Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Methane (CH₄) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH₄ budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH₄ that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska''s interior. The water column CH₄ oxidation potential for these shallow (∼2 m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH₄, whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH₄-derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.     

Diversity and activity of methanotrophs in landfill cover soils with and without landfill gas recovery systems

Aerobic CH₄ oxidation plays an important role in mitigating CH₄ release from landfills to the atmosphere. Therefore, in this study, oxidation activity and community of methanotrophs were investigated in a subtropical landfill. Among the three sites investigated, the highest CH₄ concentration was detected in the landfill cover soil of the site (A) without a landfill gas (LFG) recovery system, although the refuse in the site had been deposited for a longer time (∼14–15 years) compared to the other two sites (∼6–11 years) where a LFG recovery system was applied. In April and September, the higher CH₄ flux was detected in site A with 72.4 and 51.7 g m⁻² d⁻¹, respectively, compared to the other sites. The abundance of methanotrophs assessed by quantification of pmoA varied with location and season. A linear relationship was observed between the abundance of methanotrophs and CH₄ concentrations in the landfill cover soils (R = 0.827, P < 0.001). The key factors influencing the methanotrophic diversity in the landfill cover soils were pH, the water content and the CH₄ concentration in the soil, of which pH was the most important factor. Type I methanotrophs, including Methylococcus, Methylosarcina, Methylomicrobium and Methylobacter, and type II methanotrophs (Methylocystis) were all detected in the landfill cover soils, with Methylocystis and Methylosarcina being the dominant genera. Methylocystis was abundant in the slightly acidic landfill cover soil, especially in September, and represented more than 89% of the total terminal-restriction fragment abundance. These findings indicated that the LFG recovery system, as well as physical and chemical parameters, affected the diversity and activity of methanotrophs in landfill cover soils.     

Methanotrophs and Methanogens in Masonry

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH₄ g of stone⁻¹ day⁻¹. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH₄ g of stone⁻¹ day⁻¹ was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.     

Molecular analysis of high-affinity methane-oxidizing enrichment cultures isolated from a forest biocenosis and agrocenoses

Methane oxidation by microorganisms inhabiting aerobic soils is a key process involved in the regulation of the concentration of this significant greenhouse gas in the atmosphere; however, the microorganisms responsible for this process remain unknown. Three stable methane-oxidizing cultures were isolated from samples of forest soils (FS) and agricultural soils (AS) of Moscow oblast, as well as from soil samples collected from a Belgian agrocenosis (BS). The obtained enrichment cultures exhibit a high affinity for methane; their k_m values range from 54.2 to 176.8 nM CH₄ and are comparable to those of aerobic soils. Analysis of the fragments of the ribosomal (16S rRNA) and functional (pmoA) genes of methanotrophs by PCR— DGGE and cloning demonstrated the presence of bacteria belonging to the genera Methylocystis in FS, Methylosinus in AS and BS, and Methylocella in BS. It was established that Methylocystis and Methylosinus detected in the enrichment cultures contain the genes encoding the synthesis of the active center of two membrane-bound particulate methane monooxygenases; it is likely that one of these genes (pmoA2) is responsible for the capacity of these microorganisms for oxidation of atmospheric methane.     

Aerobic methanotrophs from the coastal thermal springs of Lake Baikal

The number, activity, and diversity of aerobic methanotrophic bacteria in the sediments of three coastal thermal springs of Lake Baikal were analyzed. The average number of methanotrophs was 10³–10⁴ cells per 1 cm³ of sediment. The highest number of methanotrophs (10⁸ cells/cm³ of silt) and the highest potential rate of methane uptake [7.7 nmol CH₄/(cm³ day)] were revealed in sediments from the Sukhaya thermal spring. The methods of molecular ecology (DGGE, FISH, analysis of pmoA gene fragments) showed the predominance in most enrichment cultures of methanotrophs of type II, i.e., of the genera Methylocystis and Methylosinus. In only one enrichment culture (from the Sukhaya thermal spring), a type I methanotroph was revealed; its similarity to Methylococcus capsulatus Bath did not exceed 80%. These results demonstrate a widespread occurrence and high activity of the aerobic methanotrophic community in the coastal thermal springs of Lake Baikal.     

