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1 Equipe Environnement et Microbiologie, IPREM UMR 5254, IBEAS Université de Pau et des Pays de l'Adour, BP1155, 64013 Pau cedex, France
2 Laboratoire de Microbiologie et Biotechnologie des Environnements Chauds, IRD, UMR_D 180 IFR-BAIM, case 925, Universités de Provence et de la Méditerranée, 163 avenue de Luminy, 13288 Marseille cedex 9, France
3 Laboratoire d'Ecologie Microbienne, IMEP UMR 6116, Université Paul Cézanne, Faculté des Sciences et Techniques de Saint-Jérôme, 13397 Marseille cedex 20, France
Correspondence
Cristiana Cravo-Laureau
cristiana.cravo-laureau{at}univ-pau.fr
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Present address: BRGM, Service Environnement et Procédés, Unité Biotechnologies, 3 av. Claude Guillemin, 45060 Orléans, France. 
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and dsrAB gene sequences of strain LM2801T are respectively DQ826724 and DQ826725.
A phase-contrast photomicrograph of cells of strain LM2801T is available as supplementary material with the online version of this paper.
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). Among these strains, five are known to oxidize alkenes (Aeckersberg et al., 1991, 1998; So & Young, 1999; Cravo-Laureau et al., 2004a, b), unsaturated hydrocarbons commonly produced in plants but also occasionally present in small quantities in crude oils (Curiale & Frolov, 1998). Among these five complete-oxidizing strains, only one degrades alkenes exclusively (Cravo-Laureau et al., 2004b). Here, we report on the isolation and characterization of a novel isolate that exclusively oxidizes long-chain alkenes and fatty acids incompletely to acetate, strain LM2801T.
Strain LM2801T was isolated from brackish sediment collected from a wastewater decantation facility that recovers wastewater from an oil refinery treatment plant (Berre, France). Sediment samples were collected using a plastic core sampler capped with a butyl stopper. Samples were stored at 4 °C until use. Enrichment and cultivation methods were described by Cravo-Laureau et al. (2004a). Enrichment cultures were supplemented with 1-eicosene (C20H40; 1.0 mM) as substrate and sodium dithionite (0.12 mM) as additional reductant (Widdel & Bak, 1992) and incubated at 30 °C in the dark. Eicosene was sterilized via hot filtration (cellulose membrane, 0.2 µm) and added hot to the culture medium. The strain was purified from an enrichment culture free of sediment as described previously (Cravo-Laureau et al., 2004a). The purity of the strain was confirmed by the absence of growth in natural lagoon-water medium supplemented with glucose (3 mM) and yeast extract (0.5 g l–1) under aerobic and anaerobic conditions and in AC medium (Difco) and by microscopic observations. Maintenance and growth of pure culture were achieved using a synthetic sulfate-reducing growth medium (Cravo-Laureau et al., 2004a) with 0.7 % (w/v) NaCl. Cultures were supplemented with 1-eicosene (1.0 mM) and sodium dithionite (0.12 mM).
Cells of strain LM2801T were short, slightly curved or vibrioid rods (see Supplementary Fig. S1, available in IJSEM Online) that were motile by means of polar flagella. Cells stained Gram-negative after the Gram staining reaction and the KOH method (Buck, 1982).
Physiological tests were performed as described by Cravo-Laureau et al. (2004a). Growth was monitored indirectly by measuring sulfide production according to Cline (1969). Results of the physiological characterization (pH, salinity and temperature) with sodium palmitate (2 mM) as growth substrate are given in the species description. Strain LM2801T used myristate (C14 : 0, 2 mM), palmitate (C16 : 0, 2 mM), stearate (C18 : 0, 2 mM), arachidate (C20 : 0, 1 mM), behenate (C22 : 0, 1 mM) and lignocerate (C24 : 0, 1 mM) as electron donors and carbon sources. Growth on the last two was particularly slow. Slight growth was obtained with 5 mM butyrate. No growth was observed on the following substrates (mM, except where stated): H2/CO2 (1 bar), H2 plus acetate (1 bar/10 mM), formate (5), acetate (10), formate plus acetate (5/10), propionate (10), isobutyrate (5), valerate (5), octanoate (2), nonanoate (2), decanoate (2), dodecanoate (2), lactate (10), pyruvate (10), citrate (5), 
-ketoglutarate (5), malate (10), succinate (10), fumarate (10), crotonate (2), tartrate (2), glycolate (2), thioglycolate (2), acetone (5), ethanol (10), methanol (10), 2-propanol (10), butanol (10), glycerol (10), glucose (5), fructose (5), gluconate (10), glycine (5), alanine (5), serine (5), threonine (10), lysine (10), cysteine (5), methionine (10), aspartate (5), glutamate (5), betaine (5), phenol (0.5), benzoate (5), gallate (5), catechol (0.5), indole (0.25), nicotinate (2), thioacetamide (2), peptone (0.5 g l–1), Casamino acids (0.5 g l–1) and yeast extract (0.5 g l–1). Malate, lactate, fumarate and pyruvate were not fermented. Sulfate (20 mM) served as an electron acceptor, whereas nitrate (10 mM), fumarate (10 mM), thiosulfate (10 mM), sulfite (5 mM), elemental sulfur (0.8 g l–1) and DMSO (10 mM) were not used. Desulfoviridin was absent. N2, NH4Cl and yeast extract were used as nitrogen sources, but not nitrate or glutamate. Vitamins were required for growth.
