we carried out a transcriptomics experiment with Methylobacter tundripaludum SV96T. We observed, at different temperatures, how varying abundances of transcripts for glycogen and protein biosynthesis relate to cellular glycogen and ribosome concentrations. Our data also demonstrated transcriptional adjustment of CH4 oxidation, oxidative phosphorylation, membrane fatty acid saturation, cell wall composition, and exopolysaccharides between temperatures. In addition, we observed differences in M. tundripaludum SV96T cell sizes at different temperatures. We conclude that thermal acclimation in Methylobacter results from transcriptional adjustment of central metabolism, protein biosynthesis, cell walls and storage. Acclimation leads to large shifts in CH4 consumption and growth efficiency, but with major differences between species. Thus, our study demonstrates that physiological adjustments to temperature change can substantially influence environmental CH4 uptake rates and that consideration of methanotroph physiology might be vital for accurate predictions of warming effects on CH4 emissions.

A combined microbial and biogeochemical dataset from high-latitude ecosystems with respect to methane cycle.

High latitudes are experiencing intense ecosystem changes with climate warming. The underlying methane (CH4) cycling dynamics remain unresolved, despite its crucial climatic feedback. Atmospheric CH4 emissions are heterogeneous, resulting from local geochemical drivers, global climatic factors, and microbial production/consumption balance. Holistic studies are mandatory to capture CH4 cycling complexity. Here, we report a large set of integrated microbial and biogeochemical data from 387 samples, using a concerted sampling strategy and experimental protocols. The study followed international standards to ensure inter-comparisons of data amongst three high-latitude regions: Alaska, Siberia, and Patagonia. The dataset encompasses different representative environmental features (e.g. lake, wetland, tundra, forest soil) of these high-latitude sites and their respective heterogeneity (e.g. characteristic microtopographic patterns). The data included physicochemical parameters, greenhouse gas concentrations and emissions, organic matter characterization, trace elements and nutrients, isotopes, microbial quantification and composition. This dataset addresses the need for a robust physicochemical framework to conduct and contextualize future research on the interactions between climate change, biogeochemical cycles and microbial communities at high-latitudes.

Genome sequence of the Arctic methanotroph Methylobacter tundripaludum SV96.

Methylobacter tundripaludum SV96(T) (ATCC BAA-1195) is a psychrotolerant aerobic methane-oxidizing gammaproteobacterium (Methylococcales, Methylococcaceae) living in High Arctic wetland soil. The strain was isolated from soil harvested in July 1996 close to the settlement Ny-Ålesund, Svalbard, Norway (78°56'N, 11°53'E), and described as a novel species in 2006. The genome includes pmo and pxm operons encoding copper membrane monooxygenases (Cu-MMOs), genes required for nitrogen fixation, and the nirS gene implicated in dissimilatory nitrite reduction to NO but no identifiable inventory for further processing of nitrogen oxides. These genome data provide the basis to investigate M. tundripaludum SV96, identified as a major player in the biogeochemistry of Arctic environments.

The Influence of Above-Ground Herbivory on the Response of Arctic Soil Methanotrophs to Increasing CH4 Concentrations and Temperatures.

Rising temperatures in the Arctic affect soil microorganisms, herbivores, and peatland vegetation, thus directly and indirectly influencing microbial CH4 production. It is not currently known how methanotrophs in Arctic peat respond to combined changes in temperature, CH4 concentration, and vegetation. We studied methanotroph responses to temperature and CH4 concentration in peat exposed to herbivory and protected by exclosures. The methanotroph activity was assessed by CH4 oxidation rate measurements using peat soil microcosms and a pure culture of Methylobacter tundripaludum SV96, qPCR, and sequencing of pmoA transcripts. Elevated CH4 concentrations led to higher CH4 oxidation rates both in grazed and exclosed peat soils, but the strongest response was observed in grazed peat soils. Furthermore, the relative transcriptional activities of different methanotroph community members were affected by the CH4 concentrations. While transcriptional responses to low CH4 concentrations were more prevalent in grazed peat soils, responses to high CH4 concentrations were more prevalent in exclosed peat soils. We observed no significant methanotroph responses to increasing temperatures. We conclude that methanotroph communities in these peat soils respond to changes in the CH4 concentration depending on their previous exposure to grazing. This "conditioning" influences which strains will thrive and, therefore, determines the function of the methanotroph community.

