International Symposium on and Their Applications 2005

Saltern crystallizer ponds as field laboratories for the study of extremely halophilic and

Aharon Oren* Department of Plant and Environmental Sciences, The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel *E-mail: [email protected]

Abstract The microbial community in salt-saturated crystallizer ponds of solar salterns is dominated by square red halophilic Archaea, Salinibacter (Bacteria), and the primary producer Dunaliella. Thanks to their often very high community densities, much information can be gained about these by analysis of samples collected directly from the field. This is illustrated by lipid and pigment studies in which specific compounds may serve as biomarkers, studies using inhibitors to differentiate between activities performed by the different groups, and studies in which utilization and metabolism of selected carbon sources is monitored to obtain information on the mode of life of the organisms in situ. These studies prove that saltern crystallizer ponds form perfect outdoor laboratories for the study of extremely halophilic microorganisms.

Keywords: Salterns; Halophilic Archaea; “Haloquadratum”; Salinibacter; Dunaliella; Lipids; carotenoids; Gas vesicles

1. Introduction

Our understanding of the world of halophilic microorganisms and the special adaptations that enable halophilic and halotolerant microorganisms to live at salt concentrations up to saturation is mostly based on the study of the microorganisms in pure culture. Many different halophilic prokaryotes and eukaryotes have been isolated from environments such as hypersaline lakes (the Dead Sea, Great Salt Lake, and others), saline soils, salted fish and other salty food products, and many of these organisms have been characterized in-depth [1]. Our understanding of the ecology of the halophilic microorganisms lags behind our knowledge on their physiology, biochemistry, and molecular biology. Field studies on the diversity, distribution, and physiology in situ in hypersaline ecosystems are relatively rare. Solar salterns, in which salt is produced by evaporation of seawater, are an especially attractive environment to study halophilic microorganisms and their adaptation to extremes of salinity. Such salterns generally consist of a series of ponds of increasing salt concentration. High densities of microorganisms develop in the brines and in the sediments of these ponds. Therefore it is not surprising that salt-saturated crystallizer ponds of solar salterns have always been favorite environments for the isolation of halophilic Archaea and other extremely halophilic and halotolerant microorganisms. These crystallizer ponds, in which halite precipitates from salt-saturated brines, are generally densely populated by halophilic microorganisms, and these impart a red color to the water (Fig. 1).

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Fig. 1 Two views of the crystallizer ponds of the salterns of the Israel Salt Industries Ltd., Eilat, Israel. Dense communities of red halophilic Archaea, dominated by flat square gas-vacuolate cells, as well as !-carotene-rich Dunaliella salina cells, impart a red-purple color to the brine.

Microscopic examination of saltern crystallizer brines (Fig. 2; see also [2]) shows the dominant organism to be extremely thin, flat, square gas-vacuolate red halophilic Archaea of the type first recognized by Walsby in 1980 in a coastal brine pool of on the Sinai Peninsula, Egypt [3]. This organism was only recently brought into culture, and the name “Haloquadratum walsbyi” was proposed for this intriguing microorganism [4,5]. A second major inhabitant of the crystallizer brines is Salinibacter, a recently characterized red pigmented extreme belonging to the domain Bacteria [6]. Development of these heterotrophs is supported by organic material supplied by the unicellular green eukaryotic alga Dunaliella salina, the sole primary producer in the crystallizer ponds. Dunaliella cells are red as well, due to their high content of !-carotene.

Fig. 2 The dominant microorganisms in the saltern crystallizer brines in Eilat: the unicellular green alga Dunaliella salina, colored red-orange due to its high content of !-carotene (A), and flat square Archaea of the type first described by Walsby [3] from a brine pool in the Sinai Peninsula, and now described as “Haloquadratum walsbyi” [4,5] (B,C). The cells in (B) had been concentrated by centrifugation, causing collapse of the gas vesicles which are visible in non-centrifugated cells only (C). The bars in part (A) and (B) represent 10 μm.

