Polar lipids and pigments as biomarkers for the study of the microbial community structure of solar salterns

Whole community lipids and pigments have been examined over a 3–5-year period in commercial salterns located in the United States, Israel, and Spain. There were significant differences in the types of lipids and pigments within the California saltern system during the 5-year period. These patterns differed seasonally despite examination of ponds with approximately the same salinities. The solar saltern in Eilat, Israel had fewer lipids on the thin-layer chromatography plates and confirmed previous analyses. The biota in the crystallizer pond in Alicante, Spain, resembled the microbial community in Israel. In the crystallizers at all three locations, phosphatidyl glycerol, methyl-phosphatidyl glycerophosphate, phosphatidyl glycerosulfate, and the sulfated diglycosyldiether lipid were identified regardless of season. This was not true for pans with salinities below 25% where no distinctive pattern was observed. Thus, we hypothesize that the more eutrophic inlet waters of the California saltern and the cooler temperatures, which result in longer retention times of water in the different pans, allow for the more diverse microbial community to develop. This is reflected then in more complex lipid and pigment patterns. However, the oligotrophic inlet waters to the Eilat saltern coupled with a drier and warmer climate result in a shorter retention time of water in the pans and a less diverse microbial community as evidenced by fewer extractable lipids and pigments.     

Comparative metabolic diversity in two solar salterns

The purpose of this study was to compare the metabolic diversity of the whole microbial community in an oligotrophic saltern (Eilat, Israel) and in a saltern with a more enriched source water (Newark, California). Between 1993 and 1998 water samples were taken from selected locations within the solar salterns of the Cargill Solar Salt Plant, Newark, California, and the Israel Salt Co. in Eilat, Israel. To examine the whole community metabolic diversity, we used the 96-well BIOLOG GN^{{ TM}} plates which contain 95 different carbon sources and a control well. Plates from samples containing greater than 15% salt were excluded from the final analyses because of a lack of reproducibility when multiple plates were inoculated with the same sample. The data were analyzed by simple matching coefficient and principal component analysis. Both methods gave similar results. Two major clusters were formed. These could be subdivided into 10 sub-clusters with only three samples from the Newark saltern in December 1997 joining at the 95% similarity level. Most of the inlet and lower salinity samples from the Cargill samples comprised one large subcluster. Several carbon sources were used by 85% of the microbial community from the California samples, while 85% of the Eilat samples had no commonly used carbon sources. These results suggest that ponds in different geographic locations may have communities with different microbial populations despite the similarities in salt content and processing for salt production.     

Microbial diversity and complexity in hypersaline environments: A preliminary assessment

The microbial communities in solar salterns and a soda lake have been characterized using two techniques: BIOLOG, to estimate the metabolic potential, and amplicon length heterogeneity analysis, to estimate the molecular diversity of these communities. Both techniques demonstrated that the halophilic Bacteria and halophilic Archaea populations in the Eilat, Israel saltern are dynamic communities with extensive metabolic potentials and changing community structures. Halophilic Bacteria were detected in Mono Lake and the lower salinity ponds at the Shark Bay saltern in Western Australia, except when the crystallizer samples were stressed by exposure to Acid Green Dye #9899. At Shark Bay, halophilic Archaea were found only in the crystallizer samples. These data confirm both the metabolic diversity and the phylogenetic complexity of the microbial communities and assert the need to develop more versatile media for the cultivation of the diversity of bacteria in hypersaline environments. Journal of Industrial Microbiology & Biotechnology (2002) 28, 48–55 DOI: 10.1038/sj/jim/7000175 Received 20 May 2001/ Accepted in revised form 15 June 2001     

A procedure for the enrichment and isolation of Halobacterium

A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of l-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community.     

Extremely halophilic Archaea from Tuz Lake,Turkey, and the adjacent Kaldirim and Kayacik salterns

