Isolation of a methane-oxidizing bacterium that bioremediates hexavalent chromium from a formerly industrialized Suburban River.

Sediment samples were taken from sediment adjacent to a suburban river in Sheffield in Northern England that had suffered heavy metal pollution due to previous activity of the steel industry (between the 17th and 19th centuries). The most abundant heavy metals found in the samples were lead, chromium, nickel, arsenic and cobalt, with maximum concentrations of 412·80, 25·232, 25·196, 8·123 and 7·66 mg kg-1 , respectively. Enrichment cultures were set up using methane as carbon and energy source, as a result of which a strain of methanotroph was isolated that was shown via 16S rRNA gene sequencing to be a strain Methylomonas koyamae and given the designation SHU1. M. koyamae SHU1 removed hexavalent chromium from an initial concentration of 10 ppm, which was inhibited by the metabolic inhibitor sodium azide or the methane monooxygenase inhibitor phenylacetylene. To the authors' knowledge, this is the first description of a strain of the widely environmentally distributed genus Methylomonas that is capable of remediating hexavalent chromium. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic methanotrophic bacteria are known for bioremediation of an increasing range of organic and inorganic pollutants, using methane as carbon and energy source. Previously, one laboratory methanotroph strain, Methylococcus capsulatus Bath, was known to bioremediate toxic chromium (VI) by reducing it to chromium (III). Here, a newly isolated methanotroph strain, Methylomonas koyamae SHU1, has been shown able to remediate chromium (VI). This indicates that chromium (VI) bioremediation is not unique to M. capsulatus and moreover adds weight to the suggestion that methanotrophs may contribute directly to chromium (VI) detoxification in nature and in polymicrobial bioremediation fed with methane.

A comparative transcriptome analysis of the novel obligate methanotroph Methylomonas sp. DH-1 reveals key differences in transcriptional responses in C1 and secondary metabolite pathways during growth on methane and methanol.

BACKGROUND: Methanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain. RESULTS: We integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden-Meyerhof-Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH. CONCLUSIONS: This study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries.

A novel methanotroph in the genus Methylomonas that contains a distinct clade of soluble methane monooxygenase.

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2-97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.

Synthesizing value-added products from methane by a new Methylomonas.

AIMS: Methane and methanol are potential carbon sources of industrial micro-organisms in addition to crop-derived bio-carbon sources. Methanotrophs that can utilize these simple, stable and large amounts chemicals are expected to be developed into 'cell factories' for the production of specific chemicals. In this study, a methanotroph that can synthesize lycopene, C30 carotenoid and exopolysaccharides (EPS) with relative better performances from C1 substrates was isolated, and its performances were evaluated. METHODS AND RESULTS: The isolated strain was identified as Methylomonas sp. ZR1 based on 16S rRNA sequence analysis. Its maximum specific growth rate achieved 0·200 h-1 under flask culture conditions, and 0·386 h-1 in bubble column reactors. ZR1 was able to utilize 35 g l-1 of methanol and even exhibited slight growth in the presence of 40 g l-1 of methanol. Furthermore, ZR1 was proved to synthesize lycopene (C40 carotenoids) besides the C30 carotenoids through HPLC-DAD and HPLC-MS/MS analysis methods. Its carotenoid extracts exhibited excellent antioxidative activities measured by the ABTS+ method. Plenty of polysaccharides were also synthesized by ZR1, the components of the polysaccharides were identified as glucose, mannose and galactose with a proportion of 1 : 2 : 1 by GC-MS, and its yield achieved 0·13 g g-1 cell dry weight. CONCLUSIONS: The isolated strain has great potential for the production of value-added bioproducts from C1 compounds because of its excellent C1 substrate utilizing abilities and its abilities to naturally synthesize lycopene, C30 carotenoids and EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a fast-growing methanotroph, its C1 carbon substrate utilizing ability is excellent in comparison with reported methanotrophs. Furthermore, besides polysaccharides and C30 carotenoids which were commonly synthesized by methanotrophs, our findings suggested that C40 lycopene could also be naturally synthesized from methane by methanotrophs.

Enrichment culture and identification of endophytic methanotrophs isolated from peatland plants.

Aerobic methane-oxidizing bacteria (MOB) are an environmentally significant group of microorganisms due to their role in the global carbon cycle. Research conducted over the past few decades has increased the interest in discovering novel genera of methane-degrading bacteria, which efficiently utilize methane and decrease the global warming effect. Moreover, methanotrophs have more promising applications in environmental bioengineering, biotechnology, and pharmacy. The investigations were undertaken to recognize the variety of endophytic methanotrophic bacteria associated with Carex nigra, Vaccinium oxycoccus, and Eriophorum vaginatum originating from Moszne peatland (East Poland). Methanotrophic bacteria were isolated from plants by adding sterile fragments of different parts of plants (roots and stems) to agar mineral medium (nitrate mineral salts (NMS)) and incubated at different methane values (1-20% CH4). Single colonies were streaked on new NMS agar media and, after incubation, transferred to liquid NMS medium. Bacterial growth dynamics in the culture solution was studied by optical density-OD600 and methane consumption. Changes in the methane concentration during incubation were controlled by the gas chromatography technique. Characterization of methanotrophs was made by fluorescence in situ hybridization (FISH) with Mg705 and Mg84 for type I methanotrophs and Ma450 for type II methanotrophs. Identification of endophytes was performed after 16S ribosomal RNA (rRNA) and mmoX gene amplification. Our study confirmed the presence of both types of methanotrophic bacteria (types I and II) with the predominance of type I methanotrophs. Among cultivable methanotrophs, there were different strains of the genus Methylomonas and Methylosinus. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged from 0.463 ± 0.067 to 5.928 ± 0.169 µmol/L CH4/mL/day.

