Halanaerobacter jeridensis sp nov., isolated from a hypersaline lake

ArticleinInternational Journal of Systematic and Evolutionary Microbiology 62(Pt 8):1970-3 · October 2011with11 Reads
Impact Factor: 2.51 · DOI: 10.1099/ijs.0.036301-0 · Source: PubMed
Abstract

An obligatory anaerobic, moderately halophilic bacterium, designated strain CEJFG43(T), was isolated from a sample of sediment collected below the salt crust on the hypersaline El Jerid lake, in southern Tunisia. The cells of this novel strain were Gram-staining-negative, non-sporulating, motile, short rods. They grew in media with 6-30% (w/v) NaCl (optimum 15%), at 20-60 °C (optimum 45 °C) and at pH 5.5-9.5 (optimum pH 8.3). The micro-organism fermented glucose, fructose, ribose, raffinose, galactose, mannose, sucrose, maltose, xylose, mannitol, pyruvate and glycerol. The products of glucose fermentation were lactate, ethanol, acetate, H(2) and CO(2). The genomic G+C DNA content of strain CEJFG43(T) was 33.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFG43(T) belonged in the genus Halanaerobacter and was most closely related to Halanaerobacter lacunarum DSM 6640(T) (95.3% gene sequence similarity) and Halanaerobacter chitinivorans DSM 9569(T) (95.3%). The predominant cellular fatty acids were non-branched (C(16:0) and C(16:1)). Based on the phylogenetic and phenotypic evidence, strain CEJFG43(T) represents a novel species in the genus Halanaerobacter for which the name Halanaerobacter jeridensis sp. nov. is proposed. The type strain is CEJFG43(T) ( = DSM 23230(T) = JCM 16696(T)).

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Halanaerobacter jeridensis sp. nov., isolated from a
hypersaline lake
M. Mezghani,
1,2
D. Alazard,
1,2
F. Karray,
1
J.-L. Cayol,
2
M. Joseph,
2
A. Postec,
2
M.-L. Fardeau,
2
J.-L. Tholozan
2
and S. Sayadi
1
Correspondence
Didier Alazard
didier.alazard@ird.fr
1
Laboratoire des Bio-Proce
´
de
´
s Environnementaux, Centre de Biotechnologie de Sfax, BP 1177,
3018 Sfax, Tunisia
2
Aix-Marseille Univ., IRD, MIO UMR 235; CNRS/INSU, MIO UMR 7294, 163 Avenue de Luminy,
13288 Marseille Cedex 9, France
An obligatory anaerobic, moderately halophilic bacterium, designated strain CEJFG43
T
, was
isolated from a sample of sediment collected below the salt crust on the hypersaline El Jerid lake,
in southern Tunisia. The cells of this novel strain were Gram-staining-negative, non-sporulating,
motile, short rods. They grew in media with 6–30 % (w/v) NaCl (optimum 15 %), at 20–60 6C
(optimum 45 6C) and at pH 5.5–9.5 (optimum pH 8.3). The micro-organism fermented glucose,
fructose, ribose, raffinose, galactose, mannose, sucrose, maltose, xylose, mannitol, pyruvate and
glycerol. The products of glucose fermentation were lactate, ethanol, acetate, H
2
and CO
2
. The
genomic G+C DNA content of strain CEJFG43
T
was 33.3 mol%. Phylogenetic analysis based
on 16S rRNA gene sequences indicated that strain CEJFG43
T
belonged in the genus
Halanaerobacter and was most closely related to Halanaerobacter lacunarum DSM 6640
T
(95.3 % gene sequence similarity) and Halanaerobacter chitinivorans DSM 9569
T
(95.3 %). The
predominant cellular fatty acids were non-branched (C
16 : 0
and C
16 : 1
). Based on the
phylogenetic and phenotypic evidence, strain CEJFG43
T
represents a novel species in the genus
Halanaerobacter for which the name Halanaerobacter jeridensis sp. nov. is proposed. The type
strain is CEJFG43
T
(5DSM 23230
T
5JCM 16696
T
).
