Chemolithoautotrophic bacteria flourish at dark water-ice interfaces of an emerged Arctic cold seep.

Below their ice shells, icy moons may offer a source of chemical energy that could support microbial life in the absence of light. In the Arctic, past and present glacial retreat leads to isostatic uplift of sediments through which cold and methane-saturated groundwater travels. This fluid reaches the surface and freezes as hill-shaped icings during winter, producing dark ice-water interfaces above water ponds containing chemical energy sources. In one such system characterized by elevated methane concentrations - the Lagoon Pingo in Adventdalen, Svalbard (~10 mg/L CH4, <0.3 mg/L O2, -0.25°C, pH 7.9), we studied amplicons of the bacterial and archaeal (microbial) 16S rRNA gene and transcripts in the water pond and overlaying ice. We show that active chemolithoautotrophic sulfur-oxidizing microorganisms (Sulfurimonas, Thiomicrorhabdus) dominated a niche at the bottom of the ice in contact with the anoxic water reservoir. There, the growing ice offers surfaces interfacing with water, and hosts favorable physico-chemical conditions for sulfide oxidation. Detection of anaerobic methanotrophs further suggests that the ice led to a steady-state dark and cold methane sink under the ice throughout winter, in two steps: first methane is oxidized to carbon dioxide and sulfates concomitantly reduced to sulfides by the activity of ANME-1a and SEEP-SRB1 consortia, in a second time energy from sulfides is used by sulfur- oxidizing microorganisms to fix carbon dioxide into organic carbon. Our results underline ice- covered and dark ecosystems as a hitherto overlooked oasis of microbial life and emphasize the need to study microbial communities in icy habitats.

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.

Microorganisms in subarctic soils are depleted of ribosomes under short-, medium-, and long-term warming.

Physiological responses of soil microorganisms to global warming are important for soil ecosystem function and the terrestrial carbon cycle. Here, we investigate the effects of weeks, years, and decades of soil warming across seasons and time on the microbial protein biosynthesis machineries (i.e. ribosomes), the most abundant cellular macromolecular complexes, using RNA:DNA and RNA:MBC (microbial biomass carbon) ratios as proxies for cellular ribosome contents. We compared warmed soils and non-warmed controls of 15 replicated subarctic grassland and forest soil temperature gradients subject to natural geothermal warming. RNA:DNA ratios tended to be lower in the warmed soils during summer and autumn, independent of warming duration (6 weeks, 8-14 years, and > 50 years), warming intensity (+3°C, +6°C, and +9°C), and ecosystem type. With increasing temperatures, RNA:MBC ratios were also decreasing. Additionally, seasonal RNA:DNA ratios of the consecutively sampled forest showed the same temperature-driven pattern. This suggests that subarctic soil microorganisms are depleted of ribosomes under warm conditions and the lack of consistent relationships with other physicochemical parameters besides temperature further suggests temperature as key driver. Furthermore, in incubation experiments, we measured significantly higher CO2 emission rates per unit of RNA from short- and long-term warmed soils compared to non-warmed controls. In conclusion, ribosome reduction may represent a widespread microbial physiological response to warming that offers a selective advantage at higher temperatures, as energy and matter can be reallocated from ribosome synthesis to other processes including substrate uptake and turnover. This way, ribosome reduction could have a substantial effect on soil carbon dynamics.

Targeting methanotrophs and isolation of a novel psychrophilic Methylobacter species from a terrestrial Arctic alkaline methane seep in Lagoon Pingo, Central Spitsbergen (78° N).

