Sami Maalej

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.

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.

Simultaneous effects of environmental factors on motile Aeromonas dynamics in an urban effluent and in the natural seawater

Seasonal dynamics of motile Aeromonas in a treated urban effluent and in natural seawater along the Sfax coast (Mediterranean sea, Tunisia) were measured over a year concurrently with seven environmental factors, and compared with those of faecal coliforms. Counts for Aeromonas from a standard plate count method, ranged from 1.48 x 10(5)CFU.100 ml(-1) to 2.2 x 10(8)CFU.100 ml(-1) in the effluent and from 7.9 x 10(3)CFU.100 ml(-1) to undetectable level in the surface marine waters. Contrary to faecal coliforms, the Aeromonas dynamics exhibited a seasonal distribution in seawater which was inverse of the seasonal distribution in the sewage: From the end of November 1998 to April 1999 (cold period), Aeromonas counts increased in the treated effluent, while it decreased very rapidly in seawater. From May to October (warm period), Aeromonas abundance decreased in the effluent but showed an increasing fluctuating trend in the marine waters with a maximum in late summer/early autumn when the temperatures were around 22-23 degrees C. Multiple correlation and regression analyses suggest, by the coefficient of determination (R(2)), that 42% of variance in Aeromonas number changes in the treated effluent, may be explained by only turbidity, radiation and Aeromonas density in the previous sample, while 37% of variance in marine ecosystem were explained by radiance and conductivity. Furthermore, the t statistics and their p values and the coefficient of partial determination (r(2)) indicated that radiance contributed the most (r(2)=0.3184, t=-3.2, p=0.0041) to the dynamics of motile Aeromonas in seawater, when combined with conductivity. The models relevant for changes in faecal coliforms abundance incorporated turbidity, radiance in the effluent and conductivity, pH, radiance, turbidity in coastal marine environment. These models explain 66% and 73% of the observed cell number fluctuation, with turbidity (r(2)=0.529, t=5.08, p=0.0001) and conductivity (r(2)=0.5407, t=4.97, p=0.0001) as dominant factors in the multivariate model proposed, respectively, for the two sampling sites. The results presented here suggest that the combination of negative effects of sunlight and conductivity in natural seawater mainly affects the colony-forming capacity and make the motile Aeromonas nonrecoverable during cold months.

Virioplankton Community Structure in Tunisian Solar Salterns

ABSTRACT 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 1010 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.

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 citys south coast is also reinforced.

Inactivation of the gene katA or sodA affects the transient entry into the viable but non-culturable response of Staphylococcus aureus in natural seawater at low temperature

We have investigated the fate of Staphylococcus aureus by starving the cells and maintaining them in natural seawater at 22 and 4 °C. At 22 °C, cells developed a long-term survival state where about 0.037% of the initial population remained culturable over more than 7 months, whereas at 4 °C, bacteria lost culturability and transiently entered into the viable but non-culturable state (VBNC). However, after 22 days of entry into the VBNC state, the number of viable cells detected via the direct viable count method decreased significantly. We show here that mutational inactivation of catalase (KatA) or superoxide dismutase (SodA) rendered strains hypersensitive to seawater stress at 4 °C and consequently, part of the seawater lethality on S. aureus at low temperature is mediated by reactive oxygen species (ROS) during microcosm-survival process. Shifting the temperature from 4 to 22 °C of totally non-culturable wild-type cells induced a partial recovery of the population. However, deficiencies in catalase or superoxide dismutase prevent resuscitation ability.

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.

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 β-carotene and reduced activity. Furthermore, the CPv-PS exhibited effective antimicrobial properties.

Characterization of Halorubrum sfaxense sp. nov., a New Halophilic Archaeon Isolated from the Solar Saltern of Sfax in Tunisia

An extremely halophilic archaeon, strain ETD6, was isolated from a marine solar saltern in Sfax, Tunisia. Analysis of the 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum among the family Halobacteriaceae, with a close relationship to Hrr. xinjiangense (99.77% of identity). However, value for DNA-DNA hybridization between strain ETD6 and Hrr.xinjiangense were about 24.5%. The G+C content of the genomic DNA was 65.1 mol% (T(m)). Strain ETD6 grew in 15–35% (w/v) NaCl. The temperature and pH ranges for growth were 20–55°C and 6–9, respectively. Optimal growth occurred at 25% NaCl, 37°C, and pH 7.4. The results of the DNA hybridization against Hrr. xinjiangense and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain ETD6 from other Hrr. species. Therefore, strain ETD6 represents a novel species of the genus Halorubrum, for which the name Hrr. sfaxense sp. nov. is proposed. The Genbank EMBL-EBI accession number is GU724599.

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.