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1 Biogas Institute of Ministry of Agriculture, Chengdu 610041, PR China
2 Center for Environmental Biotechnology, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China
Correspondence
Hui Zhang
zhanghuits{at}yahoo.com.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and mcrA gene sequences of strain ZC-1T are DQ787474 and EF026570, respectively.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), members of which grow with methanol plus H2. The family Methanosarcinaceae currently includes eight genera: Methanosarcina, Methanococcoides, Methanohalobium, Methanohalophilus, Methanolobus, Methanosalsum, Methanimicrococcus and Methanomethylovorans (Garrity & Holt, 2001; Jiang et al., 2005; Sprenger et al., 2000). Methanimicrococcus blatticola is unusual in the family because it requires both a methyl compound and H2, similar to members of the genus Methanosphaera (Sprenger et al., 2000). Members of the family Methanosaetaceae that ferment acetate as the sole source of energy include Methanosaeta concilii (Patel & Sprott, 1990), Methanothrix thermophila (Boone & Kamagata, 1998; Kamagata & Mikami, 1991) and Methanosaeta harundinacea (Ma et al., 2006). Work in our laboratory has focused on the study of anaerobes, especially methanogens, of petroleum reservoirs. Several hydrogenotrophic and methylotrophic methanogens have been isolated and identified in previous studies. Here, we describe a novel thermophilic, methylotrophic methanogen, strain ZC-1T.
Strain ZC-1T was isolated from oil-production water of block L801 in Shengli oilfield, where the oil reservoir is located 1680–1800 m below the sea floor and has a pressure of 9.24 MPa. The in situ temperature of the reservoir ranges from 75 to 80 °C and the total mineralized degree is 9794 mg l–1. Crude oil was recovered by injecting seawater and then aerobic microbial cultures. Enrichments were performed with anaerobic medium in vials sealed with butyl-rubber stoppers and aluminium caps according to Ollivier et al. (1997), modified by reducing the concentrations of MgCl2 . 6H2O and CaCl2 . 2H2O to 3 and 0.14 g l–1, respectively. The pH was adjusted to 7.0 by using 10 M KOH before autoclaving, and Na2S . 9H2O and NaHCO3 were injected from sterile stock solutions to final concentrations of 0.03 and 0.5 %, respectively, before inoculation. The gas phase consisted of H2/CO2 (80/20, v/v) at a pressure of 200 kPa. After incubation at 55 °C in the dark for 2 weeks, a large amount of methane was detected. Fresh inoculum (5 ml) was then transferred anaerobically into a new bottle of sterile M141 medium (DSMZ, 1993) with methanol (120 mmol l–1) under H2/CO2 (4/1, v/v). Ampicillin (1 mg ml–1) was added to inhibit the growth of non-methanogenic organisms. Positive cultures were diluted serially and single colonies were obtained by the Hungate roll-tube method (Hungate, 1969). The purity of isolates was checked by phase-contrast microscopy after cultivation in an enriched medium and identified medium in the absence of antibiotics (Zhang & Zhao, 1987; Zhao et al., 1986).
An Olympus BH-2 phase-contrast microscope and Olympus OM-2 camera were used routinely to observe cells. An Amray-1000B scanning electron microscope and a Hitachi-600VI transmission electron microscope were used to observe the microstructure of strain ZC-1T (Lai & Chen, 2001; Mikucki et al., 2003). Gram reaction was determined and susceptibility tests were performed as described by Boone & Whitman (1988), except that the Gram reaction was determined in PBS buffer. Strain ZC-1T occurred in clusters of two to four cells. Cysts formed during the early exponential phase, but dispersed at stationary phase (Fig. 1a, b). Cells were motile cocci, 0.7–1.0 µm in diameter (Fig. 1b, c). Most cells lysed during the Gram-staining procedure; the residual cells stained positive. Cells also lysed in SDS (0.01 %, w/v), demineralized water and TE buffer after 30 min at room temperature, but they did not lyse in PBS (0.2 M, pH 7.4) or NaCl solution (0.9 %, w/v). These results were similar to those of cells with a proteinaceous wall; the presence of such a cell wall for strain ZC-1T was confirmed by electron microscopy of ultrathin sections (Fig. 1d). Strain ZC-1T formed colonies after around 20 days incubation at 60 °C, at which time the surface colonies in agar roll tubes were about 1–5 mm in diameter, yellow, smooth, circular and convex with entire edges. Colonies fluoresced blue–green under UV light.
