# Characterization of Desulfovibrio salinus sp. nov., a slightly halophilic sulfate-reducing bacterium isolated from a saline lake in Tunisia

Zouhaier Ben Ali Gam, Abdoulaye Thioye, Jean-Luc Cayol, Manon Bartoli, Guy Fauque, Marc Labat

<figure><img src="image_1.png" /></figure>

## To cite this version:

Zouhaier Ben Ali Gam, Abdoulaye Thioye, Jean-Luc Cayol, Manon Bartoli, Guy Fauque, et al.. Characterization of Desulfovibrio salinus sp. nov., a slightly halophilic sulfate-reducing bacterium isolated from a saline lake in Tunisia. International Journal of Systematic and Evolutionary Microbiology, 2018, 68 (3), pp.715-720. 10.1099/ijsem.0.002567. hal-01981629

HAL Id: hal-01981629

https://amu.hal.science/hal-01981629

Submitted on 15 Jan 2019

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Characterization of *Desulfovibrio salinus* sp. nov., a slightly halophilic sulfate-reducing bacterium isolated from a saline lake in Tunisia

Zouhaier Ben Ali Gam,¹ Abdoulaye Thioye,¹,² Jean-Luc Cayol,¹ Manon Joseph,¹ Guy Fauque¹ and Marc Labat¹,*,

## Abstract

A novel slightly halophilic sulfate-reducing bacterium, designated strain P1BSR<sup>T</sup>, was isolated from water of a saline lake in Tunisia. Strain P1BSR<sup>T</sup> had motile (single polar flagellum), Gram-negative, rod-shaped, non-spore-forming cells, occurring singly or in pairs. Strain P1BSR<sup>T</sup> grew at temperatures between 15 and 45°C (optimum 40°C), and in a pH range between 6 and 8.5 (optimum pH 6.7). The strain required NaCl for growth (1% w/v), and tolerated high NaCl concentration (up to 12% w/v) with an optimum of 3% (w/v). Sulfate, thiosulfate and sulfite served as terminal electron acceptors, but not elemental sulfur, fumarate, nitrate and nitrite. Strain P1BSR<sup>T</sup> utilized lactate, pyruvate, formate, D-fructose and glycerol as carbon and energy sources. The main cellular fatty acid was C<sub>16:0</sub> (50.8%). The genomic DNA G+C content was 47.7 mol%. Phylogenetic analysis of 16S rRNA gene sequence similarity indicated that strain P1BSR<sup>T</sup> was affiliated to the genus *Desulfovibrio*, with the type strains *Desulfovibrio salexigens* (96.51%), *Desulfovibrio zosterae* (95.68%), *Desulfovibrio hydrothermalis* (94.81%) and *Desulfovibrio ferrireducens* (94.73%) as its closest phylogenetic relatives. On the basis of genotypic, phenotypic and phylogenetic characteristics, it is proposed to assign strain P1BSR<sup>T</sup> to a novel species of the genus *Desulfovibrio*, *Desulfovibrio salinus* sp. nov. The type strain is P1BSR<sup>T</sup> (=DSM 101510<sup>T</sup>=JCM 31065<sup>T</sup>).

Sulfate-reducing prokaryotes (SRPs) are mainly chemohe-
terotrophs, both *Bacteria* and *Archaea* (250 species of 65
genera), that can use sulfate as a terminal electron acceptor
in their energy metabolism [1, 2]. Owing to their broad met-
abolic capacities, sulfate-reducing bacteria (SRB) are
important in the mineralization of organic matter in anoxic
marine sediments. Dissimilatory sulfate reduction has been
observed in various hypersaline environments such as salt-
erns, the Dead Sea and the Great Salt Lake [3]. However,
most of the halophilic SRPs isolated so far are marine or
slightly halophilic microorganisms [with optimum salinity
ranging from 1 to 4% (w/v) NaCl] belonging to several gen-
era of SRPs, including *Desulfovibrio*, *Desulfonatronovibrio*
and *Desulfonatronobacter* species [3-6]. The first SRB spe-
cies belonging to the genus *Desulfovibrio* isolated from a
hypersaline environment was *Desulfovibrio salexigens*,
which did not grow at NaCl concentrations higher than
12% (w/v) [7]. Cord-Ruwish [8] then isolated several
strains of SRBs from hypersaline oilfield water containing
about 10% (w/v) NaCl. One isolate grew slowly up to 27%
(w/v) NaCl but has not been described in more detail since.

