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Background Managed grasslands are global sources of atmospheric methanol, which is one of the most abundant volatile organic compounds in the atmosphere and promotes oxidative capacity for tropospheric and stratospheric ozone depletion. The phyllosphere is a favoured habitat of plant-colonizing methanol-utilizing bacteria. These bacteria also occur in the rhizosphere, but their relevance for methanol consumption and ecosystem fluxes is unclear. Methanol utilizers of the plant-associated microbiota are key for the mitigation of methanol emission through consumption. However, information about grassland plant microbiota members, their biodiversity and metabolic traits, and thus key actors in the global methanol budget is largely lacking. Results We investigated the methanol utilization and consumption potentials of two common plant species (Festuca arundinacea and Taraxacum officinale) in a temperate grassland. The selected grassland exhibited methanol formation. The detection of 13C derived from 13C-methanol in 16S rRNA of the plant microbiota by stable isotope probing (SIP) revealed distinct methanol utilizer communities in the phyllosphere, roots and rhizosphere but not between plant host species. The phyllosphere was colonized by members of Gamma- and Betaproteobacteria. In the rhizosphere, 13C-labelled Bacteria were affiliated with Deltaproteobacteria, Gemmatimonadates, and Verrucomicrobiae. Less-abundant 13C-labelled Bacteria were affiliated with well-known methylotrophs of Alpha-, Gamma-, and Betaproteobacteria. Additional metagenome analyses of both plants were consistent with the SIP results and revealed Bacteria with methanol dehydrogenases (e.g., MxaF1 and XoxF1-5) of known but also unusual genera (i.e., Methylomirabilis, Methylooceanibacter, Gemmatimonas, Verminephrobacter). 14C-methanol tracing of alive plant material revealed divergent potential methanol consumption rates in both plant species but similarly high rates in the rhizosphere and phyllosphere. Conclusions Our study revealed the rhizosphere as an overlooked hotspot for methanol consumption in temperate grasslands. We further identified unusual new but potentially relevant methanol utilizers besides well-known methylotrophs in the phyllosphere and rhizosphere. We did not observe a plant host-specific methanol utilizer community. Our results suggest that our approach using quantitative SIP and metagenomics may be useful in future field studies to link gross methanol consumption rates with the rhizosphere and phyllosphere microbiome.

Background The native crop bacterial microbiota of the rhizosphere is envisioned to be engineered for sustainable agriculture. This requires the identification of keystone rhizosphere Bacteria and an understanding on how these govern crop-specific microbiome assembly from soils. We identified the metabolically active bacterial microbiota (SSU RNA) inhabiting two compartments of the rhizosphere of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), rye (Secale cereale), and oilseed rape (Brassica napus L.) at different growth stages. Results Based on metabarcoding analysis the bacterial microbiota was shaped by the two rhizosphere compartments, i.e. close and distant. Thereby implying a different spatial extent of bacterial microbiota acquirement by the cereals species versus oilseed rape. We derived core microbiota of each crop species. Massilia (barley and wheat) and unclassified Chloroflexi of group ‘KD4-96’ (oilseed rape) were identified as keystone Bacteria by combining LEfSe biomarker and network analyses. Subsequently, differential associations between networks of each crop species’ core microbiota revealed host plant-specific interconnections for specific genera, such as the unclassified Tepidisphaeraceae ‘WD2101 soil group’. Conclusions Our results provide keystone rhizosphere Bacteria derived from for crop hosts and revealed that cohort subnetworks and differential associations elucidated host species effect that was not evident from differential abundance of single bacterial genera enriched or unique to a specific plant host. Thus, we underline the importance of co-occurrence patterns within the rhizosphere microbiota that emerge in crop-specific microbiomes, which will be essential to modify native crop microbiomes for future agriculture and to develop effective bio-fertilizers.

