975

Molecular community analysis of magnesium-rich

bittern brine recovered from a Tunisian solar
saltern
Houda Baati, Raja Jarboui, Nji Gharsallah, Abdelghani Sghir, and Emna Ammar

Abstract: The microbial community of a magnesium-rich bittern brine saturated with NaCl (380400 g/L) from a Tunisian
solar saltern was investigated using a molecular approach based on 16S rRNA gene analysis and viability tests. The results
revealed the existence of microbial flora. Viability test assessment showed that 46.4% of this flora was viable but not detect-
able by culturability tests. 16S rRNA genes from 49 bacterial clones and 38 archaeal clones were sequenced and phylo-
genetically analyzed. Eleven operational taxonomic units (OTUs) determined by the DOTUR program with 97% sequence
similarity were generated for Bacteria. These OTUs were affiliated with Bacteroidetes and Gammaproteobacteria. The
archaeal community composition exhibited more diversity with 38 clones, resulting in 13 OTUs affiliated with the
Euryarchaeota phylum. Diversity measurement showed a more diverse archaeal than bacterial community at the saturated
pond.
Key words: saline, saumure amre, magnsium, gnes dARNr 16S, test de viabilit.
Rsum : Dans la saline de Sfax, la communaut microbienne de leau saumtre prleve dun bassin satur en sel (380
400 g/L) et riche en magnsium a t tudie par des approches molculaires, bases sur lanalyse des gnes codant pour
lARNr 16S et sur des tests de viabilit. Les rsultats ont montr lexistence dune flore microbienne dont 46,4 % de cette
flore est viable mais non dtectable par des tests de cultivabilit. Un total de 49 squences bactriales et 38 squences ar-
chennes a t analys. Onze units taxonomiques oprationnelles (UTO) dtermines par un programme DOTUR 97 %
de similarit ont t gnres pour le domaine Bacteria. Ces units taxonomiques appartiennent aux groupes des Bactroi-
detes et Gammaprotobactries. La communaut archenne appartenant au phylum des Euryarchaeota tait plus diversifie
et a reprsent 38 squences groupes en 13 UTO. Lanalyse de rarfaction et la mesure des indices de diversit ont montr
que la communaut des Archaea est plus diversifie que celle du domaine Bacteria.
Motscls : solar saltern, bittern brine, magnesium, 16S rRNA genes, viability test.

Introduction
Solar salterns are characterized by high rates of evapora- ity, and low toxicity (Davies and Knowles 2006). Moreover,
tion and sequential precipitation of seawater salt constituents. bittern brines could be useful in electricity cogeneration and
As a result, seawater is concentrated as it passes through a in water desalination (Ahmed et al. 2003). In Tunisia, bittern
pond series. During the progressive NaCl precipitation, the brines are used for the recovery of potassium sulfate (fertil-
ionic composition changes and the continuous Cl and Mg2+ izers), magnesium sulfate, and magnesium chloride; all have
concentrations increase (Javor 1983, 1989; Oren 2002). After some commercial value.
major salt precipitation (NaCl), the brine remaining, known A variety of microorganisms inhabit the saltern ponds
as bittern brine due to its specific taste, is highly enriched in where NaCl is dominant. Indications of life in MgCl2-rich
magnesium chloride (MgCl2). The bittern brines also contain environments were previously reported (Javor 1983, 1984,
significant amounts of impurities, including sulphate, so- 1989; Oren 1983a, 1999; Laiz et al. 2000; Antn et al.
dium, potassium, and calcium ions (Javor 1983, 1989). 2002; Butinar et al. 2005; van der Wielen et al. 2005; Burns
The salt production processes yield substantial quantities et al. 2007; Hallsworth et al. 2007). Javor (1983, 1989) iso-
of rejected bittern brines. In refrigeration systems, these lated bacteria growing in bittern brines with a MgSO4 con-
brines are used as liquid desiccants; MgCl2 is a weak desic- centration of 0.5 mol/L. The Dead Sea, supersaturated with
cant, presenting some advantages, such as low cost, availabil- NaCl (348 g/L) and enriched with magnesium and calcium
Received 22 February 2011. Revision received 2 August 2011. Accepted 4 August 2011. Published at www.nrcresearchpress.com/cjm on
22 November 2011.
H. Baati, R. Jarboui, and E. Ammar. Universit de Sfax, cole Nationale dIngnieurs de Sfax LARSEN, UR tude et Gestion des
Environnements Ctier et Urbain, B.P. 1173 - 3038 Sfax, Tunisia.
