Methylocella Silvestris Research Articles

Selection of methanotrophic platform for methanol production using methane and biogas

To develop biotechnological process for methane to methanol conversion, selection of a suitable methanotrophic platform is an important aspect. Systematic approach based on literature and public databases was developed to select representative methanotrophs Methylotuvimicrobium alcaliphilum, Methylomonas methanica, Methylosinus trichosporium and Methylocella silvestris. Selected methanotrophs were further investigated for methanol tolerance and methanol production on pure methane as well as biogas along with key enzyme activities involved in methane utilization. Among selected methanotrophs M.alcaliphilum showed maximum methanol tolerance of 6% v/v along with maximum methanol production of 307.90mg/L and 247.37mg/L on pure methane and biogas respectively. Activity of methane monooxygenase and formate dehydrogenase enzymes in M.alcaliphilum was significantly higher up to 98.40nmol/min/mg cells and 0.87 U/mg protein, respectively. Biotransformation trials in 14 L fermentor resulted in increased methanol production up to 418 and 331.20mg/L, with yield coefficient 0.83 and 0.71mg methanol/mg of pure methane and biogas respectively. The systematic selection resulted in haloalkaliphilic strain M.alcaliphilum as one of the potential methanotroph for bio-methanol production.

Novel methanotrophic and methanogenic bacterial communities from diverse ecosystems and their impact on environment

Methane oxidizing and methane releasing bacterial communities are very pioneer microbial population, abundantly present in several ecosystems. The microbial dynamics and metabolic assemblages are positively affected by these peculiar groups of bacteria, playing very important role in sustaining carbon geochemical cycle. A number of conventional methanotrophic and methanogenic communities are reported from marine and coastal environment, forest/desert ecosystem, paddy field soil, fresh water lakes, swamps and other soil ecosystems. In rice fields, both bacterial populations are complementary to each other. Methane gas is released by one community while utilized by another community through oxidation process. The present review describe novel species belonging to methanotrophic and methanogenic bacterial communities obtained through culture dependent and culture independent approach mingled with polyphasic taxonomic methods. The novel species of methanogens and methanotrophic bacterial communities have been isolated from different ecosystems and characterized for diverse biotechnological applications. To name a few, Methylobacterium phyllosphaerae sp. nov., Methanococcus aeolicus sp. nov. and Methylocella silvestris sp. nov. have been isolated from paddy field, marine environment and forest ecosystem respectively. The present review deals with biodiversity of novel species of methanogens and methanotrophs and their impact on environment.

Genomic Determinants of Phototrophy in Methanotrophic Alphaproteobacteria

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.

Facultative methanotrophs are abundant at terrestrial natural gas seeps

BackgroundNatural gas contains methane and the gaseous alkanes ethane, propane and butane, which collectively influence atmospheric chemistry and cause global warming. Methane-oxidising bacteria, methanotrophs, are crucial in mitigating emissions of methane as they oxidise most of the methane produced in soils and the subsurface before it reaches the atmosphere. Methanotrophs are usually obligate, i.e. grow only on methane and not on longer chain alkanes. Bacteria that grow on the other gaseous alkanes in natural gas such as propane have also been characterised, but they do not grow on methane. Recently, it was shown that the facultative methanotroph Methylocella silvestris grew on ethane and propane, other components of natural gas, in addition to methane. Therefore, we hypothesised that Methylocella may be prevalent at natural gas seeps and might play a major role in consuming all components of this potent greenhouse gas mixture before it is released to the atmosphere.ResultsEnvironments known to be exposed to biogenic methane emissions or thermogenic natural gas seeps were surveyed for methanotrophs. 16S rRNA gene amplicon sequencing revealed that Methylocella were the most abundant methanotrophs in natural gas seep environments. New Methylocella-specific molecular tools targeting mmoX (encoding the soluble methane monooxygenase) by PCR and Illumina amplicon sequencing were designed and used to investigate various sites. Functional gene-based assays confirmed that Methylocella were present in all of the natural gas seep sites tested here. This might be due to its ability to use methane and other short chain alkane components of natural gas. We also observed the abundance of Methylocella in other environments exposed to biogenic methane, suggesting that Methylocella has been overlooked in the past as previous ecological studies of methanotrophs often used pmoA (encoding the alpha subunit of particulate methane monooxygenase) as a marker gene.ConclusionNew biomolecular tools designed in this study have expanded our ability to detect, and our knowledge of the environmental distribution of Methylocella, a unique facultative methanotroph. This study has revealed that Methylocella are particularly abundant at natural gas seeps and may play a significant role in biogeochemical cycling of gaseous hydrocarbons.

Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

Methanotrophs are a specialized group of bacteria that can utilize methane (CH4) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1) The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris; (2) Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata; (3) The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris. These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata.

The Influence of Micro-Oxygen Addition on Desulfurization Performance and Microbial Communities during Waste-Activated Sludge Digestion in a Rusty Scrap Iron-Loaded Anaerobic Digester

In this study, micro-oxygen was integrated into a rusty scrap iron (RSI)-loaded anaerobic digester. Under an optimal RSI dosage of 20 g/L, increasing O2 levels were added stepwise in seven stages in a semi-continuous experiment. Results showed the average methane yield was 306 mL/g COD (chemical oxygen demand), and the hydrogen sulphide (H2S) concentration was 1933 ppmv with RSI addition. O2 addition induced the microbial oxidation of sulphide by stimulating sulfur-oxidizing bacteria and chemical corrosion of iron, which promoted the generation of FeS and Fe2S3. In the 6th phase of the semi-continuous test, deep desulfurization was achieved without negatively impacting system performance. Average methane yield was 301.1 mL/g COD, and H2S concentration was 75 ppmv. Sulfur mass balance was described, with 84.0%, 11.90% and 0.21% of sulfur present in solid, liquid and gaseous phases, respectively. The Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis revealed that RSI addition could enrich the diversity of hydrogenotrophic methanogens and iron-reducing bacteria to benefit methanogenesis and organic mineralization, and impoverish the methanotroph (Methylocella silvestris) to reduce the consumption of methane. Micro-oxygen supplementation could enhance the diversity of iron-oxidizing bacteria arising from the improvement of Fe(II) release rate and enrich the sulphur-oxidising bacteria to achieved desulfurization. These results demonstrated that RSI addition in combination with micro-oxygenation represents a promising method for simultaneously controlling biogas H2S concentration and improving digestion performance.

Biological Methanol Production by a Type II Methanotroph Methylocystis bryophila.

Methane (CH₄) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH₄ can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH₄; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30°C, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl₂ as a methanol dehydrogenase inhibitor, 50% CH₄ concentration, 24 h of incubation, and 9 mg of dry cell mass ml(-1) inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH₄.

Identification and characterization of trimethylamine N-oxide (TMAO) demethylase and TMAO permease in Methylocella silvestris BL2.

Methylocella silvestris, an alphaproteobacterium isolated from a forest soil, can grow on trimethylamine N-oxide (TMAO) as a sole nitrogen source; however, the molecular and biochemical mechanisms underpinning its growth remain unknown. Marker-exchange mutagenesis enabled the identification of several genes involved in TMAO metabolism, including Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting of an N-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO → 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Tdm of M. silvestris and eukaryotic Tdms have no sequence homology and contrasting characteristics. Recombinant Tdm of M. silvestris appears to be hexameric, has a high affinity for TMAO (Km = 3.3 mM; Vmax = 21.7 nmol min(-1) mg(-1) ) and only catalyses demethylation of TMAO and a structural homologue, dimethyldodecylamine N-oxide. Our study has contributed to the understanding of the genetic and biochemical mechanisms for TMAO degradation in M. silvestris.

Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris

The climate-active gas methane is generated both by biological processes and by thermogenic decomposition of fossil organic material, which forms methane and short-chain alkanes, principally ethane, propane and butane. In addition to natural sources, environments are exposed to anthropogenic inputs of all these gases from oil and gas extraction and distribution. The gases provide carbon and/or energy for a diverse range of microorganisms that can metabolize them in both anoxic and oxic zones. Aerobic methanotrophs, which can assimilate methane, have been considered to be entirely distinct from utilizers of short-chain alkanes, and studies of environments exposed to mixtures of methane and multi-carbon alkanes have assumed that disparate groups of microorganisms are responsible for the metabolism of these gases. Here we describe the mechanism by which a single bacterial strain, Methylocella silvestris, can use methane or propane as a carbon and energy source, documenting a methanotroph that can utilize a short-chain alkane as an alternative to methane. Furthermore, during growth on a mixture of these gases, efficient consumption of both gases occurred at the same time. Two soluble di-iron centre monooxygenase (SDIMO) gene clusters were identified and were found to be differentially expressed during bacterial growth on these gases, although both were required for efficient propane utilization. This report of a methanotroph expressing an additional SDIMO that seems to be uniquely involved in short-chain alkane metabolism suggests that such metabolic flexibility may be important in many environments where methane and short-chain alkanes co-occur.

