Néji Gharsallah

Prokaryotic diversity of a Tunisian multipond solar saltern

Abstract16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied.

The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia

BackgroundIn order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines.MethodsThe agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test.ResultsThe antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M. vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC50 value of 0.258 μg/ml.ConclusionThis investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential oil as anticancer agent. Further research is required to evaluate the practical values of therapeutic applications.

Olive mill wastewater stabilization in open-air ponds: Impact on clay–sandy soil

The aim of this work was to study the natural biodegradation of the stored olive mill wastewater (OMW) in ponds and the infiltration as well as the impact on soil of the effluent in the evaporation pond used for the storage over the past eight years. For this, two approaches were considered. First, a laboratory-scale column was used for the infiltration of OMW through soil (clay and sand) to predict the effect of the clayey soil in reducing OMW pollution. Second, the ponds including the effluent annually stored and having this clayey structure were investigated. At the laboratory-scale, a modification of OMW contents was noticed, with the elimination of 95% of total suspended solids (TSS), 60% of chemical oxygen demand (COD), 40% of total organic carbon (TOC), 50% of total P, 50% of phenols and 40% of minerals (K+, Mg++ and Na+). The experimented soil was able to restrain the considerable effects of OMW pollution. In the ponds, the granulometric characteristics, the physico-chemical and the biological parameters of the soil profile from the contaminated pond were compared to those of a control soil, located near the contaminated pond. Property modifications of the contaminated soil were noted, especially pH, electrical conductivity, COD and microflora. These changes can be explained by the infiltration of OMW constituents, which were noticed in the soil layers, especially phenolic compounds that have a negative effect on the ground water.

Olive mill wastewater evaporation management using PCA method Case study of natural degradation in stabilization ponds (Sfax, Tunisia)

Olive mill wastewater (OMW) evaporation ponds management was investigated in five serial evaporation open-air multiponds of 50 ha located in Sfax (Tunisia). Physico-chemical parameters and microbial flora evolution were considered. Empirical models describing the OMW characteristic changes with the operation time were established and Principal Component Analysis (PCA) described the correlation between physico-chemical and biological parameters. COD, BOD, total solids, polyphenols and electrical conductivity exhibited first-order models. Four groups exhibited high correlations. The first included temperature, density, COD, TSS, TS, BOD, VS, TOC, TKN, polyphenols and minerals. The second group was made up of yeasts and moulds. The third group was established with phenolic compounds, total sugars, fats, total phosphorous, NH(4)(+) and pH. The fourth group was constituted by exclusively aerobic bacteria. Bacterial-growth toxic effect was exhibited by high organic load, ash content and polyphenols, whereas moulds and yeasts were more adapted to OMW. During the storage, all the third group parameter values decreased and were inversely related to the others. In the last pond, COD, BOD, TS and TSS rates were reduced by 40%, 50%, 50% and 75% respectively. The evaporation and the biological activity were the main processes acting, predicting the OMW behavior during evaporation in air-open ponds.

Production of single cell protein from olive mill waste water by yeasts

Abstract Studies have been made of the growth of three yeasts Candida krusei, Saccharomyces chevalierie and Saccharomyces rouxii on olive mill wastewater (OMW). The cultures were made in a shaker flask culture and in a fermentor in order to select organisms which can produce large quantities of biomass. Three yeasts have been selected that will tolerate the polyphenols. They can be used to reduce Biological Oxygen Demand (BOD) (40–50%), and perhaps become a source of Single Cell Protein (SCP) for supplementation of animal feeds. Concentration of protein of 3.35 g 1‐1 and yield of 0.45g of biomass per g of glucose based on glucose consumption were obtained using Saccharomyces rouxii strain.

Essential oil of the leaves of Ricinus communis L.: In vitro cytotoxicity and antimicrobial properties

BackgroundThe aim of the present study was to appraise the antimicrobial activity of Ricinus communis L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines.MethodsThe agar disk diffusion method was used to study the antibacterial activity of Ricinus communis L. essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of Ricinus communis L. essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test.ResultsThe essential oil from the leaves of Ricinus communis L. was analyzed by GC–MS and bioassays were carried out. Five constituents of the oil were identified by GC–MS. The antimicrobial activity of the oil was investigated in order to evaluate its efficacy against twelve bacteria and four fungi species, using disc diffusion and minimum inhibitory concentration methods. The essential oil showed strong antimicrobial activity against all microorganisms tested with higher sensitivity for Bacillus subtilis, Staphylococcus aureus and Enterobacter cloacae. The cytotoxic and apoptotic effects of the essential oil on HeLa cell lines were examined by MTT assay. The cytotoxicity of the oil was quite strong with IC50 values less than 2.63 mg/ml for both cell lines.ConclusionThe present study showed the potential antimicrobial and anticarcinogenic properties of the essential oil of Ricinus communis L., indicating the possibilities of its potential use in the formula of natural remedies for the topical treatment of infections.

