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The composition of the microbial community formed in the course of long-term (over 60 days) continuous cultivation of an obligate methanotroph Methylococcus sp. Concept-8 on natural gas in a 50-L bioreactor at 42°C and pH 5.6 was monitored. Stable growth with high optical density of the culture (average OD540 = 13.5), specific growth rate of 0.2 h–1, and biomass yield of 4.0–4.5 g dry matter L–1 continued for 50 days. During the subsequent period of regime instability, optical density decreased sharply, down to OD540 = 4. Formate, acetate, and a number of unidentified fatty acids derived from oxidation of methane homologs were detected in the culture liquid. Microscopy revealed the highest share of satellite bacteria (10–15% of the total cell number) during the period of stable growth, while it dropped to 4.5% during growth decline. Molecular profiling of the community composition based on 16S rRNA gene sequence analysis revealed members of the genera Cohnella, Brevibacillus, Azospirillim, Thermomonas, Cupriavidus, and Paenibacillus among the major microbial satellites responsible for utilization of metabolites and the products of lysis of the methanotroph cells. During the phase of growth instability, the relative abundance of Brevibacillus decreased drastically; these organisms were most likely responsible for acetate oxidation. Isolates obtained by means of cultivation represented only a minor part of microbial diversity revealed in the community by molecular analysis. Isolation of the cultures of Brevibacillus and Cupriavidus species representing the functionally important components of the community opens the way for targeted construction of associations with strain Concept-8, which will display high productivity and stable growth.

Planctomycetes are a phylogenetic group of bacteria with complex cell organization, large genomes and a wide spectrum of genome-encoded secondary metabolites. As suggested by analysis of available planctomycete genomes, these bacteria represent a promising source of potentially novel biologically active compounds. The number of cultured planctomycetes, however, remains limited. This study was undertaken in order to assess the presence of antimicrobial activity and its spectrum in the recently described freshwater planctomycete Lacipirellula parvula PX69T representing the novel family Lacipirellulaceae. The genome of strain PX69T contained 6 gene clusters encoding type III polyketide synthases, non-ribosomal peptide synthetases, and other secondary metabolites, which displayed low similarity to those in other bacteria. Strain PX69T was shown to exhibit antimicrobial activity against a number of test microorganisms. The highest antimicrobial activity, up to 83‒87% of growth inhibition, was observed against Staphylococcus aureus and Candida albicans. The medium composition and the cultivation time have been optimized in order to maximize antimicrobial activity of strain PX69T.

The complete genome sequence of a thermotolerant obligate methanotroph Methylococcus sp. Concept-8 was determined and analyzed. This strain was obtained by long-term storage, selection, and purification of an industrial culture of Methylococcus capsulatus BSV-874, which was used for methane-based protein biosynthesis in the Soviet Union. The size of the Concept-8 genome is 3.46 Mb. Genome annotation identified 3266 open reading frames that encode proteins. The closest phylogenetic relative of strain Concept-8 is Methylococcus capsulatus Bath (98.7% identity of 16S rRNA gene sequences). Comparison of the genomic sequences of these methanotrophs revealed 88.39% average nucleotide sequence identity, indicating that Concept-8 represents a novel species of the genus Methylococcus. The genomes of the strains Bath and Concept-8 both contain two copies of rRNA operons and pmoBAC operons for particulate methane monooxygenase, as well as a single copy of the soluble methane monooxygenase operon mmoXYBZDC. Growth on methanol was possible due to the presence of two complementary methanol dehydrogenases: MxaFJGIRACKLD and XoxF. The two methanotrophs also possess highly similar sets of genes encoding enzymes of the major pathways of the metabolism of C1 compounds. The genome of Methylococcus sp. Concept-8 was found to contain two regions associated with prophages of the family Siphoviridae. The prophage regions detected in the genome of Mc. capsulatus Bath are also homologous to viral sequences of the family Siphoviridae but differ from those identified in the genome of Concept-8.

Upland forest ecosystems are recognized as net sinks for atmospheric methane (CH4), one of the most impactful greenhouse gases. Biological methane uptake in these ecosystems occurs due to the activity of aerobic methanotrophic bacteria. Russia hosts one-fifth of the global forest area, with the most extensive forest landscapes located in West Siberia. Here, we report seasonal CH4 flux measurements conducted in 2018 in three types of stands in West Siberian middle taiga–Siberian pine, Aspen, and mixed forests. High rates of methane uptake of up to −0.184 mg CH4 m−2 h−1 were measured by a static chamber method, with an estimated total growing season consumption of 4.5 ± 0.5 kg CH4 ha−1. Forest type had little to no effect on methane fluxes within each season. Soil methane oxidation rate ranged from 0 to 8.1 ng CH4 gDW−1 h−1 and was negatively related to water-filled pore space. The microbial soil communities were dominated by the Alpha- and Gammaproteobacteria, Acidobacteriota and Actinobacteriota. The major group of 16S rRNA gene reads from methanotrophs belonged to uncultivated Beijerinckiaceae bacteria. Molecular identification of methanotrophs based on retrieval of the pmoA gene confirmed that Upland Soil Cluster Alpha was the major bacterial group responsible for CH4 oxidation.