Effects of ammonium on the activity and community of methanotrophs in landfill biocover soils

The influence of NH₄⁺ on microbial CH₄ oxidation is still poorly understood in landfill cover soils. In this study, effects of NH₄⁺ addition on the activity and community structure of methanotrophs were investigated in waste biocover soil (WBS) treated by a series of NH₄⁺-N contents (0, 100, 300, 600 and 1200 mg kg⁻¹). The results showed that the addition of NH₄⁺-N ranging from 100 to 300 mg kg⁻¹ could stimulate CH₄ oxidation in the WBS samples at the first stage of activity, while the addition of an NH₄⁺-N content of 600 mg kg⁻¹ had an inhibitory effect on CH₄ oxidation in the first 4 days. The decrease of CH₄ oxidation rate observed in the last stage of activity could be caused by nitrogen limitation and/or exopolymeric substance accumulation. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. Of these, Methylocaldum was the main methanotroph in the original WBS. With incubation, a higher abundance of Methylobacter was observed in the treatments with NH₄⁺-N contents greater than 300 mg kg⁻¹, which suggested that NH₄⁺-N addition might lead to the dominance of Methylobacter in the WBS samples. Compared to type I methanotrophs, the abundance of type II methanotrophs Methylocystis and/or Methylosinus was lower in the original WBS sample. An increase in the abundance of Methylocystis and/or Methylosinus occurred in the last stage of activity, and was likely due to a nitrogen limitation condition. Redundancy analysis showed that NH₄⁺-N and the C/N ratio had a significant influence on the methanotrophic community in the WBS sample.     

Enrichment and characteristics of mixed methane-oxidizing bacteria from a Chinese coal mine

In methane-rich environments, methane-oxidizing bacteria usually occur predominantly among consortia including other types of microorganisms. In this study, artificial coal bed gas and methane gas were used to enrich mixed methanotrophic cultures from the soil of a coal mine in China, respectively. The changes in microbial community structure and function during the enrichment were examined. The microbial diversity was reduced as the enrichment proceeded, while the capacity for methane oxidation was significantly enhanced by the increased abundance of methanotrophs. The proportion of type II methanotrophs increased greatly from 7.84 % in the sampled soil to about 50 % in the enrichment cultures, due to the increase of methane concentration. After the microbial community of the cultures got stable, Methylomonas and Methylocystis became the dominant type I and type II methanotrophs, while Methylophilus was the prevailing methylotroph. The sequences affiliated with pigment-producing strains, Methylomonas rubra, Hydrogenophaga sp. AH-24, and Flavobacterium cucumis, could explain the orange appearance of the cultures. Comparing the two cultures, the multi-carbon sources in the artificial coal bed gas caused more variety of non-methanotrophic bacteria, but did not help to maintain the diversity or to increase the quantity and activity of methanotrophs. The results could help to understand the succession and interaction of microbial community in a methane-driven ecosystem.     

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

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

Characterization of Root-Associated Methanotrophs from Three Freshwater Macrophytes: Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia

Root-associated methanotrophic bacteria were enriched from three common aquatic macrophytes: Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia. At least seven distinct taxa belonging to groups I and II were identified and presumptively assigned to the genera Methylosinus, Methylocystis, Methylomonas, and Methylococcus. Four of these strains appeared to be novel on the basis of partial 16S ribosomal DNA sequence analysis. The root-methanotroph association did not appear to be highly specific, since multiple methanotrophs were isolated from each of the three plant species. Group II methanotrophs were isolated most frequently; though less common, group I isolates accounted for three of the seven distinct methanotrophs. Apparent K_m values for methane uptake by representative cultures ranged from 3 to >17 μM; for five of the eight cultures examined, apparent K_m values agreed well with apparent K_m estimates for plant roots, suggesting that these strains may be representative of those active in situ.     

Methane Oxidation and the Competition for Oxygen in the Rice Rhizosphere

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O₂ with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently <5 μM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.     