Hydrocarbon utilization was tested with 250 mg aliphatic hydrocarbons l–1 (n-alkanes, C5–C20; n-alkenes, C8–C23) and 100 mg aromatic hydrocarbons l–1 (benzene, toluene, xylene, p-cymene, naphthalene and phenanthrene). Strain LM2801T was able to oxidize and grow on n-alkenes from C14 to C23. Growth on alkenes was particularly slow; indeed, 6.5 mM sulfide was produced after 50 days of growth on eicosene whereas, in the presence of sodium palmitate, it reached 9 mM in 10 days. Growth on n-alkanes or aromatic hydrocarbons was not observed. Among sulfate-reducing bacteria able to oxidize aliphatic hydrocarbons (Widdel et al., 2007), only Desulfatibacillum alkenivorans PF2803T (Cravo-Laureau et al., 2004b) oxidizes alkenes exclusively. Nevertheless, strain LM2801T metabolizes only long-chain alkenes, whereas Desulfatibacillum alkenivorans oxidizes alkenes smaller than C14. Moreover, strain LM2801T is able to use only a very few substrates besides alkenes as electron donors and carbon sources.
Quantitative growth experiments with 1-eicosene were carried out according to Cravo-Laureau et al. (2004a). Sulfide was determined colorimetrically by the methylene blue formation reaction (Cline, 1969). The exact sulfide content of standards was determined by iodometric titration (Vogel, 1961). Sulfate analyses were performed by a turbidimetric method (Kolmert et al., 2000) after removing sulfide with zinc carbonate. Acetate concentration was determined by HPLC (Waters 600E equipped with a Phenomenex Rezex organic acids column) after removing sulfide with zinc carbonate. 1-Eicosene concentrations were determined by gas chromatography after extraction with pentane (Cravo-Laureau et al., 2004a). After 50 days of incubation, 0.94±0.04 mM eicosene and 6.03±0.74 mM sulfate were consumed, while 6.48±0.19 mM sulfide and 9.3±2.79 mM acetate were produced. These values are close to the following theoretical reaction:
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The G+C content of the DNA of strain LM2801T, determined at the DSMZ using HPLC according to a standard protocol described by Mesbah et al. (1989), was 45.5 mol%. The methods for genomic DNA purification, PCR amplification, sequence alignment and comparative analyses of genes encoding the 16S rRNA and the 
- and 
-subunits of the dissimilatory sulfite reductase (dsrAB), dendrogram construction and bootstrap analysis have been described previously (Cravo-Laureau et al., 2004a). Sequencing was done by Genome Express (Grenoble, France). Analysis of the almost-complete sequence (1353 bp) of the 16S rRNA gene of strain LM2801T revealed that this novel isolate belongs to the family Desulfobacteraceae within the class Deltaproteobacteria (Fig. 1). This analysis also shows that strain LM2801T is distantly related to other alkene-oxidizing sulfate-reducing strains, including Hxd3 (Aeckersberg et al., 1991), Pnd3 (Aeckersberg et al., 1998), AK-01 (So & Young, 1999), Desulfatibacillum aliphaticivorans CV2803T (Cravo-Laureau et al., 2004a) and Desulfatibacillum alkenivorans PF2803T (Cravo-Laureau et al., 2004b) (Fig. 1), with less than 92 % 16S rRNA gene sequence identity. Strain LM2801T grouped in a new cluster with two sulfate-reducing strains, R-ButA1 and R-CaprA1, which were isolated from rice fields (Wind et al., 1999). However, the 16S rRNA gene sequences of strain LM2801T and these two strains are only distantly related (96.2 % identity). Other known species of the Desulfobacteraceae are phylogenetically distant (less than 90 % identity) from strain LM2801T (Fig. 1). The phylogenetic position of strain LM2801T within the family Desulfobacteraceae was supported by deduced DsrAB sequence analyses (322 amino acids) (Fig. 2). Based on phylogenetic, biochemical and physiological differences (Table 1) between strain LM2801T and strains R-ButA1 and R-CaprA1, strain LM2801T is proposed as representing a novel species in a new genus, Desulfatiferula olefinivorans gen. nov., sp. nov.
