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.     

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Aim

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Methods and Results

The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.

Conclusion

The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.

Significance and Impact of the Study

This technique allowed rapid C. trachomatis genotype identification.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S 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. A. Sghir and E. Ammar have equally contributed to this work.     

Molecular community analysis of magnesium-rich bittern brine recovered from a Tunisian solar saltern

The microbial community of a magnesium-rich bittern brine saturated with NaCl (380-400 g/L) from a Tunisian solar saltern was investigated using a molecular approach based on 16S rRNA gene analysis and viability tests. The results revealed the existence of microbial flora. Viability test assessment showed that 46.4% of this flora was viable but not detectable by culturability tests. 16S rRNA genes from 49 bacterial clones and 38 archaeal clones were sequenced and phylogenetically analyzed. Eleven operational taxonomic units (OTUs) determined by the DOTUR program with 97% sequence similarity were generated for Bacteria. These OTUs were affiliated with Bacteroidetes and Gammaproteobacteria. The archaeal community composition exhibited more diversity with 38 clones, resulting in 13 OTUs affiliated with the Euryarchaeota phylum. Diversity measurement showed a more diverse archaeal than bacterial community at the saturated pond.     

First Report of Tomato Yellow Leaf Curl Virus-Israel Species Infecting Tomato, Pepper and Bean in Tunisia

Tomato yellow leaf curl virus disease (TYLCVD) has been observed in Tunisia for more than 20 years. Until year 2004, only the Tomato yellow leaf curl Sardinia virus‐Sicily (TYLCSV‐[Sic]) was detected in tomato, pepper and bean crops. In the Sahel region, some tomato samples showing severe TYLCVD symptoms were collected from greenhouses in 2004 and 2005. Typing of these isolates revealed for the first time the presence of the TYLCV Israel in Tunisia. This result was confirmed by using several sets of specific primers and by sequencing. This species has also been detected on pepper and bean collected from fields in the same region. The sequencing of a tomato and a bean isolate showed that they both share more than 97% of sequence identity with the TYLCV from Dominican Republic ( AF024715 ). The TYLCV has been found in single and mixed infection with the TYLCSV‐[Sic].     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

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.