Proteomic response to phosphorus deficiency and aluminum stress of three aluminum-tolerant phosphobacteria isolated from acidic soils.

Aluminum (Al)-tolerant phosphobacteria enhance plant growth in acidic soils by improving Al complexing and phosphorus (P) availability. However, the impact of Al stress and P deficiency on bacterial biochemistry and physiology remains unclear. We investigated the single and mutual effects of Al stress (10 mM) and P deficiency (0.05 mM) on the proteome of three aluminum-tolerant phosphobacteria: Enterobacter sp. 198, Enterobacter sp. RJAL6, and Klebsiella sp. RCJ4. Cultivated under varying conditions, P deficiency upregulated P metabolism proteins while Al exposure downregulated iron-sulfur and heme-containing proteins and upregulated iron acquisition proteins. This demonstrated that Al influence on iron homeostasis and bacterial central metabolism. This study offers crucial insights into bacterial behavior in acidic soils, benefiting the development of bioinoculants for crops facing Al toxicity and P deficiency. This investigation marks the first proteomic study on the interaction between high Al and P deficiency in acid soils-adapted bacteria.

Identification of diverse antibiotic resistant bacteria in agricultural soil with H218O stable isotope probing combined with high-throughput sequencing.

BACKGROUND: We aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled "heavy" and the unlabelled "light" SIP fractions were sequenced. RESULTS: An increase of the 16S rRNA copy numbers in the "heavy" fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) were highly abundant after two days of incubation with antibiotics. Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. In comparison, only two ARGs from the unbinned-assembled light fractions were identified. CONCLUSIONS: The results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

Thrive or survive: prokaryotic life in hypersaline soils.

BACKGROUND: Soil services are central to life on the planet, with microorganisms as their main drivers. Thus, the evaluation of soil quality requires an understanding of the principles and factors governing microbial dynamics within it. High salt content is a constraint for life affecting more than 900 million hectares of land, a number predicted to rise at an alarming rate due to changing climate. Nevertheless, little is known about how microbial life unfolds in these habitats. In this study, DNA stable-isotope probing (DNA-SIP) with 18O-water was used to determine for the first time the taxa able to grow in hypersaline soil samples (ECe = 97.02 dS/m). We further evaluated the role of light on prokaryotes growth in this habitat. RESULTS: We detected growth of both archaea and bacteria, with taxon-specific growth patterns providing insights into the drivers of success in saline soils. Phylotypes related to extreme halophiles, including haloarchaea and Salinibacter, which share an energetically efficient mechanism for salt adaptation (salt-in strategy), dominated the active community. Bacteria related to moderately halophilic and halotolerant taxa, such as Staphylococcus, Aliifodinibius, Bradymonadales or Chitinophagales also grew during the incubations, but they incorporated less heavy isotope. Light did not stimulate prokaryotic photosynthesis but instead restricted the growth of most bacteria and reduced the diversity of archaea that grew. CONCLUSIONS: The results of this study suggest that life in saline soils is energetically expensive and that soil heterogeneity and traits such as exopolysaccharide production or predation may support growth in hypersaline soils. The contribution of phototrophy to supporting the heterotrophic community in saline soils remains unclear. This study paves the way toward a more comprehensive understanding of the functioning of these environments, which is fundamental to their management. Furthermore, it illustrates the potential of further research in saline soils to deepen our understanding of the effect of salinity on microbial communities.

Diversity and assembly of active bacteria and their potential function along soil aggregates in a paddy field.