Intense evaporation from inland salt lakes can leave the
water in such lakes extremely saline, with salt contents
close to saturation. In Tunisia, these hypersaline lakes are
called sebkhat or chott. Although usually appearing dry, with
a thin crust of salt covering a layer of spongy, brine-soaked
soil, heavy winter rains can result in surface water. The largest
salt lake in Tunisia, El Jerid, covers about 5000 km
2
in the
south of the country. Although the salts in El Jerid lake come
from the geological formations of the surrounding moun-
tains, the lake contains similar ions to seawater, with a salt
concentration generally varying between 250 and 330 g l
21
(Kbir-Ariguib et al., 2001). Salt lakes are inhabited by
halophilic and halotolerant micro-organisms. The anaerobic
halophilic fermentative bacteria represent a diverse but
closely related group of micro-organisms. Such bacteria have
been placed in a single order, the Halanaerobiales.Thisorder
is currently divided into two families: the Halanaerobiaceae
(Oren et al., 1984) and the Halobacteroidaceae (Rainey et al.,
1995). At the time of writing, the genus Halanaerobacter
(in the family Halobacteroidaceae) comprises just three
species: Halanaerobacter chitinivorans (Liaw & Mah, 1992),
Halanaerobacter lacunarum (formerly Halobacteroides lacu-
naris;Zhilinaet al., 1991) and Halanaerobacter salinarius
(Moune
´
et al., 1999). The definition of this genus, like those of
other genera in the order Halanaerobiales, has been largely
based on the results of phylogenetic analyses of 16S rRNA
gene sequences.
In this report, we describe a strain of anaerobic, halophilic,
fermentative bacterium isolated from a sample of sedi-
ment from El Jerid lake. This strain, des ignated CEJFG43
T
,
was found to represent a novel species in the genus
Halanaerobacter.
Strain CEJFG43
T
was isolated from a sample of black
sediment collected below the salt crust on the edge of El
Jerid lake, in southern Tunisia. The sample was collected in
a steril e glass bottle that was completely filled with water
from the lake and kept at 4
u
C until the isolation attempt.
The water used to fill the bottle was at pH 7.6 and highly
saline, with a total mineral content of 370 g l
21
. Standard
anaerobic techniques were used throughout the study
(Hungate, 1969; Balch et al., 1979 ). A small subsample of
sediment (1 g) was inoculated into an enrichment medium
containing (l
21
): 1 g N H
4
Cl, 0.5 g K
2
HPO
4
, 0.8 g KCl,
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain CEJFG43
T
(5DSM 23230
T
5JCM 16696
T
)is
GU265900.
International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1970–1973 DOI 10.1099/ijs.0.036301-0
1970 036301
G
2012 IUMS Printed in Great Britain
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0.1 g CaCl
2
, 10 g MgSO
4
.7H
2
O, 15 g MgCl
2
.7H
2
O, 1 mg
NaBr, 250 g NaCl, 0.5 g yeast extract, 0.5 g tryptone, 0.5 g
cysteine-HCl, 0.001 g resazurin and 10 ml trace element
solution (Balch et al., 1979). The pH of the medium was
adjusted to 7.6 with 1 M KOH before the medium was
boiled under a stream of O
2
-free N
2
gas and then cooled to
room temperature. The cooled medium was dispensed into
Hungate tubes (at 5 ml per tube) and serum bottles (at
20 ml per bottle), under a stream of N
2
/CO
2
(80 : 20 %,
v/v) gas, and then sterilized by autoclaving at 110
u
C for
45 min. Prior to inoculation of the medium in each bottle,
0.4 ml 10 % (w/v) NaHCO
3
, 0.4 ml 2 % (w/v) Na
2
S and
0.4 ml 1 M glucose were injected into the bottle, from sterile
stock solutions. The medium in each Hungate tube was
similarly supplemented, using 0.1 ml of each stock solution.