Aerobic methanotrophic bacteria are an ecologically important group of microorganisms, which are functionally specialized in oxidation of the greenhouse gas methane. Recent insights into the growing pool of available genome sequences from methanotrophs revealed a number of as-yet-unknown metabolic capabilities of these bacteria. Thus, the genes indicative of aerobic anoxygenic photosynthesis by means of the photosystem II characteristic of purple bacteria were revealed in the genome of an obligate methanotroph Methylocapsa palsarum NE2T. Advanced search for genomic determinants of phototrophy in other methanotrophs confirmed their occurrence in a number of methanotrophic Alphaproteobacteria, including Methylocella silvestris BL2T and TVC, Methylocystis rosea SV97T and GW6, as well as Methylocystis spp. strains SB2 and MitZ-2018. Genomes of these methanotrophs contained the pufABCLM gene clusters encoding the light-harvesting complex, bch/chl genes responsible for bacteriochlorophyll biosynthesis, and the pucC gene essential for bacteriochlorophyll transport, as well as the crtFDC, crtL and crtB genes responsible for carotenoid biosynthesis. Organization of these gene clusters was conserved within each methanotroph species and was highly similar in Methylocapsa and Methylocella strains. A number of rearrangements, including inverse localization of the genes encoding bacteriochlorophyll and carotenoid biosynthesis, were observed in the genomes of Methylocystis species. The presence of pufLM genes was also revealed in a new isolate of Methylocapsa palsarum, strain NSB8, which was obtained in this study from a tundra wetland of European Northern Russia. The presence of phototrophy-related genes in all available strains of the abovementioned species indicates their functional importance for these bacteria and suggests realization of the phototrophic potential under certain environmental conditions.

An isolate of aerobic, Gram-stain-negative, rod-shaped, non-motile and light-pink pigmented bacteria, designated SBC68T, was obtained from slightly decomposed thalli of the lichen Cladonia sp. collected from the forested tundra of north-western Siberia. Cells of this isolate occurred singly, in pairs or in rosettes. These bacteria were acidophilic (optimum growth at pH 4.3–5.6) and mesophilic (optimum growth at 20–30 °C) but were also capable of growth at low temperatures, down to 7 °C. The preferred growth substrates were sugars, some organic acids and lichenan. The major fatty acids were iso-C15 : 0, C16 : 1ω7c, C16 : 0, Cω7t, and 13,16-dimethyl octacosanedioic acid. The only quinone was MK-8, and the G+C content of the DNA was 54.7 mol%. SBC68T represented a member of the family Acidobactericeae; the closest taxonomically described relatives were Edaphobacter dinghuensis DHF9T and Granulicella aggregans TPB6028T (97.2 and 97.1 % 16S rRNA gene sequence similarity, respectively). In 16S rRNA gene-based trees, SBC68T clustered together with species of the genus Edaphobacter . However, this isolate differed from all previously described species of the genus Edaphobacter with respect to the pink pigmentation, formation of cell rosettes and substrate utilization pattern. On the basis of these data, strain SBC68T should be considered to represent a novel species of acidobacteria, for which the name Edaphobacter lichenicola sp. nov. is proposed. The type strain is SBC68T (=DSM 104462T=VKM B-3208T).

This study assessed the microbial diversity, activity, and composition of methane-oxidizing communities of a subarctic wetland in Russia with mosaic cover of Sphagnum mosses and lichens of the genera Cladonia and Cetraria. Potential methane-oxidizing activity of peat sampled from lichen-dominated wetland sites was higher than that in the sites dominated by Sphagnum mosses. In peat from lichen-dominated sites, major bacterial groups identified by high-throughput sequencing of the 16S rRNA genes were the Acidobacteria (35.4–41.2% of total 16S rRNA gene reads), Alphaproteobacteria (19.1–24.2%), Gammaproteobacteria (7.9–11.1%), Actinobacteria (5.5–13.2%), Planctomycetes (7.2–9.5%), and Verrucomicrobia (5.1–9.5%). The distinctive feature of this community was high proportion of Subdivision 2 Acidobacteria, which are not characteristic for boreal Sphagnum peat bogs. Methanotrophic community composition was determined by molecular analysis of the pmoA gene encoding particulate methane monooxygenase. Most (~80%) of all pmoA gene fragments revealed in peat from lichen-dominated sites belonged to the phylogenetic lineage represented by a microaerobic spiral-shaped methanotroph, “Candidatus Methylospira mobilis”. Members of the genus Methylocystis, which are typical inhabitants of boreal Sphagnum peat bogs, represented only a minor group of indigenous methanotrophs. The specific feature of a methanotrophic community in peat from lichen-dominated sites was the presence of uncultivated USCα (Upland Soil Cluster alpha) methanotrophs, which are typical for acidic upland soils showing atmospheric methane oxidation. The methanotrophic community composition in lichen-dominated sites of a tundra wetland, therefore, was markedly different from that in boreal Sphagnum peat bogs.

