Diversity of halophilic and halotolerant bacteria in the largest seasonal hypersaline lake (Aran-Bidgol-Iran)

Abstract

Purpose

In this study, the culturable halophilic and halotolerant bacterial diversity was determined in Aran-Bidgol as a thalassohaline seasonal hypersaline lake in Iran.

Methods

Thirty water, soil, sediments, coastal mud, multi-color brines and salt crystals samples were extracted and cultured using different media and incubation conditions. Totally 958 isolates were obtained and 87 isolates were selected for further studies, based on morphological, physiological and biochemical tests, representing different morphotypes.

Results

Based on 16S rRNA gene sequence analyses, the isolates exhibited 94.6–100% sequence similarity to the closest known species of the genera Bacillus, Halomonas, Oceanobacillus, Salinicoccus, Thalassobacillus, Ornithinibacillus, Halobacillus, Salicola, Virgibacillus, Aerococcus, Arthrobacter, Idiomarina, Paraliobacillus, Staphylococcus, Acinetobacter, Aneurinibacillus, Brevibacillus, Brevundimonas, Chromohalobacter, Gracilibacillus, Jeotgalicoccus, Kocuria, Marinilactibacillus, Marinobacter, Microbacterium, Paenibacillus, Paracoccus, Piscibacillus, Pseudomonas and Sediminibacillus and also, comparison of ARDRA patterns among the sequenced strains, using AluI, Bst UI and Hpa II enzymes showed that these patterns are in accordance with the phylogenetic position of these strains.

Conclusion

The PCR-RFLP analyses suggested that ARDRA possess a functional potential for distinguishing halophilic bacteria to be used for further studies in elementary steps of isolation to reduce the tedious duplication of isolates.

Introduction

Hypersaline environments, those with oversaturated salinities higher than seawater, are extreme environments in which the microbial diversity is drastically limited by the high salt concentrations. Contingent upon the creation and cause of the composition and origin of salts, two sorts of hypersaline environments are distinguished: thalassohaline environments, originated by the evaporation of seawater, having NaCl mostly, and athalassohaline environments, which originated by the evaporation of non-marine salted water, and their ionic composition are quite different from seawater salts [1]. Based on the optimal salt concentration in order to live, microorganisms are classified as halophilic (growing best on media containing more than 0.2 M salt) and non-halophilic (growing best in media containing less than 0.2 M salt) categories. Halophilic microorganisms with optimum growth in media containing 0.2–0.5 M, 0.5–2.5 M and 2.5–5.2 M salt are considered as slight, moderate and extreme halophiles, respectively, while halotolerant microorganisms are those non-halophilic microorganisms, able to grow at high salt concentrations [2, 3]. Halophilic and halotolerant prokaryotes are found both within the Archaeal and the Bacterial domains. Most extreme halophiles belong to the class Halobacteria, whereas most moderately halophilic and halotolerant prokaryotes are scattered in diverse phylogenetic branches of the domain Bacteria [4]. As a result, bacteria are dominant in environments with salinities up to 10–15%, whereas Archaea tend to be known as the main responsible of the pink to red brine and salt layers at higher salt concentrations [5, 6]. Iran has an extensive diversity of hypersaline environments including salt mines, desert saline soils, hypersaline rivers and especially hypersaline lakes. Seasonal hypersaline lakes are abundant in Iran; Aran-Bidgol, is the largest, followed by Howz-Soltan, Bakhtegan, Tashk, and Maharloo are among the biggest lakes. Urmia is the only permanent hypersaline lake of Iran and the second largest hypersaline lake in the world. Regardless of the substantial abundance and diversity of hypersaline ecosystems in Iran, little information is currently available regarding the biodiversity of halophilic bacteria and archaea of these hypersaline lakes. Microbial biodiversity can be studied using culture-dependent and culture-independent approaches. Despite the fact that culture-dependent techniques are the best way for the acquisition of a more developed comprehension of the ecological function and physiology of microorganisms, they are time consuming, demanding toil and display a low proficiency, as merely 0.1–1% of prokaryotic world is predicted to be cultured [7]. Furthermore, culture-independent techniques leave the microorganism outside of achieving, because all acquired is a sign of its existence and not the living cell. Molecular techniques which can identify microorganisms at the first steps of isolation can be helpful in saving time and decreasing the number of duplicate isolates in culture-based studies. Amplified Ribosomal DNA Restriction Analysis (ARDRA) is one of those techniques which seem to be very useful in primary identification and classification of bacteria using culture-dependent and culture-independent approaches. The current study aimed to determine the culturable halophilic and halotolerant bacterial diversity associated with Aran-Bidgol, a thalassohaline seasonal hypersaline lake in Iran.