Numerous studies have found that soil microbiomes differ at the aggregate level indicating they provide spatially heterogeneous habitats for microbial communities to develop. However, an understanding of the assembly processes and the functional profile of microbes at the aggregate level remain largely rudimentary, particularly for those active members in soil aggregates. In this study, we investigated the diversity, co-occurrence network, assembly process and predictive functional profile of active bacteria in aggregates of different sizes using H218O-based DNA stable isotope probing (SIP) and 16S rRNA gene sequencing. Most of the bacterial reads were active with 91 % of total reads incorporating labelled water during the incubation. The active microbial community belonged mostly of Proteobacteria and Actinobacteria, with a relative abundance of 55.32 % and 28.12 %, respectively. Assembly processes of the active bacteria were more stochastic than total bacteria, while the assembly processes of total bacteria were more influenced by deterministic processes. Furthermore, many functional profiles such as environmental information processing increased in active bacteria (19.39 %) compared to total bacteria (11.22 %). After incubation, the diversity and relative abundance of active bacteria of certain phyla increased, such as Proteobacteria (50.70 % to 59.95 %), Gemmatimonadetes (2.63 % to 4.11 %), and Bacteroidetes (1.50 % to 2.84 %). In small macroaggregates (SMA: 0.25-2 mm), the active bacterial community and its assembly processes differed from that of other soil aggregates (MA: microaggregates, <0.25 mm; LMA: large macroaggregates, 2-4 mm). For functional profiles, the relative abundance of important functions, such as amino acid metabolism, signal transduction and cell motility, increased with incubation days and/or in SMA compared to other aggregates. This study provides robust evidence that the community of active bacteria and its assembly processes in soil aggregates differed from total bacteria, and suggests the importance of dominant active bacteria (such as Proteobacteria) for the predicted functional profiles in the soil ecosystem.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Multimodal correlative imaging and modelling of phosphorus uptake from soil by hyphae of mycorrhizal fungi.

Phosphorus (P) is essential for plant growth. Arbuscular mycorrhizal fungi (AMF) aid its uptake by acquiring P from sources distant from roots in return for carbon. Little is known about how AMF colonise soil pore-space, and models of AMF-enhanced P-uptake are poorly validated. We used synchrotron X-ray computed tomography to visualize mycorrhizas in soil and synchrotron X-ray fluorescence/X-ray absorption near edge structure (XRF/XANES) elemental mapping for P, sulphur (S) and aluminium (Al) in combination with modelling. We found that AMF inoculation had a suppressive effect on colonisation by other soil fungi and identified differences in structure and growth rate between hyphae of AMF and nonmycorrhizal fungi. Our results showed that AMF co-locate with areas of high P and low Al, and preferentially associate with organic-type P species over Al-rich inorganic P. We discovered that AMF avoid Al-rich areas as a source of P. Sulphur-rich regions were found to be correlated with higher hyphal density and an increased organic-associated P-pool, whilst oxidized S-species were found close to AMF hyphae. Increased S oxidation close to AMF suggested the observed changes were microbiome-related. Our experimentally-validated model led to an estimate of P-uptake by AMF hyphae that is an order of magnitude lower than rates previously estimated - a result with significant implications for the modelling of plant-soil-AMF interactions.

Disproportionate CH4 Sink Strength from an Endemic, Sub-Alpine Australian Soil Microbial Community.

Soil-to-atmosphere methane (CH4) fluxes are dependent on opposing microbial processes of production and consumption. Here we use a soil-vegetation gradient in an Australian sub-alpine ecosystem to examine links between composition of soil microbial communities, and the fluxes of greenhouse gases they regulate. For each soil/vegetation type (forest, grassland, and bog), we measured carbon dioxide (CO2) and CH4 fluxes and their production/consumption at 5 cm intervals to a depth of 30 cm. All soils were sources of CO2, ranging from 49 to 93 mg CO2 m-2 h-1. Forest soils were strong net sinks for CH4, at rates of up to -413 µg CH4 m-2 h-1. Grassland soils varied, with some soils acting as sources and some as sinks, but overall averaged -97 µg CH4 m-2 h-1. Bog soils were net sources of CH4 (+340 µg CH4 m-2 h-1). Methanotrophs were dominated by USCα in forest and grassland soils, and Candidatus Methylomirabilis in the bog soils. Methylocystis were also detected at relatively low abundance in all soils. Our study suggests that there is a disproportionately large contribution of these ecosystems to the global soil CH4 sink, which highlights our dependence on soil ecosystem services in remote locations driven by unique populations of soil microbes. It is paramount to explore and understand these remote, hard-to-reach ecosystems to better understand biogeochemical cycles that underpin global sustainability.

Towards a microbial process-based understanding of the resilience of peatland ecosystem service provisioning - A research agenda.