Each enrichment culture was incubated at 35
u
C for
2 weeks. A novel strain, designated CEJFG43
T
,was
obtained and this was purified by two rounds of culture
using the roll-tube method (Hungate, 1969) and enrichment
medium that had been solidified by the addition of 2 %
(w/v) agar. After 6 days in roll tubes, on the solid medium,
the novel strain formed cream-coloured colonies with
regular edges and diameters of 0.8–1.0 mm. The strain grew
as motile, short rods that measured approximately 1.262.5–
6.0
mm and occurred singly or in pairs (Fig. 1). These rods
were Gram-staining-negative. Spores were never observed.
Examination of ultrathin sections of the rods, by transmis-
sion electron microscopy, revealed that each had a tri-
layered cell wall and an envelope that was typical of
Gram-staining-negative bacteria (Fig. 1). The purity of the
isolate was checked by microscopic observations.
Strain CEJFG43
T
was strictly anaerobic and moderately
halophilic, growing in media containing 6–30 % (w/v)
NaCl (optimum 15 %). No growth was observed in the
absence of NaCl. It grew at pH 5.5–9.5 (optimum pH 8.3)
and at 20–60
u
C (optimum 45
u
C). Substrates were tested
at a final concentration of 20 mM in the basal medium
(with 0.2 g yeast extract and 0.2 g tryptone l
21
). Experi-
ments were conducted in duplicate and, in each case, two
successive subcultures were performed under the same
conditions. To test for electron acceptors, sodium thiosul-
fate, sodium sulfate, sodium sulfite, elemental sulfur and
nitrate were added to the medium at final concentrations of
20 mM, 20 mM, 2 mM, 2 % (w/v) and 10 mM, respectively.
Strain CEJFG43
T
used glucose, fructose, sucrose, maltose,
mannose, raffinose, ribose, xylose, trehalose, starch, manni-
tol, pyruvate and glycerol. No growth was observed on
cellobiose, sorbose, arabinose, lactose, rhamnose, acetate,
lactate, succinate, fumarate, Casamino acids, betaine,
trimethylamine or peptone. The end products from the
fermentation of glucose (20 mM) were determined by
HPLC, using an Aminex HPX-87H column (Bio-Rad) with
5mMH
2
SO
4
as the mobile phase. Strain CEJFG43
T
formed
almost equal amounts of lactate and ethanol (13 and
12 mM, respectively) as well as smaller amounts of acetate
(1.5 mM), CO
2
(1.6 mM) and H
2
.
The cellular fatty acid composition of strain CEJFG43
T
was
determined by the Identification Service of the Deutsche
Sammlung von Mikroorganismen und Zellkulturen
(DSMZ), Braunschweig, Germany. After cells had been
grown in basal liquid medium at 45
u
C for 5 days, fatty acid
methyl esters were produced using the method of Miller
(1982), with the modifications of Kuykendall et al. (1988),
before being separated and identified using version 4.0 of the
Fig. 1. Phase-contrast light micrograph of cells of strain
CEJFG43
T
(a) and a transmission electron micrograph of an
ultrathin section of part of a cell of the same strain (b). Bars, 5 mm
(a) and 100 nm (b).
Table 1. Cellular fatty acid contents of strain CEJFG43
T
Fatty acid Content (%)
C
9:0
0.2
C
10 : 0
6.1
C
10 : 0
3-OH 0.4
C
14 : 0
7.9
C
15 : 1
v9c 0.5
C
15 : 0
1.1
C
16 : 1
v7c 4.5
C
16 : 1
v9c 42.5
C
16 : 1
v11c 2.3
C
16 : 0
21.7
C
16 : 1
v9c DMA 0.6
C
16 : 0
DMA 0.3
C
18 : 1
v11c 1.1
C
18 : 0
1.1
Summed feature*
2 8.2
4 0.9
8 0.6
*Summed features represent groups of two or three fatty acids that
cannot be separated by GLC with the MIDI system. Summed feature 2
comprises C
12 : 0
3-OH and/or C
13 : 0
DMA. Summed feature 4
comprised C
15 : 2
and/or C
15 : 1
v7c. Summed feature 8 comprised
C
17 : 1
v9c and/or C
17 : 2
.