Tuz Lake is a hypersaline lake located in Central Anatolia, Turkey. The lake and its salterns, Kaldirim and Kayacik, are the major sources of solar salt for industrial applications in Turkey, especially in the food and leather industries. Use of the crude solar salt often results in microbial deterioration of the products. We therefore initiated a thorough characterization of the microbial communities in Tuz Lake and its adjacent salterns, and we present here the results of investigations on diversity of extremely halophilic Archaea. Twenty-seven colonies of aerobic red or pink Archaea (family Halobacteriaceae) were selected according to colony shape, size, consistency and pigmentation, and characterized according to their phenotypic characteristics, polar lipid contents, and antibiotic sensitivities. Furthermore, 16S rRNA genes of the isolates were screened by DGGE analysis and partially sequenced. Phylogenetic analysis showed that most isolates belonged to the genera Haloarcula, Halorubrum and Halobacterium. Haloarcula was found to be dominant both in Tuz Lake and in the saltern samples. Halorubrum species were isolated from Tuz Lake and from the Kaldirim saltern, and Halobacterium species were recovered from Tuz Lake and from the Kayacik saltern. All strains showed various activities of hydrolytic enzymes (proteases, amylases, cellulases, and others), activities which are responsible for the detrimental effects of the crude salt in food and leather products.     

Characterization of the endoevaporitic microbial communities in a hypersaline gypsum crust by fatty acid analysis

We have used fatty acid analyses to study the community structure of a layered endoevaporitic microbial community within a gypsum crust that covers the bottom of a saltern evaporation pond in Eilat, Israel. This community, living at a salinity of 218–238 g l⁻¹ total dissolved salts, consists of an upper brown layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, a red-purple layer with both Chromatium and Ectothiorhodospira/Halorhodospira type of purple sulfur bacteria, and a black layer in which dissimilatory sulfate reduction occurs. An olive-green layer is sometimes present below the red-purple layer. Analysis by gas chromatography/mass spectrometry of the fatty acid methyl esters prepared from the different fractions showed characteristic patterns in each layer, and these could be related to fatty acid composition data from the literature and to fatty acid analyses of representative halophilic microorganisms isolated from the site. The nature of the fatty acids in the green layer suggests that the cyanobacteria present there use the oxygen-independent pathway for production of unsaturated fatty acids, a pathway only occasionally encountered in filamentous cyanobacteria. The facultative anaerobic nature of the cyanobacteria in the green layer was confirmed by their ability to perform anoxygenic photosynthesis with sulfide as electron donor. Specific signature fatty acids identified for each layer corresponded well with the microscopic and functional analysis of the biota present. Guest Editor: John M. Melack Saline Waters and their Biota     

Microbial communities and processes within a hypersaline gypsum crust in a saltern evaporation pond (Eilat,Israel)

Gypsum crusts containing multicolored, stratified microbial communities develop in the evaporation ponds of a commercial saltern in Eilat, Israel at salt concentrations between 190 and 240 g l⁻¹. The upper 0.5–2 cm of the crust is densely populated by orange-brown unicellular cyanobacteria. Below, a layer of green-colored filamentous cyanobacteria is found. Underneath, a bright purple layer of anoxygenic phototrophs is present, below which a reduced black layer is found. We have investigated the biological properties of this crust using a wide variety of techniques, and we here review the results of these interdisciplinary studies. The tests performed included microscopic examination of the biota, phylogenetic analyses based on 16S rRNA gene clone libraries and denaturing gradient gel electrophoresis, fatty acid analysis, light intensity and light quality measurements, microelectrode studies of oxygen profiles and oxygen evolution, determination of sulfate reduction using radioisotope methods, and measurement of methane evolution. The stable vertical stratification in the system enabled separate analyses of the different layers with a high spatial resolution. It was therefore possible to combine the different approaches and obtain information on the activities of the different types of oxygenic and anoxygenic phototrophs, dissimilatory sulfate reducers and methanogens in the different layers, as well as phylogenetic information on the nature of the microorganisms responsible for these processes. The gypsum crust thus becomes a paradigm for the study of a wide variety of microbial processes and their interrelationships in the presence of high salt concentrations. Guest Editors: J. John & B. Timms Salt Lake Research: Biodiversity and Conservation—Selected papers from the 9th Conference of the International Society for Salt Lake Research     

Structure of euhalothece-362, a novel red-shifted mycosporine-like amino acid, from a halophilic cyanobacterium (Euhalothece sp.)

The unicellular cyanobacterium Euhalothece sp. strain LK-1, isolated from a gypsum crust on the bottom of a hypersaline saltern pond in Eilat, Israel, contains high concentrations of two mycosporine-like amino acids with maximum absorbance at 331 and 362 nm when grown at high light intensities. The 331 nm-absorbing compound has previously been identified as mycosporine-2-glycine. Here, we confirm this identification and document the elucidation of the structure of the 362 nm absorbing compound ('euhalothece-362'), using liquid chromatography/mass spectrometry combined with other techniques, as a novel compound, 2-(E)-3-(E)-2,3-dihydroxyprop-1-enylimino-mycosporine-alanine.     

