Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle

Here I describe how losses of fixed nitrogen can occur in riparian zones by the activity of denitrifying bacteria associated with methane-oxidizing (methanotrophic) bacteria. Several methanotrophs catalyze nitrogen cycle processes that can occur in riparian buffer zones, including nitrification and nitrogen fixation. Methanotrophs can produce nitric and nitrous oxides during oxidation of ammonium (nitrification), but they cannot carry out denitrification. However, there is good evidence that denitrifying bacteria can be associated with methanotrophs and can use simple carbon compounds released by the methanotrophs as substrates for the denitrification reactions and for growth. Evidence is presented that denitrifiers isolated from methanotrophic gel-stabilized oxygen gradient systems can use methanol, formaldehyde, and formate, all methane oxidation intermediates, to support their denitrification. Such denitrification associated with methanotrophs can release dinitrogen and so contributes to losses of fixed nitrogen, and may also produce the important atmospheric trace gases nitric and nitrous oxides. Data presented also show that some methanotrophs produce nitrogen oxides, including nitrite, nitric oxide, and nitrous oxide, during growth on nitrate. Assimilatory reduction of nitrate appears to be a requirement for the release of these products.     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase ( pmoA ) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum . Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.     

Molecular characterization of a microbial consortium involved in methane oxidation coupled to denitrification under micro‐aerobic conditions

Methane can be used as an alternative carbon source in biological denitrification because it is nontoxic, widely available and relatively inexpensive. A microbial consortium involved in methane oxidation coupled to denitrification (MOD) was enriched with nitrite and nitrate as electron acceptors under micro‐aerobic conditions. The 16S rRNA gene combined with pmoA phylogeny of methanotrophs and nirK phylogeny of denitrifiers were analysed to reveal the dominant microbial populations and functional microorganisms. Real‐time quantitative polymerase chain reaction results showed high numbers of methanotrophs and denitrifiers in the enriched consortium. The 16S rRNA gene clone library revealed that Methylococcaceae and Methylophilaceae were the dominant populations in the MOD ecosystem. Phylogenetic analyses of pmoA gene clone libraries indicated that all methanotrophs belonged to Methylococcaceae, a type I methanotroph employing the ribulose monophosphate pathway for methane oxidation. Methylotrophic denitrifiers of the Methylophilaceae that can utilize organic intermediates (i.e. formaldehyde, citrate and acetate) released from the methanotrophs played a vital role in aerobic denitrification. This study is the first report to confirm micro‐aerobic denitrification and to make phylogenetic and functional assignments for some members of the microbial assemblages involved in MOD.     

Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in non-amended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.     

Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing

Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.     

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.     

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.     

Revealing the community and metabolic potential of active methanotrophs by targeted metagenomics in the Zoige wetland of the Tibetan Plateau

The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy-making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using ¹³C-labelled methane to label the genomes of active methanotrophs, examine the methane oxidation potential and recover metagenome-assembled genomes (MAGs) of active methanotrophs. We found that gammaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase coding genes, and participate in methane oxidation via H₄MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA-SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming.     

Long-term effects of mineral versus organic fertilizers on activity and structure of the methanotrophic community in agricultural soils

Agricultural practices, such as mineral nitrogen fertilization, have an impact on the soil's ability to oxidize methane, but little is known about the shifts in the methanotrophic community composition associated with these practices. Therefore, the long-term effect of both mineral (NH4NO3) and organic (manure and GFT-compost) fertilizer applications on the soil methanotrophic community activity and structure were investigated. Both high and low affinity methane oxidation rates were lower in the soil treated with mineral fertilizer compared to the other soils. An enhanced nitrate concentration was observed in the mineral fertilized soil but nitrate did not show a direct affect on the high affinity methane oxidation. In contrast, the low affinity methane oxidation was slowed down by increased nitrate concentrations, which suggests a direct effect of nitrate on low affinity methane oxidation. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments specific for methanotrophs revealed a distinct community between the mineral and organic fertilized soils as extra Type I methanotrophic bands (phylotypes) became visible in the organic fertilized soils. These phylotypes were not visible in the patterns of the added organic fertilizers suggesting an indirect effect of the organic fertilizers on the methanotrophic community. Additionally, a molecular analysis was performed after the low affinity methane oxidation test. The enhanced methane concentrations used in the test enriched certain low affinity methanotrophs in the organic fertilized soils but not in the mineral fertilized soil. Supporting the molecular and functional observations, fatty acids characteristic for methanotrophs were less abundant in the soil treated with mineral fertilizer compared to the soil treated with compost. In conclusion, the function and molecular and chemical composition of the methanotrophic community are all altered in soil fertilized with mineral fertilizer.     

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.     