Most of the methane produced on our planet gets naturally oxidized by a group of methanotrophic microorganisms before it reaches the atmosphere. These microorganisms are able to oxidize methane, both aerobically and anaerobically, and use it as their sole energy source. Although methanotrophs have been studied for more than a century, there are still many unknown and uncultivated groups prevalent in various ecosystems. This study focused on the diversity and adaptation of aerobic methane-oxidizing bacteria in different environments by comparing their phenotypic and genotypic properties. We used lab-scale microcosms to create a countergradient of oxygen and methane for preenrichment, followed by classical isolation techniques to obtain methane-oxidizing bacteria from a freshwater environment. This resulted in the discovery and isolation of a novel methanotroph with interesting physiological and genomic properties that could possibly make this bacterium able to cope with fluctuating environmental conditions., Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named “Methylotetracoccus oryzae” C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C16:1ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems. IMPORTANCE Most of the methane produced on our planet gets naturally oxidized by a group of methanotrophic microorganisms before it reaches the atmosphere. These microorganisms are able to oxidize methane, both aerobically and anaerobically, and use it as their sole energy source. Although methanotrophs have been studied for more than a century, there are still many unknown and uncultivated groups prevalent in various ecosystems. This study focused on the diversity and adaptation of aerobic methane-oxidizing bacteria in different environments by comparing their phenotypic and genotypic properties. We used lab-scale microcosms to create a countergradient of oxygen and methane for preenrichment, followed by classical isolation techniques to obtain methane-oxidizing bacteria from a freshwater environment. This resulted in the discovery and isolation of a novel methanotroph with interesting physiological and genomic properties that could possibly make this bacterium able to cope with fluctuating environmental conditions.

Three strains of aerobic psychrotolerant methanotrophic bacteria Methylovulum psychrotolerans, isolated from geographically remote low-temperature environments in Northern Russia, were grown at three different growth temperatures, 20, 10 and 4°C and were found to be capable of oxidizing methane at all temperatures. The three M. psychrotolerans strains adapted their membranes to decreasing growth temperature by increasing the percent of unsaturated fatty acid (FAs), both for the bulk and intact polar lipid (IPL)-bound FAs. Furthermore, the ratio of βOH-C16:0 to n-C16:0 increased as growth temperature decreased. The IPL head group composition did not change as an adaption to temperature. The most notable hopanoid temperature adaptation of M. psychrotolerans was an increase in unsaturated hopanols with decreasing temperature. As the growth temperature decreased from 20 to 4°C, the percent of unsaturated M. psychrotolerans bulk-FAs increased from 79 to 89 % while the total percent of unsaturated hopanoids increased from 27 to 49 %. While increased FA unsaturation in response to decreased temperature is a commonly observed response in order to maintain the liquid-crystalline character of bacterial membranes, hopanoid unsaturation upon cold exposition has not previously been described. In order to investigate the mechanisms of both FA and hopanoid cold-adaption in M. psychrotolerans we identified genes in the genome of M. psychrotolerans that potentially code for FA and hopanoid desaturases. The unsaturation of hopanoids represents a novel membrane adaption to maintain homeostasis upon cold adaptation.

This study examined potential disturbances of methanotrophic communities playing a key role in reducing methane emissions from the peat bog Tasin Borskoye (Vladimir oblast, Russia), as a result of the wildfire in 2007. The potential activity of the methane-oxidizing filter in the burned peatland sites and the abundance of indigenous methanotrophic bacteria were significantly reduced in comparison to the undisturbed sites. Molecular analysis of the methanotrophic community structure by means of PCR amplification and cloning of the pmoA gene encoding particulate methane monooxygenase revealed the replacement of typical peat-inhabiting, acidophilic type II methanotrophic bacteria with type I methanotrophs, which are less active in acidic environments. In summary, both the structure and the activity of the methane-oxidizing filter in burned peatland sites underwent significant changes, which were clearly pronounced even after 7 years of the natural ecosystem recovery. These results point to the long-term character of the disturbances caused by wildfire in peatlands.

Methylovulum psychrotolerans Sph1T is an aerobic, obligate methanotroph, which was isolated from cold methane seeps in West Siberia. This bacterium possesses only a particulate methane monooxygenase and is widely distributed in low-temperature environments. Strain Sph1T has the genomic potential for biosynthesis of hopanoids required for the maintenance of intracytoplasmic membranes.

