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Int J Syst Evol Microbiol 54 (2004), 1639-1642; DOI  10.1099/ijs.0.63104-0
© 2004 International Union of Microbiological Societies

Desulfatibacillum alkenivorans sp. nov., a novel n-alkene-degrading, sulfate-reducing bacterium, and emended description of the genus Desulfatibacillum

Cristiana Cravo-Laureau1,{dagger}, Robert Matheron1, Catherine Joulian2, Jean-Luc Cayol2 and Agnès Hirschler-Réa1,{dagger}

1 Laboratoire de Microbiologie, IMEP CNRS UMR 6116, Faculté des Sciences et Techniques de Saint-Jérôme, case 452, 13397 Marseille cedex 20, France
2 Laboratoire de Microbiologie IRD, UR 101, IFR-BAIM case 925, Universités de Provence et de la Méditerranée, 163 avenue de Luminy, 13288 Marseille cedex 9, France

Correspondence
Cristiana Cravo-Laureau
c.cravo-laureau{at}univ.u-3mrs.fr


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An alkene-degrading, sulfate-reducing bacterium, strain PF2803T, was isolated from oil-polluted sediments (Fos Harbour, France). The cells were found to be Gram-negative, non-sporulating, non-motile and to have a slightly curved rod shape. Optimum growth occurred at 1 % (w/v) NaCl, pH 6·8 and 28–30 °C. Strain PF2803T oxidized alkenes (from C8 to C23). The G+C content of the genomic DNA was 57·8 mol% (HPLC). On the basis of 16S rRNA gene sequence analyses, strain PF2803T belongs to the family ‘Desulfobacteraceae’ in the class ‘Deltaproteobacteria’, with Desulfatibacillum aliphaticivorans as its closest relative (99·6 % identity). Comparative sequence analyses of the dissimilatory sulfite reductase (dsrAB) gene supported the affiliation of strain PF2803T to the genus Desulfatibacillum. DNA–DNA hybridization with its closest taxon demonstrated 48·4 % similarity. On the basis of the results of physiological and genetic analyses, strain PF2803T is identified as a novel species of the genus Desulfatibacillum, for which the name Desulfatibacillum alkenivorans sp. nov. is proposed. The type strain is PF2803T (=DSM 16219T=ATCC BAA-924T).

The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene and dsrAB gene sequences of strain PF2803T are respectively AY493562 and AY504426.

A phase-contrast micrograph of cells of strain PF2803T is available as supplementary material in IJSEM Online.

{dagger}Present address: Laboratoire de Microbiologie IRD, UR 101, IFR-BAIM case 925, Universités de Provence et de la Méditerranée, 163 avenue de Luminy, 13288 Marseille cedex 9, France. Back


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To date, 12 sulfate-reducing bacterial strains that degrade hydrocarbons have been isolated (Spormann & Widdel, 2000Go; Cravo-Laureau et al., 2004Go). Of these strains, only five were able to oxidize aliphatic hydrocarbons (Aeckersberg et al., 1991Go, 1998Go; Rueter et al., 1994Go; So & Young, 1999Go; Cravo-Laureau et al., 2004Go). One of them was recently described as a member of a novel genus with the species name Desulfatibacillum aliphaticivorans (Cravo-Laureau et al., 2004Go). Here we report on the isolation and characterization of a novel species within this genus, for which we propose the name Desulfatibacillum alkenivorans sp. nov.

Several sulfate-reducing bacterial strains were isolated from oil-polluted sediments of a sewage plant of Fos Harbour (France). Prior to isolation, sediments (3/4) recovered by water (1/4) under a small gas phase were stored at room temperature for about 5 years in a plastic bottle under natural light conditions until use. The bottle was hermetically sealed and oxygen diffusion was limited. Enrichment and culture methods were as previously described (Cravo-Laureau et al., 2004Go). The sulfidogenic enrichment culture was obtained by incubating a homogeneous fraction (12 ml) of the plastic-bottle contents with natural sea-water medium (48 ml) (Cravo-Laureau et al., 2004Go) under a gas mixture (N2/CO2, 90 : 10) using anaerobic techniques (Pfennig et al., 1981Go). Enrichment culture was supplemented with 1-tetradecene (1·4 mM final concentration) as the energy and carbon source. Sodium dithionite (0·12 mM) was added as an additional reducing agent (Widdel & Bak, 1992Go). The enrichment culture was incubated at 30 °C in the dark, without agitation. After several subcultures with 1-tetradecene, pure cultures were obtained by repeated passage through a deep-agar dilution series according to Pfennig & Trüper (1981)Go, with sodium octanoate (2 mM) as substrate. The purity of the strains was confirmed by the absence of growth in natural sea-water medium supplemented with glucose (3 mM) and yeast extract (0·5 g l–1) under aerobic and anaerobic conditions, as well as in Ac medium (Difco), and by microscopic observations. Pure cultures were routinely maintained in a synthetic sulfate-reducing bacterial culture medium (Cravo-Laureau et al., 2004Go) with 1·5 % (w/v) NaCl. Cultures were supplemented with 1-tetradecene (1·4 mM) and sodium dithionite (0·12 mM).

