Hydrolytic enzyme screening and carotenoid production evaluation of halophilic archaea isolated from highly heavy metal-enriched solar saltern sediments.

This paper aimed to screen the enzymatic activities and evaluate the carotenoid production level of twenty-two halophilic archaea isolated from Sfax solar saltern sediments. The molecular identification performed by sequencing the 16S rRNA genes showed that all strains have a high similarity degree (99.7-100%) with Halobacterium salinarum NRC-1. The strains were screened for the presence of eight hydrolase activities using agar plate-based assays. The most detected enzyme was gelatinase (77.27% of total strains), followed by protease (63.63%) and amylase activities (50%). The carotenoid production yields of the strains ranged between 2.027 and 14.880 mg/l. The UV-Visible spectroscopy of pigments revealed that it was a bacterioruberin type. When evaluated and compared to the standard ß-carotene, the antioxidant capacities of these pigments showed a scavenging activity of more than 75% at a concentration of 5 µg/ml for three strains (AS16, AS17, and AS18). Then a sequence of one-step optimization processes was performed, using the one-factor-at-a-time approach, to define the optimum conditions for growth and carotenoid production of the highest carotenoid producing strain (AS17). Different environmental factors and nutritional conditions were tested. Variations in these factors were found to deeply influence growth and carotenoid production. A maximum carotenoid production (16.490 mg/l), higher than that of the control (14.880 mg/l), was observed at 37 °C, pH 7, 250 g/l of salinity, with 80% air phase in the flask at 110 rpm, in presence of light and in culture media containing (g/l) 10, yeast extract; 7.5, casamino acid; 20, MgSO4; 4, KCl; and 3, trisodium citrate.

Genomic analysis of heavy metal-resistant Halobacterium salinarum isolated from Sfax solar saltern sediments.

The draft genome sequences of five archaeal strains, isolated from Sfax solar saltern sediments and affiliated with Halobacterium salinarum, were analyzed in order to reveal their adaptive strategies to live in hypersaline environments polluted with heavy metals. The genomes of the strains (named AS1, AS2, AS8, AS11, and AS19) are found to contain 2,060,688; 2,467,461; 2,236,624; 2,432,692; and 2,428,727 bp respectively, with a G + C content of 65.5, 66.0, 67.0, and 66.2%. The majority of these genes (43.69-55.65%) are annotated as hypothetical proteins. Growth under osmotic stress is possible by genes coding for potassium uptake, sodium efflux, and kinases, as well as stress proteins, DNA repair systems, and proteasomal components. These strains harbor many genes responsible for metal transport/resistance, such as: copper-translocating P-type ATPases, ABC transporter, and cobalt-zinc-cadmium resistance protein. In addition, detoxification enzymes and secondary metabolites are also identified. The results show strain AS1, as compared to the other strains, is more adapted to heavy metals and may be used in the bioremediation of multi-metal contaminated environments. This study highlights the presence of several commercially valuable bioproducts (carotenoids, retinal proteins, exopolysaccharide, stress proteins, squalene, and siderophores) and enzymes (protease, sulfatase, phosphatase, phosphoesterase, and chitinase) that can be used in many industrial applications.

Resistance of a Halobacterium salinarum isolate from a solar saltern to cadmium, lead, nickel, zinc, and copper.

The current study focuses on the tolerance of a strain of Halobacterium salinarum isolated from Sfax solar saltern (Tunisia) towards cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn), and copper (Cu) by using agar dilution methods in complex and minimal media. The results showed the least inhibitory metals based on Minimum Inhibitory Concentrations (MICs) were lead (MIC = 4.5 mM), cadmium (MIC = 4 mM), and nickel (MIC = 2.5 mM) in complex medium. The MICs of these metals were more inhibitory (MIC < 2 mM) in the other tested media. The archaeal strain revealed a high sensitivity for copper and zinc, with MICs below 0.5 mM for both metals. Growth kinetics in complex and minimal media showed the strain to be more sensitive to the metals in liquid media than in solid media. The growth kinetic assays indicated the presence of selected heavy metals resulted in a lower growth rate and lower total cell mass relative to the control. Despite that cadmium and lead are nonessential and have no nutrient value, they were the most tolerated metals by H. salinarum strain. In addition, pigment intensity in the strain was inhibited by the presence of the heavy metals relative to the control.