The (d)evolution of methanotrophy in the Beijerinckiaceae—a comparative genomics analysis

The alphaproteobacterial family Beijerinckiaceae contains generalists that grow on a wide range of substrates, and specialists that grow only on methane and methanol. We investigated the evolution of this family by comparing the genomes of the generalist organotroph Beijerinckia indica, the facultative methanotroph Methylocella silvestris and the obligate methanotroph Methylocapsa acidiphila. Highly resolved phylogenetic construction based on universally conserved genes demonstrated that the Beijerinckiaceae forms a monophyletic cluster with the Methylocystaceae, the only other family of alphaproteobacterial methanotrophs. Phylogenetic analyses also demonstrated a vertical inheritance pattern of methanotrophy and methylotrophy genes within these families. Conversely, many lateral gene transfer (LGT) events were detected for genes encoding carbohydrate transport and metabolism, energy production and conversion, and transcriptional regulation in the genome of B. indica, suggesting that it has recently acquired these genes. A key difference between the generalist B. indica and its specialist methanotrophic relatives was an abundance of transporter elements, particularly periplasmic-binding proteins and major facilitator transporters. The most parsimonious scenario for the evolution of methanotrophy in the Alphaproteobacteria is that it occurred only once, when a methylotroph acquired methane monooxygenases (MMOs) via LGT. This was supported by a compositional analysis suggesting that all MMOs in Alphaproteobacteria methanotrophs are foreign in origin. Some members of the Beijerinckiaceae subsequently lost methanotrophic functions and regained the ability to grow on multicarbon energy substrates. We conclude that B. indica is a recidivist multitroph, the only known example of a bacterium having completely abandoned an evolved lifestyle of specialized methanotrophy.