Novel prokaryotic diversity in sediments of Tunisian multipond solar saltern.

The phylogenetic diversity of a sediment microbial community from two ponds having different salinities, 150-200 g/l (M2) and 250-300 g/l (TS38), of an Sfax (Tunisia) solar saltern, was investigated using 16S rRNA clone libraries. The 16S rRNA genes from 135 bacterial clones and 105 archaeal clones were sequenced and phylogenetically analyzed. 32 operational taxonomic units (OTUs) were generated for Bacteria and 64 for Archaea. The bacterial community in M2 sediment was affiliated only with Bacteroidetes, while that in TS38 sediment was dominated by clones affiliated with Bacteroidetes, (gamma, alpha, delta) Proteobacteria and unclassified bacteria; these represented 56.52, 26.08, 4.34, 4.34 and 8.7% of the OTUs, respectively. In the M2 and TS38 sediments, 44.44 and 43.47% of the bacterial OTUs, respectively, were novel. All archaeal sequences fell into the Euryarchaeota phylum. In both sediments, 38.46 and 72.55% of the OTUs had less than 97% 16S rRNA sequence identity, representing novel OTUs. Two sequences, retrieved from TS38 sediment, were found to be affiliated with the candidate division MSBL-1 defining two OTUs. The sediment phylogenetic study revealed the presence of a highly diverse microbial population in highly salty media.

Isolation and Characterization of Moderately Halophilic Bacteria from Tunisian Solar Saltern

Bacterial screenings from solar saltern in Sfax (Tunisia) lead to the isolation of 40 moderately halophilic bacteria which were able to grow optimally in media with 5–15% of salt. These isolates were phylogenetically characterized using 16S rRNA gene sequencing. Two groups were identified including 36 strains of Gamma-Proteobacteria (90%) and 4 strains of Firmicutes (10%). The Gamma-Proteobacteria group consisted of several subgroups of the Halomonadaceae (52.5%), the Vibrionaceae (15%), the Alteromonadaceae (10%), the Idiomarinaceae (7.5%), and the Alcanivoracaceae (5%). Moreover, three novel species: 183ZD08, 191ZA02, and 191ZA09 were found, show <97% sequence similarity of the 16S rRNA sequences while compared to previously published cultivated species. Most of these strains (70%) were able to produce hydrolases: amylases, proteases, phosphatases, and DNAases. Over the isolates, 60% produced phosphatases, 15.0% proteases, 12.5% amylases and DNAases equally. This study showed that the solar saltern of Sfax is an optimal environment for halophilic bacterial growth, where diverse viable bacterial communities are available and may have many industrial applications.

Time-dependent evolution of olive mill wastewater sludge organic and inorganic components and resident microbiota in multi-pond evaporation system

The physico-chemical and microbiological characterizations of olive mill wastewater sludge (OMWS) were investigated in five OMW evaporation ponds of the open-pond system in Sfax (Tunisia), during the olive oil production period in 2004. Time-dependent changes in both physico-chemical parameters and the microbiota were investigated. Mathematical models and principal component analysis (PCA) were used to establish the correlations between the studied parameters. During the effluent time-dependent changes in the ponds, the result of OMWS analysis showed an increase of sludge index (SI), ash content, total solids (TS), volatile solids (VS), ethyl acetate extractive (EAE) and total phosphorus (Total P), as well as microbial flora especially the yeasts and moulds. The SI, TS, VS and Total P changes with time fit a simple linear equation, while EAE, phenols and NH(4)(+) fit a second-degree polynomial model. The PCA analysis exhibited three correlated groups. The first group included temperature, ash content, evaporation, SI, TS, VS, Total P, EAE, yeasts and moulds. The second group was made by bacteria and moisture; and the third group by NH(4)(+), oil and phenol. Such modelling might be of help in the prediction of OMW changes in natural evaporation ponds.

Chemical constituents and antioxidant activity of the essential oil from aerial parts of Artemisia herba-alba grown in Tunisian semi-arid region

Essential oils and their components are becoming increasingly popular as naturally occurring antioxidant agents. In this work, the composition of essential oil in Artemisia herba-alba from southwest Tunisia, obtained by hydrodistillation was determined by GC/MS. Eighteen compounds were identified with the main constituents namely, α-thujone (24.88%), germacrene D (14.48%), camphor (10.81%), 1,8-cineole (8.91%) and β-thujone (8.32%). The oil was screened for its antioxidant activity with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, β-carotene bleaching and reducing power assays. The essential oil of A. herba-alba exhibited a good antioxidant activity with all assays with dose dependent manner and can be attributed to its presence in the oil. Key words: Artemisia herba alba, essential oil, chemical composition, antioxidant activity.