Methods and materials

Chemicals

All the culture media, organic and inorganic compounds for culture techniques and DNA extraction were obtained from Merck (Darmstadt, Germany) unless stated otherwise. Restriction enzymes, lysozyme, proteinase, DNA Ladder and PCR solutions were purchased from Fermentas (St. Leon-Rot, Germany).

Description of Aran-Bidgol lake

Aran-Bidgol hypersaline lake, likewise called Masileh, Qom and Namak lake, is a natural thalassohaline ecosystem, situated in central part of Iran (34 °18′-34 °45′N, 51 °33′-52 °10′E) (Fig. 1). It has an altitude of about 800 m above sea level and an area of about 2400 km² (depending on evaporation and precipitation rate). Marshlands are broad around the lake, especially in the western part. This lake is nearly dry in the summer while salt layers of 5 to 54 m’ depth are covering its bed, separated with clay layers. High evaporation and low precipitation led to the creation of high salinities up to immersion, in the lake. Noteworthy hexagonal shapes are framed on salt crusts when they got dry which is a special feature of the lake. Shoor, Karaj, Hableh Rood, Jajrood and Ghareh-Soo rivers are the main sources of the lake’s water supply. Brines and salt layers are hued in orange to dull red, green and black by microbial communities. Representatives of the genera Bacillus, Thalassobacillus, Salinicoccus, Halobacillus, Halomonas, Staphylococcus, Marinococcus, Idiomarina and Salicola have been previously isolated from these colored brines and salt crystals [8].

Fig. 1.

Aran-Bidgol hypersaline lake. Sampling sites selected for this study are indicated as A, B, C, D, E and H

Sample collection

A total of 30 samples were obtained from brines, mud, salt crystals and shallow sediments (top 40 cm) of six ecologically most different locations in Aran-Bidgol lake in September 2007 and February 2008. The six sampling sites are shown in Fig. 1. Eastern part of the lake was not available for sampling and no specimen was collected from this part. At the time of sampling, the lake possessed a temperature range of 34–45 °C (annual temperature ranges from −12 °C to 48 °C), a salinity of 18–40%, a pH of 6.8–8 and high concentrations of Cl⁻ as major anion and Na⁺ as major cation followed by SO₄²⁻ and Mg²⁺, respectively.

Samples were collected in replicates, based on standard microbiological methods. The salinity and pH of the samples were determined in situ with Seven Multi dual meter pH/conductivity (Mettler Toledo, Greifensee, Switzerland). Direct plating and serial dilutions both on liquid and solid media were carried out directly on site. 0.5 ml liquid or 0.5 g solid samples were inoculated on 5 ml liquid media (in 50 ml falcon tubes). Soil samples were serially diluted and inoculated. For each sample, 3% seawater nutrient medium (SN) [9], 7.5% halophilic medium (HM) [10], 10-fold diluted SN 3%, 10-fold diluted HM 7.5% and solar salt nutrient 3% and 7.5% were used both as liquid and solid media on site. Diluted 7.5% HM and 3% SN media were prepared by 10-fold dilution of nutrients of HM and SN media, respectively and used according to Janssen suggestion of diluted media for improved isolation of oligotrophic bacteria [11]; 3% and 7.5% solar salt (from Aran-Bidgol lake) was also added to nutrient agar to cover possible unknown salt demands of some native bacteria. The samples were placed in a polythene bag, marked and tied. They were kept on ice during the transportation to a commercial water chemistry laboratory for analysis of the salts composition.

Isolation and cultivation of bacteria

Inoculated solid and liquid media were transported to the laboratory and incubated at 34 °C for up to 3 weeks. Incubation of broth media was carried out on an orbital shaking incubator (Vision Scientific) at 150 r.p.m. After 7 days of incubation, 100 μl of these cultures were streaked on agar plates of the same media and incubated. Representatives of different colony morphotypes were streaked on the same media. Pure cultures were isolated after repeated re-streaking, examined microscopically (model CX31; Olympus) and maintained both on slopes of saline nutrient agar 3% and 7.5% at 4–6 °C and as frozen 30% (v/v) glycerol cultures at −80 °C. Growth at different salt concentrations (0, 1, 3, 5.0, 7.5, 10, 15, 20, 25 and 30%, w/v) was tested on medium containing 5 g/l yeast extract at pH 7.5 and monitored by turbidity at OD₆₀₀ using a spectroscopic method (model UV-160 A; Shimadzu). Isolates showing the first growth at 3–15%, 1–3% and lower than 1% salinities were considered as moderate halophiles, slight halophile and halotolerant, respectively. In order to distinguish between morphologically similar isolates some microscopy, physiological and biochemical features, namely Gram stain reaction, endospore formation, motility, catalase and oxidase were performed.