Peatlands are wetland ecosystems with great significance as natural habitats and as major global carbon stores. They have been subject to widespread exploitation and degradation with resulting losses in characteristic biota and ecosystem functions such as climate regulation. More recently, large-scale programmes have been established to restore peatland ecosystems and the various services they provide to society. Despite significant progress in peatland science and restoration practice, we lack a process-based understanding of how soil microbiota influence peatland functioning and mediate the resilience and recovery of ecosystem services, to perturbations associated with land use and climate change. We argue that there is a need to: in the short-term, characterise peatland microbial communities across a range of spatial and temporal scales and develop an improved understanding of the links between peatland habitat, ecological functions and microbial processes; in the medium term, define what a successfully restored 'target' peatland microbiome looks like for key carbon cycle related ecosystem services and develop microbial-based monitoring tools for assessing restoration needs; and in the longer term, to use this knowledge to influence restoration practices and assess progress on the trajectory towards 'intact' peatland status. Rapid advances in genetic characterisation of the structure and functions of microbial communities offer the potential for transformative progress in these areas, but the scale and speed of methodological and conceptual advances in studying ecosystem functions is a challenge for peatland scientists. Advances in this area require multidisciplinary collaborations between peatland scientists, data scientists and microbiologists and ultimately, collaboration with the modelling community. Developing a process-based understanding of the resilience and recovery of peatlands to perturbations, such as climate extremes, fires, and drainage, will be key to meeting climate targets and delivering ecosystem services cost effectively.

Magnetic resonance sounding dataset of a hard-rock headwater catchment for assessing the vertical distribution of water contents in the subsurface.

Magnetic Resonance Sounding (MRS) measurements are acquired at 16 stations in the Strengbach headwater catchment (Vosges Mountains - France). These data, rendering the vertical distribution of water contents in the subsurface, are used to show their potential in conditioning a hydrological model of the catchment, as described in the article "Magnetic resonance sounding measurements as posterior information to condition hydrological model parameters: Application to a hard-rock headwater catchment" - Journal of Hydrology (2020). Acquisition protocols follow a free induction decay scheme. Data are filtered by applying a band-pass filter at the Larmor frequency. A filter removing the 50 Hz noise is also applied with the exception of data at a Larmor frequency close to the 50 Hz harmonic. The signal envelopes are then fitted by a decaying exponential function over time to estimate the median characteristic relaxation time of each MRS sounding.

Imagery of internal structure and destabilization features of active volcano by 3D high resolution airborne electromagnetism.

Present-day volcano imaging and monitoring relies primarily on ground surface and satellite remote sensing observations. The overall understanding of the volcanic edifice and its dynamics is thus limited by surface investigation, spatial resolution and penetration depth of the ground methods, but also by human and material resources, and harsh environments. Here, we show for the first time that an airborne electromagnetic survey provides a 3D global resistivity model of an active volcano. The high-resolution survey acquired at the Piton de la Fournaise volcano on La Réunion Island, Indian Ocean, shows unprecedented details of the internal structure of the edifice, highlighting the upwelling hydrothermal system below the craters, magma intrusion pathways and inherited faults. Together with surface monitoring, such airborne imagery have a high potential to better characterize volcano internal structure and magmatic processes, and therefore to better anticipate catastrophic events such as phreato-magmatic eruptions or volcano destabilizations.

Targeted Metatranscriptomics of Soil Microbial Communities with Stable Isotope Probing.

Metatranscriptomics is a powerful tool for capturing gene expression patterns in microbial communities and investigating their responses to environmental conditions. Stable isotope probing (SIP) is a method to target specific functional groups of microorganisms in environmental samples. The combination of RNA-SIP with metatranscriptomic analysis enhances the detection and identification of mRNA from target microorganisms. In this chapter we provide a protocol for RNA-SIP, mRNA enrichment, and mRNA preparation for high-throughput sequencing using an example of targeting methanotrophs in rice field soil.

The response of methanotrophs to additions of either ammonium, nitrate or urea in alpine swamp meadow soil as revealed by stable isotope probing.

Different forms of nitrogen (N) are deposited on the Qinghai-Tibetan plateau (QTP), while their differential effects on soil methanotrophs and their activity remain elusive. We constructed microcosms amended with different N fertilizers (ammonia, nitrate and urea) using the soils sampled from a swamp meadow on the QTP. The responses of active methanotrophs to different forms of nitrogen were determined by stable isotope probing with 5% 13C-methane. At the early stage of incubation, all N fertilizers, especially urea, suppressed methane oxidation compared with the control. The methane oxidation rate increased during the incubation, suggesting an adaptation and stimulation of some methanotrophs to elevated methane. At the onset of the incubation, the type II methanotrophs Methylocystis were most abundant, but decreased during the incubation and were replaced by the type Ia methanotrophs Methylomonas. Ammonia and urea had similar effects on the methanotroph communities, both characterized by an elevation in the proportion of Methylobacter and more diverse methanotroph communities. Nitrate had less effect on the methanotroph community. Our results uncovered the active methanotrophs responding to different nitrogen forms, and suggested that urea-N might have large effects on methanotroph diversity and activity in swamp meadow soils on the QTP.