N. Gharsallah. Facult des Sciences de Sfax, Laboratoire de biotechnologie microbienne, B.P. 802 - 3018 Sfax, Tunisia.
A. Sghir. Universit dEvry Val dEssonne, 2, rue Gaston Crmieux - 91057 Evry, France; Centre National de la Recherche Scientifique
Unit mixte de recherche, 8030-CE, France; Commissariat lnergie Atomique Genoscope, France.
Corresponding author: Emna Ammar (e-mail: ammarenis@yahoo.fr).

Can. J. Microbiol. 57: 975981 (2011) doi:10.1139/W11-088 Published by NRC Research Press
976
Table 1. Physicochemical characteristics
of brine collected from L3 pond.
Characteristic Value
Physical parameters
Temperature (C) 29.0
pH 5.4
Salinity (g/L) 380.0
Density 1.3
Turbidity (NTU) 35.8
Redox potential (mV) 96.1
Water activity 0.68
Major elements
Na+ (g/L) 2.7
K+ (g/L) 1.6
Ca2+ (g/L) 0.0
Mg2+ (g/L) 96.7
Cl (g/L) 256.9
SO42 (g/L) 33.6
MgCl2 (mol/L) 3.6
cations, is considered one of the most extreme environments
on Earth. It is dominated by halophilic Archaea, particularly
the Halobacterium genus, by halophilic microalgae Duna-
liella, and by fungi (Oren 1983b, 1999; Burns et al. 2007).
In Spain, Laiz et al. (2000) cultivated microbes from building
efflorescences of epsomite at 25% magnesium sulfate. Anton
et al. (2002) isolated viable microbes from saltern crystallizer
ponds at 0.6 mol/L MgCl2. More recently, many yeast spe-
cies were isolated from the water rich in MgCl2 (bitterns) in
the La Trinitat saltern (Butinar et al. 2005).
In the Mediterranean Sea, Discovery Basin, one of the
most extreme saline environments known (MgCl2 = 5 mol/L;
aw < 0.4), was recently considered regarding live limits in
MgCl2-containing environments and enigma of prokaryotic
life in such marine environment (van der Wielen et al.
2005; Hallsworth et al. 2007). In such a marine environ-
ment, van der Wielen et al. (2005) evidenced the presence
of a metabolically active microbial community. However,
Hallsworth et al. (2007) showed that at MgCl2 concentra-
tions exceeding 2.3 mol/L, microbes may not be metabol-
ically active and (or) alive or viable and that the loss of
metabolic activity was due to Mg salt, which may act as a
chaotrope, weakening electrostatic interactions and destabi-
lizing biological macromolecules.
The aims of the present work were to determine the geo-
chemical conditions influencing the bittern brine microbial
communities and to assess the eventual life in the bittern
brine, using molecular approaches and viability test.
Materials and methods
Sample collection
The samples were collected aseptically in sterile flasks
submerged 10 cm below the water surface of L3 pond of the
solar saltern located in the coastal area of Sfax city (Tunisia)
(3439N and 1042E) in May 2006 (Baati et al. 2008). One nomic DNA extraction was performed as described by
litre of the investigated sample was the mean of 1 L of 10
subsamples, spanning the whole pond. The representative
sample designed for molecular and microbial analysis was
Can. J. Microbiol. Vol. 57, 2011
immediately kept on ice for transport back to the laboratory
within 2 h of collection. The sample was centrifuged at
12 000g for 10 min. The resulting pellet was washed with
phosphate-buffered saline, which contained (per litre) KCl,
0.2 g; NaCl, 8 g; KH2PO4, 0.2 g; Na2HPO412H2O, 29 g
(pH 7.4), and was stored at 20 C. The pond L3 is charac-
terized by high salt concentration (380 g/L) (Table 1). Salt
concentration was determined at 120 C by drying 50 mL of
brine water in a crystallizing dish, and then the total salt con-
centration was calculated by determining the difference in
masses before and after evaporation. Chlorine content was
determined by titration (Skoog et al. 1996). Ca2+ and Mg2+
contents were determined by the volumetric method after
complexation (Harris 1997), SO42 by the gravimetric method
(Belcher et al. 1954), and K+ and Na+ by flame photometry
(Sherwood 410) (Golterman and Clymo 1971). The brine
water activity was determined over a range of concentrations
at 25 C, using a water activity apparatus (Novasina Sprint
TH-500).