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Mesophilic sulfate-reducing bacteria. Gram-negative cells, motile by polar flagella. Rod-shaped, non-sporulating cells. Desulfoviridin is not detected. Organic substrates are oxidized incompletely. The type species is Desulfatiferula olefinivorans.
Description of Desulfatiferula olefinivorans sp. nov.
Desulfatiferula olefinivorans [o.le.fi.ni.vo'rans. N.L. n. olefinum olefin; L. part. adj. vorans devouring; N.L. part. adj. olefinivorans olefin (alkene) devouring].
Cells are slightly curved or vibrioid rods (0.45x0.8–5.0 µm). Growth occurs at 16–38 °C (optimum 30–36 °C) and pH 6.6–8.3 (optimum pH 7.5). Growth occurs at NaCl concentrations of 0–50 g l–1 (optimum 6–10 g NaCl l–1), and at least 0.5 g MgCl2 . 6H2O l–1 is required for growth. Only sulfate is used as an electron acceptor. C14 to C24 fatty acids serve as electron donors as well as alkenes (C14–C23). Vitamins are required. The DNA G+C content of the type strain is 45.5 mol% (HPLC).
The type strain, LM2801T (=DSM 18843T =JCM 14469T), was isolated from oil-polluted sediment (Berre lagoon, France).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aeckersberg, F., Rainey, F. A. & Widdel, F. (1998). Growth, natural relationships, cellular fatty acids and metabolic adaptation of sulfate-reducing bacteria that utilize long-chain alkanes under anoxic conditions. Arch Microbiol 170, 361–369.[CrossRef][Medline]
Buck, J. D. (1982). Nonstaining (KOH) method for determination of Gram reactions of marine bacteria. Appl Environ Microbiol 44, 992–993.
Cline, J. D. (1969). Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr 14, 454–458.
Cravo-Laureau, C., Matheron, R., Cayol, J.-L., Joulian, C. & Hirschler-Réa, A. (2004a). Desulfatibacillum aliphaticivorans gen. nov., sp. nov., an n-alkane- and n-alkene-degrading sulfate-reducing bacterium. Int J Syst Evol Microbiol 54, 77–83.
Cravo-Laureau, C., Matheron, R., Joulian, C., Cayol, J.-L. & Hirschler-Réa, A. (2004b). Desulfatibacillum alkenivorans sp. nov., a novel n-alkene-degrading, sulfate-reducing bacterium, and emended description of the genus Desulfatibacillum. Int J Syst Evol Microbiol 54, 1639–1642.
Curiale, J. A. & Frolov, E. B. (1998). Occurrence and origin of olefins in crude oils. A critical review. Org Geochem 29, 397–408.[CrossRef]
Kolmert, A., Wikström, P. & Hallberg, K. B. (2000). A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures. J Microbiol Methods 41, 179–184.[CrossRef][Medline]
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
So, C. M. & Young, L. Y. (1999). Isolation and characterization of a sulfate-reducing bacterium that anaerobically degrades alkanes. Appl Environ Microbiol 65, 2969–2976.
Vogel, A. I. (1961). A Text-Book of Quantitative Inorganic Analysis, 3rd edn. London: Longmans.
Widdel, F. & Bak, F. (1992). Gram-negative mesophilic sulfate-reducing bacteria. In The Prokaryotes, 2nd edn, vol. 4, pp. 3352–3378. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Widdel, F., Musat, F., Knittel, K. & Galushko, A. (2007). Anaerobic degradation of hydrocarbons with sulphate as electron acceptor. In Sulfate-Reducing Bacteria: Environmental and Engineered Systems, pp. 265–303. Edited by L. L. Barton & W. A. Hamilton. Cambridge: Cambridge University Press.
Wind, T., Stubner, S. & Conrad, R. (1999). Sulfate-reducing bacteria in rice field soil and on rice roots. Syst Appl Microbiol 22, 269–279.[Medline]
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