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Diel Migrations of Microorganisms within a Benthic, Hypersaline Mat Community.

We studied the diel migrations of several species of microorganisms in a hypersaline, layered microbial mat. The migrations were quantified by repeated coring of the mat with glass capillary tubes. The resulting minicores were microscopically analyzed by using bright-field and epifluorescence (visible and infrared) microscopy to determine depths of coherent layers and were later dissected to determine direct microscopic counts of microorganisms. Microelectrode measurements of oxygen concentration, fiber optic microprobe measurements of light penetration within the mat, and incident irradiance measurements accompanied the minicore sampling. In addition, pigment content, photosynthesis and irradiance responses, the capacity for anoxygenic photosynthesis, and gliding speeds were determined for the migrating cyanobacteria. Heavily pigmented Oscillatoria sp. and Spirulina cf. subsalsa migrated downward into the mat during the early morning and remained deep until dusk, when upward migration occurred. The mean depth of the migration (not more than 0.4 to 0.5 mm) was directly correlated with the incident irradiance over the mat surface. We estimated that light intensity at the upper boundary of the migrating cyanobacteria was attenuated to such an extent that photoinhibition was effectively avoided but that intensities which saturated photosynthesis were maintained through most of the daylight hours. Light was a cue of paramount importance in triggering and modulating the migration of the cyanobacteria, even though the migrating phenomenon could not be explained solely in terms of a light response. We failed to detect diel migration patterns for other cyanobacterial species and filamentous anoxyphotobacteria. The sulfide-oxidizing bacterium Beggiatoa sp. migrated as a band that followed low oxygen concentrations within the mat during daylight hours. During the nighttime, part of this population migrated toward the mat surface, but a significant proportion remained deep.

Journal Article↗

Organic osmolytes in aerobic bacteria from mono lake, an alkaline, moderately hypersaline environment.

The identity and concentrations of intracellular organic solutes were determined by nuclear magnetic resonance spectroscopy for two strains of aerobic, gram-negative bacteria isolated from Mono Lake, Calif., an alkaline, moderately hypersaline lake. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was the major endogenous solute in both organisms. Concentrations of ectoine varied with external NaCl levels in strain ML-D but not in strain ML-G, where the level was high but invariant from 1.5 to 3.0 M NaCl. Hydroxyectoine also occurred in strain ML-D, especially at elevated NaCl concentrations (2.5 and 3.0 M), but at levels lower than those of ectoine. Exogenous organic solutes that might occur in Mono Lake were examined for their effects on the de novo synthesis of ectoine. Dimethylsulfoniopropionate (DMSP) (0.1 or 1 mM) did not significantly lower ectoine levels in either isolate, and only strain ML-G showed any capacity for DMSP accumulation. With nitrogen limitation, however, DMSP (0.1 mM) substituted for ectoine in strain ML-G and became the main organic solute. Glycine betaine (GB) was more effective than DMSP in affecting ectoine levels, principally in strain ML-D. Strain ML-D accumulated GB to 50 or 67% of its organic solute pool at 2.5 M NaCl, at an external level of 0.1 or 1 mM GB, respectively. Strain ML-D also accumulated arsenobetaine. The methylated zwitterionic compounds, probably metabolic products of phytoplankton (DMSP and GB) or brine shrimps (arsenobetaine) in Mono Lake, may function as osmolytes for indigenous bacteria when present at high concentrations or under conditions of nitrogen limitation or salt stress.

Journal Article↗

Anoxygenic photosynthesis and nitrogen fixation by a microbial mat community in a bahamian hypersaline lagoon.

