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1 Laboratoire de Microbiologie IRD, UMR 180, Universités de Provence et de la Méditerranée, ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
2 Laboratoire d'Ecologie et de Technologie Microbienne, INSAT, 1080 Tunis, Tunisia
3 Instituto Mexicano del Petróleo, Programa de Biotecnología del Petróleo, 07730 México DF, Mexico
Correspondence
D. Alazard
didier.alazard{at}univmed.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains E-2T and IMP-2 are respectively DQ365924 and DQ365925.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). They are defined by their ability to use sulfate as a terminal electron acceptor during anaerobic respiration. Phylogenetically, they may be divided into four major groups, based on 16S rRNA gene sequence analysis. They include the Gram-negative SRB of the Deltaproteobacteria, the Gram-positive spore-forming SRB, the deeply branching thermophilic SRB and the thermophilic archaeal sulfate reducers (Castro et al., 2000; Fauque & Ollivier, 2004; Stackebrandt et al., 1995). The mesophilic members of the Deltaproteobacteria represent the largest group of SRB, now including around 40 genera. Members of the genus Desulfovibrio have been isolated frequently from marine environments, including Desulfovibrio acrylicus (van der Maarel et al., 1996), D. africanus (Campbell et al., 1966), D. giganteus (Esnault et al., 1988), D. gigas (Le Gall, 1963) and D. inopinatus (Reichenbecher & Schink, 1997). Halotolerant to halophilic Desulfovibrio species have also been recovered from oilfield environments (Birkeland, 2005); these include Desulfovibrio vietnamensis (Dang et al., 1996) and D. longus (Magot et al., 1992), considered as halotolerant, and Desulfovibrio gabonensis (Tardy-Jacquenod et al., 1996), D. capillatus (Miranda-Tello et al., 2003), D. bastinii (Magot et al., 2004), D. indonesiensis (Feio et al., 1998) and D. gracilis, considered as moderate halophiles. Although most of these latter species were isolated from oilfield production waters, it is difficult to reach conclusions about the indigenous character of these bacteria with regard to oil reservoirs: are they contaminants or common inhabitants of deep oil reservoirs? This question remains unanswered because of possible contamination through drilling or water-flooding processes (Magot, 2005). In this study, we report on the isolation of two moderately halophilic strains of SRB that were recovered from marine sediments contaminated by industrial activities and from an oil–water separation system treating oilfield production fluids. Their phenotypic, genotypic and phylogenetic characteristics suggest that they represent a novel species of the genus Desulfovibrio.
Strain E-2T was isolated from marine sediments contaminated by industrial activities (phosphogypsum disposal resulting from phosphoric acid production) near Sfax (Tunisia), while strain IMP-2 was isolated from an oil–water separation tank treating production fluids from an offshore production platform in the Gulf of Mexico. Samples were collected in sterile glass bottles and kept at room temperature until used. Standard anaerobic techniques were used throughout this study (Balch et al., 1979; Hungate, 1969). Enrichment and isolation were performed using basal SRB growth medium, containing (per litre distilled water) 1 g NH4Cl, 0.3 g K2HPO4, 0.3 g KH2PO4, 0.1 g KCl, 0.1 g CaCl2, 0.2 g MgSO4 . 7H2O, 30 g NaCl, 0.2 g yeast extract, 0.5 g cysteine hydrochloride, 1 mg resazurin and 10 ml trace mineral element solution (Balch et al., 1979). The pH was adjusted to 7 with 10 M KOH and the medium was boiled under a stream of O2-free N2 gas and cooled to room temperature. Aliquots were dispensed into Hungate tubes (5 ml) and serum bottles (20 ml) under a stream of N2/CO2 (80 : 20, v/v) gas and the sealed vessels were autoclaved for 45 min at 110 °C. Prior to inoculation, Na2S . 9H2O and NaHCO3 were injected from anaerobic sterile stock solutions to respective final concentrations of 0.04 and 0.2 % (w/v). Liquid cultures were incubated at 35 °C for 1 week. Pure cultures were obtained by repeated application of the agar roll-tube dilution method (Hungate, 1969). Purity of the isolates was checked by microscope observation and inoculation in sulfate-free media containing yeast extract and sugars. D. gabonensis DSM 10636T and D. indonesiensis DSM 15121T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) and cultivated according to the procedures recommended by the DSMZ. In order to characterize the two isolates phenotypically, standard and specific tests for SRB were performed, including Gram reaction, cell morphology, motility and determination of the electron donors and acceptors used and the presence of desulfoviridin and c-type cytochromes (Postgate, 1984), as well as other tests as shown in Table 1 or included in the species description. Substrates were tested at a final concentration of 20 mM in SRB medium. To test for electron acceptors, sodium thiosulfate, sodium sulfate, sodium sulfite, elemental sulfur and nitrate were added to the medium at final concentrations of 20 mM, 20 mM, 2 mM, 2 % (w/v) and 10 mM, respectively.
