Diversity of the particulate methane monooxygenase gene in methanotrophic samples from different rice field soils in China and the Philippines

Methanotrophic bacteria play a crucial role in regulating the emission of CH4 from rice fields into the atmosphere. We investigated the CH4 oxidation activity together with the diversity of methanotrophic bacteria in ten rice field soils from different geographic locations. Upon incubation of aerated soil slurries under 7% CH4, rates of CH4 oxidation increased after a lag phase of 1-4 days and reached values of 3-10 micromol d(-1) g-dw(-1) soil. The methanotrophic community was assayed by retrieval of the pmoA gene which encodes the a subunit of the particulate methane monooxygenase. After extraction of DNA from actively CH4-oxidizing soil samples and PCR-amplification of the pmoA, the community was analyzed by Denaturant Gradient Gel Electrophoresis (DGGE) and Terminal Restriction Fragment Length Polymorphism (T-RFLP). DGGE bands were excised, the pmoA re-amplified, sequenced and the encoded amino acid sequence comparatively analyzed by phylogenetic treeing. The analyses allowed the detection of pmoA sequences related to the following methanotrophic genera: the type-I methanotrophs Methylobacter, Methylomicrobium, Methylococcus and Methylocaldum, and the type-II methanotrophs Methylocystis and Methylosinus. T-RFLP analysis detected a similar diversity, but type-II pmoA more frequently than DGGE. All soils but one contained type-II in addition to type-I methanotrophs. Type-I Methylomonas was not detected at all. Different combinations of methanotrophic genera were detected in the different soils. However, there was no obvious geographic pattern of the distribution of methanotrophs.     

Effect of temperature on composition of the methanotrophic community in rice field and forest soil

Temperature change affects methane consumption in soil. However, there is no information on possible temperature control of methanotrophic bacterial populations. Therefore, we studied CH(4) consumption and populations of methanotrophs in an upland forest soil and a rice field soil incubated at different temperatures between 5 and 45 degrees C for up to 40 days. Potential methane consumption was measured at 4% CH(4). The temporal progress of CH(4) consumption indicated growth of methanotrophs. Both soils showed maximum CH(4) consumption at 25-35 degrees C, but no activity at >40 degrees C. In forest soil CH(4) was also consumed at 5 degrees C, but in rice soil only at 15 degrees C. Methanotroph populations were assessed by terminal restriction fragment length polymorphism (T-RFLP) targeting particulate methane monooxygenase (pmoA) genes. Eight T-RFs with relative abundance >1% were retrieved from both forest and rice soil. The individual T-RFs were tentatively assigned to different methanotrophic populations (e.g. Methylococcus/Methylocaldum, Methylomicrobium, Methylobacter, Methylocystis/Methylosinus) according to published sequence data. Two T-RFs were assigned to ammonium monooxygenase (amoA) gene sequences. Statistical tests showed that temperature affected the relative abundance of most T-RFs. Furthermore, the relative abundance of individual T-RFs differed between the two soils, and also exhibited different temperature dependence. We conclude that temperature can be an important factor regulating the community composition of methanotrophs in soil.     

Methanotrophic communities in the soils of Russian northern taiga and subarctic tundra

The PCR analysis of DNA extracted from soil samples taken in Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved alpha-subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the alpha-subunit of particulate methane monooxygenase, and the mxaF gene encoding the alpha-subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

The active methanotrophic community in hydromorphic soils changes in response to changing methane concentration

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.     