The structural changes in the bacterial community composition of the Galitskii Mokh mesotrophic peatland (Tver oblast) after the 2010 wildfire were analyzed. Burned sites demonstrated an increase in peat water pH and mineralization, with elevated nitrate and sulfate concentrations, which resulted in doubling of the numbers of culturable sulfate reducers. Although the rate of methane oxidation by peat samples from burned sites decreased, methane emission did not increase. Enumeration of bacterial phylogenetic groups by fluorescent in situ hybridization revealed doubling of the numbers of Alphaproteobacteria and Bacteroidetes in burned areas compared to an undamaged site, while a reverse pattern was observed for Deltaproteobacteria and Planctomycetes. Comparative analysis of the 16S rRNA clone libraries revealed shifts in the bacterial community composition in the peat from burned sites. Slowly growing bacteria of the phyla Verrucomicrobia and Planctomycetes were replaced by fast-growing colonizers from the Proteobacteria. The originally diverse Actinobacterial community was replaced by the one dominated by the phylotypes related to thermophilic Aciditerrimonas ferrireducens. In the Acidobacteria, a typical group of peat-inhabiting bacteria, the subgroups 3, 6, and 17 were replaced by subgroups 7 and 10. These changes in the structure of peatland microbial communities could result in disturbances of some biospheric functions of these ecosystems.

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are potentially important (although poorly studied) sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3–5°C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with prevalence of type I methanotrophs. Among the latter, microorganisms related to Methylobacter psychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two methanotrophic isolates were determined. Methylobacter sp. CMS7 exhibited active growth at 4–10°C, while Methylocystis sp. SB12 grew better at 20°C. Experimental results confirmed the major role of methanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.

The waters of small lakes located in swampy catchment areas of upper Volga contain considerable amounts of ultrasmall microbial cells that pass through 0.22-μm-pore-size filters. As shown in our previous study [1], most of these cells represent the bacterial genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia of the class Betaproteobacteria, as well as euryarchaea of the uncharacterized clade LDS. The aim of the present study was to investigate the possible effect of the waters draining swampy areas on the composition of the filterable microbial fraction in lakes fed by swampy catchments. To address this question molecular identification was performed of prokaryotic ultramicroforms in the peat of the ombrotrophic Sphagnum bog Obukhovskoe, located, like the lakes studied previously [1], in the Mologa-Sheksna catchment area. The number of filterable microorganisms in 1 g wet peat was 3.8 × 106 cells, or 0.5% of total microbial cell number in the peat. From the DNA of the filterable cell fraction, 100 clones of bacterial and 77 clones of archaeal 16S rRNA genes were obtained. The bacterial clone library contained 16S rRNA gene sequences representing the classes Beta- and Gammaproteobacteria (the genera Janthinobacterium and Pseudomonas, respectively) and the phylum Bacteroidetes (the genera Chryseobacterium and Epilithonimonas) and differed significantly from the clone library of bacterial ultramicroforms of lake water. By contrast, the pools of filterable archaea in bogs and lakes were essentially similar. They were represented by the euryarchaeal clade LDS and methanogens of the orders Methanobacteriales and Methanosarcinales. Additionally, the pool of filterable archaea of the bog included methanogens of the order Methanomicrobiales and representatives of the uncharacterized euryarchaeal clade RC-V (Rice Cluster V) and of the phylum Thaumarchaeota.

Planctomycetes are common inhabitants of northern wetlands. A significant proportion of these bacteria revealed in peat with the Planctomycetes-specific probes PLA46 and PLA886 is represented by filamentous forms which have not been cultured under laboratory conditions. In the present work, one of such organisms was isolated from a Sphagnum peat bog in a monoculture. The organism had large spherical cells assembled in long filaments. It could grow only in associations with bacterial satellites; attempts to isolate it in pure culture were unsuccessful. The organism was identified by PCR amplification, cloning, and subsequent analysis of its 16S rRNA gene fragment. Comparative sequence analysis revealed its affiliation with the Isosphaera-Singulisphaera group within the order Planctomycetales. The nucleotide sequence of the 16S rRNA gene of the new organism exhibited 94–96% similarity to those of the unicellular peat-inhabiting planctomycete Singulisphaera acidiphila and uncharacterized filamentous planctomycete “Nostocoida limicola III” from activated sludge. The new planctomycete utilized heteropolysaccharides of microbial origin as growth substrates and could grow at the low pH and temperatures typical of the northern wetlands.

Wetland ecosystems are the natural centers of freshwater formation in northern Russia lowland landscapes. The humic acidic waters formed in bogs feed the numerous lakes of the northern regions. One milliliter of the water in these lakes contains up to 104 ultrasmall microbial cells that pass through “bacterial” filters with a pore size of 0.22 μm. The vast majority of these cells do not grow on nutrient media and cannot be identified by routine cultivation-based approaches. Their identification was performed by analysis of clone libraries obtained by PCR amplification of archaeal and bacterial 16S rRNA genes from the fraction of cells collected from water filtrates of acidic lakes. Most of the obtained bacterial 16S rRNA gene sequences represented the class Betaproteobacteria and exhibited the highest homology of (94–99%) with 16S rRNA genes of representatives of the genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia. The archaeal 16S rRNA gene clone library comprised genes of Euryarchaeota representatives. One-third of these genes exhibited 97–99% homology to the 16S rRNA genes of taxonomically described organisms of the orders Methanobacteriales and Methanosarcinales. The rest of the cloned archaeal 16S rRNA genes were only distantly related (71–74% homology) to those in all earlier characterized archaea.

