Genome analysis of the salt-resistant Paludifilum halophilum DSM 102817T reveals genes involved in flux-tuning of ectoines and unexplored bioactive secondary metabolites.

Paludifilum halophilum DSM 102817T is the first member of the genus Paludifilum in the Thermoactinomycetaceae family. The thermohalophilic bacterium was isolated from the solar saltern of Sfax, Tunisia and was shown to be able to produce ectoines with a relatively high-yield and to cope with salt stress conditions. In this study, the whole genome of P. halophilum was sequenced and analysed. Analysis revealed 3,789,765 base pairs with an average GC% content of 51.5%. A total of 3775 genes were predicted of which 3616 were protein-coding genes and 73 were RNA genes. The genes encoding key enzymes for ectoines (ectoine and hydroxyectoine) synthesis (ectABCD) were identified from the bacterial genome next to a gene cluster (ehuABCD) encoding a binding-protein-dependent ABC transport system responsible for ectoines mobility through the cell membrane. With the aid of KEGG analysis, we found that the central catabolic network of P. halophilum comprises the pathways of glycolysis, tricarboxylic acid cycle, and pentose phosphate. In addition, anaplerotic pathways replenishing oxaloacetate and glutamate synthesis from central metabolism needed for high ectoines biosynthetic fluxes were identified through several key enzymes. Furthermore, a total of 18 antiSMASH-predicted putative biosynthetic gene clusters for secondary metabolites with high novelty and diversity were identified in P. halophilum genome, including biosynthesis of colabomycine-A, fusaricidin-E, zwittermycin A, streptomycin, mycosubtilin and meilingmycin. Based on these data, P. halophilum emerged as a promising source for ectoines and antimicrobials with the potential to be scaled up for industrial production, which could benefit the pharmaceutical and cosmetic industries.

Flavonoid-rich fraction attenuates permethrin-induced toxicity by modulating ROS-mediated hepatic oxidative stress and mitochondrial dysfunction ex vivo and in vivo in rat.

The present study explores the antioxidant, anti-microbial, and hepatoprotective potentials of flavonoid-rich fractions from Fumaria officinalis against permethrin-induced liver damage ex vivo/in vivo in rat. However, HPLC-DAD analysis revealed the richness of 6 components in ethyl acetate fraction (EAF) where ferulic acid, rosmarinic acid, and myricetin are the most abundant. The in vitro assays showed that EAFs have impressive antioxidant and anti-microbial properties. Ex vivo, permethrin (PER) (100 µM) induced a decrease of hepatic AST and ALT activities and 25-OH vitamin D and vitamin C levels and an increase of ALP and LDH activities, TBARS, and ϒ-GT levels with a disturbance of oxidative status. The hepatoprotective effect of EAF (1 mg/mL) against PER was confirmed by the amelioration of oxidative stress profile. In vivo, permethrin was found to increase absolute and relative liver weights, plasma transaminase activities, lactate-to-pyruvate ratio, hepatic and mitochondrial lipid peroxidation, and protein oxidation levels. This pesticide triggered a decrease of Ca2+ and Mg2+-ATPases and mitochondrial enzyme activities. The co-treatment with EAF reestablished the hepatic and mitochondrial function, which could be attributed to its richness in phenolic compounds.

The saltern-derived Paludifilum halophilum DSM 102817T is a new high-yield ectoines producer in minimal medium and under salt stress conditions.

In the present study, the growth conditions and accumulation of ectoines (ectoine and hydroxyectoine) by Paludifilum halophilum DSM 102817T under salt stress conditions have been investigated. The productivity assay of this strain for ectoines revealed that the highest cellular content was reached in the minimal glucose sea water medium (SW-15) within 15% salinity. The addition of 0.1% (w/v) aspartic acid to the medium allowed an average of four times higher biomass production, and a dry mycelial biomass of 1.76 g L-1 was obtained after 6 days of growth in shake flasks at 40 °C and 200 rpm. Among the inorganic cations supplemented to the glucose SW-15 medium, the addition of 1 mM Fe2+ yielded the highest amount of mycelial biomass (3.45 g L-1) and total ectoines content (119 mg g-1), resulting in about 410 mg L-1 of products at the end of exponential growth phase. After 1 h of incubation in an osmotic downshock solution containing 2% NaCl, 70% of this content was released by the mycelium, and recovering cells maintained a high survival, with a maximal growth rate (µ max) of about 93% of the control population exposed to 15% NaCl. During growth at optimal salinity and temperature (15% NaCl and 40 °C), P. halophilum developed a compact and circular pellets that were easy to separate by simple decantation from both fermentation media and after hypoosmotic shock. Overall, the ectoines excreting P. halophilum could be a promising resource for ectoines production in a commercially valuable culture medium and at a large-scale fermentation process.