Genomic DNA extraction

Genomic DNA was extracted from a single colony of each isolate on agar plates following the protocol by Wilson [12]. The results were checked by 0.7% Agarose gel electrophoresis in TAE buffer 1X at 100 V.

Amplified ribosomal DNA restriction analysis (ARDRA)

Primer and enzyme selection

Prediction of primer matching and rational choice of optimal restriction enzymes is a critical step to an efficient and informative ARDRA analysis which can be performed in-silico. Here, we used in-silico simulation of ARDRA analysis on a set of existing valid sequences of halophilic and halotolerant bacteria. Sequences were retrieved from RDP (Ribosomal Database Project, (www.rdp.cme.msu.edu), checked for the validity of their reference and aligned using Clustal X [13]. Universal primers were checked against sequences and primers 341 F and 1408 R were found to match a maximum number of sequences. Restriction analysis of the simulated PCR products was carried out by the use of PCR-RFLP. An algorithm, based on Fuzzy logics, the theory of Graphs and Back-tracing, was employed to find the best enzyme set, which can distinguish the highest number of sequence pairs and yet, has the rational number of enzymes [14]. All combinations of 1 to 4 enzyme sets from AluI (AG/CT), BstUI (CG/CG), DdeI (C/TNAG), HaeIII (GG/CC), HhaI (GCG/C), HinfI (G/ANTC), HpaII (C/CGG), MboI (/GATC), RsaI (GT/AC), Sau96I (G/GNCC) and TaqI (T/CGA) were checked. For in-gel analysis, we used a Fuzzy Logic definition for the difference. A real “difference value” between 0 and 1 was assigned for a pair of electrophoresis gel patterns. For each pair of bands, if they are not distinguishable at all, the value 0, and if they are fully distinguishable, value 1 was assigned. In the case of minor differences, a value between 0 and 1 was given regarding the distinguishable part. This method would be reproducible in any sequence set to introduce a set of optimal restriction enzymes on the basis of defined sequences available. A database of predicted RFLP pattern was made for further application as a reference for the differentiation of our bacteria.

ARDRA analysis

For ARDRA analysis, amplification of 16S rRNA gene sequences was carried out using the bacterial forward primer 341 F and universal reverse primer 1408 R. Amplification reaction contained 20 pm of each primer, PCR Master Mix (Fermentas, Germany) and 100 ng of appropriate genomic DNA as a template in a 50 μl system. The amplification was performed by initial denaturation at 95 °C for 5 min followed by 25 cycles of 93 °C for 1 min, 65 °C for 45 s and 71 °C for 45 s and a final extension at 71 °C for 5 min. The results were checked with the same method as mentioned for DNA extraction. Amplicons were purified using PCR product purification kit (Bioneer, Korea) and digested separately with restriction enzymes AluI, BstUI and HpaII (Fermentas, Germany) as per the manufacturer’s instructions using R buffer and 8 μl of purified PCR product in a final volume of 20 μl at 37 °C for 3 h. Digested bands were separated by gel electrophoresis using a 2% agarose gel in 1 × TAE buffer at 100 V, stained with 1 μg ml⁻¹ Ethidium bromide and visualized under UV light at 254 nm. The resulting patterns were analyzed and checked manually. The presence or absence of each band was assigned as 1 and 0, respectively. Consequent matrices were further analyzed based on the UPGMA analysis and Dice’s coefficient of similarity using Denro-UPGMA program (http://genomes.urv.cat/UPGMA) [15].

