The ecology and taxonomy of aerobic chemoorganotrophic halophilic eubacteria

Abstract There exists a wide diversity of halophilic eubacteria with chemoorganotrophic-aerobic metabolism. Most of them have a more moderate salt response than halophilic archaebacteria, falling into the category of moderately halophilic bacteria. Although mostly isolated from salted food, their natural habitats are hypersaline waters of intermediate levels of salt concentration, and hypersaline soils. In hypersaline waters, the taxonomic groups found are the ones that also predominate in ocean waters, such as representatives of the genera Vibrio, Pseudomonas and Flavobacterium . However, in hypersaline soils, the taxonomic groups present are those typical of normal soils, such as Pseudomonas, Bacillus and Gram-positive cocci. The halophilic bacteria from soils are also more resistant to exposure to low salt concentrations than the organisms isolated from waters. Therefore, it seems that the general characteristics of the hypersaline environments drastically affect the types of halophilic bacteria present, and that the halophilic character has arisen in many phylogenetic groups of eubacteria.     

Salt to conserve: a review on the ecology and preservation of hypersaline ecosystems

When it comes to the investigation of key ecosystems in the world, we often omit salt from the ecological recipe. In fact, despite occupying almost half of the volume of inland waters and providing crucial services to humanity and nature, inland saline ecosystems are often overlooked in discussions regarding the preservation of global aquatic resources of our planet. As a result, our knowledge of the biological and geochemical dynamics shaping these environments remains incomplete and we are hesitant in framing effective protective strategies against the increasing natural and anthropogenic threats faced by such habitats. Hypersaline lakes, water bodies where the concentration of salt exceeds 35 g/l, occur mainly in arid and semiarid areas resulting from hydrological imbalances triggering the accumulation of salts over time. Often considered the ‘exotic siblings’ within the family of inland waters, these ecosystems host some of the most extremophile communities worldwide and provide essential habitats for waterbirds and many other organisms in already water-stressed regions. These systems are often highlighted as natural laboratories, ideal for addressing central ecological questions due to their relatively low complexity and simple food web structures. However, recent studies on the biogeochemical mechanisms framing hypersaline communities have challenged this archetype, arguing that newly discovered highly diverse communities are characterised by specific trophic interactions shaped by high levels of specialisation. The main goal of this review is to explore our current understanding of the ecological dynamics of hypersaline ecosystems by addressing four main research questions: (i) why are hypersaline lakes unique from a biological and geochemical perspective; (ii) which biota inhabit these ecosystems and how have they adapted to the high salt conditions; (iii) how do we protect biodiversity from increasing natural and anthropogenic threats; and (iv) which scientific tools will help us preserve hypersaline ecosystems in the future? First, we focus on the ecological characterisation of hypersaline ecosystems, illustrate hydrogeochemical dynamics regulating such environments, and outline key ecoregions supporting hypersaline systems across the globe. Second, we depict the diversity and functional aspects of key taxa found in hypersaline lakes, from microorganisms to plants, invertebrates, waterbirds and upper trophic levels. Next, we describe ecosystem services and discuss possible conservation guidelines. Finally, we outline how cutting-edge technologies can provide new insights into the study of hypersaline ecology. Overall, this review sheds further light onto these understudied ecosystems, largely unrecognised as important sources of unique biological and functional diversity. We provide perspectives for key future research avenues, and advocate that the conservation of hypersaline lakes should not be taken with ‘a grain of salt’.     