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Thanks to their often very high community densities, their simple community structure, and also in most cases their easy accessibility, the saltern crystallizer ponds form an ideal site to study the behavior of communities of halophilic microorganisms in their natural environment. As the examples presented below will show, much can be learned about the life of halophilic Archaea and Bacteria by the study of the saltern brines in situ or by the laboratory analysis of saltern brine samples. The information thus obtained nicely complements laboratory studies with pure cultures.

2. How much of the heterotrophic activity in the crystallizer ponds is due to the Archaea?

In our early studies we exploited the fundamental differences in properties of the halophilic Archaea as compared to the Bacteria to assess what part of the heterotrophic activity measured can be attributed to the bacterial component of the community, to the archaeal component, or to both. These studies were based on the use of specific inhibitors. Halobacterium and other non-coccoid members of the are lysed by low concentrations of bile acids (deoxycholate, taurocholate). When measuring amino acids incorporation in saltern ponds of different salinity in Eilat, Israel, we found only little inhibition by 50 mg/l taurocholate in ponds with salt concentrations below 250 g/l, but in the crystallizers inhibition was complete [7,8]. A similar observation was made in salterns in Spain [9]. Likewise, we exploited the difference in sensitivity to antibiotics between Archaea and Bacteria to differentiate between the groups. Protein synthesis in halophilic Archaea is effectively blocked by anisomycin; Bacteria are generally sensitive to chloramphenicol and erythromycin, and protein synthesis by the eukaryal ribosome is sensitive to cycloheximide as well as to anisomycin. In crystallizer ponds, incorporation of labeled amino acids was more than 95% inhibited by anisomycin [10]. Chloramphenicol and erythromycin also caused some inhibition [9,10]. Whether this was due to a limited sensitivity of the halophilic Archaea to these antibiotics or to the activity in situ of extremely halophilic Bacteria such as Salinibacter (see section 4) remains to be ascertained [8]. Aphidicolin, a potent inhibitor of halobacterial DNA polymerase, completely abolished incorporation of [methyl-3H]thymidine in the saltern crystallizer ponds in Eilat [11].

3. What types of halophilic Archaea are found in the crystallizers and what is their mode of life in situ?

3.1 Lipids as biomarkers to characterize the halophilic Archaea

Polar lipids are excellent biomarkers, and the types of archaeal phytanyl ether lipids present in the microbial community in the crystallizer ponds can be exploited to obtain information on the species composition. Especially valuable are the number and types of glycolipids (diglycosyl, triglycosyl and tetraglycosyl diether lipids, whether or not carrying sulfate groups on the sugar moieties), the presence or absence of the diether lipid derivative of phosphatidylglycerosulfate, and the presence or absence of sesterterpenyl chains replacing one of the phytanyl chains in certain species. The lipids can be characterized by thin-layer chromatography [12-15] or by mass spectrometry techniques such as electrospray ionization mass spectrometry [16] (see also section 4, Fig. 5).

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Long before the square Archaea dominating the crystallizer ecosystem were brought into culture, information was available on their polar lipid composition. Brines dominated by the square cells showed a simple lipid pattern, with the diphytanyl derivatives of phosphatidylglycerol (PG), phosphatidylglycerosulfate (Me-PGP), the methyl ester of phosphatidylglycerophosphate (PGS), and a single glycolipid, chromatographically identical - to S-DGD-1 (1-O-[!-D-mannose-(2-SO4 )-(1'"4')-!-D-glucose]-2,3-di-O-phytanyl-sn- glycerol) [12,13]. Electrospray ionization mass spectrometry also enabled the detection of archaeal cardiolipin derivatives in the biomass of the salterns of Margharita di Savoia (Italy) [16].