Enrichment and isolation
The halophilic bacteria enrichments and isolation were per-
formed on two media. The first medium was prepared using
the brine sampled from the solar saltern (L3 pond) enriched
with yeast extract (5 g/L) in a 500 mL Erlenmeyer flask. The
second medium was synthetic and contained the following
(per litre): NaCl, 250 g; KCl, 2 g; MgSO47H2O, 20 g; yeast
extract, 5 g; trisodium citrate, 3 g; and agar, 20 g (Elevi et al.
2004). Each studied medium was inoculated with 30 mL of
the brine sample. The two media were incubated on a rotary
shaker at 200 r/min for 7 days at 37 C. Both media were
adjusted to a pH of 7.2. A 100 L volume of each culture
and its decimal dilutions were spread onto the two solid me-
dia. The plates were incubated at 37 C in a water-saturated
atmosphere for 4 weeks.
Viability test
In the studied sample, bacterial viability was assessed by
acridine orange as described by Hobbie et al. (1977),
Zimmermann et al. (1978), and Naganuma and Miura
(1997). The viable cells were enumerated in a Malassez
counting chamber, using a confocal laser-scanning micro-
scope (LSM 5 PASCAL; Zeiss) with the 100 objective (via-
ble cells were observed with a 488 nm excitation and a
535 nm bandpass emission filter; dead bacteria were ob-
served with a 488 nm excitation and a 580 nm emission
longpass filter). The viable fluorescent cells were green,
whereas nonviable fluorescent cells were red. LSM Imaging
Browser software was used for image analysis.
Genomic DNA extraction
Cell biomass from L3 pond was collected by water sample
centrifugation (30 mL) at 12 000g for 15 min at 4 C. Pellets
were diluted in 200 L of TE buffer (10 mmol/L TrisHCl
and 1 mmol/L EDTA, pH 8) and then incubated at 90 C for
10 min. After ice cooling, various enzymes (lysozyme, pro-
nase, mutanolysine, RNase, and lipase) were added and ge-
Chouari et al. (2003). The extracted DNA was then visual-
ized by electrophoresis on 1% agarose gel with ethidium bro-
mide staining.
Published by NRC Research Press
Baati et al.
Fig. 1. Neighbor-joining phylogenetic tree representing bacterial operational taxonomic units (OTUs) recovered from L3 bittern brine. The
tree was calculated using the ARB software package. The total number of sequences is indicated in brackets. The scale bar represents the
mean number of substitutions per site. Thermofilum pendens (X14835) was used as outgroup.
PCR amplification of 16S rRNA genes
The 16S rRNA genes were amplified by using bacteria-
specific forward primer 008F (Hicks et al. 1992) or archaea-
specific primer 21F (DeLong 1992), combined with the uni-
versal reverse primer 1390R (Zheng et al. 1996). The PCR
thermal profile was as follows: initial denaturation at 94 C
for 5 min, primer annealing at 59 C for 1 min, and extension
at 72 C for 1.5 min. The final elongation step was extended
to 15 min. Under these conditions, a single PCR product of
1.4 kb was obtained and purified by the QIAquick Qiagen kit
(QIAGEN, Hilden, Germany). The resulting PCR product
was quantified by gel electrophoresis before cloning.