Halanaerobacter jeridensis sp. nov.
http://ijs.sgmjournals.org 1971
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Microbial Identification System (MIDI; Sasser, 1990). The
predominant fatty acids of strain CEJFG43
T
, like those of the
recognized members of the genus Halanaerobacter (Oren,
2006), were found to be C
16 : 0
and C
16 : 1
(Table 1).
The genomic DNA G+C content of the novel strain
(33.3 mol%) was also determined by the Identi fication
Service of the DSMZ, by using HPLC to investigate DNA
that had been extracted using the Wizard Genomic DNA
Purification kit (Promega).
The 16S rRNA gene of strain CEJFG43
T
was amplified by
PCR, using the universal primers Rd1 and Fd1 (Weisburg
et al., 1991). The nearly complete sequence of the 16S rRNA
gene (1423 nt) was aligned with the corresponding
sequences of closely related taxa from the GenBank database
by using programs provided by the Ribosomal Database
Project II (Maidak et al., 2001). All the sequences were
imported and verified manually using version 5.0.9 of the
BioEdit sequence editor (Hall, 1999). Overall, after positions
of sequence and alignment uncertainty were omitted, 1228
positions of alignment were computed using the method of
Jukes & Cantor (1969). Phylogenetic trees were then
produced in the
TREECON (Van de Peer & De Wachter,
1994) and
PHYLIP software packages (Felsenstein, 1993) by
using the neighbour-joining, maximum-likelihood and
maximum-parsimony algorithms. In the neighbour-joining
tree (Fig. 2), as in the maximum-likelihood and maximum-
parsimony trees (data not shown), strain CEJFG43
T
was
clustered with members of the genus Halanaerobacter.In
pairwise comparisons, the novel strain appeared most
closely related to H. lacunarum DSM 6640
T
(95.3 % 16S
rRNA gene sequence similarity) and H. chitinivorans
DSM 9569
T
(also 95.3 %). Unlike H. lacunarum and H.
chitinivorans, however, strain CEJFG43
T
was able to ferment
xylose, raffinose, pyruvate and glycerol (Table 2). Glycerol is
considered to be one of the main carbon and energy sources
for heterotrophic bacterial communities in hypersaline
environments such as salterns because the microalgae
(Dunaliella species) in such environments produce large
amounts of this compound in response to the extracellular
osmotic pressure (Ben-Amotz & Avron, 1973).
Based on the phylogenetic and phenotypic evidence, strain
CEJFG43
T
represents a novel species in the genus Hala-
naerobacter for which the name Halanaerobacter jeridensi s
sp. nov. is proposed.
0.05
Halanaerobium praevalens ATCC 33744
T
(M59123)
Selenihalanaerobacter shriftii
ATCC BAA-73
T
(AF310247)
Halanaerobacter jeridensis
DSM 23230
T
(GU265900)
Halobacteroides elegans DSM 6639
T
(AJ238119)
Halanaerobacter lacunarum DSM 6640
T
(U32593)
Halanaerobacter chitinivorans DSM 9569
T
(X89076)
Halanaerobacter salinarius DSM 12146
T
(Y14212)
Halanaerobaculum tunisiense DSM 19997
T
(EU327343)
Halobacteroides halobius DSM 150
T
(U32595)
100
97
100
99
100
99
Fig. 2. Neighbour-joining phylogenetic tree
based on 16S rRNA gene sequences, show-
ing the relationship between strain CEJFG43
T
and closely related species in the family
Halobacteroidaceae. Halanaerobium praeva-
lens ATCC 33744
T
was used as an outgroup.
