Investigation of the methanotrophic communities of the hot springs of the Uzon caldera,Kamchatka, by molecular ecological techniques

The methane-oxidizing microbial communities inhabiting the bottom sediments of 36 hot springs of the Uzon caldera (Kamchatka, Russia) located in the thermal fields Vostochnoe, Oranzhevoe, and Severnoe, as well as near the lakes Fumarol’noe and Khloridnoe and the Izvilistyi stream, were studied. Methanotrophic bacteria were detected by PCR and FISH in only 8 hot springs. The highest numbers of copies of the pmoA gene (molecular marker of methanotrophy) (2.8 × 10⁷ and 1.1 × 10⁷ copies/mL sediment) were detected in the Kul’turnyi and Kvadrat springs; however, in other springs, the numbers of the pmoA gene copies were significantly lower (5.4 × 10³–2.8 × 10⁶ copies/mL sediment). By using the FISH method, only type I methanotrophs were detected in these springs, with their percentage ranging from 0.3 to 20.5% of the total number of eubacteria. PCR-DGGE analysis of the pmoA gene showed that the diversity of methanotrophs was extremely low (no more that two components). Analysis of the deduced PmoA amino acid sequences demonstrated that methanotrophic bacteria of the genus Methylothermus, closely related to representatives of two valid species, widely occurred in the thermal springs near Lake Fumarol’noe. Other bacteria differing considerably from the detected Methylothermus species were detected as well. In the springs with low pH values (2.6–3.8), methanotrophic Gammaproteobacteria most closely related to the genera Methylomonas and Methylobacter were detected for the first time.     

Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov.

Two methanotrophic bacteria with optimum growth temperatures above 40° C were isolated. Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring. When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus (∼ 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the γ-subgroup of the Proteobacteria related to extant Type I methanotrophs. Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase. The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis. PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs. We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14L^T and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile. Received: 2 April 1997 / Accepted: 23 July 1997     

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.     

Tobermolite effects on methane removal activity and microbial community of a lab-scale soil biocover

Three identical lab-scale biocovers were packed with an engineered soil (BC 1), tobermolite only (BC 2), and a mixture of the soil and tobermolite (BC 3), and were operated at an inlet load of 338–400 g-CH₄ m^?2 d^?1 and a space velocity of 0.12 h^?1. The methane removal capacity was 293 ± 47 g-CH₄ m^?2 d^?1 in steady state in the BC 3, which was significantly higher than those in the BC 1 and BC 2 (106 ± 24 and 114 ± 48 g-CH₄ m^?2 d^?1, respectively). Quantitative PCR indicated that bacterial and methanotrophic densities (6.62–6.78 × 10⁷ 16S rDNA gene copy number g-dry sample^?1 and 1.37–2.23 × 10⁷ pmoA gene copy number g-dry sample^?1 in the BC 1 and BC 3, respectively) were significantly higher than those in the BC 2. Ribosomal tag pyrosequencing showed that methanotrophs comprised approximately 60 % of the bacterial community in the BC 2 and BC 3, while they only comprised 43 % in the BC 1. The engineered soil favored the growth of total bacteria including methanotrophs, while the presence of tobermolite enhanced the relative abundance of methanotrophs, resulting in an improved habitat for methanotrophs as well as greater methane mitigation performance in the mixture. Moreover, a batch experiment indicated that the soil and tobermolite mixture could display a stable methane oxidation level over wide temperature (20–40 °C, at least 38 μmol g-dry sample^?1 h^?1) and pH (5–8, at least 61 μmol g-dry sample^?1 h^?1) ranges. In conclusion, the soil and tobermolite mixture is promising for methane mitigation.     

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.     

Bacterial Community Structure in Two Permafrost Wetlands on the Tibetan Plateau and Sanjiang Plain,China

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10¹⁰ 16S rRNA gene copies per gram of wet soil in both wetlands, with 10⁸ pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.     

Characterization of C1-Metabolizing Prokaryotic Communities in Methane Seep Habitats at the Kuroshima Knoll, Southern Ryukyu Arc, by Analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA Genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (~34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the δ-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 × 10⁶ copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 × 10⁶ copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in γ-Proteobacteria and had a high copy number of mxaF (7.1 × 10⁶ copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 × 10² copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C₁ metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

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.     

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).     

Comparison of RNA- and DNA-based bacterial communities in a lab-scale methane-degrading biocover

Methanotrophs must become established and active in a landfill biocover for successful methane oxidation. A lab-scale biocover with a soil mixture was operated for removal of methane and nonmethane volatile organic compounds, such as dimethyl sulfide (DMS), benzene (B), and toluene (T). The methane elimination capacity was 211?±?40 g?m^?2 d^?1 at inlet loads of 330–516 g?m^?2 d^?1. DMS, B, and T were completely removed at the bottom layer (40–50 cm) with inlet loads of 221.6?±?92.2, 99.6?±?19.5, and 23.4?±?4.9 mg m^?2 d^?1, respectively. The bacterial community was examined based on DNA and RNA using ribosomal tag pyrosequencing. Interestingly, methanotrophs comprised 80 % of the active community (RNA) while 29 % of the counterpart (DNA). Types I and II methanotrophs equally contributed to methane oxidation, and Methylobacter, Methylocaldum, and Methylocystis were dominant in both communities. The DNA vs. RNA comparison suggests that DNA-based analysis alone can lead to a significant underestimation of active members.     

