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Isolation and characterization of halophilic bacteria from the dead sea coast, Jordan.

Introduction

In spite of its high salinity, the Dead Sea is the natural habitat for a variety of microorganisms [1, 2, 3]. These include unicellular green algae of the genus Dunaliella, which is the sole primary producers in the lake, and a variety of heterotrophic prokaryotes, aerobes, anaerobes as well as Archaea [4, 5]. A number of Dead Sea microorganisms have been isolated and were maintained in culture such as Archaea, Haloarcula marismortui [3]. A similar strain was isolated from the Dead Sea in the late 1960s [6]. This organism has become a popular model for the study of halophilic enzymes, and its genome was recently sequenced [7]. Haloferax volcanii isolated in the early 1970s from surface sediments [8]. Halorubrum sodomense isolated from the archaeal bloom that developed in the Dead Sea in 1980 [9]. Halobaculum gomorrense, isolated during the bloom of 1992 [10]. Additional archaeal isolates have been obtained in the course of the years from the Dead Sea [11]. These include a number of strains isolated from the Jordanian side of the lake. Other archaeal strains have recently been revived from old enrichment cultures set up by Volcani in the 1930s, and these are awaiting a full characterization [12, 13].

Two moderately halophilic Bacteria isolated by Volcani have been preserved: Halomonas halmophila and Chromohalobacter marismortui [2]. Both grew at salt concentrations as low as 5-10 g [l.sup.-1], but tolerate up to 200-300 g [l.sup.-1]. Chromohalobacter israelensis was isolated from crude solar salt from a Dead Sea evaporation pond [14, 15, 16]. Recent attempts to isolate live bacteria from Volcani's old enrichments led to the isolation of Salibacillus marismortui [17]. This endospore -forming bacterium grows between 50 and 250 g 1-1 salt, with an optimum at 100 g [l.sup.-1]. Number of obligatory halophilic Bacteria have been recovered from the sediments of the Dead Sea: Halobacteroides halobius [18], a species of slender, flexible rods that ferment simple sugars to ethanol, acetic acid, hydrogen, and C[O.sub.2], Sporohalobacter lortetii produced gas vesicles that remain attached to the mature endospores [19, 20]. Orenia marismortui another endospore-forming fermentative anaerobe was also isolated [20]. Selenihalanaerobacter shriftii an organism that lives by anaerobic respiration which oxidizes glycerol or glucose to acetate and C[O.sub.2], while reducing selenate to a mixture of selenite and elemental selenium. Nitrate and trimethylamine N-oxide are also used as electron acceptors.

To what extent the isolates listed above represent the truly important components of the Dead Sea biota is unknown. Moreover, quantitative data on the microbial communities in the Dead Sea are highly incomplete. All of Volcani's early work was of a qualitative nature, based on enrichment cultures. We have no quantitative data on the community densities of algae, Archaea and Bacteria in the lake prior to the 1979 mixing event, with the exception of a few isolated measurements based on samples collected in 1963-1964 [21].

We have also a reasonable insight in the dynamics of the microbial communities in the Dead Sea from 1980 onwards [22-27, 10, 28, 29, 30]. Dense blooms of Dunaliella and of halophilic Archaea developed in the upper few meters of the water column in 1980 and in 1992, in both cases triggered by a significant dilution of the upper water layers by massive amounts of fresh water that entered the lake through the Jordan River and rain floods from the catchment area. In both years the entire Dead Sea was colored red due to the dense communities of carotenoid-rich Archaea. On the other hand, the monomictic periods (1983-1991 and 1996 until present) were characterized by very low community densities of microorganisms, to the extent that it is now difficult to detect any life at all in the water column. This study aimed to isolate and characterize extreme halophilic bacteria from the Jordanian side of the Dead Sea and compare these isolates with other previously isolates halophilic bacteria.

Material and Methods

Area of study

The study area is located in the Southwest coast of Dead sea in the municipality of Al Karak (lat. 31[degrees]18' N, long. 36[degrees]01'E) (fig 1). The area is characterized by low precipitation (less than 50 mm) and salinity (30-35%) The samples (soils and sediments) were collected from a variety locations in the Dead Sea in 21-26/2/2006 using Oga machine. These sites were located in the (Al; AL-hadeetha area) which long away from southern shore of the Dead Sea 200m and (A2; AL-mazra'ah area) which long away from southern shore of the Dead Sea 100 m. the samples depth ranged from surface, 25cm to 40cm. The samples collected in sterile glass jars and stored until they reached the lab.

Isolation and cultivation

In total, seven isolates halophilic bacteria were collected from salty environments including hypersaline soils, hypersaline sediment. All strains were grown in a saline nutrient broth (HiMedia Laboratories) with a final total NaCl salt concentration of 0.5, 1, 2, 3M. The salts solution composition was prepared according to [31]. When necessary, the medium was solidified by adding 2% (w/v) Agar (Scharlau Chemie). The cultures were incubated at 37[degrees]C on an orbital shaking incubator (orbital incubator SI 50, Stuart scientific) at 150 rpm for 48 h with the pH adjusted to 7.2 before autoclaving.

