Water bodies with NaCl concentrations approaching saturation are often populated by dense microbial communities. Red halophilic Archaea of the family Halobacteriaceae dominate in such environments. The application of molecular biological techniques, in particular the use of approaches based on the characterization of ribosomal RNA sequences, has greatly contributed to our understanding of the community structure of halophilic Archaea in hypersaline ecosystems. Analyses of lipids extracted from the environment have also provided useful information. This article reviews our present understanding of the community structure of halophilic Archaea in saltern crystallizer ponds, in the Dead Sea, in African hypersaline soda lakes, and in other hypersaline water bodies. It was recently shown that red heterotrophic Bacteria of the genus Salinibacter, which are no less salt-dependent and salt-tolerant than the most halophilic among the Archaea, may coexist with the halophilic archaeal community. Our latest insights into their distribution in hypersaline ecosystems are presented as well.
Environments with NaCl concentrations approaching saturation are often populated by dense microbial communities. As a result of the lack of predation and the often high nutrient levels, densities of 107–108 cells ml−1 and higher are not unusual. Many halophilic microorganisms have a high content of carotenoid pigments, and as a result the waters of the Great Salt Lake (Utah), crystallizer ponds of solar salterns, and hypersaline soda lakes such as Lake Magadi (Kenya) are often bright red. Red waters are even sometimes found in the Dead Sea.
Red halophilic Archaea of the family Halobacteriaceae  dominate in these environments. At the time of writing (August 2001), the family Halobacteriaceae consisted of 15 genera with 40 species. Most are pigmented red due to a high content of C-50 carotenoid pigments (α-bacterioruberin and derivatives) in their membrane, in some cases accompanied by the purple retinal pigment bacteriorhodopsin.
Most ecological studies in the past have been restricted to isolation and characterization of microorganisms from the environment. This approach has yielded valuable information on the biodiversity present . However, the percentage of the microorganisms recovered as colonies on agar plates is generally low, and therefore this type of data provides little information on the true community structure.
The application of molecular biological techniques to microbial ecology, in particular the use of approaches based on the characterization of ribosomal RNA sequences, has shown that the cultured organisms are generally different from those that dominate in the natural environment. In addition, we know little about the growth rates of halophilic Archaea in situ and about the factors that lead to their death.
This review intends to provide an overview of the experimental approaches used in recent years to increase our understanding of the ecology of halophilic Archaea in ecosystems with salt concentrations approaching saturation. Most of these investigations have been performed in saltern crystallizer ponds, and therefore the insights obtained from these studies are presented first. Then follows a discussion of a number of additional hypersaline environments in which molecular techniques have been applied to obtain information on the nature of the halophilic archaeal communities.
It was recently discovered that red heterotrophic Bacteria, no less salt-dependent and salt-tolerant than the most halophilic among the Archaea, may coexist with the halophilic archaeal community . Information about their distribution in hypersaline ecosystems is presented as well.
Ecology of extremely halophilic Archaea
Saltern crystallizer ponds
Multi-pond solar salterns present a gradient of salinities, from seawater salinity to halite saturation. The salt concentration in each pond is kept relatively constant, and microbial community densities are generally high. Although salterns are superficially similar all over the world, they do differ with respect to nutrient status and retention time of the water, depending on climatic conditions .
Many species of halophilic Archaea have been isolated from crystallizer ponds, the NaCl-saturated ponds in which halite is deposited. Isolates include the type strains of Haloferax mediterranei, Haloferax gibbonsii, Haloferax denitrificans, Halogeometricum borinquense, Halococcus saccharolyticus, Haloterrigena thermotolerans, Halorubrum saccharovorum, Halorubrum coriense, Haloarcula hispanica and Haloarcula japonica. Halobacterium salinarum could be grown from brine samples from crystallizer ponds in Eilat, Israel and Newark, CA, USA in anaerobic enrichment cultures in the presence of l-arginine . From saltern ponds near Alicante, Spain, species of Haloarcula, Haloferax, Halorubrum and Halobacterium have been recovered at a high frequency [6,7]. The significance of such results is limited as the number of colonies obtained is only a small fraction of the total numbers of prokaryotes present.