One year later, Trüper and Galinski [9] isolated a few SRB
strains from hot brines in the Red Sea that were similar to
*Desulfovibrio halophilus*, a moderately halophilic sulfate
reducer isolated by Caumette *et al*. [10] from the hypersa-
line Solar Lake in Sinai. The latter isolate grew in salinity
ranging from 3 to 18% (w/v), and optimally at 6-7% (w/v)
NaCl. However, since 1991, no novel SRB species belonging
to the genus *Desulfovibrio* has been characterized that grows
in salt concentrations above 10% NaCl (w/v). No pure cul-
ture of extreme halophilic SRB (i.e. growing under saturat-
ing-salt conditions) has been isolated until now.

In this study, we report the isolation and characterization of
a mesophilic, slightly halophilic SRB isolated from water
samples of a Tunisian saline lake that is able to grow in up
to 12% NaCl (w/v), but grows optimally at 3% (w/v). This
SRB isolate is proposed to represent a novel species of the
genus *Desulfovibrio*.

Water samples were collected from a continental saline
lake located in the middle-east of Tunisia and transported
to the laboratory at ambient temperature. Bacteria were

**Author affiliations:** ¹Laboratoire de Microbiologie IRD, Aix-Marseille Université, Université du Sud Toulon-Var, CNRS/INSU, IRD, MIO UM110, 163 avenue de Luminy, case 925, F-13288 Marseille cedex 9, France; ²Laboratoire de Microbiologie Appliquée et de Génie Industriel, Ecole Supérieure Polytechnique, Université Cheikh Anta Diop, BP 5005 Dakar-Fann, Dakar, Sénégal.

*Correspondence: Marc Labat, marc.labat@mio.osupytheas.fr

Keywords: *Desulfovibrio*; slightly halophilic; sulfate-reducing bacterium; saline lake; Tunisia.

Abbreviations: SRB, sulfate-reducing bacteria; SRP, sulfate-reducing prokaryote.

The Genbank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain P1BSR<sup>T</sup> is KT767983.
isolated and cultivated under strict anaerobiosis according
to the Hungate technique [11], modified for use with syrin-
ges [12, 13].

The basal culture medium (BM) for isolation included (per
litre): 0.3 g KH₂PO₄, 0.2 g K₂HPO₄, 0.3 g NH₄Cl, 30 g NaCl,
0.5 g KCl, 0.1 g CaCl₂, 0.4 g MgCl₂, 0.5 g cysteine HCl, 0.5 g
yeast extract (Difco), 1 ml mineral element solution and
1 ml 0.1% (w/v) resazurin [14]. The pH was adjusted to 7.2
with 10 M KOH solution and the medium was boiled under
a stream of O₂-free N₂ gas and cooled to room temperature.
Aliquots of 5 ml were dispensed into Hungate tubes under
N₂/CO₂ (80:20, v/v) and subsequently sterilized by
autoclaving at 120 °C for 20 min. Before inoculation, 0.1 ml
10% (w/v) NaHCO₃, 0.1 ml 2% (w/v) Na₂S.9H₂O, 20 mM
lactate and 20 mM sulfate were injected from sterile stock
solutions into the tubes. Na₂S.9H₂O as a reducing agent was
omitted when H₂S production was determined [8].

Enrichments were performed in Hungate tubes or serum bottles inoculated with 10% (v/v) sample and incubated at 30°C. The culture was purified by repeated use of the Hungate roll tube method, using agar solid medium (0.8% w/v), and transferred to liquid medium.

The pH, temperature and NaCl concentration ranges for
growth were determined in duplicate experiments using BM
supplemented with lactate (20 mM) as electron donor and
sulfate (20 mM) as previously described [15]. The pH (from
5 to 9) of the culture medium was adjusted by injecting ali-
quots of anaerobic stock solution into Hungate tubes con-
taining 100 mM HCl for low pH or either 10% NaHCO₃
(w/v) or 8% Na₂CO₃ (w/v) for high pH. Water baths were
used to incubate bacterial cultures from 15 to 50 °C. NaCl
requirement was determined by directly weighing NaCl into
the Hungate tubes before dispensing the medium. Cultures
were subcultured at least twice under the same experimental
conditions before determination of growth rates.