PCR and sequencing of 16S rRNA gene

For sequencing purpose, 16S rRNA gene was PCR amplified using the bacterial forward primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) or 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and the reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′) or 1488R (5′-CGGTTACCTTGTTACGACTTCACC-3′), which enables the amplification of nearly the entire 16S rRNA gene. The PCR reaction program was performed by initial denaturation at 95 °C for 5 min followed by 25 cycles of 93 °C for 1 min, 48–57 °C for 1 min, 72 °C for 1.5 min and a final extension at 72 °C for 5 min. A typical PCR mixture (in 25 μl volume) contained the following components: 0.5–2 mM MgCl₂, 50 μM concentration of each deoxynucleoside triphosphate, a 0.5 μM concentration of each primer, 1.25 U of Taq DNA polymerase and buffer as recommended by the manufacturer (MBI Fermentas). The purified PCR product was sequenced using an automated sequencer by Seq Lab Laboratory (Germany).

Nucleotide sequence analysis

In this study, the similarity of 16S rRNA gene sequences was obtained for the known sequences in Gen Bank (www.ncbi.nlm.nih.gov) using BLASTN and through the Ez-Bio-Cloud. Phylogenetic analysis was performed with program MEGA 5.1 [16] using neighbor-joining, maximum parsimony and maximum likelihood methods [17, 18]. After multiple alignments by SINA [19], the tree topologies were evaluated by bootstrap analyses based on 100 replicates [20].

Nucleotide sequence accession numbers

Partial or full 16S rRNA gene sequences, obtained from this study, have been submitted to the NCBI GeneBank database under accession numbers GU332638, GU397375-GU397409, GU474982-GU474989 and HQ433437- HQ433478.

Results

Sampling and isolation of strains

Properties of each isolation site and the total number of sequenced isolates obtained from each site are shown in Table 1. A total of 958 isolates were obtained, among them, 87 isolates were selected for further characterization as representatives of the isolated culturable diversity according to their distinct morphological characteristics. Regardless of previous studies of most aquatic environments, almost 80% of isolated bacteria were Gram-stain-positive. Besides, 11% of the isolates were Gram-stain-positive cocci while only one Gram-stain-negative coccus was isolated.

Table 1.

Geographical and physical conditions of the sampling sites studied as well as a total number of isolates obtained from each sampling site

	Isolation site	GPS	Sample types	pH	Salinity (%)	Total sequenced isolates	Total isolates
							Gram-stain-positive	Gram-stain-negative
A	Shoreline	N: 34.31265 E: 51.7667	Soil	7.2	18	29	210	78
B	Sargardan island	N: 34.37411 E: 51.83416	Soil Solar salt	6.8	31	18	112	36
C	Parsiansolar saltern	N: 34.50792 E: 51.72439	Brine Solar salt	8.0	30	12	180	60
D	Kansar solar saltern	N: 34.50106 E: 51.76385	Soil Solar salt Brine	7.3	37	2	48	12
E	Eighteenth kilometer saltern	N: 34.64378 E: 51.83838	Brine Solar salt	7.9	40	11	12	18
H	Western marshland	Not determined	Salt sludge	7.8	25	15	150	42

About 49% of the isolates showed optimum NaCl concentrations for growth at 3–15%, whereas for the remaining 51% the optimum growth occurred in 0–1% NaCl. Salt tolerance range occurred in the range of 0–25% NaCl depending on the particular isolate; many showed the ability to grow at a wide range of NaCl concentrations. Isolates T5B and B1B grew at a salinity range as wide as 0% to 25% NaCl. None of the isolates grew optimally at 1–3% NaCl, which is typical for slight halophiles. The selected 87 isolates were subjected to 16S rRNA sequencing and ARDRA analyses.

Phylogenetic diversity based on 16S rRNA gene sequence analyses

The 16S rRNA gene sequence comparative studies showed that the bacterial isolates belong to Firmicutes, Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. The majority of strains investigated belong to the genus Bacillus and related genera (almost 61%) in comparison to 23% that belongs to the Proteobacteria. The strains were related to the genera Bacillus (28), Halomonas (8), Oceanobacillus (5), Salinicoccus (5), Thalassobacillus (5), Ornithinibacillus (4), Halobacillus (3), Salicola (3), Virgibacillus (3), Aerococcus (2), Arthrobacter (2), Idiomarina (2), Paraliobacillus (2), Staphylococcus (2), Acinetobacter (1), Aneurinibacillus (1), Brevibacillus (1), Brevundimonas (1), Chromohalobacter (1), Gracilibacillus (1), Jeotgalicoccus (1), Kocuria (1), Marinilactibacillus (1), Marinobacter (1), Microbacterium (1), Paenibacillus (1), Paracoccus (1), Piscibacillus (1), Pseudomonas (1) and Sediminibacillus (1). The phylogenetic tree based on 16S rRNA gene sequences of the isolates was constructed using the neighbour-joining method and showed their relationship to different genera (Figs. 2, 3 and 4). Similar tree topologies were obtained when the maximum-parsimony or maximum-likelihood algorithms were used (data not shown).

