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1 Université de Pau et des Pays de l'Adour, IBEAS, Laboratoire d'Ecologie Moléculaire, EA3525, F-64013 Pau, France
2 11 Place Garibaldi, F-42000 Saint Etienne, France
Correspondence
Michel Magot
michel.magot{at}univ-pau.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of SRL4225T, SRL6146T and Desulfovibrio longus DSM 6739T are AY359868, U53464 and AY359867, respectively.
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). Among other approaches, it was decided to isolate, characterize and identify all cultivable bacteria from a set of representative samples of production water. During this work, a large number of previously undescribed moderately halophilic, mesophilic, strictly anaerobic bacterial species have been isolated and described (Magot et al., 1997a, b; Ollivier et al., 1997; Ravot et al., 1997, 1999). Several strains of sulfate-reducing bacteria that could not be related to any known species on a phylogenetic basis have also been isolated. Among them, strains SRL4225T and SRL6146T were included in the genus Desulfovibrio; however, they are distant enough from known Desulfovibrio species, either phylogenetically or physiologically, to be described as novel species, for which we propose the names Desulfovibrio bastinii sp. nov. (SRL4225T) and Desulfovibrio gracilis sp. nov. (SRL6146T).
Strain SRL4225T was isolated from a water sample collected in the oil pipeline linking offshore production platforms of the Emeraude offshore oilfield (Congo) to onshore treatment facilities. The total bacterial count in the water sample (acridine orange direct count) indicated the presence of 2·5x104 bacteria ml–1. Most probable number counts of sulfate-reducing bacteria were done as described previously (Magot et al., 1992) and showed the presence of 2·5x102 cultivable sulfate-reducing bacteria ml–1. In this experiment, the total salinity of the culture medium and the incubation temperature were adjusted so that they corresponded to the in situ conditions: total salinity of 53 g l–1 and a temperature of 37 °C. Isolation of pure strains from the highest dilution of the most probable number count was done on solid medium in Petri dishes. Round black colonies (2–3 mm in diameter) were obtained after 2 weeks incubation. Several colonies were picked and their isolation was repeated twice. Cell morphology and restriction patterns of the amplified 16S rRNA genes allowed detection of strain SRL4225T, potentially representing a previously unknown species. Cells of strain SRL4225T were actively motile vibrios occurring singly or in pairs and were 2·0–3·0x0·5 µm in size (Fig. 1a).
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). Electron microscopic observations indicated that cells of both strains were motile by means of a single polar flagellum (not shown). Gram staining of both strains was negative.
The physiological and metabolic characterization of the strains included tests to determine utilization of different carbon substrates and energy sources, utilization of a variety of electron acceptors, fermentative growth, temperature, pH and salinity optima, vitamin requirements and pigment content; the tests used have been described previously (Magot et al., 1992; Tardy-Jacquenod et al., 1996).
The energy and carbon sources used by SRL4225T and SRL6146T are shown in Table 1 and indicate that both isolates used a rather limited number of substrates. After five transfers, neither strain grew in the absence of vitamins or yeast extract.
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Both strains are moderately halophilic bacteria displaying a similar salt requirement that is in agreement with the salinity of their oilfield-water habitat (Table 1). Strain SRL4225T can grow between pH 5·2 and 7·4, with an optimum at pH 5·8–6·2: such tolerance of low pH is uncommon among sulfate-reducing bacteria. According to these characteristics, this strain can be considered as a moderately acidophilic sulfate-reducing bacterium, a physiological description that corresponds with the pH of Emeraude Oilfield water in situ (pH 5·5–6·5), but which is quite uncommon in this bacterial group.
The optimum temperatures for growth of both strains (see descriptions of the species) are also in accordance with the in situ temperature, which varies from 35 to 42 °C in the different production zones of the oilfield.
Under optimal conditions with lactate as electron donor and sulfate as electron acceptor, the doubling times of strains SRL4225T and SRL6146T were 15 and 22 h, respectively.
Determination of the G+C content of the DNA of strains SRL4225T and SRL6146T was done by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. The results are reported in Table 1.
The 16S rRNA genes of SRL4225T and SRL6146T were amplified and their sequences determined and analysed as previously described (Tardy-Jacquenod et al., 1996). A new sequence of the 16S rRNA gene of Desulfovibrio longus DSM 6739T was determined, for the purpose of this work, by using the same procedure.
The results of FASTA analysis of the 1416-nt 16S rRNA gene sequence of strain SRL4225T against sequences in GenBank, and the construction of a phylogenetic tree as previously described (Tardy-Jacquenod et al., 1996), revealed that strain SRL4225T forms a distinct cluster with the undescribed Desulfovibrio species strain ASPO3 (93·6 % similarity, distance matrix value), Desulfovibrio hydrothermalis (92·6 %), Desulfovibrio salexigens (91·8 %) and Desulfovibrio zosterae (91·7 %) (Fig. 2). These results suggested that SRL4225T could represent a novel species within the genus Desulfovibrio, although many of its characteristics (except its pH tolerance) overlap with those of known Desulfovibrio strains.