Bacterial growth was monitored by measuring the increase
in turbidity at 580 nm after inserting Hungate tubes into the
cuvette holder of a Cary 50 UV-vis spectrophotometer
(Varian). H₂S production was determined photometrically
as colloidal CuS following the method described in [8].

Morphological characteristics and purity of strains were
checked under an Optiphot (Nikon) phase-contrast micro-
scope. Presence of spores was analysed by phase-contrast
microscopy observations of cultures and after pasteurization
tests performed for 10 and 20 min at 80, 90 and 100 °C.

Culture of the isolate (strain P1BSR<sup>T</sup>) was stopped at the
end of the exponential phase and sent to the Deutsche
Sammlung von Mikroorganismen und Zellkulturen
(DSMZ) for cellular fatty acid analysis. Fatty acids were
extracted using the Miller method [16] as modified by
Kuykendall et al. [17], and the cellular fatty acid profile was
analysed by gas chromatography using the Microbial Iden-
fication System (MIDI, Sherlock Version 6.1; database,
TSBA40; gas chromatograph, model 6890 N; Agilent

Technologies) G+C content was determined by HPLC as
described by [18].

Substrate utilization was tested in the presence of 0.5 g
yeast extract 1⁻¹. Substrates (20 mM except where indi-
cated) included lactate, pyruvate, acetate, formate, fuma-
rate, malate, succinate, propionate, methanol (0.1 % w/v),
ethanol (0.1 % w/v), butanol (0.1 % w/v), glycerol (0.1 %
w/v), D-fructose, D-glucose and H₂/CO₂ (20 : 80 v/v). Ele-
mental sulfur (1 % w/v), sulfate (20 mM), thiosulfate
(20 mM), sulfite (2 mM), fumarate (10 mM), nitrate
(10 mM) and nitrite (2 mM) were tested as potential termi-
nal electron acceptors.

Extraction and purification of total DNA followed by ampli-
fication and sequencing of the 16S rRNA gene were per-
formed as previously described [15]. The 16S rRNA gene
sequence was then compared with available sequences
found in GenBank using the BLASTN search [19]. Evolution-
ary history was inferred using the neighbour-joining
method [20]. A multiple alignment was built using the
MUSCLE program [21] implemented in MEGA6 [22]. Sequence
positions with alignment uncertainty and gaps were omitted
from the analysis. Evolutionary analyses were conducted in
MEGA6 using the neighbour-joining method. Evolutionary
distances were computed using the maximum-composite-
likelihood method [23]. The final dataset consisted of a total
of 1213 positions. Branch robustness was estimated by the
non-parametric bootstrap procedure implemented in
MEGA6 (1000 replicates of the original dataset). The tree was

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Transmission electron micrograph of an ultrathin section of a cell of strain P1BSR<sup>T</sup> showing a thin peptidoglycan layer and an outer crenated membrane; bar, 100 nm.</figcaption></figure>
drawn to scale, with branch lengths measured in number of
substitutions per site [24].

Several colonies developed after incubation at 30 °C and
were picked separately. Colonies were black and circular
with diameters ranging from 1.0 to 2.0 mm after 1 week of
incubation at 30 °C. The serial dilution process was
repeated several times until the isolates were deemed to be
axenic. Purity of the isolates was checked by microscopy
and inoculation in sulfate-free medium containing yeast
extract and D-glucose to confirm the absence of contamina-
tion by fermentative microorganisms. Several strains were
isolated that showed similar morphology, size and meta-
bolic profiles, and the same phylogenic inference was
obtained for all of them. One strain, designated P1BSR<sup>T</sup>,
was selected and used for further metabolic and physiologi-
cal characterization.

Cells of strain P1BSR<sup>T</sup> were Gram-stain-negative rods (0.7–0.9×2.3 µm) when grown on medium containing lactate as an electron donor and sulfate as a terminal electron acceptor. Ultrathin sections showed a typical Gram-negative cell wall with a thin peptidoglycan layer and a crenated outer membrane (Fig. 1).