Termite mounds contain soil-derived methanotroph communities kinetically adapted to elevated methane concentrations

Termite mounds have recently been confirmed to mitigate approximately half of termite methane (CH₄) emissions, but the aerobic CH₄ oxidising bacteria (methanotrophs) responsible for this consumption have not been resolved. Here, we describe the abundance, composition and CH₄ oxidation kinetics of the methanotroph communities in the mounds of three distinct termite species sampled from Northern Australia. Results from three independent methods employed show that methanotrophs are rare members of microbial communities in termite mounds, with a comparable abundance but distinct composition to those of adjoining soil samples. Across all mounds, the most abundant and prevalent methane monooxygenase sequences were affiliated with upland soil cluster α (USCα), with sequences homologous to Methylocystis and tropical upland soil cluster (TUSC) also detected. The reconstruction of a metagenome-assembled genome of a mound USCα representative highlighted the metabolic capabilities of this group of methanotrophs. The apparent Michaelis–Menten kinetics of CH₄ oxidation in mounds were estimated from in situ reaction rates. Methane affinities of the communities were in the low micromolar range, which is one to two orders of magnitude higher than those of upland soils, but significantly lower than those measured in soils with a large CH₄ source such as landfill cover soils. The rate constant of CH₄ oxidation, as well as the porosity of the mound material, were significantly positively correlated with the abundance of methanotroph communities of termite mounds. We conclude that termite-derived CH₄ emissions have selected for distinct methanotroph communities that are kinetically adapted to elevated CH₄ concentrations. However, factors other than substrate concentration appear to limit methanotroph abundance and hence these bacteria only partially mitigate termite-derived CH₄ emissions. Our results also highlight the predominant role of USCα in an environment with elevated CH₄ concentrations and suggest a higher functional diversity within this group than previously recognised.Subject terms: Soil microbiology, Biogeochemistry     

Abundance and diversity of methanotrophic Gammaproteobacteria in northern wetlands

Numeric abundance, identity, and pH preferences of methanotrophic Gammaproteobacteria (type I methanotrophs) inhabiting the northern acidic wetlands were studied. The rates of methane oxidation by peat samples from six wetlands of European Northern Russia (pH 3.9–4.7) varied from 0.04 to 0.60 μg CH₄ g^?1 peat h^?1. The number of cells revealed by hybridization with fluorochrome labeled probes M84 + M705 specific for type I methanotrophs was 0.05–2.16 × 10⁵ cells g^?1 dry peat, i.e., 0.4–12.5% of the total number of methanotrophs and 0.004–0.39% of the total number of bacteria. Analysis of the fragments of the pmoA gene encoding particulate methane monooxygenase revealed predominance of the genus Methylocystis (92% of the clones) in the studied sample of acidic peat, while the proportion of the pmoA sequences of type I methanotrophs was insignificant (8%). PCR amplification of the 16S rRNA gene fragments of type I methanotrophs with TypeIF-Type IR primers had low specificity, since only three sequences out of 53 analyzed belonged to methanotrophs and exhibited 93–99% similarity to those of Methylovulum, Methylomonas, and Methylobacter species. Isolates of type I methanotrophs obtained from peat (strains SH10 and 83A5) were identified as members of the species Methylomonas paludis and Methylovulum miyakonense, respectively. Only Methylomonas paludis SH10 was capable of growth in acidic media (pH range for growth 3.8–7.2 with the optimum at pH 5.8–6.2), while Methylovulum miyakonense 83A5 exhibited the typical growth characteristics of neutrophilic methanotrophs (pH range for growth 5.5–8.0 with the optimum at pH 6.5–7.5).     

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10⁶ cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10⁴ type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10⁶ type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 10⁶ cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.     