Breeding biology of the Madagascar Paradise Flycatcher,Terpsiphone mutata,with special reference to plumage variation in males

This study was undertaken to understand the migratory strategies of the Dunlins (Calidris alpina) caught in Eilat, Israel, before and after they accomplish the crossing of the combined ecological barrier of the Sinai, Sahara and Sahel deserts. Between 1999–2001, a total of 410 adults and 342 juveniles were banded. The significant difference in mean wing length between birds caught in autumn and spring reflects the degree of abrasion of the outer primaries during over-wintering in Africa. Dunlins caught in Eilat in autumn and early winter had a mean wing length 1.4–1.9mm longer than in the spring. The rate of body mass increase was comparatively high and the mean body mass of the heaviest 10% of Dunlin at Eilat was 56.2g (SE ± 0.6, N = 80). The heaviest birds from Eilat carried on average about 10g of fat with a lipid index (fat mass as a percentage of total body mass) of 18%. These reserves allow a flight of approximately 1 000km, which is probably sufficient for continued migration to more southerly wintering grounds.     

Migratory Masked Shrikes,Lanius nubicus staging at the desert edge: phenology,and sex- and age-related differences in body mass

Migratory Masked Shrikes (Lanius nubicus) were mist-netted during the spring and autumn migrations at Eilat, Israel. Nearly four times more shrikes were trapped during spring than in autumn. This could be explained by either the species assorting to loop migration, or a higher capture rate owing to poor body condition after crossing the Sahara in spring. In spring, no differences in phenology were observed between the sex- and age-groups. In contrast, in autumn juvenile males arrived significantly later than the rest of the population. Significant differences in wing chord length were found between the four sex and age classes (adult male, adult female, juvenile male, juvenile female) both in spring and autumn. However, significant differences in body mass were found only in autumn. In spring, in both sexes, wing length and body mass changed significantly with date of arrival. Masked Shrike in spring were lighter than in autumn. The results suggest that migration over desert is energetically costly and Eilat is a very important staging place for migratory Masked Shrikes.     

Dihydroxyacetone metabolism in <Emphasis Type="Italic">Salinibacter ruber</Emphasis> and in <Emphasis Type="Italic">Haloquadratum walsbyi</Emphasis>

The extremely halophilic bacterium Salinibacter ruber inhabits saltern crystallizer ponds worldwide, together with the square archaeon Haloquadratum walsbyi. Cultures of Salinibacter have been shown to convert up to 20% of the glycerol added to a not previously characterized overflow product. We here identify this product of incomplete glycerol oxidation by Salinibacter as dihydroxyacetone. Genomic information suggests that H. walsbyi possesses an efficient uptake system for dihydroxyacetone, and we show here that dihydroxyacetone is indeed metabolized by Haloquadratum cultures, as well as by the heterotrophic prokaryotic community of the saltern crystallizer ponds in Eilat, Israel, dominated by Haloquadratum-like cells. In the absence of glycerol, Salinibacter also takes up dihydroxyacetone. Degradation of glycerol, produced in hypersaline lakes as an osmotic solute by the green alga Dunaliella salina may thus involve dihydroxyacetone as an intermediate, which can then be taken up by different types of heterotrophs present in the environment.     

Environmental adaptation factors of two yeasts isolated from the leachate of a uranium mineral heap

Mycosporine-like amino acids (MAAs) were extracted from a unicellular cyanobacterium (Euhalothece sp.) isolated from a gypsum crust on the bottom of a hypersaline saltern pond in Eilat, Israel. When grown at high light intensities, this isolate contained high concentrations of two MAAs, one showing maximum optical density at 331 nm and one at 362 nm. The compound absorbing at 331 nm was purified by preparative high performance liquid chromatography, and its structure was elucidated by one-dimensional ((1)H and (13)C) and two-dimensional nuclear magnetic resonance, mass spectrometry and amino acid analysis, and identified as mycosporine-2-glycine. This is the first report of mycosporine-2-glycine in cyanobacteria.     

