Journal of Basic Microbiology 2012, 52, 1–6 1

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Research Paper

Isolation and identification of selenite reducing archaea from Tuz (salt) Lake In Turkey

Kıymet Güven, Mehmet Burçin Mutlu, Ceyhun Çırpan and Hatice Mehtap Kutlu

In this study, Tuz lake brine samples were investigated for isolation and identification of selenite resistant halophilic prokaryotes. Among the 20 strains of extremely halophilic Bacteria and Archaea, a Gram negative rod designated as strain 106, showed high capacity in the resistance to selenite (25 mM) under aerobic conditions. Phenotypic characterizations and phylogenetic analyses based on 16S rDNA sequence comparison indicated that strain 106 was Halorubrum xinjiangense.

The ability of strain 106 to deposite selenium-containing particles were investigated by Transmission Electron Microscopy (TEM). Electron micrographs shows intact cells after selenite reduction and large amounts of selenium-containing particles are present in the culture medium indicating that strain 106 is able to efficiently transport elemental selenium out of the cell.

Keywords: Selenite / Archaea / Tuz Lake / TEM / Halorubrum sp.

Received: January 09, 2012; accepted: January 10, 2012

DOI 10.1002/jobm.201200008

Introduction*

Selenium (Se), a metalloid, occupies a unique position as regard to continuing conflicting aspects of its toxico-logical and nutritional significance. It is a naturally occurring element and is essential for biological sys-tems at low concentrations and toxic at higher levels [1]. Under aerobic conditions Se is present in several redox forms including the elemental form Se. But, se-lenate (Se (VI), SeO4

2–) and selenite (Se(IV), SeO32–) exist

predominantly in aquatic environments and occur as the high valence soluble oxyanions [2]. The roles of Se in the biosphere is both beneficial and detrimental and it is becoming evident that microor-ganisms play a major role in the global Se cycle. It has been shown that phylogenetically diverse bacteria from genera Rhodospirillium [3], Bacillus sp. [4], Pseudoalteromo-nas [1, 5], Ralstonia [6], rhizobacteria [7], bacilli [8] reduce selenite to elemental selenium (insoluble and nontoxic) as a strategy of cellular detoxification. Certain strains that are resistant to selenium oxyan-ions and reduce selenite or selenate to elemental sele- Correspondence: Kıymet Güven, Anadolu University, Faculty of Science Department of Biology Eskisehir, Turkey E-mail: kguven@anadolu.edu.tr Phone: +90 222 3350580/4724 Fax: +90 222 3204910

nium could be potentially used for the bioremediation of contaminated soils, sediments, industrial effluents and agricultural drainage waters [4]. The biogenic Se nanospheres have potentials for use in diverse applications of industrial devices such as rectifiers, solar cells, photographic exposure meters and xerography due to their photoelectric and semi-conducter properties [9]. It is also a component of sele-nium nanowires [10, 11]. Se reducing bacteria produce extracellular granules of Se as stable and uniform mono-clinic crystalline nanospheres which is not achieved by chemical synthesis [12]. Halophilic microorganisms are considered suitable canditates for screening their potentials and tolereance to toxic substances for both bioremediation and bio-transformation studies because of their capability of growing in exceptionally high concentrations of anions and cations. They are not only naturally tolerant of elements that are commonly toxic for other microor-ganisms, but also require these elements [13]. Tuz Lake is an inland hypersaline water body (salin-ity above 30% NaCl), located in the central plateau of Turkey, 120 km south of Ankara. It is the second largest lake of Turkey, with a lenght of 90 km a width of 35 km and a total surface of 1,665 km2 within a closed basin. Water flows into the Tuz Lake through

2120715 MIK 03/12 MIK00008u.doc Kraus/Schambach/Pfü.VMWare: CS3

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Melendiz Stream and drainage channels of Konya plain. In summer, the lake dries out and a 30 cm layer of salt forms because of the evaporation. In winter, water is not more than 2 m deep. Three hundred thousand tons of salt, which is the 60% of the total salt produc-tion in Turkey, are obtained per year from the lake [14, 15]. In this study, we investigated selenite resistant halo-philic prokaryotes isolated from Tuz (Salt) lake brine samples. We characterized the selenite reducing strain by phenotypically and phylogenetic analysis based on 16S rDNA sequence comparison.