Enhancement of Antibacterial Activity of Paludifilum halophilum and Identification of N-(1-Carboxy-ethyl)-phthalamic Acid as the Main Bioactive Compound.

The aim of this study was to determine the combined effect of fermentation parameters and enhance the production of cellular biomass and antibacterial compounds from Paludifilum halophilum SMBg3 using the response surface methodology (RSM). Eight variables were screened to assess the effects of fermentation parameters on growth and metabolite production by Taguchi experimental design. Among these, the initial pH, temperature, and the percentage of MgSO4·7H2O in the medium were found to be most influential. The Box-Behnken design was applied to derive a statistical model for the optimization of these three fermentation parameters. The optimal parameters were initial pH: 8.3, temperature growth: 44°C, and MgSO4·7H2O: 1.6%, respectively. The maximum yield of biomass and metabolite production were, respectively, 11 mg/mL dry weight and 15.5 mm inhibition zone diameter against Salmonella enterica, which were in agreement with predicted values. The bioactive compounds were separated by the thick-layer chromatography technique and analyzed by GC/MS, NMR (1D and 2D), and Fourier-transform infrared spectroscopy (FT-IR). In addition to several fatty acids, N-(1-carboxy-ethyl)-phthalamic acid was identified as the main antibacterial compound. This element exhibited a potent activity against the ciprofloxacin-resistant Salmonella enterica CIP 8039 and Pseudomonas aeruginosa ATCC 9027 with a minimum inhibitory concentration (MIC) value range of 12.5-25 µg/mL. Results demonstrated that P. halophilum strain SMBg3 is a promising resource for novel antibacterial production due to its high-level yield potential and the capacity for large-scale fermentation.

The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins.

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.

Antagonistic Properties of Some Halophilic Thermoactinomycetes Isolated from Superficial Sediment of a Solar Saltern and Production of Cyclic Antimicrobial Peptides by the Novel Isolate Paludifilum halophilum.

This study has focused on the isolation of twenty-three halophilic actinomycetes from two ponds of different salinity and the evaluation of their ability to exert an antimicrobial activity against both their competitors and several other pathogens. From the 23 isolates, 18 strains showed antagonistic activity, while 19 showed activities against one or more of the seven pathogen strains tested. Six strains exhibited consistent antibacterial activity against Gram-negative and Gram-positive pathogens characterized at the physiological and molecular levels. These strains shared only 94-95% 16S rRNA sequence identity with the closely related species of the Thermoactinomycetaceae family. Among them, the potent strain SMBg3 was further characterized and assigned to a new genus in the family for which the name Paludifilum halophilum (DSM 102817T) is proposed. Sequential extraction of the antimicrobial compounds with ethyl acetate revealed that the crude extract from SMBg3 strain had inhibitory effect on the growth of the plant pathogen Agrobacterium tumefaciens and the human pathogens Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa. Based on the HRESI-MS spectral data, the cyclic lipopeptide Gramicidin S and four cyclic dipeptides (CDPs) named cyclo(L-4-OH-Pro-L-Leu), cyclo(L-Tyr-L-Pro), cyclo(L-Phe-L-Pro), and cyclo(L-Leu-L-Pro) were detected in the fermentation broth of Paludifilum halophilum. To our knowledge, this is the first report on the isolation of these compounds from members of the Thermoactinomycetaceae family.

Box-Behnken design for extraction optimization of crude polysaccharides from Tunisian Phormidium versicolor cyanobacteria (NCC 466): Partial characterization, in vitro antioxidant and antimicrobial activities.