Production of uracil from methane by a newly isolated Methylomonas sp. SW1.

Methane is an abundant, inexpensive one-carbon feedstock and one of the most powerful greenhouse gases. Because it does not compete with food demand, it is considered a promising carbon feedstock for the production of valuable products using methanotrophic bacteria. Here, we isolated a novel methanotrophic bacterium, Methylomonas sp. SW1, from a sewage sample obtained from Wonju City Water Supply Drainage Center, Republic of Korea. The conditions for uracil production by Methylomonas sp. SW1, such as Cu2+ concentration and temperature were investigated and optimized. As a result, Methylomonas sp. SW1 produced uracil from methane as a sole carbon source with a titer of 2.1mg/L in 84h without genetic engineering under the optimized condition. The results in this study demonstrate the feasibility of using Methylomonas sp. SW1 for the production of uracil from methane. This is the first report of uracil production from gas feedstock by methanotrophic bacteria.

Methylomonas lenta sp. nov., a methanotroph isolated from manure and a denitrification tank.

Two methanotrophic bacteria, strains R-45377(T) and R-45370, were respectively isolated from a slurry pit of a cow stable and from a denitrification tank of a wastewater treatment plant in Belgium. The strains showed 99.9 % 16S rRNA gene sequence similarity. Cells were Gram-negative, motile rods containing type I methanotroph intracytoplasmic membranes. Colonies and liquid cultures appeared white to pale pink. The pmoA gene encoding particulate methane monooxygenase (pMMO) and the nifH gene encoding nitrogenase were present. Soluble methane monooxygenase (sMMO) activity, the presence of the mmoX gene encoding sMMO and the presence of the pxmA gene encoding a sequence-divergent pMMO were not detected. Methane and methanol were utilized as sole carbon sources. The strains grew optimally at 20 °C (range 15-28 °C) and at pH 6.8-7.3 (range pH 6.3-7.8). The strains grew in media supplemented with up to 1.2 % NaCl. The major cellular fatty acids were C16 : 1ω8c, C16 : 1ω5c, C16 : 1ω7c, C14 : 0, C15 : 0 and C16 : 0 and the DNA G+C content was 47 mol%. 16S rRNA gene- and pmoA-based phylogenetic analyses showed that the isolates cluster among members of the genus Methylomonas within the class Gammaproteobacteria, with pairwise 16S rRNA gene sequence similarities of 97.5 and 97.2 % between R-45377(T) and the closest related type strains, Methylomonas scandinavica SR5(T) and Methylomonas paludis MG30(T), respectively. Based on phenotypic characterization of strains R-45377(T) and R-45370, their low 16S rRNA gene sequence similarities and the formation of a separate phylogenetic lineage compared with existing species of the genus Methylomonas, we propose to classify these strains in a novel species, Methylomonas lenta sp. nov., with R-45377(T) ( = LMG 26260(T) = JCM 19378(T)) as the type strain.

Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas.

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.

Methylomonas paludis sp. nov., the first acid-tolerant member of the genus Methylomonas, from an acidic wetland.

An aerobic methanotrophic bacterium was isolated from an acidic (pH 3.9) Sphagnum peat bog in north-eastern Russia and designated strain MG30(T). Cells of this strain were Gram-negative, pale pink-pigmented, non-motile, thick rods that were covered by large polysaccharide capsules and contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme (pMMO) and utilized only methane and methanol. Carbon was assimilated via the ribulose-monophosphate pathway; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strain MG30(T) was able to grow at a pH range of 3.8-7.3 (optimum pH 5.8-6.4) and at temperatures between 8 and 30 °C (optimum 20-25 °C). The major cellular fatty acids were C16:1ω5t, C16:1ω8c, C16:1ω7c and C14:0; the DNA G+C content was 48.5 mol%. The isolate belongs to the family Methylococcaceae of the class Gammaproteobacteria and displayed 94.7-96.9% 16S rRNA gene sequence similarity to members of the genus Methylomonas. However, strain MG30(T) differed from all taxonomically characterized members of this genus by the absence of motility, the ability to grow in acidic conditions and low DNA G+C content. Therefore, we propose to classify this strain as representing a novel, acid-tolerant species of the genus Methylomonas, Methylomonas paludis sp. nov. Strain MG30(T) (=DSM 24973(T)=VKM B-2745(T)) is the type strain.

