International Journal of Systematic and Evolutionary Microbiology (2013), 63, 4237–4242 DOI 10.1099/ijs.0.

051664-0

Isolation and characterization of Desulfocurvus

Correspondence
Marie-Laure Fardeau
marie-laure.fardeau@univ-amu.fr
thunnarius sp. nov., a sulfate-reducing bacterium
isolated from an anaerobic sequencing batch
reactor treating cooking wastewater
Olfa Hamdi,1,2 Wajdi Ben Hania,1,2 Anne Postec,1 Manon Bartoli,1
Moktar Hamdi,2 Hassib Bouallagui,2 Guy Fauque,1 Bernard Ollivier1
and Marie-Laure Fardeau1
1
Laboratoire de Microbiologie IRD, Aix-Marseille Université, Université du Sud Toulon-Var, CNRS/
INSU, IRD, MIO, UM 110, case 925, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France
2
Laboratoire d’Ecologie et de Technologie Microbienne, Institut National des Sciences Appliquées
et de Technologie, Centre Urbain Nord, BP 676, 1080 Tunis, Université de Carthage, Tunisia

A novel anaerobic, chemo-organotrophic, sulfate-reducing bacterium, designated strain Olac 40T,

was isolated from a Tunisian wastewater digestor. Cells were curved, motile rods or vibrios (5.0–
7.0¾0.5 mm). Strain Olac 40T grew at temperatures between 15 and 50 6C (optimum 40 6C),
and between pH 5.0 and 9.0 (optimum pH 7.1). It did not require NaCl for growth but tolerated it
up to 50 g l”1 (optimum 2 g l”1). In the presence of sulfate or thiosulfate, strain Olac 40T used
lactate, pyruvate and formate as energy sources. Growth was observed on H2 only in the
presence of acetate as carbon source. In the presence of sulfate or thiosulfate, the end products
of lactate oxidation were acetate, sulfide and CO2. Sulfate, thiosulfate and sulfite were used as
terminal electron acceptors, but not elemental sulfur, nitrate or nitrite. The genomic DNA
G+C content of strain Olac 40T was 70 mol%. The profile of polar lipids consisted of
phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and four phospholipids. The
main fatty acids were C16 : 0, anteiso-C15 : 0 and iso-C15 : 0. Phylogenetic analysis of the 16S rRNA
gene sequence indicated that strain Olac 40T was affiliated with the family Desulfovibrionaceae
within the class Deltaproteobacteria. On the basis of 16S rRNA gene sequence comparisons and
physiological characteristics, strain Olac 40T is proposed to be assigned to a novel species of the
genus Desulfocurvus, for which the name Desulfocurvus thunnarius is proposed. The type strain
is Olac 40T (5DSM 26129T5JCM 18546T).

Sulfate-reducing bacteria (SRB) are versatile in their use
of various electron acceptors and electron donors and they
can also thrive in a range of different environmental
conditions (Fauque & Ollivier, 2004; Muyzer & Stams,
2008; Barton & Fauque, 2009). They are ubiquitous, and can
be found in many natural terrestrial, marine and sub-
terrestrial ecosystems together with engineered ones, such as
anaerobic wastewater treatment plants, when sulfate is
present (Birkeland, 2005; Ben Dov et al., 2007; Ollivier et al.,
2007; Muyzer & Stams, 2008; Ollivier & Guyot, 2009).
Sulfate reduction may account, particularly in marine
anoxic sediments, for more than 50 % of the mineralization
of organic matter (Jørgensen, 1977) thus being indicative of
the significant ecological role to be played by SRB regarding
the C an S cycles on Earth. It is only recently that a novel
species of a novel genus of SRB, Desulfocurvus vexinensis,
has been reported (Klouche et al., 2009). This bacterium,
isolated from a well that collected water from a deep saline
aquifer used for underground gas storage at a depth of
830 m in the Paris Basin, France (Basso et al., 2009; Klouche
et al., 2009), belongs to the phylum Proteobacteria,
class Deltaproteobacteria, order Desulfovibrionales, family
Desulfovibrionaceae. It is an anaerobic, chemo-organotrophic,
incomplete-oxidizing, SRB.

Abbreviation: SRB, sulfate-reducing bacteria.