好氧甲烷氧化菌生态学研究进展

PDF HTML阅读 XML下载 导出引用 引用提醒 好氧甲烷氧化菌生态学研究进展 DOI: 10.5846/stxb201207171013 作者: 作者单位: 中国科学院研究生院,中国科学院研究生院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金资助项目（41271277/D010504） Ecology of aerobic methane oxidizing bacteria (methanotrophs) Author: Affiliation: Graduate university of Chinese Academy of Sciences,Graduate university of Chinese Academy of Sciences Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:好氧甲烷氧化菌是以甲烷为碳源和能源的细菌。好氧甲烷氧化菌在自然环境中分布广泛，人类已从土壤、淡水和海洋沉积物、泥炭沼泽、热泉、海水和南极环境分离到甲烷氧化菌的纯培养。好氧甲烷氧化菌可分为14个属，包括研究较为深入的隶属于变形菌门Alpha和Gamma纲的细菌，以及属于疣微菌门的极端嗜热嗜酸甲烷氧化菌。最近，好氧甲烷氧化菌还被发现存在于苔藓类植物（尤其是泥炭苔藓）共生体中，兼性营养好氧甲烷氧化菌也被发现。通过对好氧甲烷氧化菌的分类、生理生化特征、分子生物学检测方法以及微生物生态学中的研究成果的总结与分析，以及对甲烷氧化菌研究所面临的问题进行讨论，以期为今后进一步开展好氧甲烷氧化菌及其在碳循环中的作用研究提供参考。 Abstract:Aerobic methane oxidizing bacteria (methanotrophs) are a fascinating group of bacteria that have the unique ability to grow on methane as their sole carbon and energy source. They appear to be widespread in nature and have been isolated from a number of different environments. There are now 14 recognized genera of methanotrophs belong to two phyla, Proteobacteria and thermoacidiphilic Verrucomicrobia.The former was well studied and separated into two classes, TypeⅠ and TypeⅡ methanotrophs, which belong to Alpha and Gamma Proteobacteria. Extremely thermophilic, acidophilic methanotrophs from the phylum Verrucomicrobia have been isolated, thus expanding both the taxonomic diversity and physiological range of aerobic methanotrophy. The discovery of the facultative methanotroph Methylocella silvestris has changed the view that methanotrophs were obligate organism. They can cooxidize a considerable number of organic compounds and also have considerable potential in biotechnology. A wide variety of methanotrophic symbionts in and on the mosses were recently detected, and showing the global prevalence of this symbiosis. Traditional way used cultivation to enrichment or isolation to study methanotrophs in the environment. Molecular ecology techniques applied in the last few decades have greatly expanded our knowledge of methanotroph ecology. The most obvious marker for detecting methanotrophs in various environments is the 16S rRNA gene, due to the large database of sequences available. Primers and probes targeting different genera or species have been designed and used extensively in combination with polymerase chain reaction (PCR) based clone library analysis, denaturing gradient gel electrophoresis (DGGE) analysis, and fluorescent in situ hybridization (FISH) analysis. Several functional genes have also been used for the detection of methanotrophs in environmental samples, including pmoA (encoding the key subunits of particulate methane monooxygenase), mmoX (encoding the key subunits of soluble methane monooxygenase), mxaF (encoding the key subunits of methanol dehydrogenase), nifH (encoding the dinitrogenase reductase), and genes involved in C1 transfer pathways. To understand the active community of methanotrophs in the environment, stable isotope probing (SIP) techniques have been developed, including DNA-SIP, RNA-SIP, mRNA-SIP, and phospholipid fatty acid (PLFA)-SIP. SIP has also been combined with metagenomics to discover novel methanotrophs. Other very powerful molecular techniques have been developed in the last few years, including microautoradiography (MAR)-FISH, isotope array, Raman-FISH, nano-secondary ion mass spectrometry (NanoSIMS), and microfluidic digital PCR, these techniques can now be used in the analyses of methanotrophs. Both cultivation and cultivation independent molecular methods have been used intensively in last few decades to study the diversity, distribution, and abundance in environments of methanotrophs, such as soils, freshwater, marine sediments, acid peat bogs, hot springs, seawater and extreme environments. In the microcosm of soil, the growth and diversity of methanotrophs are also influenced by several environmental factors. This review highlights recent progress in the research of the taxonomy, of the discovery of novel aerobic methanotrophs, of the biochemistry, of the molecular techniques and the environment impacts on methanotrophs, we also emphasize deficiencies and issues need to be solved in future studies. This review will provide theoretical foundation for future methanotrophic ecology study and explain the key role methanotrophs play in carbon cycle. 参考文献 相似文献 引证文献

Comparison of One- and Two-dimensional Liquid Chromatography Approaches in the Label-free Quantitative Analysis of Methylocella silvestris

The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase-reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.

Acetate Repression of Methane Oxidation by Supplemental Methylocella silvestris in a Peat Soil Microcosm

Methylocella spp. are facultative methanotrophs that grow on methane and multicarbon substrates, such as acetate. Acetate represses transcription of methane monooxygenase of Methylocella silvestris in laboratory culture. DNA stable-isotope probing (DNA-SIP) using (13)C-methane and (12)C-acetate, carried out with Methylocella-spiked peat soil, showed that acetate also repressed methane oxidation by Methylocella in environmental samples.

γ-Glutamylmethylamide Is an Essential Intermediate in the Metabolism of Methylamine by Methylocella silvestris

Methylocella silvestris BL2, a facultative methane utilizer, can grow on monomethylamine (MMA) as a sole carbon and nitrogen source. No activity of MMA dehydrogenase was detectable. Instead, this bacterium utilizes a methylated amino acid pathway (gamma-glutamylmethylamide [GMA] and N-methylglutamate [NMG]) for MMA metabolism. The activities of the two key enzymes in this pathway, GMA synthetase and NMG dehydrogenase, were found when the bacterium was grown on MMA. GMA was detected by high-performance liquid chromatography-mass spectrometry only when the bacterium was grown on MMA but not when it was grown on methanol. Proteomic analysis of soluble and membrane fractions of the proteome from MMA- and methanol-grown cultures revealed that an eight-gene cluster (Msil2632 to Msil2639) was induced by MMA and cotranscribed as an operon, as shown by reverse transcription-PCR. GMA-dissimilating enzyme activity was also detected when it was grown on MMA. Formaldehyde and ammonium production from GMA was dependent on glutamate but not on alpha-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (gmas, Msil2635) within this eight-gene cluster, with a kanamycin gene cassette, abolished growth of M. silvestris on MMA as either a sole carbon or a sole nitrogen source. Overall, our results suggest that gmas is essential in MMA metabolism by M. silvestris.

