Molecular Analyses of Novel Methanotrophic Communities in Forest Soil That Oxidize Atmospheric Methane

Forest and other upland soils are important sinks for atmospheric CH₄, consuming 20 to 60 Tg of CH₄ per year. Consumption of atmospheric CH₄ by soil is a microbiological process. However, little is known about the methanotrophic bacterial community in forest soils. We measured vertical profiles of atmospheric CH₄ 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 α 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 CH₄ in situ and in vitro at all times. In winter, atmospheric CH₄ 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 μg 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 CH₄ 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 CH₄ consumption rates also exhibited more bands of pmoA in DGGE, indicating that high CH₄ 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 CH₄ consumption.     

Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase

Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.     

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.     

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.     

Effects of O2 and CH4 on presence and activity of the indigenous methanotrophic community in rice field soil

The activity and distribution of methanotrophs in soil depend on the availability of CH4 and O2. Therefore, we investigated the activity and structure of the methanotrophic community in rice field soil under four factorial combinations of high and low CH4 and O2 concentrations. The methanotrophic population structure was resolved by denaturant gradient gel electrophoresis (DGGE) with different PCR primer sets targeting the 16S rRNA gene, and two functional genes coding for key enzymes in methanotrophs, i.e. the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Changes in the biomass of type I and II methanotrophic bacteria in the rice soil were determined by analysis of phospholipid-ester-linked fatty acid (PLFA) biomarkers. The relative contribution of type I and II methanotrophs to the measured methane oxidation activity was determined by labelling of soil samples with 14CH4 followed by analysis of [14C]-PLFAs. CH4 oxidation was repressed by high O2 (20.5%), and enhanced by low O2 (1%). Depending on the CH4 and O2 mixing ratios, different methanotrophic communities developed with a higher diversity at low than at high CH4 concentration as revealed by PCR-DGGE. However, a prevalence of type I or II populations was not detected. The [14C]-PLFA fingerprints, on the other hand, revealed that CH4 oxidation activity was dominated by type I methanotrophs in incubations with low CH4 mixing ratios (1000 p.p.m.v.) and during initiation of CH4 consumption regardless of O2 or CH4 mixing ratio. At high methane mixing ratios (10 000 p.p.m.v.), type I and II methanotrophs contributed equally to the measured CH4 metabolism. Collectively, type I methanotrophs responded fast and with pronounced shifts in population structure and dominated the activity under all four gas mixtures. Type II methanotrophs, on the other hand, although apparently more abundant, always present and showing a largely stable population structure, became active later and contributed to CH4 oxidation activity mainly under high CH4 mixing ratios.     

Vertical distribution of the methanotrophic community after drainage of rice field soil

Anoxic soils, such as flooded rice fields, are major sources of the greenhouse gas CH(4) while oxic upland soils are major sinks of atmospheric CH(4). Nevertheless, CH(4) is also consumed in rice fields where up to 90% of the produced CH(4) is oxidized in a narrow oxic zone around the rice roots and in the soil surface layer before it escapes into the atmosphere. After 1 day drainage of rice field soil, CH(4) oxidation was detected in the top 2-mm soil layers, but after 8 days drainage the zone of CH(4) oxidation extended to 8 mm depth. Simultaneously, the potential for CH(4) production decreased, but some production was still detectable after 8 days drainage throughout the soil profile. The vertical distribution of the methanotrophic community was also monitored after 1 and 8 days drainage using denaturing gradient gel electrophoresis after PCR amplification with primer sets targeting two regions on the 16S rRNA gene that are relatively specific for methylotrophic alpha- and gamma-Proteobacteria, and targeting two functional genes encoding subunits of key enzymes in all methanotrophs, i.e. the genes for the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Drainage stimulated the methanotrophic community. Eight days after drainage, new methanotrophic populations appeared and a distinct methanotrophic community developed. The population structure of type I and II methanotrophs was differently affected by drainage. Type II methanotrophs (alpha-Proteobacteria) were present throughout the soil core directly after drainage (1 day), and the community composition remained largely unchanged with depth. Only two new type II populations appeared after 8 days of drainage. Drainage had a more pronounced impact on the type I methanotrophic community (gamma-Proteobacteria). Type I populations were not or only weakly detected 1 day after drainage. However, after 8 days of drainage, a large diversity of type I methanotrophs were detected, altough they were not evenly distributed throughout the soil core but dominated at different depths. A distinct type I community structure had developed within each soil section between 0 and 20 mm soil depth, indicating the widening of suitable habitats for methanotrophs in the rice field soil within 1 week of drainage.     

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.     

Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays

Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.     

Regulation of microbial methane production and oxidation by intermittent drainage in rice field soil

