Prokaryotic biodiversity of halophilic microorganisms isolated from Sehline Sebkha Salt Lake ( Tunisia )

North of Tunisia consists of numerous ecosystems including extreme hypersaline environments in which the microbial diversity has been poorly studied. The Sehline Sebkha is an important source of salt for food. Due to its economical importance with regards to its salt value, a microbial survey has been conducted. The purpose of this research was to examine the phenotypic features as well as the physiological and biochemical characteristics of the microbial diversity of this extreme ecosystem, with the aim of screening for metabolites of industrial interest. Four samples were obtained from 4 saline sites for physico-chemical and microbiological analyses. All samples studied were hypersaline (NaCl concentration ranging from 150 to 260 g/L). A specific halophilic microbial community was recovered from each site and initial characterization of isolated microorganisms was performed by using both phenotypic and phylogenetic approaches. The 16S rRNA genes from 77 bacterial strains and two archaeal strains were isolated and phylogenetically analyzed and belonged to two phyla Firmicutes and gamma-proteobacteria of the domain Bacteria. The results show that the Sehline Lake harbored novel prokaryotic diversity, never reported before for solar salterns. In addition, diversity measurement indicated an increase of bacterial diversity with rising salinity gradient, which is probably due to competition between bacteria and others species.


INTRODUCTION
Previous studies on the microbiology of hypersaline environments showed that halophilic members of the domain Archaea were dominant, whereas those of the domain Bacteria represented limited components (Litchfield and Gillevet, 2002;Ochsenreiter et al., 2002;Baati et al., 2008;Hedi et al., 2009).Nevertheless, other studies have demonstrated that members of the bacteria domain play an important role in hypersaline environments (Antoin et al., 2000).Such extremophiles were described by their ability to produce compounds of industrial interest and biotechnological products (biopolymers, exopolysaccharides, hydrolases, amylases, cellulases, proteases and lipases).In addition, halophilic organisms play important roles in fermenting fish sauces and in transforming and degrading waste and organic pollutants (Grant et al., 1998;Boone and Garrity, 2001).Although molecular diversity studies have been carried out in other hypersaline environments, this work represents the first one where such a study was performed on the anoxic sediment underlying microbial mats in Mediterranean salterns.North of Tunisia consists of numerous ecosystems including extreme (e.g.hypersaline) environments (Monastir, Tunisia) in which the microbial diversity has been poorly studied.The largest saline lake named Sehline Sebkha, located in North of Tunisia covers nearly 16 km 2 .Sehline Sebkha salt lake is a hypersaline environment in the north east part of Tunisia which is considered as a thalassohaline habitat, putting it in the same category as the Great Salt Lake or solar salterns (Rodriguez-Valera et al., 1988).Physico-chemical conditions of the Sebkha revealed that this extreme environment showed high salinity, high radiations (U-V) and changes in temperatures and dryness which make it relevant to be studied by microbiologists.The saltern lake is also an important source of salt for food, providing a wide set of ecological niches for halophilic microorganisms.
However, no study with regards to its microbial diversity has been undertaken so far.The purpose of this research was to chemically analyse salt and brine samples collected from the lake, to isolate novel extremely halophilic aerobic or facultative anaerobic microorganisms that develop in it, and to examine their phenotypic features and their physiological and biochemical characteristics with the aim to screen for metabolites of industrial interest produced by the novel halophilic isolates.

Sample collection
The strains were isolated aseptically from mixed water and sediments of the Sehline Sebkha (Figure 1).Taking into account the in situ physico-chemical conditions and level of wastewater pollutants, the Sebkha was divided into four experimental sites.Water and sediment samples were collected at the surface and at various depths (0.1, 0.2 and 0.3 m), within each site.

Physico-chemical analysis of the samples
The physico-chemical analysis of water and soil samples from the hypersaline Sehline Sebkha (Tunisia), performed by standard methods (Trussel et al., 1989); are reported in Table 1.Cl − was quantified by titration with AgNO3, Mg 2+ was quantified by atomic absorption spectrophotometry, Na + was quantified by flame spectrophotometry, and Ca 2+ was quantified by complexometry using EDTA.Temperature and pH were measured in situ.