The optimal temperature for growth was 40 °C (range 15–45 °C) and the optimum pH was 6.7 (range pH 6–8.5). The strain required a minimum of 1 % (w/v) NaCl for growth, and tolerated up to 12 % (w/v); optimum growth occurred with 3 % (w/v) NaCl. All substrates were used by strain P1BSR<sup>T</sup> with incomplete oxidation. Amongst the substrates tested, only lactate, pyruvate, formate, glycerol and D-fructose were used as electron donors. End products of pyruvate, fumarate and malate were not fermented. Thiosulfate, sulfate and sulfite, but not fumarate, elemental sulfur, nitrate or nitrite served as terminal electron acceptors. Malate, succinate, methanol, ethanol, butanol, D-glucose, acetate, propionate and H<sub>2</sub>/CO<sub>2</sub> did not support growth. The presence of yeast extract in minimal medium with lactate as the only energy and carbon source was required for growth.

Based on the nearly complete (1536 bp) 16S rRNA gene sequence of strain P1BSR<sup>T</sup>, the phylogenetic tree obtained by the neighbour-joining method, as shown in Fig. 2, confirms that strain P1BSR<sup>T</sup> is an SRB belonging to the genus *Desulfovibrio*, sharing 96.51 % 16S rRNA gene sequence identity with its closest phylogenetic relatives, i.e. *Desulfovibrio salexigens* DSM 2638<sup>T</sup> [4, 7, 25] isolated from sling mud (British Guiana), 95.68 % with *Desulfovibrio zosterae*

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Phylogenetic dendrogram based on 1368 unambiguous base pairs of 16S rRNA gene sequence data, indicating the position of strain P1BSR<sup>T</sup> and its closest relative sequences validated at a species level belonging to the genus *Desulfovibrio*. The tree was reconstructed using the neighbour-joining algorithm based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 2000 replications) greater than 60 % are shown at branch points. *Desulfofotulus sapovorans* 33892<sup>T</sup> was used as outgroup. Numbers in parentheses are Genbank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences. Bar, 2 % estimated sequence divergence.</figcaption></figure>
DSM 11974<sup>T</sup> [26], 94.81% with *Desulfovibrio hydrothermolis* DSM 14728<sup>T</sup> [27] and 94.73% with *Desulfovibrio ferrireducens* DSM 16995<sup>T</sup> [28], thus indicating that strain P1BSRT<sup>T</sup> represents a novel species of the genus *Desulfovibrio*. Further evidence resulted from several phenotypic and biochemical differences, including cell morphology, but also comparison of the utilization of carbon and energy sources with its two most-closely-related *Desulfovibrio* species (*Desulfovibrio salexigens* DSM 2638<sup>T</sup> and *Desulfovibrio zosterae* DSM 11974<sup>T</sup>) (Table 1). Indeed, in contrast to *Desulfovibrio salexigens* showing typically vibrio-shaped cells, as observed for most *Desulfovibrio* species, P1BSRT<sup>T</sup> cells were rod-shaped and occurred singly or in pairs. These three *Desulfovibrio* strains were mesophilic with different optimum temperatures (Table 1). Strain P1BSRT<sup>T</sup> and *Desulfovibrio salexigens* DSM 2638<sup>T</sup> have similar NaCl requirement (0.6 to 1% w/v) and can tolerate up to 12% (w/v) NaCl (Table 1). The three strains share similar genomic DNA G+C content (42.7 to 47.7mol%). The three strains differed slightly in the utilization of growth substrates and terminal electron acceptors (Table 1). Strain P1BSRT<sup>T</sup> was unable to

use H<sub>2</sub>/CO<sub>2</sub> and ethanol as electron donors in contrast to *Desulfovibrio salexigens* DSM 2638<sup>T</sup>. Strain P1BSRT<sup>T</sup> was unable to use fumarate and malate as electron donors, in contrast to *Desulfovibrio salexigens* DSM 2638<sup>T</sup> and *Desulfovibrio zosterae* DSM 11974<sup>T</sup> (Table 1). All the three strains reduced sulfate, thiosulfate and sulfite, but in contrast to P1BSRT<sup>T</sup>, *Desulfovibrio salexigens* DSM 2638<sup>T</sup> utilized fumarate and nitrate as terminal electron acceptors (Table 1). The main cellular fatty acids of strain P1BSRT<sup>T</sup> were C<sub>16:0</sub> (50.8%), iso-C<sub>15:0</sub> (9.4%), iso-C<sub>17:1</sub> C (9%) and C<sub>16:1</sub> cis9 (8.8%) (Table 2). The fatty acid pattern of strain P1BSRT<sup>T</sup> clearly distinguished it from its closest relative *Desulfovibrio salexigens* (Table 2). *Desulfovibrio salexigens* showed a predominance of iso-C<sub>17:1</sub> C (25.5% versus 9% in strain P1BSRT<sup>T</sup>), whereas strain P1BSRT<sup>T</sup> showed a predominance of C<sub>16:0</sub> (50.8 vs 21.6% in *Desulfovibrio salexigens*) (Table 2).