Clone library construction and sequencing
The bacterial and archaeal PCR products were cloned us-
ing a TA cloning kit (pGEM-T Easy vector; Promega) in ac-
cordance with the manufacturers instructions. Successful
transformants were used to inoculate 150 L of Luria
Bertani medium (Difco) supplemented with 5% glycerol and
ampicillin (100 g/mL) (Chouari et al. 2003). For each clone,
a PCR for the insert-size check consisted of 1 L of Luria
Bertani broth containing overnight-grown transformants,
1 L each of SP6 primer (0.5 mmol/L) (ATTTAGGTGA-
CACTATAGAATC) and T7 primer (0.5 mmol/L) (TAATAC-
GACTCACTATAGGGCGA), 5 L of reaction buffer,
0.25 L of TaKaRa polymerase (5 U/mL, Promega), and
977
water to a final volume of 50 L. The clones without insert
or those containing incorrect-sized inserts were excluded
from the sequencing. The PCR products were visualized on
a 1% agarose gel to ensure the presence of the expected-size
inserts. Plasmid extraction and 16S rRNA gene sequencing in
both directions of all correct-sized inserts obtained were per-
formed as described by Artiguenave et al. (2000).
Phylogenetic analyses
The resulting 16S rRNA sequences obtained after assem-
blage with Phrap (http://www.phrap.org) were chimera
checked using the procedure described by Juretschko et al.
(2002) and were then compared using BLAST with those
available in public databases (GenBank, RDP (http://rdp.
cme.msu.edu/) and Greengenes (http://greengenes.lbl.gov)).
The sequences from databases with the best BLAST scores
were imported into the ARB data set (http://www.arb-home.
de) (2005 version) when necessary. All sequences having
more than 1200 nucleotides were imported into the ARB
database. An automatic alignment was performed, which was
manually checked and corrected when necessary. Phyloge-
netic placement was done by comparison with reference se-
quences, representing the main descent lines in Bacteria and
Archaea domains. Using the ARB program and database
package, phylogenetic trees were constructed by the neighbor-
joining method incorporating JukesCantor corrections.
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978
Fig. 2. Neighbor-joining phylogenetic tree representing archaeal operational taxonomic units (OTUs) recovered from L3 bittern brine. The tree
was calculated using the ARB software package. The total number of sequences is indicated in brackets. The scale bar represents the mean
number of substitutions per site. Thermotoga maritime (AJ401021) was used as outgroup.
Sequences statistical analyses and population diversity
DOTUR software (http://www.bio.umass.edu/micro/schloss/
software/dotur.html) was used to group sequences into
OTUs and to calculate various diversity indices and rich-
ness estimations (Chao 1987; Magurran 1996; Hughes et
al. 2001; Schloss and Handelsman 2005). Sequences show-
ing more than 97% homology were considered to belong to
the same OTU. Clone libraries coverage was calculated as
described by Good (1954). All sequence data were accumu-
lated in collectors curves established to compare the rela-
tive diversity and coverage of each library by plotting the
number of OTUs versus the number of clones.
Nucleotide sequence accession numbers
In this study, the reported sequences have been submitted
to EMBL, GenBank databases under accession Nos.
FN994899FN994947 for the bacterial clone sequences, and
FN994948FN994985 for the archaeal clones sequences.
Results
Physicochemical and microbiological analyses
The physicochemical characteristics of brine from L3 pond
are presented in Table 1. Brine was pale yellow in color and
viscous. The average temperature at the sampling site was
29 C. The pH was acidic (5.4). The studied brine contained
Can. J. Microbiol. Vol. 57, 2011
substantial concentrations of Mg2+ and Cl, giving rise to
MgCl2 (3.6 mol/L). However, Na+ concentration was low, since
NaCl was previously precipitated. The water activity (aw) value
of bittern brine was 0.68. This is above the minimum value al-
lowing microbial growth (aw = 0.6) (Brown 1990; Grant 2004).
Acridine orange used to estimate bacterial viability showed that
the prokaryotic cell concentration was above 1.4 107 cells/
mL, while the survival rate was about 46.4%.
Bacterial 16S rRNA sequences phylogenetic distribution
After the enrichment in the two different media, trials of
microorganism cultivation were unsuccessful and no isolate
was grown. Therefore, PCR-based cultivation-independent
methods were explored to obtain information about available
microbial flora in the L3 pond. The phylogenetic affiliation
of the obtained sequences and their relationship with other
bacterial and archaeal strains were determined, including en-
vironmental sequences from the GenBank database. The 16S
rRNA gene sequence phylogenetic trees obtained are pre-
sented in Figs. 1 and 2.
