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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P. R. China
2 Graduate School, Chinese Academy of Sciences, Beijing 100049, P. R. China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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ABSTRACT |
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-subunit of methyl-coenzyme M reductase, and of the deduced amino acids of mcrA, between strain 8AcT and Methanosaeta concilii DSM 3671T were 36 and 78·9 %, respectively. Based on the phylogenetic and phenotypic analyses, the novel species Methanosaeta harundinacea sp. nov. is proposed, with strain 8AcT (=JCM 13211T=CGMCC 1.5026T) as the type strain.
Published online ahead of print on 9 September 2005 as DOI 10.1099/ijs.0.63887-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Methanosaeta harundinacea 8AcT and 6Ac are AY817738 and AY970347, respectively, and those for the partial sequences of mcrA of Methanosaeta harundinacea 8AcT and 6Ac are AY970348 and AY970349, respectively.
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). All described acetate-using methanogens belong to the order Methanosarcinales. Among these, Methanosaeta species can be distinguished from members of the other genera by their exclusive use of acetate as a substrate for producing methane and by their rod-shaped cells, instead of coccoid or pseudosarcinal cells such as those of Methanosarcina species. In addition, in contrast to Methanosarcina species, Methanosaeta strains are favoured in environments with low concentrations of acetate (Huser et al., 1982). To date, only two species have been described in the genus Methanosaeta, Methanosaeta concilii (Patel & Sprott, 1990) and ‘Methanosaeta thermophila’ (Kamagata et al., 1992; Boone & Kamagata, 1998), although investigations based on culture-independent approaches have indicated that Methanosaeta strains occur widely (Lueders et al., 2001; Zengler et al., 1999). Ultrastructural analysis of granules from upflow anaerobic sludge blanket (UASB) reactors using scanning and transmission electron microscopy has also shown numerous Methanosaeta-like cells, implying that they could play important roles in methanogenesis (Guiot et al., 1992; MacLeod et al., 1990; Gonzalez-Gil et al., 2001). During a survey of the microbial community of a mesophilic methane-producing UASB reactor in Beijing, China, we isolated two strains of rod-shaped methanogens that produced methane exclusively from acetate. Phylogenetically, the strains were affiliated to the genus Methanosaeta; however, they were distantly related to the existing species of the genus. Moreover, their cells were rod-shaped instead of in a filament. Based on the phylogenetic and phenotypic data, a novel species of Methanosaeta is proposed.
Pre-reduced basal medium was prepared as described by Huser et al. (1982) and sodium acetate (20 mM), yeast extract (0·05 %, w/v) and peptone (0·05 %, w/v) were added to form complete medium. The medium was dispensed in screw-capped tubes sealed with butyl rubber stoppers and the gas phase was N2/CO2 (80 : 20, 1·01x105 Pa) for routine cultivation unless indicated. All inoculations and transfers were done with syringes and needles and all cultures were incubated at 37 °C in the dark.
For enrichment of the acetate-using methanogenic culture, UASB granules for treating beer-manufacture wastewater in Beijing were inoculated and suspended with a vortex, and then serially diluted in complete medium containing 0·5 g penicillin l–1 (final concentration) to inhibit bacterial growth. Methanogenic dilution cultures were verified by methane production and acetate consumption after cultivation for a few weeks. This manipulation was repeated several times until pure cultures were obtained. Culture purity was confirmed periodically by monitoring cell morphology using normal bright-field microscopy, as well as by lack of growth in a rich medium, such as peptone/yeast extract/glucose liquid.
Cells of strain 8AcT in exponential growth phase were used for morphological examination under a transmission electron microscope (H-600A; Hitachi), after being coated with palladium/iridium alloy with a high vacuum evaporator (HUS-5GB; Hitachi). Cell motility was observed with a phase-contrast microscope (BH-2; Olympus). Cells from an exponential-phase culture were incubated in 1 % SDS and distilled water as a hypotonic solution to check the susceptibility to lysis (Boone & Whitman, 1988) and with proteinase K (50 µg ml–1) to break the cell-enclosure sheath. Cell lysis or sheath breakage was determined microscopically, with untreated cells as a control. Physiological studies, such as substrate utilization, growth factor requirement, pH range, growth temperature range and NaCl tolerance were performed as described previously (Ma et al., 2005). Methane production and substrate degradation were measured as described by Zhang et al. (2004). Formate cleavage was determined by measuring formate after culture for 20 days in basal medium containing formate (10 mM). Formate was detected at 242 nm by HPLC (Waters model 1525). A reverse-phase HPLC (ODS Hypersil; Hewlett Packard) mobile phase of acetonitrile/water (30 : 70, v/v) was used at a flow rate of 0·7 ml min–1.
Genomic DNA extraction and purification were performed according to Jarrell et al. (1992), with a 1 l culture. The G+C content was determined by using the thermal denaturation method (Owen & Pitcher, 1985), with Escherichia coli K-12 as the reference, with a UV800 spectrophotometer (Beckman).