Therefore, based on its phenotypic, chemotaxonomic and phylogenetic characteristics, we propose strain P1BSRT<sup>T</sup> as a novel species of the genus *Desulfovibrio*, for which the name *Desulfovibrio salinus* sp. nov. is proposed.

<caption>**Table 1.** Physiological and biochemical characteristics of strain P1BSRT<sup>T</sup> in comparison with its two most-closely-related *Desulfovibrio* species</caption>

Strains: 1, P1BSRT<sup>T</sup> (data from this study); 2, *Desulfovibrio salexigens* (DSM 2638<sup>T</sup>) [4, 7, 25]; 3, *Desulfovibrio zosterae* (DSM 11974<sup>T</sup>) [26]. +, Positive; —, negative; NR, not reported.

<table><thead><tr><th>Characteristic</th><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>Shape</td><td>Rod</td><td>Vibrio</td><td>Curved rod</td></tr><tr><td>Cell size (µm)</td><td>0.7–0.9×2–3</td><td>0.5–0.8×1.3–2.5</td><td>0.5×3.0</td></tr><tr><td>Habitat</td><td>Saline lake</td><td>Marine or estuarine mud</td><td>Roots</td></tr><tr><td>Temperature range (°C)</td><td>15–45</td><td>Up to 45</td><td>5–34.5</td></tr><tr><td>Optimum temperature</td><td>40</td><td>30–36</td><td>32.5–34.5</td></tr><tr><td>pH range</td><td>6–8.5</td><td>NR</td><td>5.5–7.5</td></tr><tr><td>Optimum pH</td><td>6.7</td><td>7.8</td><td>6.8–7.3</td></tr><tr><td>NaCl range (%)</td><td>1–12</td><td>0.5–12</td><td>0–3.5</td></tr><tr><td>Optimum NaCl (%)</td><td>3</td><td>2.0–4.0</td><td>1.2</td></tr><tr><td>DNA G+C content (mol%)</td><td>47.7</td><td>47.1</td><td>42.7</td></tr><tr><td>Electron donor (with sulfate):</td><td></td><td></td><td></td></tr><tr><td>H<sub>2</sub>/CO<sub>2</sub></td><td>—</td><td>+</td><td>—</td></tr><tr><td>Lactate</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Malate</td><td>—</td><td>+</td><td>+</td></tr><tr><td>Fumarate</td><td>—</td><td>+</td><td>+</td></tr><tr><td>Formate</td><td>+</td><td>+</td><td>—</td></tr><tr><td>Acetate</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Propionate</td><td>—</td><td>—</td><td>—</td></tr><tr><td>D-Fructose</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Ethanol</td><td>—</td><td>+</td><td>+</td></tr><tr><td>Glycerol</td><td>+</td><td>+</td><td>NR</td></tr><tr><td>Electron acceptor with lactate as energy and carbon source:</td><td></td><td></td><td></td></tr><tr><td>Sulfate</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Thiosulfate</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Sulfite</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Elemental sulfur</td><td>—</td><td>—</td><td>+</td></tr><tr><td>Nitrate</td><td>—</td><td>+</td><td>—</td></tr><tr><td>Fumarate</td><td>—</td><td>+</td><td>+</td></tr></tbody></table>
<caption>**Table 2.** Comparison of the main fatty acids (%) of strain P1BSRT with its closest relative *Desulfovibrio salexigens* (Method TSBA40; Calcul method ANAER6)</caption>

Strains: 1, P1BSRT (data from this study); 2, *Desulfovibrio salexigens* (DSM 2638T) (data from [26]).