Simultaneous measurements of photosynthesis (both oxygenic and anoxygenic) and N(inf2) fixation were conducted to discern the relationships between photosynthesis, N(inf2) fixation, and environmental factors potentially regulating these processes in microbial mats in a tropical hypersaline lagoon (Salt Pond, San Salvador Island, Bahamas). Major photoautotrophs included cyanobacteria, purple phototrophic bacteria, and diatoms. Chemosystematic photopigments were used as indicators of the relative abundance of mat phototrophs. Experimental manipulations consisted of light and dark incubations of intact mat samples exposed to the photosystem II inhibitor DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a dissolved organic carbon source (D-glucose), and normal seawater (37(permil)). Photosynthetic rates were measured by both O(inf2) and (sup14)C methods, and nitrogenase activity (NA) was estimated by the acetylene reduction assay. Moderate reductions in salinity (from 74 to 37(permil)) had no measurable effect on photosynthesis, O(inf2) consumption, or NA. CO(inf2) fixation in DCMU-amended samples was (symbl)25% of that in the control (nonamended) samples and demonstrated photosynthetic activity by anoxygenic phototrophs. NA in DCMU-amended samples, which was consistently higher (by a factor of 2 to 3) than the other (light and dark) treatments, was also attributed to purple phototrophic bacteria. The ecological implication is that N(inf2) fixation by anoxygenic phototrophs (purple phototrophic bacteria and possibly cyanobacteria) may be regulated by the activity of oxygenic phototrophs (cyanobacteria and diatoms). Consortial interactions that enhance the physiological plasticity of the mat community may be a key for optimizing production, N(inf2) fixation, and persistence in these extreme environments.

Journal Article↗

Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar lake, Sinai, Egypt).

The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the Halobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the genera Methanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus Thermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.

Archaea↗

Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.

Chlorobi↗

Evaluation of the LIVE/DEAD BacLight kit for detection of extremophilic archaea and visualization of microorganisms in environmental hypersaline samples.

Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.

Archaea↗

The halophilic fungus Hortaea werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular cation concentrations in hypersaline environments.

Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na+ even when grown at high NaCl concentrations and can be thus considered Na+ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K+ or Na+ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H. werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.

Adaptation, Physiological↗

Genetic variance in the composition of two functional groups (diazotrophs and cyanobacteria) from a hypersaline microbial mat.

Examination of variation in ecological communities can lead to an understanding of the forces that structure communities, the consequences of change at the ecosystem level, and the relevant scales involved. This study details spatial and seasonal variability in the composition of nitrogen-fixing and cyanobacterial (i.e., oxygenic photosynthetic) functional groups of a benthic, hypersaline microbial mat from Salt Pond, San Salvador Island, Bahamas. This system shows extreme annual variability in the salinity of the overlying water and the extent of water coverage. Analysis of molecular variance and F(ST) tests of genetic differentiation of nifH and cyanobacterial 16S rRNA gene clone libraries allowed for changes at multiple taxonomic levels (i.e., above, below, and at the species level) to inform the conclusions regarding these functional groups. Composition of the nitrogen-fixing community showed significant seasonal changes related to salinity, while cyanobacterial composition showed no consistent seasonal pattern. Both functional groups exhibited significant spatial variation, changing with depth in the mat and horizontally with distance from the shoreline. The patterns of change suggest that cyanobacterial composition was more insensitive to water stress, and consequently, cyanobacteria dominated the nitrogen-fixing community during dry months but gave way to a more diverse community of diazotrophs in wet months. This seasonal pattern may allow the mat community to respond quickly to water-freshening events after prolonged dry conditions (system recovery) and maintain ecosystem function in the face of disturbance during the wet season (system resilience).

Bahamas↗

Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat.

We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (> or =97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O(2) and H(2)S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity. The sequences determined in this study have been submitted to the GenBank database and assigned accession numbers DQ 329539 to DQ 331020, and DQ 397339 to DQ 397511.

Bacteria↗

Determination of biological and physicochemical parameters of Artemia franciscana strains in hypersaline environments for aquaculture in the Colombian Caribbean.