The GenBank /EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain Olac 40T is KC513819.
One supplementary figure is available with the online version of this
paper.
In this study, a novel mesophilic, halotolerant, SRB pertaining
to the genus Desulfocurvus and isolated from sludge samples
of a Tunisian anaerobic bioreactor is described.
The samples were collected under anaerobic conditions
from the sludge of a 2 l anaerobic sequencing batch reactor

051664 G 2013 IUMS Printed in Great Britain 4237

O. Hamdi and others
(temperature: 37 uC, pH: 7.6, flow rate: 100 ml day21) fed
continuously with cooking tuna wastewater at a flow rate
of 100 ml per day (30 g NaCl l21) and transported to the
laboratory at ambient temperature. Micro-organisms were
isolated and cultivated under strict anaerobiosis, according
to the Hungate technique (Hungate, 1969). The basal
medium (BM) for isolation contained (g l21): NH4Cl (1.0),
K2HPO4 (0.3), KH2PO4 (0.3), KCl (0.1), CaCl2 . 2H2O
(0.1), NaCl (40), yeast extract (Difco) (0.1), cysteine-
hydrochloride (0.5), thiosulfate (3.16); 1 ml trace mineral
element solution (Widdel & Pfennig, 1982) and 1 ml 0.1 %
resazurin. The pH was adjusted to 7.6 with 10 M KOH
solution. The BM was boiled under a stream of O2-free N2
gas, cooled to room temperature. Aliquots of 5 ml were
dispensed into Hungate tubes, degassed under N2/CO2
(80 : 20, v/v) and subsequently sterilized by autoclaving at
120 uC for 20 min. Prior to inoculation, 0.1 ml 10 % (w/v)
NaHCO3, 0.1 ml 2 % (w/v) Na2S . 9H2O, 0.1 ml 15 % (w/v)
MgCl2 . 6H2O and 20 mM lactate were injected from
sterile stock solutions into the tubes. Desulfocurvus
vexinensis As36T (5DSM 17965T) was cultivated in the
same medium.
Enrichments were performed in Hungate tubes containing
BM inoculated with 10 % of sample and incubated at
37 uC. The culture was purified by repeated use of the
Hungate Roll tubes method, using 1.6 % agar solid
medium, and then transferred into liquid medium
(Hungate, 1969). The pH, temperature and NaCl concen-
tration ranges for growth were determined using BM
supplemented with 20 mM lactate. For studies of NaCl
requirements, NaCl was weighed directly in the tubes
for concentrations ranging from 0 to 5 % NaCl before
dispensing BM exempt of NaCl. To determine the
optimum pH for growth, the pH of BM in Hungate tubes
(5 ml) was adjusted by injecting appropriate aliquots of
0.1 M HCl, (acidic pH), 10 % NaHCO3 or 8 % Na2CO3
(basic pH) from sterile anaerobic stock solutions to give a
pH range between pH 5.2 and 9.2. Bacterial cultures were
incubated from 15 to 55 uC (at 5 uC intervals).
Cultures were subcultured into fresh medium at least twice
under the same experimental conditions before determin-
ing growth rates and substrates use.
The Gram reaction was determined with heat-fixed liquid
cultures stained with Difco kit reagents.
For electron microscopy, exponentially grown cells were
negatively stained with 1 % sodium phosphotungstic acid
(pH 7.0) as described by Fardeau et al. (1997). Whole
cells were observed with a Hitachi model H 600 electron
microscope at an accelerating voltage of 75 kV.
Methanol, ethanol, propanol, butanol, glycerol, glucose,
fructose, acetate, propionate, butyrate, succinate, fumarate,
malate, citrate, formate, lactate, pyruvate and Casamino
acids were tested as substrates in BM at a final
concentration of 20 mM and H2/CO2 (80 : 20, v/v) and
H2/CO2+2 mM acetate at 2 bar. To test for electron
4238
acceptors, sodium thiosulfate (20 mM), sodium sulfate
(20 mM), sodium sulfite (2 mM), elemental sulfur
(10 g l21), nitrate (20 mM) and nitrite (2 mM) were
added to the medium. H2S production was determined
photometrically as colloidal CuS according to Cord-
Ruwisch (1985). End products of metabolism were
measured by HPLC after 2 days of incubation at 37 uC
(Fardeau et al., 2000). Growth of strain Olac 40T and strain
As36T was stopped at the end of exponential phase and
cultures were sent to DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig,
Germany) for fatty acid analysis. Fatty acids were extracted
using the method of Miller (1982), with the modifications
of Kuykendall et al. (1988), and the profile of cellular fatty
acids was analysed by GC using the Microbial
Identification System (MIDI, Sherlock version 6.1; data-
base, TSBA40; gas chromatograph, model 6890N, Agilent).
Analysis of the polar lipids was carried out by the
Identification Service of the DSMZ for the two strains,
Olac 40Tand As36T. Polar lipids were separated by two-
dimensional TLC and detected using molybdatophospho-
ric acid and heating at 200 uC for 10 min. The presence of
c-type cytochromes and desulfoviridin (the dissimilatory
high-spin bisulfite reductase) were determined on the
crude bacterial extract according to the method described
by Postgate (1956).
The determination of the G+C content of the DNA and
DNA–DNA hybridization were performed at the DSMZ.
Genomic DNA for analysis of the base composition and
DNA–DNA hybridization was isolated after disruption of
bacterial cells by using a French press (Thermo Spectronic)
and purified by chromatography on hydroxyapatite using
the procedure of Cashion et al. (1977). The DNA G+C
content was determined by using HPLC as described
by Mesbah et al. (1989). DNA–DNA hybridization was
carried out as described by De Ley et al. (1970) under
consideration of the modifications described by Huß et al.
(1983) using a model Cary 100 Bio UV/VIS-spectropho-
tometer equipped with a Peltier-thermostated 666 multi-
cell changer and a temperature controller with in situ
temperature probe (Varian).
The extraction and purification of total DNA followed by
the amplification and sequencing of the 16S rRNA
gene were previously described (Khelifi et al., 2010).
The 16S rRNA gene sequence was then compared with
available sequences in the GenBank database using the
BLASTN search (Altschul et al., 1997). A multiple alignment
was built using the MUSCLE program (Edgar, 2004)
implemented in MEGA5 (Tamura et al., 2011). Positions
of sequences with alignment uncertainty and gaps
were omitted from the analysis. Evolutionary analyses
were conducted in MEGA5 using the maximum-likelihood
method based on the Kimura two-parameter model
(Kimura, 1980). A discrete Gamma distribution was
used to model evolutionary rate differences among sites
[5 categories (+G, parameter50.3965)]. The analysis
involved 14 nucleotide sequences. There were a total of
International Journal of Systematic and Evolutionary Microbiology 63