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. Potential growth-stimulating compounds were tested in basal medium [KCl, 0.34 g; NH4Cl, 0.25 g; K2HPO4, 0.2 g; NaCl, 24 g; MgCl2 . 6H2O, 10.2 g; yeast extract (Oxoid), 2 g; resazurin, 0.001 g; cysteine/HCl . H2O, 0.5 g; distilled water, 1 l; pH approx. 7.0], with addition of sludge fluids (5 ml) or coenzyme M (HS-coM; 0.0025 g) as appropriate. Cells of strain ZC-1T were strictly anaerobic and used only methanol, methylamine and trimethylamine as substrates for methanogenesis. No growth or methanogenesis was observed on sodium formate, sodium acetate, ethanol, dimethylsulfide, secondary alcohols, H2/CO2 or H2/CO. Strain ZC-1T did not grow when MgCl2 or both yeast extract and trypticase were removed from the M141 medium. Growth was much slower with trypticase than with yeast extract in basal medium (data not shown). The MgCl2 concentration for good growth ranged from 0.05 to 0.20 M. Sludge fluid and HS-coM stimulated growth dramatically. After 156 h incubation, total methane production reached 0.41 mM with HS-coM, whereas the control reached just 0.04 mM. Cells also grew faster when sludge fluid was added (data not shown). To investigate the sensitivity of strain ZC-1T to antibiotics, kanamycin, streptomycin, ampicillin, chloramphenicol, rifampicin (all at 200 µg ml–1) and erythromycin (125 and 500 µg ml–1) were tested in basal medium with methanol (120 mM) at 65 °C. Growth was determined from total methane production. Growth of strain ZC-1T was inhibited completely by chloramphenicol (200 µg ml–1) and erythromycin (500 µg ml–1); kanamycin (200 µg ml–1) and erythromycin (125 µg ml–1) caused partial inhibition of growth. The other antibiotics had no effects on the growth of strain ZC-1T.
Effects of pH, temperature and NaCl concentration on strain ZC-1T were determined in triplicate in basal medium with methanol (120 mM). Specific growth rates were measured according to Powell (1983). Gradient pH was adjusted according to Takai et al. (2002). Strain ZC-1T grew between pH 5.5 and 8.0; no growth was observed at pH 5.0 or 8.5. The temperature range for growth was between 50 and 70 °C; growth was not observed at 45 or 75 °C. Strain ZC-1T required 0.1–1.1 M NaCl for growth. Optimal growth, with a doubling time of around 5 h, occurred at pH 6.0–6.5, 65 °C, 0.3–0.5 M NaCl and 0.05–0.20 M MgCl2.
Genomic DNA was isolated and purified by using the method of Marmur (1961) as modified by Jarrell et al. (1992), and its G+C content was determined by the thermal-denaturation method (Marmur & Doty, 1962), using a Beckman DU 800 spectrophotometer. Escherichia coli K12 DNA was used as a reference. The G+C content of the genomic DNA of strain ZC-1T was 56 mol%, which was higher than those of members of the genus Methanosarcina, but similar to those of members of the genus Methanosaeta.
