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1 Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany
2 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia
3 Institute of Microbiology, Russian Academy of Sciences, Moscow 117811, Russia
Correspondence
Peter F. Dunfield
dunfield{at}staff.uni-marburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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7. Strain BL2T showed only 21–22 % DNA–DNA hybridization with Methylocella palustris KT. The data therefore suggest that strains BL2T and A1 represent a novel species of Methylocella; the name Methylocella silvestris sp. nov. is proposed, with strain BL2T (=DSM 15510T=NCIMB 13906T) as the type strain.
Published online ahead of print on 13 January 2003 as DOI 10.1099/ijs.0.02481-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the partial gene sequences of mmoX, mxaF and nifH of Methylocella silvestris strain BL2T are respectively AJ491847–AJ491850.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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- and 
-subclasses of the Proteobacteria; these are respectively designated as type I or type II methanotrophs. The two types differ physiologically in several ways, most notably in their carbon-assimilation pathways [ribulose monophosphate (RuMP) versus serine pathway] and the arrangement of intracytoplasmic membranes (ICMs) (Hanson & Hanson, 1996
).
The existence of methanotrophs adapted to high or low extremes of temperature, pH and salinity has been demonstrated by cultivation-independent molecular ecology techniques and by the isolation and characterization of pure cultures (Murrell et al., 1998; Trotsenko & Khmelenina, 2002). Much of the Earth's surface, including most wetlands and forest soils of the temperate and boreal zones, is acidic. Recently, novel acidophilic methanotrophs were isolated from Sphagnum peat bogs and described as the new genera and species Methylocella palustris (Dedysh et al., 1998, 2000) and Methylocapsa acidiphila (Dedysh et al., 2002). These isolates grow optimally at about pH 5·5. They belong to the -subclass of the Proteobacteria but do not form a monophyletic group with the previously known type II methanotrophs of the genera Methylosinus and Methylocystis. Instead, they are closely related to the acidophilic, non-methanotrophic bacterium Beijerinckia indica. Methylocapsa acidiphila B2T, like most other known methanotrophs, possesses the particulate methane monooxygenase enzyme (pMMO) and contains a well-developed ICM system in which pMMO is bound. Methylocella palustris does not contain an ICM system, which makes it unique among methanotrophs. The genes encoding the pMMO enzyme, which are present in all other methanotrophs, could not be detected in Methylocella palustris with either a PCR assay considered universal for the pmoA gene or hybridization with a pmoA gene fragment of Methylococcus capsulatus Bath. Methylocella palustris, therefore, appears to possess a soluble form of methane monooxygenase (sMMO) only.
Here, we describe a third organism belonging to this acidophilic group of methanotrophs. This organism was isolated from acidic forest soil rather than from Sphagnum peat.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Intact cores were sealed at the bottom and placed in a gas-tight chamber containing 20 % (v/v) CH4 in air. The headspace in the chamber was replaced periodically.
After 6 months, soil crumbs from the mineral A horizon (8–13 cm from the soil surface) of the cores were sprinkled on plates of dilute nitrate mineral salts (DNMS) medium. This was nitrate mineral salts medium, as described previously for the isolation of methanotrophic bacteria (Hanson et al., 1991), diluted 1 : 5 with distilled water and containing 1 mM NaH2PO4/Na2HPO4 buffer (pH 5·8). DNMS was solidified with Bacto agar (15 g l-1; Difco). After incubation for 2 months in an atmosphere containing 20 % CH4, slimy, semi-transparent masses developed around most soil crumbs on the plates. These were carefully picked with a loop and restreaked onto fresh DNMS plates. Single colonies were successively selected from plates for restreaking until pure cultures were obtained. Two such cultures were designated BL2T and A1.
Reference bacterial strains.
Methylocella palustris KT (=ATCC 700799T), Methylocapsa acidiphila B2T (=DSM 13967T=NCIMB 13765T) and B. indica subsp. indica ATCC 9039T were used as reference strains. Methylocapsa acidiphila B2T was grown on half-strength nitrogen-free M1 medium (Dedysh et al., 2002). Methylocella palustris KT was cultivated on M1 medium supplemented with 250 mg KNO3 l-1 and solidified with 1·5 % (w/v) agar. Both methanotrophs were grown under a gas phase of 10 % (v/v) CH4 in air. B. indica subsp. indica ATCC 9039T was grown on nitrogen-free mineral medium containing glucose (Becking, 1984).