Thermophilic methane production and oxidation in compost

Methane cycling within compost heaps has not yet been investigated in detail. We show that thermophilic methane oxidation occurred after a lag phase of up to one day in 4-week old, 8-week old and mature (>10-week old) compost material. The potential rate of methane oxidation was between 2.6 and 4.1 micromol CH4(gdw)(-1)h(-1). Profiles of methane concentrations within heaps of different ages indicated that 46-98% of the methane produced was oxidised by methanotrophic bacteria. The population size of thermophilic methanotrophs was estimated at 10(9) cells (gdw)(-1), based on methane oxidation rates. A methanotroph (strain KTM-1) was isolated from the highest positive step of a serial dilution series. This strain belonged to the genus Methylocaldum, which contains thermotolerant and thermophilic methanotrophs. The closest relative organism on the basis of 16S rRNA gene sequence identity was M. szegediense (>99%), a species originally isolated from hot springs. The temperature optimum (45-55 degrees C) for methane oxidation within the compost material was identical to that of strain KTM-1, suggesting that this strain was well adapted to the conditions in the compost material. The temperatures measured in the upper layer (0-40 cm) of the compost heaps were also in this range, so we assume that these organisms are capable of effectively reducing the potential methane emissions from compost.     

Effect of Afforestation and Reforestation of Pastures on the Activity and Population Dynamics of Methanotrophic Bacteria

We investigated the effect of afforestation and reforestation of pastures on methane oxidation and the methanotrophic communities in soils from three different New Zealand sites. Methane oxidation was measured in soils from two pine (Pinus radiata) forests and one shrubland (mainly Kunzea ericoides var. ericoides) and three adjacent permanent pastures. The methane oxidation rate was consistently higher in the pine forest or shrubland soils than in the adjacent pasture soils. A combination of phospholipid fatty acid (PLFA) and stable isotope probing (SIP) analyses of these soils revealed that different methanotrophic communities were active in soils under the different vegetations. The C₁₈ PLFAs (signature of type II methanotrophs) predominated under pine and shrublands, and C₁₆ PLFAs (type I methanotrophs) predominated under pastures. Analysis of the methanotrophs by molecular methods revealed further differences in methanotrophic community structure under the different vegetation types. Cloning and sequencing and terminal-restriction fragment length polymorphism analysis of the particulate methane oxygenase gene (pmoA) from different samples confirmed the PLFA-SIP results that methanotrophic bacteria related to type II methanotrophs were dominant in pine forest and shrubland, and type I methanotrophs (related to Methylococcus capsulatus) were dominant in all pasture soils. We report that afforestation and reforestation of pastures caused changes in methane oxidation by altering the community structure of methanotrophic bacteria in these soils.     

Activity of a Methanotrophic Consortium Isolated from Landfill Cover Soil: Response to Temperature,pH, CO2, and Porous Adsorbent

A robust, naturally evolving methanotrophic community in landfill cover soil (LFCS) can be the simplest way to mitigate landfill methane emission. In this study, bacterial community composition in LFCS and methane oxidation potential of enriched methanotrophic consortium, in comparison to that of axenic Methylosinus sporium, was investigated. Growth and methane oxidation of the consortium was studied in liquid phase batch experiments under varying temperature (20–40°C), pH (5–10), headspace CO2, and in presence of porous adsorbent (1.3 cm³ sponge cubes). The 16S rRNA gene analysis revealed presence of both type-I and type-II methanotrophs along with few obligate methylotroph in LFCS. Though the optimal growth condition of the consortium was at 30°C and pH 7, it was more resilient in comparison to M. sporium. With increasing availability of porous adsorbent, methane consumption by the consortium was significantly improved (p < 0.001) reaching a maximum specific methane oxidation rate of 11.4 μmol mg^?1 biomass h^?1. Thus, inducing naturally thriving methanotrophs in LFCS is a better alternative to axenic methanotrophic culture in methane emission management.     

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 in cold seeps of the floodplains of northern rivers

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.     

Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments

The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.     

Thermophilic and thermotolerant aerobic methanotrophs

The review generalizes the modern data on the taxonomic, structural, and functional diversity of aerobic methanotrophs growing at 25–50°C (Methylococcus capsulatus), 30–62°C (Methylocaldum szegediense, Methylocaldum gracile, and Methylocaldum tepidum), and 50–65°C (Methylothermus thermalis), which belong mainly to the Gammaproteobacteria. The specific features of adaptation of these methanotrophs to the temperature influences are considered on the metabolic and genetic levels. The recent sensational reports on the discovery and primary characterization of thermoacidophilic methanotrophs of the phylum Verrucomicrobia surviving at extreme pH (1–2) and temperature (65°C) values, corresponding to extremely low levels of CH₄ and O₂ solubility, are analyzed. The possibilities of implementation of the biotechnological potential of thermophilic and thermotolerant methanotrophs are discussed.