Widespread soil bacterium that oxidizes atmospheric methane.

The global atmospheric level of methane (CH4), the second most important greenhouse gas, is currently increasing by ∼10 million tons per year. Microbial oxidation in unsaturated soils is the only known biological process that removes CH4 from the atmosphere, but so far, bacteria that can grow on atmospheric CH4 have eluded all cultivation efforts. In this study, we have isolated a pure culture of a bacterium, strain MG08 that grows on air at atmospheric concentrations of CH4 [1.86 parts per million volume (p.p.m.v.)]. This organism, named Methylocapsa gorgona, is globally distributed in soils and closely related to uncultured members of the upland soil cluster α. CH4 oxidation experiments and 13C-single cell isotope analyses demonstrated that it oxidizes atmospheric CH4 aerobically and assimilates carbon from both CH4 and CO2 Its estimated specific affinity for CH4 (a0s) is the highest for any cultivated methanotroph. However, growth on ambient air was also confirmed for Methylocapsa acidiphila and Methylocapsa aurea, close relatives with a lower specific affinity for CH4, suggesting that the ability to utilize atmospheric CH4 for growth is more widespread than previously believed. The closed genome of M. gorgona MG08 encodes a single particulate methane monooxygenase, the serine cycle for assimilation of carbon from CH4 and CO2, and CO2 fixation via the recently postulated reductive glycine pathway. It also fixes dinitrogen and expresses the genes for a high-affinity hydrogenase and carbon monoxide dehydrogenase, suggesting that atmospheric CH4 oxidizers harvest additional energy from oxidation of the atmospheric trace gases carbon monoxide (0.2 p.p.m.v.) and hydrogen (0.5 p.p.m.v.).

Physiological basis for atmospheric methane oxidation and methanotrophic growth on air.

Atmospheric methane oxidizing bacteria (atmMOB) constitute the sole biological sink for atmospheric methane. Still, the physiological basis allowing atmMOB to grow on air is not well understood. Here we assess the ability and strategies of seven methanotrophic species to grow with air as sole energy, carbon, and nitrogen source. Four species, including three outside the canonical atmMOB group USCα, enduringly oxidized atmospheric methane, carbon monoxide, and hydrogen during 12 months of growth on air. These four species exhibited distinct substrate preferences implying the existence of multiple metabolic strategies to grow on air. The estimated energy yields of the atmMOB were substantially lower than previously assumed necessary for cellular maintenance in atmMOB and other aerobic microorganisms. Moreover, the atmMOB also covered their nitrogen requirements from air. During growth on air, the atmMOB decreased investments in biosynthesis while increasing investments in trace gas oxidation. Furthermore, we confirm that a high apparent specific affinity for methane is a key characteristic of atmMOB. Our work shows that atmMOB grow on the trace concentrations of methane, carbon monoxide, and hydrogen present in air and outlines the metabolic strategies that enable atmMOB to mitigate greenhouse gases.

Thermal acclimation of methanotrophs from the genus Methylobacter.

Methanotrophs oxidize most of the methane (CH4) produced in natural and anthropogenic ecosystems. Often living close to soil surfaces, these microorganisms must frequently adjust to temperature change. While many environmental studies have addressed temperature effects on CH4 oxidation and methanotrophic communities, there is little knowledge about the physiological adjustments that underlie these effects. We have studied thermal acclimation in Methylobacter, a widespread, abundant, and environmentally important methanotrophic genus. Comparisons of growth and CH4 oxidation kinetics at different temperatures in three members of the genus demonstrate that temperature has a strong influence on how much CH4 is consumed to support growth at different CH4 concentrations. However, the temperature effect varies considerably between species, suggesting that how a methanotrophic community is composed influences the temperature effect on CH4 uptake. To understand thermal acclimation mechanisms widely we carried out a transcriptomics experiment with Methylobacter tundripaludum SV96T. We observed, at different temperatures, how varying abundances of transcripts for glycogen and protein biosynthesis relate to cellular glycogen and ribosome concentrations. Our data also demonstrated transcriptional adjustment of CH4 oxidation, oxidative phosphorylation, membrane fatty acid saturation, cell wall composition, and exopolysaccharides between temperatures. In addition, we observed differences in M. tundripaludum SV96T cell sizes at different temperatures. We conclude that thermal acclimation in Methylobacter results from transcriptional adjustment of central metabolism, protein biosynthesis, cell walls and storage. Acclimation leads to large shifts in CH4 consumption and growth efficiency, but with major differences between species. Thus, our study demonstrates that physiological adjustments to temperature change can substantially influence environmental CH4 uptake rates and that consideration of methanotroph physiology might be vital for accurate predictions of warming effects on CH4 emissions.