The microbial diversity associated with terrestrial groundwater seepage through permafrost soils is tightly coupled to the geochemistry of these fluids. Terrestrial alkaline methane seeps from Lagoon Pingo, Central Spitsbergen (78°N) in Norway, with methane-saturated and oxygen-limited groundwater discharge providing a potential habitat for methanotrophy. Here, we report on the microbial community's comparative analyses and distribution patterns at two sites close to Lagoon Pingo's methane emission source. To target methane-oxidizing bacteria from this system, we analysed the microbial community pattern of replicate samples from two sections near the main methane seepage source. DNA extraction, metabarcoding and subsequent sequencing of 16S rRNA genes revealed microbial communities where the major prokaryotic phyla were Pseudomonadota (42-47%), Gemmatimonadota (4-14%) and Actinobacteriota (7-11%). Among the Pseudomonadota, members of the genus Methylobacter were present at relative abundances between 1.6 and 4.7%. Enrichment targeting the methane oxidising bacteria was set up using methane seep sediments as inoculum and methane as the sole carbon and energy source, and this resulted in the isolation of a novel psychrophilic methane oxidizer, LS7-T4AT. The optimum growth temperature for the isolate was 13 °C and the pH optimum was 8.0. The morphology of cells was short rods, and TEM analysis revealed intracytoplasmic membranes arranged in stacks, a distinctive feature for Type I methanotrophs in the family Methylomonadaceae of the class Gammaproteobacteria. The strain belongs to the genus Methylobacter based on high 16S rRNA gene similarity to the psychrophilic species of Methylobacter psychrophilus Z-0021T (98.95%), the psychrophilic strain Methylobacter sp. strain S3L5C (99.00%), and the Arctic mesophilic species of Methylobacter tundripaludum SV96T (99.06%). The genome size of LS7-T4AT was 4,338,157 bp with a G + C content of 47.93%. The average nucleotide identities (ANIb) of strain LS7-T4AT to 10 isolated strains of genus Methylobacter were between 75.54 and 85.51%, lower than the species threshold of 95%. The strain LS7-T4AT represents a novel Arctic species, distinct from other members of the genus Methylobacter, for which the name Methylobacter svalbardensis sp. nov. is proposed. The type of strain is LS7-T4AT (DSMZ:114308, JCM:39463).

Abrupt permafrost thaw triggers activity of copiotrophs and microbiome predators.

Permafrost soils store a substantial part of the global soil carbon and nitrogen. However, global warming causes abrupt erosion and gradual thaw, which make these stocks vulnerable to microbial decomposition into greenhouse gases. Here, we investigated the microbial response to abrupt in situ permafrost thaw. We sequenced the total RNA of a 1 m deep soil core consisting of up to 26 500-year-old permafrost material from an active abrupt erosion site. We analysed the microbial community in the active layer soil, the recently thawed, and the intact permafrost, and found maximum RNA:DNA ratios in recently thawed permafrost indicating a high microbial activity. In thawed permafrost, potentially copiotrophic Burkholderiales and Sphingobacteriales, but also microbiome predators dominated the community. Overall, both thaw-dependent and long-term soil properties significantly correlated with changes in community composition, as did microbiome predator abundance. Bacterial predators were dominated in shallower depths by Myxococcota, while protozoa, especially Cercozoa and Ciliophora, almost tripled in relative abundance in thawed layers. Our findings highlight the ecological importance of a diverse interkingdom and active microbial community highly abundant in abruptly thawing permafrost, as well as predation as potential biological control mechanism.

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.

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.

Down-regulation of the bacterial protein biosynthesis machinery in response to weeks, years, and decades of soil warming.

How soil microorganisms respond to global warming is key to infer future soil-climate feedbacks, yet poorly understood. Here, we applied metatranscriptomics to investigate microbial physiological responses to medium-term (8 years) and long-term (>50 years) subarctic grassland soil warming of +6°C. Besides indications for a community-wide up-regulation of centralmetabolic pathways and cell replication, we observed a down-regulation of the bacterial protein biosynthesis machinery in the warmed soils, coinciding with a lower microbial biomass, RNA, and soil substrate content. We conclude that permanently accelerated reaction rates at higher temperatures and reduced substrate concentrations result in cellular reduction of ribosomes, the macromolecular complexes carrying out protein biosynthesis. Later efforts to test this, including a short-term warming experiment (6 weeks, +6°C), further supported our conclusion. Down-regulating the protein biosynthesis machinery liberates energy and matter, allowing soil bacteria to maintain high metabolic activities and cell division rates even after decades of warming.

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 [...].

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.

Decoupling of microbial community dynamics and functions in Arctic peat soil exposed to short term warming.

Temperature is an important factor governing microbe-mediated carbon feedback from permafrost soils. The link between taxonomic and functional microbial responses to temperature change remains elusive due to the lack of studies assessing both aspects of microbial ecology. Our previous study reported microbial metabolic and trophic shifts in response to short-term temperature increases in Arctic peat soil, and linked these shifts to higher CH4 and CO2 production rates (Proceedings of the National Academy of Sciences of the United States of America, 112, E2507-E2516). Here, we studied the taxonomic composition and functional potential of samples from the same experiment. We see that along a high-resolution temperature gradient (1-30°C), microbial communities change discretely, but not continuously or stochastically, in response to rising temperatures. The taxonomic variability may thus in part reflect the varied temperature responses of individual taxa and the competition between these taxa for resources. These taxonomic responses contrast the stable functional potential (metagenomic-based) across all temperatures or the previously observed metabolic or trophic shifts at key temperatures. Furthermore, with rising temperatures we observed a progressive decrease in species diversity (Shannon Index) and increased dispersion of greenhouse gas (GHG) production rates. We conclude that the taxonomic variation is decoupled from both the functional potential of the community and the previously observed temperature-dependent changes in microbial function. However, the reduced diversity at higher temperatures might help explain the higher variability in GHG production at higher temperatures.