Microscopic examination of saltern crystallizer brines worldwide generally shows that flat, square or rectangular, gas-vacuolated cells dominate in the community [8–10] (Fig. 1). Such square halophiles were first described by Walsby  from a brine pool on the coast of the Sinai peninsula, Egypt. They were reported to contain bacteriorhodopsin . Unfortunately, this type of square flat gas-vacuolated cells has not yet been brought into culture.
Molecular, culture-independent rRNA-based studies have been performed to characterize the archaeal communities in saltern crystallizer ponds. These studies indicated that neither of the species recovered on agar plates represents a major part of the archaeal community in those ponds. In a study of Spanish salterns, 5S rRNA was extracted from the microbial assemblages without prior amplification, and electrophoretically compared with 5S rRNAs from cultured halophilic Archaea. The crystallizer ponds yielded two bands, neither of which matched with that of any of the cultured halophilic Archaea . Another approach used was based on comparison of restriction digests of 16S rDNA amplified from the DNA extracted from the biomass. Restriction fragment length polymorphism was determined by amplification with Archaea- or Bacteria-specific primers, followed by digestion with AluI, HinfI and MboI. Bacterial diversity was found to decrease with salinity, while archaeal diversity increased .
More detailed information on the nature of the Archaea present in crystallizer ponds was obtained by sequencing 16S rDNA genes amplified from DNA isolated from the biomass. Most of these studies were performed in the Santa Pola salterns near Alicante, Spain. From the crystallizer ponds, one archaeal phylotype was recovered almost exclusively. It is only distantly related to Haloferax, its closest cultured relative [7,15–17]. The same phylotype also dominated the archaeal community in the crystallizers of the Eilat salterns . Similar techniques have been employed in a study of the microbial diversity in the microbial mats covering the sediments of saltern ponds on the Mediterranean coast of France (Salin-de-Giraud). Two concentrator ponds were sampled, one with a salinity that fluctuated between 90 and 158 g l−1, and a second in which the salt concentration varied between 164 and 228 g l−1 (i.e. salinities much below those of the crystallizer ponds). Fourteen and 23 sequences of Halobacteriaceae from the respective sites were characterized. These sequences were very diverse, and spread all over the phylogenetic tree of the family .
The technique of fluorescent in situ hybridization (FISH) now enables a direct characterization of the archaeal communities in saltern ponds, while exploiting 16S rRNA sequence information derived from cultured halophiles or from environmental samples. A probe designed to react with the dominant phylotype obtained from the Alicante crystallizers reacted with the yet uncultured, flat, square, gas-vacuolated cells, again confirming their abundance in the ecosystem . No cultures of this organism exist as yet; more or less square Archaea obtained in culture such as the motile strain 801030/1 isolated from the Sinai brine pool  and described as Haloarcula quadrata are phylogenetically unrelated.
Polar lipids are excellent biomarkers that can be exploited to obtain information on the nature of the microbial communities inhabiting hypersaline environments. The lipids of the halophilic Archaea are easy to differentiate from bacterial or eukaryal lipids. The structure of the archaeal lipids is based on phytanyl groups bound by ether linkages to the glycerol backbone, with various types of substituents on the third carbon of the glycerol. A considerable diversity in polar lipid structure exists among the genera and species of the Halobacteriaceae . As polar lipids can easily be characterized by thin-layer chromatography, they can conveniently be used to characterize not only pure cultures of halophilic Archaea, but natural communities as well.