Verification of strain purity.
Culture purity was ensured by examination under phase-contrast and electron microscopy and by plating on media containing the following different organic substrates: nutrient agar (Difco), nutrient agar diluted 1 : 10 or DNMS supplemented with either 0·1 % (w/v) sucrose or glucose. Plates were incubated in an atmosphere not supplemented with CH4. To ensure detection of slowly growing heterotrophic satellites, growth was assessed after incubation for 4 weeks at 25 °C.
Finally, DNA was extracted from two separate batches of strain BL2T and PCR-amplified partial fragments of the 16S rRNA gene were analysed by denaturing gradient gel electrophoresis to determine whether only a single gene product was detectable in the cultures. All steps were performed as described previously (Henckel et al., 1999) except that the PCR mixtures all contained premix F (Epicenter Technologies) instead of 1·5 mM MgCl2, and the denaturing gradient gel electrophoresis gel was run at 150 V for 6 h.
Morphological observations.
Cell morphology was examined in batch cultures grown in liquid DNMS for 2 weeks (in vials sealed with butyl-rubber stoppers and injected with CH4 at 10 %, v/v). The presence of cysts or exospores was examined using stationary-phase liquid and plate cultures (up to 4–5 months old) (Dedysh et al., 2000). The Gram-stain reaction and lysis in 3 % (w/v) KOH (Gerhardt, 1981) were performed on both 1- and 3-week-old cultures. Lysis by 0·2 and 2 % (w/v) SDS was determined by direct microscopic observation.
Electron microscopy.
Cells were collected by centrifugation and fixed for 1 h at 4 °C in 0·15 M cacodylate buffer (pH 7·3) containing 0·1 % (w/v) ruthenium red and 1·2 % (w/v) glutaraldehyde. Cells were then washed three times with 0·15 M cacodylate buffer (pH 7·3) by centrifugation (3 min at 3000 g). Cells were additionally fixed for 3 h at 20 °C in a solution containing 1·5 % (w/v) OsO4 and 0·1 % (w/v) ruthenium red in 0·05 M cacodylate buffer (pH 7·3) and then washed with 0·15 M cacodylate buffer (pH 7·3). Thin-sectioning, staining and transmission electron microscopy were performed as described previously (Dedysh et al., 2000).
Physiological tests.
To test the pH range, DNMS containing 1·25 mM phosphate buffer was adjusted to pH 4–7·5 (in steps of 0·5 pH units) with H3PO4. Duplicate 120 ml serum vials (40 ml medium) at each pH were capped with butyl-rubber stoppers and injected with 10 % (v/v) CH4. The OD600 was measured in an Eppendorf BioPhotometer at 2-day intervals for 2 weeks. Growth at temperatures from 4 to 45 °C and tolerance of NaCl concentrations from 0·01 to 1 % (w/v) were also determined in liquid DNMS cultures monitored for 2 weeks (Dedysh et al., 2000).
Nitrogen fixation was assayed using the acetylene-reduction procedure of Takeda (1988). Batch cultures in nitrogen-free DNMS medium containing 0·5 % (v/v) methanol were assayed in the mid-exponential growth phase. Incubations with acetylene were performed under both aerobic and microaerobic conditions, using the protocol described for Methylocella palustris (Dedysh et al., 2000).
Range of utilizable carbon and nitrogen sources.
The range of potential growth substrates was examined using 0·05 % (w/v) concentrations of the following carbon sources: formate, formamide, methylamine, dimethylamine, trimethylamine, glucose, fructose, sucrose, lactose, galactose, xylose, sorbose, maltose, raffinose, arabinose, ribose, lactate, oxalate, citrate, mannitol and sorbitol. The capacity to utilize methanol at concentrations from 0·01 to 6 % (v/v) was determined in DNMS liquid medium supplemented with methanol (vials were capped with butyl-rubber stoppers to prevent loss of methanol by vaporization). Nitrogen sources were tested by replacing KNO3 in DNMS with 0·05 % (w/v) NaNO2, NH2OH, (NH4)2SO4, NH4Cl, glycine, L-alanine, L-serine, L-isoleucine, L-proline, L-cystine, L-methionine, L-glutamine, L-histidine, L-arginine, L-lysine or yeast extract. Growth was examined, after incubation for 30 days, in comparison with a negative control (i.e. growth on nitrogen-free DNMS medium).