Simultaneous Oxidation of Atmospheric Methane, Carbon Monoxide and Hydrogen for Bacterial Growth.

The second largest sink for atmospheric methane (CH4) is atmospheric methane oxidizing-bacteria (atmMOB). How atmMOB are able to sustain life on the low CH4 concentrations in air is unknown. Here, we show that during growth, with air as its only source for energy and carbon, the recently isolated atmospheric methane-oxidizer Methylocapsa gorgona MG08 (USCα) oxidizes three atmospheric energy sources: CH4, carbon monoxide (CO), and hydrogen (H2) to support growth. The cell-specific CH4 oxidation rate of M. gorgona MG08 was estimated at ~0.7 × 10-18 mol cell-1 h-1, which, together with the oxidation of CO and H2, supplies 0.38 kJ Cmol-1 h-1 during growth in air. This is seven times lower than previously assumed necessary to support bacterial maintenance. We conclude that atmospheric methane-oxidation is supported by a metabolic flexibility that enables the simultaneous harvest of CH4, H2 and CO from air, but the key characteristic of atmospheric CH4 oxidizing bacteria might be very low energy requirements.

Increased microbial expression of organic nitrogen cycling genes in long-term warmed grassland soils.

Global warming increases soil temperatures and promotes faster growth and turnover of soil microbial communities. As microbial cell walls contain a high proportion of organic nitrogen, a higher turnover rate of microbes should also be reflected in an accelerated organic nitrogen cycling in soil. We used a metatranscriptomics and metagenomics approach to demonstrate that the relative transcription level of genes encoding enzymes involved in the extracellular depolymerization of high-molecular-weight organic nitrogen was higher in medium-term (8 years) and long-term (>50 years) warmed soils than in ambient soils. This was mainly driven by increased levels of transcripts coding for enzymes involved in the degradation of microbial cell walls and proteins. Additionally, higher transcription levels for chitin, nucleic acid, and peptidoglycan degrading enzymes were found in long-term warmed soils. We conclude that an acceleration in microbial turnover under warming is coupled to higher investments in N acquisition enzymes, particularly those involved in the breakdown and recycling of microbial residues, in comparison with ambient conditions.

Environmental patterns of brown moss- and Sphagnum-associated microbial communities.

Northern peatlands typically develop through succession from fens dominated by the moss family Amblystegiaceae to bogs dominated by the moss genus Sphagnum. How the different plants and abiotic environmental conditions provided in Amblystegiaceae and Sphagnum peat shape the respective moss associated microbial communities is unknown. Through a large-scale molecular and biogeochemical study spanning Arctic, sub-Arctic and temperate regions we assessed how the endo- and epiphytic microbial communities of natural northern peatland mosses relate to peatland type (Sphagnum and Amblystegiaceae), location, moss taxa and abiotic environmental variables. Microbial diversity and community structure were distinctly different between Amblystegiaceae and Sphagnum peatlands, and within each of these two peatland types moss taxon explained the largest part of microbial community variation. Sphagnum and Amblystegiaceae shared few (< 1% of all operational taxonomic units (OTUs)) but strikingly abundant (up to 65% of relative abundance) OTUs. This core community overlapped by one third with the Sphagnum-specific core-community. Thus, the most abundant microorganisms in Sphagnum that are also found in all the Sphagnum plants studied, are the same OTUs as those few shared with Amblystegiaceae. Finally, we could confirm that these highly abundant OTUs were endophytes in Sphagnum, but epiphytes on Amblystegiaceae. We conclude that moss taxa and abiotic environmental variables associate with particular microbial communities. While moss taxon was the most influential parameter, hydrology, pH and temperature also had significant effects on the microbial communities. A small though highly abundant core community is shared between Sphagnum and Amblystegiaceae.

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.

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.

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.).

Characterization of the cecum microbiome from wild and captive rock ptarmigans indigenous to Arctic Norway.

