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1 Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China
2 Department of Geology, Peking University, Beijing 100871, China
3 Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
Correspondence
Xiao-Lei Wu
xiaolei_wu{at}tsinghua.edu.cn
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ABSTRACT |
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7c, C16 : 0 and C19 : 0 cyclo 8c. The G+C content of the DNA was 66.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SL014B-85T belonged to the genus Halomonas in the Gammaproteobacteria, with highest sequence similarity of 98.1 and 97.8 % to Halomonas alimentaria DSM 15356T and Halomonas ventosae DSM 15911T, respectively. DNA–DNA relatedness values were below 40 % with members of closely related Halomonas species. Results of phenotypic, biochemical and phylogenetic analyses revealed that strain SL014B-85T could be classified as representing a novel species of the genus Halomonas, for which the name Halomonas shengliensis sp. nov. is proposed. The type strain is SL014B-85T (=CGMCC 1.6444T=LMG 23897T).
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), H. campisalis (Mormile et al., 1999)], production of exopolysaccharides [Halomonas eurihalina (Mellado et al., 1995), H. maura (Bouchotroch et al., 2001), H. ventosae (Martínez-Cánovas et al., 2004)] and degradation of aromatic compounds (Halomonas organivorans; García et al., 2004). Because of the diverse functions of these moderately halophilic bacteria, in recent years we have focused on the characterization of the moderately halophilic bacterial community that is able to degrade petroleum hydrocarbons in several saline oilfields of China. Bacterial strains that could use crude oil as the sole carbon source were isolated (Gu et al., 2007; Wang et al., 2007), among which was strain SL014B-85T. Based on its phenotypic, physiological, biochemical and phylogenetic characteristics, strain SL014B-85T is considered to represent a novel species of the genus Halomonas.
Strain SL014B-85T was isolated from an oil-polluted saline soil from the coastal Shengli oilfield, in Shangdong Province in eastern China, by a 10-fold dilution plating technique on oil-produced water (OPW) agar plates (Wang et al., 2007) at 30 °C. As the organic and mineral contents of the oil-producing water were constantly changing, cultivation of strain SL014B-85T for identification purposes was carried out in artificial seawater (ASW) medium, consisting of 5 g peptone, 1 g yeast extract, 4 g Na2SO4, 0.68 g KCl, 0.1 g KBr, 0.025 g H3BO3, 5.4 g MgCl2.H2O, 1.5 g CaCl2.2H2O, 0.024 g SrCl2.6H2O, 0.2 g NaHCO3, 0.04 g Na2HPO4, 0.5 g NH4Cl and 0.002 g NaF, per litre of water, with 2.4 % (w/v) NaCl (pH 8.0) (Eguchi et al., 1996).
After growth in ASW medium for 2 days at 30 °C, cell morphology was examined via transmission electron microscopy. Salt requirement for growth was tested by using ASW medium with NaCl concentrations ranging from 0 to 30 % (w/v) (pH 8.0, at 30 °C) (Bouchotroch et al., 2001). pH and temperature requirements for growth were determined in ASW medium by adjusting pH values between 2.0 and 12.0 (30 °C) and by incubation at 4–50 °C (pH 8.0).
Oxidase activity was tested as described by Smibert & Krieg (1994) and catalase activity was determined by use of a 3 % (v/v) hydrogen peroxide solution. Nitrite and nitrate reduction were tested in ASW medium by growing the cells separately in the presence of nitrite and nitrate (Berendes et al., 1996). Denitrification was tested by growing the cells anaerobically in the presence of nitrate (Zumft, 1992). Hydrolysis of starch, gelatin and Tween 80, urease activity, and growth on sole carbon sources were examined according to the procedures of Williams et al. (1983) on ASW medium without organic compounds at 30 °C for 5–7 days. H2S production was tested in ASW medium supplemented with 0.01 % L-cysteine, the indicator being a strip of paper impregnated with lead acetate placed in the neck of the tube (Clarke, 1953; Mata et al., 2002).
Strain SL014B-85T, Halomonas alimentaria DSM 15356T and H. ventosae DSM 15911T were grown in ASW medium for 3 days at 30 °C; their cellular fatty acids were analysed as described by Komagata & Suzuki (1987), and then tested by using GC/MS following the instructions of the Microbial Identification System (MIDI; Microbial ID Inc.). Polar lipid analyses were performed following a standard extraction procedure, and polar lipids were then tested by one- and two-dimensional TLC on Merck silica gel 60 F254 aluminium-backed thin-layer plates according to the methods of Kates (1986) and Collins et al. (1980). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987), by using an HPLC fitted with a reversed-phase column (Shim-pack, VP-ODS; Shimadzu).