Preparation of cell-free extract.
Cells grown under CH4 (1–2 weeks) were harvested by centrifugation (15 000 g, 20 min) and washed in 0·05 M Tris/HCl buffer (pH 7·2) containing 5 mM MgCl2. Cells (1 g fresh weight) were resuspended in 5 ml of the same buffer; 1 ml of a solution containing 1 mg lysozyme (Sigma) was added and the resulting mixture was incubated for 3 h at 20 °C. The suspension was centrifuged (15 000 g, 20 min), washed in 0·05 M Tris/HCl buffer (pH 7·2) containing 5 mM MgCl2 and 0·1 mM PMSF, resuspended in the same buffer, sonicated (3x 30 s) and centrifuged (15 000 g, 40 min). The soluble fraction was used to measure enzyme activities.
Enzyme assays.
Enzymic studies were performed as described for Methylocella palustris KT (Dedysh et al., 2000). To test for sMMO activity, naphthol formation from naphthalene was monitored with tetrazotized o-dianisidine according to the procedure of Phelps et al. (1992). sMMO activity was measured in whole cells grown in either copper-deficient medium (with no added copper) or at copper excess (2·5 µM Cu2+). MMO activity was also estimated as the velocity of radioactivity incorporation from 14CH4 into both cells and CO2. Cells were grown to an OD600 of 0·3–0·5, harvested by centrifugation (10 000 g, 20 min), washed in 10 mM potassium phosphate buffer (pH 6·5) and then resuspended in the same buffer containing 10 mM sodium formate. Aliquots (1 ml) of the cell suspension were placed into 15 ml vials, which were capped and sealed; 100 µl 14CH4 (2 µCi µmol-1) was injected into the gas phase and the vials were incubated at 20 °C. After 10, 20 or 30 min, 1 ml 2 M NaOH was added with a syringe to stop all metabolic processes and to absorb all 14CO2 evolved. After 1 day, 0·2 ml culture samples were taken, placed onto Whatman GF/F glass-fibre papers (1·5x1·5 cm) and air-dried. Papers were rinsed with 0·2 ml distilled water and dried again. Radioactivity was measured on an Intertechnique SL-30 liquid scintillation spectrometer.
Comparative sequence analysis.
DNA to be used in PCRs was extracted from strains BL2T and A1 using a mechanical disruption procedure (Henckel et al., 1999). PCR-mediated amplification of the 16S rRNA gene from position 28 to 1491 (numbering according to the International Union of Biochemistry nomenclature for Escherichia coli 16S rRNA), as well as of a partial fragment of the mxaF gene, was carried out as described previously (Dedysh et al., 2000). Partial fragments of the mmoX gene (encoding a subunit of sMMO) and of the nifH gene (encoding dinitrogenase reductase H) were amplified using primers and PCR programs described, respectively, by Auman et al. (2000) and Auman et al. (2001). In each case, PCR mixtures contained 0·5 µM each primer, 1x Premix F (Epicenter Technologies) and 1 U Taq polymerase (QBioGene). Sequencing was performed as described previously (Dedysh et al., 2000) and comparative phylogenetic analysis was carried out using the ARB program package (Strunk & Ludwig, 1996).
DNA base composition and DNA–DNA hybridization.
Cultures of strain BL2T and Methylocella palustris KT were grown on DNMS plates for 8 weeks. Cells were separated from their capsular matrix by a combination of heating to 50 °C and vigorous shaking. DNA extraction and DNA–DNA hybridization of the two strains were performed as described by Martin et al. (1997). The G+C content of the DNA was analysed as described by Groth et al. (1996).
Analysis of fatty acid methyl esters.
Fatty acid methyl esters were extracted from cells and analysed by GC as described by Kämpfer & Kroppenstedt (1996).