In this study, response surface methodology (RSM) based on Box-Behnken design (BBD) was employed to optimize the aqueous extraction of crude polysaccharides from Tunisian cyanobacteria Phormidium versicolor (NCC 466). The optimal extraction conditions with an extraction yield of 21.56±0.92% were as follows: extraction temperature at 81.05°C, extraction time of 3.99h, and water to raw material ratio of 21.52mLg-1. Crude Phormidium versicolor polysaccharides (CPv-PS) are found to be a hetero-sulfated-anionic polysaccharides that contained carbohydrate (79.37±1.58%), protein (0.45±0.11%), uronic acids (4.37±0.19%) and sulfate (6.83±0.28%). The carbohydrate fraction was composed of arabinose, xylose, ribose, rhamnose, N-acetyl glucosamine, galactose, glucose, mannose, glucuronic acid and saccharose with corresponding mole percentages of 2.41, 14.58, 2.18, 6.23, 7.04, 28.21, 26.04, 3.02, 0.86 and 5.07, respectively. Evaluation of the antioxidant activity in vitro suggested that CPv-PS strongly scavenged radicals, prevented bleaching of ß-carotene and reduced activity. Furthermore, the CPv-PS exhibited effective antimicrobial properties.

Biochemical and physiological responses of halophilic nanophytoplankton (Dunaliella salina) from exposure to xeno-estrogen 17α-ethinylestradiol.

The environmental impacts of various pollutants on the entire levels of organisms are under investigation. Among these pollutants, endocrine-disrupting compounds (EDCs) present a serious hazard, even though the environmental significance of these compounds remains basically unknown. To drop some light on this field, we assessed the effects of a 11-day exposure of 17α-ethinylestradiol (EE2) on the growth, metabolic content, antioxidant response, oxidative stress, and genetic damage of Dunaliella salina, isolated from Tunisian biotopes. The results showed that at 10 ng L-1, EE2 could stimulate the growth of D. salina and increase its cellular content of photosynthetic pigments and metabolites; however, it did not significantly increase the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) or the level of malondialdehyde (MDA) and hydrogen peroxide (H2O2). In contrast, exposure to high levels of EE2 concentrations significantly inhibited the growth of D. salina (P < 0.05), decreased the cellular content of photosynthetic pigments, increased the cellular content of all of the metabolites and the SOD activity, and inhibited CAT and GPx activities. Nevertheless, the balance between oxidant and antioxidant enzymes was disrupted because H2O2 content along with MDA content simultaneously increased. Contrary to expected results, DNA damage (strand breaks) decreased after the exposure of algae to EE2. The results of this study suggest that EE2 toxicity could result in environmental impacts with consequences on the whole aquatic community. Graphical abstract.

Descriptive and multivariate analyses of four Tunisian wastewater treatment plants: A comparison between different treatment processes and their efficiency improvement.

This study was undertaken to evaluate the performance of four wastewater treatment plants/processes over a 4 year period. The wastewater flow evolution, energy consumption, and quality indicator parameters (BOD5, COD and TSS) at the inlet and outlet sites of the plants were determined. In comparing three domestic WWTPs with different wastewater treatment processes, the multivariate analyses (RDA and ANOVA) showed that although the Agareb plant received the highest pollution load, it displayed a high level of removal efficiency especially for COD, BOD, TSS, TKN and NH4+. It also revealed that the fluctuations in the wastewater composition and its contamination by varied industrial discharge could lead to the decrease in performance of the WWTP with activated sludge process as observed for the Southern Sfax plant. However, the electrolysis of the outlet water of Southern Sfax plant showed a significant improvement in COD removal.

Paludifilum halophilum gen. nov., sp. nov., a thermoactinomycete isolated from superficial sediment of a solar saltern.