Of the two sulfate-reducing isolates, strain PF2803T was chosen for taxonomic description. The cell walls of strain PF2803T produced a Gram-negative reaction in classical Gram staining and with the KOH method (Buck, 1982Go). The cells were non-motile, polymorphic rod-shaped and sometimes slightly curved (see supplementary figure in IJSEM Online).

Physiological characteristics (pH, salinity and temperature ranges) were determined as previously described (Cravo-Laureau et al., 2004Go). The results are presented in the species description. Under optimal growth conditions, the doubling time, with sodium octanoate as substrate, was about 22 h. The doubling time of strain PF2803T growing on hydrocarbon could not be determined because of the aggregation of the cells to hydrocarbon droplets. Therefore measurements of optical density, as well as centrifugation of cells, were not possible. The results of electron-donor and carbon-source utilization are presented in Table 1Go. Malate and pyruvate were not fermented. Strain PF2803T used sulfate (20 mM), thiosulfate (10 mM) and sulfite (5 mM) but not nitrate (10 mM), fumarate (10 mM) or elemental sulfur (0·8 g l–1) as electron acceptors. Cells of the new isolate did not contain desulfoviridin. Disproportionation of thiosulfate and sulfite was not observed. N2, NH4Cl and yeast extract were each used as a nitrogen source, but neither nitrate nor glutamate was used. Vitamins were not essential for growth.


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  		Table 1. DNA G+C contents and physiological characteristics of strains PF2803T and D. aliphaticivorans DSM 15576T

+, Growth; –, no growth; (+), slight growth. All strains were positive for utilization of the following (mM, except where stated otherwise): H2/CO2 (1 bar), pyruvate (10), crotonate (2), isobutyrate (5), butyrate (5), valerate (5), octanoate (2), nonanoate (2), palmitate (5), stearate (2) and butanol (10). All strains were negative for utilization of the following (mM, except where stated otherwise): formate+acetate (5/10), lactate (10), citrate (5), 2-oxoglutarate (5), tartrate (2), glycolate (2), thioglycolate (2), acetone (5), ethanol (10), methanol (10), 2-propanol (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).

 
Aliphatic hydrocarbons (n-alkanes, C5 to C20; n-alkenes, C8 to C23) were added at a final concentration of 250 p.p.m., whereas aromatic hydrocarbons (benzene, toluene, xylene, p-cymene, naphthalene and phenanthrene) were added at a final concentration of 100 p.p.m. Strain PF2803T was able to oxidize n-alkenes from C8 to C23. Growth on n-alkanes or aromatic hydrocarbons was not observed. This is the first report of a sulfate-reducing bacterium that, of all the hydrocarbons, exclusively degrades alkenes. Quantitative growth experiments on 1-tetradecene were carried out according to Cravo-Laureau et al. (2004)Go. Sulfide was determined colorimetrically (Cline, 1969Go). The exact sulfide content of standards was determined by iodometric titration (Vogel, 1961Go). Measurements of sulfate were performed using the turbidimetric method (Tabatabai, 1974Go) after removal of sulfide with zinc carbonate. The concentrations of 1-tetradecene were determined by GC after extraction with pentane (Cravo-Laureau et al., 2004Go). After 50 days incubation, strain PF2803T oxidized 0·92±0·13 mM tetradecene, consumed 12·01±1·41 mM sulfate and produced 11·5±1·15 mM sulfide. The complete oxidation of 1-tetradecene corresponds to the following theoretical reaction: C14H28+10·5 {ije541639E001}->10·5 S2–+14 H2O+14 CO2.

Our experimental results give values that are close to this reaction, showing that strain PF2803T completely oxidized hydrocarbon.