In saltern crystallizer ponds, lipid patterns were found to be relatively simple and to be quite similar in different geographic locations. Four main polar lipid fractions were detected in the Eilat salterns: the diphytanyl derivatives of phosphatidylglycerol (PG), and of the methyl ester of phosphatidylglycerophosphate (Me-PGP), the diphytanyl derivative of phosphatidylglycerosulfate (PGS), and a single glycolipid, chromatographically identical to S-DGD-1 (1-O-[α-d-mannose-(2-SO−4)-(1′→4′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol) . As this lipid pattern was found in a community dominated by square, flat, gas-vacuolated cells, it may be assumed that this is also the polar lipid composition of this organism . Lipid extracts of biomass collected from crystallizers of the more nutrient-enriched Newark, CA salterns showed a higher complexity than the oligotrophic salterns of Eilat .
[methyl-3H]Thymidine was used to estimate the growth rates of prokaryote communities in the saltern of Eilat . Calculated doubling times of the heterotrophic community in the crystallizer ponds were between 6 and 22.6 days. Similar values were reported in a study of saltern ponds in Spain, with estimated doubling times between 2.5 and 5 days at salinities between 250 and 372 g l−1. Incorporation of [3H]leucine has also been used to assess the growth rate of Bacteria and Archaea in Spanish salterns. Doubling times thus estimated in high-salinity ponds (250–380 g l−1) were generally between 2 and 5 days, but occasionally as long as 70 days .
We still know little about the factors responsible for the death of halophilic Archaea in their natural environment. Protozoa have never been encountered in large numbers, if at all, in saltern crystallizer ponds. Death by lysis due to bacteriophages, however, may occur, as appeared from studies in Spanish salterns [8,25]. Numbers of presumed viruses as high as 109 ml−1 were observed in NaCl-saturated brines examined in the electron microscope. Between 1% and 10% of the flat square Archaea had visible phages inside; the estimated burst size was more than 200 viruses per cell. However, calculations showed that viruses did not exert a strong control over the prokaryotic abundance and growth rate: at the highest salinities the percentage of cells lost daily by viral lysis was calculated to be lower than 5%.
Attempts have been made to assess whether halophilic archaeocins (halocins) excreted by halophilic Archaea may inhibit the growth of other archaeal species in the saltern crystallizers, and thus regulate the archaeal community size and composition in these ponds. No indication was obtained that halocins are an important ecological factor in this ecosystem .
The Dead Sea
The Dead Sea presents unique challenges to the halophilic microorganisms inhabiting it because of its peculiar ionic composition. Divalent cations dominate over monovalent cations (presently about 1.9 M Mg2+ and 0.4 M Ca2+, with in addition 1.7 M Na+ and 0.2 M K+). The pH is relatively low (about 6.0). Microbial blooms occur in the lake only after winter rain floods cause the formation of a diluted upper water layer. A dilution of 10–20% is required to trigger the development of a bloom of the unicellular green alga Dunaliella, accompanied by large numbers of halophilic Archaea. Thus, a prokaryotic community of up to 1.9×107 cells ml−1 was observed in 1980, and even higher numbers (3.5×107 ml−1) were measured in 1992. In both those years the lake was colored red due to the archaeal carotenoids. At other times algae are absent from the water column, and hardly any prokaryotic microorganisms can be detected .
Bacteriorhodopsin may also be present in the Dead Sea biota. Biomass collected from the lake in 1981 had the characteristic purple color of bacteriorhodopsin. The report of its abundance in the community (up to 0.6 nmol l−1 or 0.4 nmol mg protein−1)  was the first account of the occurrence of this pigment in any natural community of halophilic Archaea. For comparison, Javor  found 2.2 nmol l−1 bacteriorhodopsin in the archaeal community of an oligotrophic saltern crystallizer pond in Baja California (Mexico), while in a more eutrophic saltern in California the retinal pigment could not be detected. At least one Dead Sea isolate, Halorubrum sodomense, can synthesize purple membrane. In the autumn of 1981, a time at which halophilic Archaea were still abundant but very few Dunaliella cells were found, the low level of light-dependent CO2 fixation was probably driven by bacteriorhodopsin rather than by chlorophyll. Evidence for this was obtained from the action spectrum of the process and by the use of specific inhibitors . Different mechanisms have been suggested to explain the nature of the bacteriorhodopsin-driven CO2 photoassimilation, such as carboxylation of propionyl-CoA to yield α-ketobutyrate or reactions leading to the biosynthesis of glycine.