A novel filamentous, halophilic, thermotolerant bacterium, strain SMBg3T was isolated from superficial sediment of a solar saltern in Sfax, Tunisia. The isolate is Gram-staining-positive, aerobic, catalase- and oxidase-positive. Optimum growth occurred at 40-45 °C, with 10 % (w/v) NaCl and at pH 8.0-9.0. Long and well developed aerial and substrate mycelia, with long chains of fluorescent and circular spores, were observed on all tested media. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMBg3T belongs to an independent phylogenetic lineage of the family Thermoactinomycetaceae and shows a gene sequence similarity of 94 % with Desmospora activa DSM 45169T 94.2 % with Kroppenstedtia eburnea DSM 45196T, 94.3 % with Kroppenstedtia guangzhouensis KCTC 29149T, 94.3 % with Melghirimyces algeriensisDSM 45474T and 94.5 % with Salinithrix halophila CECT 8506T. The predominant menaquinone is MK-7, but MK-8 and some minor unidentified components are also present in trace amounts. The major cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0. In addition to four major polar lipids identified as phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmethylethanolamine, five minor unknown lipids were detected in cell membranes. The DNA G+C content of strain SMBg3T is 51.2 mol%. Strain SMBg3T is distinct from recognized genera of the family Thermoactinomycetaceae by morphological, biochemical and chemotaxonomic characteristics. On the basis of physiological and phylogenetic data, strain SMBg3T represents a novel species of a new genus in the family Thermoactinomycetaceae for which the name Paludifilum halophilum gen. nov., sp. nov. is proposed. The type strain of the type species is SMBg3T (=DSM 102817T=CCUG 68698T).

Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

Characterization of halo-alkaline and thermostable protease from Halorubrum ezzemoulense strain ETR14 isolated from Sfax solar saltern in Tunisia.

A total of 54 halophilic strains were isolated from crystallizer TS18 (26 strains) and non-crystallizer M1 (28 strains) ponds and screened for their ability to produce protease, amylase, and lipase activities. Enzymatic assays allowed the selection of thirty-two active strains, among them, the ETR14 strain from TS18 showed maximum protease production yields and therefore, selected for further analysis. The results from 16S rRNA gene sequence analysis revealed that the strain belonged to Halorubrum ezzemoulense (Hrr. ezzemoulense) species. Further results indicated that optimum growth and protease production yields were obtained with 10-15% NaCl concentrations in the DSC-97 medium. The enzyme was also able to maintain high levels of protease activity at salt concentrations of up to 25%. While readily available carbon sources were noted to significantly reduce protease production, the combination between yeast extract and peptone enhanced protease excretion, which reached a maximum of 284 U ml(-1) at the end of the exponential growth phase. The enzyme exhibited optimum activity at pH 9 and 60 °C. The halophilic protease retained 87% of its initial activity after 1 h incubation at 70 °C and showed high stability over a wide range of pH, ranging from 7 to 10. This protease exhibited good temperature, pH, and salinity tolerance, which distinguishes it from other proteases previously described from other members of the holoarchaea genera and makes it a promising candidate for application in various industries.

Spatial and seasonal variability of pico-, nano- and microphytoplankton at the bottom seawater in the north coast of Sfax, Eastern Mediterranean Sea.

In the frame of the Taparura Project, we studied the distribution of pico-, nano- and microphytoplankton communities in relation to environmental variables at 18 stations sampled during four coastal cruises conducted between October 2009 and July 2010 at the bottom, on the north coast of Sfax (Tunisia, Eastern Mediterranean Sea). The restoration effect on coastal ultraphytoplankton (<10 µm) and microphytoplankton (<200 µm) was investigated using conventional flow cytometry and inverted microscopy. Flow cytometry analysis of ultraphytoplankton resolved six groups (Prochlorococcus, Synechococcus, nanoeukaryotes and three distinct subgroups within picoeukaryotes). In addition to these autotrophic groups, two unknown groups were characterised on the north coast. Picophytoplankton abundance shifted from a summer dominance of Synechococcus to a dominance of picoeukaryotes and Prochlorococcus during spring. Nanoeukaryotes were the most abundant in spring. Microphytoplankton was resolved into five groups, labelled Bacillariophyceae, Dinophyceae, Cyanobacteriae, Euglenophyceae and Chlorophyceae. A total of 90 microphytoplankton species were identified in all stations, with an overwhelming abundance of large diatoms, a typical trait of benthic communities (Coscinodiscus sp., Grammatophora sp., Navicula sp., Pleurosigma sp., Striatella unipunctata …). Results collected in this study are favouring a beneficial impact on the ecosystem of the Sfax north coast restoration achieved by the Taparura Project.

Diversity of cultivable halophilic archaea and bacteria from superficial hypersaline sediments of Tunisian solar salterns.