Biochemical and molecular characterization of methanotrophs in soil from a pristine New Zealand beech forest

Methane (CH4) oxidation and the methanotrophic community structure of a pristine New Zealand beech forest were investigated using biochemical and molecular methods. Phospholipid-fatty acid-stable-isotope probing (PLFA-SIP) was used to identify the active population of methanotrophs in soil beneath the forest floor, while terminal-restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the pmoA gene were used to characterize the methanotrophic community. PLFA-SIP suggested that type II methanotrophs were the predominant active group. T-RFLP and cloning and sequencing of the pmoA genes revealed that the methanotrophic community was diverse, and a slightly higher number of type II methanotrophs were detected in the clone library. Most of the clones from type II methanotrophs were related to uncultured pmoA genes obtained directly from environmental samples, while clones from type I were distantly related to Methylococcus capsulatus. A combined data analysis suggested that the type II methanotrophs may be mainly responsible for atmospheric CH4 consumption. Further sequence analysis suggested that most of the methanotrophs detected shared their phylogeny with methanotrophs reported from soils in the Northern Hemisphere. However, some of the pmoA sequences obtained from this forest had comparatively low similarity (<97%) to known sequences available in public databases, suggesting that they may belong to novel groups of methanotrophic bacteria. Different methods of methanotrophic community analysis were also compared, and it is suggested that a combination of molecular methods with PLFA-SIP can address several shortcomings of stable isotope probing.     

Effect of afforestation and reforestation of pastures on the activity and population dynamics of methanotrophic bacteria

We investigated the effect of afforestation and reforestation of pastures on methane oxidation and the methanotrophic communities in soils from three different New Zealand sites. Methane oxidation was measured in soils from two pine (Pinus radiata) forests and one shrubland (mainly Kunzea ericoides var. ericoides) and three adjacent permanent pastures. The methane oxidation rate was consistently higher in the pine forest or shrubland soils than in the adjacent pasture soils. A combination of phospholipid fatty acid (PLFA) and stable isotope probing (SIP) analyses of these soils revealed that different methanotrophic communities were active in soils under the different vegetations. The C18 PLFAs (signature of type II methanotrophs) predominated under pine and shrublands, and C16 PLFAs (type I methanotrophs) predominated under pastures. Analysis of the methanotrophs by molecular methods revealed further differences in methanotrophic community structure under the different vegetation types. Cloning and sequencing and terminal-restriction fragment length polymorphism analysis of the particulate methane oxygenase gene (pmoA) from different samples confirmed the PLFA-SIP results that methanotrophic bacteria related to type II methanotrophs were dominant in pine forest and shrubland, and type I methanotrophs (related to Methylococcus capsulatus) were dominant in all pasture soils. We report that afforestation and reforestation of pastures caused changes in methane oxidation by altering the community structure of methanotrophic bacteria in these soils.     

Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the alpha subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.     

Molecular analyses of novel methanotrophic communities in forest soil that oxidize atmospheric methane

Forest and other upland soils are important sinks for atmospheric CH(4), consuming 20 to 60 Tg of CH(4) per year. Consumption of atmospheric CH(4) by soil is a microbiological process. However, little is known about the methanotrophic bacterial community in forest soils. We measured vertical profiles of atmospheric CH(4) oxidation rates in a German forest soil and characterized the methanotrophic populations by PCR and denaturing gradient gel electrophoresis (DGGE) with primer sets targeting the pmoA gene, coding for the alpha subunit of the particulate methane monooxygenase, and the small-subunit rRNA gene (SSU rDNA) of all life. The forest soil was a sink for atmospheric CH(4) in situ and in vitro at all times. In winter, atmospheric CH(4) was oxidized in a well-defined subsurface soil layer (6 to 14 cm deep), whereas in summer, the complete soil core was active (0 cm to 26 cm deep). The content of total extractable DNA was about 10-fold higher in summer than in winter. It decreased with soil depth (0 to 28 cm deep) from about 40 to 1 microg DNA per g (dry weight) of soil. The PCR product concentration of SSU rDNA of all life was constant both in winter and in summer. However, the PCR product concentration of pmoA changed with depth and season. pmoA was detected only in soil layers with active CH(4) oxidation, i.e., 6 to 16 cm deep in winter and throughout the soil core in summer. The same methanotrophic populations were present in winter and summer. Layers with high CH(4) consumption rates also exhibited more bands of pmoA in DGGE, indicating that high CH(4) oxidation activity was positively correlated with the number of methanotrophic populations present. The pmoA sequences derived from excised DGGE bands were only distantly related to those of known methanotrophs, indicating the existence of unknown methanotrophs involved in atmospheric CH(4) consumption.     