Genomic DNA was extracted from cells grown in ASW medium for 2 days at 30 °C according to the method of Marmur (1961). Purity was assessed based on A280/A260 and A230/A260 ratios (Johnson, 1994). The G+C content of the genomic DNA was determined by thermal denaturation (Marmur & Doty, 1962) with DNA from Escherichia coli K-12 as a control. DNA–DNA hybridization experiments were performed in triplicate following the methods of De Ley et al. (1970) and Huß et al. (1983). The 16S rRNA gene was amplified (Embley, 1991) with universal bacterial primers corresponding to E. coli positions 8F (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-GGTTACCTTGTTACGACTT). The 16S rRNA gene sequence of strain SL014B-85T was aligned with those of related Halomonas species by using MEGA software (Kumar et al., 2004). Phylogenetic trees were constructed via the neighbour-joining method (Saitou & Nei, 1987) and maximum-parsimony algorithm of MEGA, version 5.0 (Kumar et al., 2004), and re-evaluated with the interior branch test of phylogeny.
Cells of strain SL014B-85T were Gram-negative, short-rods (0.6–0.8x1.0–1.6 µm) with several lateral flagella (Fig. 1). Colonies on ASW agar plates were creamy and circular. Growth occurred at 0–15 % (w/v) NaCl (optimum 5–15 %), pH 8.0–9.0 (optimum pH 8.5) and 10–42 °C (optimum 30 °C). The strain was positive for oxidase, catalase and urease activities, aerobic nitrate and nitrite reduction, as well as for anaerobic nitrate reduction, but negative for hydrolysis of starch, gelatin and Tween 80 and H2S production (Table 1). Strain SL014B-85T was able to use glucose, sucrose, lactose, mannose, trehalose, galactose, dextrin, gluconate, malate, malonate, succinate and sorbitol, but not ribose, arabinose, fructose, cellobiose, maltose, sorbose, xylose, mannitol, inositol or lactate as sole carbon sources in ASW medium (Table 1). In addition, it was able to grow with the crude oil from Shengli oilfield as the sole carbon source.
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7c (43.05 % of total fatty acids), C16 : 0 (24.81 %), C19 : 0 cyclo 8c (10.14 %), C12 : 0 3-OH (8.41 %), C16 : 17c and/or iso-C15 : 0 2-OH (6.98 %), C12 : 0 (2.70 %) and C10 : 0 (1.73 %) (Table 2), and ubiquinone with nine isoprene units (Q9) was the only isoprenoid quinone. The predominant polar lipids of strain SL014B-85T were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine, which are characteristic of members of the genus Halomonas. The G+C content of the DNA of strain SL014B-85T was 66.6 mol%, within the range of 52–68 mol% reported for the genus Halomonas by Franzmann & Tindall (1990), but distinct from the most closely related species H. alimentaria (63.0 mol%; Yoon et al., 2002) and H. ventosae (74.3 mol%; Martínez-Cánovas et al., 2004).
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; Dobson & Franzmann, 1996) and Halomonas halodenitrificans DSM 735T (Dobson & Franzmann, 1996) (Fig. 2). Strain SL014B-85T showed 16S rRNA gene sequence similarity of 98.1 and 97.8 %, respectively, to the type strains of the most closely related species H. alimentaria and H. ventosae. DNA–DNA hybridization experiments were carried out in triplicate and results showed mean levels of relatedness of 30.5±0.06 % to H. alimentaria DSM 15356T and 15.5±0.07 % to H. ventosae DSM 15911T, indicating that strain SL014B-85T was genotypically distinct from these type strains (Wayne et al., 1987).
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Description of Halomonas shengliensis sp. nov.
Halomonas shengliensis (sheng.li.en'sis. N.L. fem. adj. shengliensis pertaining to Shengli oilfield, China, where the type strain was isolated).
Cells are moderately halophilic, Gram-negative, crude-oil-utilizing short rods (0.6–0.8x1.0–1.6 µm) with several lateral flagella. Colonies on ASW agar are creamy and circular. Grows at 0–15 % (w/v) NaCl, pH 8.0–9.0 and 10–42 °C; optimum growth occurs at 5–15 % (w/v) NaCl, pH 8.5 and 30 °C. Utilizes glucose, sucrose, lactose, mannose, galactose, dextrin, gluconate, malate, malonate, succinate and sorbitol as sole carbon sources. Oxidase-, urease- and catalase-positive, but negative for hydrolysis of starch, Tween 80 and gelatin and production of H2S. It is able to reduce nitrate anaerobically as well as nitrate and nitrite aerobically. Q9 is the only isoprenoid quinone and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine are the predominant polar lipids. The main cellular fatty acids are C18 : 17c (43.05 %), C16 : 0 (24.81 %) and C19 : 0 cyclo 8c (10.14 %). The DNA G+C content of the type strain is 66.6 mol%.
The type strain, SL014B-85T (=CGMCC 1.6444T=LMG 23897T), was isolated from a saline soil from the coastal Shengli oilfield in eastern China.
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ACKNOWLEDGEMENTS |
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