Identification of the isolate

Morphological and physiological characterizations were defined in basal culture media containing NaCl ranged from 5% to 15%(w/v). Bacteria were grown either on salty nutrient broth or agar medium. Gram reaction, motility, shape and color of colony, oxidase activities, nitrate reduction, esculin, tween 20 and 80 hydrolyzes, and indol production were checked as recommended [32]. Acid production from carbohydrates and sugars and utilization of carbon and nitrogen sources were evaluated as in [33].

[FIGURE 1 OMITTED]

Sensitivity to antimicrobial agents.

Susceptibility to antimicrobial agents was tested on either HM 20 or HM 12 media using sensitivity discs containing:

1. Ampicilin (2[micro]g)

2. Nalidic acid (30[micro]g)

3. Carbenicilline (100[micro]g)

4. Tetracycline (30[micro]g)

5. Vancomycin (30[micro]g)

6. Penicilline (5[micro]g)

7. Oxacillin (1[micro]g)

8. Ofloxacin (5[micro]g)

9. Amoxicillin (30[micro]g).

10. Cefuperazone (75[micro]g)

11. Oleandomycin (15[micro]g)

12. Rifampicin (30[micro]g)

13. Cefuroxime (30[micro]g)

14. Streptomycin (l0[micro]g)

Antimicrobial discs were laid on plates of HM (20 or 12) which had been surface inoculated with 0.2m1 24hrs grown test strain [11].

Results and discussion

A large number of orange-red and creamy colonies were obtained and found to be extremely and moderately halophilic bacteria respectively. Two isolates of extreme halophilic bacteria (orange-red), were selected depending on colony and cell morphologies, biochemical tests and optimal condition growth. Six isolates of moderate halophilic bacteria were selected depending on same tests as mentioned above. Table (1) shows the names of isolates chosen and their sites of origin.

The isolates were cultured with optimal pH 7, temperature 37[degrees]C, and NaCl concentration 20% for group one (H4 and H5) isolates, 8% for group 2 (H1, H3, H6 and H7) isolates and 15% for group 3(H2) isolate. All isolates grew exponentially with a specific growth rate of 0.056 [h.sup.-1] (Fig. 2, 3 and 4). Growth decelerated between 10 and 20 h, after which the culture entered into a stationary phase.

The isolates were divided into three different groups according to their morphological, biochemical and physiological characterizations: Group one isolates H4 and H5 were isolated from sediment of the eastern-south of the Dead Sea. They could be extreme halophilic bacteria because it can grow in media contain more than 3M NaCl. The main characterizations of H4 and H5: Gram negative, pleomorphic, orange to red colonies, non motile, no spore, optimum temp. 37-40[degrees]C, optimum pH 7, range of NaCl concentration 0.5-3.5M, needed Mg[Cl.sub.2], optimum Mg[Cl.sub.2], 20%, utilize glucose, fructose, sucrose, citrate, Mannitol, maltose and lactose, hydrolyze DNA, Oxidase and Catalase were produced; resistance for antibiotics Oleandomycin and Oxacillin, H5 produced HZS. According to the above results our isolates could be compared with species which were previously isolated from the Dead Sea coast. From morphological and biochemical tests; our isolates were found to be similar to Haloferax volcani and halobacterium sodomense, but most similar to Haloferax volcani with little differences such as H. volcani can hydrolyze starch, and can't grow anaerobically, and optimal temp. 45[degrees]C (Table 2).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Group two isolates; H1, H3, H6 and H7; rod cells, motile, creamy circular entire raised colonies, utilize organic compounds; glucose, fructose, and can hydrolyze gelatin and Aesculin, Oxidase positive, optimum NaCl concentration 8%, optimum pH 7, optimum temperature 37[degrees]C.

Group three isolate H2, facultative anaerobic, rod cell, non motile, cream, utilize organic compounds; glucose, fructose, sucrose, can hydrolyze starch, tween 80, Aesculin, and casein, Oxidase positive, can reduction nitrate, optimum NaCl concentration 15%, optimum pH 7, optimum temperature 37[degrees]C.

Groups two and three are moderately halophilic bacteria and there are differences between them and other known species isolated from the Dead Sea; Flavobacterium halmephilum, Chromobacterium marismortui, Halomonas israelensis.

The susceptibility test showed that some of the isolates were resistant against all the tested antibiotics, whereas the others were sensitive to all tested antibiotics except Cefuroxim (Table 3). The exact mechanism behind this resistant pattern was unknown. In general, the bacterial resistance to antibiotics indicate that this bacteria was previously exposed to antibiotics earlier. However this phenomenon may not applicable for the isolates as there was no possible means of antibiotic contamination in Dead Sea. Since the isolate showed a broad-spectrum sensitivity pattern, it could be used as test organism to screen broadspectrum antibacterial agents instead of screening for a battery of test organisms.