In the L3 bittern brine, the 49 bacterial sequences recov-
ered were grouped into 11 OTUs, which were affiliated with
the Gammaproteobacteria and Bacteroidetes divisions
(Fig. 1). Bacteroidetes were dominated by Sphingobacter-
iales represented by the genus Salinibacter, one of the micro-
organisms frequently found in the salterns.
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Baati et al.
Table 2. Diversity indices for bacterial and archaeal libraries.
Brine samples from Baati et al. 2008
M2 pond (150 g/L) TS38 pond (250 g/L)
Index Bacteria Archaea Bacteria
Coverage (%) 79.0 83.3 91.5
Dominance (1/D)* 11.0 2.1 4.2
Evenness (J) 2.0 1.2 1.5
Species richness 38.0 23.0 23.0
(Chao)
*Reciprocal of Simpson index.
Archaeal 16S rRNA sequences phylogenetic distribution
A total of 38 16S rRNA sequences were obtained from the
archaeal clone library, resulting in 13 OTUs. A phylogenetic
analysis showed that all the sequences found could be classi-
fied as the Euryarchaeota group with a diverse assemblage of
Halobacteriales (Fig. 2). These were affiliated with the Halo-
bacteriaceae, such as Haloquadratum, Halorubrum, Halor-
habdus, and Halobacterium, described for the first time in
such magnesium concentrated brine. It should be mentioned
that no sequences affiliated with Crenarchaeota were detected.
Diversity measurement and rarefaction analyses
The relatively high coverage values of bacterial and ar-
chaeal clone libraries exhibited a large microbial community
proportion sampled. Rarefaction analysis performed by plot-
ting the number of OTUs clustered at 97% similarity against
the number of clones sequenced (Fig. 3) showed that the
archaeal sequence population was more diverse statistically
compared with the bacterial groups. This observation was
confirmed by the diversity parameters, suggesting a clear
archaeal population increase (Table 2).
Discussion
Prokaryotic diversity analysis based on the 16S rRNA
technique has been used to study diverse microbial commun-
ities in extreme environments. The microbial diversity in
these environments makes for interesting study and allows
for identification of novel microorganisms and understanding
of ecosystem functioning (Lizama et al. 2001). This approach
prevents bias by culturing microbial biota leading to vast new
phylogenetic lineage (Amann et al. 1995).
In L3 pond bittern brine, phylogenetic analysis of bacterial
and archaeal sequences showed that this pond supported only
limited flora, mainly represented by some genera of the ar-
chaeal domain, with the genus Haloquadratum (58% of the
sequences) dominating. Nevertheless, a few bacterial domain
representatives were observed.
In saturated NaCl solutions, the abundance of Haloquadra-
tum walsbyi may be explained by its good growth and toler-
ance to high magnesium concentrations that may be found
after NaCl precipitation, in relation with sequential seawater
evaporation (Burns et al. 2007). Haloquadratum walsbyi is
the most hyperhalophilic organism known, as further magne-
sium salts concentration leads to sterility of the brines (Javor
1984). Bolhuis et al. (2004) showed that this genus requires
at least 180 g/L total salts and can tolerate high MgCl2 con-
centration (exceeding 2 mol/L MgCl2). This extreme MgCl2
tolerance may reflect the adaptation of the square Archaea
979
Present study
S5 pond (310 g/L) L3 pond (380 g/L)
Archaea Bacteria Archaea Bacteria Archaea
60.0 95.4 63.0 98.6 86.8
9.0 1.8 11.6 1.1 9.5
1.9 0.7 1.9 0.4 1.8
80.0 11.0 106.6 13.0 42.0
Fig. 3. Rarefaction curves generated for 16S rRNA genes in bacter-
ial and archaeal clones libraries from L3 bittern brine.
(Haloquadratum) to NaCl-saturated and MgCl2-rich condi-
tions in the ponds where this microorganism dominates
(Bolhuis et al. 2004). Indeed, the Haloquadratum sp. genome
sequence revealed several unique adaptive traits that allow
this organism to thrive in its specific and extreme niche
(Bolhuis et al. 2006; Legault et al. 2006). Haloquadratum
walsbyi expresses a water-enriched capsule by encoding halo-
mucin, a large protein that plays an important role in tissue
protection against dessication (Bolhuis et al. 2006). In addi-
tion, it synthesizes a poly-gamma-glutamate capsule that con-
tributes to the cell wall rigidity and maintenance of the
unique square morphology (Lobasso et al. 2008).