Materials and methods

Collection of samples Samples were taken from two different locations (38°46′25″ N, 33°12′57″ E and 38°45′33″ N, 33°08′10″ E) of the Tuz Lake. The total salt concentration of each sample was determined in situ with a hand refractome-ter (Eclipse).

Isolation of microorganisms Isolation of strains was performed on Sea Water ((SW) medium (g l–1): NaBr 0.65; NaHCO3 0.167; KCl 5; CaCl2 0.723; MgSO4 ⋅ 7 H2O: 49.49; MgCl2 ⋅ 6 H2O 34.567; NaCl 195; yeast extract (YE), 1; agar, 20) [16]. One ml of sev-eral dilutions (from 10–1 to 10–5) of the original water samples were used to inoculate the plates by a “spread plating” technique. After incubation of the plates for 15–20 d at 37 °C, colonies grown were restreaked on the same medium for single colony isolation.

Determination of selenite resistance Sodium selenite was obtained from Aldrich (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The stock solutions were prepared in distilled water and main-tained at 4 °C following sterilization by microbiological filter (0.22 µm). Working solutions were prepared and used immediately before using. The resistance of strains to selenite was measured by the agar dilution method [13]. 20 ml of melted Sea Wa-ter agar medium was mixed with various concentra-tions of selenite (1 mM to 50 mM). Then 10 µl of the bacterial suspension (108 cfu/ml) was inoculated onto each plate by spread plating. After incubation at 37 °C for 3 weeks colonies resistant for selenite was deter-mined. Each plate was prepared in triplicate.

Measurements of bacterial growth rate in the presence of increasing Se concentrations The cultures were grown aseptically in flasks contain-ing 50 ml of medium. The medium for the selenate experiment was the same as isolation medium and it contained; no sodium selenate or in range of 5 mM to 0.5 M sodium selenate. All flasks were placed in a shaker mainteined at 120 rpm for 1 week at 37 °C. The absorbance of each culture flask was measured spec-trophotometrically at 600 nm as an indicator growth. We also inoculated our isolate to the SW agar plates containing various concentrations of selenite (5 mM to 50 mM). After one week incubation at 37 °C colonies were counted.

Identification of the isolate and phylogenetic analysis Extraction of genomic DNA from isolates, PCR amplifi-cation of 16S rRNA gene and direct sequencing of the purified PCR products were performed as previously described in detail [17]. 16S rDNA sequences (>1350 bases) of closely related taxa of the bacterial isolates were retrieved from the NCBI database by using BLAST (Basic Local Aligment Search Tool). These sequences were aligned using CLUSTAL X program [18] and a neighbour-joining phy-logenetic tree was constructed using TREECON pro-gram [19].

Transmission Electron microscopy Strains growing on a selenite containing medium that showed dark red colony color were collected by scrap-ing and processed for transmission electron micro-scopic analysis. Sample morphology and size were determined using TEM (FEI, Tecnai). Cells were fixed with % 2.5 gluteral-dehyde in 0.1 M phosphate buffer (pH 7.4) and left in phosphate buffered saline (PBS) overnight at 4 °C. After being embedded in agar and post fixed in 2% osmium tetroxide cells were dehydrated in graded ethanol: 70, 90, 96 and 100%. Then the cells were embedded in EPON 812 epoxy. They were thin sectioned using a diamond knife to a maximum thickness of 80 nm–100 nm. The sections were stained with lead citrate and uranyl acetate [20].

Results

The isolation of halophilic strains resistant to selenite was performed using a culture medium maintaining rapid bacterial growth. Among the 20 strains isolated

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(A) (B)

Figure 1. Colonies of strain 106 in SW agar (A) without selenite and (B) with 25 mM selenite.

from Tuz Lake in Turkey only one isolate was resistant to selenite. Strain 106 showed maximum resistance to selenite (25 mM). The resistance of strain 106 was asso-ciated with reduction of selenite to selenium and the appearance of orange to dark red colonies in the se-lenite containing medium. The strain 106 was Gram negative, non-sporulating, aerobic rod and, produced oxidase and catalase. The

colonies on SW agar medium appeared round, small orange-reddish colonies. When the cells were grown in the presence of high levels of sodium selenite, cells accumulated intracellular deposits of red, amorphous, elemental Se, giving cultures a dark-red color (Fig. 1A and B). The organism was capable of growth over a wide range of NaCl concentration (10–25%, w/v). The strain

Figure 2. 16S rRNA gene sequence-based dendogram showing the phylogenetic position of the selenium resistant strain 106 and its relationship with other halophilic Archaea.