In conclusion, it should be said that the assignment of new isolates to known genera based on chemotaxonomic and fingerprinting methods is not an easy task, but these methods are potentially useful for assessing biodiversity. The aim of this study was to investigate the diversity of haloarchaea. The environmental genomic approach can provide much new information. Much new knowledge has been obtained about the structure of microbial communities by characterization of small subunit rRNA genes, and this approach is nowadays one of the methods most widely used to characterize communities of Bacteria and Archaea. The method has never yet been applied to the Dead Sea. When the proper equipment and finances are available, "complete" sequencing of the DNA isolated from the environment now also belongs to the possibilities. This was recently demonstrated by Craig Venter's genomic studies of plankton collected from the Sargasso Sea [34]. The Dead Sea is probably a much simpler ecosystem than the open ocean as far as microbial diversity is concerned, and therefore similar studies in the Dead Sea may be relatively easy to perform. The fact that the complete genome of one Dead Sea archaeon (Haloarcula marismortui) is already known [7], and a second genome (Haloferax volcanii) will soon be published, will be very helpful here.

References

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(1) Khaled A. Tarawneh, (2) Mohammed Wedyan, (1) Mahmoud A. Al-zou'bi, (1) Khaled M. Khleifat and (1) Amjed Tarawneh

(1) Department of Biological Sciences, Mutah University, Mutah, Jordan.

(2) Department of Biology, Al Hussein Bin Talal University, Ma'an, Jordan.

Corresponding Author:

Mohammed Wedyan, Department of Biology, Al Hussein Bin Talal University, Ma'an, Jordan. E-mail: mwedyan@ahu.edu.jo
Table 1: Isolates and site of origin.

Isolates Site of origin

H1 AL-Hadeetha
H2 AL-Hadeetha
H3 AL-Mazra'ah
H4 Sediment of the southern east shore of the Dead Sea
H5 Sediment of the southern east shore of the Dead Sea
H6 Sediment of the southern east shore of the Dead Sea
H7 Sediment of the southern east shore of the Dead Sea

Table 2: Shows the biochemical characteristics
and morphological studies of the isolates.

Characteristic H1 H2 H3 H4

Colony
 pigmentation Creamy Creamy Creamy Red orange
Cell
 morphology Rods Pleomorphic Rods Pleomorphic
Gram stain - - - -
Motility + - + -

 pH for growth

Range 6-8 6-8 6-8 6-9
Optimum 7 7 7 7
NaCl range for
 growth(%) 1-15 1-25 1-15 5-35

 Temperature for growth ([degrees]C)

Range 25-50 25-50 25-45 25-50

 Acid production from

Maltose + + + +
Glucose + + + +
Sucrose + + + +

 Hydrolysis of

Casein + + + -
Gelatin + - + -
Tween 20 - - - -
Tween 80 + + - -
Starch - + + +
Nitrate reduction + + + +
Hydrolysis of
 Aesculin + + + -
Hydrogen sulfide
 test - - - -

Characteristic H5 H6 H7

Colony
 pigmentation Red orange Creamy Creamy
Cell
 morphology Pleomorphic Rods Rods
Gram stain - - -
Motility - + +

 pH for growth

Range 6-9 6-8 6-8
Optimum 7 7 7
NaCl range for 5-35 1-15 1-20
 growth(%)

 Temperature for growth ([degree]C)

Range 25-50 25-50 25-45

 Acid production from

Maltose + + +
Glucose + + +
Sucrose + + +

Casein - - +
Gelatin - + +
Tween 20 - - -
Tween 80 - - -
Starch - - +
Nitrate reduction + + +
Hydrolysis of
 Aesculin - + +
Hydrogen sulfide
 test + - -

+ Positive; -negative.

Table 3: Sensitivity to antimicrobial agents.

Antimicrobial agents ([micro]g) H1 H2 H3 H4 H5 H6 H7

Amoxacillin (30) BBL S S S S S S S
Ampicilin(10) Oxoid S S S S R S S
Carbenicilline (100) Oxoid S S S S S S S
Cefoperazone (75) BBL S S S I I S S
Cefuroxime (30) BBL R R R I I R R
Nalidic acid(30) BBL I I I S S R S
Oflaxicin (5) Oxoid S S S S S S S
Oleandomycin (15) Oxoid S S S R R S S
Oxacillin (1) BBL S I I R R I S
Penicilline (5) Oxoid S I I S S I I
Rifampicin (30) Oxoid S S S S S S S
Streptomycin (10) Bioanalyse S S I S S I S
Tetracycline(30) Bioanalyse I S I S S I I
Vancomycin (3) BBL S S S I S S S

R: Resistance. S: Susceptible. I: Intermediate
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Title Annotation:Original Article
Author:Tarawneh, Khaled A.; Wedyan, Mohammed; Al-zou'bi, Mahmoud A.; Khleifat, Khaled M.; Tarawneh, Amjed
Publication:Advances in Environmental Biology
Article Type:Report
Date:May 1, 2008
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