Enrichment and isolation
Considering the importance of salinity within the Sebkha, we focused our isolation procedures particularly on extreme halophilic archaea and bacterial microbiota.
Enrichment cultures and isolation procedures to recover aerobic or facultative anaerobic extremely halophilic microorganisms were performed in medium containing (per liter): NaCl, 250 g; MgCl2.6H2O, 13 g; MgSO4.7H2O,20 g; KCl, 4 g; CaCl2.2H2O, 1 g; NaBr, 0.5 g; NaHCO3, 0.2 g; yeast extract, 5 g; tryptone, 8 g; and glucose, 1 g.This medium was selected because it contains the majority of components which can be utilized by halophilic microorganisms.The pH was adjusted to 7.2 and 8.2 with 10 M NaOH before autoclaving.Enrichment cultures were subcultured several times under the same conditions.
Strains were grown in 100 mL of medium in 250 mL Erlenmeyer flasks in a rotatory shaker under agitation at 150 rpm.The adequate temperature chosen for growth was 37°C (average temperature at the sampling sites for isolation at 12 h in the morning).Aliquots (100 µL) of 10 -1 -10 -4 dilutions were plated onto agar medium.
After two weeks of incubation (at least) at 37°C (plates incubated in a humid steam room adding distilled water to avoid dryness), red, orange-red, pale-pink, yellowish, cream, white, and also transparent colonies were observed.Different colonies were picked and restreaked several times (three times at least) to obtain pure cultures.

Characterization and identification of isolates
The isolates that showed different phenotypic characteristics and phylogenetic signatures (Amplified rDNA Restriction Analysis; ARDRA, 16S rRNA gene sequences), were chosen for further characterization.Isolated strains were examined for colony and cell morphologies and cell motility.Colonial morphologies were described by using standard microbiological criteria such as pigmentation, colonial elevation, consistency and opacity.Gram-staining was carried out with the method described by Dussault (1955).The temperature for cultures growth was 37°C and NaCl concentration growth was limited to 25%.The pH tolerance of each isolate was tested in medium with pH values of 7.2 and 8.2.

Biochemical tests of bacteria
Chitinase, cellulase, xylanase, protease and curdlanase activities were analysed for each representative species of the bacteria using colonies of the strains.For each test, a mixture of 50% agar (4% agar dissolved in sodium acetate 0.1 M, pH 5) and 50% enzymatic substrate such CM-Curdlan-RBB, CM-Cellulose-RBB, CM-Xylan-RBB or CM-Chitine-RBV (LOEWE Biochemica GmBH Laboratory), was prepared in plate.Test protease of bacteria was examined in medium containing 50% nutrient agar and 50% half-skim milk.All the tests were supplemented with 10% NaCl.Microorganisms showing clearing zones after 48 h of incubation at 37°C were considered as enzymatic producers.
The reactions were put in a thermal reactor thermocycler (BIOMetra, Leusden, The Netherlands), denatured for 1 min at 95°C and subjected to 30 cycles for 20 s at 95°C, 30 s at 55°C, and 1 min and 30 s at 72°C.This was followed by a final elongation step for 5 min at 72°C.The PCR products were analyzed on 1% (w/v) agarose gels and sent to GATC (Germany) for sequencing using universal primers Fd1 and Rd1 described previously.Sequence data were imported into the BioEdit version 5.0.9 sequence editor (Hall, 1999); base-calling was examined, and a contiguous sequence was obtained.The full sequence was aligned using the RDP Sequence Aligner program (Maidak et al., 2001).The consensus sequence was manually adjusted to conform to the 16S rRNA gene secondary structure model (Winker and Woese, 1991).A nonredundant BLAST search (Altschul et al., 1997) identified its closest relatives.Sequences used in the phylogenetic analysis were obtained from the RDP (Maidak et al., 2001) and GenBank databases (Benson et al., 1999).Sequence positions and alignment ambiguities were eliminated and pairwise evolutionary distances were calculated using the method of Jukes and Cantor (1969).A dendrogram was constructed using the neighbour-joining method (Saitou and Nei, 1987).Confidence in tree topology was determined using 100-bootstrapped trees (Felsenstein, 1985).