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus / Methylocystis , type I methanotrophs related to Methylobacter / Methylosoma and Methylococcus , and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using ¹³CH₄ were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella , which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium , were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.     

A novel pmoA lineage represented by the acidophilic methanotrophic bacterium Methylocapsa acidophila B2

A fragment of the functional gene pmoA, which encodes the active-site polypeptide of particulate methane monooxygenase (pMMO), was PCR-amplified from DNA of the recently described acidophilic methanotrophic bacterium Methylocapsa acidiphila [corrected] B2. This methanotroph was isolated from an acidic Sphagnum peat bog and possesses a novel type III arrangement of intracytoplasmic membranes. Comparative sequence analysis revealed that the inferred peptide sequence of pmoA of Methylocapsa acidiphila [corrected] B2 belongs to a novel PmoA lineage. This lineage was only distantly related to the PmoA sequence cluster of type II methanotrophs, with identity values between 69.5% and 72%. The identity values between the PmoA of Methylocapsa acidiphila [corrected] B2 and PmoA sequences of type I methanotrophs ranged from 55.5 to 68%. However, the PmoA of this acidophilic methanotroph was more closely affiliated with the inferred peptide sequences of pmoA clones retrieved from various acidic upland soils showing atmospheric methane consumption. The PmoA identity values with these clones were 79.5-81%. Nonetheless, the apparent affinity for methane exhibited by Methylocapsa acidiphila [corrected] B2 was 1-2 microM, which is similar to values measured in other methanotrophic bacteria. This finding suggests that the pMMO of Methylocapsa acidiphila [corrected] B2 is not a novel enzyme specialized to have a high affinity for methane and that apparent "high-affinity" methane uptake is either the result of particular culture conditions or is performed by another pMMO type.     

Diversity and activity of methanotrophic bacteria in different upland soils

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the "forest sequence cluster" (USC alpha), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel "upland soil cluster gamma" (USC gamma) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with (13)CH(4) at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of (13)C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC gamma sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC alpha sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.     

Molecular characterization of methanotrophic communities in forest soils that consume atmospheric methane

Methanotroph abundance was analyzed in control and long-term nitrogen-amended pine and hardwood soils using rRNA-targeted quantitative hybridization. Family-specific 16S rRNA and pmoA/amoA genes were analyzed via PCR-directed assays to elucidate methanotrophic bacteria inhabiting soils undergoing atmospheric methane consumption. Quantitative hybridizations suggested methanotrophs related to the family Methylocystaceae were one order of magnitude more abundant than Methyloccocaceae and more sensitive to nitrogen-addition in pine soils. 16S rRNA gene phylotypes related to known Methylocystaceae and acidophilic methanotrophs and pmoA/amoA gene sequences, including three related to the upland soil cluster Alphaproteobacteria (USCalpha) group, were detected across different treatments and soil depths. Our results suggest that methanotrophic members of the Methylocystaceae and Beijerinckiaceae may be the candidates for soil atmospheric methane consumption.     

Activity and diversity of methanotrophs in the soil–water interface and rhizospheric soil from a flooded temperate rice field