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The G+C content of the DNA was determined by HPLC at the Identification Service of the DSMZ as 60.4 mol% for strain E-2T and 62.7 mol% for strain IMP-2. The 16S rRNA genes of strains E-2T and IMP-2 were amplified and sequenced as described elsewhere (Maidak et al., 2001; Miranda-Tello et al., 2003; Weisburg et al., 1991). Sequences of 1524 and 1478 nucleotides, respectively, of the 16S rRNA genes of strains E-2T and IMP-2 were determined and aligned manually using the alignment editor BioEdit version 5.0.9 (Hall, 1999). The 16S rRNA gene sequences of the new isolates shared 99.0 % similarity, suggesting that they are closely related phylogenetically. The strains belonged to the genus Desulfovibrio, of the class Deltaproteobacteria, with D. indonesiensis Ind 1T (98.3 % similarity) and D. gabonensis SEBR 2840T (97.1 % similarity) as their closest phylogenetic relatives (Fig. 1).
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and DNA–DNA hybridization was performed as described by De Ley et al. (1970) with the modification described by Huß et al. (1983) and Escara & Hutton (1980), using a Gilford System model 2600 spectrometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program of Jahnke (1992). A quite high DNA–DNA reassociation value of 64.5±3.8 % (mean of four values) was obtained between strain E-2T and IMP-2. This value is close to the threshold value of 70 % for assignment of strains to the same species (Wayne et al., 1987). Moreover, the few physiological differences that exist between the two isolated strains are not sufficient to differentiate them at the species level (Table 1
). Strains E-2T and IMP-2 should therefore be assigned to the same species of the genus Desulfovibrio. DNA–DNA hybridization data revealed relatedness of only 15.7 % between strain E-2T and D. indonesiensis DSM 15121T and 26.0 % between strain E-2T and D. gabonensis DSM 10636T. Strain IMP-2 showed 12.4 % relatedness to D. indonesiensis DSM 15121T and 35.2 % relatedness to D. gabonensis DSM 10636T. Among the phenotypic differences that exist between D. indonesiensis and the two isolates, the former did not use ethanol, malate or fructose as electron donors or elemental sulfur as a terminal electron acceptor and did not ferment fumarate. In contrast to our isolates, D. gabonensis oxidized butanol and used fumarate as an electron acceptor (Table 1). Based on the phenotypic, genotypic and phylogenetic characteristics of our isolates, we propose to assign them to a novel species, Desulfovibrio marinus sp. nov. Finally, our results indicated that phylogenetically similar SRB inhabit both marine and oilfield environments. In this respect, marine SRB may be possible contaminants of oil reservoirs through water-flooding processes in particular, thus contributing to oil souring and corrosion problems in the oil industry (Crolet, 2005; Vance & Thrasher, 2005).
Description of Desulfovibrio marinus sp. nov.
Desulfovibrio marinus (ma.ri'nus. L. masc. adj. marinus of or belonging to the sea, marine).
Cells are strictly anaerobic, vibrio-shaped or sigmoid, 0.5x1.5–2.5 µm, occurring singly and in chains. Motile by a polar flagellum. Grows at 20–50 °C, with optimum growth at 37 °C. Grows in the presence of NaCl at 0.5 to 11 %, with optimum growth around 2.5–5 %. The optimum pH for growth is 7.0; growth occurs at pH 6.5–8.5. Uses H2, formate, fumarate, lactate, malate, pyruvate, succinate and fructose as electron donors. Lactate is converted to acetate. Substrates that are not used include acetate, benzoate, butyrate, citrate, propionate, valerate, butanol, glycerol, 2-propanol, methanol, glucose, Casamino acids and peptone. Pyruvate and fumarate are fermented. Uses elemental sulfur, sulfate, thiosulfate and sulfite but not fumarate, nitrate or nitrite as electron acceptors. Desulfoviridin and c-type cytochromes are present. The G+C content of DNA of the type strain is 60.4 mol% (HPLC).
The type strain, strain E-2T (=DSM 18311T =JCM 14040T), was isolated from seawater near Sfax (Tunisia). A second strain of the species, IMP-2, was isolated from oilfield production fluids in the Gulf of Mexico.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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