1403 positions in the final dataset. Branch robustness of
the resulting maximum-likelihood tree was estimated by
the non-parametric bootstrap procedure implemented in
MEGA5 (500 replicates of the original dataset). The tree was
drawn to scale, with branch lengths measured in the
number of substitutions per site.
Several colonies developed after incubation at 37 uC and
were picked separately. Colonies were black and circular
with diameters ranging from 1.0 to 2.0 mm after 3–5 days
of incubation at 37 uC. The process of serial dilution was
repeated several times until the isolates were deemed to
be axenic. Several strains were isolated; their morphology
and metabolic profiles were similar and the same
phylogenic inference was obtained for all of them. One
strain, designated Olac40T, was selected and used for
further metabolic and physiological characterization.
Cells of strain Olac 40T were curved rods or vibrios staining
Gram-negative (5–7 mm by 0.5 mm) when grown on a
medium containing lactate as electron donor and thio-
sulfate as terminal electron acceptor (Fig. 1a). Ultrathin
sections showed a typical Gram-negative cell wall with a
thin peptidoglycan layer and an outer membrane (Fig. 1b).
Cells were motile by a single polar flagellum (Fig. 1c).
Strain Olac 40T was anaerobic but tolerated up to 1 % O2.
The physiological optimal growth conditions were deter-
mined in duplicate experiments conducted in BM contain-
ing lactate (20 mM) and thiosulfate (20 mM) as previously
described (Fardeau et al., 2000). The optimal temperature
for growth was 40 uC (range 15–50 uC). Optimum pH was
7.1 (range pH 5.0–9.0). The strain could grow without
NaCl, but tolerated it up to 50 g l21 with an optimum at
2 g l21. Substrate utilization was tested with 1 g yeast
extract l21 added to BM. Very few of the tested compounds
for growth were oxidized. They include only lactate,
(a) (b)
10 µm 200 nm
Fig. 1. (a) Phase-contrast photomicrograph showing cells of strain Olac 40T (bar, 10 mm). (b) Thin section electron micrograph
of strain Olac 40T showing the Gram-negative type of cell wall (bar, 0.2 mm). (c) Electron micrograph of strain Olac 40T
showing a single polar flagellum (bar, 0.5 mm).
http://ijs.sgmjournals.org
Desulfocurvus thunnarius sp. nov.
pyruvate, formate and H2, which was used only in the
presence of acetate (2 mM) as carbon source. Thiosulfate,
sulfate and sulfite, but not elemental sulfur, nitrate or
nitrite served as terminal electron acceptors. They were
reduced to sulfide. Methanol, ethanol, propanol, butanol,
glycerol, glucose, fructose, acetate, propionate, butyrate,
succinate, fumarate, malate, citrate and Casamino acids did
not support growth. Under optimal conditions with lactate
as electron donor and thiosulfate as electron acceptor, the
maximum growth rate of strain Olac 40T was 0.26 h21.
Although growth occurred in minimal medium with
lactate as the only energy and carbon sources, yeast extract,
biotrypcase and also vitamins (Balch et al., 1979) improved
growth on this substrate.
Visible absorption spectra of a cell-free extract of strain
Olac 40T showed the presence of low redox potential c-type
cytochromes (very likely the tetraheme cytochrome c3)
with absorption peaks at 522, 551 and 418 nm in the
dithionite reduced form. The characteristic absorption
band (at 628 nm) of desulfoviridin (the dissimilatory
high-spin bisulfite reductase characteristic of the genus
Desulfovibrio) was not detected in the cell-free extract.
The reddish colour of the crude extract may possibly
indicate the presence of desulforubidin, the other cyto-
plasmic dissimilatory high-spin bisulfite reductase isolated
in mesophilic non-sporulating species of SRB (Fauque
et al., 1991; Fauque & Barton, 2012).
The major fatty acids of strain Olac 40T were: C16 : 0
(29.2 %), anteiso-C15 : 0 (21.2 %) and iso-C15 : 0 (19.3 %);
for Desulfocurvus vexinensis As36T, major fatty acids were
anteiso-C15 : 0 (25.0 %), C16 : 0 (22.3 %), iso-C15 : 0 (21.1 %)
(Table 1). The polar lipid profile of strain Olac 40T
consisted of phosphatidylglycerol, four phospholipids,
phosphatidylethanolamine and aminophospholipid. The
(c)
0.5 µm
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O. Hamdi and others