Fig. 2.

Neighbor-joining tree showing the phylogenetic position of the isolates related to Bacillaceae and Paenibacillaceae. Bootstrap values based on 100 resamplings are shown at the branch points. Only bootstrap values greater than 50% are indicated. Bar 0.1 substitutions per nucleotide position

Fig. 3.

Neighbor-joining tree showing the phylogenetic position of the isolates related to Staphylococcaceae, Lactobacillales and Micrococcineae. Bootstrap values based on 100 resamplings are shown at the branch points. Only bootstrap values greater than 50% are indicated. Bar 0.1 substitutions per nucleotide position

Fig. 4.

Neighbor-joining tree showing the phylogenetic relationships of the isolates belonging to Proteobacteria. Bootstrap values based on 100 resamplings are shown at the branch points. Only bootstrap values greater than 50% are indicated. Bar 0.1 substitutions per nucleotide position

In silico study and ARDRA analysis

A total of 1437 complete 16S rRNA gene sequences, related to halophilic and halotolerant strains, were obtained from the RDP database. Of these, 1374 sequences were matched by the primer set 341F and 1408R, which was the primer set matching the maximum number of sequences. Among different combinations of commercial 4 bp recognizing restriction enzymes, results showed that AluI, BstUI and HpaII set showed the optimum differentiation. This set can differentiate 97% of sequences if two strains were considered as “differentiated” when they show two different bands in electrophoresis. If having four bands in each differentiated pair (two bands in each sequence) was considered as the limit, differentiation came to 93%. ARDRA analysis with the three enzymes, by means of aforementioned primers, was validated for identification and differentiation of most known halophilic and halotolerant bacteria. The 16S rRNA gene digestion of the 87 isolates selected as representative strains led to the production of 17, 13 and 16 fragments of different sizes for AluI, BstUI, and HpaII, respectively. Fragments of 50–1000 bp, according to DNA ladder on electrophoresis, were considered in the analysis. On the basis of these fragments distribution, 27, 21 and 29 distinguishable patterns were recognized for AluI, BstUI and HpaII enzymes, respectively. The frequencies by which the patterns were observed in different isolates were not the same, for example one group of AluI comprises 19 isolates whereas many other patterns in each enzyme were unique among the strains. Considering the three digestion patterns for each isolate, the 87 isolates were grouped into 58 RFLP profiles. UPGMA dendrogram based on total RFLPs is presented in Fig. 5. Fifteen profiles are representatives of more than one isolate, while other 43 patterns are unique. The dominant group in the UPGMA tree has five isolates among which four are the closest to the type strain of Thalassobacillus devorans G191. Although this dendrogram is not fully equivalent to the phylogenetic tree based on sequence analysis, results show that the RFLP pattern can be used as a useful estimation for identification of duplicates in isolation processes.

Fig. 5.

UPGMA dendrogram showing the RFLP relationship of the strains. The Dice’s coefficient of similarity was used for comparison