<table><thead><tr><th rowspan="2">Fatty acid</th><th colspan="2">Percentage (w/v) of total fatty acids</th></tr><tr><th>1</th><th>2</th></tr></thead><tbody><tr><td>C<sub>14</sub>:0</td><td>1.6</td><td>1.0</td></tr><tr><td>iso-C<sub>15</sub>:0</td><td>9.4</td><td>15.8</td></tr><tr><td>iso-C<sub>16</sub>:1 H</td><td>1.2</td><td>0.3</td></tr><tr><td>iso-C<sub>16</sub>:0</td><td>1.6</td><td>0.3</td></tr><tr><td>C<sub>16</sub>:1cis9</td><td>8.8</td><td>9.8</td></tr><tr><td>C<sub>16</sub>:0</td><td>50.8</td><td>21.6</td></tr><tr><td>iso-C<sub>17</sub>:1 C</td><td>9.0</td><td>25.5</td></tr><tr><td>anteiso-C<sub>17</sub>:1 C</td><td>2.3</td><td>4.0</td></tr><tr><td>iso-C<sub>17</sub>:0</td><td>3.0</td><td>5.4</td></tr><tr><td>anteiso-C<sub>17</sub>:0</td><td>1.0</td><td>1.7</td></tr><tr><td>C<sub>18</sub>:1cis9</td><td>0.5</td><td>0.3</td></tr><tr><td>C<sub>18</sub>:1cis11/trans9/trans6</td><td>1.0</td><td>5.1</td></tr><tr><td>C<sub>18</sub>:0</td><td>4.4</td><td>1.4</td></tr></tbody></table>

DESCRIPTION OF DESULFOVIBRIO SALINUS SP. NOV.

*Desulfovibrio salinus* (sa.li'nus. N. L. masc. adj. salinus salty).

Rod-shaped cells, 0.7–0.9 µm wide and 2–3 µm long, occurring singly or in pairs and motile by a single polar flagellum. pH range for growth: 6–8.5, optimum at pH 6.7. Temperature range for growth: 15–45 °C, optimum at 40 °C. NaCl 1% (w/v) is required for growth. NaCl range for growth: 1–12% NaCl (w/v), with optimum growth at 3% (w/v). All substrates are used with incomplete oxidation. Formate, d-fructose, lactate, pyruvate and glycerol are utilized as electron donors. Substrates not utilized include malate, succinate, methanol, ethanol, d-glucose, acetate, propionate and H₂/CO₂. Pyruvate is not fermented. Utilizes sulfate, thiosulfate and sulfite as terminal electron acceptors but not elemental sulfur, fumarate, nitrate or nitrite.

The type strain is P1BSRT (=DSM 101510T =JCM 31065T), isolated from water collected from a saline lake in Tunisia. The G+C content of the DNA of the type strain is 47.7 mol%.

Funding information

The authors received no specific grant from any funding agency.

Conflicts of interest

The authors declare that there are no conflicts of interest.

References

1. Barton LL, Fauque G. Biochemistry, physiology and biotechnology of sulfate-reducing bacteria. In: Laskin Al, Sariaslani S and Gadd GM (editors). *Advances in Applied Microbiology*. San Diego: Academic Press; 2009. pp. 41–98.

2. Rabus R, Venceslau SS, Wöhlbrand L, Voordouw G, Wall JD et al. A post-genomic view of the ecophysiology, catabolism and biotechnological relevance of sulphate-reducing prokaryotes. *Adv Microb Physiol* 2015;66:55–321.

3. Fauque G. Ecology of sulfate-reducing bacteria. In: Barton LL (editor). *Biotechnology Handbooks Sulfate-Reducing Bacteria*. New York: Plenum Press; 1995. pp. 217–241.

4. Caumette P. Ecology and physiology of phototrophic bacteria and sulfate-reducing bacteria in marine salterns. *Experientia* 1993;49: 473–481.

5. Sorokin DY, Tourova TP, Abbas B, Suhacheva MV, Muyzer G. *Desulfonatronovibrio halophilus* sp. nov., a novel moderately halophilic sulfate-reducing bacterium from hypersaline chloride-sulfate lakes in Central Asia. *Extremophiles* 2012;16:411–417.

6. Sorokin DY, Chernyh NA, Poroshina MN. *Desulfonatronobacter acetoxydans* sp. nov.: a first acetate-oxidizing, extremely salt-tolerant alkaliphilic SRB from a hypersaline soda lake. *Extremophiles* 2015;19:899–907.

7. Postgate JR, Campbell LL. Classification of *Desulfovibrio* species, the nonsporulating sulfate-reducing bacteria. *Bacteriol Rev* 1966; 30:732–738.