Fluorescent in situ hybridization (FISH) with rRNA-specific oligonucleotide probes was used to assess the numbers and phylogenetic diversity of prokaryotic microorganisms in the water of small boreal lakes and peatland catchments of the swampy upper Volga basin. The abundance of bacterioplankton in lake water was found to vary from 1.6 to 8.7 × 106 cells ml−1, with the highest values detected in neutral eutrophic lakes. The total cell numbers in the peat of ombrotrophic bogs were 3.9–4.3 × 108 cells g−1 of wet peat. The proportion of bacteria identified by the group-specific probes decreased from 79–85% in neutral (pH 6.6–6.9) mesotrophic and eutrophic lakes to 65–69% in acidic (pH 4.4–5.5) dystrophic lakes and to 51–58% in the peat of acidic (pH 3.6–3.9) ombrotrophic bogs. The diversity of bacterial communities was highest in lakes with neutral water. These communities were dominated by members of the phylum Actinobacteria (31–44% of the total bacterial number), while the contribution of Alphaproteobacteria (16–19%), Bacteroidetes (6–16%), Betaproteobacteria (6–7%), Planctomycetes (2–8%), and Gammaproteobacteria (4–5%) was also significant. In acidic dystrophic lakes, Actinobacteria (25–35%) and Betaproteobacteria (25–34%) predominated, while peatland catchments were dominated by the Alphaproteobacteria (20–23%). The presence of acidobacteria and some planctomycetes common for bogs in the water of acidic dystrophic lakes, as well as the high proportion of bacteria (31–49%) that were not identified by the group-specific probes, suggest the impact of microbial processes in peatland catchments on the microbial composition of the receiving waters.

Representatives of the genus Methylocystis are traditionally considered to be obligately methanotrophic bacteria, which are incapable of growth on multicarbon substrates. Here, we describe a novel member of this genus, strain H2s, which represents a numerically abundant and ecologically important methanotroph population in northern Sphagnum-dominated wetlands. This isolate demonstrates a clear preference for growth on methane but is able to grow slowly on acetate in the absence of methane. Strain H2s possesses both forms of methane monooxygenase (particulate and soluble MMO) and a well-developed system of intracytoplasmic membranes (ICM). In cells grown for several transfers on acetate, these ICM are maintained, although in a reduced form, and mRNA transcripts of particulate MMO are detectable. These cells resume their growth on methane faster than those kept for the same period of time without any substrate. Growth on acetate leads to a major shift in the phospholipid fatty acid composition. The re-examination of all type strains of the validly described Methylocystis species showed that Methylocystis heyeri H2T and Methylocystis echinoides IMET10491T are also capable of slow growth on acetate. This capability might represent an important part of the survival strategy of Methylocystis spp. in environments where methane availability is variable or limited.

An aerobic, methanotrophic bacterium, designated KYGT, was isolated from a forest soil in Germany. Cells of strain KYGT were Gram-negative, non-motile, slightly curved rods that multiplied by binary fission and produced yellow colonies. The cells contained intracellular granules of poly-β-hydroxybutyrate at each cell pole, a particulate methane monooxygenase (pMMO) and stacks of intracytoplasmic membranes (ICMs) packed in parallel along one side of the cell envelope. Strain KYGT grew at pH 5.2–7.2 and 2–33 °C and could fix atmospheric nitrogen under reduced oxygen tension. The major cellular fatty acid was C18 : 1 ω7c (81.5 %) and the DNA G+C content was 61.4 mol%. Strain KYGT belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the obligate methanotroph Methylocapsa acidiphila B2T (98.1 % 16S rRNA gene sequence similarity and 84.7 % pmoA sequence similarity). Unlike Methylocapsa acidiphila B2T, which grows only on methane and methanol, strain KYGT was able to grow facultatively on acetate. Facultative acetate utilization is a characteristic of the methanotrophs of the genus Methylocella, but the genus Methylocella does not produce pMMO or ICMs. Strain KYGT differed from Methylocapsa acidiphila B2T on the basis of substrate utilization pattern, pigmentation, pH range, cell ultrastructure and efficiency of dinitrogen fixation. Therefore, we propose a novel species, Methylocapsa aurea sp. nov., to accommodate this bacterium. The type strain is KYGT (=DSM 22158T =VKM B-2544T).