3.2 Conversion of organic substrates by the archaeal community in the crystallizers

Dunaliella cells accumulate molar concentrations of glycerol as osmotic solute. Therefore it may be assumed that glycerol is one of the most abundantly available substrates for the heterotrophic community in the crystallizer ponds. Glycerol is rapidly taken up by saltern crystallizer brines, and we measured short turnover times of a few hours only, for this compound in the Eilat salterns [17]. However, microautoradiography combined with fluorescence in situ hybridization suggested that the square Archaea in Spanish saltern ponds did not take up glycerol [18]. Glycerol is incompletely metabolized by the community: rather than incorporation into the cells and respiration to CO2 only, a significant part of the glycerol added, even at concentrations as low as 1.3-3 μM, was found to be converted into organic acids: acetate and D-lactate [19]. Formation of these acids from glycerol and sugars was earlier known from pure cultures of Haloferax, Haloarcula and Halorubrum species. In saltern brines the lactate formed from radiolabeled glycerol was degraded within a day after depletion of the substrate, but the amount of labeled acetate decreased only very slowly. Recently we have started to use poly-!-hydroxybutyric acid, produced as a storage polymer by the square Archaea [4], as a marker to follow the use of different substrates by the community. We thus showed that, in contrast to earlier reports [18], glycerol is taken up by these organisms (P. Khristo, R. Elevi Bardavid and A. Oren, unpublished results.

3.3 Are halocins important in the interspecies competition between different types of Archaea in the crystallizers?

Many halophilic Archaea of the family Halobacteriaceae have been documented to excrete halocins (halophilic bacteriocins) that inhibit the growth of other members of the family. Although it can be speculated that the excretion of such halocins may be advantageous when competing with related species, the true value of halocin production in the ecology of the halophilic Archaea has never been documented. As the saltern crystallizer pond are inhabited by very dense communities of halophilic Archaea – commonly between 107 and 108 cells/ml, the saltern brines are a perfect experimental system to test for the presence of halocins. We did not detect any halocin activity against a range of species of Halobacteriaceae in saltern brines from different locations in Israel, Spain, and the USA, not even after concentration of compounds of molecular mass >1000 or >5000 Da by ultrafiltration. These concentrates also did not significantly inhibit heterotrophic activity, as measured by uptake of radiolabeled glycerol or amino acids, in a variety of test strains. It was concluded that the contribution of halocins in the competition between different halobacteria in hypersaline aquatic environments is probably negligible [20].

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3.4 What advantage do the gas vesicles bestow on the square Archaea in the crystallizers?

Possession of gas vesicles is generally considered to be advantageous to halophilic Archaea: the vesicles are assumed to enable the cells to float, and thus reach high oxygen concentrations and high light intensities at the surface of the brine. This hypothesis was, however, never yet supported by evidence from field studies: there are no reports in the literature confirming that indeed such cells do float toward the surface in their natural environments. We have therefore initiated studies to assess the ecological advantage of the gas vesicles of the square Archaea [3-5, see also Fig. 2C], so abundant in the Eilat saltern crystallizer ponds. During static incubation in the laboratory, no flotation of the square cells toward the brine surface was observed. Moreover, we performed “accelerated flotation” experiments in which brine samples were centrifuged at speeds insufficient to cause collapse of the gas vesicles. After 12 h of centrifugation at an acceleration of 26 x g at the bottom of the tube, equivalent to incubation for 13 days at normal gravity, the cells were still homogeneously distributed throughout the tubes. However, when the gas vesicles had first been collapsed by pressurization, they slowly sank in the tubes (Fig. 3) (A. Oren, N. Pri-El, O. Shapiro, and N. Siboni, unpublished results). Thus, the gas vesicles appear to bestow neutral rather than positive buoyancy to the cells. The assumption that the gas vesicles enable the square Archaea to float to the surface of the brines in which they live, resulting in an ecological advantage, was therefore not supported by experimental evidence, and the true function of the gas vesicles remains enigmatic.

Fig. 3 Vertical distribution of prokaryotes from an Eilat crystallizer brine sample (upper panel) and an identical sample in which the gas vesicles had been collapsed by pressurization (lower panel), following centrifugation for 12 h in 10 ml portions at 500 rpm in a SS-34 rotor (acceleration at the bottom of the tube: 26 x g).

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4. How abundant is Salinibacter in the crystallizer ponds?