Upland Soil Cluster Gamma dominates methanotrophic communities in upland grassland soils.

Aerobic methanotrophs in upland soils consume atmospheric methane, serving as a critical counterbalance to global warming; however, the biogeographic distribution patterns of their abundance and community composition are poorly understood, especial at a large scale. In this study, soils were sampled from 30 grasslands across >2000 km on the Qinghai-Tibetan Plateau to determine the distribution patterns of methanotrophs and their driving factors at a regional scale. Methanotroph abundance and community composition were analyzed using quantitative PCR and Illumina Miseq sequencing of pmoA genes, respectively. The pmoA gene copies ranged from 8.2 × 105 to 1.1 × 108 per gram dry soil. Among the 30 grassland soil samples, Upland Soil Cluster Gamma (USCγ) dominated the methanotroph communities in 26 samples. Jasper Ridge Cluster (JR3) was the most dominant methanotrophic cluster in two samples; while Methylocystis, cluster FWs, and Methylobacter were abundant in other two wet soil samples. Interestingly, reanalyzing the pmoA genes sequencing data from existing publications suggested that USCγ was also the main methanotrophic cluster in grassland soils in other regions, especially when their mean annual precipitation was <500 mm. Canonical Analysis of Principal Coordinates including all soil samples indicated that the methanotrophic community composition was significantly correlated with local environmental factors, among which mean annual precipitation and pH showed the strongest correlations. Variance partitioning analysis showed that environmental factors and spatial distance were significant factors affecting the community structure of methanotrophs, and environmental properties were more important factors. Collectively, these findings indicate that atmospheric methane may be mainly oxidized by USCγ in upland soils. They also highlight the key role of water availability and pH in determining the abundance and community profiles of grassland soil methanotrophs.

Methylococcaceae are the dominant active aerobic methanotrophs in a Chinese tidal marsh.

Although coastal marshes are net carbon sinks, they are net methane sources. Aerobic methanotrophs in coastal marsh soils are important methane consumers, but their activity and populations are poorly characterized. DNA stable-isotope probing followed by sequencing was used to determine how active methanotrophic populations differed in the main habitats of a Chinese coastal marsh. These habitats included mudflat, native plant-dominated, and alien plant-dominated habitats. Methylococcaceae was the most active methanotroph family across four habitats. Abundant methylotroph sequences, including methanotrophs and non-methane-oxidizing methylotrophs (Methylotenera and Methylophaga), constituted 50-70% of the 16S rRNA genes detected in the labeled native plant-dominated and mudflat soils. Methylotrophs were less abundant (~ 20%) in labeled alien plant-dominated soil, suggesting less methane assimilation into the target community or a different extent of carbon cross-feeding. Canonical correspondence analysis indicated a significant correlation between the active bacterial communities and soil properties (salinity, organic carbon, total nitrogen, pH, and available phosphorus). Importantly, these results highlight how changing vegetation or soil features in coastal marshes may change their resident active methanotrophic populations, which will further influence methane cycling.

Rhizosphere Bacterial Communities Differ According to Fertilizer Regimes and Cabbage (Brassica oleracea var. capitata L.) Harvest Time, but Not Aphid Herbivory.

Rhizosphere microbial communities are known to be highly diverse and strongly dependent on various attributes of the host plant, such as species, nutritional status, and growth stage. High-throughput 16S rRNA gene amplicon sequencing has been used to characterize the rhizosphere bacterial community of many important crop species, but this is the first study to date to characterize the bacterial and archaeal community of Brassica oleracea var. capitata. The study also tested the response of the bacterial community to fertilizer type (organic or synthetic) and N dosage (high or low), in addition to plant age (9 or 12 weeks) and aphid (Myzus persicae) herbivory (present/absent). The impact of aboveground herbivory on belowground microbial communities has received little attention in the literature, and since the type (organic or mineral) and amount of fertilizer applications are known to affect M. percicae populations, these treatments were applied at agricultural rates to test for synergistic effects on the soil bacterial community. Fertilizer type and plant growth were found to result in significantly different rhizosphere bacterial communities, while there was no effect of aphid herbivory. Several operational taxonomic units were identified as varying significantly in abundance between the treatment groups and age cohorts. These included members of the S-oxidizing genus Thiobacillus, which was significantly more abundant in organically fertilized 12-week-old cabbages, and the N-fixing cyanobacteria Phormidium, which appeared to decline in synthetically fertilized soils relative to controls. These responses may be an effect of accumulating root-derived glucosinolates in the B. oleracea rhizosphere and increased N-availability, respectively.

