Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.     

Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria

Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs, while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcus genus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type II (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments where selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobacter sp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment.     

Aerobic methanotrophs from the coastal thermal springs of Lake Baikal

The number, activity, and diversity of aerobic methanotrophic bacteria in the sediments of three coastal thermal springs of Lake Baikal were analyzed. The average number of methanotrophs was 10³–10⁴ cells per 1 cm³ of sediment. The highest number of methanotrophs (10⁸ cells/cm³ of silt) and the highest potential rate of methane uptake [7.7 nmol CH₄/(cm³ day)] were revealed in sediments from the Sukhaya thermal spring. The methods of molecular ecology (DGGE, FISH, analysis of pmoA gene fragments) showed the predominance in most enrichment cultures of methanotrophs of type II, i.e., of the genera Methylocystis and Methylosinus. In only one enrichment culture (from the Sukhaya thermal spring), a type I methanotroph was revealed; its similarity to Methylococcus capsulatus Bath did not exceed 80%. These results demonstrate a widespread occurrence and high activity of the aerobic methanotrophic community in the coastal thermal springs of Lake Baikal.     

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°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.     

Effects of ammonium on the activity and community of methanotrophs in landfill biocover soils

The influence of NH₄⁺ on microbial CH₄ oxidation is still poorly understood in landfill cover soils. In this study, effects of NH₄⁺ addition on the activity and community structure of methanotrophs were investigated in waste biocover soil (WBS) treated by a series of NH₄⁺-N contents (0, 100, 300, 600 and 1200 mg kg⁻¹). The results showed that the addition of NH₄⁺-N ranging from 100 to 300 mg kg⁻¹ could stimulate CH₄ oxidation in the WBS samples at the first stage of activity, while the addition of an NH₄⁺-N content of 600 mg kg⁻¹ had an inhibitory effect on CH₄ oxidation in the first 4 days. The decrease of CH₄ oxidation rate observed in the last stage of activity could be caused by nitrogen limitation and/or exopolymeric substance accumulation. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. Of these, Methylocaldum was the main methanotroph in the original WBS. With incubation, a higher abundance of Methylobacter was observed in the treatments with NH₄⁺-N contents greater than 300 mg kg⁻¹, which suggested that NH₄⁺-N addition might lead to the dominance of Methylobacter in the WBS samples. Compared to type I methanotrophs, the abundance of type II methanotrophs Methylocystis and/or Methylosinus was lower in the original WBS sample. An increase in the abundance of Methylocystis and/or Methylosinus occurred in the last stage of activity, and was likely due to a nitrogen limitation condition. Redundancy analysis showed that NH₄⁺-N and the C/N ratio had a significant influence on the methanotrophic community in the WBS sample.     

Influence of temperature on the δ13C values and distribution of methanotroph‐related hopanoids in Sphagnum‐dominated peat bogs

Methane emissions from peat bogs are mitigated by methanotrophs, which live in symbiosis with peat moss (e.g. Sphagnum). Here, we investigate the influence of temperature and resultant changes in methane fluxes on Sphagnum and methanotroph‐related biomarkers, evaluating their potential as proxies in ancient bogs. A pulse‐chase experiment using ¹³C‐labelled methane in the field clearly showed label uptake in diploptene, a biomarker for methanotrophs, demonstrating in situ methanotrophic activity in Sphagnum under natural conditions. Peat cores containing live Sphagnum were incubated at 5, 10, 15, 20 and 25°C for two months, causing differences in net methane fluxes. The natural δ¹³C values of diploptene extracted from Sphagnum showed a strong correlation with temperature and methane production. The δ¹³C values ranged from ?34‰ at 5°C to ?41‰ at 25°C. These results are best explained by enhanced expression of the methanotrophic enzymatic isotope effect at higher methane concentrations. Hence, δ¹³C values of diploptene, or its diagenetic products, potentially provide a useful tool to assess methanotrophic activity in past environments. Increased methane fluxes towards Sphagnum did not affect δ¹³C values of bulk Sphagnum and its specific marker, the C₂₃ n‐alkane. The concentration of methanotroph‐specific bacteriohopanepolyols (BHPs), aminobacteriohopanetetrol (aminotetrol, characteristic for type II and to a lesser extent type I methanotrophs) and aminobacteriohopanepentol (aminopentol, a marker for type I methanotrophs) showed a non‐linear response to increased methane fluxes, with relatively high abundances at 25°C compared to those at 20°C or below. Aminotetrol was more abundant than aminopentol, in contrast to similar abundances of aminotetrol and aminopentol in fresh Sphagnum. This probably indicates that type II methanotrophs became prevalent under the experimental conditions relative to type I methanotrophs. Even though BHP concentrations may not directly reflect bacterial activity, they may provide insight into the presence of different types of methanotrophs.     