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RESULTS |
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Cell morphology and ultrastructure
Cells of strains BL2T and A1 were Gram-negative, non-motile, short, slightly curved rods, 0·6–0·8 µm in width and 1·2–1·5 µm in length. They reproduced by normal cell division. Similarly to Methylocella palustris, the cells had a distinctive bipolar appearance which was more or less pronounced depending on the culture conditions. Cells occurred singly or as aggregates in which each cell was separated from the others by the capsular material. This material stained with ruthenium red (Luft, 1964) and was therefore polysaccharidic in nature (Fig. 1a–c). The formation of rosettes was not observed. Although both plate and liquid cultures up to 5 months old were thoroughly examined, no spores or other resting stages were observed.
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). Ovoid vesicles were occasionally combined into chains (Fig. 1e). It is possible that these ovoid vesicles originated through division of the long tube-shaped vesicles. All three types of vesicle (flattened, tube-shaped and ovoid) were bounded by three-layered membranes, and each contained a homogeneous matrix of lower electron density with respect to the cytoplasm. In methanol-grown cells, small ovoid vesicles combined into chains on the cell periphery were predominant (Fig. 1a, b). These ICM structures were similar to those of Methylocella palustris (Dedysh et al., 2000), except that the vesicles in Methylocella palustris were smaller and more spherical. Methanol-grown cells contained one large inclusion of poly-
-hydroxybutyrate per cell (Fig. 1f).
Culture purity
Both isolates displayed a uniform cell morphology in cultures of different age. No growth was observed either on any of the complex organic plating media tested or on various multicarbon substrates in liquid culture. Finally, only a single 16S rRNA gene product (as determined by denaturing gradient gel electrophoresis analysis) could be amplified by PCR from DNA of cultures of strain BL2T.
Physiological characteristics
Strains BL2T and A1 grew on methane or methanol as the sole carbon and energy source. Slow growth was also observed on ammonium formate and on methylamine. In contrast to Methylocella palustris, which did not grow at methanol concentrations above 0·3 %, the isolates from forest soil utilized methanol at a wide range of concentrations (0·01–5 %). The optimum was between 0·5 and 2 %. Cells grown at higher methanol concentrations (3–5 %) had large capsules up to 5–8 µm in diameter. Growth did not occur on the other C1 or multicarbon compounds tested. Growth factors were not required. Both strains utilized ammonium salts, nitrates and yeast extract as nitrogen sources. Weak growth also occurred when L-alanine, L-serine, L-cystine, L-glutamine and L-proline were provided as nitrogen sources. Although the isolates were also capable of growth on nitrogen-free agar medium, they grew poorly under aerobic conditions in liquid nitrogen-free medium. Acetylene-reduction activity by strains BL2T and A1 was detectable only in microaerobic conditions and ranged from 4·0 to 5·5 nmol C2H4 (mg dry biomass)-1 h-1. The amplification of a nifH gene from strain BL2T provides additional evidence that the genetic potential for nitrogen fixation is present.
The pH of the medium was altered by the growth of the culture, so the strict pH dependence was difficult to determine. However, no growth occurred at pH 4 or 7·5. The fastest growth occurred at pH 5·5 and, after growth for 2 weeks in media with initial pH values between 5 and 6·5, the final pH tended to be about 5·8. The temperature range for growth was 4–30 °C, with the optimum at 15–25 °C. No growth occurred at 37 °C. The specific growth rate of culture under CH4 (10 %, v/v), calculated from increases in OD600 in the exponential phase (10 days) of growth (a mean of 10 vials), was 0·33 day-1 (equal to a doubling time of 2·1 days). Like Methylocella palustris, the forest isolates grew better on diluted media, but the optimum salt concentration was about twice as high (0·4–1·0 g salts l-1) (Dedysh et al., 2000). Growth was inhibited completely by NaCl concentrations above 0·8 %.
Cellular fatty acid profiles
Overall, the cellular fatty acid composition of strain BL2T was quite similar to those of Methylocella palustris KT and Methylocapsa acidiphila B2T (Table 1). The distinguishing features of the phospholipid fatty acid profile of strain BL2T compared with Methylocella palustris KT were the presence of the i17 : 0, i18 : 0 and 19 : 0 fatty acids and a lower total content of C16 fatty acids.