Three obligately aerobic, heterotrophic bacteria, designated strains WM1T, TPB606T and TPB621, were isolated from acidic Sphagnum-dominated tundra and Siberian wetlands in Russia. Cells of these isolates were Gram-negative, non-motile coccobacilli that occurred singly, in pairs or in chains, and were covered by large capsules. The novel strains were moderately acidophilic and psychrotolerant organisms capable of growth at pH 3.0-7.6 and 2-30 degrees C. Cells contained numerous intracellular poly-beta-hydroxybutyrate granules (3-4 per cell). The major cellular fatty acid was cyclo C19:0omega8c and the predominant quinone was Q-10. Strains TPB606T and TPB621, isolated from Siberian wetland, possessed almost identical 16S rRNA gene sequences and shared 97.2% sequence similarity with tundra strain WM1T. The three strains were shown to belong to the Alphaproteobacteria, but were related only distantly to the type strains of acidophilic bacteria Acidisphaera rubrifaciens (93.4-94.3% 16S rRNA gene sequence similarity), Rhodopila globiformis (92.2-93.3%), and members of the genera Acidiphilium (91.3-93%) and Acidocella (91.8-92.4%). The DNA G+C contents of the novel strains were 60.5-61.9 mol%. The low levels of DNA-DNA relatedness (37%) and a number of phenotypic differences between the Siberian strains TPB606T and TPB621 and the tundra strain WM1T indicated that they represent two separate species. As the three isolates are clearly distinct from all recognized acidophilic members of the Alphaproteobacteria, they are considered to represent two novel species of a new genus, for which the names Acidisoma tundrae gen. nov., sp. nov. and Acidisoma sibiricum sp. nov. are proposed. The type strain of Acidisoma sibiricum is TPB606T (=DSM 21000T=VKM B-2487T) and the type strain of Acidisoma tundrae is WM1T (=DSM 19999T=VKM B-2488T).

Two isolates of aerobic methanotrophic bacteria, strains Sph1T and Sph2, were obtained from cold methane seeps in a floodplain of the river Mukhrinskaya, Irtysh basin, West Siberia. Another morphologically and phenotypically similar methanotroph, strain OZ2, was isolated from a sediment of a subarctic freshwater lake, Archangelsk region, northern Russia. Cells of these three strains were Gram-stain-negative, light-pink-pigmented, non-motile, encapsulated, large cocci that contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme and utilized only methane and methanol. Strains Sph1T, Sph2 and OZ2 were able to grow at a pH range of 4.0–8.9 (optimum at pH 6.0–7.0) and at temperatures between 2 and 36 °C. Although their temperature optimum was at 20–25 °C, these methanotrophs grew well at lower temperatures, down to 4 °C. The major cellular fatty acids were C16 : 1ω5c, C16 : 1ω6c, C16 : 1ω7c, C16 : 1ω8c, C16 : 0 and C14 : 0; the DNA G+C content was 51.4–51.9 mol%. Strains Sph1T, Sph2 and OZ2 displayed nearly identical (99.1–99.7 % similarity) 16S rRNA gene sequences and belonged to the family Methylococcaceae of the class Gammaproteobacteria . The most closely related organism was Methylovulum miyakonense HT12T (96.0–96.5 % 16S rRNA gene sequence similarity and 90 % pmoA sequence similarity). The novel isolates, however, differed from Methylovulum miyakonense HT12T by cell morphology, pigmentation, absence of soluble methane monooxygenase, more active growth at low temperatures, growth over a broader pH range and higher DNA G+C content. On the basis of these differences, we propose a novel species, Methylovulum psychrotolerans sp. nov., to accommodate these methanotrophs. Strain Sph1T (=LMG 29227T=VKM B-3018T) is the type strain.

Slow degradation of organic matter in acidic Sphagnum peat bogs suggests a limited activity of organotrophic microorganisms. Monitoring of the Sphagnum debris decomposition in a laboratory simulation experiment showed that this process was accompanied by a shift in the water color to brownish due to accumulation of humic substances and by the development of a specific bacterial community with a density of 2.4 × 107 cells ml−1. About half of these organisms are metabolically active and detectable with rRNA-specific oligonucleotide probes. Molecular identification of the components of this microbial community showed the numerical dominance of bacteria affiliated with the phyla Alphaproteobacteria, Actinobacteria, and Planctomycetes. The population sizes of the Firmicutes and Bacteroidetes, which are believed to be the main agents of bacterially-mediated decomposition in eutrophic wetlands, were low. The numbers of planctomycetes increased at the final stage of Sphagnum decomposition. The representative isolates of the Alphaproteobacteria were able to utilize galacturonic acid, the only low-molecular-weight organic compound detected in the water samples; the representatives of the Planctomycetes were able to decompose some heteropolysaccharides, which points to the possible functional role of these groups of microorganisms in the community under study. Thus, the composition of the bacterial community responsible for Sphagnum decomposition in acidic and low-mineral oligotrophic conditions seems to be fundamentally different from that of the bacterial community which decomposes plant debris in eutrophic ecosystems at neutral pH.

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 10 7 and 1.1 × 10 7 cells g −1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes . This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

Bacteria of the genus Burkholderia are a typical component of the microbial complex of Sphagnum peat bogs and constitute a substantial portion of the aerobic chemoorganotrophic isolates which are routinely obtained from these environments on an acidic nutrient media. The ecophysiological characteristics of the 27 strains of such organisms, which were isolated from the peat of acidic Sphagnum bogs of the boreal and tundra zones of Russia, Canada, and Estonia, were investigated in the present study. Most of the Burkholderia strains isolated from these bogs were phylogenetically close to the species B. glathei, B. phenazinium, B. fungorum, and B. caryophylli, the typical inhabitants of soil and plant rhizosphere. The bog isolates utilized a broad range of substrates as carbon and energy sources, including organic acids, sugars, polyalcohols, and certain aromatic compounds. All the strains studied were capable of growth on nitrogen-free media. They developed in the pH range of 3.5 to 7.4 and from 3 to 37°C, with the optima at pH 5–7 and 11–23°C, respectively. They were therefore moderately acidophilic, psychroactive, dinitrogen-fixing microorganisms well adapted to the conditions of acidic northern Sphagnum bogs.