Bootstrap values .80 % (based on 1000
resampled datasets) are shown at nodes. Bar,
0.05 substitutions per nucleotide position.
Table 2. Differentiating physiological and biochemical char-
acteristics of strain CEJFG43
T
and its closest relatives in the
genus Halanaerobacter
Strains: 1, CEJFG43
T
(data from this study); 2, H. lacunarum DSM
6640
T
(Zhilina et al., 1991); 3, H. chitinivorans DSM 9569
T
(Liaw &
Mah, 1992). +, Positive; 2, negative;
W, weakly positive; ND, no data
available.
Characteristic 1 2 3
Temperature range for growth (
u
C) 20–60 25–52 23–50
Optimum temperature (
u
C) 45 35–40 30–45
pH range for growth 5.5–9.5 6–8 6–8
Optimum pH 8.3 6.5–7 7
NaCl range for growth (%, w/v) 6–30 10–30 3–30
Optimum NaCl (%, w/v) 15 15–18 12–18
Assimilation of:
Ribose + 2
ND
Cellobiose 2 ++
Raffinose + 22
Galactose + 22
Sorbose 2 + 2
Xylose + 22
Pyruvate + 22
Glycerol + 22
Peptone 22
W
Elemental sulphur as electron
acceptor
2 + ND
DNA G+C content (mol%) 33.3 32.4 34.8
M. Mezghani and others
1972 International Journal of Systematic and Evolutionary Microbiology 62
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Description of Halanaerobacter jeridensis
sp. nov.
Halanaerobacter jeridensis (je.ri.den9sis. N.L. masc. adj.
jeridensis of El Jerid, the lake from which the type strain
was recovered).
Cells are strictly anaerobic, motile rods (1.262.5–6
mm)
that occur singly or in pairs. Mesophilic, with a maximum
temperature for growth of 60
u
C (optimum 45
u
C). Grows
with 6–30 % (w/v) NaCl (optimum 15 %) and at pH 5.5–
9.5 (optimum pH 8.3). Ferments glucose, fructose, ribose,
raffinose, galactose, mannose, sucrose, maltose, mannitol,
xylose, trehalose, starch, pyruvate and glycerol but not
arabinose, cellobiose, rhamnose, sorbose, lactose, succinate,
fumarate, acetate, choline, glycine, leucine, serine, Casamino
acids or peptone. Glucose is converted into lactate, ethanol,
acetate, H
2
and CO
2
. Does not use elemental sulfur, sulfate,
thiosulfate, sulfite, fumarate, nitrate or nitrite as electron
acceptors. The predominant cellular fatty acids are C
16 : 1
and
C
16 : 0
.
The type strain, CEJFG43
T
(5DSM 23230
T
5JCM 16696
T
),
was isolated from sediment collected from El Jerid lake in
southern Tunisia. The gen omic DNA G+C content of the
type strain is 33.3 mol%.
Acknowledgements
The authors thank Professor J. Euze
´
by for his help with etymology of
the novel species. M. M. was supported by the city of Marseille via a
research grant (Accueil Master Recherche).