Studies with specific inhibitors such as bile acids and antibiotics affecting protein synthesis (see also Section 3.1) showed that in 1988 (a period in which only low numbers of microorganisms were present in the lake's water column) all heterotrophic activity could be attributed to halophilic Archaea [30,31].
A variety of archaeal halophiles have been isolated from the Dead Sea, including Haloferax volcanii, Haloarcula marismortui, Hrr. sodomense, and Halobaculum gomorrense. Little is known about the importance of these and possibly other, as yet uncultured species. Analyses of polar lipids extracted from the community that formed the bloom in 1992 showed a simple pattern. Only three polar lipid fractions were detected: PG, Me-PGP, and a single glycolipid, chromatographically identical to S-DGD-1; PGS was absent . Such a lipid composition is characteristic of the genus Haloferax and also of Hbl. gomorrense, a species isolated from the bloom.
We know little about the factors responsible for the decline in bacterial numbers following the occasional massive blooms. The finding of large numbers of virus-like particles in the lake  suggests that bacteriophages may be involved in controlling the community size of prokaryotes in the Dead Sea.
Solar Lake is a small lake on the Sinai peninsula (Egypt) on the shore of the Gulf of Aqaba. In summer the water column of the pond is hypersaline (about 200 g l−1) and aerobic down to the bottom (maximum depth 4.5–5 m). In winter the lake is stratified, with a layer of less saline (about 60 g l−1) water floating on top of the heavier bottom waters (180–200 g l−1). The hypolimnion rapidly turns anaerobic, and heats up to temperatures of 55–60°C and higher due to heliothermal heating.
The archaeal biodiversity of the water column of Solar Lake was recently studied throughout the annual cycle. Archaeal 16S rDNA was amplified from the biomass, separated by denaturing gradient gel electrophoresis, and sequenced. Archaea were abundantly detected in the water column, both during summer mixing and during winter stratification, including in the hot anaerobic, sulfide-rich hypolimnion . Of the 165 archaeal clones analyzed, 144 belonged to the Halobacteriaceae, including 92 out of the 104 clones obtained from the anaerobic layer during stratification. Two clusters of clones of Halobacteriaceae sequences recovered shared 94% sequence identity. Their closest cultivated relative is Haloferax (89% identity), but an even closer relationship was found with the phylotype most abundant in saltern crystallizers [15–17], now assigned to the gas-vacuolated flat square Archaea . Representatives of this cluster were found both in the aerobic and in the anaerobic parts of the water column, and at temperatures ranging from 15 to 55°C .
Antarctic hypersaline lakes
The hypersaline lakes of the Vestfold Hills lake system of Eastern Antarctica have been the subject of a number of studies on microbial distribution. Recently, 16S rDNA sequencing techniques have been included in these studies. One of these lakes is Deep Lake, a monomictic, 36 m deep lake with a salinity of 320 g l−1 and temperatures between −14 and −18°C. The biodiversity in Deep Lake is low, and is dominated by Archaea of the family Halobacteriaceae. The predominant phylotype was closely related to Halorubrum lacusprofundi, a cold-tolerant halophilic Archaeon originally isolated from this lake. Furthermore, three deep-branching clusters of novel types of Archaea were detected . Analysis of polar lipids recovered from the lake's sediments yielded a lipid fingerprint very similar to that of Hrr. lacusprofundi.
African alkaline hypersaline lakes
Lake Magadi (Kenya), an alkaline lake in the East African Rift Valley, is salt-saturated, and contains a precipitate of trona (sodium sesquicarbonate). The pH of the brine is about 10. Alkaliphilic members of the Halobacteriaceae dominate the microbial community.