Active Methanotrophs in Two Contrasting North American Peatland Ecosystems Revealed Using DNA-SIP

The active methanotroph community was investigated in two contrasting North American peatlands, a nutrient-rich sedge fen and nutrient-poor Sphagnum bog using in vitro incubations and ¹³C-DNA stable-isotope probing (SIP) to measure methane (CH₄) oxidation rates and label active microbes followed by fingerprinting and sequencing of bacterial and archaeal 16S rDNA and methane monooxygenase (pmoA and mmoX) genes. Rates of CH₄ oxidation were slightly, but significantly, faster in the bog and methanotrophs belonged to the class Alphaproteobacteria and were similar to other methanotrophs of the genera Methylocystis, Methylosinus, and Methylocapsa or Methylocella detected in, or isolated from, European bogs. The fen had a greater phylogenetic diversity of organisms that had assimilated ¹³C, including methanotrophs from both the Alpha- and Gammaproteobacteria classes and other potentially non-methanotrophic organisms that were similar to bacteria detected in a UK and Finnish fen. Based on similarities between bacteria in our sites and those in Europe, including Russia, we conclude that site physicochemical characteristics rather than biogeography controlled the phylogenetic diversity of active methanotrophs and that differences in phylogenetic diversity between the bog and fen did not relate to measured CH₄ oxidation rates. A single crenarchaeon in the bog site appeared to be assimilating ¹³C in 16S rDNA; however, its phylogenetic similarity to other CO₂-utilizing archaea probably indicates that this organism is not directly involved in CH₄ oxidation in peat.     

Methanotrophic Bacteria of Acidic Sphagnum Peat Bogs

Acidic Sphagnum peat bogs cover a considerable part of the territory of Russia and are an important natural source of biogenic methane, which is formed in their anaerobic layers. A considerable portion of this methane is consumed in the aerobic part of the bog profile by acidophilic methanotrophic bacteria, which comprise the methane filter of Sphagnum peat bogs and decrease CH₄ emission to the atmosphere. For a long time, these bacteria escaped isolation, which became possible only after the elucidation of the optimal conditions of their functioning in situ: pH 4.5–5.5; temperature, from 15 to 20°C; and low salt concentration in the solution. Imitation of these conditions and rejection of earlier used media with a high content of biogenic elements allowed methanotrophic bacteria of two new genera and species—Methylocella palustris and Methylocapsa acidiphila—to be isolated from the peat of Sphagnum peat bogs of European northern Russia and western Siberia. These bacteria are well adapted to the conditions in cold, acidic, oligotrophic Sphagnum peat bogs. They grow in a pH range of 4.2–7.5 with an optimum at 5.0–5.5, prefer moderate temperatures (15–25°C) and media with a low content of mineral salts (200–500 mg/l), and are capable of active dinitrogen fixation. Design of fluorescently labeled 16S rRNA–targeted oligonucleotide probes for the detection of Methylocella palustris and Methylocapsa acidiphila and their application to the analysis of sphagnum peat samples showed that these bacteria represent dominant populations of methanotrophs with a density of 10⁵–10⁶ cells/g peat. In addition to Methylocella and Methylocapsa populations, one more abundant population of methanotrophs was revealed (10⁶ cells/g peat), which were phylogenetically close to the genus Methylocystis.     

Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.     

Microbial community composition and methanotroph diversity of a subarctic wetland in Russia

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.     