Biological properties of carotenoids extracted from Halobacterium halobium isolated from a Tunisian solar saltern.

BACKGROUND: Bioactive molecules have received increasing attention due to their nutraceutical attributes and anticancer, antioxidant, antiproliferative and apoptosis-inducing properties. This study aimed to investigate the biological properties of carotenoids extracted from Archaea. METHODS: Halophilic Archaea strains were isolated from the brine of a local crystallizer pond (TS7) of a solar saltern at Sfax, Tunisia. The most carotenoid-producing strain (M8) was investigated on heptoma cell line (HepG2), and its viability was assessed by the MTT-test. The cells were incubated with different sub-lethal extract rates, with carotenoid concentrations ranging from 0.2 to 1.5 µM. Antioxidant activity was evaluated through exposing the cells to sub-lethal extract concentrations for 24 hours and then to oxidative stress induced by 60 µM arachidonic acid and 50 µM H2O2. RESULTS: Compared to non-treated cells, bacterial carotenoid extracts inhibited HepG2 cell viability (50%). A time and dose effect was observed, with cell viability undergoing a significant (P < 0.05) decrease with extract concentration. After exposure to oxidative stress, control cells underwent a significant (P < 0.05) decrease in viability as compared to the non-treated cells. CONCLUSIONS: The bacterial extracts under investigation were noted to exhibit the strongest free radical scavenging activity with high carotenoid concentrations. The carotenoid extract also showed significant antiproliferative activity against HepG2 human cancer cell lines.

Assessment of sediment quality in the Mediterranean Sea-Boughrara lagoon exchange areas (southeastern Tunisia): GIS approach-based chemometric methods.

Concentrations of selected heavy metals (Cd, Pb, Zn, Cu, Mn, and Fe) in surface sediments from 66 sites in both northern and eastern Mediterranean Sea-Boughrara lagoon exchange areas (southeastern Tunisia) were studied in order to understand current metal contamination due to the urbanization and economic development of nearby several coastal regions of the Gulf of Gabès. Multiple approaches were applied for the sediment quality assessment. These approaches were based on GIS coupled with chemometric methods (enrichment factors, geoaccumulation index, principal component analysis, and cluster analysis). Enrichment factors and principal component analysis revealed two distinct groups of metals. The first group corresponded to Fe and Mn derived from natural sources, and the second group contained Cd, Pb, Zn, and Cu originated from man-made sources. For these latter metals, cluster analysis showed two distinct distributions in the selected areas. They were attributed to temporal and spatial variations of contaminant sources input. The geoaccumulation index (I (geo)) values explained that only Cd, Pb, and Cu can be considered as moderate to extreme pollutants in the studied sediments.

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.

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.

Microbial community of salt crystals processed from Mediterranean seawater based on 16S rRNA analysis.

Phylogenetic analysis of 16S rRNA was used to investigate for the first time the structure of the microbial community that inhabits salt crystals retrieved from the bottom of a solar saltern, located in the coastal area of the Mediterranean Sea (Sfax, Tunisia). This community lives in an extremely salty environment of 250-310 g/L total dissolved salt. A total of 78 bacterial 16S rRNA clone sequences making up to 21 operational taxonomic units (OTUs), determined by the DOTUR program to 97% sequence similarity, was analyzed. These OTUs were affiliated to Bacteroidetes (71.4% of OTUs), and gamma-Proteobacteria and alpha-Proteobacteria (equally represented by 14.2% of the OTUs observed). The archaeal community composition appeared more diverse with 68 clones, resulting in 44 OTUs, all affiliated with the Euryarchaeota phylum. Of the bacterial and archaeal clones showing <97% 16S rRNA sequence identity with sequences in public databases, 47.6% and 84.1% respectively were novel clones. Both rarefaction curves and diversity measurements (Simpson, Shannon-Weaver, Chao) showed a more diverse archaeal than bacterial community at the Tunisian solar saltern pond. The analysis of an increasing clone's number may reveal additional local diversity.

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.

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.