Correction: Rainer et al. The Influence of Above-Ground Herbivory on the Response of Arctic Soil Methanotrophs to Increasing CH4 Concentrations and Temperatures. Microorganisms 2021, 9, 2080.

The authors wish to make the following corrections to this paper [...].

Impacts of inter- and intralaboratory variations on the reproducibility of microbial community analyses.

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.

Methanotroph populations and CH4 oxidation potentials in high-Arctic peat are altered by herbivory induced vegetation change.

Methane oxidizing bacteria (methanotrophs) within the genus Methylobacter constitute the biological filter for methane (CH4) in many Arctic soils. Multiple Methylobacter strains have been identified in these environments but we seldom know the ecological significance of the different strains. High-Arctic peatlands in Svalbard are heavily influenced by herbivory, leading to reduced vascular plant and root biomass. Here, we have measured potential CH4 oxidation rates and identified the active methantrophs in grazed peat and peat protected from grazing by fencing (exclosures) for 18 years. Grazed peat sustained a higher water table, higher CH4 concentrations and lower oxygen (O2) concentrations than exclosed peat. Correspondingly, the highest CH4 oxidation potentials were closer to the O2 rich surface in the grazed than in the protected peat. A comparison of 16S rRNA genes showed that the majority of methanotrophs in both sites belong to the genus Methylobacter. Further analyses of pmoA transcripts revealed that several Methylobacter OTUs were active in the peat but that different OTUs dominated the grazed peat than the exclosed peat. We conclude that grazing influences soil conditions, the active CH4 filter and that different Methylobacter populations are responsible for CH4 oxidation depending on the environmental conditions.

The Impact of Methane on Microbial Communities at Marine Arctic Gas Hydrate Bearing Sediment.

Cold seeps are characterized by high biomass, which is supported by the microbial oxidation of the available methane by capable microorganisms. The carbon is subsequently transferred to higher trophic levels. South of Svalbard, five geological mounds shaped by the formation of methane gas hydrates, have been recently located. Methane gas seeping activity has been observed on four of them, and flares were primarily concentrated at their summits. At three of these mounds, and along a distance gradient from their summit to their outskirt, we investigated the eukaryotic and prokaryotic biodiversity linked to 16S and 18S rDNA. Here we show that local methane seepage and other environmental conditions did affect the microbial community structure and composition. We could not demonstrate a community gradient from the summit to the edge of the mounds. Instead, a similar community structure in any methane-rich sediments could be retrieved at any location on these mounds. The oxidation of methane was largely driven by anaerobic methanotrophic Archaea-1 (ANME-1) and the communities also hosted high relative abundances of sulfate reducing bacterial groups although none demonstrated a clear co-occurrence with the predominance of ANME-1. Additional common taxa were observed and their abundances were likely benefiting from the end products of methane oxidation. Among these were sulfide-oxidizing Campilobacterota, organic matter degraders, such as Bathyarchaeota, Woesearchaeota, or thermoplasmatales marine benthic group D, and heterotrophic ciliates and Cercozoa.

Anaerobic oxidation of methane and associated microbiome in anoxic water of Northwestern Siberian lakes.