Aims:  To combine molecular and cultivation techniques to characterize the methanotrophic community in the soil–water interface (SWI) and rhizospheric soil from flooded rice fields in Uruguay, a temperate region in South America.
Methods and Results:  A novel type I, related to the genus Methylococcus , and three type II methanotrophs were isolated from the highest positive dilution steps from the most probable number (MPN) counts. Potential methane oxidation activities measured in slurried samples were higher in the rhizospheric soil compared to the SWI and were stimulated by N-fertilization. PmoA (particulate methane monooxygenase) clone libraries were constructed for both rice microsites. SWI clones clustered in six groups related to cultivated and uncultivated members from different ecosystems of the genera Methylobacter , Methylomonas , Methylococcus and a novel type I sublineage while cultivation and T-RFLP (terminal restriction fragment length polymorphism) analysis confirmed the presence of type II methanotrophs.
Conclusions:  Cultivation techniques, cloning analysis and T-RFLP fingerprinting of the pmoA gene revealed a diverse methanotrophic community in the rice rhizospheric soil and SWI.
Significance and Impact of the Study:  This study reports, for the first time, the analysis of the methanotrophic diversity in rice SWI and this diversity may be exploited in reducing methane emissions.     

Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing

A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.     

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Study of nucleotide sequences of nifH genes in methanotrophic bacteria

Using a previously developed primer system, nifH gene fragments 450 nucleotides long were amplified, cloned, and sequenced for representatives of nitrogen-fixing methanotrophic bacteria of the genera Methylococcus, Methylocystis and Methylosinus. Fragments of nifH genes were also detected and sequenced in representatives of the genera Methylomonas and Methylobacter, which were not considered diazotrophs until recently. Phylogenetic analysis revealed remoteness of nifH genes sequences of methanotroph types I and II. At the same time, close relationship was found between nifH of type I methanotrophs and representatives of gamma-proteobacteria and between nifH genes of type II methanotrophs and representatives of alpha-proteobacteria. The results obtained in this study are in good accordance with the data of phylogenetic analysis based on 16S rRNA sequence comparison with the only exception of Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes of Methylocystis and Methylosinus representatives. Our findings extend the database of primary sequences of nifH genes and allow the contribution of methanotrophs to the process of nitrogen fixation to be estimated.     

Differential effects of nitrogenous fertilizers on methane-consuming microbes in rice field and forest soils

The impact of environmental perturbation (e.g., nitrogenous fertilizers) on the dynamics of methane fluxes from soils and wetland systems is poorly understood. Results of fertilizer studies are often contradictory, even within similar ecosystems. In the present study the hypothesis of whether these contradictory results may be explained by the composition of the methane-consuming microbial community and hence whether methanotrophic diversity affects methane fluxes was investigated. To this end, rice field and forest soils were incubated in microcosms and supplemented with different nitrogenous fertilizers and methane concentrations. By labeling the methane with 13C, diversity and function could be coupled by analyses of phospholipid-derived fatty acids (PLFA) extracted from the soils at different time points during incubation. In both rice field and forest soils, the activity as well as the growth rate of methane-consuming bacteria was affected differentially. For type I methanotrophs, fertilizer application stimulated the consumption of methane and the subsequent growth, while type II methanotrophs were generally inhibited. Terminal restriction fragment length polymorphism analyses of the pmoA gene supported the PLFA results. Multivariate analyses of stable-isotope-probing PLFA profiles indicated that in forest and rice field soils, Methylocystis (type II) species were affected by fertilization. The type I methanotrophs active in forest soils (Methylomicrobium/Methylosarcina related) differed from the active species in rice field soils (Methylobacter/Methylomonas related). Our results provide a case example showing that microbial community structure indeed matters, especially when assessing and predicting the impact of environmental change on biodiversity loss and ecosystem functioning.     

Isolation and properties of new strains of obligate methanotrophs

New strains of obligate methanotrophic bacteria which assimilate only methane or methanol as the source of carbon and energy have been isolated. According to their morphology, ultrastructure, cultural and physiologo-biochemical characteristics, the bacteria were classed as Methylobacter vinelandii, Methylobacter bovis, Methylobacter chroococcum and Mehylosinus sporium. A new species Methylocystis echinoides sp. nov. is described; it differs from other methanotrophs in certain morphological and physiological properties. The subspecies Methylosinus trichosporium var. methanolicum is recommended to be termed as Methylocytis methanolicus sp. nov.