Unravelling the Identity, Metabolic Potential and Global Biogeography of the Atmospheric Methane-Oxidizing Upland Soil Cluster α.

Understanding of global methane sources and sinks is a prerequisite for the design of strategies to counteract global warming. Microbial methane oxidation in soils represents the largest biological sink for atmospheric methane. However, still very little is known about the identity, metabolic properties and distribution of the microbial group proposed to be responsible for most of this uptake, the uncultivated upland soil cluster α (USCα). Here, we reconstructed a draft genome of USCα from a combination of targeted cell sorting and metagenomes from forest soil, providing the first insights into its metabolic potential and environmental adaptation strategies. The 16S rRNA gene sequence recovered was distinctive and suggests this crucial group as a new genus within the Beijerinckiaceae, close to Methylocapsa. Application of a fluorescently labelled suicide substrate for the particulate methane monooxygenase enzyme (pMMO) coupled to 16S rRNA fluorescence in situ hybridisation (FISH) allowed for the first time a direct link of the high-affinity activity of methane oxidation to USCα cells in situ. Analysis of the global biogeography of this group further revealed its presence in previously unrecognized habitats, such as subterranean and volcanic biofilm environments, indicating a potential role of these environments in the biological sink for atmospheric methane.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Salinity Affects the Composition of the Aerobic Methanotroph Community in Alkaline Lake Sediments from the Tibetan Plateau.

Lakes are widely distributed on the Tibetan Plateau, which plays an important role in natural methane emission. Aerobic methanotrophs in lake sediments reduce the amount of methane released into the atmosphere. However, no study to date has analyzed the methanotroph community composition and their driving factors in sediments of these high-altitude lakes (>4000 m). To provide new insights on this aspect, the abundance and composition in the sediments of six high-altitude alkaline lakes (including both freshwater and saline lakes) on the Tibetan Plateau were studied. The quantitative PCR, terminal restriction fragment length polymorphism, and 454-pyrosequencing methods were used to target the pmoA genes. The pmoA gene copies ranged 104-106 per gram fresh sediment. Type I methanotrophs predominated in Tibetan lake sediments, with Methylobacter and uncultivated type Ib methanotrophs being dominant in freshwater lakes and Methylomicrobium in saline lakes. Combining the pmoA-pyrosequencing data from Tibetan lakes with other published pmoA-sequencing data from lake sediments of other regions, a significant salinity and alkalinity effect (P = 0.001) was detected, especially salinity, which explained ∼25% of methanotroph community variability. The main effect was Methylomicrobium being dominant (up to 100%) in saline lakes only. In freshwater lakes, however, methanotroph composition was relatively diverse, including Methylobacter, Methylocystis, and uncultured type Ib clusters. This study provides the first methanotroph data for high-altitude lake sediments (>4000 m) and shows that salinity is a driving factor for the community composition of aerobic methanotrophs.

Identification of active aerobic methanotrophs in plateau wetlands using DNA stable isotope probing.

Sedge-dominated wetlands on the Qinghai-Tibetan Plateau are methane emission centers. Methanotrophs at these sites play a role in reducing methane emissions, but relatively little is known about the composition of active methanotrophs in these wetlands. Here, we used DNA stable isotope probing to identify the key active aerobic methanotrophs in three sedge-dominated wetlands on the plateau. We found that Methylocystis species were active in two peatlands, Hongyuan and Dangxiong. Methylobacter species were found to be active only in Dangxiong peat. Hongyuan peat had the highest methane oxidation rate, and cross-feeding of carbon from methanotrophs to methylotrophic Hyphomicrobium species was observed. Owing to a low methane oxidation rate during the incubation, the labeling of methanotrophs in Maduo wetland samples was not detected. Our results indicate that there are large differences in the activity of methanotrophs in the wetlands of this region.