Restriction endonuclease digestions
The PCR amplification and restriction endonuclease digestions were performed as previously described (Hedi et al., 2009).Enzymatic digestions were performed by incubating 5 μL of the PCR products with 10 U of each endonuclease and the corresponding enzyme buffer.Digestions were incubated for one hour at 37°C for AluI, HaeIII, and RsaI and products were analyzed on 2% (w/v) agarose gels.

Physico-chemical analysis of the samples
The temperature at the sampling sites was 21°C at 8 h in the morning.The pH of sediment samples was between 8.6 and 9.1 and may be considered as weakly alkaline.The highest values of moisture and salt saturation content were found in the S2 sample.Sodium and chloride were the most abundant ions.Sulfate and magnesium content were found higher also in the four samples when compared with others ions (Table 1).The total salt composition of the S4 sampling site was higher than the other sampling sites (Table 1).Total ionic composition of the lake differed depending on the area sampled.Taking into account the mineral composition of the lake, with regard to its concentration in Na and Cl -, it is clear that halophilic microorganisms should inhabit this lake, thus justifying studies on the microbial survey of it.

Microbiological analyses
After several dilutions and subculturing in the same liquid medium under aerobic conditions, colonies were isolated in the agar medium containing 25% NaCl.The total number of extremely halophilic bacteria in the salt samples of sites 1 and 3 (3.49x 10 4 -7.3 x 10 5 CFU/g, respectively) was higher than in the salt samples of sites 2 and 4 (4.1 x 10 3 -3.2x 10 4 CFU/g, respectively).The high concentration of salt limited the number of strains.The 126 strains isolated belong to 11 different genera within Bacteria and Archaea domains (Table 3).The total strains give an idea about the distribution of major microbial groups that inhabit the Sebkha, taking into account the salinity of the culture medium used for isolation.The number of genera found decreased specially in sites 2 and 4, because of increase of ecosystem salinity (Rodriguez-Valera, 1993).
On the basis of phenotypic characteristics (macro and microscopic analysis), physiological analysis (NaCl, pH), biochemical tests (chitinase, cellulase, curdlanase, xylanase and protease) and molecular approaches [16S analysis, ARDRA], only 79 isolates were selected for characterization and examined in greater details.These strains have been identified by analyzing sequences of genes encoding for the 16S rRNA (Figure 2).The others strains were a repeated isolates showing the same ARDRA profiles analyses.

Colonial and cell morphology
The dominant bacterial population in hypersaline environment comprised motile or non motile, Gram-positive microorganisms and most of them were spore-forming bacteria.Most colonies on agar media were 0.5 -2 mm in diameter after three weeks of incubation.These colonies were smooth, circular, low-convex, transparent or translucent and entire.Cells of all strains isolated were short, long and swollen rods and occurred in singles, pairs or short chains.Colonial pigmentation from these samples ranged from blood-red to pale-pink.Optimum growth occured at, 25% (w/v) NaCl, 37°C, and two pH (7.2 and 8.2) (Table 2).No growth was observed at NaCl concentrations of less than 15% (w/v) for the majority of isolates, thus suggesting that these isolates should be considered as extremely halophilic according to the definition of Ventosa et al. (1998).

Biochemical tests
To identify and characterize the enzymatic capabilities of the isolated strains, some biochemical tests were conducted.Large zones of clearing around the growing bacteria were observed.Others strains were also unable to form clearing zones.These results suggested that enzymes may be secreted by the strains into the culture medium.Results reveal also that many isolates were able to produce chitinase and curdlanase (Table 4).Moreover, most of them exhibited cellulase activity.A few number of the strains chosen were able to produce protease and xylanase.In addition, all isolates showed variability in degradation of enzymes that reflect inter-and intraspecific polymorphism.