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http://ijs.sgmjournals.org 1973
Page 4
    • "This is the first report describing the microbial populations present in the water and sediment of Chott El Jerid using molecular methods [denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR]. Few ecological studies of this environment based on the cultivation approach have been investigated (Ben Abdallah et al. 2015; Mezghani et al. 2012). The main purpose of this research is to obtain an overview of the abundance, the structure, and microbial diversity of both archaeal and bacterial communities in water and sediment samples of Chott El Jerid using culture-dependent and culture-independent techniques. "
    [Show abstract] [Hide abstract] ABSTRACT: Prokaryotic diversity was investigated in a Tunisian salt lake, Chott El Jerid, by quantitative real-time PCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting methods targeting the 16S rRNA gene and culture-dependent methods. Two different samples S1-10 and S2-10 were taken from under the salt crust of Chott El Jerid in the dry season. DGGE analysis revealed that bacterial sequences were related to Firmicutes, Proteobacteria, unclassified bacteria, and Deinococcus-Thermus phyla. Anaerobic fermentative and sulfate-reducing bacteria were also detected in this ecosystem. Within the domain archaea, all sequences were affiliated to Euryarchaeota phylum. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of bacteria was 5 × 10(6) DNA copies g(-1) whereas archaea varied between 5 × 10(5) and 10(6) DNA copies g(-1) in these samples. Eight anaerobic halophilic fermentative bacterial strains were isolated and affiliated with the species Halanaerobium alcaliphilum, Halanaerobium saccharolyticum, and Sporohalobacter salinus. These data showed an abundant and diverse microbial community detected in the hypersaline thalassohaline environment of Chott El Jerid.
    Full-text · Article · Jan 2016 · Extremophiles
  • [Show abstract] [Hide abstract] ABSTRACT: The order Halanaerobiales, families Halanaerobiaceae and Halobacteroidaceae, consists of obligatory anaerobic, moderately halophilic bacteria that require NaCl concentrations between 0.5 and 3.4 M for optimal growth. Representatives have been isolated from anaerobic sediments of salt lakes worldwide, from brines associated with oil reservoirs, and also from fermented salted foods. Some species are thermophilic or alkaliphilic. Although phylogenetically affiliated with the low G+C branch of the Firmicutes, the cells show a Gram-negative wall structure, and most species stain Gram-negative. Some representatives of the Halobacteroidaceae produce endospores. Most species ferment carbohydrates to acetate, ethanol, H2, CO2, and other fermentation products. Within the Halobacteroidaceae, a greater metabolic diversity is found, with some species displaying a homoacetogenic metabolism; growth by anaerobic respiration using different electron acceptors including nitrate, trimethylamine.-oxide, selenate, arsenate, or Fe(III); or chemolithoautotrophic growth on hydrogen and elemental sulfur. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.
    No preview · Article · Nov 2013
  • [Show abstract] [Hide abstract] ABSTRACT: Halophilic, obligately anaerobic, Gram-negative bacterial strains were isolated from a sediment sample taken under salt crust of El-Jerid hypersaline lake located in southern Tunisia by using tryptone or glucose as substrate. One of them (strain CEJFT1BT) was characterized phenotypically and phylogenetically. Cells were non-motile, non-spore forming, short rods. Strain CEJFT1BT was able to grow in the presence of 5-30 % (w/v) NaCl (optimum 20 %) and at 30-60 °C (optimum 45 °C). It grew at pH 5.5-7.75 and the optimum pH for growth was 6.75. The isolate required yeast extract for growth. Substrates utilized by strain CEJFT1BT included glucose, fructose, sucrose, pyruvate, casaminoacids and starch. Individual amino acid such as glutamate, lysine, methionine, serine, tyrosine and amino acid mixtures by Stickland reaction such as alanine-glycine, valine-proline, leucine-proline, isoleucine-proline were also utilized. Products of glucose fermentation were acetate (major product), butyrate, isobutyrate, H2 and CO2. The DNA G+C content of strain CEJFT1BT was 32.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFT1BT could be assigned to the genus Sporohalobacter. The sequence similarity between strain CEJFT1BT and Sporohalobacter lortetii was 98.5%, but DNA-DNA hybridization between the two strains revealed relatedness value of 56.4%, indicating that they are not related at the species level. The combination of phylogenetic analysis, DNA-DNA hybridization data, and differences in substrate utilization supported the view that strain CEJFT1BT represents a new species of the genus Sporohalobacter, for which the name Sporohalobacter salinus sp. nov. is proposed. The type strain is strain CEJFT1BT (DSM 26781 = JCM 19279).
    No preview · Article · Nov 2014 · International Journal of Systematic and Evolutionary Microbiology