The microbial population of sphagnum peat bogs of northern Russia was analyzed with respect to the presence and cell numbers of representatives of particular phylogenetic groups of prokaryotes by means of in situ hybridization with fluorescently labeled group-specific rRNA-targeted oligonucleotide probes with broad detection spectra. The total number of cells that hybridized with universal Archaea- and Bacteria-specific probes varied, in peat samples of different bogs, from 45 to 83% of the number of cells revealed by DAPI staining. Down the bog profiles, the total number of prokaryotes and the fraction of archaea among them increased. Application of a set of oligonucleotide probes showed that the number of microorganisms belonging to such phylogenetic lineages of the domain Bacteria as the phyla Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, and Planctomycetes constituted, in total, 14.0–26.5% of the number of eubacteria detected in the samples. Among the bacteria identified in the peat samples, the most abundant were representatives of the classes Alphaproteobacteria and Betaproteobacteria and the phyla Acidobacteria, Bacteroidetes, and Actinobacteria.

A novel species, Methylocella tundrae, is proposed for three methanotrophic strains (T4T, TCh1 and TY1) isolated from acidic Sphagnum tundra peatlands. These strains are aerobic, Gram-negative, non-motile, dinitrogen-fixing rods that possess a soluble methane monooxygenase and utilize the serine pathway for carbon assimilation. Strains T4T, TCh1 and TY1 are moderately acidophilic organisms capable of growth between pH 4·2 and 7·5 (optimum 5·5–6·0) and between 5 and 30 °C (optimum 15 °C). The major phospholipid fatty acid is 18 : 1ω7c. The DNA G+C content of strain T4T is 63·3 mol%. The three strains possess almost identical 16S rRNA gene sequences and are most closely related to two previously identified species of Methylocella, Methylocella palustris (97 % similarity) and Methylocella silvestris (97·5 % similarity). DNA–DNA hybridization values of strain T4T with Methylocella palustris KT and Methylocella silvestris BL2T were respectively 27 and 36 %. Thus, the tundra strains represent a novel species, for which the name Methylocella tundrae sp. nov. is proposed. Strain T4T (=DSM 15673T=NCIMB 13949T) is the type strain.

An aerobic methanotrophic bacterium was isolated from a collapsed palsa soil in northern Norway and designated strain NE2T. Cells of this strain were Gram-stain-negative, non-motile, non-pigmented, slightly curved thick rods that multiplied by normal cell division. The cells possessed a particulate methane monooxygenase enzyme (pMMO) and utilized methane and methanol. Strain NE2T grew in a wide pH range of 4.1–8.0 (optimum pH 5.2–6.5) at temperatures between 6 and 32 °C (optimum 18–25 °C), and was capable of atmospheric nitrogen fixation under reduced oxygen tension. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C16 : 1ω7c, and the DNA G+C content was 61.7 mol%. The isolate belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the facultative methanotroph Methylocapsa aurea KYGT (98.3 % 16S rRNA gene sequence similarity and 84 % PmoA sequence identity). However, strain NE2T differed from Methylocapsa aurea KYGT by cell morphology, the absence of pigmentation, inability to grow on acetate, broader pH growth range, and higher tolerance to NaCl. Therefore, strain NE2T represents a novel species of the genus Methylocapsa, for which we propose the name Methylocapsa palsarum sp. nov. The type strain is NE2T ( = LMG 28715T = VKM B-2945T).

The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size. The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density. In this work, Pirt's model was tested on two bacteria—Alcaligenes sp. and Pseudomonas fluorescens—having markedly distinct cultural properties and grown on an agarized medium with pyruvate. The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (105–106 cells), and for different depths of the agar layer, determining the amount of available substrate. A linear growth mode was observed only with P. fluorescens and only in the case of growth from a microdroplet. When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of Kr (the rate of colony radius increase) occurring after 2–3 days of growth. The growth of P. fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp. turned out to be directly related to the medium layer thickness. The departure from linearity is consistently explained by a new kinetic scheme stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center. The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by the age of cells. The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.