Abundance and diversity of methanotrophic Gammaproteobacteria in northern wetlands

Numeric abundance, identity, and pH preferences of methanotrophic Gammaproteobacteria (type I methanotrophs) inhabiting the northern acidic wetlands were studied. The rates of methane oxidation by peat samples from six wetlands of European Northern Russia (pH 3.9–4.7) varied from 0.04 to 0.60 μg CH₄ g^?1 peat h^?1. The number of cells revealed by hybridization with fluorochrome labeled probes M84 + M705 specific for type I methanotrophs was 0.05–2.16 × 10⁵ cells g^?1 dry peat, i.e., 0.4–12.5% of the total number of methanotrophs and 0.004–0.39% of the total number of bacteria. Analysis of the fragments of the pmoA gene encoding particulate methane monooxygenase revealed predominance of the genus Methylocystis (92% of the clones) in the studied sample of acidic peat, while the proportion of the pmoA sequences of type I methanotrophs was insignificant (8%). PCR amplification of the 16S rRNA gene fragments of type I methanotrophs with TypeIF-Type IR primers had low specificity, since only three sequences out of 53 analyzed belonged to methanotrophs and exhibited 93–99% similarity to those of Methylovulum, Methylomonas, and Methylobacter species. Isolates of type I methanotrophs obtained from peat (strains SH10 and 83A5) were identified as members of the species Methylomonas paludis and Methylovulum miyakonense, respectively. Only Methylomonas paludis SH10 was capable of growth in acidic media (pH range for growth 3.8–7.2 with the optimum at pH 5.8–6.2), while Methylovulum miyakonense 83A5 exhibited the typical growth characteristics of neutrophilic methanotrophs (pH range for growth 5.5–8.0 with the optimum at pH 6.5–7.5).     

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Methane (CH₄) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH₄ budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH₄ that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska''s interior. The water column CH₄ oxidation potential for these shallow (∼2 m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH₄, whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH₄-derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.     

Analysis of Methane Monooxygenase Genes in Mono Lake Suggests That Increased Methane Oxidation Activity May Correlate with a Change in Methanotroph Community Structure

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.     

Selection of Type I and Type II methanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions

Type II methanotrophs produce polyhydroxybutyrate (PHB), while Type I methanotrophs do not. A laboratory-scale fluidized bed reactor was initially inoculated with a Type II Methylocystis-like dominated culture. At elevated levels of dissolved oxygen (DO, 9 mg/L), pH of 6.2–6.5 with nitrate as the N-source, a Methylobacter-like Type I methanotroph became dominant within the biofilms which did not produce PHB. A shift to biofilms capable of PHB production was achieved by re-inoculating with Type II Methylosinus culture, providing dissolved N₂ as the N-source, and maintaining a low influent DO (2.0 mg/L). The resulting biofilms contained both Types I and II methanotrophs. Batch tests indicated that biofilm samples grown with N₂ became dominated by Type II methanotrophs and produced PHB. Enrichments with nitrate or ammonium were dominated by Type I methanotrophs without PHB production capability. The key selection factors favoring Type II were N₂ as N-source and low DO.     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4–2.0 × 10⁷ cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

Molecular Ecological Analysis of Methanogens and Methanotrophs in Blanket Bog Peat

Abstract Methane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences. Received: 9 March 1999; Accepted: 21 June 1999     

Methanotrophs and Methanogens in Masonry

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH₄ g of stone⁻¹ day⁻¹. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH₄ g of stone⁻¹ day⁻¹ was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.     

Culturable psychrotolerant methanotrophic bacteria in landfill cover soil

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10°C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20°C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40°C from a sample collected in May (the temperature of the cover soil was 11.5–12.5°C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S rRNA genes with the type strain SV96^T) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15°C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.     

High abundance of planctomycetes in anoxic layers of a Sphagnum peat bog

The depth distribution of planctomycete abundance has been examined in six different sites of the Sphagnum peat bog Bakchar, Tomsk oblast, Russia. In situ hybridization of peat with the fluorescently labeled oligonucleotide probes PLA46 and PLA886, reported to be group-specific for representatives of the phylum Planctomycetes, revealed two distinct population maxima of these bacteria in all of the profiles examined. The first population maximum was detected in the uppermost, oxic layer of the bog profile, while the second maximum was located at a depth of 30 cm below the water table level. The population sizes of planctomycetes in the uppermost layer and at a depth of 30 cm were of the same order of magnitude and comprised 0.5–1.5 × 10⁷ and 0.4?0.7 × 10⁷ cells per g^?1 of wet peat, respectively. Only 25–30% of the total number of planctomycete cells in the anoxic layer could be detected if the probe PLA886, whose target specificity is restricted to taxonomically characterized aerobic planctomycetes of the genera Gemmata, Planctomyces, Pirellula, and Isosphaera, was used alone. Other planctomycete cells in this layer were detected only with the probe PLA46, which possesses a much wider scope. This suggests the affiliation of these organisms with a yet undescribed phylogenetic subgroup within the Planctomycetes.