A Comparison of Labeling and Label-Free Mass Spectrometry-Based Proteomics Approaches

The proteome of the recently discovered bacterium Methylocella silvestris has been characterized using three profiling and comparative proteomics approaches. The organism has been grown on two different substrates enabling variations in protein expression to be identified. The results obtained using the experimental approaches have been compared with respect to number of proteins identified, confidence in identification, sequence coverage and agreement of regulated proteins. The sample preparation, instrumental time and sample loading requirements of the differing experiments are compared and discussed. A preliminary screen of the protein regulation results for biological significance has also been performed.

Facultative Methanotrophs Revisited

Facultative Methanotrophs Revisited

Aerobic methanotrophic bacteria of cold ecosystems

This review summarizes the recent advances in understanding the ecophysiological role and structure–function features of methanotrophic bacteria living in various cold ecosystems. The occurrence of methanotrophs in a majority of psychrosphere sites was verified by direct measurement of their methane-utilizing activity, by electron microscopy and immunofluorescent observations, and analyses of specific signatures in cellular phospholipids and total DNAs extracted from environmental samples. Surprisingly, the phenotypic and genotypic markers of virtually all extant methanotrophs were detected in various cold habitats, such as underground waters, Northern taiga and tundra soils, polar lakes and permafrost sediments. Also, recent findings indicated that even after long-term storage in permafrost, some methanotrophs can oxidize and assimilate methane not only at positive but also at subzero temperatures. Pure cultures of psychrophilic and psychrotolerant methanotrophs were isolated and characterized as new genera and species: Methylobacter psychrophilus, Methylosphaera hansonii, Methylocella palustris, Methylocella silvestris, Methylocella tundrae, Methylocapsa acidiphila and Methylomonas scandinavica. However, our knowledge about their adaptive mechanisms and survival in cold ecosystems remains limited and needs to be established using both traditional and molecular microbiological methods.

Methylocella tundrae sp. nov., a novel methanotrophic bacterium from acidic tundra peatlands.

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 degrees C (optimum 15 degrees C). The major phospholipid fatty acid is 18:1omega7c. 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.

Methylocella silvestris sp. nov., a novel methanotroph isolated from an acidic forest cambisol.

Two strains of Gram-negative, aerobic, non-pigmented, non-motile, rod-shaped, methane-oxidizing bacteria were isolated from an acidic forest cambisol near Marburg, Germany, and were designated as strains BL2(T) and A1. These bacteria were morphologically and phenotypically similar to Methylocella palustris K(T). The cells possess a highly specific bipolar appearance. They lack the intracytoplasmic membranes common to all methane-oxidizing bacteria except Methylocella, but contain a vesicular membrane system connected to the cytoplasmic membrane. A soluble methane monooxygenase was present, but no particulate methane monooxygenase could be detected. These bacteria utilize the serine pathway for carbon assimilation. Strains BL2(T) and A1 are moderately acidophilic, mesophilic organisms capable of growth at pH values between 4.5 and 7 (with an optimum at pH 5.5) and at temperatures between 4 and 30 degrees C. Compared with Methylocella palustris K(T), these strains have greater tolerance of cold temperatures, dissolved salts and methanol. On the basis of 16S rRNA gene sequence identity, of species with validly published names, strain BL2(T) is most closely related to Methylocella palustris K(T) (97.3 % identity), Beijerinckia indica subsp. indica ATCC 9039(T) (97.1 %) and Methylocapsa acidiphila B2(T) (96.2 %). The DNA G+C content is 60 mol% and the major phospholipid fatty acid is 18 : 1omega7. Strain BL2(T) showed only 21-22 % DNA-DNA hybridization with Methylocella palustris K(T). The data therefore suggest that strains BL2(T) and A1 represent a novel species of Methylocella; the name Methylocella silvestris sp. nov. is proposed, with strain BL2(T) (=DSM 15510(T)=NCIMB 13906(T)) as the type strain.