Hindawi Publishing Corporation International Journal of Microbiology Volume 2009, Article ID 731786, 17 pages doi:10.1155/2009/731786 Research Article Studies on the Biodiversity of Halophilic Microorganisms Isolated from El-Djerid Salt Lake (Tunisia) under Aerobic Conditions Abdeljabbar Hedi,1, 2 Najla Sadfi,1 Marie-Laure Fardeau,2 Hanene Rebib,1 Jean-Luc Cayol,2 Bernard Ollivier,2 and Abdellatif Boudabous1 1 Laboratoire Microorganismes et Biomol´ecules Actives, Facult´e des Sciences de Tunis, Universit´e de Tunis El Manar, 2092 Tunis, Tunisia 2 Laboratoire de Microbiologie et de Biotechnologie des Environnements Chauds, UMR180, IRD, Universit´es de Provence et de la M´editerran´ee, ESIL case 925, 13288 Marseille cedex 9, France Correspondence should be addressed to Jean-Luc Cayol, [email protected] Received 8 April 2009; Accepted 27 August 2009 Recommended by Thomas L. Kieft Bacterial and archaeal aerobic communities were recovered from sediments from the shallow El-Djerid salt lake in Tunisia, and their salinity gradient distribution was established. Six samples for physicochemical and microbiological analyses were obtained from 6 saline sites in the lake for physico-chemical and microbiological analyses. All samples studied were considered hypersaline with NaCl concentration ranging from 150 to 260 g/L. A specific halophilic microbial community was recovered from each site, and characterization of isolated microorganisms was performed via both phenotypic and phylogenetic approaches. Only one extreme halophilic organism, domain Archaea, was isolated from site 4 only, whereas organisms in the domain Bacteria were recovered from the five remaining sampling sites that contained up to 250 g/L NaCl. Members of the domain Bacteria belonged to genera Salicola, Pontibacillus, Halomonas, Marinococcus,andHalobacillus, whereas the only member of domain Archaea isolated belonged to the genus Halorubrum. The results of this study are discussed in terms of the ecological significance of these microorganisms in the breakdown of organic matter in Lake El-Djerid and their potential for industry applications. Copyright © 2009 Abdeljabbar Hedi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction brines of Wadi Natrun (Egypt), and Lake Magadi (Kenya) [7–9]. It is noteworthy that low taxonomic biodiversity is Hypersaline environments are found in a wide variety of observed in all these saline environments [10, 11], most aquatic and terrestrial ecosystems. They are inhabited by probably due to the highly salt concentrations measured in halotolerant microorganisms but also halophilic microor- these environments. ganisms ranging from moderate halophiles with higher To adapt to high saline conditions, halophilic microor- growth rates in media containing between 0.5 M and 2.5 M ganisms have developed various biochemical strategies, NaCl to extreme halophiles with higher growth rates in including compatible solute synthesis to maintain cell struc- media containing over 2.5 M NaCl [1]. Aerobic, anaerobic, ture and function [12–14]. These solutes (e.g., ecto¨ıne) plus and facultative anaerobic microbes belonging to domains other compounds (bacteriorhodopsins, exopolysaccharides, Archaea and Bacteria have been recovered from these extreme hydrolases, biosurfactants) produced by halophilic microbes ecosystems, where they participate in overall organic matter are clearly of industrial interest. Besides these metabolical oxidation [2–6]. and physiological features, halophilic microorganisms are Moderate and extreme halophiles have been isolated not known to play important roles in fermenting fish sauces and only from hypersaline ecosystems (salt lakes, marine salterns in transforming and degrading waste and organic pollutants and saline soils) but also from alkaline ecosystems (alkaline in saline wastewaters [15–17]. lakes). The most widely studied ecosystems are the Great Southern Tunisia features numerous ecosystems includ- Salt Lake (Utah, USA), the Dead Sea (Israel),¨ the alkaline ing extreme (hypersaline) environments in which microbial 2 International Journal of Microbiology S1 S2 S3 S4 S5 S6 2.3. Enrichment and Isolation. Enrichment cultures and isolation procedures to recover aerobic or facultatively anaer- obic moderately to extremely halophilic microorganisms GAFSA were performed in medium containing (per liter): NaCl, 250 g; MgCl2 6H2O, 13 g; MgSO4 7H2O, 20 g; KCl, 4 g; CaCl2 2H2O,1g;NaBr,0.5g;NaHCO3, 0.2 g; yeast extract, 5 g; GABES tryptone, 8 g; and glucose, 1 g. pH was adjusted to 7.2 with S1S2 S3 10 M NaOH before autoclaving. Enrichment cultures were S4 S5 subcultured several times under the same conditions. Strains S6 were grown in 100 mL of medium in 250-mL Erlenmeyer flasks in a rotary shaker at 37◦C under agitation at 150 rpm. Aliquots (100 μl) of 10−1–10−4 dilutions were plated onto agar medium. After two weeks of incubation at 37◦C, there were red, orange-red, pale-pink, yellowish, cream, white, 15 Km and transparent colonies. Different colonies were picked and restreaked several times to obtain pure cultures. Microbial ◦ Figure 1: Site map of the El-Djerid lake (Tunisia) and sampling cultures were stored at −80 C in the isolation medium points. supplemented with 50% glycerol. 