Growth
All strains shared more than 97% identity with their closest phylogenetic relative (Table 3) thus suggesting that they may be considered at the same species level until the results of DNA/DNA hybridization studies will be performed to validate or not their affiliation (work under progress).
Only two strain representatives of domain Archaea were identified as Haloferax sp. and Natrinema sp., but these microorganisms have not been further characterized

DISCUSSION
In recent years, a number of extremophilic microorganisms ranging from aerobes to anaerobes have been isolated.Research on microorganisms from extreme environments also intensified with the recognition of a third domain of life (Archaea) by Woese and Fox (1977).Investigations on the microbial ecology of various hypersaline environments have been largely extended during the last decades.In contrast to halotolerant microorganism which do not require NaCl for growth but can grow under saline conditions, halophiles must have NaCl for growth (Ventosa et al., 1998).Both molecular and microbiological studies revealed the presence of moderately to extremely halophilic microorganisms in a wide range of these saline environments (Cayol et al., 1994;Oren, 2002a, b;Demergasso et al., 2004;Ventosa, 2006).
In the present study, we described microbial diversity among Bacteria and Archaea domains within four sites of two pH media (7.2 and 8.2) and high salinity (25%).The microbial communities in the four studied sites were different in terms of diversity and phylogenetic distribution of the 16S rRNA sequences.The differences between the samples indicated that microbial diversity may be strongly influenced by physical and chemical parameters in the four sites, particularly differences in salt concentration and in ions specification.Analysis of soil samples from the four sites studied is reported in Table 1.They differ from those of the other hypersaline environments studied so far (Hedi et al., 2009).Sodium and chloride concentrations in the four sites are higher than those of the Dead Sea in Israel, in particular (Oren, 1993).Waters of the Dead Sea and the Great Salt Lake in the USA, are slightly acidic (pH between 6 and 7), but the pH of the four sites studied are up to 8, and should be therefore considered as weakly alkaline (Oren, 1993).On the other side, the pH of Lakes Wadi Natrun and Magadi (in Kenya) are considered as highly alkaline environments (pH 11) (Jeon et al., 2005).
A total of 126 extremely halophilic strains have been isolated.Among them, 79 strains (77 Bacteria, 2 Archaea) with different pigmentations (cream, white, yellowish, and reddish-orange) as observed with colonies on agar plates have been further characterized.All bacterial strains were found as Gram-positive rods.The phylogenetic analysis indicate that all isolates were members of ten genera of the domain Bacteria including Salicola, Pontibacillus, Halomonas, Marinococcus, Bacillus, Gracibacillus, Halobacillus, Yeomjeonicoccus,   habitats in Spain and Marocco focused on the screening of new exopolysaccharide (EPS)-producing bacteria, several strains were isolated from saline soils and described as new species belonging to the genus Halomonas (Thongthai et al., 1992;Bouchotroch et al., 2001;Jones, 2001;Martínez-Cánovas et al., 2004).
Similar to what we observed in our experiments, a minority of these micro-organisms isolated were identified as members of genera Gracibacillus, Halovibrio, Marinococcus and Pseudomonas.Several other aerobic or facultative anaerobic, moderately halophilic bacteria have been classified within genera related to the order Bacillales (Spring et al., 1996).The use of these microorganisms has been underlined (production of compatible solutes, halophilic enzymes, biopolymers and bioremediation processes) and reviewed in detail (Ventosa et al., 1998;Margesin and Schinner, 2001;Mellado and Ventosa, 2003).The potential industrial use of this collection of halophiles that we have got will be screened for molecules of industrial interest than enzymes (e.g.EPS and PHA, etc) (work in progress).Finally, we believe that studies on these bacteria should be emphasized as they constitute a source of halostable enzymes (Table 4) which may be used in different pharmaco-chemical industries (Jones, 2004;Quesada et al., 2004).

Figure 1 .
Figure 1.Location map of the Sebkha Sehline Lake (Tunisia) and sampling points.

Figure 2 .
Figure 2. 16S rRNA gene-based phylogenetic trees of the bacterial domain, including the 16S rDNA sequences from sediments sample of Sehline Sebkha.The topologies of phylogenetic trees build using the maximum-likehood and maximum-parsimony algorithms were similar to those of the tree constructed by neighbour-joining analyses.

Table 1 .
Physico-chemical characteristics of the sediment samples.

Table 2 .
Phenotypic features of the 24 representatives strains studied.

Table 3 .
Distribution and taxonomic characteristics of micro-organisms isolated from the 4 sites sampled in Sehline Sebkha Lake.
a Data not shown.