However, other Halobacteriaceae genera, such as Haloru-
brum and Halobaculum members, have been revealed to be
adapted to life at magnesium concentrations exceeding
2.5 mol/L, but they can only achieve this in the presence of
significant concentrations of NaCl (Oren 1983a, 1983b,
1995; Oren et al. 1984), suggesting that neither water activity
reduction nor osmotic stress would be the primary MgCl2 in-
hibitory property. One clone similar to Halorhabdus utahen-
sis (92.6% similarity) was also detected. The latter can
tolerate up to 0.8 mol/L MgCl2 (Rao et al. 1997) and has
been also previously detected in Discovery Basin brine (van
der Wielen et al. 2005).
The comparative analysis of the microbial diversity in bit-
tern brine collected from L3 pond (380 g/L) with that of
three previously studied brine ponds (M2, TS38, S5; Baati et
al. 2008) with gradually increasing salinity ranging from
150 to 310 g/L showed that the bacterial diversity decreased
with increasing salinity (from 150 to 380 g/L), whereas the
archaeal diversity increased from M2 to S5 ponds but de-
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980
creased in L3 pond (Table 2). Thus, the L3 bittern brine mi-
crobial community was lower than that in the other brines
(Baati et al. 2008), the salt crystal (Baati et al. 2010a), and
the sediments (Baati et al. 2010b) from Tunisian saltern. The
most striking difference between the geochemistry of bittern
brine collected from L3 pond and the other three ponds
(M2, TS38, and S5) (Baati et al. 2008) was the extremely
high concentration of Cl and Mg2+ and the low concentra-
tion of Na+ in L3 bittern brine (Table 1). Furthermore, the
low microbial diversity detected in the bittern brine studied
has been attributed to its high salinity (380400 g/L NaCl)
and its saturation with MgCl2 (3.6 mol/L), a chaotropic
solute. This solute weakens electrostatic interactions and de-
stabilizes biological macromolecules, or kosmotropes
(Hallsworth et al. 2003, 2007). In the L3 pond, life requires
specific adaptations, resulting in microbial communities that
are distinctly different from the ones in the other Tunisian so-
lar saltern ponds. Thus, in the L3 pond, hypersaline condi-
tions and MgCl2 abundance subject the microbial flora to
severe osmotic and chaotropic stress.
Moreover, our study confirmed the life contingency in bit-
tern brines from L3 pond, with its very high magnesium con-
centrations. While studying the Discovery Basin brine, one of
the most well-known extreme saline environments (MgCl2 =
5 mol/L; aw < 0.4), van der Wielen et al. (2005) evidenced
the presence of a metabolically active microbial community.
The latter contributes to carbon and sulfur biogeochemical
cycling, based on microbial activity measurement (such as
ectoenzymatic activities, glutamic acid uptake, sulfate reduc-
tion, and methane production rates). More recently, another
Discovery Basin brine study revealed that the mRNA bio-
marker was not detected in the saturated brine and suggested
that detected microbes may not be metabolically active or
alive at MgCl2 concentrations exceeding 2.3 mol/L
(Hallsworth et al. 2007). Consequently, cellular life may not
be possible in the Discovery Basin brine, and the microbes
detected at 5 mol/L MgCl2 may be dormant in situ. Indeed,
bacteria are either dead or viable but not metabolically active
when MgCl2 concentration is higher than 2.3 mol/L.
In conclusion, this study evidenced the existence of micro-
bial flora in bittern brine by using a molecular approach, and
almost the half of this flora (46.4%) was viable but not de-
tectable by currently used methods (culturability tests). Mo-
lecular analysis of the recovered clones showed that Archaea
populations seem to be the dominant inhabitants.
Acknowledgements
The authors would like to thank the COTUSAL staff for
technical help, especially Ridha Amdouni and the Genoscope
sequencing and bioinformatic teams. We are very grateful to
Ali Gargouri (Centre of Biotechnology in Sfax, Tunisia) for
his help in cell counting by confocal laser-scanning micro-
scopy, and to Hela Chabouni Fourati, an English teacher
trainer in the area of Sfax, for English language correction.
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