The role of glycerol in the nutrition of halophilic archaeal communities: a study of respiratory electron transport

Abstract Respiratory electron transport activity in the Dead Sea and saltern crystallizer ponds, hypersaline environments inhabited by dense communities of halophilic archaea and unicellular green algae of the genus Dunaliella , was assayed by measuring reduction of 2-( p -iodophenyl)-3( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Typical rates obtained were in the order of 5.5–17.7 nmol INT reduced h ⁻¹ per 10⁶ cells at 35 ° C. In Dead Sea water samples, respiratory activity was stimulated more than two-fold by addition of glycerol, but not by any of the other carbon compounds tested, including sugars, organic acids, and amino acids, or by addition of inorganic nutrients. Stimulation by glycerol had a half-saturation constant of 0.75 μM. A similar respiratory activity was also found when Dead Sea water samples were diluted with distilled water and incubated in the light. As Dunaliella cells did not reduce INT, it is suggested that photosynthetically produced glycerol leaking from the algae is the preferred carbon and energy source for the development of halophilic archaea in hypersaline environments. In samples from saltern crystallizer pond stimulation of INT reduction by glycerol was much less pronounced, probably because the community was less severely carbon-limited.     

The solitary ascidian Herdmania momus: native (Red Sea) versus non-indigenous (Mediterranean) populations

During 2005 monthly samples of the solitary ascidian Herdmania momus were collected along the Mediterranean and Red Sea coasts of Israel, in order to investigate possible differences in life history strategies of the two populations. The samples were preserved, dissected, and measurements were made of length, total weight, gonad weight, oocyte diameter and the occurrence of symbionts was recorded. Additionally, field surveys showed that in the Mediterranean H. momus exclusively inhabit artificial substrates, and are common at greater depths than in Eilat (Red Sea). Individuals of H. momus in Eilat reproduced year round. Although individuals from the Mediterranean were significantly larger than individuals collected in Eilat their gonad indices and oocyte diameter measurements indicate that they have a short reproductive season. Copepods were found in 50% of the samples from both sites, while a pontoniine shrimp was found in 14% of the samples from Eilat only. The marked differences between the native vs. non-indigenous populations of H. momus are attributed to differential food availability, water temperature, currents and wave exposure. The increasing evidence of negative effects of non-indigenous ascidians on natural fauna from other regions in the world emphasizes the need for additional research regarding the ecology of ascidians along the coasts of Israel.     

STOPOVER AND FAT DEPOSITION BY BLACKCAPS SYLVIA ATRICAPILLA FOLLOWING SPRING MIGRATION OVER THE SAHARA

Maitav, A. & Izhaki, I. 1994. Stopover and fat deposition by Blackcaps Sylvia atricapilla following spring migration over the Sahara. Ostrich 65:160-166.

Length of stopover and rate of weight change were studied in Blackcaps that stopover in Eilat (southern Israel) in spring after a trans-Saharan flight. The birds studied had already completed 2000 km of desert crossing and faced just 200 km more. The potential predicted flight range for these transients suggests that the fat depleted Blackcaps (fat class=0) could not reach the east Mediterranean scrublands, which offer more extensive foraging opportunities than the desert area around Eilat, without refueling. However, 46% of the Blackcaps had sufficient fat (fat class > 0) to reach central or northern Israel in a single flight in still air. Recaptured birds stayed in Eilat for 2,4±1,9 days. Although the body mass of an average recaptured bird increased by 0,27±1,44 g/day, which was an increase of 0,8%±7,0% of its body mass per day during stopover, these results were not significantly different from zero. Stopover length, mass change during stopover, and the rate of change in mass did not differ between males and females and between those who stopped over for only one day to those which stayed > 1 day. However, there was a tendency of these lean birds to stay for a shorter period in Eilat than relatively fat birds. There was also some tendency for lean birds to gain more mass during their stopover than fat birds. It seems that in spring, when less time is allotted for the whole migration program, the migrants resume their journey before fat reserves have been replenished.     

Presence of two novel cardiolipins in the halophilic archaeal community in the crystallizer brines from the salterns of Margherita di Savoia (Italy) and Eilat (Israel)

Two novel cardiolipin derivatives were recently detected in Halobacterium salinarum, namely an archaeal analog of bisphosphatidylglycerol (BPG) and a glycocardiolipin (GlyC). GlyC was found to be tightly bound to bacteriorhodopsin. To obtain information on the presence and distribution of these archaeal cardiolipins, we have analyzed the lipids extracted from the crystallizer ponds of the salterns of Margherita di Savoia (Italy) and Eilat (Israel) and from cultures of representative species of the Halobacteriaceae by electrospray ionization mass spectrometry. BPG was present as a minor lipid component in the lipids extracted from the biomass of the Margherita di Savoia and the Eilat salterns, while GlyC was detected only in the extract of the biomass of Margherita di Savoia. Both compounds were enriched in the membrane fraction obtained by dialysis of the cells against distilled water. We detected BPG in all members of the Halobacteriaceae tested, but GlyC has so far been found only in the genera Halobacterium and Haloarcula. A sulfated diglycosyl diether was the major glycolipid detected in the biomass of both salterns.     