A number of halophilic alkaliphilic Archaea have been isolated from the lake and characterized (Natronobacterium gregoryi, Natrialba magadii, Halorubrum vacuolatum, Natronococcus occultus). The archaeal community populating the crystallizer ponds of the alkaline (pH about 12) solar salterns on the shore of the lake has recently been characterized by 16S rDNA sequencing of clones obtained after PCR amplification from DNA extracted from the biomass [37,38]. Most sequences recovered shared more than 95% identity to each other, but only 88–90% to Natronomonas pharaonis, their closest relative among the cultivated haloalkaliphilic Archaea. Two other clones retrieved were only 76% similar to any known archaeal sequence, showing that also in this extreme environment the microorganisms that dominate the community are still awaiting isolation.
Ecology of extremely halophilic Bacteria
Activity of halophilic Bacteria at the highest salt concentrations
Until recently it was assumed that at salt concentrations at or near NaCl saturation, Archaea of the family Halobacteriaceae are the only active aerobic heterotrophs. This assumption was based in part on culture experiments: red colonies of organisms that did not grow below 100–150 g l−1 salt were generally the only colony type recovered. Secondly, activity measurements in the presence of inhibitors specific for either archaeal or bacterial activities suggested that above about 250 g l−1 salt essentially all heterotrophic activity could be attributed to Archaea.
Already in 1956 it was proposed that bile acids (Bacto-Oxgall) may be a useful agent to differentiate between red archaeal halophiles (not yet recognized as such at the time) and other types of halophilic microorganisms . The ability of bile acids (deoxycholate, taurocholate) at low concentrations to lyse halophilic Archaea was exploited in studies of the uptake of radioactively labeled amino acids in saltern brines in Eilat. Above 300 g l−1 salt, 50 mg l−1 taurocholate caused complete inhibition . Similarly, in Spanish salterns of the Ebro delta and near Alicante, taurocholate completely inhibited incorporation of [3H]leucine at the highest salinities, while below 200 g/l relatively little inhibition was observed .
Antibiotics specifically affecting protein synthesis in Archaea or in Bacteria have also been employed in ecological studies. Specially useful are anisomycin (inhibiting Archaea and Eucarya) and chloramphenicol or erythromycin, inhibiting the bacterial protein synthesis machinery. Incorporation of labeled amino acids by samples collected from crystallizer ponds in Eilat was inhibited more than 95% by anisomycin [30,31]. Erythromycin has been employed in similar experiments in saltern ponds in Spain. While as expected [3H]leucine incorporation was fully inhibited by erythromycin in the lower salt concentration range, only half of the activity was inhibited in the Archaea-dominated crystallizer ponds . In the Eilat salterns most of the amino acid uptake at the highest salinities was resistant to chloramphenicol [30,31].
Also DNA synthesis can be targeted by specific antibiotics. Aphidicolin, a potent inhibitor of halobacterial DNA polymerase, completely abolished incorporation of [methyl-3H]thymidine in saltern crystallizer ponds in Eilat .
Salinibacter ruber, an extremely halophilic Bacterium
In spite of the above, it is now becoming clear that Bacteria may also contribute to the aerobic heterotrophic prokaryotic community at the highest salt concentrations. PCR amplification of 16S rDNA from biomass collected from saltern crystallizers in Spain, using Bacteria-specific primers, yielded sequences distantly related to Rhodothermus marinus (Cytophaga/Flavobacterium/Bacteroides phylum) . Similar sequences have also been recovered from salterns in the south of France . Using fluorescent oligonucleotide probes designed to detect this phylotype, the organism was shown to be rod-shaped (Fig. 1), and to be very abundant: in the crystallizer ponds on Ibiza and Mallorca between 18 and 27% of all prokaryotes belonged to this type, in crystallizers on the Canary Islands they were less abundant (5–8%) . It may be noted that the finding of a specific type of Bacteria adapted to life at the highest salt concentrations was already predicted by an earlier study of restriction fragment length polymorphism of 16S rDNA amplified from the Spanish salterns using Bacteria-specific primers . Bacterial restriction fragment patterns were obtained from the crystallizer ponds, and these were very different from those retrieved from lower salinity ponds. The statement by Martinez-Murcia et al. that the crystallizer environment ‘probably represents an extremely specialized niche for Bacteria’ predates the isolation of such bacteria by 5 years.