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Cell-free extracts of strain BL2T displayed activities of serine-pathway-specific enzymes (hydroxypyruvate reductase and serine-glyoxylate aminotransferase) but not of the key RuMP-cycle enzyme hexulosephosphate synthase or of key enzymes of the ribulose 1,5-bisphosphate cycle (ribulose bisphosphate carboxylase/oxygenase and phosphoribulokinase) (Table 2). Activity of 2-oxoglutarate dehydrogenase was also detected, indicating that the complete tricarboxylic acid cycle operates in this methanotroph. However, glyoxylate-cycle enzymes were absent. Pyrophosphate-dependent 6-phosphofructokinase and fructose-1,6-bisphosphate aldolase were present. Also, high activities of the pentose-phosphate-cycle enzymes (glucose-6-phosphate and 6-phosphogluconate dehydrogenases) were measured in cell extracts. In general, the metabolic pattern of BL2T is similar to those of Methylocella palustris and Methylocapsa acidiphila (Dedysh et al., 2000, 2002).
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) indicated that strains BL2T and A1 belong to the -subclass of the Proteobacteria. The closest neighbours are Methylocella palustris KT (2·7 % sequence difference), B. indica subsp. indica ATCC 9039T (2·9 %) and Methylocapsa acidiphila B2T (3·8 %). The DNA–DNA hybridization value of strain BL2T with its closest phylogenetic neighbour (Methylocella palustris KT) was therefore determined and was found to be only 21–22 %. DNA–DNA hybridization was not carried out with B. indica because this bacterium is a non-methanotrophic heterotroph. This critical phenotypic difference alone is sufficient to rule out the inclusion of the isolates in the genus Beijerinckia.
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). The partial mxaF nucleotide sequences of strains BL2T and A1 differed from that of Methylocella palustris KT by 18·8–20·5 % (14·9–15·9 % derived amino acid sequence difference) and it was not evident from mxaF sequence-based phylogeny that they were closer phylogenetically to Methylocella palustris than to other methylotrophic bacteria (data not shown).
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), A189/mb661r (Costello & Lidstrom, 1999) and A189/A648b (Dunfield et al., 2002). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2000). The isolation of Methylocella strains BL2T and A1 from a forest soil extends the known phylogenetic and ecological diversity of this genus. The forest strains were phenotypically and phylogenetically most similar to Methylocella palustris among known bacteria. However, minor phenotypic differences were evident between Methylocella palustris KT and strain BL2T, sequence-based phylogenetic identities were low compared with identities among other closely related methanotrophic genera and the DNA–DNA hybridization was only 21–22 %, indicating that the forest strains should be considered as representing a novel species (Wayne et al., 1987; Stackebrandt & Goebel, 1994).
Bacteria of the genus Methylocella lack an extensive ICM system and appear to consume methane primarily via sMMO instead of the pMMO that is associated with the ICM systems of other methanotrophs. The sMMO activity in strain BL2T was only partially decreased by a level of copper (2·5 µM) that completely inhibited sMMO activity in Methylosinus trichosporium OB3bT (Morton et al., 2000). It is reasonable that inhibition or repression of sMMO should not occur in Methylocella strains if they do not contain pMMO as an alternative to sMMO. However, we cannot rule out the presence of pMMO in strain BL2T. In 14CH4 experiments, MMO activity remained constant under conditions that lowered sMMO activity by 50 %.
In contrast to alkaliphilic species, which are represented mostly by type I methanotrophs and grow well at high external salinities (Trotsenko & Khmelenina, 2002), acidophilic methanotrophs grow only in media with a low dissolved salt content. This interesting feature deserves further study, which may lead to a better understanding of the main principles of adaptation and survival of acidophilic and alkaliphilic methanotrophs.