Background: Managed grasslands are global sources of atmospheric methanol, which is one of the most abundant volatile organic compounds (VOCs) in the atmosphere and promotes oxidative capacity for tropospheric and stratospheric ozone depletion. The phyllosphere is a favoured habitat of plant-colonizing methanol-utilizing bacteria. These bacteria also occur in the rhizosphere, but their relevance for methanol consumption and ecosystem fluxes is unclear. The estimated global methanol emission rate is considerably higher than those escaped into the atmosphere. Thus, methanol utilizers in the plant microbiota might be key for the mitigation of methanol emission through consumption. However, information about grassland plant microbiota members, their biodiversity and metabolic traits, and thus key actors in the global methanol budget is largely lacking.Results: We investigated the methanol utilization and consumption potentials of two common plant species (Festuca arundinacea and Taraxacum officinale) in a temperate and fertilized grassland. The selected grassland exhibited net methanol emission. The detection of 13C derived from 13C-methanol in 16S rRNA of the plant microbiota by stable isotope probing (SIP) revealed distinct methanol utilizer communities in the phyllosphere, roots and rhizosphere but not between plant host species. The phyllosphere was colonized by members of Gamma- and Betaproteobacteria. In the rhizosphere, 13C-labelled Bacteria were affiliated with Deltaproteobacteria, Gemmatimonadates, and Verrucomicrobiae. Less-abundant 13C-labelled Bacteria were affiliated with well-known methylotrophs of Alpha-, Gamma-, and Betaproteobacteria. Additional metagenome analyses of both plants were consistent with the SIP results and revealed Bacteria with methanol dehydrogenases (e.g., MxaF1 and XoxF1-5) of known but also unusual genera (i.e., Methylomirabilis, Methylooceanibacter, Gemmatimonas, Verminephrobacter). 14C-methanol tracing of alive plant material revealed divergent methanol oxidation rates in both plant species but similarly high rates in the rhizosphere and phyllosphere.Conclusions: The rhizosphere has been shown to be an overlooked hotspot for methanol consumption in grasslands. We also identified unusual methanol utilizers in the phyllosphere and rhizosphere. We did not observe a plant host-specific methanol utilizer community. Our results suggest a model for methanol turnover in which both the sources (plants) and sinks (microbiota) of a volatile are separated but in the same ecological unit.

Background: Chloromethane (CH3Cl) is the most abundant chlorinated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. Results and Conclusions: We investigated chloromethane-degrading taxa associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13C-labelled CH3Cl combined with metagenomic DNA sequencing. Metagenome assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterised methylotrophic cmuA-independent pathway likely drives CH3Cl degradation in the investigated tree ferns.

A novel strain was isolated from grassland soil that has the potential to assimilate ammonium by the reduction of nitrate in the presence of oxygen. Whole genome sequence analysis revealed the presence of an assimilatory cytoplasmic nitrate reductase gene nasA and the assimilatory nitrite reductase genes nirBD which are involved in the sequential reduction of nitrate to nitrite and further to ammonium, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate represents a member of the genus Pseudomonas . The closest phylogenetic neighbours based on 16S rRNA gene sequence analysis are the type strains of Pseudomonas peli (98.17%) and Pseudomonas guineae (98.03%). In contrast, phylogenomic analysis revealed a close relationship to Pseudomonas alcaligenes . Computation of the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) with the closest phylogenetic neighbours of S1-A32-2T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. On the basis of these results, it was concluded that the soil isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas campi sp. nov. (type strain S1-A32-2T=LMG 31521T=DSM 110222T) is proposed.

International audience; Chloromethane (CH3 Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3 Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3 Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3 Cl-degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13 C-labelled CH3 Cl combined with metagenomics. Metagenome-assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3 Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3 Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3 Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA-independent pathway may drive CH3 Cl degradation in the investigated tree ferns.

Methanol is an abundant atmospheric volatile organic compound (VOC) that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope-probing (SIP) experiment under methylotrophic (only [13C1]-methanol was supplemented) and combined substrate conditions ([12C1]-methanol and alternative multi-carbon [13Cu]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the Beijerinckiaceae (Bacteria) that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different Beijerinckiaceae-affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from Bacteria, we also identified the yeasts Cryptococcus and Trichosporon as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove methylotrophy of these fungi.