The 16S rRNA gene was amplified from the genomic DNA as described previously (Furlong et al., 2002). A partial fragment of the gene encoding the -subunit of methyl-coenzyme M reductase (mcrA) was amplified according to Lueders et al. (2001). Purified PCR products of the 16S rRNA gene of 1400 bp in length and a partial fragment of mcrA of 500 bp in length were cloned into the vector pUCm-T, and sequenced by Bioasia Company (Beijing).
Sequences in GenBank similar to those of the 16S rRNA gene and mcrA partial fragment were determined using the BLASTN algorithm. The best matching sequences were retrieved from the database and aligned and similarity analysis was performed with CLUSTAL_X (Thompson et al., 1997). Phylogenetic trees were constructed using MEGA 2.1 software (Kumar et al., 2001).
Cells of strains 8AcT and 6Ac were rod-shaped (0·8–1·0x3–5 µm; Fig. 1) and occurred singly or in pairs; however, at high cell density when grown in high concentrations of acetate (>100 mM), the cells were arranged as a chain within a common sheath. Cells of strains 8AcT and 6Ac were non-motile, Gram-variable and resisted disruption by 1 % SDS (w/v) or hypotonic solution. However, cell-chain sheaths were partly destroyed by proteinase K (50 µg ml–1), indicating a proteinaceous composition. The two strains were strictly anaerobic and growth was completely inhibited in air. Acetate was the only substrate tested to support growth and methane production, at concentrations of acetate of 2–350 mM. None of the following substrates tested was used for growth or methane production: H2/CO2, methanol, ethanol, trimethylamine, isobutanol and isopropanol (each 20 mM). Moreover, formate was not cleaved to hydrogen and carbon dioxide. Yeast extract (0·05–1 %, w/v) or peptone (0·05–1 %, w/v) was required for growth, whereas vitamins were not essential, in contrast to some Methanosaeta strains, for which yeast extract might be inhibitory (Touzel et al., 1988). Growth of strains 8AcT and 6Ac was observed at 25–45 °C, with the fastest growth occurring at 34–37 °C. The pH range for growth was 6·5–9·0, with the fastest growth at pH 7·2–7·6. The specific growth rate of strain 8AcT in complete medium at 37 °C was 0·030 h–1 (doubling time 28 h), whereas the specific growth rate in acetate-containing basal medium with addition of yeast extract, peptone or tryptone (0·05 %, w/v) was 0·016, 0·013 and 0·0045 h–1, respectively. The G+C content of the genomic DNA of strain 8AcT was 55·7 mol%.
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) showed 99·5 % similarity between the two novel strains 8AcT and 6Ac, indicating that they represented a single species; however, similarities of only 92·5 and 87·3 % were obtained between strain 8AcT and Methanosaeta concilii DSM 3013 and ‘Methanosaeta thermophila’ DSM 6194, respectively. In addition to 16S rRNA gene sequence analysis, mcrA gene sequences have also been used as a phylogenetic tool for the order Methanosarcinales; similarity levels among different species of the same genus range from 87·5 to 93·8 % (Springer et al., 1995). A phylogenetic tree (Fig. 3) was constructed based on deduced amino acid sequences of the mcrA gene, including those of strain 8AcT and related methanogens. The similarities of the partial fragments of mcrA and deduced amino acid sequences between strain 8AcT and Methanosaeta concilii DSM 3671T were 36 and 78·9 %, respectively. Phylogenetic analysis based on either the 16S rRNA gene or mcrA indicated that strain 8AcT represented a novel species of the genus Methanosaeta. In addition, strain 8AcT also differed from other species in cell shape and G+C content. Differential phenotypic characteristics of strain 8AcT and other Methanosaeta species are shown in Table 1.
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Description of Methanosaeta harundinacea sp. nov.
Methanosaeta harundinacea (ha.run.di.na'ce.a. L. fem. adj. harundinacea like a reed, referring to the cell shape of a reed stem).
Cells are rod-shaped (0·8–1·0x3–5 µm), occurring singly or in pairs; however, they are arranged as a chain within a common sheath at high cell density when grown in high concentrations of acetate (>100 mM). Gram-variable. Non-motile. Cells are resistant to lysis by 1 % SDS (w/v) and hypotonic solution, whereas the sheath of the long chain is partly destroyed by proteinase K (50 µg ml–1). Methanogenic. Acetate is the exclusive tested substrate for growth and methanogenesis. No growth on or methane production from H2/CO2, formate, methanol, ethanol, trimethylamine, isobutanol or isopropanol. Formate is not cleaved to hydrogen and carbon dioxide. Yeast extract or peptone are required for growth, but not vitamins. The temperature range for growth is 25–45 °C, with optimal growth at 34–37 °C. The pH range for growth is 6·5–9·0 and the optimum pH is 7·2–7·6. The DNA G+C content of the type strain is 55·7 mol% (Tm).
The type strain, 8AcT (=JCM 13211T=CGMCC 1.5026T), was isolated from granules from a UASB anaerobic digester treating beer-manufacture wastewater.
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ACKNOWLEDGEMENTS |
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