Batch Experiments Demonstrating a Two-Stage Bacterial Process Coupling Methanotrophic and Heterotrophic Bacteria for 1-Alkene Production From Methane.

Methane (CH4) is a sustainable carbon feedstock for value-added chemical production in aerobic CH4-oxidizing bacteria (methanotrophs). Under substrate-limited (e.g., oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as feedstocks for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Type I and Type II methanotrophs, Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8-7.0% of consumed carbon from CH4 (C-CH4), while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7-1.8% of consumed C-CH4. For the first time, this study demonstrated the use of organic acid-rich spent media of methanotrophs cultivating engineered Acinetobacter baylyi ADP1 'tesA-undA cells for 1-alkene production. The highest yield of 1-undecene was obtained from the spent medium of M. tundripaludum SV96 at 68.9 ± 11.6 µmol mol Csubstrate -1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.

Genome sequence of the Arctic methanotroph Methylobacter tundripaludum SV96.

Methylobacter tundripaludum SV96(T) (ATCC BAA-1195) is a psychrotolerant aerobic methane-oxidizing gammaproteobacterium (Methylococcales, Methylococcaceae) living in High Arctic wetland soil. The strain was isolated from soil harvested in July 1996 close to the settlement Ny-Ålesund, Svalbard, Norway (78°56'N, 11°53'E), and described as a novel species in 2006. The genome includes pmo and pxm operons encoding copper membrane monooxygenases (Cu-MMOs), genes required for nitrogen fixation, and the nirS gene implicated in dissimilatory nitrite reduction to NO but no identifiable inventory for further processing of nitrogen oxides. These genome data provide the basis to investigate M. tundripaludum SV96, identified as a major player in the biogeochemistry of Arctic environments.

Simultaneous Oxidation of Atmospheric Methane, Carbon Monoxide and Hydrogen for Bacterial Growth.

The second largest sink for atmospheric methane (CH4) is atmospheric methane oxidizing-bacteria (atmMOB). How atmMOB are able to sustain life on the low CH4 concentrations in air is unknown. Here, we show that during growth, with air as its only source for energy and carbon, the recently isolated atmospheric methane-oxidizer Methylocapsa gorgona MG08 (USCα) oxidizes three atmospheric energy sources: CH4, carbon monoxide (CO), and hydrogen (H2) to support growth. The cell-specific CH4 oxidation rate of M. gorgona MG08 was estimated at ~0.7 × 10-18 mol cell-1 h-1, which, together with the oxidation of CO and H2, supplies 0.38 kJ Cmol-1 h-1 during growth in air. This is seven times lower than previously assumed necessary to support bacterial maintenance. We conclude that atmospheric methane-oxidation is supported by a metabolic flexibility that enables the simultaneous harvest of CH4, H2 and CO from air, but the key characteristic of atmospheric CH4 oxidizing bacteria might be very low energy requirements.

Impacts of inter- and intralaboratory variations on the reproducibility of microbial community analyses.

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.

Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system.

Abstract A new method for isolation of methane oxidising bacteria (methanotrophs) is presented. Soil samples from a wetland area and a landfill were plated on polycarbonate membranes, which were incubated in a methane-air atmosphere using a non-sterile soil suspension as the medium. The membrane acted as a permeable growth support. The membrane method resulted in selective growth conditions, which allowed isolation of methane oxidising bacteria. The method resulted in isolation of both type I and type II methanotrophs from natural wetland and landfill soils. The isolates obtained from the landfill were dominated by type II methanotrophs and included several isolates carrying the gene for soluble methane monooxygenase (sMMO). Repetitive element sequence-based PCR fingerprinting documented genotypic diversity at the strain level. The presented method is a promising tool for easy and rapid isolation of different indigenous methanotrophs from an environment of interest.