Summary The impact of temperature on the largely unresolved intermediary ecosystem metabolism and associated unknown microbiota that link cellulose degradation and methane production in soils of a moderately acidic (pH 4.5) fen was investigated. Supplemental [13C]cellulose stimulated the accumulation of propionate, acetate and carbon dioxide as well as initial methane production in anoxic peat soil slurries at 15°C and 5°C. Accumulation of organic acids at 15°C was twice as fast as that at 5°C. 16S rRNA [13C]cellulose stable isotope probing identified novel unclassified Bacteria (79% identity to the next cultured relative Fibrobacter succinogenes), unclassified Bacteroidetes (89% identity to Prolixibacter bellariivorans), Porphyromonadaceae, Acidobacteriaceae and Ruminococcaceae as main anaerobic degraders of cellulose-derived carbon at both 15°C and 5°C. Holophagaceae and Spirochaetaceae were more abundant at 15°C. Clostridiaceae dominated the degradation of cellulose-derived carbon only at 5°C. Methanosarcina was the dominant methanogenic taxa at both 15°C and 5°C. Relative abundance of Methanocella increased at 15°C whereas that of Methanoregula and Methanosaeta increased at 5°C. Thaumarchaeota closely related to Nitrosotalea (presently not known to grow anaerobically) were abundant at 5°C but absent at 15°C indicating that Nitrosotalea sp. might be capable of anaerobic growth at low temperatures in peat.

International audience; Soils, paleosols and terrestrial sediments serve as archives for studying climate change, and represent important terrestrial carbon pools. Archive functioning relies on the chronological integrity of the respective units. Incorporation of younger organic matter (OM) e.g. by plant roots and associated microorganisms into deep subsoil and underlying soil parent material may reduce reliability of paleoenvironmental records and stability of buried OM. Long-term effects of sedimentary characteristics and deep rooting on deep subsoil microbial communities remain largely unknown. We characterized fossil and living microbial communities based on molecular markers in a Central European Late Pleistocene loess-paleosol sequence containing recent and ancient roots with ages of several millenia. The molecular approach, comprising free and phospholipid fatty acids (FAs), core and intact polar glycerol dialkyl glycerol tetraethers (GDGTs), as well as 16S rRNA genes from bacterial DNA, revealed the presence of living microorganisms along the sequence, with bacterial community composition comparable to that of modern topsoils. Up to 88% redundancy between bacterial genetic fingerprint and molecular signature of fossil microorganisms suggested a time-integrated signal of the molecular markers accumulated over a time span potentially lasting from sedimentation over one or more rooting phases until today. Free FAs, core GDGTs and DNA, considered as remains of fossil microorganisms, corresponded with ancient and recent root quantities, whereas phospholipid FAs and intact polar GDGTs, presumably derived from living microorganisms, correlated only with living roots. The biogeochemical and ecological disequilibrium induced by postsedimentary rooting may entail long-term microbial processes like OM mineralization, which may continue even millenia after the lifetime of the root. Deep roots and their fossil remains have been observed in various terrestrial settings, and roots as well as associated microorganisms cause both, OM incorporation and mineralization. Therefore, these findings are crucial for improved understanding of OM dynamics and carbon sequestration potential in deep subsoils.

Cellulose is the most abundant biopolymer in terrestrial ecosystems and is degraded by microbial communities in soils. However, relatively little is known about the diversity and function of soil prokaryotes that might participate in the overall degradation of this biopolymer. The active cellulolytic and saccharolytic Bacteria in an agricultural soil were evaluated by 16S rRNA (13)C-based stable isotope probing. Cellulose, cellobiose and glucose were mineralized under oxic conditions in soil slurries to carbon dioxide. Under anoxic conditions, these substrates were converted primarily to acetate, butyrate, carbon dioxide, hydrogen and traces of propionate and iso-butyrate; the production of these fermentation end-products was concomitant with the apparent reduction of iron(III). [(13)C]-cellulose was mainly degraded under oxic conditions by novel family-level taxa of the Bacteroidetes and Chloroflexi, and a known family-level taxon of Planctomycetes, whereas degradation under anoxic conditions was facilitated by the Kineosporiaceae (Actinobacteria) and cluster III Clostridiaceae and novel clusters within Bacteroidetes. Active aerobic sub-communities in oxic [(13)C]-cellobiose and [(13)C]-glucose treatments were dominated by Intrasporangiaceae and Micrococcaceae (Actinobacteria) whereas active cluster I Clostridiaceae (Firmicutes) were prevalent in anoxic treatments. A very large number (i.e. 28) of the detected taxa did not closely affiliate with known families, and active Archaea were not detected in any of the treatments. These collective findings suggest that: (i) a large uncultured diversity of soil Bacteria was involved in the utilization of cellulose and products of its hydrolysis, (ii) the active saccharolytic community differed phylogenetically from the active cellulolytic community, (iii) oxygen availability impacted differentially on the activity of taxa and (iv) different redox guilds (e.g. fermenters and iron reducers) compete or interact during cellulose degradation in aerated soils.