Prokaryotes in the superficial sediments are ecologically important microorganisms that are responsible for the decomposition, mineralization and subsequent recycling of organic matter. The aim of this study was to explore the phylogenetic and functional diversity of halophilic archaea and bacteria isolated from the superficial sediments of solar salterns at Sfax, Tunisia. Sixty four strains were isolated from crystallizer (TS18) and non-crystallizer (M1) ponds and submitted to genotypic characterization and evaluation by amplified ribosomal RNA restriction analysis (ARDRA) techniques. Our findings revealed that the archaeal diversity observed for 29 isolates generated five distinct patterns from the non-crystallizer M1 pond, with Halorubrum chaoviator as the most prevalent cultivable species. However, in the TS18 crystallizer pond, ten restriction patterns were observed, with the prevalence of haloarchaea EB27K, a not yet identified genotype. The construction of a neighbour-joining tree of 16S rRNA gene sequences resulted in the division of the potential new species into two major groups, with four strains closely related to the sequence of the unculturable haloarchaeon EB27K and one strain to the recently described Halovenus aranensis strain. The 35 bacterial strains observed in this work were present only in the non-crystallizer pond (M1) and presented two distinct ARDRA patterns. These strains belonged to the γ-proteobacteria subdivision, with members of Salicola marasensis (83%) being the most predominant species among the isolates. 16S rRNA gene sequencing revealed that Salicola strains displayed different degrees of homogeneity. The results from pulsed field gel electrophoresis assays showed that the Salicola isolates could be clustered in two distinct groups with different genome sizes.

Seasonal distribution of ultraphytoplankton and heterotrophic prokaryotes in relation to abiotic variables on the north coast of Sfax after restoration.

The Taparura project was set up to restore the north Sfax coast (Tunisia) by shutting down the northern phosphate plant responsible for chronic pollution and uncontrolled phosphogypsum dumping. The restoration effect on coastal ultraphytoplankton (<10 µm) and heterotrophic prokaryotes was investigated using conventional flow cytometry over four successive seasons during 2009-2010. Cell concentrations were generally higher than values reported for the open sea, both in the western and eastern Mediterranean basins. One striking point was that chl a concentration on the north Sfax coast was unchanged after restoration but was still one order of magnitude higher than in the Gulf of Gabès. Restoration of pH, following the shutdown of the phosphate processing plants on the north coast, appeared to reach normal levels for seawater during the study, whereas seawater acidification persisted on the south coast where plants are still in operation. The largest ultraphytoplankton biomass was from an unknown cell group, whose identity and role needs to be established.

Spring plankton community structure and distribution in the north and south coasts of Sfax (Tunisia) after north coast restoration.

The first phase of the Taparura Project aimed at restoring the north coast of Sfax (Tunisia), highly polluted by phosphate industry and uncontrolled phosphogypsum dumping. Before restoration of the north coast of Sfax, we investigated the state of the ecosystem in related coastal waters. To establish the impact of the Taparura Project, we conducted a similar study both after restoration. To discriminate natural changes over time, we extended the study to the south coast of Sfax, submitted to the same industrial pressure but not yet restored. The present study, conducted in May 2010 at 36 stations (18 on each coast, north and south), covered the spatial distribution of the microbial assemblage, nutrients, and abiotic parameters by collecting seawater samples at the surface and the water-sediment interface. Results revealed a striking difference between the two coasts regarding pH, with strong acidification of seawater in the south, likely generated by industrial activity. Suspended matter was higher in the north than in the south. Flow cytometry analysis of ultraphytoplankton (<10 µm) resolved six groups (Prochlorococcus, Synechococcus, nanoeukaryotes and three distinct subgroups within picoeukaryotes). In addition to these autotrophic groups, two unknown groups were characterised on the south coast. Heterotrophic prokaryotes were resolved into three groups, labelled LNA (low nucleic acid content), HNA1 and HNA2 (high nucleic acid content). Prochloroccocus, pico-nano-microphytoplankton, heterotrophic prokaryotes and ciliates were more abundant in the north, whereas Synechococcus and unknown species were more abundant in the south where chl a concentration was also higher. The results show that restoration had positive effects on the microbial assemblage of the north coast; they also highlight the strong acidification still prevalent in the south that may be responsible for the lower development of most phytoplankton groups and the occurrence of unknown species. The case for restoration of the city's south coast is also reinforced.

Restoration impact of an uncontrolled phosphogypsum dump site on the seasonal distribution of abiotic variables, phytoplankton and zooplankton along the near shore of the south-western Mediterranean coast.