Table 1. Comparison of the main fatty acids (%) of strain Olac Desulfocurvus vexinensis possesses desulfoviridin (Kuever
40T and Desulfocurvus vexinensis As36T et al., 2005; Fauque & Barton, 2012). DNA–DNA
hybridization experiments revealed that strain Olac 40T
Cellular fatty acid Olac 40T As36T showed only 41.2 % reassociation with Desulfocurvus
C14 : 0 2.2 1.5 vexinensis thus indicating that strain Olac 40T represents
iso-C15 : 0 19.3 21.1 a distinct species within the genus Desulfocurvus.
anteiso-C15 : 0 21.2 25.0 Strain Olac 40T is therefore proposed as a representative
C16 : 0 29.2 22.3 of a novel species of the genus Desulfocurvus. This is
iso-C16 : 0 1.6 0.8 supported by the DNA G+C content of strain Olac 40T,
anteiso-C17 : 0 5.0 4.5 which is slightly higher than that of Desulfocurvus
iso-C17 : 0 11.7 13.6 vexinensis (Klouche et al., 2009) (Table 2). Similarly to
Summed feature 10 3.2 3.0 Desulfocurvus vexinensis, strain Olac 40T used a limited
C16 : 1v7c 2.6 4.7 range of substrates including lactate, pyruvate and
C18 : 0 2.3 1.7
formate, but unlike Desulfocurvus vexinensis oxidized
hydrogen. Strain Olac 40T also differed from
Summed feature 10 consisted of C18 : 1v7c.
Desulfocurvus vexinensis by its tolerance to 1 % O2. The
profiles of fatty acids and polar lipids were slightly different
for the two strains. A comparison of the main character-
polar lipid profile was the same for Desulfocurvus vexinensis istics of strain Olac 40T and Desulfocurvus vexinensis is
As36T but with only one phospholipid (Fig. S1, available in given in Tables 1 and 2. Based on phenotypic, and genetic
IJSEM Online). The DNA G+C content of strain Olac 40T characteristics of strain Olac 40T, we propose it to be
was 70 mol%. assigned to a novel species of the genus Desulfocurvus,
Desulfocurvus thunnarius sp. nov.
The phylogenetic tree obtained by the maximum-like-
lihood method, as shown in Fig. 2, showed that strain Olac Description of Desulfocurvus thunnarius sp. nov.
40T is a new member of the genus Desulfocurvus, sharing
99 % sequence similarity with the single species of the Desulfocurvus thunnarius [thun.na9ri.us. L. masc. adj.
genus, Desulfocurvus vexinensis. Its other two closest thunnarius of or belonging to a tunny (cooking tuna
relatives were the proposed species ‘Desulfovibrio ferrophi- wastewater, Tunisia), where the species was first recovered].
lus’ DSM 15579 (Dinh et al., 2004) with 93.9 % similarity Cells are anaerobic, motile rods or vibrios (5–760.5 mm),
and Desulfovibrio senezii DSM 8436T (Tsu et al., 1998) with neutrophilic and slightly halotolerant (0–50 g NaCl l21,
90.8 % similarity. However, in contrast to species of the optimum at 2 g l21). The temperature range for growth is
genus Desulfovibrio, neither strain Olac 40T nor 20–50 uC, with an optimum of 40 uC. The optimum pH is

