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 ␣), 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 ␥" (USC ␥) 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 ␥ 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 ␣ sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.Methane (CH 4 ) is present in the atmosphere at a mixing ratio of about 1.7 ppm of volume (ppmv). An estimated 30 Tg of CH 4 from the atmosphere year Ϫ1 is oxidized by aerobic methanotrophic bacteria (MB) in upland soils, accounting for about 6% of the global atmospheric CH 4 sink (21, 31). Bender and Conrad (2) suggested that MB active in upland soils are specialized oligotrophs adapted to the trace level of atmospheric CH 4 and possess a methane monooxygenase (MMO) with a higher substrate affinity than that of cultivated MB. It was later demonstrated that the application of single-reactant Michaelis-Menten kinetics to MMO is not always appropriate and that the apparent affinity for CH 4 varies depending on the cultivation conditions (13). Nevertheless, recent studies indicate that MB in at least some soils that oxidize atmospheric CH 4 are indeed taxonomically distinct from known MB (28).The 13 recognized genera of MB are divided into two groups, type I (further divided into types I and X) and type II. These differ in phylogenetic affiliation (Gammaproteobacteria versus Alphaproteobacteria) and in diverse biochemical characteristics (21). Identification of MB in soils is often performed by the cultivation-independent detection of a fragment of pmoA, a gene encoding the active-site subunit of particulate MMO (22,26,30,35,38). This marker gene is ...

Permafrost-affected soils of the Siberian Arctic were investigated with regard to identification of nitrite oxidizing bacteria active at low temperature. Analysis of the fatty acid profiles of enrichment cultures grown at 41C, 101C and 171C revealed a pattern that was different from that of known nitrite oxidizers but was similar to fatty acid profiles of Betaproteobacteria. Electron microscopy of two enrichment cultures grown at 101C showed prevalent cells with a conspicuous ultrastructure. Sequence analysis of the 16S rRNA genes allocated the organisms to a so far uncultivated cluster of the Betaproteobacteria, with Gallionella ferruginea as next related taxonomically described organism. The results demonstrate that a novel genus of chemolithoautotrophic nitrite oxidizing bacteria is present in polygonal tundra soils and can be enriched at low temperatures up to 171C. Cloned sequences with high sequence similarities were previously reported from mesophilic habitats like activated sludge and therefore an involvement of this taxon in nitrite oxidation in nonarctic habitats is suggested. The presented culture will provide an opportunity to correlate nitrification with nonidentified environmental clones in moderate habitats and give insights into mechanisms of cold adaptation. We propose provisional classification of the novel nitrite oxidizing bacterium as 'Candidatus Nitrotoga arctica'.

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Summary Previously uncultured nitrite‐oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira‐like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira‐like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml−1. The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA‐targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira‐like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘Candidatus Nitrospira defluvii’.

SummaryFor the understanding and assessment of recent and future carbon dynamics of arctic permafrost soils the processes of CH 4 production and oxidation, the community structure and the quality of dissolved organic matter (DOM) were studied in two soils of a polygonal tundra. Activities of methanogens and methanotrophs differed significantly in their rates and distribution patterns among the two investigated profiles. Community structure analysis showed similarities between both soils for ester-linked phospholipid fatty acids (PLFAs) and differences in the fraction of unsaponifiable PLFAs and phospholipid ether lipids. Furthermore, a shift of the overall composition of the microbiota with depth at both sites was indicated by an increasing portion of iso-and anteiso-branched fatty acids related to the amount of straight-chain fatty acids. Although permafrost soils represent a large carbon pool, it was shown that the reduced quality of organic matter leads to a substrate limitation of the microbial metabolism. It can be concluded from our and previous findings first that microbial communities in the active layer of an Arctic polygon tundra are composed by members of all three domains of life, with a total biomass comparable to temperate soil ecosystems, and second that these microorganisms are well adapted to the extreme temperature gradient of their environment.

Methanotrophic communities were studied in several periodically water-saturated gleyic soils. When sampled, each soil had an oxic upper layer and consumed methane from the atmosphere (at 1.75 ppmv). In most gleyic soils the K(m(app)) values for methane were between 70 and 800 ppmv. These are higher than most values observed in dry upland soils, but lower than those measured in wetlands. Based on cultivation-independent retrieval of the pmoA-gene and quantification of partial pmoA gene sequences, type II (Alphaproteobacteria) methanotrophs of the genus Methylocystis spp. were abundant (> 10(7) pmoA target molecules per gram of dry soil). Type I (Gammaproteobacteria) methanotrophs related to the genera Methylobacter and Methylocaldum/Methylococcus were detected in some soils. Six pmoA sequence types not closely related to sequences from cultivated methanotrophs were detected as well, indicating that diverse uncultivated methanotrophs were present. Three Gleysols were incubated under different mixing ratios of (13)C-labelled methane to examine (13)C incorporation into phospholipid fatty acids (PLFAs). Phospholipid fatty acids typical of type II methanotrophs, 16:0 and 18:1omega7c, were labelled with (13)C in all soils after incubation under an atmosphere containing 30 ppmv of methane. Incubation under 500 ppmv of methane resulted in labelling of additional PLFAs besides 16:0 and 18:1omega7c, suggesting that the composition of the active methanotrophic community changed in response to increased methane supply. In two soils, 16:1 PLFAs typical of type I methanotrophs were strongly labelled after incubation under the high methane mixing ratio only. Type II methanotrophs are most likely responsible for atmospheric methane uptake in these soils, while type I methanotrophs become active when methane is produced in the soil.

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Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.