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Marinobacter Gudaonensis Sp. Nov., Isolated from an Oil-Polluted Saline Soil in a Chinese Oilfield

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Marinobacter Gudaonensis Sp. Nov., Isolated from an Oil-Polluted Saline Soil in a Chinese Oilfield 中国科技论文在线 http://www.paper.edu.cn International Journal of Systematic and Evolutionary Microbiology (2007), 57, 250–254 DOI 10.1099/ijs.0.64522-0 Marinobacter gudaonensis sp. nov., isolated from an oil-polluted saline soil in a Chinese oilfield Jun Gu,1 Hua Cai,1 Su-Lin Yu,1 Ri Qu,1 Bin Yin,2 Yu-Feng Guo,1 Jin-Yi Zhao3 and Xiao-Lei Wu1 Correspondence 1Department of Environmental Science and Engineering, State Joint Key Laboratory of Xiao-Lei Wu Environmental Simulation and Pollution Control, Tsinghua University, Beijing 100084, China [email protected] 2State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China 3Daqing Oilfield Company Ltd, Daqing 163712, China Two novel strains, SL014B61AT and SL014B11A, were isolated from an oil-polluted saline soil from Gudao in the coastal Shengli Oilfield, eastern China. Cells of strains SL014B61AT and SL014B11A were motile, Gram-negative and rod-shaped. Growth occurred at NaCl concentrations of between 0 and 15 % and at temperatures of between 10 and 45 6C. Strain SL014B61AT had Q9 as the major respiratory quinone and C16 : 0 (21.2 %), C18 : 1v9c (20.3 %), C16 : 1v7c (7.3 %) and C16 : 1v9c (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). The genus Marinobacter was proposed by Gauthier et al. characterization of two novel strains, SL014B61AT and (1992) with a single species Marinobacter hydrocarbonoclas- SL014B11A, that were isolated from an oil-polluted saline ticus. The genus currently contains 14 species with validly soil in a coastal oilfield in eastern China. The results indicate published names (Gorshkova et al., 2003; Martı´n et al., 2003; the two isolates represent a novel species of the genus Shieh et al., 2003; Yoon et al., 2003, 2004; Romanenko et al., Marinobacter. 2005; Shivaji et al. 2005; Green et al. 2006; Kim et al. 2006; Liebgott et al. 2006). The type species, Marinobacter An oil-polluted soil was sampled from a ditch containing hydrocarbonoclasticus, is able to utilize various hydrocarbons discharged oil recovery wastewater in Gudao Oil-Product, as the sole source of carbon and energy (Gauthier et al., a coastal Shengli Oilfield in Shandong Province, eastern 1992). The second species of the genus to be recognized, China. The temperature of the soil was around 30 uC all year Marinobacter aquaeolei, was isolated from an oil-producing round and the salinity of the soil was around 1 % NaCl (w/w). The main organic compounds in the soil were well on an offshore platform in southern Vietnam (Nguyen T et al., 1999). However, research by Ma´rquez and Ventosa has petroleum hydrocarbons. Strains SL014B61A and suggested that M. aquaeolei is a later heterotypic synonym of SL014B11A were isolated from the soil by a 10-fold dilution M. hydrocarbonoclasticus based on fatty acid composition, plating technique on inorganic salts agar containing (w/v) DNA G+C content and DNA–DNA hybridization studies 0.5 % NaCl, 0.1 % NH4H2PO4, 0.1 % (NH4)2SO4, 0.02 % (Ma´rquez & Ventosa, 2005). In this study, we report the 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 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA incubated for 3–5 days at 30 uC. gene sequences of strains SL014B61AT and SL014B11A are DQ414419 and DQ629025, respectively. After the strains had grown to late exponential phase on Electron micrographs of cells of strain SL014B61AT are available as a marine agar 2216 (MA), cell morphology and flagellum type supplementary figure in IJSEM Online. were examined using transmission and scanning electron 250 64522 G 2007 IUMS Printed in Great Britain 转载 中国科技论文在线 http://www.paper.edu.cn Marinobacter gudaonensis sp. nov. microscopy. Carbon source assimilation was tested using 1–2 mm) were produced on MA after incubation at 30 uC the mineral medium solution of Shivaji et al. (2005). Each for 3–5 days. Colonies were smooth, uniformly circular, flat carbon source was added at a concentration of 0.2 % (w/v) and a little transparent. The pH range and NaCl concen- after the mineral base solution had been autoclaved. Growth trations for growth were pH 6.0–9.5 (optimum pH, 7.5–8.0) was examined after incubation at 30 uC for 1, 7, 10 and and 0 %–15 % NaCl (w/v) (optimum NaCl 2.0–3.0 %). 