Methylomonas koyamae sp. nov., a type I methane-oxidizing bacterium from floodwater of a rice paddy field.

A novel methane-oxidizing bacterium, strain Fw12E-Y(T), was isolated from floodwater of a rice paddy field in Japan. Cells of strain Fw12E-Y(T) were Gram-negative, motile rods with a single polar flagellum and type I intracytoplasmic membrane arrangement. The strain grew only on methane or methanol as sole carbon and energy source. It was able to grow at 10-40 °C (optimum 30 °C), at pH 5.5-7.0 (optimum 6.5) and with 0-0.1% (w/w) NaCl (no growth above 0.5% NaCl). 16S rRNA gene sequence analysis showed that strain Fw12E-Y(T) is related most closely to members of the genus Methylomonas, but at low levels of similarity (95.0-95.4%). Phylogenetic analysis of pmoA and mxaF genes indicated that the strain belongs to the genus Methylomonas (97 and 92 % deduced amino acid sequence identities to Methylomonas methanica S1(T), respectively). The DNA G+C content of strain Fw12E-Y(T) was 57.1 mol%. Chemotaxonomic data regarding the major quinone (MQ-8) and major fatty acids (C(16:1) and C(14:0)) also supported its affiliation to the genus Methylomonas. Based on phenotypic, genomic and phylogenetic data, strain Fw12E-Y(T) is considered to represent a novel species of the genus Methylomonas, for which the name Methylomonas koyamae sp. nov. is proposed. The type strain is Fw12E-Y(T) ( = JCM 16701(T) = NBRC 105905(T) = NCIMB 14606(T)).

Complete genome sequence of the aerobic marine methanotroph Methylomonas methanica MC09.

Methylomonas methanica MC09 is a mesophilic, halotolerant, aerobic, methanotrophic member of the Gammaproteobacteria, isolated from coastal seawater. Here we present the complete genome sequence of this strain, the first available from an aerobic marine methanotroph.

[Comparative characterization of cultured methane-oxidizing bacteria by serological and molecular methods].

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.

Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling.

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.

Analysis of fae and fhcD genes in Mono Lake, California.

Genes for two enzymes of the tetrahydromethanopterin-linked C(1) transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).

[Physicochemical and biological factors affecting atmospheric methane oxidation in gray forest soils].

The decline of methane oxidizing activities in gray forest soil upon its conversion into arable land was shown to be caused by major changes in biotic and physicochemical properties of soil. Using the method of immune serums, methane-oxidizing bacteria were detected in both forest and agricultural soils, but their populations differed significantly in both abundance and composition. In the forest soil, the number of methanotrophs was an order of magnitude higher than in arable soil, amounting to 3.5 x 10(8) and 0.24 x 10(8) cells/g soil, respectively. All methane-oxidizing bacteria identified in the forest soil belonged to the genus Methylocystis, and 94% of these were represented by a single species, M. parvus. The arable soil was dominated by type I methanotrophs (Methylobacter and Methylomonas, 67.6%), occurring along with bacteria of the genus Methylocystis. In addition, arable soil is characterized by a low content of microbial biomass, lower porosity and water permeability of soil aggregates, and the predominance of nitrogen mineralization processes over those of nitrogen immobilization. These factors can also contribute to lower rates of methane oxidation in arable soil as compared to forest soil.

[Methanotrophic communities in the soils of Russian northern taiga and subarctic tundra]. / Metanotrofnye soobshchestva pochv severnoi taigi i subarkticheskoi tundry Rossii.

The PCR analysis of DNA extracted from soil samples taken in Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved alpha-subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the alpha-subunit of particulate methane monooxygenase, and the mxaF gene encoding the alpha-subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.

Molecular characterization of methanotrophic isolates from freshwater lake sediment.

Profiles of dissolved O(2) and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 micromol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed for mmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.

Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.

Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden.

Methane-utilizing bacteria were enriched from deep igneous rock environments and affiliated by amplification of functional and phylogenetic gene probes. Type I methanotrophs belonging to the genera Methylomonas and Methylobacter dominated in enrichment cultures from depths below 400 m. A pure culture of an obligate methanotroph (strain SR5) was isolated and characterized. Pink-pigmented motile rods of the new isolate contained intracytoplasmic membranes as stacks of vesicles, assimilated methane via the ribulose monophosphate pathway and had an incomplete tricarboxylic acid cycle. Phosphatidyl glycerol, methylene ubiquinone and cytochrome c552 were prevailing. The DNA G+C content is 53.3 mol %. Strain SR5 grew at temperatures between 5 and 30 degrees C with optimum at 15 degrees C, close to its in situ temperature. Analyses of 16S rRNA gene, whole cell protein, enzymatic and physiological analyses of strain SR-5 revealed significant differences compared to the other representatives of Type I methanotrophs. Based on pheno- and genotypic characteristics we propose to refer the strain SR5 as to a new species, Methylomonas scandinavica.