Coniferous forest soils often consume less of the greenhouse gas methane (CH 4 ) than deciduous forest soils. The reasons for this phenomenon have not been resolved. It might be caused by differences in the diffusive flux of CH 4 through the organic layer, pH or different concentrations of potentially inhibitory compounds. Soil samples were investigated from three adjacent European beech (Fagus sylvatica) and Norway spruce (Picea abies) stands in Germany. Maximal CH 4 oxidation velocities (V max(app) ) and Michaelis Menten constants (K M(app) ), retrieved from intact soil cores at constant CH 4 concentrations, temperature and matric potential, were twice as great in beech as in spruce soils. Also atmospheric CH 4 oxidation rates measured in homogenized soil samples displayed the same trend. Greatest atmospheric CH 4 oxidation rates were detected in the O a horizon or in the upper 5 cm of the mineral soil. In contrast to the beech soils, the O a horizon of the spruce soils consumed no CH 4 . A differential effect due to divergent diffusive flux through the litter layer was not found. pH and ammonium concentration were similar in samples from both forest soil types. Ethylene accumulation in all soils was negligible under oxic conditions. These collective results suggest that the different atmospheric CH 4 uptake by beech and spruce soils is caused by different CH 4 oxidizing capacities of methanotrophic communities in the O a horizon and top mineral soil.

Anoxic micro zones that occur in soil aggregates of oxic soils may be temporarily extended after rainfall and thus facilitate the anaerobic degradation of organic compounds in soils. The microbial degradation of glucose by anoxic slurries of a forest soil yielded acetate, CO2, H2, succinate, and ethanol, products indicative of mixed acid fermentation. Prokaryotes involved in this process were identified by time-resolved 16S rRNA gene-targeted stable isotope probing with [13C-U]-glucose. All labeled phylotypes from the 13C-enriched 16S rRNA gene were most closely related to Rahnella and Ewingella, enterobacterial genera known to catalyze mixed acid fermentation. These results indicate that facultative aerobes, in particular Enterobacteriaceae, (1) can outcompete obligate anaerobes when conditions become anoxic in forest soils and (2) may be involved in the initial decomposition of monosaccharides in anoxic micro zones of aerated forest soils.

Biological oxidation of atmospheric methane (CH4) in forest soils of the northern hemisphere is an important sink in the global CH4 cycle. The effect of tree species on atmospheric CH4 oxidation is not well understood. Previous studies suggest that soils under European beech (Fagus sylvatica) consume more atmospheric CH4 than soils under Norway spruce (Picea abies). A major difference in the chemical constituents of these tree species is the production of monoterpenes. The objectives of the present study were (i) to quantify monoterpenes in leaves, needles, organic horizons (Oi, Oe, Oa), and mineral soil of a Norway spruce site and an adjacent European beech site of a temperate forest (Steigerwald, Germany) and (ii) to evaluate the potential of abundant monoterpenes to inhibit atmospheric CH4 oxidation. Major compounds were α- and β-pinene, limonene, and camphene. Highest concentrations were measured in Norway spruce samples (up to 63.9 μmol g dw − 1 ). In European beech samples, monoterpene concentrations were close to or below the detection limit (≤0.00015 μmol g dw − 1 ). For limonene, α- and β-pinene, the dose-dependent inhibition on atmospheric CH4 oxidation was determined. β-Pinene had the highest inhibition efficiency, followed by limonene and α-pinene. Norway spruce roots and mineral soil samples displayed similar monoterpene profiles, suggesting that roots can be considered as a source for inhibitory monoterpenes in Norway spruce soils. Monoterpene addition was always coupled to an increased carbon dioxide production. This indicates that monoterpenes may be microbially mineralized in these soils. In summary, the study revealed that the release of monoterpenes by both roots and litter may be sources in soil and that in situ monoterpene concentrations in spruce soil are high enough to explain reduced atmospheric CH4 uptake.