The active methanotrophic community in a wetland from the High Arctic.

The dominant terminal process of carbon mineralization in most freshwater wetlands is methanogenesis. With methane being an important greenhouse gas, the predicted warming of the Arctic may provide a positive feedback. However, the amount of methane released to the atmosphere may be controlled by the activity of methane-oxidizing bacteria (methanotrophs) living in the oxic surface layer of wetlands. Previously, methanotrophs have been isolated and identified by genetic profiling in High Arctic wetlands showing the presence of only a few genotypes. Two isolates from Solvatnet (Ny-Ålesund, Spitsbergen; 79°N) are available: Methylobacter tundripaludum (type I) and Methylocystis rosea (type II), raising the question whether the low diversity is a cultivation effect. We have revisited Solvatnet applying stable isotope probing (SIP) with (13) C-labelled methane. 16S rRNA profiling revealed active type I methanotrophs including M. tundripaludum, while no active type II methanotrophs were identified. These results indicate that the extant M. tundripaludum is an active methane oxidizer at its locus typicus; furthermore, Methylobacter seems to be the dominant active genus. Diversity of methanotrophs was low as compared, e.g. to wetland rice fields in the Mediterranean. This low diversity suggests a high vulnerability of Arctic methanotroph communities, which deserves more attention.

Methylobacter tundripaludum sp. nov., a methane-oxidizing bacterium from Arctic wetland soil on the Svalbard islands, Norway (78 degrees N).

A Gram-negative, rod-shaped, non-motile, non-spore forming bacterium (SV96T) was isolated from wetland soil near Ny-Alesund, Svalbard. On the basis of 16S rRNA gene sequence similarity, strain SV96T was shown to belong to the Gammaproteobacteria, related to Methylobacter psychrophilus Z-0021T (99.1 %), Methylobacter luteus ATCC 49878T (97.3 %), Methylobacter marinus A45T (97.0 %) and Methylobacter whittenburyi ATCC 51738T (95.8 %); the closest related species within the genus Methylomicrobium with a validly published name was Methylomicrobium album ATCC 33003T (95.0 %). Chemotaxonomic data (including the major fatty acids: 16 : 1omega8, 16 : 1omega7 and 16 : 1omega5t) supported the affiliation of strain SV96T to the genus Methylobacter. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV96T from the four Methylobacter species mentioned above. Strain SV96T therefore represents a novel species, for which the name Methylobacter tundripaludum sp. nov. is proposed (type strain SV96T = DSM 17260T = ATCC BAA-1195T).

Methylocystis rosea sp. nov., a novel methanotrophic bacterium from Arctic wetland soil, Svalbard, Norway (78 degrees N).

A Gram-negative, rod-shaped, non-motile, non-spore-forming, pink-pigmented bacterium, SV97T, was isolated from a wetland soil near Ny-Alesund, Svalbard Islands, Norway (78 degrees N). On the basis of 16S rRNA gene sequence similarity, strain SV97T was shown to belong to the Alphaproteobacteria and was highly related to a number of non-characterized Methylocystis strains with GenBank accession nos AJ458507 and AJ458502 (100 %) and AF177299, AJ458510, AJ458467, AJ458471, AJ431384, AJ458475, AJ458484, AJ458501 and AJ458466 (99 %). The most closely related type strains were Methylocystis parvus OBBP(T) (97.2 %) and Methylocystis echinoides IMET 10491T (97%). The closest related recognized species within the genus Methylosinus was Methylosinus sporium NCIMB 11126T (96.0% similarity). Chemotaxonomic and phenotypic data (C(18:1)omega8 as the major fatty acid, non-motile, no rosette formation) supported the affiliation of strain SV97T to the genus Methylocystis. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV97(T) from the two recognized Methylocystis species. Strain SV97T therefore represents a novel species, for which the name Methylocystis rosea sp. nov. is proposed, with the type strain SV97T (= DSM 17261T = ATCC BAA-1196T).

Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway)--denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures.

The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78 degrees N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 degrees C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.