BACKGROUND: Artemia (Crustacea, Anostraca), also known as brine shrimp, are typical inhabitants of extreme environments. These hypersaline environments vary considerably in their physicochemical composition, and even their climatic conditions and elevation. Several thalassohaline (marine) environments along the Colombian Caribbean coast were surveyed in order to contribute to the knowledge of brine shrimp biotopes in South America by determining some vital biological and physicochemical parameters for Artemia survival. Additionally, cyst quality tests, biometrical and essential fatty acids analysis were performed to evaluate the economic viability of some of these strains for the aquaculture industry. RESULTS: In addition to the three locations (Galerazamba, Manaure, and Pozos Colorados) reported in the literature three decades ago in the Colombian Caribbean, six new locations were registered (Salina Cero, Kangaru, Tayrona, Bahía Hondita, Warrego and Pusheo). All habitats sampled showed that chloride was the prevailing anion, as expected, because of their thalassohaline origin. There were significant differences in cyst diameter grouping strains in the following manner according to this parameter: 1) San Francisco Bay (SFB-Control, USA), 2) Galerazamba and Tayrona, 3) Kangarú, 4) Manaure, and 5) Salina Cero and Pozos Colorados. Chorion thickness values were smaller in Tayrona, followed by Salina Cero, Galerazamba, Manaure, SFB, Kangarú and Pozos Colorados. There were significant differences in naupliar size, grouping strains as follows (smallest to largest): 1) Galerazamba, 2) Manaure, 3) SFB, Kangarú, and Salina Cero, 4) Pozos Colorados, and 5) Tayrona. Overall, cyst quality analysis conducted on samples from Manaure, Galerazamba, and Salina Cero revealed that all sites exhibited a relatively high number of cysts.g-1. Essential fatty acids (EFA) analysis performed on nauplii from cyst samples from Manaure, Galerazamba, Salina Cero and Tayrona revealed that cysts from all sites exhibited high arachidonic acid:20:4(n-6) (ArA) and eicosapentaenoic acid: 20:5(n-3) (EPA) levels comparable to the control sample (SFB). In contrast, most cysts collected (including SFB) at different locations, and during different months, presented low docosahexaenoic acid: 22:6(n-3) (DHA) levels (Manaure was the only exception with high DHA levels). Some variations in EPA and ArA levels were observed in all sites, contrasting with the much lower DHA levels which remained constant for all locations, except for Manaure which exhibited variable DHA levels. DHA/EPA ratio was overall very low for all sites compared to SFB cysts. All strains had a low DHA/ArA, but a high EPA/ArA ratio, including the control. CONCLUSION: The Colombian A. franciscana habitats analyzed were determined to be thalassohaline, and suitable for A. franciscana development. EFA profiles demonstrated that Tayrona, Galerazamba, Manaure and Salina Cero strains are suitable food for marine fish and crustacean culture because of their high EPA/ArA ratio, but might have to be fortified with DHA rich emulsions depending on the nutritional requirements of the species to be cultured, because of their overall low DHA content. The relatively small nauplii are appropriate for marine larvaeculture. In contrast, the strains from Tayrona, Kangarú, Salina Cero, and Pozos Colorados may be of use but limited to Artemia small biomass production quantities, because of the small surface area of their respective locations; Artemia could be exploited at these locations for local aquaculture applications. In general, cyst quality evaluation for Manaure, Salina Cero and Galerazamba cysts revealed that cysts from these three locations could improve their quality by concentrating efforts on cyst processing techniques. Finally, most locations had great A. franciscana production potential and require different degrees of water quality and/or infrastructure management.

Journal Article↗

Physiological, biochemical and morphological indicators of osmoregulatory stress in 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water.