The discovery of Salinibacter ruber, a novel type of extremely halophilic prokaryotes affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, and first isolated from Spanish saltern ponds [6,8], raises the question to what extent this organism contributes to the biomass and activity of the heterotrophic community in the salterns. Salinibacter can easily be isolated from crystallizer brine samples, and a selective isolation and enumeration procedure has been developed, based on colony counts on selective media [21]. Furthermore, fluorescence in situ hybridization has been successfully applied to the quantification of the contribution of Salinibacter in salterns in Spain [22]. Two specific molecules have been identified in Salinibacter that can conveniently be used as biomarkers to monitor its presence in hypersaline environments: a unique carotenoid pigment and a novel type of sulfonolipid. The red color of Salinibacter is caused by a single red pigment with an absorption maximum at 478 nm and a shoulder at about 510 nm. Its structure has been elucidated as a C40-carotenoid glycoside, esterified with a fatty acid (C15:0 iso), and the pigment was named salinixanthin [23] (Fig. 4a). It can easily be separated from the archaeal C50 bacterioruberin carotenoids by HPLC, and this enables the separate quantification of the carotenoids of the extremely halophilic Archaea and Bacteria. It was thus established that approximately 5% of the total prokaryotic pigments extracted from a crystallizer pond near Alicante could be attributed to Salinibacter [24] (Fig. 4b).

Fig. 4 The structure of salinixanthin, the red carotenoid of Salinibacter ruber (A), and HPLC separation and characterization of carotenoids extracted from the saltern crystallizers of Santa Pola, Alicante, Spain in March 2000 (B). The upper left panel of part B presents the elution pattern as measured at 450 nm. Fractions I, II, III and IV are bacterioruberin derivatives originating from halophilic Archaea; fraction VI can be identified as salinixanthin on the basis of its retention time and its absorption spectrum, and fraction XI is !-carotene from the green alga Dunaliella. Modified from [22] (A) and [23] (B).

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The second biomarker is a unique sulfonolipid that represents about 10% of the cellular lipids of Salinibacter. Its structure has recently been elucidated as 2-carboxy-2-amino-3-O- (13’-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid [25] (Fig. 5A). Electrospray ionization mass spectrometry analysis of a lipid extract of the biomass collected from the saltern crystallizer ponds of Margherita di Savoia, Italy, showed presence of a peak at m/z = 660.4 [16] (Fig. 5B), that can be attributed to the presence of the Salinibacter sulfonolipid.

Fig. 5 The structure of the novel sulfonolipid of Salinibacter ruber (A), and electrospray ionization mass spectrometry analysis (negative ion) of a total lipid extract of the biomass collected from the saltern crystallizer ponds of Margherita di Savoia, Italy (B), showing evidence for the presence of the Salinibacter sulfonolipid in these salterns (peak at m/z = 660.4). Most of the remaining peaks can be attributed to phospholipids and glycolipids of halophilic Archaea. The peak at m/z = 805.6 is archaeal PG, 885.6 = PGS, 899.7 = Me-PGP, and 1055.9 = S-DGD-1 (see also section 3.1). Modified from [24] (A) and [16] (B).

3. Conclusion

The crystallizer ponds of solar salterns worldwide provide the microbiologist with an ideal natural laboratory for microbial ecology studies at the extremes of salinity. Community diversity is low, being restricted to halophilic Archaea of the family Halobacteriaceae, accompanied by halophilic Bacteria of the genus Salinibacter and the unicellular eukaryotic alga Dunaliella as primary producer. Due to the extremely high densities at which these microorganisms occur in the system, even small samples provide sufficient biomass for laboratory analyses. In addition, salterns are generally easily accessible. These properties, as illustrated by the examples provided above, make the saltern crystallizers an ideal environment to study many different aspects of hypersaline microbial ecology and of the adaptation of microorganisms to life at high salt concentrations.

4. Acknowledgment

My current work on the salterns, with special emphasis on the function of Salinibacter in the saltern ecosystem, is supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (grant 504/03).

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5. References

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