The objective of the study was to elucidate the depth distribution and community composition of Archaea in a temperate acidic forest soil. Numbers of Archaea and Bacteria were measured in the upper 18 cm of the soil, and soil cores were sampled on two separate occasions using quantitative PCR targeting 16S rRNA genes. Maximum numbers of Archaea were 0.6–3.8 × 108 16S rRNA genes per gram of dry soil. Numbers of Bacteria were generally higher, but Archaea always accounted for a high percentage of the total gene numbers (12–38%). The archaeal community structure was analysed by the construction of clone libraries and by terminal restriction length polymorphism (T-RFLP) using the same Archaea-specific primers. With the reverse primer labelled, T-RFLP analysis led to the detection of four T-RFs. Three had lengths of 83, 185 and 218 bp and corresponded to uncultured Crenarchaeota. One (447 bp) was assigned to Thermoplasmales. Labelling of the forward primer allowed further separation of the T-RF into Crenarchaeota Group I.1c and Group I.1b, and indicated that Crenarchaeota of the Group I.1c were the predominant 16S rRNA genotype (≤85%) in the soil. The abundance of Archaea and concentration of ammonia and nitrate decreased with soil depth. Hence it is unclear if the detected Crenarchaeota Group I.1c participated in ammonia oxidation or had another phenotype.

Methanotrophic communities were studied in several periodically water-saturated gleyic soils. When sampled, each soil had an oxic upper layer and consumed methane from the atmosphere (at 1.75 ppmv). In most gleyic soils the K(m(app)) values for methane were between 70 and 800 ppmv. These are higher than most values observed in dry upland soils, but lower than those measured in wetlands. Based on cultivation-independent retrieval of the pmoA-gene and quantification of partial pmoA gene sequences, type II (Alphaproteobacteria) methanotrophs of the genus Methylocystis spp. were abundant (> 10(7) pmoA target molecules per gram of dry soil). Type I (Gammaproteobacteria) methanotrophs related to the genera Methylobacter and Methylocaldum/Methylococcus were detected in some soils. Six pmoA sequence types not closely related to sequences from cultivated methanotrophs were detected as well, indicating that diverse uncultivated methanotrophs were present. Three Gleysols were incubated under different mixing ratios of (13)C-labelled methane to examine (13)C incorporation into phospholipid fatty acids (PLFAs). Phospholipid fatty acids typical of type II methanotrophs, 16:0 and 18:1omega7c, were labelled with (13)C in all soils after incubation under an atmosphere containing 30 ppmv of methane. Incubation under 500 ppmv of methane resulted in labelling of additional PLFAs besides 16:0 and 18:1omega7c, suggesting that the composition of the active methanotrophic community changed in response to increased methane supply. In two soils, 16:1 PLFAs typical of type I methanotrophs were strongly labelled after incubation under the high methane mixing ratio only. Type II methanotrophs are most likely responsible for atmospheric methane uptake in these soils, while type I methanotrophs become active when methane is produced in the soil.

Soil microeukaryotes may trophically benefit from plant biopolymers. However, carbon transfer from cellulose into soil microeukaryotes has not been demonstrated so far. Microeukaryotes assimilating cellulose-derived carbon in oxic and anoxic soil slurries were therefore examined by rRNA-based stable-isotope probing. Bacteriovorous flagellates and ciliates and, likely, mixotrophic algae and saprotrophic fungi incorporated carbon from supplemental [U- 13 C]cellulose under oxic conditions. A previous study using the same soil suggested that cellulolytic Bacteria assimilated 13 C of supplemental cellulose. Thus, it can be assumed that ciliates, cercozoa, and chrysophytes assimilated carbon by grazing upon and utilizing metabolic products of Bacteria that hydrolyzed cellulose in the soil slurries.

