Isolation of sulfate-reducing bacteria from Tunisian marine sediments and description of Desulfovibrio bizertensis sp. nov.

In marine ecosystems, sulfate-reducing bacteria (SRB) contribute significantly (50 %) to the mineralization of organic matter (Jørgensen, 1982). Hydrogenotrophic SRB of the family Desulfovibrionaceae within the Deltaproteobacteria have been found as common inhabitants of these ecosystems (Jørgensen & Bak, 1991; Widdel & Hansen, 1992). Within the Desulfovibrionaceae, many marine Desulfovibrio species have been isolated. They include 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), all recovered from shallow marine ecosystems, and D. profundus (Bale et al., 1997) and D. hydrothermalis (Alazard et al., 2003) recovered from deep marine environments.

We have undertaken microbiological studies to isolate, in particular, hydrogenotrophic SRB from various marine sediments recovered near Tunis, Korbous and Bizerte, Tunisia. In addition to hydrogen, peptone and yeast extract were also used as energy sources in the presence of sulfate as the terminal electron acceptor. Using these cultures, we isolated only members of the genus Desulfovibrio. One of these organisms, designated strain MB3^T, is suggested to represent a novel species of the genus Desulfovibrio.

Sediment samples were collected in sterile plastic bottles from the sea off Korbous, Tunis and Bizerte, and kept at 5 °C until inoculation. The in situ temperature, pH and conductivity of samples were 30 °C, 7.8 and 62 mS cm⁻¹, respectively.

The Hungate technique (Hungate, 1969) was then used throughout for cultivation. The basal medium contained (per litre of distilled water): 0.3 g KH₂PO₄, 0.3 g K₂HPO₄, 1.0 g NH₄Cl, 23 g NaCl, 3 g Na₂SO₄, 0.1 g KCl, 0.1 g CaCl₂.2H₂O, 0.1 g yeast extract (Difco), 0.5 g cysteine hydrochloride, 1 ml trace mineral element solution (Widdel & Pfennig, 1984) and 1 ml 0.1 % resazurin; pH was adjusted to 7.2 with 10 M KOH. The basal medium was boiled under a stream of O₂-free N₂ gas and cooled to room temperature and 5 ml aliquots were distributed in Hungate tubes under a stream of O₂-free N₂ gas. The N₂ gas phase was replaced with N₂/CO₂ (80 : 20, v/v) and the tubes were autoclaved for 45 min at 110 °C. Prior to inoculation, 0.1 ml 2 % Na₂S.9H₂O, 0.1 ml 10 % NaHCO₃ and 0.1 ml 15 % MgCl₂.6H₂O were added.

Enrichments were performed in Hungate tubes containing 5 ml medium and inoculated with sample diluted to 10 %. H₂+CO₂ [80 : 20 (v/v), 2 bar], peptone (10 g l⁻¹) or yeast extract (0.2 g l⁻¹) were used as substrates. Acetate (2 mM) was added as the carbon source in the presence of hydrogen as electron donor. The tubes were incubated at 30 °C for 3 days. Three enrichment series were performed. Cultures were purified by repeated use of the Hungate roll-tube method with medium solidified with 2.5 % (w/v) agar (Difco). Several colonies that developed were picked and cultured in the corresponding culture medium. The process of isolation was repeated several times until isolates were deemed to be axenic. Physiological optimal growth conditions (for strain MB3^T only) were determined in duplicate experiments conducted in basal medium containing lactate (20 mM) and thiosulfate (20 mM) as described by Fardeau et al. (1993). Growth was measured by inserting tubes directly into a model Cary 50 Scan spectrophotometer (Varian) and measuring the OD₅₈₀. Sulfide was determined photometrically as colloidal CuS following the method of Cord-Ruwisch (1985).

Genomic DNA was extracted according to the protocol described for the Wizard Genomic DNA purification kit (Promega). 16S rRNA genes were amplified by using primers Fd1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and Rd1 (5-AAGGAGGTGATCCAGCC-3′) and by using the following reaction conditions: 1 min at 94 °C, 30 cycles of 30 s at 94 °C, 1 min at 50 °C and 2 min at 72 °C, and a final extension step of 10 min at 72 °C. PCR fragments were then cloned into pGEM-T-easy (Promega). Recombinant clones, with inserts of the correct length, were sequenced by using primers SP6 (5′-ATTTAGGTGACACTATAGAA-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′) (Genome Express). The nucleotide sequences of the 16S rRNA genes were compared with reference sequences from the GenBank database (Benson et al., 1999). The 16S rRNA gene sequence of strain MB3^T was aligned with reference sequences of various Desulfovibrio species using programs provided by the Ribosomal Database Project II (Maidak et al., 2001). Sequence alignment was verified manually using the program bioedit (Hall, 1999). Positions of sequence and alignment uncertainty were omitted from the analysis. Pairwise evolutionary distances based on an unambiguous stretch of 1274 bp were computed by using the Jukes & Cantor (1969) method. The dendrogram was constructed by using the neighbour-joining method (Saitou & Nei, 1987). Confidence in the tree topology was determined by bootstrap analysis based on 100 resamplings (Felsenstein, 1985).