The organism harboring this novel phylotype has recently been isolated and described as a new genus and species: S. ruber. Recognition of bacterial colonies was based either on their polar lipid pattern or on hybridization with a specific fluorescent 16S rRNA-targeted probe . Salinibacter is a motile rod, pigmented red by a pigment (probably a carotenoid) with an absorption maximum at 482 nm and a shoulder at 506–510 nm. Because of the red pigmentation of the colonies this type of Bacterium has probably been overlooked in the past, red colonies growing at 250 g l−1 salt having been considered to be archaeal. The organism is no less halophilic than the archaeal halophiles: no growth was obtained below 100 g l−1 NaCl, and for optimal growth concentrations between 150 and 230 g l−1 are required. The physiological properties of Salinibacter are unusual: the organism apparently uses KCl to provide osmotic balance, while lacking high concentrations of organic osmotic solutes . Thus, its physiology resembles that of the halophilic Archaea more than that of other aerobes within the domain Bacteria.
HPLC analysis of pigments extracted from Spanish saltern crystallizer ponds enabled the identification and quantitative assessment of the red pigment of Salinibacter. Approximately 5% of the total prokaryotic pigment absorbance could be attributed to the presence of Salinibacter. The pigment was not detected in samples collected from crystallizers of Eilat, and possibly traces of it were found in the salterns in San Francisco Bay near Newark, CA .
The above survey shows that our understanding of the community structure of aerobic halophilic microorganisms in hypersaline lakes is still limited. Application of rRNA-based characterization techniques has demonstrated that those microorganisms known in culture are not dominant in the natural environment, and that most of the ecologically important organisms are still awaiting isolation. Hbt. salinarum, the type strain and the best-known representative of the family Halobacteriaceae, is not a quantitatively important component of the microbial community in any hypersaline lake investigated, although it can be isolated from such lakes by means of a selective enrichment procedure . Similarly, probably none of the other halophilic Archaea brought in culture can be claimed to be a dominant component of the natural community in the lake or saltern from which it was isolated, Hrr. lacusprofundi from Deep Lake, Antarctica, being a possible exception .
The recent isolation of the extremely halophilic red Bacterium S. ruber from saltern crystallizers  presents us with a rare example of the isolation of an organism whose abundance and ecological relevance had been previously recognized on account of its 16S rRNA sequence . It is to be hoped that other ecologically relevant organisms, known thus far only from their 16S rRNA sequences, will soon be isolated and characterized. At present, microbiologists still dream of growing and characterizing the square, flat, halophilic Archaeon, first described in 1980  and now known to be the dominant archaeal form in saltern crystallizers [15–17,19], and possibly in other hypersaline environments as well .
Hypersaline environments can be expected to have a relatively simple ecosystem structure as the number and metabolic diversity of the known microorganisms adapted to life at high salt concentrations is rather limited. Salt lakes and other ecosystems with salt concentrations at or approaching saturation are therefore convenient model systems for studies in microbial ecology. However, the data that have accumulated in recent years, especially thanks to the introduction of molecular biological methods, have provided ample evidence that even such ‘simple’ ecosystems are considerably more complex than had been assumed before. As yet uncultured species dominate in hypersaline ecosystems. In this respect is our present level of understanding of these ecosystem not greatly different from our understanding of more conventional marine and terrestrial ecosystems. However, as the case of the discovery of Salinibacter shows, breakthroughs are possible, so that it still may be relatively easy to obtain a proper understanding of the microbial ecology of hypersaline environments in the near future.