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

Footnotes

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., 2004). 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., 2004) under a gas mixture (N₂/CO₂, 90 : 10) using anaerobic techniques (Pfennig et al., 1981). 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, 1992). 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), 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 l¹) 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., 2004) 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 PF2803^T was chosen for taxonomic description. The cell walls of strain PF2803^T produced a Gram-negative reaction in classical Gram staining and with the KOH method (Buck, 1982). 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., 2004). 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 PF2803^T 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 1. Malate and pyruvate were not fermented. Strain PF2803^T 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 l¹) 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.

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 l1), Casamino acids (0·5 g l1) and yeast extract (0·5 g l1).

Our experimental results give values that are close to this reaction, showing that strain PF2803^T completely oxidized hydrocarbon.

The G+C content of the DNA of strain PF2803^T, 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), 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 α- and β-subunits of the dissimilatory-sulfite-reductase (dsrAB) genes have been described previously (Cravo-Laureau et al., 2004). 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. 1). The phylogenetic position of the novel isolate within the genus Desulfatibacillum was supported by dsrAB sequence analyses (329 aa) (Fig. 2). DNADNA hybridization studies with strain PF2803^T and D. aliphaticivorans (DSM 15576^T) were performed at the DSMZ by the method of De Ley et al. (1970), as modified by Escara & Hutton (1980) and Huß et al. (1983). 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 1), it is significantly below the value of 70 % recommended by Wayne et al. (1987) for strains of the same species. On the basis of physiological differences (Table 1) between strain PF2803^T and D. aliphaticivorans, DNADNA hybridization results and DNA G+C contents, it is justified to consider strain PF2803^T as a novel species of the genus Desulfatibacillum, for which the name Desulfatibacillum alkenivorans is proposed.

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 (0·68x1·24·5 µm). Growth occurs at 2238 °C (optimum, 2830 °C) and pH 6·28·0 (optimum, pH 6·8). Growth occurs at NaCl concentrations of 550 g l¹ (optimum, 10 g NaCl l¹). 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 PF2803^T (=DSM 16219^T=ATCC BAA-924^T). Isolated from oil-polluted sediments of a sewage plant at Fos Harbour (France).