Changes in Activity and Community Structure of Methane-Oxidizing Bacteria over the Growth Period of Rice

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.     

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.     

The Methanol Dehydrogenase Gene,mxaF, as a Functional and Phylogenetic Marker for Proteobacterial Methanotrophs in Natural Environments

The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.     

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.     

Comparing field and microcosm experiments: a case study on methano- and methylo-trophic bacteria in paddy soil

Methane-oxidising bacteria (MOB) play an important role in the reduction of methane emissions from rice agriculture. In rice fields, they are subjected to many environmental and field management parameters, which may have a significant impact on their community composition. To study this in greater detail, the community structure of methano- and methylo-trophic bacteria was investigated in a rice field in northern Italy during the summer 1999 and compared to a microcosm study described previously. We used PCR-based denaturing gradient gel electrophoresis applying 16S rDNA (9alpha and 10gamma) and mxaF (methanol-dehydrogenase) primer sets. In parallel, population size and activity of MOB were determined. This study provides the first comprehensive investigation of different compartments (bulk soil, rhizosphere, rhizoplane, and homogenate) throughout an entire rice-growing season in the field. Lower cell numbers of MOB were detected in the field compared to the microcosms, possibly due to lower CH4 concentrations in the soil pore water. In both studies, growth of MOB occurred predominantly at the root surface (rhizoplane) and in the root (homogenate), whereas cell numbers in bulk soil showed only minor changes throughout the season. Molecular analysis detected only few changes in alpha-proteobacterial methylotrophs during the season, whereas a higher variability was detected in gamma-proteobacteria. Nevertheless, the sequences of electrophoretic bands showed that the diversity in the field study and in the microcosms was comparable. Activity patterns of MOB and the population structure of methylotrophic bacteria agreed well between both studies, even though the detected quantities differed. Extrapolations of microcosm data to the field scale are thus possible, but should be used carefully when concerning quantitative changes.     

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.     

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 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.     

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 α 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.     

长期不同施肥下水稻土甲烷氧化能力及甲烷氧化菌多样性的变化

稻田内源甲烷的氧化是稻田甲烷减排的重要途径。而甲烷氧化菌是土壤中甲烷氧化的主要施动者,在长期不同施肥条件下,土壤微生物群落的演变是否影响到土壤甲烷氧化菌群落结构及其活性,进而影响到田土壤CH4向大气的实际排放强度还不清楚。为此,选择太湖地区一个长期肥料试验的稻田土壤为研究对象,分析长期不同肥料施用对土壤甲烷氧化能力的影响及其与土壤中甲烷氧化菌群落结构变化的可能关系。结果表明,长期不同的施肥措施下稻田土壤对甲烷的氧化能力产生了明显差异,伴随着土壤中甲烷氧化菌（MOBI和MOBII）的基因群落多样性的显著变化。长期单一施用氮肥为主的化肥显著降低了土壤对甲烷的氧化能力,同时显著降低了稻田土壤甲烷氧化菌的多样性和丰富度;不同施肥下甲烷氧化菌多样性的变化与土壤的甲烷氧化能力的变化趋势相一致。因此,研究显示长期不同施肥处理下甲烷氧化菌群落结构的改变可能是引起水稻土甲烷氧化能力变化的一个主要因素,有机无机配合施用可以明显降低稻田土壤甲烷的大气释放潜能。但长期不同施肥处理下甲烷氧化菌活性的变化还有待于进一步研究。     

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.     

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.     

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.     

Sulfate-reducing bacteria in rice field soil and on rice roots.

Rice plants that were grown in flooded rice soil microcosms were examined for their ability to exhibit sulfate reducing activity. Washed excised rice roots showed sulfate reduction potential when incubated in anaerobic medium indicating the presence of sulfate-reducing bacteria. Rice plants, that were incubated in a double-chamber (phylloshpere and rhizosphere separated), showed potential sulfate reduction rates in the anoxic rhizosphere compartment. These rates decreased when oxygen was allowed to penetrate through the aerenchyma system of the plants into the anoxic root compartment, indicating that sulfate reducers on the roots were partially inhibited by oxygen or that sulfate was regenerated by oxidation of reduced S-compounds. The potential activity of sulfate reducers on rice roots was consistent with MPN enumerations showing that H2-utilizing sulfate-reducing bacteria were present in high numbers on the rhizoplane (4.1 x 10(7) g-1 root fresh weight) and in the adjacent rhizosperic soil (2.5 x 10(7) g-1 soil dry weight). Acetate-oxidizing sulfate reducers, on the other hand, showed highest numbers in the unplanted bulk soil (1.9 x 10(6) g-1 soil dry weight). Two sulfate reducing bacteria were isolated from the highest dilutions of the MPN series and were characterized physiologically and phylogenetically. Strain F1-7b which was isolated from the rhizoplane with H2 as electron donor was related to subgroup II of the family Desulfovibrionaceae. Strain EZ-2C2, isolated from the rhizoplane on acetate, grouped together with Desulforhabdus sp. and Syntrophobacter wolinii. Other strains of sulfate-reducing bacteria originated from bulk soil of rice soil microcosms and were isolated using different electron donors. From these isolates, strains R-AcA1, R-IbutA1, R-PimA1 and R-AcetonA170 were Gram-positive bacteria which were affiliated with the genus Desulfotomaculum. The other isolates were members of subgroup II of the Desulfovibrionaceae (R-SucA1 and R-LacA1), were related to Desulforhabdus sp. (strain BKA11), Desulfobulbus (R-PropA1), or culstered between Desulfobotulus sapovorans and Desulfosarcina variabilis (R-ButA1 and R-CaprA1).     

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.     

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.     

Response of methanotrophic activity and community structure to temperature changes in a diffusive CH4/O2 counter gradient in an unsaturated porous medium

Microbial methane oxidation is a key process in the global methane cycle. In the context of global warming, it is important to understand the responses of the methane-oxidizing microbial community to temperature changes in terms of community structure and activity. We studied microbial methane oxidation in a laboratory-column system in which a diffusive CH₄/O₂ counter gradient was maintained in an unsaturated porous medium at temperatures between 4 and 20 °C. Methane oxidation was highly efficient at all temperatures, as on average 99 ± 0.5% of methane supplied to the system was oxidized. The methanotrophic community that established in the model system after initial inoculation appeared to be able to adapt quickly to different temperatures, as methane emissions remained low even after the system was subjected to abrupt temperature changes. FISH showed that Type I as well as Type II methanotrophs were probably responsible for the observed activity in the column system, with a dominance of Type I methanotrophs. Cloning and sequencing suggested that Type I methanotrophs were represented by the genus Methylobacter while Type II were represented by Methylocystis . The results suggest that in an unsaturated system with diffusive substrate supply, direct effects of temperature on apparent methanotrophic activity and community may be of minor importance. However, this remains to be verified in the field.     

Comparison of Aerobic Methanotrophic Communities in Littoral and Profundal Sediments of Lake Constance by a Molecular Approach

Analysis of pmoA and 16S rRNA gene clone libraries of methanotrophic bacteria in Lake Constance revealed an overall dominance of type I methanotrophs in both littoral and profundal sediments. The sediments exhibited minor differences in their methanotrophic community structures. Type X methanotrophs made up a significant part of the clone libraries only in the profundal sediment and were also found only there as a prominent peak by T-RFLP analyses.     

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 O(2) 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 microM). 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.