Halobacteria: the evidence for longevity

Subterranean salt deposits are the remains of ancient hypersaline waters that presumably supported dense populations of halophilic microorganisms including representatives of the haloarchaea (halobacteria). Ancient subterranean salt deposits (evaporites) are common throughout the world, and the majority sampled to date appear to support diverse populations of halobacteria. The inaccessibility of deep subsurface deposits, and the special requirements of these organisms for survival, make contamination by halobacteria from surface sites unlikely. It is conceivable that these subterranean halobacteria are autochthonous, presumably relict populations derived from ancient hypersaline seas that have been revived from a state of dormancy. One would predict that halobacteria that have been insulated and isolated inside ancient evaporites would be different from comparable bacteria from surface environments, and that it might be possible to use a molecular chronometer to establish if the evolutionary position of the subsurface isolates correlated with the geological age of the evaporite. Extensive comparisons have been made between the 16S rRNA genes of surface and subsurface halobacteria without showing any conclusive differences between the two groups. A further phylogenetic comparison exploits an unusual feature of one particular group of halobacteria that possess at least two heterogeneous copies of the 16S rRNA gene, the sequences of which may have been converging or diverging over geological time. However, results to date have yet to show any gene sequence differences between surface and evaporite-derived halobacteria that might arguably be an indication of long-term dormancy. Received: January 22, 1998 / Accepted: February 16, 1998     

Hypersaline waters in salterns - natural ecological niches for halophilic black yeasts

Hypersaline waters in salterns have so far been considered to be populated only with halophilic algae and bacteria and completely lacking halophilic fungi. In this paper we present population dynamics of polymorphic black yeasts, isolated from hypersaline waters (3-30% NaCl) of a saltern, in relation to different physicochemical parameters. Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, Aureobasidium pullulans and Cladosporium spp. were detected with the highest frequency just before the peak of halite (NaCl) concentration. Since H. werneckii, P. triangularis and T. salinum are not known outside saline environments, these results suggest that hypersaline water is their natural ecological niche.     

Adaptations to life in a hypersaline water‐body: Adaptations at the egg and early embryonic stage of Tanytarsus barbitarsis freeman (Diptera,chironomidae)

Oviposition, egg sinking rates, ion excretion in eggs and early embryos, and egg permeability and resistance to desiccation were studied in Tanytarsus barbitarsis a chironomid inhabiting highly saline waters in southern Australia. The aim was to investigate the nature of adaptations displayed by eggs and early embryonic stages of this species to life in hypersaline waters.

Adaptations appear to be of two major sorts: one sort relates to the physiological stress of life in a hypersaline medium, the other to the need to minimize fish predation. The importance of selective forces other than salinity in determining the fauna of salt lakes is highlighted.     

Halotolerant and halophilic fungi

Extreme environments have for long been considered to be populated almost exclusively by prokaryotic organisms and therefore monopolized by bacteriologists. Solar salterns are natural hypersaline environments characterized by extreme concentrations of NaCl, often high concentrations of other ions, high uv irradiation and in some cases extremes in pH. In 2000 fungi were first reported to be active inhabitants of solar salterns. Since then many new species and species previously known only as food contaminants have been discovered in hypersaline environments around the globe. The eukaryotic microorganism most studied for its salt tolerance is Saccharomyces cerevisiae. However, S. cerevisiae is rather salt sensitive and not able to adapt to hypersaline conditions. In contrast, some species like Debaryomyces hansenii, Hortaea werneckii, and Wallemia ichthyophaga have been isolated globally from natural hypersaline environments. We believe that all three are more suitable model organisms to study halotolerance in eukaryotes than S. cerevisiae. Furthermore, they belong to different and distant taxonomic groups and have developed different strategies to cope with the same problems of ion toxicity and loss of water.     

Two Fixed Ratio Dilutions for Soil Salinity Monitoring in Hypersaline Wetlands

Highly soluble salts are undesirable in agriculture because they reduce yields or the quality of most cash crops and can leak to surface or sub-surface waters. In some cases salinity can be associated with unique history, rarity, or special habitats protected by environmental laws. Yet in considering the measurement of soil salinity for long-term monitoring purposes, adequate methods are required. Both saturated paste extracts, intended for agriculture, and direct surface and/or porewater salinity measurement, used in inundated wetlands, are unsuited for hypersaline wetlands that often are only occasionally inundated. For these cases, we propose the use of 1:5 soil/water (weight/weight) extracts as the standard for expressing the electrical conductivity (EC) of such soils and for further salt determinations. We also propose checking for ion-pairing with a 1:10 or more diluted extract in hypersaline soils. As an illustration, we apply the two-dilutions approach to a set of 359 soil samples from saline wetlands ranging in ECe from 2.3 dS m^-1 to 183.0 dS m^-1. This easy procedure will be useful in survey campaigns and in the monitoring of soil salt content.     