'In connection with the Taparura Project, we studied the distribution of phytoplankton and zooplankton communities in relation to environmental variables at 18 stations sampled during four coastal cruises conducted between October 2009 and July 2010 on the north coast of Sfax (Tunisia, western Mediterranean Sea). The inshore location was largely dominated by diatoms (66 %) represented essentially by members of the genera Navicula, Grammatophora, and Licmophora. Dinophyceae were numerically the second largest group and showed an enhanced species richness. Cyanobacteriae developed in association with an important proliferation of colonial Trichodesmium erythraeum, contributing 39.4 % of total phytoplankton abundances. The results suggest that phytoplankters are generally adapted to specific environmental conditions. Copepods were the most abundant zooplankton group (82 %) of total zooplankton. A total of 21 copepod species were identified in all stations, with an overwhelming abundance of Oithona similis in autumn and summer, Euterpina acutifrons in winter, and Oncaea conifera in spring. The phosphogypsum restoration had been acutely necessary allowing dominant zooplankton species to exploit a wide range of food resources including phytoplankton and thus improving water quality.

Virioplankton community structure in Tunisian solar salterns.

The microbial community inhabiting Sfax solar salterns on the east coast of Tunisia has been studied by means of different molecular and culture-dependent tools that have unveiled the presence of novel microbial groups as well as a community structure different from that of other coastal hypersaline environments. We have focused on the study of the viral assemblages of these salterns and their changes along the salinity gradient and over time. Viruses from three ponds (C4, M1, and TS) encompassing salinities from moderately hypersaline to saturated (around 14, 19, and 35%, respectively) were sampled in May and October 2009 and analyzed by transmission electron microscopy (TEM) and pulsed-field gel electrophoresis (PFGE). Additionally, for all three October samples and the May TS sample, viral metagenomic DNA was cloned in fosmids, end sequenced, and analyzed. Viral concentration, as well as virus-to-cell ratios, increased along the salinity gradient, with around 10(10) virus-like particles (VLPs)/ml in close-to-saturation ponds, which represents the highest viral concentration reported so far for aquatic systems. Four distinct morphologies could be observed with TEM (spherical, tailed, spindled, and filamentous) but with various proportions in the different samples. Metagenomic analyses indicated that every pond harbored a distinct viral assemblage whose G+C content could be roughly correlated with that of the active part of the microbial community that may have constituted the putative hosts. As previously reported for hypersaline metaviromes, most sequences did not have matches in the databases, although some were conserved among the Sfax metaviromes. BLASTx, BLASTp, and dinucleotide frequency analyses indicated that (i) factors additional to salinity could be structuring viral communities and (ii) every metavirome had unique gene contents and dinucleotide frequencies. Comparison with hypersaline metaviromes available in the databases indicated that the viral assemblages present in close-to-saturation environments located thousands of kilometers apart presented some common traits among them in spite of their differences regarding the putative hosts. A small core metavirome for close-to-saturation systems was found that contained 7 sequences of around 100 nucleotides (nt) whose function was not hinted at by in silico search results, although it most likely represents properties essential for hyperhalophilic viruses.

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia.

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of "species" during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.

Impacts of an uncontrolled phosphogypsum dumpsite on summer distribution of phytoplankton, copepods and ciliates in relation to abiotic variables along the near-shore of the southwestern Mediterranean coast.

In connection with the Taparura Project, studies of spatial distribution of the crustacean zooplankton community, nutrients, phytoplankton and ciliates were conducted in July 2007 at 45 stations spread over fifteen transects along the coast north of Sfax. The results showed that the N/P ratio was lower than the Redfield ratio, suggesting potential N limitation. Phytoplankton was characterised by the proliferation of several diatoms, while ciliates were largely dominated by spirotrichs. Copepods were the most abundant zooplankton present during the entire study period, comprising 61% of the total zooplankton community. Twelve copepod families were identified at every station, with a high percentage of Oithonidae (77% of copepods) dominated by Oithona nana. The abundance of this species was correlated with that of diatoms, Cocoolithophorideae and ciliated Colpodea, suggesting that O. nana may feed on a wide range of prey. Despite human pressure and industrial activities, the coastal waters north of Sfax showed a wide diversity of phytoplankton, ciliates and zooplankton.