The G+C content of the DNA of strain PF2803T, determined at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) using HPLC according to a standard protocol described by Mesbah et al. (1989)Go, was 57·8 mol%. The methods used for extraction and purification of genomic DNA, for PCR amplification, for sequence alignment and for comparative analyses of 16S rRNA genes and {alpha}- and {beta}-subunits of the dissimilatory-sulfite-reductase (dsrAB) genes have been described previously (Cravo-Laureau et al., 2004Go). Sequencing was carried out by Genome Express (Grenoble, France). 16S rRNA gene sequence analysis (1295 bp) revealed that the novel isolate belonged to the class ‘Deltaproteobacteria’ and that its closest relative is D. aliphaticivorans (99·6 % identity) (Fig. 1Go). The phylogenetic position of the novel isolate within the genus Desulfatibacillum was supported by dsrAB sequence analyses (329 aa) (Fig. 2Go). DNA–DNA hybridization studies with strain PF2803T and D. aliphaticivorans (DSM 15576T) were performed at the DSMZ by the method of De Ley et al. (1970)Go, as modified by Escara & Hutton (1980)Go and Huß et al. (1983)Go. The relatedness value between the two strains is 48·4 %. Although this value is relatively high with regard to the fact that the G+C (% mol) contents of the DNAs are very different (Table 1Go), it is significantly below the value of 70 % recommended by Wayne et al. (1987)Go for strains of the same species. On the basis of physiological differences (Table 1Go) between strain PF2803T and D. aliphaticivorans, DNA–DNA hybridization results and DNA G+C contents, it is justified to consider strain PF2803T as a novel species of the genus Desulfatibacillum, for which the name Desulfatibacillum alkenivorans is proposed.



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  		Fig. 1. Phylogenetic tree based on comparative analyses of 16S rRNA gene sequences of strain PF2803T and its relatives. Bar, 2 nucleotide substitutions per 100 nucleotides.

 


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  		Fig. 2. Phylogenetic tree based on comparative analyses of amino acid sequences deduced from dsrAB genes of strain PF2803T and its relatives. Bar, 5 substitutions per 100 amino acids.

 
Emended description of the genus Desulfatibacillum Cravo-Laureau et al. 2004Go
Alkanes and/or alkenes are used as substrates. The type species is Desulfatibacillum aliphaticivorans.

Description of Desulfatibacillum alkenivorans sp. nov.
Desulfatibacillum alkenivorans (al.ke.ni'vo.rans. N.L. n. alkenum alkene, a specific hydrocarbon molecule; L. v. voro to devour; N.L. part. adj. alkenivorans devouring alkenes).

Cells are slightly curved rods (0·68x1·2–4·5 µm). Growth occurs at 22–38 °C (optimum, 28–30 °C) and pH 6·2–8·0 (optimum, pH 6·8). Growth occurs at NaCl concentrations of 5–50 g l–1 (optimum, 10 g NaCl l–1). Sulfate, sulfite and thiosulfate are used as electron acceptors. H2, pyruvate, malate, crotonate, isobutyrate, fatty acids (C4 to C18), butanol and glycerol serve as electron donors. Able to grow autotrophically. Alkenes (C8 to C23) are oxidized. No vitamins are required. The G+C content of the DNA is 57·8 mol% (HPLC).

The type strain is PF2803T (=DSM 16219T=ATCC BAA-924T). Isolated from oil-polluted sediments of a sewage plant at Fos Harbour (France).


   ACKNOWLEDGEMENTS
 
We thank V. Calvert for technical assistance, P. Caumette for help with the nomenclature of the strain and L. Casalot for her careful reading of the English. This work was supported by a grant from the Centre National de la Recherche Scientifique and TotalFinaElf through research group Hycar 1123. We acknowledge the comments of Dr P. Vandamme and the anonymous reviewers who helped to improve the manuscript.


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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. (2004). Desulfatibacillum aliphaticivorans gen. nov., sp. nov., an n-alkane- and n-alkene-degrading, sulfate-reducing bacterium. Int J Syst Evol Microbiol 54, 77–83.[Abstract/Free Full Text]

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]

Escara, J. F. & Hutton, J. R. (1980). Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of the renaturation rate. Biopolymers 19, 1315–1327.[CrossRef][Medline]

Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.

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.

Pfennig, N. & Trüper, H. G. (1981). Isolation of members of the families Chromatiaceae and Chlorobiaceae. In The Prokaryotes, vol. 1, pp. 279–289. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel. Berlin: Springer.

Pfennig, N., Widdel, F. & Trüper, H. G. (1981). The dissimilatory sulfate-reducing bacteria. In The Prokaryotes, vol. 1, pp. 926–940. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel. Berlin: Springer.

Rueter, P., Rabus, R., Wilkes, H., Aeckersberg, F., Rainey, F. A., Jannash, H. W. & Widdel, F. (1994). Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria. Nature 372, 455–457.[CrossRef][Medline]

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