100 Desulfocurvus thunnarius Olac 40T (KC513819)

87 Desulfocurvus vexinensis VNs36T (DQ841177)
97 ‘Desulfovibrio ferrophilus’ IS5 (AY274449)
0.05
Desulfovibrio senezii CVLT (AF050100)
Desulfovibrio alkalitolerans RT2T (AY649785)
99 Desulfovibrio burkinensis HDvT (AF053752)
Desulfovibrio magneticus RS-1T (D43944)
88 100 Desulfovibrio alcoholivorans DSM 5433T (AF053751)

71 Desulfovibrio fructosivorans DSM 3604T (AF050101)

70 Desulfovibrio marrakechensis EMSSDQ4T (AM947130)
Desulfomicrobium norvegicum DSM 1741T (AJ277897)
Desulfonatronum cooperativum Z-7999T (AY725424)
Desulfonatronum thiodismutans MLF1T (AF373920)
Desulfotomaculum australicum ACM 3917T (M96665)

Fig. 2. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences of strain Olac 40T and related species
of the genera Desulfocurvus, Desulfovibrio, Desulfomicrobium and Desulfonatronum. The tree was rooted by using
Desulfotomaculum australicum as an outgroup. Numbers at nodes represent bootstrap values (.50 %) inferred by MEGA5 from
500 replicates. Bar, 5 nt changes per 100 nt.

4240 International Journal of Systematic and Evolutionary Microbiology 63


Table 2. Comparison of the main characteristics of strain Olac 40T and Desulfocurvus vexinensis As36T
Cells of both strains were vibrios or curved rods.
Characteristic
Size (mm)
DNA G+C content (mol%)
Temperature range for growth (optimum) (uC)
pH range for growth (optimum)
Salinity range for growth (optimum) (%)
Hydrogen as electron donor
7.1 (range pH 5–9). Lactate, pyruvate, formate and H2,
only in the presence of acetate as carbon source, are used as
energy sources. The main end product of lactate catabolism
is acetate. Methanol, ethanol, propanol, butanol, glycerol,
glucose, fructose, acetate, propionate, butyrate, succinate,
fumarate, malate, citrate and Casamino acids are not used
as substrates. Sulfate, sulfite and thiosulfate serve as
electron acceptors. The main fatty acids are C16 : 0,
anteiso-C15 : 0 and iso-C15 : 0. c-Type cytochromes are
present and desulfoviridin is absent in the crude extract.
The type strain is Olac 40T (5DSM 26129T5JCM 18546T),
which was isolated from an anaerobic sequencing batch
reactor treating tuna cooking wastewater. The genomic
DNA G+C content of the type strain is 70 mol%.
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