The protocol for PCR amplification was as follows: a single colony was dissolved in 100 µl sterile demineralized water for 10 min at 99 °C and centrifuged at 10 000 r.p.m. in a microfuge for 5 min. Supernatant (5 µl) served as the DNA template. The 16S rRNA and mcrA genes were amplified with a TaKaRa Thermocycler Dice TP600, using a TaKaRa 16S rDNA Bacterial Identification PCR kit. The primers for 16S rRNA gene amplification were the archaeon-specific primers 8F/1492R and 109F/915R (Banning et al., 2005). The primers for mcrA gene amplification were ME1/ME2 (Hales et al., 1996). PCR products were purified with a TaKaRa Agarose Gel DNA Purification kit version 2.0 and sequenced directly with an ABI PRISM BigDye Terminator v3.1 Cycle Sequencing kit and an ABI PRISM 3730XL DNA sequencer. A partial 16S rRNA gene sequence (1346 bp) and the DNA (669 bp) and inferred amino acid sequences of the mcrA gene were obtained and compared with sequences deposited in GenBank by using the BLAST program (Altschul et al., 1990), and then aligned with those of related methanogens in the order Methanosarcinales by using CLUSTAL_X software (Thompson et al., 1997). Phylogenetic trees were constructed by using the neighbour-joining method in MEGA3.1 software (Kumar et al., 2004). Bootstrap values were calculated after 1000 replications. The 16S rRNA gene sequence of strain ZC-1T had 90.6 % similarity to that of Methanosaeta harundinacea 8AcT and 89.1 % similarity to those of Methanohalobium evestigatum Z-7303T and Methanomethylovorans hollandica DMS1T (Fig. 2). The inferred amino acid sequence of the mcrA gene of strain ZC-1T showed 76.6 % similarity to that of Methanothrix thermophila PTT and 70.3 % similarity to that of Methanohalobium evestigatum Z-7303 T(Fig. 3).
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; Ollivier et al., 1984; Touzel et al., 1985; Zhilina & Zavarzin, 1987; Zinder & Mah, 1979). However, these species are all moderate thermophiles, with temperature optima equal to or below 55 °C. In contrast, strain ZC-1T is the first reported methylotrophic methanogen with a temperature optimum as high as 65 °C. It can also survive at 70 °C. Phylogenetic analyses of the 16S rRNA and mcrA genes suggest that strain ZC-1T is related more closely to the family Methanosaetaceae than the family Methanosarcinaceae. However, strain ZC-1T does not ferment acetate, which is a special characteristic of the family Methanosaetaceae. In addition, 16S rRNA gene sequence similarities of 88–93 % are common for members of different families within the methanogens (Garrity & Holt, 2001). Based upon the low-level sequence similarity for the 16S rRNA gene with the most closely related methanogens and the morphological and physiological characteristics described in Table 1, we propose that strain ZC-1T is a representative of a novel family within the order Methanosarcinales. The novel genus and species Methermicoccus shengliensis gen. nov., sp. nov. and the family Methermicoccaceae fam. nov. are proposed to accommodate strain ZC-1T.
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Small, thermophilic cocci. Methanol, methylamine and trimethylamine are used as substrates for methanogenesis. The definition of the family is determined primarily by phylogenetic analyses of the 16S rRNA gene. The DNA G+C content is >50 mol%. The family currently contains a single genus.
The type genus of the family is Methermicoccus.
Description of Methermicoccus gen. nov.
Methermicoccus (Me.ther.mi.coc'cus. N.L. masc. n. Methermicoccus arbitrary name referring to a small, thermophilic, methane-producing coccus).
Small cocci with a mean diameter of 0.7 µm. Growth occurs at temperatures up to 70 °C; no growth occurs below 50 °C. Methanol, methylamine and trimethylamine are used as substrates for methanogenesis. Mg2+ is required for growth. The DNA G+C content is >50 mol%.
The type species of the genus is Methermicoccus shengliensis.
Description of Methermicoccus shengliensis sp. nov.
Methermicoccus shengliensis (shen.gli.en'sis. N.L. masc. adj. shengliensis pertaining to Shengli oilfield, where the type strain was isolated).
Cells are small, motile cocci with a diameter of 0.7–1.0 µm. Cells occur in clusters of two to four cells, and sometimes form cysts during the exponential phase. Cells are lysed in SDS (0.01 %, w/v) and H2O, but not in PBS (0.2 M) or NaCl solution (0.9 %, w/v). HS-coM stimulates cell growth dramatically. Only methanol, methylamine and trimethylamine are used as substrates for methanogenesis. Fast growth occurs at pH 6.0–6.5 (range for growth, pH 5.5–8.0), 65 °C (range for growth, 50–70 °C), 0.3–0.5 M NaCl (range for growth, 0.2–1.1 M) and 0.05–0.20 M MgCl2. The DNA G+C content is 56 mol%.
The type strain, ZC-1T (=CGMCC 1.5056T=DSM 18856T), was isolated from oil-production water of Shengli oilfield, China.
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ACKNOWLEDGEMENTS |
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