14 days. Hydrolysis of starch, gelatin and Tween 80 was Growth was observed at temperatures of 10–45 uC, but assessed as described by Smibert & Krieg (1994). Nitrate and not at 4 uCor50uC. The novel isolates gave a positive nitrite reduction were assessed as described by Lanyi (1987). reaction in tests for catalase and oxidase and reduced Optimum pH and temperature for growth were determined nitrate to nitrite. Nitrite was not reduced to N2. Starch and using marine broth 2216. The requirement for and tolerance Tween 80 were hydrolysed, but no hydrolysis of urea or of various NaCl concentrations were determined in a medium gelatin was detected. Both strains were susceptible to 21 containing (l ): 1.0 g MgCl2.6H2O, 5.0 g MgSO4.7H2O, kanamycin, tetracycline, ampicillin, chloramphenicol, 0.7 g KCl, 0.15 g CaCl2.2H2O, 0.5 g NH4Cl,0.1gKBr,0.27g streptomycin, erythromycin and gentamicin. The other KH2PO4,0.04gSrCl2.6H2O, 0.025 g H3BO3,5.0gpeptone main characteristics that differentiate the novel strains from and 1.0 g yeast extract (pH 8.0) with various NaCl con- the type strains of species of the genus Marinobacter are centrations (0, 0.5, 1, 3, 5, 10, 15, 18, 20 and 25 %). Sensitivity listed in Table 1. to various antibiotics (kanamycin, tetracycline, chloramphe- nicol, ampicillin, streptomycin, erythromycin and gentami- Almost complete 16S rRNA gene sequences were deter- T cin) was tested by using the method described by Cho & mined for strains SL014B61A and SL014B11A. Analysis of Giovannoni (2003). the 16S rRNA gene sequences revealed that strain SL014B61AT was a member of the class Gammaproteo- Cells of strain SL014B61AT, Marinobacter bryozoorum DSM bacteria and had a close phylogenetic relationship with T T 15401 and Marinobacter lipolyticus SM19 were grown on species of the genus Marinobacter; 16S rRNA gene sequence MA at 28 uC for 3 days for cellular fatty acid analyses. similarity ranged from 94.2 to 97.9 % (Fig. 1). The 16S Cellular fatty acid methyl esters were prepared and analysed rRNA gene sequence of strain SL014B61AT had 100 % using GC according to the instructions of the Microbial similarity to that of strain SL014B11A. The novel strains Identification System (MIDI). Fatty acid profiles were were most closely related to M. bryozoorum DSM 15401T analysed by the Sherlock system (Microbial ID). Lipo- (97.9 %) and M. lipolyticus SM19T (97.8 %). Lower quinones were extracted from lyophilized cells with chloro- similarity values were observed with other Marinobacter form/methanol (2 : 1, v/v) as described by Tindall (1990). species, such as M. hydrocarbonoclasticus ATCC 49840T Respiratory lipoquinones were analysed using reversed- (94.9 %) and Marinobacter litoralis SW-45T (94.2 %). 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, 59-AGAGTTTGA- TCCTGGCTCAG-39 and 1492r, 59-GGTTACCTTGTTAC- GACTT-39. 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 resamp- ling 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 uCin26 SSC buffer (0.15 M NaCl buffered with Fig. 1. Phylogenetic position of Marinobacter gudaonensis 0.015 M trisodium citrate, pH 7.0). sp. nov. and other Marinobacter species according to 16S rRNA gene sequence analysis. The topology shown was obtained by The two novel isolates were Gram-negative, rod-shaped and using neighbour-joining methods. Bootstrap values (expressed as motile with a polar flagellum (see Supplementary Fig. S1a, b percentages of 1000 replications) greater than 50 % are shown in IJSEM Online). The small creamy colonies (about at branch points. Bar, 1 % sequence divergence. http://ijs.sgmjournals.org 251 中国科技论文在线 http://www.paper.edu.cn J. Gu and others Table 1. Characteristics that differentiate Marinobacter gudaonensis sp. nov. from related species of the genus Marinobacter Strains: 1, M.
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