Phylogenetic and Functional Diversity Within Toluene-Degrading,Sulphate-Reducing Consortia Enriched from a Contaminated Aquifer

Three toluene-degrading microbial consortia were enriched under sulphate-reducing conditions from different zones of a benzene, toluene, ethylbenzene and xylenes (BTEX) plume of two connected contaminated aquifers. Two cultures were obtained from a weakly contaminated zone of the lower aquifer, while one culture originated from the highly contaminated upper aquifer. We hypothesised that the different habitat characteristics are reflected by distinct degrader populations. Degradation of toluene with concomitant production of sulphide was demonstrated in laboratory microcosms and the enrichment cultures were phylogenetically characterised. The benzylsuccinate synthase alpha-subunit (bssA) marker gene, encoding the enzyme initiating anaerobic toluene degradation, was targeted to characterise the catabolic diversity within the enrichment cultures. It was shown that the hydrogeochemical parameters in the different zones of the plume determined the microbial composition of the enrichment cultures. Both enrichment cultures from the weakly contaminated zone were of a very similar composition, dominated by Deltaproteobacteria with the Desulfobulbaceae (a Desulfopila-related phylotype) as key players. Two different bssA sequence types were found, which were both affiliated to genes from sulphate-reducing Deltaproteobacteria. In contrast, the enrichment culture from the highly contaminated zone was dominated by Clostridia with a Desulfosporosinus-related phylotype as presumed key player. A distinct bssA sequence type with high similarity to other recently detected sequences from clostridial toluene degraders was dominant in this culture. This work contributes to our understanding of the niche partitioning between degrader populations in distinct compartments of BTEX-contaminated aquifers.     

Comparative characterization of methanotrophic enrichments by serological and molecular methods

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A, were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.     

Degradation of organic pollutants by methane grown microbial consortia

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge (AAE-A2), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent K_m and V_max for DBP degradation by AAE-A2 at 20 °C was 3.1 ± 1.2 mg l^–1 and 8.7 ± 1.1 mg DBP (g protein × h)^–1, respectively. AAE-A2 also showed fast degradation of BTX (230 ± 30 nmol benzene (mg protein × h)^–1 at 20 °C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83–92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).     

Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau

Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH₄ under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2–3.4 × 10⁸ pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH₄ oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH₄–N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH₄ oxidation, and the alpine meadow has great potential to consume more CH₄ under future warmer and grazing conditions on the Tibetan Plateau.     

Metaproteomic Identification of Diazotrophic Methanotrophs and Their Localization in Root Tissues of Field-Grown Rice Plants

In a previous study by our group, CH₄ oxidation and N₂ fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH₄ oxidation and N₂ fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH₄ oxidation and N₂ fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH₄ oxidation and N₂ fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.     

Atmospheric Methane Consumption by Forest Soils and Extracted Bacteria at Different pH Values

The effect of pH on atmospheric methane (CH₄) consumption was studied with slurries of forest soils and with bacteria extracted from the same soils. Soil samples were collected from a mixed hardwood stand in New Hampshire, from jackpine and aspen stands at the BOREAS (Boreal Ecosystem Atmosphere Study) site near Thompson, northern Manitoba, from sites in southern Québec, including a beech stand and a meadow, and from a site in Ontario (cultivated humisol). Consumption of atmospheric CH₄ (concentration, approximately 1.8 ppm) occurred at depths of >5 cm in both acidic (pH 4.5 to 5.2) and alkaline (pH 7.2 to 7.8) soils. In slurries of acidic soils, maximum activity occurred at different pH values (pH 4.0 to 6.5). Bacteria extracted from these soils by high-speed blending and density gradient centrifugation showed pH responses different from the pH responses of the slurries. In all cases, these bacteria had a methanotrophy pH optimum of 5.8 and exhibited no activity at pH 6.8 to 7.0, the pH optimum range for known methanotrophs. This difference in pH responses could be useful in modifying media currently used for isolation of these organisms. Methanotrophic activity was induced in previously non-CH₄-consuming soils by preincubation with 5% (vol/vol) CH₄ (50,000 μl of CH₄ per liter) or by liquid enrichment with 20% CH₄. The bacteria showed pH responses typical of known methanotrophs and not typical of preexisting consumers of ambient CH₄. Furthermore, methanotrophs induced by high CH₄ levels were more readily extracted from soil than preexisting ambient CH₄ consumers were. In the alkaline soils, preexisting activity either was destroyed or resisted extraction by the procedure used. The results support the hypothesis that consumers of ambient CH₄ in soils are physiologically distinct from the known methanotrophs.