好氧甲烷氧化菌生态学研究进展

好氧甲烷氧化菌是一群以甲烷为碳源和能源的细菌。好氧甲烷氧化菌在自然环境中分布广泛,人类已从土壤、淡水和海洋沉积、泥炭沼泽、热泉、海水和南极环境分离到甲烷氧化菌的纯培养。好氧甲烷氧化菌可分为14个属,包括研究较为深入的隶属于变形菌门Alpha和Gamma纲的细菌,以及属于疣微菌门的极端嗜热嗜酸甲烷氧化菌。最近,好氧甲烷氧化菌还被发现存在于苔藓类植物（尤其是泥炭苔藓）共生体中,兼性营养好氧甲烷氧化菌也被发现。本文通过对好氧甲烷氧化菌的分类、生理生化特征、分子生物学检测方法以及微生物生态学中的研究成果的总结与分析,以及对甲烷氧化菌研究所面临的问题进行讨论,以期为今后进一步开展好氧甲烷氧化菌及其在碳循环中的作用研究提供参考。     

Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin

Methane vents are of significant geochemical and ecological importance. Notable progress has been made toward understanding anaerobic methane oxidation in marine sediments; however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs and distantly related to established methanotrophic clades. Water column-associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.     

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.     

Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating ¹³C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient ¹³C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of ¹³CH₄ with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of ¹³CH₄ was slow and some cross-feeding of ¹³C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of ¹³CH₄, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of ¹³C into active methanotrophs while avoiding the cross-feeding of ¹³C.     

Microbiological activities contributing to nitrogen removal with methane: effects of methyl fluoride and tungstate

When methane (CH(4)) and O(2) are present, nitrogen can be removed from wastewater that does not contain other organic carbon sources. In this study, microbial activities during methane-dependent denitrification (MDD) were investigated by adding inhibitors of methane-oxidation and denitrification. Sludge susceptible to MDD showed methane oxidation activity in the presence of CH(4) and O(2), and denitrification activity with methanol and acetate under anoxic conditions. Methyl fluoride (CH(3)F) is known to inhibit methane oxidation. When CH(3)F was present, MDD did not occur, perhaps because methane oxidation was inhibited. Tungstate (WO(4)(2-)), a known inhibitor of nitrate reduction, also lowered denitrification activity in the sludge, and partly inhibited methane oxidation. When WO(4)(2-) was added to the medium, MDD almost ceased, perhaps because of a synergic inhibitory effect on denitrification and methane oxidation. These results show that both methane oxidation and denitrification contribute to MDD.     

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.     

Large‐scale biogeography and environmental regulation of methanotrophic bacteria across boreal inland waters

Aerobic methanotrophic bacteria (methanotrophs) use methane as a source of carbon and energy, thereby mitigating net methane emissions from natural sources. Methanotrophs represent a widespread and phylogenetically complex guild, yet the biogeography of this functional group and the factors that explain the taxonomic structure of the methanotrophic assemblage are still poorly understood. Here, we used high‐throughput sequencing of the 16S rRNA gene of the bacterial community to study the methanotrophic community composition and the environmental factors that influence their distribution and relative abundance in a wide range of freshwater habitats, including lakes, streams and rivers across the boreal landscape. Within one region, soil and soil water samples were additionally taken from the surrounding watersheds in order to cover the full terrestrial–aquatic continuum. The composition of methanotrophic communities across the boreal landscape showed only a modest degree of regional differentiation but a strong structuring along the hydrologic continuum from soil to lake communities, regardless of regions. This pattern along the hydrologic continuum was mostly explained by a clear niche differentiation between type I and type II methanotrophs along environmental gradients in pH, and methane concentrations. Our results suggest very different roles of type I and type II methanotrophs within inland waters, the latter likely having a terrestrial source and reflecting passive transport and dilution along the aquatic networks, but this is an unresolved issue that requires further investigation.     

A methane-driven microbial food web in a wetland rice soil

Methane oxidation is a key process controlling methane emission from anoxic habitats into the atmosphere. Methanotrophs, responsible for aerobic methane oxidation, do not only oxidize but also assimilate methane. Once assimilated, methane carbon may be utilized by other organisms. Here we report on a microbial food web in a rice field soil driven by methane. A thin layer of water-saturated rice field soil was incubated under opposing gradients of oxygen and (13)C-labelled methane. Bacterial and eukaryotic communities incorporating methane carbon were analysed by RNA-stable isotope probing (SIP). Terminal restriction fragment length polymorphism (T-RFLP) and cloning showed that methanotrophs were the most prominent group of bacteria incorporating methane carbon. In addition, a few Myxobacteria-related sequences were obtained from the 'heavy' rRNA fraction. Denaturing gradient gel electrophoresis (DGGE) targeting 18S rRNA detected various groups of protists in the 'heavy' rRNA fraction including naked amoeba (Lobosea and Heterolobosea), ciliates (Colpodea) and flagellates (Cercozoa). Incubation of soil under different methane concentrations in air resulted in the development of distinct protozoan communities. These results suggest that methane carbon is incorporated into non-methanotrophic pro- and microeukaryotes probably via grazing, and that methane oxidation is a shaping force of the microeukaryotic community depending on methane availability.     

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.