Hypersaline waters - a potential source of foodborne toxigenic aspergilli and penicillia

Previous studies of hypersaline environments have revealed the dominant presence of melanized yeast-like fungi and related Cladosporium spp. In this study, we focused on the genera Aspergillus and Penicillium and their teleomorphic forms. From oligotrophic and eutrophic hypersaline waters around the world, 60 different species were identified, according to their morphological characteristics and extrolite profiles. For the confirmation of five new species, additionally, sequence analysis of the internal transcribed spacer region, the partial large subunit-rDNA and the partial β-tubulin gene was performed. The species Aspergillus niger, Eurotium amstelodami and Penicillium chrysogenum were detected with the highest frequencies at all of the sampled sites; thus, they represent the pan-global stable mycobiota in hypersaline environments. Possible candidates were also Aspergillus sydowii and Eurotium herbariorum, as they were quite evenly distributed among the sampled sites, and Aspergillus candidus, which was abundant, but more locally distributed. These species and their byproducts can accumulate downstream following evaporation of brine, and they can become entrapped in the salt crystals. Consequently, marine salt used for consumption can be a potential source of food-borne fungi and their byproducts. For example, ochratoxin-A-producing species Penicillium nordicum was recovered from brine, salt and salted meat products.     

Does the potentially toxic cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> exist in the soda lakes of East Africa?

Presently, the food chains of the famous saline alkaline flamingo-lakes of East Africa are the focus of intense scientific discussion as the lakes host toxic cyanobacteria, which when consumed by Lesser Flamingos, weaken the birds and therefore make them susceptible to attacks by infective diseases. The distribution, genetic and toxicological aspects of Microcystis in Kenya has been studied extensively. Although there are reports on the occurrence of Microcystis in Kenya’s hypersaline alkaline lakes, they have not been confirmed. Our investigations carried out over a 10-year period in about 50 inland waters showed that Microcystis occurs exclusively in freshwaters, but never in the hypersaline alkaline lakes. Microscopic examinations of the phytoplankton of these lakes revealed the presence of Anabaenopsis abijatae (Nostococales) whose lumpy structure makes it roughly similar to Microcystis when viewed under an inverted microscope. We conclude that the possible occurrence of Microcystis in hypersaline alkaline lakes is doubtful and, as such, confirmatory studies including microphotographic documentation of findings should be carried out.     

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Añana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.     

Anaerobic degradation of organic compounds at high salt concentrations

A number of obligately anaerobic fermentative bacteria are known to degrade a variety of organic substrates such as sugars, amino acids, and others, in the presence of high salt concentrations (up to 3–4 M) to products such as hydrogen, CO₂, acetate and higher fatty acids, and ethanol. Our understanding of the fate of these products in hypersaline environments is still extremely limited. The occurrence of bacterial sulfate reduction is well established at salt concentrations of up to 24%; however, the bacteria involved have not yet been isolated in pure culture, and the range of electron donors used is unknown. Halophilic or halotolerant methanogenic bacteria using hydrogen/CO₂ or acetate as energy source are notably absent; methanogenesis under hypersaline conditions is probably limited to such substrates as methanol and methylamines, which cannot be expected to be major products of anaerobic degradation of most organic compounds.     