Several SRB were isolated from marine sediments recovered near Tunis, Korbous and Bizerte in the presence of hydrogen, peptone or yeast extract as substrates and sulfate as the terminal electron acceptor. All isolates were found to be phylogenetically related to members of the genus Desulfovibrio (Table 1⇓). Strains LB2 and MB2, isolated from marine sediments recovered near Bizerte, were found to be closely related (>99 % 16S rRNA gene sequence similarity) to an uncharacterized Desulfovibrio strain, TBP-1 (Boyle et al., 1999). Strain SIJ23, isolated from marine sediments recovered near Tunis, and strain LB4, isolated from marine sediments recovered near Bizerte, were phylogenetically related (99–100 % similarity) to Desulfovibrio alaskensis and Desulfovibrio senezii, respectively (Feio et al., 2004; Tsu et al., 1998). Four other strains isolated from marine sediments recovered near Korbous (strain KM2), Bizerte (strains LB3 and MB3^T) and Tunis (strain LT3) also had D. senezii as their closest phylogenetic relative, but with low 16S rRNA gene sequence similarity (<97 %), indicating that they may represent novel species of the genus Desulfovibrio. Only strain MB3^T was characterized further.

Strain MB3^T was strictly anaerobic and mesophilic; optimal temperature for growth was 40 °C (range 15–45 °C). For pH studies, the medium was adjusted to the desired pH using anaerobically prepared stock solutions of NaHCO₃ (10 %) or Na₂CO₃ (10 %). Strain MB3^T was neutrophilic; the optimum pH for growth was 7 and growth occurred between pH 6.0 and 8.1. For determination of NaCl requirements, NaCl was weighed directly in the tubes at concentrations of 0–130 g l⁻¹ before dispensing to NaCl-free basal medium. The isolate was slightly halophilic and grew in the presence of NaCl concentrations ranging from 5 to 50 g l⁻¹, with optimum growth at 20 g l⁻¹.

The following substrates (20 mM) were used as carbon and energy sources: lactate, ethanol, pyruvate, malate, fumarate, succinate, H₂/CO₂ with acetate (2 mM), butanol and propanol. Acetate, propionate, butyrate, Casamino acids (0.1 %), fructose, glucose and methanol (40 mM) were also tested but did not support growth. Sulfate (20 mM), thiosulfate (20 mM), elemental sulfur (0.1 %), sulfite (2 mM) and fumarate (20 mM) were used as terminal electron acceptors, but not nitrate (20 mM) or nitrite (2 mM). Strain MB3^T fermented pyruvate into acetate, hydrogen and CO₂. Fumarate was fermented only weakly. Fermentation products were determined as described by Fardeau et al. (1993). The end products from lactate metabolism in the presence of thiosulfate as terminal electron acceptor were acetate, CO₂ and H₂S.

Growth of strain MB3^T was inhibited by the addition of chloramphenicol (50 μg ml⁻¹), ampicillin (100 μg ml⁻¹) and vancomycin (300 μg ml⁻¹).

The presence of bisulfite reductase (desulfoviridin) was confirmed by measuring the absorbance of cell-free extracts at 630 nm (Badziong et al., 1978). In addition, c-type cytochromes were detected by reduction of extracts with sodium dithionite, with two peaks occurring at 418 and 550 nm.

The G+C content of the DNA (determined by the Identification Service of the DSMZ, Braunschweig, Germany) was 51 mol% based on the method of Mesbah et al. (1989).

Analysis of the almost complete sequence (1274 bp) of the 16S rRNA gene of strain MB3^T revealed that it grouped with members of the family Desulfovibrionaceae, order Desulfovibriales, in the Deltapoteobacteria. The phylogenetic tree constructed is shown in Fig. 1⇓. As indicated above, strain MB3^T clustered with D. senezii DSM 8436^T, an isolate recovered from a solar saltern in California (Tsu et al., 1998), with a 16S rRNA gene sequence similarity of 91.3 %. Phenotypic differences were observed between strain MB3^Tand D. senezii, including the range of substrates utilized, salt tolerance and DNA G+C content (Table 2⇓). In addition to the taxonomic significance of strain MB3^T as a novel representative of the SRB within the Deltaproteobacteria, it is noteworthy that the enrichments described herein, performed with marine sediment samples originating from different locations in Tunisia, have led to the isolation of only Desulfovibrio-like strains when using hydrogen as the substrate. However, the prominent isolation of these Desulfovibrio strains may result from their known ability to grow rapidly when using hydrogen as substrate (Jørgensen & Bak, 1991) as compared with the growth of other hydrogenotrophic SRB species. Interestingly, among the Desulfovibrio strains recovered from Tunisian marine sediments, we have isolated some using peptone as substrate, providing further evidence that SRB may play a decisive role in the regulation of electron flow in protein amino-acid turnover through sulfate reduction in marine ecosystems (Hansen & Blackburn, 1995; Baena et al., 1998).

On the basis of its phenotypic, genotypic and phylogenetic characteristics, strain MB3^T is proposed as the type strain of a novel species, Desulfovibrio bizertensis sp. nov.