Rock ptarmigans (Lagopus muta) are gallinaceous birds inhabiting arctic and sub-arctic environments. Their diet varies by season, including plants or plant parts of high nutritional value, but also toxic plant secondary metabolites (PSMs). Little is known about the microbes driving organic matter decomposition in the cecum of ptarmigans, especially the last steps leading to methanogenesis. The cecum microbiome in wild rock ptarmigans from Arctic Norway was characterized to unveil their functional potential for PSM detoxification, methanogenesis and polysaccharides degradation. Cecal samples were collected from wild ptarmigans from Svalbard (L. m. hyperborea) and northern Norway (L. m. muta) during autumn/winter (Sept-Dec). Samples from captive Svalbard ptarmigans fed commercial pelleted feed were included to investigate the effect of diet on microbial composition and function. Abundances of methanogens and bacteria were determined by qRT-PCR, while microbial community composition and functional potential were studied using 16S rRNA gene sequencing and shotgun metagenomics. Abundances of bacteria and methanogenic Archaea were higher in wild ptarmigans compared to captive birds. The ceca of wild ptarmigans housed bacterial groups involved in PSM-degradation, and genes mediating the conversion of phenol compounds to pyruvate. Methanomassiliicoccaceae was the major archaeal family in wild ptarmigans, carrying the genes for methanogenesis from methanol. It might be related to increased methanol production from pectin degradation in wild birds due to a diet consisting of primarily fresh pectin-rich plants. Both wild and captive ptarmigans possessed a broad suite of genes for the depolymerization of hemicellulose and non-cellulosic polysaccharides (e.g. starch). In conclusion, there were no physiological and phenotypical dissimilarities in the microbiota found in the cecum of wild ptarmigans on mainland Norway and Svalbard. While substantial differences in the functional potential for PSM degradation and methanogenesis in wild and captive birds seem to be a direct consequence of their dissimilar diets.

Holistic Assessment of Rumen Microbiome Dynamics through Quantitative Metatranscriptomics Reveals Multifunctional Redundancy during Key Steps of Anaerobic Feed Degradation.

Ruminant livestock is a major source of the potent greenhouse gas methane. The complex rumen microbiome, consisting of bacteria, archaea, and microbial eukaryotes, facilitates anaerobic plant biomass degradation in the cow rumen, leading to methane emissions. Using an integrated approach combining multidomain quantitative metatranscriptomics with gas and volatile fatty acid (VFA) profiling, we aimed at obtaining the most comprehensive picture of the active rumen microbiome during feed degradation to date. Bacterial, archaeal, and eukaryotic biomass, but also methane emissions and VFA concentrations, increased drastically within an hour after feed intake. mRNA profiling revealed a dynamic response of carbohydrate-active enzyme transcripts, transcripts involved in VFA production and methanogenesis. While the relative abundances of functional transcripts did not mirror observed processes, such as methane emissions, transformation to mRNA abundance per gram of rumen fluid echoed ruminant processes. The microbiome composition was highly individual, with, e.g., ciliate, Neocallimastigaceae, Prevotellaceae, Succinivibrionaceae, and Fibrobacteraceae abundances differing between cows. Microbiome individuality was accompanied by inter- and intradomain multifunctional redundancy among microbiome members during feed degradation. This likely enabled the robust performance of the anaerobic degradation process in each rumen. Neocallimastigaceae and ciliates contributed an unexpectedly large share of transcripts for cellulose- and hemicellulose-degrading enzymes, respectively. Methyl-reducing but not CO2-reducing methanogens were positively correlated with methane emissions. While Methanomassiliicoccales switched from methanol to methylamines as electron acceptors, Methanosphaera became the dominating methanol-reducing methanogen. This study for the first time linked rumen meta-omics with processes and enabled holistic insights into the contribution of all microbiome members to feed degradation. IMPORTANCE Ruminant animals, such as cows, live in a tight symbiotic association with microorganisms, allowing them to feed on otherwise indigestible plant biomass as food sources. Methane is produced as an end product of the anaerobic feed degradation in ruminants and is emitted to the atmosphere, making ruminant animals among the major anthropogenic sources of the potent greenhouse gas methane. Using newly developed quantitative metatranscriptomics for holistic microbiome analysis, we here identified bacterial, archaeal, and eukaryotic key players and the short-term dynamics of the rumen microbiome during anaerobic plant biomass degradation and subsequent methane emissions. These novel insights might pave the way for novel ecologically and economically sustainable methane mitigation strategies, much needed in times of global climate change.