Biodegradation of organic pollutants by halophilic bacteria and archaea

Hypersaline environments are important for both surface extension and ecological significance. As all other ecosystems, they are impacted by pollution. However, little information is available on the biodegradation of organic pollutants by halophilic microorganisms in such environments. In addition, it is estimated that 5% of industrial effluents are saline and hypersaline. Conventional nonextremophilic microorganisms are unable to efficiently perform the removal of organic pollutants at high salt concentrations. Halophilic microorganisms are metabolically different and are adapted to extreme salinity; these microorganisms are good candidates for the bioremediation of hypersaline environments and treatment of saline effluents. This literature survey indicates that both the moderately halophilic bacteria and the extremely halophilic archaea have a broader catabolic versatility and capability than previously thought. A diversity of contaminating compounds is susceptible to be degraded by halotolerant and halophile bacteria. Nevertheless, significant research efforts are still necessary in order to estimate the true potential of these microorganisms to be applied in environmental processes and in the remediation of contaminated hypersaline ecosystems. This effort should be also focused on basic research to understand the overall degradation mechanism, to identify the enzymes involved in the degradation process and the metabolism regulation.     

Stock delineation of pink snapper and tailor from Western Australia by analysis of stable isotope and strontium/calcium ratios in otolith carbonate

The ¹⁸O/¹⁶O and ¹³C/¹²C ratios in the otolith carbonate of pink snapper Pagrus auratus and tailor (bluefish) Pomatomus saltatrix, from several locations along the Western Australian coast, indicated that pink snapper stocks are location specific but that tailor stocks are less so. The hypersaline Shark Bay, on the coast of Western Australia, generated strongly characteristic isotopic signatures which serve as natural tags. Otolith carbonate from pink snapper from normal oceanic waters just north of Shark Bay showed no evidence that the fish had been in hypersaline water. Similarly, pink snapper from the hypersaline bay showed no evidence of having spent time at normal oceanic salinity. By contrast, some tailor from oceanic waters showed evidence of having spent considerable time in the bay, and some fish from the bay had oceanic signatures. This suggested that tailor were more migratory than snapper. The similarity in the distribution of the isotopic signatures (from oceanic to hypersaline) in otolith carbonate from tailor from oceanic waters north of Shark Bay (Koks Island), and from those within Shark Bay, indicated a single stock in this region (in contrast to pink snapper). Moreover, tailor from coastal south-western Australia and from the Shark Bay area could be considered seperately for some management purposes. For pink snapper stocks from oceanic waters, oxygen isotope signatures were clearly related to water temperature although the temperature relationship was obscured for fish within Shark Bay because of the strength of the signal generated by the hypersaline water. For tailor the temperature relationship was not obvious, probably because migrations of tailor smeared the temperature effect, and the hypersaline Shark Bay waters dominated, and, possibly, at the southern extemity of the range, the freshwater in some estuaries influenced the isotopic signatures of the otolith carbonate. Strontium/calcium ratios in pink snapper otoliths also indicated a separation of stocks, but for tailor overlap of signatures again suggested migratory behaviour.     

Archaea with square cells

Two groups of microbiologists have independently isolated 'Walsby's square bacterium' from salt crystallizer ponds; its growth depends on pyruvate. Genetic analysis shows that the squares, discovered 25 years ago on the Sinai Peninsula, are archaea rather than bacteria. These transparent tile-like cells might have been dismissed as surface artefacts of salt crystals but for their gas vesicles--structures peculiar to prokaryotic organisms. Paradoxically, the square archaea are the dominant microorganisms in some hypersaline environments and might be globally important.     

Genotypic diversity of oscillatoriacean strains belonging to the genera Geitlerinema and Spirulina determined by 16S rDNA restriction analysis

Genotypic diversity of several cyanobacterial strains mostly isolated from marine or brackish waters, belonging to the genera Geitlerinema and Spirulina, was investigated by amplified 16S ribosomal DNA restriction analysis and compared with morphological features and response to salinity. Cluster analysis was performed on amplified 16S rDNA restriction profiles of these strains along with profiles obtained from sequence data of five Spirulina-like strains, including three representatives of the new genus Halospirulina. Our strains with tightly coiled trichomes from hypersaline waters could be assigned to the Halospirulina genus. Among the uncoiled strains, the two strains of hypersaline origin clustered together and were found to be distant from their counterparts of marine and freshwater habitat. Moreover, another cluster, formed by alkali-tolerant strains with tightly coiled trichomes, was well delineated.     