The salinity tolerance of the 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum), a current inhabitant of the hypersaline Salton Sea in California, USA, was investigated to identify osmoregulatory stress indicators for possible use in developing a model of salinity tolerance. Seawater-acclimated (35 g l(-1)) tilapia hybrids were exposed to salinities from 35-95 g l(-1), using gradual and direct transfer protocols, and physiological (plasma osmolality, [Na+], [Cl-], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical (Na+, K(+)-ATPase) and morphological (number of mature, accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress were measured. Tilapia tolerated salinities ranging from 35 g l(-1) to 65 g l(-1) with little or no change in osmoregulatory status; however, in fish exposed to 75-95 g l(-1) salinity, plasma osmolality, [Na+], [Cl-], Na+, K(+)-ATPase, and the number of apoptotic chloride cells, all showed increases. The increase in apoptotic chloride cells at salinities greater than 55 g l(-1), prior to changes in physiological and biochemical parameters, indicates that it may be the most sensitive indicator of osmoregulatory stress. Oxygen consumption decreased with salinity, indicating a reduction in activity level at high salinity. Finally, 'California' Mozambique tilapia have a salinity tolerance similar to that of pure Mozambique tilapia; however, cellular necrosis at 95 g l(-1) indicates they may be unable to withstand extreme salinities for extended periods of time.

Analysis of Variance↗

Mortality of waterfowl on a hypersaline wetland as a result of salt encrustation.

Approximately 300 geese, primarily lesser Canada geese (Branta canadensis parvipes) were found unable to fly or dead on a small hypersaline lake (conductivity 77,000-90,000 mumhos/cm) in western Saskatchewan in September 1985. The birds were heavily encrusted with sodium sulfate crystals. Dead birds that were necropsied had aspirated lake water and had evidence of acute muscle degeneration. The live geese (155) were captured and moved to nearby freshwater wetlands where most apparently survived. Some birds died of severe myopathy after translocation. Five northern shovelers (Anas clypeata) were found encrusted with salt and unable to fly on the lake approximately 10 days later. Salt encrustation apparently occurred when rapid cooling of the lake resulted in supersaturation and crystallization of the dissolved salt. A local resident recalled similar events occurring on the lake in autumn on at least two other occasions during the past 50 yr.

Animals↗

Numerical taxonomy of heavy metal-tolerant nonhalophilic bacteria isolated from hypersaline environments.

A total of 232 metal-tolerant bacterial strains were isolated from water and sediment samples collected in different hypersaline environments located in Cádiz, Huelva and Morón de la Frontera (Spain). They were isolated on a medium containing mercury, chromium, cadmium, copper or zinc. These halotolerant isolates were analyzed by numerical taxonomy techniques by using the simple matching (SSM) and Jaccard (SJ) coefficients; clustering was achieved using the unweighted pair group method with averages (UPGMA) algorithm. At the 81% and 83% similarity level, different numbers of phenons were obtained for Gram-negative and Gram-positive halotolerant microorganisms. Most of the 48 Gram-negative metal-tolerant strains studied were grouped into nine phenons, representing the genera Pseudoalteromonas, Alteromonas, Xanthomonas, Pseudomonas, Alcaligenes and Enterobacteria. The 72 Gram-positive metal-tolerant strains grouped into eight phenons, with only 15 strains left unassigned. Most of the isolates were assigned to the genus Bacillus (seven phenons), and one phenon comprised microorganisms with phenotypic characteristics similar to those of the genus Celullomonas.

Algorithms↗

Pulsed-field gel electrophoresis analysis of virus assemblages present in a hypersaline environment.

A method for analyzing virus assemblages in aquatic environments was developed and used for studying the highest-salinity ponds (from 13.4 to 35% salinity) from a multi-pond solar saltern in Alicante, Spain. The protocol consisted of a series of concentration and purification steps including tangential flow filtration and ultracentrifugation, followed by the preparation of total viral nucleic acids that were subsequently separated by pulsed-field gel electrophoresis. For every sample analyzed, a characteristic DNA pattern was obtained, whose complexity was related to viral diversity. The comparison of our results with a similar analysis carried out with marine virus assemblages shows that, as expected, the viral diversity corresponding to the analyzed hypersaline environment is considerably lower than that of a marine environment.

Bacteriophages↗

Performance of fungi in low temperature and hypersaline environments.

