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1 Department of Environmental Science and Engineering, State Joint Key Laboratory of Environmental Simulation and Pollution Control, Tsinghua University, Beijing 100084, China
2 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
3 Daqing Oilfield Company Ltd, Daqing 163712, China
Correspondence
Xiao-Lei Wu
xiaolei_wu{at}tsinghua.edu.cn
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ABSTRACT |
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9c (20.3 %), C16 : 17c (7.3 %) and C16 : 19c (6.4 %) as predominant fatty acids. The G+C content of the DNA was 57.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SL014B61AT belonged to the genus Marinobacter in the class Gammaproteobacteria. Strain SL014B61AT showed the highest 16S rRNA gene sequence similarity with Marinobacter bryozoorum (97.9 %) and showed 97.8 % sequence similarity to Marinobacter lipolyticus. DNA–DNA relatedness to the reference strains Marinobacter bryozoorum and Marinobacter lipolyticus was 35.5 % and 33.8 %, respectively. On the basis of these data, it is proposed that strains SL014B61AT and SL014B11A represent a novel species, Marinobacter gudaonensis sp. nov. The type strain is strain SL014B61AT (=DSM 18066T=LMG 23509T=CGMCC 1.6294T).
Electron micrographs of cells of strain SL014B61AT are available as a supplementary figure in IJSEM Online.
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with a single species Marinobacter hydrocarbonoclasticus. The genus currently contains 14 species with validly published names (Gorshkova et al., 2003; Martín et al., 2003; Shieh et al., 2003; Yoon et al., 2003, 2004; Romanenko et al., 2005; Shivaji et al. 2005; Green et al. 2006; Kim et al. 2006; Liebgott et al. 2006). The type species, Marinobacter hydrocarbonoclasticus, is able to utilize various hydrocarbons as the sole source of carbon and energy (Gauthier et al., 1992). The second species of the genus to be recognized, Marinobacter aquaeolei, was isolated from an oil-producing well on an offshore platform in southern Vietnam (Nguyen et al., 1999). However, research by Márquez and Ventosa has suggested that M. aquaeolei is a later heterotypic synonym of M. hydrocarbonoclasticus based on fatty acid composition, DNA G+C content and DNA–DNA hybridization studies (Márquez & Ventosa, 2005). In this study, we report the characterization of two novel strains, SL014B61AT and SL014B11A, that were isolated from an oil-polluted saline soil in a coastal oilfield in eastern China. The results indicate the two isolates represent a novel species of the genus Marinobacter. An oil-polluted soil was sampled from a ditch containing discharged oil recovery wastewater in Gudao Oil-Product, a coastal Shengli Oilfield in Shandong Province, eastern China. The temperature of the soil was around 30 °C all year round and the salinity of the soil was around 1 % NaCl (w/w). The main organic compounds in the soil were petroleum hydrocarbons. Strains SL014B61AT and SL014B11A were isolated from the soil by a 10-fold dilution plating technique on inorganic salts agar containing (w/v) 0.5 % NaCl, 0.1 % NH4H2PO4, 0.1 % (NH4)2SO4, 0.02 % MgSO4.7H2O, 0.3 % KNO3, 0.1 % K2HPO4 and distilled oil recovery wastewater instead of pure water. The isolates were purified by restreaking on plates of inorganic salts agar incubated for 3–5 days at 30 °C.
After the strains had grown to late exponential phase on marine agar 2216 (MA), cell morphology and flagellum type were examined using transmission and scanning electron microscopy. Carbon source assimilation was tested using the mineral medium solution of Shivaji et al. (2005). Each carbon source was added at a concentration of 0.2 % (w/v) after the mineral base solution had been autoclaved. Growth was examined after incubation at 30 °C for 1, 7, 10 and 14 days. Hydrolysis of starch, gelatin and Tween 80 was assessed as described by Smibert & Krieg (1994). Nitrate and nitrite reduction were assessed as described by Lanyi (1987). Optimum pH and temperature for growth were determined using marine broth 2216. The requirement for and tolerance of various NaCl concentrations were determined in a medium containing (l–1): 1.0 g MgCl2.6H2O, 5.0 g MgSO4.7H2O, 0.7 g KCl, 0.15 g CaCl2.2H2O, 0.5 g NH4Cl, 0.1 g KBr, 0.27 g KH2PO4, 0.04 g SrCl2.6H2O, 0.025 g H3BO3, 5.0 g peptone and 1.0 g yeast extract (pH 8.0) with various NaCl concentrations (0, 0.5, 1, 3, 5, 10, 15, 18, 20 and 25 %). Sensitivity to various antibiotics (kanamycin, tetracycline, chloramphenicol, ampicillin, streptomycin, erythromycin and gentamicin) was tested by using the method described by Cho & Giovannoni (2003).