Irreversible habitat specialization does not constrain diversification in hypersaline water beetles

Specialization to extreme environments is often considered an evolutionary dead‐end, leading to irreversible adaptations and reduced evolvability. There is, however, mixed evidence for this macroevolutionary pattern, and limited data from speciose lineages. Here, we tested the effect of habitat specialization to hypersaline waters in the diversification rates of aquatic beetles of the genus Ochthebius (Coleoptera, Hydraenidae), using a molecular phylogeny with more than 50% of the 546 recognized species, including representatives of all but one of the nine recognized subgenera and 17 species groups. Phylogenies were built combining mitochondrial and nuclear genes, with the addition of 42 mitochondrial genomes. Using Bayesian methods of character reconstruction, we show that hypersaline tolerance is an irreversible ecological specialization that arose multiple times. Two lineages of Ochthebius experienced a significant increase in diversification rates, one of them inhabiting hypersaline waters, but there was no overall correlation with habitat or any significant decrease in diversification rates despite the irreversibility of hypersaline tolerance. Our study tested for the first time the impact of hypersaline habitat specialization on diversification rates, finding no support for it to be an evolutionary dead‐end. On the contrary, multiple and ancient lineages fully adapted to these extreme osmotic conditions have persisted and diversified over a long evolutionary timescale.     

Ciliates along Oxyclines of Permanently Stratified Marine Water Columns

Studies of microbial communities in areas of the world where permanent marine water column oxyclines exist suggest they are “hotspots” of microbial activity, and that these water features and the anoxic waters below them are inhabited by diverse protist taxa, including ciliates. These communities have minimal taxonomic overlap with those in overlying oxic water columns. Some ciliate taxa have been detected in multiple locations where these stable water column oxyclines exist; however, differences in such factors as hydrochemistry in the habitats that have been studied suggest local selection for distinct communities. We compare published data on ciliate communities from studies of deep marine water column oxyclines in Caricao Basin, Venezuela, and the Black Sea, with data from coastal, shallower oxycline waters in Framvaren and Mariager fjords, and from several deep‐sea hypersaline anoxic basins in the Eastern Mediterranean Sea. Putative symbioses between Bacteria, Archaea, and ciliates observed along these oxyclines suggests a strategy of cooperative metabolism for survival that includes chemosynthetic autotrophy and exchanges of metabolic intermediates or end products between hosts and their prokaryotic partners.     

Salt stress and plasma-membrane fluidity in selected extremophilic yeasts and yeast-like fungi

We have investigated changes in plasma-membrane fluidity in relation to NaCl concentrations in yeasts and yeast-like fungi that were isolated from either subglacial ice or hypersaline waters. In both of these natural environments, these organisms are exposed to low water activity, due to either high NaCl concentrations or low temperatures. Our data indicate that the fluidity of the plasma membrane can be used as an indicator of fitness for survival in extreme environments. Fungi that can survive in such extreme environments, such as Hortaea werneckii in the hypersaline waters of salterns, and Cryptococcus liquefaciens in subglacial environments, showed similar profiles of plasma-membrane fluidity in response to raised salinity. The same was seen for ubiquitous fungi, which are generally adapted for different types of stress, such as Aureobasidium pullulans and Rhodotorula mucilaginosa. Representatives of both of these groups modulated their plasma-membrane fluidity differently. When salinity exceeded their optimal range, the ubiquitous stress-tolerant species (A. pullulans, Rh. mucilaginosa) showed increased plasma-membrane fluidity, whereas in the dominant extremophiles (H. werneckii, Cr. liquefaciens), it decreased. On the other hand, the plasma membranes of the fungi with a narrow ecological amplitude (Arctic A. pullulans and Rhodosporium diobovatum) showed different responses.