Microhabitats, thermal heterogeneity, and patterns of physiological stress in the rocky intertidal zone.

Thermal stress has been considered to be among the most important determinants of organismal distribution in the rocky intertidal zone. Yet our understanding of how body temperatures experienced under field conditions vary in space and time, and of how these temperatures translate into physiological performance, is still rudimentary. We continuously monitored temperatures at a site in central California for a period of two years, using loggers designed to mimic the thermal characteristics of mussels, Mytilus californianus. Model mussel temperatures were recorded on both a horizontal and a vertical, north-facing microsite, and in an adjacent tidepool. We periodically measured levels of heat shock proteins (Hsp70), a measure of thermal stress, from mussels at each microsite. Mussel temperatures were consistently higher on the horizontal surface than on the vertical surface, and differences in body temperature between these sites were reflected in the amount of Hsp70. Seasonal peaks in extreme high temperatures ("acute" high temperatures) did not always coincide with peaks in average daily maxima ("chronic" high temperatures), suggesting that the time history of body temperature may be an important factor in determining levels of thermal stress. Temporal patterns in body temperature during low tide were decoupled from patterns in water temperature, suggesting that water temperature is an ineffective metric of thermal stress for intertidal organisms. This study demonstrates that spatial and temporal variability in thermal stress can be highly complex, and "snapshot" sampling of temperature and biochemical indices may not always be a reliable method for defining thermal stress at a site.     

Contrasting photo- and thermoperiod-induced changes in abscisic acid and lipid contents in leaves of mungbean seedlings

Seedlings of Phaseolus aureus ROXB were grown under 12/12 h light/dark cycles with the light period at 32.5°C and darkness at 10°C (normal conditions N) or with light at 10°C and darkness at 32.5°C (inverse conditions, I). I-conditions affected the level of chlorophyll and carotenoids (very low), monogalactosyldiacylgycerol (low) and phosphatidylinositol (high) in the leaves. Leaves of I-seedlings showed a sharp and durable decline of relative water content during the low temperature phase. For the N-seedlings, loss of water was restricted to the end of this period. The loss of water was accompanied by visible symptoms of wilting at specific times of day. Although the pigment content remained nearly unchanged, ABA content of leaves of both N-and I-seedlings increased during water stress. Upon return to the warm period, ABA level continued to increase after the leaves had regained turgor, this 'after stress'increase being more pronounced in the leaves of I-seedlings. Exogenous application of ABA induced a slight increase in the content of phospholipids in N- and I-leaves and a decrease in free fatty acids, whereas monogalactosyldiacylglycerol content was significantly reduced in N-leaves after application of ABA. Upon transfer of I-plants to 20°C for 12 h during the light period, pigment and chloroplastic lipid content increased rapidly whereas upon a further exposure to 10°C in light, pigments and especially monogalactosyldiacylglycerol were lost. The control of pigment and lipid metabolism and the role of ABA during chilling stress are discussed.     

Estimation of the contribution of halobacterial to the bacterial biomass and activity in solar salterns by the use of bile salts

Abstract Bile salts (deoxycholate, taurocholate) were used to estimate the contribution of bacteria of the Halobacterium group to bacterial community size and activity at different salinities as found in a multi-pond saltern. Low concentrations of bile salts cause lysis of halophilic archaebacteria of the Halobacterium group, while halophilic eubacteria and halococci remain microscopically intact. Upon addition of bile salts, total bacterial numbers (as estimated microscopically) in saltern ponds at salinities below 250 g/l did not decrease, while above this salinity bacterial numbers decreased by 30–50%. To estimate the contribution of halobacteria to overall heterotrophic activity, the effect of bile salt addition was tested on the incorporation of labelled amino acids. In saltern ponds of a salinity below 250 g/l activity was not greatly inhibited by taurocholate, while at salinity above 300 g/l taurocholate completely abolished incorporation of amino acids.