During the past ten years we have observed a broad array of stress capabilities in common fungi including ability to grow in aqueous ammonia and other alkaline solutions, in acids, in the presence of heavy metals, and in various salt media at low temperature. This report is concerned primarily with (a) the performance of Aspergillaceae in a variety of saturated salts, (b) distinctive roles for K+ and Rb+ ions, and (c) the lowest temperatures at which growth in nutrient brines has been observed, namely 267 degrees K in as little as 14 days. We also describe a novel solid medium based upon gelatin, glycerol and water in which fungal cultures growing at 248 degrees K can be directly examined under oil-immersion magnification. The performance capabilities of the fungi show that tolerance or adaptability to harsh and extreme physical-chemical environments cannot be considered a unique feature of prokaryotic life forms. Salt flats, brine pools and other natural hypersaline environments have long been recognized as real ecological niches harboring a range of biota from pseudomonad bacteria and green algae to specialized crustaceans. A notable omission in this ecological record is the fungi, although the group is known to include marine forms.

Aspergillus↗

Pilot-scale studies on biological treatment of hypersaline wastewater at low temperature.

In order to investigate the feasibility of biological treatment of hypersaline wastewater produced from toilet flushing with seawater at low temperature, pilot-scale studies were established with plug-flow activated sludge process at low temperature (5-9 degrees C) based on bench-scale experiments. The critical salinity concentration of 30 g/L, which resulted from the cooperation results of the non-halophilic bacteria and the halophilic bacteria, was drawn in bench-scale experiments. Pilot-scale studies showed that high COD removal efficiency, higher than 80%, was obtained at low temperature when 30 percent seawater was introduced. The salinity improved the settleability of activated sludge, and average sludge value dropped down from 38% to 22.5% after adding seawater. Seawater salinity had a strong negative effect on notronomonas and nitrobacter growth, but much more on the nitrobacter. The nitrification action was mainly accomplished by nitrosomonas. Bench-scale experiments using two SBRs were carried out for further investigation under different conditions of salinities, ammonia loadings and temperatures. Biological nitrogen removal via nitrite pathway from wastewater containing 30 percent seawater was achieved, but the ammonia removal efficiency was strongly related not only to the influent ammonia loading at different salinities but also to temperature. When the ratio of seawater to wastewater was 30 percent, and the ammonia loading was below the critical value of 0.15 kgNH4+-N/(kgMLSS.d), the ammonia removal efficiency via nitrite pathway was above 90%. The critical level of ammonia loading was 0.15, 0.08 and 0.03 kgNH4+-N/(kgMLSS.d) respectively at the different temperature 30 degrees C, 25 degrees C and 20 degrees C when the influent ammonia concentration was 60-80 mg/L and pH was 7.5-8.0.

Ammonia↗

[Number of bacteria and features of their activity in hypersaline reservoirs of the Crimea].

The incidence of bacteria, their biomass production, and heterotrophic assimilation of CO2 by bacterioplankton were studied in the Crimean hypersaline water reservoirs from May to October of 1974. The total incidence of bacteria in the natural brine of these reservoirs varied from 20 to 70 x 10(6) cells per 1 ml. Such a high bacterial number may be caused by the combined action of water evaporation which increased the concentration of bacterial cells and active growth of microflora. Low values of bacterial production and heterotrophic CO2 assimilation should be attributed to weak activity of microflora in the reservoirs.

Seasons↗

[The depressor effect of intracerebroventricular injection of BQ123 and endothelin (ET)--antiserum on hypertensive rats produced by aorta stenosis and hypersaline uptake].

An experimental model of hypertensive rat was made by abdominal aorta stenosis and hypersaline uptake. The blood pressure was significantly decreased by intracerebroventricular injection (i.c.v.) of BQ123 a potent antagonist of ET-1 receptor, in a dose-dependent manner. Negative chronotropic effect on the heart was produced by high dose of BQ123, while the depressor effect was produced by i.c.v. ET-antiserum. These results indicate that endothelin in CNS may play an important role in the pathological physiology of hypertension.

Animals↗