Arctic lakes emit methane (CH4) to the atmosphere. The magnitude of this flux could increase with permafrost thaw but might also be mitigated by microbial CH4 oxidation. Methane oxidation in oxic water has been extensively studied, while the contribution of anaerobic oxidation of methane (AOM) to CH4 mitigation is not fully understood. We have investigated four Northern Siberian stratified lakes in an area of discontinuous permafrost nearby Igarka, Russia. Analyses of CH4 concentrations in the water column demonstrated that 60 to 100% of upward diffusing CH4 was oxidized in the anoxic layers of the four lakes. A combination of pmoA and mcrA gene qPCR and 16S rRNA gene metabarcoding showed that the same taxa, all within Methylomonadaceae and including the predominant genus Methylobacter as well as Crenothrix, could be the major methane-oxidizing bacteria (MOB) in the anoxic water of the four lakes. Correlation between Methylomonadaceae and OTUs within Methylotenera, Geothrix and Geobacter genera indicated that AOM might occur in an interaction between MOB, denitrifiers and iron-cycling partners. We conclude that MOB within Methylomonadaceae could have a crucial impact on CH4 cycling in these Siberian Arctic lakes by mitigating the majority of produced CH4 before it leaves the anoxic zone. This finding emphasizes the importance of AOM by Methylomonadaceae and extends our knowledge about CH4 cycle in lakes, a crucial component of the global CH4 cycle.

Methane-fuelled biofilms predominantly composed of methanotrophic ANME-1 in Arctic gas hydrate-related sediments.

Sedimentary biofilms comprising microbial communities mediating the anaerobic oxidation of methane are rare. Here, we describe two biofilm communities discovered in sediment cores recovered from Arctic cold seep sites (gas hydrate pingos) in the north-western Barents Sea, characterized by steady methane fluxes. We found macroscopically visible biofilms in pockets in the sediment matrix at the depth of the sulphate-methane-transition zone. 16S rRNA gene surveys revealed that the microbial community in one of the two biofilms comprised exclusively of putative anaerobic methanotrophic archaea of which ANME-1 was the sole archaeal taxon. The bacterial community consisted of relatives of sulphate-reducing bacteria (SRB) belonging to uncultured Desulfobacteraceae clustering into SEEP-SRB1 (i.e. the typical SRB associated to ANME-1), and members of the atribacterial JS1 clade. Confocal laser scanning microscopy demonstrates that this biofilm is composed of multicellular strands and patches of ANME-1 that are loosely associated with SRB cells, but not tightly connected in aggregates. Our discovery of methanotrophic biofilms in sediment pockets closely associated with methane seeps constitutes a hitherto overlooked and potentially widespread sink for methane and sulphate in marine sediments.

Draft Genome Sequence of Methylocapsa palsarum NE2T, an Obligate Methanotroph from Subarctic Soil.

Methylocapsa palsarum NE2T is an aerobic, mildly acidophilic, obligate methanotroph. Similar to other Methylocapsa species, it possesses only a particulate methane monooxygenase and is capable of atmospheric nitrogen fixation. The genome sequence of this typical inhabitant of subarctic wetlands and soils also contains genes indicative of aerobic anoxygenic photosynthesis.

Draft Genome Sequences of Two Gammaproteobacterial Methanotrophs Isolated from Rice Ecosystems.

The genomes of the aerobic methanotrophs "Methyloterricola oryzae" strain 73aT and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73aT represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum.

A new cell morphotype among methane oxidizers: a spiral-shaped obligately microaerophilic methanotroph from northern low-oxygen environments.

Although representatives with spiral-shaped cells are described for many functional groups of bacteria, this cell morphotype has never been observed among methanotrophs. Here, we show that spiral-shaped methanotrophic bacteria do exist in nature but elude isolation by conventional approaches due to the preference for growth under micro-oxic conditions. The helical cell shape may enable rapid motility of these bacteria in water-saturated, heterogeneous environments with high microbial biofilm content, therefore offering an advantage of fast cell positioning under desired high methane/low oxygen conditions. The pmoA genes encoding a subunit of particulate methane monooxygenase from these methanotrophs form a new genus-level lineage within the family Methylococcaceae, type Ib methanotrophs. Application of a pmoA-based microarray detected these bacteria in a variety of high-latitude freshwater environments including wetlands and lake sediments. As revealed by the environmental pmoA distribution analysis, type Ib methanotrophs tend to live very near the methane source, where oxygen is scarce. The former perception of type Ib methanotrophs as being typical for thermal habitats appears to be incorrect because only a minor proportion of pmoA sequences from these bacteria originated from environments with elevated temperatures.

Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine Ecosystems.

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.