Soil drainage is one of the most promising approaches to mitigate methane (CH(4) ) emission from paddy fields. The microbial mechanism for the drainage effect on CH(4) emission, however, remains poorly understood. In the present study, we determined the effect of short (four drainages of 5-6 days each) and long drainage cycles (two drainages of 10-11 days each) on CH(4) emission and analyzed the response of the structure and abundance of methanogens and methanotrophs in a Chinese rice field soil at the DNA level. Rice biomass production was similar between drainage and the practice of continuous flooding. The rate of CH(4) emission, however, was reduced by 59% and 85% for the long and short drainage cycles, respectively. Quantitative (real-time) PCR analysis revealed that the total abundance of archaeal populations decreased by 40% after multiple drainages, indicating the inhibitory effects on methanogen growth. The structure of the methanogen community as determined by terminal restriction fragment length polymorphism analysis, however, remained unaffected by drainages, although it varied among rhizosphere, bulk and surface soils. Quantitative PCR analysis of the methanotrophic functional pmoA genes revealed that the total abundance of methanotrophs in rhizosphere soil increased two to three times after soil drainages, indicating a stimulation of methanotroph growth. The CH(4) oxidation potential in the rhizosphere soil also increased significantly. Furthermore, drainages caused a shift of the methanotrophic community, with a significantly increase of type II methanotrophic bacteria in the rhizosphere and surface soil. Thus, both inhibition of methanogens and stimulation of methanotrophs were partly responsible for the reduction of CH(4) emissions. The methanotroph community, however, appeared to react more sensitively to soil drainage compared with the methanogen community.     

Shifts in identity and activity of methanotrophs in arctic lake sediments in response to temperature changes

Methane (CH(4)) flux to the atmosphere is mitigated via microbial CH(4) oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH(4) emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH(4) oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH(4) oxidation potential of 37.5 μmol g(-1) day(-1) in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR of pmoA and of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in (13)C-labeled DNA obtained by SIP demonstrated that the type I methanotrophs Methylobacter, Methylomonas, and Methylosoma dominated carbon acquisition from CH(4) in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH(4), especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship (R = 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.     

CO2浓度增加对长白山森林土壤甲烷氧化影响

大气CO2浓度升高可能对森林土壤的甲烷(CH4)氧化速率产生影响.本文采用开顶箱技术,对连续6年高浓度CO2(500 μmol·mol-1)处理的长白山森林典型树种蒙古栎树下土壤CH4氧化速率进行研究,并利用CH4氧化菌的16S rRNA特异性引物以及CH4单加氧酶功能基因引物分析了土壤中CH4氧化菌的群落结构与数量.结果表明:CO2浓度增高后,生长季土壤甲烷氧化量与对照和裸地相比分别降低了4％和22％;基于16S rRNA特异性引物的DGGE分析表明,CO2浓度增高导致两类甲烷氧化菌的多样性指数降低;CO2浓度增高对土壤中Ⅰ类甲烷氧化菌数量无显著影响,而使土壤中Ⅱ类甲烷氧化菌数量显著减少,功能基因pmoA拷贝数与对照和裸地相比分别降低了15％和46％.CO2浓度增高导致森林土壤甲烷氧化菌数量与活性降低,土壤含水量的增加可能是导致这一现象的主要原因.     

Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in different soils.

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1omega8c (up to 9.0% of the total (14)C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.     

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.     

CO2浓度增加对长白山森林土壤甲烷氧化影响

大气CO2浓度升高可能对森林土壤的甲烷(CH4)氧化速率产生影响.本文采用开顶箱技术,对连续6年高浓度CO2（500 μmol·mol-1）处理的长白山森林典型树种蒙古栎树下土壤CH4氧化速率进行研究,并利用CH4氧化菌的16S rRNA特异性引物以及CH4单加氧酶功能基因引物分析了土壤中CH4氧化菌的群落结构与数量.结果表明： CO2浓度增高后,生长季土壤甲烷氧化量与对照和裸地相比分别降低了4%和22%;基于16S rRNA特异性引物的DGGE分析表明,CO2浓度增高导致两类甲烷氧化菌的多样性指数降低;CO2浓度增高对土壤中Ⅰ类甲烷氧化菌数量无显著影响,而使土壤中Ⅱ类甲烷氧化菌数量显著减少,功能基因pmoA拷贝数与对照和裸地相比分别降低了15%和46%.CO2浓度增高导致森林土壤甲烷氧化菌数量与活性降低,土壤含水量的增加可能是导致这一现象的主要原因.     

Identification of the functionally active methanotroph population in a peat soil microcosm by stable-isotope probing

The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing. "Heavy" (13)C-labeled DNA, produced after microbial growth on (13)CH(4), was separated from naturally abundant (12)C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH(4) oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating that these methanotrophs were active in peat exposed to 8% (vol/vol) CH(4). The (13)C-DNA libraries also contained clones that were related to beta-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH(4) cycling in this soil.     

Responses of oxidation rate and microbial communities to methane in simulated landfill cover soil microcosms

CH4 oxidation capacities and microbial community structures developed in response to the presence of CH4 were investigated in two types of landfill cover soil microcosms, waste soil (fine material in stabilized waste) and clay soil. CH4 emission fluxes were lower in the waste soil cover over the course of the experiment. After exposure to CH4 flow for 120 days, the waste soil developed CH4 oxidation capacity from 0.53 to 11.25-13.48micromol CH4gd.w.(-1)h(-1), which was ten times higher than the clay soil. The topsoils of the two soil covers were observed dried and inhibited CH4 oxidation. The maximum CH4 oxidation rate occurred at the depth of 10-20cm in the waste soil cover (the middle layer), whereas it took place mainly at the depth of 20-30cm in the clay soil cover (the bottom layer). The amounts of the phospholipid fatty acid (PLFA) biomarks 16:1omega8c and 18:1omega8c for type I and II methanotrophs, respectively, showed that type I methanotrophic bacteria predominated in the clay soil, while the type II methanotrophic bacteria were abundant in the waste soil, and the highest population in the middle layer. The results also indicated that a greater active methanotrophic community was developed in the waste soil relative to the clay soil.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.