Discussion

Iran has a great diversity of hypersaline environments including salt mines, desert saline soil, hypersaline soils and especially hypersaline lakes whose microbial communities needs to be unveiled. Playa or seasonal hypersaline lakes are abundant in Iran, of which Aran-Bidgol lake with an area of about 2400 km² is the largest hypersaline playa in Iran. The detailed study of such unknown and yet diverse ecosystems would allow to determine not only the microbial diversity but also the gene pools and potential use of this information for biotechnological applications. Soil samples of areas A and C have the lower salinity in comparison to other areas, which could justify the observed higher diversity. In spite of the fact that the total number of isolates was relatively low, the percentage of Gram-stain-negative bacteria in sampling site E (18th kilometers Saltern) was 60% whereas this ratio was 21–27% in other sites. A salinity of saturated 40% was also observed in sampling site E. Representatives of Salicola species and endospore-forming bacilli were dominant among the Gram-stain-negative and the Gram-stain-positive representatives, respectively. This may be explained by the ability of Salicola species to grow at high salinities being considered as an extreme halophile [14, 21, 22]. Most strains were able to grow in a wide range of salinities. This pattern may be a consequence of the highly variable conditions of the lake which experiences seasonal and day/night changes in water level and temperature. The majority of tested strains belong to the genus Bacillus and related genera (almost 61%) in comparison to 23% that belongs to the Proteobacteria. The dominance of endospore-forming bacilli is not the usual case in most hypersaline lakes [3, 23, 24] and this fact may be due to the brutal and rapidly changing environmental conditions of this seasonal lake where temperature, salinity and water activity dramatically vacillate between day and night likewise between seasons. Their high metabolic rate and the ability to form dormant shapes may prompt a higher survival of members of the endospore-forming bacilli. Among Gram-stain-negative bacteria, members of the Halomonadaceae were dominant and this is consistent with other studies in hypersaline lakes [3, 23, 24]. Representatives of the genera Bacillus, Thalassobacillus, Ornithinibacillus and Virgibacillus within the Firmicutes and Halomonas in the Proteobacteria were the most frequently isolated strains. Salinivibrio species are among the most common inhabitants isolated from hypersaline lakes [10, 25, 26] but they were not isolated in our study. This result is in accordance with previous studies of Howz Soltan, Urmia lake and Incheh Broun, other permanent and seasonal hypersaline lakes in Iran [22] and it might be due to the high salinity of the habitats at the time of sampling. Representatives of Idiomarina, a common inhabitant of saline waters were also isolated from A and C sampling sites. Less than 1% of the current microbial diversity is assumed to be cultured whereas culturing is the only way to “gain” a strain and the best way to understand its physiology, biochemistry and function. One way to expand our knowledge of the possible features of microorganisms is to obtain more microbial types and study their features and ecological roles. Previously unstudied ecosystems are great sources for new microbial species and thus new potential biotechnological applications [27–29]. In our study, 16S rRNA gene sequence analysis showed that many of the isolates could be considered as potential representatives of new species. Six strains have less than 97% 16S rRNA gene sequence similarity to any known bacterial species and are probably representatives of new taxa. Complete polyphasic characterization has been performed on some of the retrieved strains in order to determine their exact taxonomic position. Amongst the studied strains X4B, P4B, A76 and B6B represent new taxa at the genus level, and have been described as Saliterribacilluspersicus, Alteribacillusbidgolensis, Aliicoccuspersicus and Aquibacillushalophilus, respectively. Besides seven additional strains represent new species which were described as Bacillus iranensis, B. persicus, Ornithinibacillus halophilus, B. halosaccharovorans, Marinobacterpersicus, B. salsus and Oceanobacilluslimi, Oceanobacillushalophilus, and Oceanobacillus longus [1, 4, 8, 14, 29]. Further studies on other potential new taxa are under evaluation. Besides the culturable study of the microbial diversity, investigations using culture-independent methods would be of great help to reveal the actual community structure of the lake. We used ARDRA analysis to check its ability to discriminate halophilic and halotolerant microorganisms in primary steps of culturing. This method is simple, rapid and universally applicable, which make it a suitable tool for first steps of differentiation. Although the dendrogram obtained from ARDRA profiles is not comparable to phylogenetic trees, the overall separation layout proves to be of great use and would avoid duplicate isolates in the primary steps of taxonomic studies. The efficiency and accuracy of restriction fragment analysis depend on several factors, including the number of restriction enzymes used, the particular restriction enzymes employed, the resolution of the gel to perform fragment differentiation and laboratory conditions [30–32]. Rational choice of primers, restriction enzymes and electrophoretic conditions were considered using a computer simulated program (data not shown) which permitted us to select AluI, HpaII and BstUI enzymes as the best enzyme set to distinguish halophilic and halotolerant microorganisms, considering 50–1000 bp bands on 2% Agarose gel electrophoresis.

Conclusion

Results showed that ARDRA possess a functional potential for distinguishing halophilic bacteria. This method is simple, rapid and universally applicable as a suitable tool for first steps of differentiation. A total of 58 patterns were obtained using this set up which are not far away from the total differentiated isolates on the basis of 16S rRNA gene sequences.

Author contributions

Mohammad Ali Amoozegar and Antonio Ventosa planned the experiments. Maryam Didari, Maryam Bagheri, Dr. Saied Bouzari, Hamid Babavalian, Hamid Tebyanian and Mehdi Hassanshahian carried out the experimental work and prepared the draft manuscript. All authors revised and contributed to the preparation of the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animal experiments by any of the authors.