8. Cord-Ruwisch R. A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. *J Microbiol Methods* 1985;4:33–36.

9. Trüper HG, Galinski EA. Concentrated brines as habitats for microorganisms. *Experientia* 1986;42:1182–1187.

10. Caumette P, Cohen Y, Matheron R. Isolation and characterization of *Desulfovibrio halophilus* sp. nov., a halophilic sulfate-reducing bacterium isolated from Solar Lake (Sinai). *Syst Appl Microbiol* 1991;14:33–38.

11. Hungate RE. A roll-tube method for the cultivation of strict anaerobes. *Methods Microbiol* 1969;3B:117–132.

12. Macy JM, Snellen JE, Hungate RE. Use of syringe methods for anaerobiosis. *Am J Clin Nutr* 1972;25:1318–1323.

13. Miller TL, Wolin MJ. A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. *Appl Microbiol* 1974; 27:985–987.

14. Widdel F, Pfennig N. Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of *Desulfobacter postgatei* gen. nov., sp. nov. *Arch Microbiol* 1981;129:395–400.

15. Ben Ali Gam Z, Queslati R, Abdelkafi S, Casalot L, Tholozan JL et al. *Desulfovibrio tunisiensis* sp. nov., a novel weakly halotolerant, sulfate-reducing bacterium isolated from exhaust water of a Tunisian oil refinery. *Int J Syst Evol Microbiol* 2009;59:1059–1063.

16. Miller LT. Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. *J Clin Microbiol* 1982;16:584–586.

17. Kuykendall LD, Roy MA, O'Neill JJ, Devine TE. Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of *Bradyrhizobium japonicum*. *Int J Syst Bacteriol* 1988; 38:358–361.

18. Mesbah M, Premachandran U, Whitman WB. Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. *Int J Syst Bacteriol* 1989;39: 159–167.

19. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. *J Mol Biol* 1990;215:403–410.

20. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. *Mol Biol Evol* 1987;4:406–425.

21. Edgar RC. Local homology recognition and distance measures in linear time using compressed amino acid alphabets. *Nucleic Acids Res* 2004;32:380–385.
22. **Tamura K, Stecher G, Peterson D, Filipski A, Kumar S.** MEGA6: molecular evolutionary genetics analysis version 6.0. *Mol Biol Evol* 2013;30:2725-2729.

23. **Tamura K, Nei M, Kumar S.** Prospects for inferring very large phylogenies by using the neighbor-joining method. *Proc Natl Acad Sci USA* 2004;101:11030-11035.

24. **Felsenstein J.** Confidence limits on phylogenies: an approach using the bootstrap. *Evolution* 1985;39:783-791.

25. **Kuever J.** The family *Desulfovibrionaceae*. In: Rosenberg E, Delong E, Stackebrandt E and Thompson F (editors). *The Prokaryotes, Deltaproteobacteria and Epsilonproteobacteria*. Heidelberg: Springer; 2014. pp. 107-133.

26. **Nielsen JT, Liesack W, Finster K.** *Desulfovibrio zosterae* sp. nov., a new sulfate reducer isolated from surface-sterilized roots of the seagrass *Zostera marina*. *Int J Syst Bacteriol* 1999;49:859-865.

27. **Alazard D, Dukan S, Urios A, Verhé F, Bouabida N et al.** *Desulfovibrio hydrothermalis* sp. nov., a novel sulfate-reducing bacterium isolated from hydrothermal vents. *Int J Syst Evol Microbiol* 2003; 53:173-178.

28. **Vandieken V, Knoblauch C, Jørgensen BB.** *Desulfovibrio frigidus* sp. nov. and *Desulfovibrio ferrireducens* sp. nov., psychrotolerant bacteria isolated from Arctic fjord sediments (Svalbard) with the ability to reduce Fe(III). *Int J Syst Evol Microbiol* 2006;56: 681-685.

**Five reasons to publish your next article with a Microbiology Society journal**

1. The Microbiology Society is a not-for-profit organization.

2. We offer fast and rigorous peer review – average time to first decision is 4–6 weeks.

3. Our journals have a global readership with subscriptions held in research institutions around the world.

4. 80% of our authors rate our submission process as 'excellent' or 'very good'.

5. Your article will be published on an interactive journal platform with advanced metrics.

**Find out more and submit your article at microbiologyresearch.org.**