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Figure 3. Growth rates of the cultures that grown in various concentration of selenite.

was capable of growth over a wide range of pH values (3–11), with an optimal pH of 7–8. Optimum growth temperature was 37 °C, minimum was approximately 25 °C and maximum was approximately 55 °C. The isolate used a wide variety of organic carbon sources as the sole source of carbon for aerobic growth such as glucose, fructose, galactose, sucrose, trehalose, manni-tol. Different responses were obtained against antibiot-ics, and strain 106 was resistant to penicillin G, ampicil-lin, vancomycin, tetracycline and chloramphenicol. The strain was sensitive to bacitracin and novobiocin. Iden-fication of the isolate was carried out by PCR amplifica-tion and sequencing of 16S rRNA genes. The strain was phylogenetically related to Halorubrum xinjiangense (99%

16S rRNA gene similarity). The phylogenetic tree (Fig. 2) constructed by neighbour joining method indicated that the isolate 106 clustered within the genus Halobac-terium [21]. The closest relative of the isolate was Halo-rubrum xinjiangense. Therefore, isolate 106 was identified as Halorubrum xinjiangense. There is no significant differences between the growth curves of the cultures that were grown in vari-ous concentration of selenite (Fig. 3). Maximal densities were very similar whether or not selenite in varying concentrations was present in the culture medium. In the presence of metalloids cells accumulated Se intracellularly and release of these particles were indi-cated in ultrathin electron microscope sections (Fig. 4).

(A) (B)

Figure 4. Transmission electron micrographs of the strain 106 in the medium containing 25 mM selenite (A) and selenite-free control (B).

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Discussion

Halophilic microorganisms tolerate high concentra-tions of toxic metals and therefore they can be used for bioremediation and biotransformation of toxic metals. Furthermore, such information could be used as bioas-say indicator organisms in saline polluted environ-ments [22]. Toxic heavy metal and oxyanion resistance was reported in halophilic microorganisms, e.g. Halo-monas [22–24], halophilic spore-forming bacilli [13], Salinicoccus sp. [25] and Pseudoalteromonas [5]. In this study, isolate 106 was identified as Halorubrum xinjian-gense which is a member of Halobacteriacea and plays important roles in the carbon and nitrogen cycles of hypersaline environments [26]. Likely mechanisms of bacterial resistance to selenite include export, methylation or volatilization and pre-cipitation of insoluble selenium metal [27]. During se-lenite reduction by microorganisms, elemental Se de-posits in the periplasm, in the cytoplasm and outside the cell [28–30]. Particles containing elemental Se found outside cells may be released by cell lysis [31] or the particles may be expelled by a membrane efflux pump as a result of membrane-associated reductase [30]. It is known that high level of selenium does not ef-fect the growth rate of the cultures because of it’s non-toxic nature. Elemental selenium, highly insoluble, represents a nontoxic storage form for Ralstonia metal-lidurans CH34 [32]. Amoozegar et al. [13] reported that increase in salinity from 5% (w/v) to 15% (w/v) en-hanced tolerance to toxic oxyanions in moderately halophilicspore-forming rod bacteria. The resulting colonies were grown in the varying degrees of concen-trations of selenite with saline medium in this study indicates that Halorubrum xinjiangense adapted it’s me-tabolism to selenite stress. In conclusion, in the present study, we report for the first time that an archaea isolated from the hypersaline Tuz Lake is capable of reducing selenite to selenium. Further studies including the effect of pH, temperature and incubation time should be determined for maxi-mum reduction of selenite to selenium. Bacteria capa-ble of reducing selenite could be useful in the applied biometallurgy of selenite, rare and expensive metals used extensively for their properties as semiconductors [1]. The genes involved in selenite resistance have not been identified and the exact mechanism of selenite bioreduction is still unknown. In future studies, this strain could be geneticaly manipulated and exploited for in situ bioremediation of selenium-containing min-ing and industrial wastewaters containing higher amounts of salts.

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