The cambisol from which strains BL2T and A1 were obtained has a pH of 3·8–4·3. This is typical of most forested soils of the temperate zone, so it is possible that members of the genus Methylocella are widespread geographically, in terrestrial soil as well as in wetlands. The methanotroph community of this cambisol has been characterized using cultivation-independent molecular methods (Henckel et al., 2000). This study failed to detect methanotrophs related to Methylocella species. However, the primer systems used targeted only (i) the pmoA gene, which is not detectable in strain BL2T, and (ii) the 16S rRNA gene, from which only the most abundant members of the entire bacterial community are expected to be retrieved. Cultivation of Methylocella strains adds to the known diversity of the methanotroph community in this forest soil; it also demonstrates the method-inherent limitations of cultivation-independent techniques in the exhaustive characterization of a microbial community. Cultivation-independent analysis did identify a pmoA sequence cluster somewhat related to the pmoA of Methylocapsa acidiphila B2T (Henckel et al., 2000), leading to the hypothesis that more, as yet uncultivated, acidophilic methanotrophs exist.
Forest soils periodically produce methane in anaerobic microsites, especially when water-saturated (e.g. Yavitt et al., 1990; Wang & Bettany, 1997; Andersen et al., 1998), and are therefore a potential habitat for methanotrophic bacteria. These soils also consume methane from the atmosphere, but it is unlikely that methanotrophs possessing only sMMO are responsible for this activity. The pMMO form of the enzyme has a higher affinity for methane than does sMMO (Hanson & Hanson, 1996) and is a more probable candidate for the consumption of the trace (1·7 p.p.m.v.) level of CH4 in the atmosphere. When batch cultures of strain BL2T were cultivated in closed flasks, the threshold CH4 concentration below which no more consumption occurred was always >100 p.p.m.v. (data not shown). On the basis of the growth conditions used, it is therefore unlikely that strains BL2T and A1 are capable of surviving on, or even of transiently consuming, atmospheric methane. We propose that these strains live on methanol or on methane produced in anaerobic soil microsites.
Emended description of Methylocella Dedysh et al. 2000
Methylocella (Me.thy.lo.cel'la. N.L. n. methylum the methyl group; L. n. cella a chamber, a cell; N.L. n. Methylocella methyl-using cell).
Cells are Gram-negative, polymorphic, straight or curved short rods, 0·6–1·0 µm wide by 1·0–2·5 µm long, with rounded ends. Produce large, highly refractile, intracellular poly--hydroxybutyrate granules, one at each pole. Reproduce by normal cell division. Cells occur singly or in irregularly shaped aggregates, but do not form rosettes. Non-motile. Encapsulated. Resting cells (if they occur) are exospores. Cells are not lysed by 2 % SDS. The ICM system is different from those of both types I and II methanotrophic bacteria. Cells contain an extensive periplasmic space and a vesicular membrane system connected to the cytoplasmic membrane. Possess sMMO. The temperature range for growth is 4–30 °C with an optimum at 15–25 °C; no growth occurs at 37 °C. Growth occurs at pH 4·5–7·0. Highly sensitive to salt stress: prefer diluted media with a low salt content. Utilize C1 compounds via the serine pathway. Do not contain the enzymes of the RuMP and ribulose 1,5-bisphosphate pathways. The tricarboxylic acid cycle is complete. Fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. The major phospholipid fatty acids are 18 : 1 acids. The G+C content of the DNA is 60–61 mol%. Phylogenetically related to, but clearly distinct from, the type II methanotroph genera Methylocystis and Methylosinus in the -subclass of the Proteobacteria; more closely affiliated with the acidophilic methanotrophic bacterium Methylocapsa acidiphila and the acidophilic heterotrophic bacterium B. indica. Distinguished from B. indica by the ability to consume methane. Habitats are acidic wetlands, particularly Sphagnum peat bogs, and acidic soils. The type species is Methylocella palustris.
Description of Methylocella silvestris sp. nov.
Methylocella silvestris (sil.ves'tris. L. adj. silvestris of the forest).
The description is as for the genus, with the following additional traits. Optimal growth occurs at 15–25 °C and at pH 5·5. Capable of slow growth at 4 °C. Carbon sources used include methane, methanol and methylamines. Utilizes methanol in a wide concentration range, from 0·01 to 5 % (v/v). NaCl inhibits growth at concentrations above 0·8 % (w/v). The type strain is strain BL2T (=DSM 15510T=NCIMB 13906T), which was isolated from an acidic cambisol under a beech-dominated forest near Marburg, Germany. The species also includes strain A1.
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ACKNOWLEDGEMENTS |
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