Cells of strain SL014B61AT, Marinobacter bryozoorum DSM 15401T and Marinobacter lipolyticus SM19T were grown on MA at 28 °C for 3 days for cellular fatty acid analyses. Cellular fatty acid methyl esters were prepared and analysed using GC according to the instructions of the Microbial Identification System (MIDI). Fatty acid profiles were analysed by the Sherlock system (Microbial ID). Lipoquinones were extracted from lyophilized cells with chloroform/methanol (2 : 1, v/v) as described by Tindall (1990). Respiratory lipoquinones were analysed using reversed-phase HPLC (Shim-pack, VP-ODS, Shimadzu). Genomic DNA was extracted and purified by the method of Marmur (1961) and DNA purity was assessed by the A280/A260 and A230/A260 ratios (Johnson, 1994). The DNA G+C content was determined by thermal denaturation (Marmur & Doty, 1962) using DNA from Escherichia coli K-12 as a control. The 16S rRNA gene was amplified as described previously (Rainey et al., 1996), except that the following pair of bacterial universal primers was used: 8f, 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492r, 5'-GGTTACCTTGTTACGACTT-3'. 16S rRNA gene sequence alignments were performed with the CLUSTAL_X program (version 1.64b; Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) and evaluated by bootstrap analysis based on 1000 resampling replicates with the SEQBOOT, DNADIST, NEIGHBOR and CONSENSE programs of the PHYLIP software package version 3.6 (Felsenstein, 2004). DNA–DNA hybridization was performed in triplicate by the thermal denaturation and renaturation method of Huß et al. (1983), modified from that of De Ley et al. (1970). The temperature of renaturation was 76.5 °C in 2x SSC buffer (0.15 M NaCl buffered with 0.015 M trisodium citrate, pH 7.0).
The two novel isolates were Gram-negative, rod-shaped and motile with a polar flagellum (see Supplementary Fig. S1a, b in IJSEM Online). The small creamy colonies (about 1–2 mm) were produced on MA after incubation at 30 °C for 3–5 days. Colonies were smooth, uniformly circular, flat and a little transparent. The pH range and NaCl concentrations for growth were pH 6.0–9.5 (optimum pH, 7.5–8.0) and 0 %–15 % NaCl (w/v) (optimum NaCl 2.0–3.0 %). Growth was observed at temperatures of 10–45 °C, but not at 4 °C or 50 °C. The novel isolates gave a positive reaction in tests for catalase and oxidase and reduced nitrate to nitrite. Nitrite was not reduced to N2. Starch and Tween 80 were hydrolysed, but no hydrolysis of urea or gelatin was detected. Both strains were susceptible to kanamycin, tetracycline, ampicillin, chloramphenicol, streptomycin, erythromycin and gentamicin. The other main characteristics that differentiate the novel strains from the type strains of species of the genus Marinobacter are listed in Table 1.
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). The 16S rRNA gene sequence of strain SL014B61AT had 100 % similarity to that of strain SL014B11A. The novel strains were most closely related to M. bryozoorum DSM 15401T (97.9 %) and M. lipolyticus SM19T (97.8 %). Lower similarity values were observed with other Marinobacter species, such as M. hydrocarbonoclasticus ATCC 49840T (94.9 %) and Marinobacter litoralis SW-45T (94.2 %).
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The results of the cellular fatty acid content analysis are given in Table 2. Fatty acids C12 : 0 3-OH, C16 : 0, C16 : 19c and C18 : 19c have been reported to be predominant in other known Marinobacter species (Spröer et al., 1998; Nguyen et al., 1999; Martín et al., 2003; Yoon et al., 2003, 2004).The predominant cellular fatty acids of strain SL014B61AT were C16 : 0 (21.2 %), C18 : 19c (20.3 %), C18 : 36c (6, 9, 12) (8.5 %), C16 : 17c (7.3 %) and C16 : 19c (6.4 %). This differed from those of the reference species M. bryozoorum DSM 15401T, but was similar to those of M. lipolyticus SM19T. The G+C content of strain SL014B61AT was 57.9 mol% (Tm).
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Description of Marinobacter gudaonensis sp. nov.
Marinobacter gudaonensis (gu.dao.nen'sis. N.L. masc. adj. gudaonensis pertaining to Gudao of the Shengli Oilfield, P. R. China, from where the type strain was first isolated.)
Cells are Gram-negative, rod-shaped (0.3–0.5x1.2–1.8 µm) and motile with a polar flagellum on semi-solid medium. Growth occurs in 0–15 % NaCl at temperatures of between 10 and 45 °C. Colonies on MA are smooth, uniformly circular, flat and a little transparent after 3–5 days. Positive results in tests for catalase, oxidase and nitrate-reducing activities and for the hydrolysis of starch and Tween 80. Negative results in tests for gelatin hydrolysis and urease and nitrite-reducing activities. The following substrates are utilized as a sole carbon source for growth: D-maltose, L-proline, dextrin, D-glucose, citrate, D-xylose, L-alanine, propionate, ethanol, D-fructose, pyruvate, D-trehalose, sucrose, D-sorbitol, acetate, succinate and D-galactose. Sensitive to kanamycin (10 µg), tetracycline (5 µg), chloramphenicol (10 µg), ampicillin (10 µg), streptomycin (10 µg), erythromycin (10 µg) and gentamicin (10 µg). Q9 is a main respiratory quinone and C16 : 0 (21.2 %), C18 : 19c (20.3 %), C18 : 36c (6, 9, 12) (8.5 %), C16 : 17c (7.3 %) and C16 : 19c (6.4 %) are the predominant fatty acids. The G+C content of the DNA is 57.9 mol% (Tm).
The type strain, SL014B61AT (=DSM 18066T=LMG 23509T=CGMCC 1.6294T), was isolated from an oil-polluted saline soil in Gudao in the coastal Shengli Oilfield, eastern China. Strain SL014B11A is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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