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), and to the recently described species ‘Desulfovibrio capillatus’ (98·2 % similarity), isolated from an oilfield separator (Miranda-Tello et al., 2003). Since it is also closely related to Desulfovibrio longus (97·5 % similarity), the results of DNA–DNA reassociation experiments, done by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen, were analysed to assess the species status of SRL6146T. The relatedness between SRL6146T and D. longus (23·1 %; this study) and that between SRL6146T and ‘D. capillatus’ (28·6 %; Miranda-Tello et al., 2003) clearly indicated that the three strains represent three distinct species.
Thus, SRL6146T, D. longus and ‘D. capillatus’ form a distinct cluster of closely related bacteria isolated from oilfield waters. Strain SRL6146T closely resembles D. longus in terms of its morphological, physiological and biochemical characteristics. Apart from the 3·3 mol% difference in the G+C contents of their DNAs, the main difference between these species lies in their salt tolerance. SRL6146T is a true moderately halophilic bacterium, since it requires at least 20 g NaCl l–1, whereas D. longus can grow in the absence of salt (Table 1). This major physiological difference is also reflected by the upper limit of salt tolerance of 120 g l–1, as opposed to 80 g l–1 for D. longus, and its optimum salinity of 50–60 g NaCl l–1, which compares with 10–20 g l–1 for D. longus.
SRL6146T is phenotypically different from the other related species, ‘D. capillatus’ (Miranda-Tello et al., 2003). These two bacteria differ in morphology, use different electron donors, differ in the fermentation of pyruvate and differ in the use of fumarate as electron acceptor (Table 1).
On the basis of these observations, we propose to assign strains SRL4225T and SRL6146T to the genus Desulfovibrio as novel species, Desulfovibrio bastinii sp. nov. and Desulfovibrio gracilis sp. nov., respectively.
The two novel bacterial strains isolated during this work display several characteristics that suggest that they might be indigenous to the oil reservoir. Their moderately halophilic character, with optimal salinities for growth between 40 and 60 g l–1, correspond perfectly to the in situ water salinity of 53 g l–1. In addition, the optimal growth temperatures and pH values are in good agreement with the physico-chemical conditions prevailing in the reservoir, i.e. 37–42 °C and pH 5·5–6·5. For these reasons, we consider that SRL4225T and SRL6146T represent novel bacterial strains originating from a deep subsurface environment (Magot et al., 2000).
Description of Desulfovibrio bastinii sp. nov.
Desulfovibrio bastinii (bas.ti'ni.i. N.L. gen. n. bastinii of Bastin, named after the American microbiologist Edson S. Bastin, who studied sulfate-reducing bacteria from oilfields at the beginning of the 20th century).
Cells are vibrioid, 0·5x2–3 µm, occur singly or in pairs and are motile by means of a single polar flagellum. Gram-negative and non-spore-forming. Colonies are round with entire edges, smooth, and grey to dark grey. Moderately halophilic. Optimal NaCl concentration for growth is 40 g l–1; growth occurs between 10 and 120 g NaCl l–1. Mesophilic. Optimal growth temperature is 35–40 °C; range for growth is 20–50 °C. Moderately acidophilic. pH range is 5·2–7·4; optimum is pH 5·8–6·2. Vitamins are required for growth. Strictly anaerobic. Sulfate, sulfite, thiosulfate and elemental sulfur serve as electron acceptors, whereas nitrate and fumarate do not. No autotrophic growth. Lactate, pyruvate, malate, fumarate, ethanol, butanol, glycerol and hydrogen in the presence of acetate are used as growth substrates. Lactate is oxidized to acetate and CO2. Pyruvate is fermented. Desulfoviridin is present. The G+C content of the DNA is 44·6 mol%.
The type strain is SRL4225T (=DSM 16055T=ATCC BAA-903T), isolated from oilfield production water collected from a pipeline of Emeraude Oilfield, Congo.
Description of Desulfovibrio gracilis sp. nov.
Desulfovibrio gracilis (gra'cil.is. L. masc. adj. gracilis slim, slender, thin).
Cells are rod-shaped, 0·3x4·5–9·0 µm, straight, curved or vibrioid and thin, occur singly and are motile by means of a single polar flagellum. Gram-negative and non spore-forming. Colonies are round with entire edges, smooth and grey to dark grey. Moderately halophilic. Optimal NaCl concentration for growth is 50–60 g l–1; growth occurs between 20 and 120 g NaCl l–1. Mesophilic. Optimal growth temperature is 37–40 °C; range for growth is 20–40 °C. pH range is 5·4–8·4; optimum is pH 6·8–7·2. Vitamins are required for growth. Strictly anaerobic. Sulfate, sulfite, thiosulfate, elemental sulfur and fumarate serve as electron acceptors; nitrate is not used. No autotrophic growth. Lactate and pyruvate are used as growth substrates. Lactate is oxidized to acetate and CO2. Fumarate and hydrogen are used in the presence of acetate. Glucose, fructose, succinate, malate, fumarate, pyruvate, lactate, cysteine and glycerol are not fermented. Desulfoviridin is present. The G+C content of the DNA is 59·0 mol%.
The type strain is SRL6146T (=DSM 16080T=ATCC BAA-904T), isolated from oilfield production water collected at the E153 wellhead of Emeraude Oilfield, Congo.
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ACKNOWLEDGEMENTS |
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