The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 10(6)-10(8) gsoilDW(-1). Methanol dissimilation was measured based on conversion of supplemented (14)C-methanol, and occurred at concentrations down to 0.002 μmol methanol gsoilDW(-1). Tested soils exhibited specific affinities to methanol (a(0)s=0.01 d(-1)) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (0.6 μmol methanol gsoilDW(-1)) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant-methylotroph interactions were determinative for soil methylotrophs.

Norway spruce ( Picea abies ) forests exhibit lower annual atmospheric methane consumption rates than do European beech ( Fagus sylvatica ) forests. In the current study, pmoA (encoding a subunit of membrane-bound CH 4 monooxygenase) genes from three temperate forest ecosystems with both beech and spruce stands were analyzed to assess the potential effect of tree species on methanotrophic communities. A pmoA sequence difference of 7% at the derived protein level correlated with the species-level distance cutoff value of 3% based on the 16S rRNA gene. Applying this distance cutoff, higher numbers of species-level pmoA genotypes were detected in beech than in spruce soil samples, all affiliating with upland soil cluster α (USCα). Additionally, two deep-branching genotypes (named 6 and 7) were present in various soil samples not affiliating with pmoA or amoA . Abundance of USCα pmoA genes was higher in beech soils and reached up to (1.2 ± 0.2) × 10 8 pmoA genes per g of dry weight. Calculated atmospheric methane oxidation rates per cell yielded the same trend. However, these values were below the theoretical threshold necessary for facilitating cell maintenance, suggesting that USCα species might require alternative carbon or energy sources to thrive in forest soils. These collective results indicate that the methanotrophic diversity and abundance in spruce soils are lower than those of beech soils, suggesting that tree species-related factors might influence the in situ activity of methanotrophs.

Methanol is an atmospheric compound that is primarily released from plant polymers and impacts ozone formation. The global methanol emission rate from terrestrial ecosystems is of the same order of magnitude (4.9 x 10(12) mol year(-1)) as that of methane (10 x 10(12) mol year(-1)). The major proportion of the annual plant-released methanol does not enter the atmosphere, but may be reoxidized by biological methanol oxidation, which is catalyzed by methanol-oxidizing prokaryotes. Fifty-six aerobic methanol-oxidizing species have been isolated from soils. These methylotrophs belong to the Alpha-, Beta-, and Gammaproteobacteria, Verrucomicrobia, Firmicutes, and Actinobacteria. Their ecological niches are determined by oxygen and methanol concentration, temperature, pH, the capability to utilize nitrate as an electron acceptor, and the spectrum of nitrogen sources and utilizable multicarbon substrates. Recently discovered interactions with eukaryotes indicate that their ecological niches may not solely be defined by physicochemical parameters. Nonetheless, there are still gaps in knowledge; based on global methanol budgets, methanol oxidation in soil is important, but has not been addressed adequately by biogeochemical studies. Ratios of above-ground and soil-internal methanol oxidation are not known. The contribution to methanol-oxidation by aerobic and anaerobic methylotrophs in situ also needs further research.

The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 microg CH4 m(-2) h(-1)) of atmospheric CH4 at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real-time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia-oxidizing bacteria. As yet uncultured methanotrophs of a group previously named 'forest soil cluster' or 'USC alpha' were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group 'Cluster I' were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10(6) pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia-oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH4 uptake rates due to their low cell-specific CH4 oxidation activity. Based on CH4 flux and bacterial abundance data, estimated cell-specific CH4 oxidation rates of the detected methanotrophic bacteria were 540-800 x 10(-18) mol cell(-1) h(-1), which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell-specific CH4 oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH4 alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USC alpha methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA transcripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USC alpha and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell-specific CH4 oxidation activities.