2.4. Characterization and Identification of Isolates. Among ff diversity has been poorly studied. El-Djerid Sebkha, the the 130 strains isolated, only 36 showed di erent phenotypic largest saline lake (5000 km2) in southern Tunisia, is an characteristics and phylogenetic signatures (ARDRA, 16S important source of salt for food, but its microbial diversity rRNA gene sequences). These were chosen for further has never yet been studied. Given its economic value for characterization. Isolates were examined for colony and the region as a salt source, we conducted a microbial survey cell morphology and motility. Colonial morphologies were to gain better knowledge of the microbial diversity thriving described using standard microbiological criteria, with spe- in this extreme ecosystem. The purpose of this research cial emphasis on pigmentation, diameter, colonial elevation, was to chemically analyse salt and brine samples collected consistency, and opacity [19]. These characteristics were from the lake, isolate any novel extremely halophilic aero- described for cultures grown at optimum temperature, pH, bic or facultative anaerobic microorganisms, and examine and salt concentration. For biochemical tests, the strains were grown in flasks their phenotypic features and physiological and biochemical ◦ characteristics with a view to screening for metabolites and cultures were incubated at 37 C. The optimal ionic of industrial interest produced by the novel halophilic content (per liter: 4 g of KCl, 13 g of MgCl2 6H2O, 1 g of isolates. CaCl2 2H2O, 20 g of MgSO4 7H2O, 0.5 g of NaBr, 0.2 g of NaHCO3, 250 g of NaCl) was used in all the biochemical test media. Oxidase reaction was performed according to Kovacs 2. Material and Methods (1956) [20]. Catalase was determined by adding 10 volumes of H2O2 to each strain culture (after 18 hour incubation 2.1. Sample Collections. The studied strains were isolated at 37◦C) on solid medium. Gelatinase, β-galactosidase, from water and sediments of the El-Djerid Sebkha, a urease, indol production, and Voges-Proskauer tests were shallow lake located in southern Tunisia. According to in performed using standard procedures. Other phenotypic situ physico-chemical conditions and level of wastewater characteristics were determined using API 20E and API 20NE pollutants, the Sebkha was divided into six experimental sites kits (BioMerieux,´ Marcy l’Etoile, France) according to Logan (Figure 1). The samples were collected in February 2006. and Berkeley (1984) [21]. Water and sediment samples were collected at the surface and at various depths (0.1, 0.2, 0.3 m) in each site. All samples were collected into sterile bottles and stored in ice boxes in 2.5. PCR Amplification of 16S rDNA. The DNA from bac- the laboratory. terial cultures was extracted using a Wizard Genomic DNA Purification Kit. The 16S rRNA gene of the isolate strain was amplified by adding 1 μL of cell culture to a thermocycler 2.2. Physicochemical Analysis of the Samples. pH, moisture microtube containing 5 μLof10× taq buffer, 0.5 μLofeach + + 2+ 2+ − μ content, and Na ,K,Ca ,Mg ,andCl content of 50 nM Fd1 and Rd1 primers, 5 Lof25mMMgCl2 6H2O, the salt and sediment samples were measured according to 0.5 μLof25mMdNTPs,0.5μL of Taq polymerase (5U μL−1), − standard methods of Trussel et al. [18]; Cl was quantified and 38 μL of sterilized distilled water. Universal primers 2+ by titration with AgNO3,Mg was quantified by atomic Fd1andRd1(Fd1,5-AGAGTTTGATCCTGGCTCAG-3 absorption spectrophotometry, Na+ was quantified by flame and Rd1, 5-AAGGAGGTGATCCAGCC-3) were used to spectrophotometry, and Ca2+ was quantified by complexom- obtain a PCR product of ∼1.5 kb corresponding to base etry using EDTA. Temperature and pH were measured in positions 8-1542 based on Escherichia coli numbering of the situ. 16S rRNA gene [22]. The sample was placed in a hybrid International Journal of Microbiology 3 10% Halobacillus trueperi DSM 10404T Halobacillus aidingensis JCM 12771T Halobacillus karajensis DSM 14948T Halobacillus dabanensis JCM 12772T Halobacillus yeomjeoni DSM 17110T Halobacillus litoralis DSM 10405T Halobacillus locisalis