Representatives of the genus Methylocystis are traditionally considered to be obligately methanotrophic bacteria, which are incapable of growth on multicarbon substrates. Here, we describe a novel member of this genus, strain H2s, which represents a numerically abundant and ecologically important methanotroph population in northern Sphagnum-dominated wetlands. This isolate demonstrates a clear preference for growth on methane but is able to grow slowly on acetate in the absence of methane. Strain H2s possesses both forms of methane monooxygenase (particulate and soluble MMO) and a well-developed system of intracytoplasmic membranes (ICM). In cells grown for several transfers on acetate, these ICM are maintained, although in a reduced form, and mRNA transcripts of particulate MMO are detectable. These cells resume their growth on methane faster than those kept for the same period of time without any substrate. Growth on acetate leads to a major shift in the phospholipid fatty acid composition. The re-examination of all type strains of the validly described Methylocystis species showed that Methylocystis heyeri H2(T) and Methylocystis echinoides IMET10491(T) are also capable of slow growth on acetate. This capability might represent an important part of the survival strategy of Methylocystis spp. in environments where methane availability is variable or limited.

Three strains of budding, ellipsoid-shaped and rosette-forming bacteria were isolated from acidic Sphagnum-dominated boreal wetlands of northern Russia and were designated strains MPL7T, MOB77 and SB2. The presence of crateriform pits and numerous fibrillar appendages on the cell surface and an unusual spur-like projection on one pole of the cell indicated a planctomycete morphotype. These isolates are moderately acidophilic, mesophilic organisms capable of growth at pH values between 4.2 and 7.5 (with an optimum at pH 5.0–6.2) and at temperatures between 4 and 32 °C (optimum 15–26 °C). The major fatty acids are C16 : 0 and C16 : 1 ω7c; the major quinone is MK-6. The G+C content of the DNA is 54.4–56.5 mol%. Strains MPL7T, MOB77 and SB2 possess nearly identical 16S rRNA gene sequences and belong to the planctomycete lineage defined by the genus Planctomyces, being most closely related to Planctomyces limnophilus DSM 3776T (86.9–87.1 % sequence similarity). However, strain MPL7T showed only 28 % DNA–DNA hybridization with P. limnophilus DSM 3776T. Compared with currently described members of the genus Planctomyces, the isolates from northern wetlands do not form long and distinctive stalks, have greater tolerance of acidic conditions and low temperatures, are more sensitive to NaCl, lack pigmentation and degrade a wider range of biopolymers. The data therefore suggest that strains MPL7T, MOB77 and SB2 represent a novel genus and species, for which the name Schlesneria paludicola gen. nov., sp. nov., is proposed. Strain MPL7T (=ATCC BAA-1393T =VKM B-2452T) is the type strain of Schlesneria paludicola.

A novel species is proposed for two strains of methanotrophic bacteria (H2T and Sakb1) isolated from an acidic (pH 4.3) Sphagnum peat bog lake (Teufelssee, Germany) and an acidic (pH 4.2) tropical forest soil (Thailand), respectively. Cells of strains H2T and Sakb1 were aerobic, Gram-negative, non-motile, straight or curved rods that were covered by large polysaccharide capsules and contained an intracytoplasmic membrane system typical of type II methanotrophs. They possessed both a particulate and a soluble methane monooxygenase and utilized the serine pathway for carbon assimilation. They were moderately acidophilic organisms capable of growth between pH 4.4 and 7.5 (optimum 5.8–6.2). The most unique characteristic of these strains was the phospholipid fatty acid profile. In addition to the signature fatty acid of type II methanotrophs (18 : 1ω8c), the cells also contained large amounts of what was previously considered to be a signature fatty acid of type I methanotrophs, 16 : 1ω8c. The DNA G+C contents of strains H2T and Sakb1 were 61.5 and 62.1 mol%, respectively. The 16S rRNA gene sequences possessed 96–98 % similarity to sequences of other type II methanotrophs in the genera Methylosinus and Methylocystis. 16S rRNA gene sequence and pmoA phylogeny demonstrated that the strains form a novel lineage within the genus Methylocystis. DNA–DNA hybridization values of strain H2T with Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T were 18 and 25 %, respectively. Thus, it is proposed that these two strains represent a novel species, Methylocystis heyeri sp. nov. Strain H2T (=DSM 16984T=VKM B-2426T) is the type strain.

Aspects of archaeal diversity in peat soil samples from climatically and geographically distinct wetlands (subarctic: West Siberia Bog, Russia; temperate: Akaiyachi Mire, Japan; subtropical: Okefenokee Swamp, USA) were studied by molecular phylogenetic techniques. DNA was extracted directly from the soil samples and 16S rRNA genes were amplified by polymerase chain reaction. Partial sequences of the amplified 16S rDNAs (total 426 clones) were compared with known sequences from GenBank and the Ribosome Database Project (RDP). Peat-derived sequences were mostly related to Euryarchaeota, principally methanogens. Sets of sequences (operational taxonomic unit; OTU) were created for each wetland (21 OTUs for West Siberia; 22 OTUs for Akaiyachi; 33 OTUs for Okefenokee). The majority of the OTUs clustered in and showed low similarities to the Methanosarcinales family (West Siberia) or the Methanomicrobiales family (Akaiyachi and Okefenokee). In terms of the Shannon-Weaver diversity index, the archaeal community diversity in Okefenokee Swamp was greater than that of the other wetlands.