Extraction and characterization of chitin, chitosan, and protein hydrolysate from the invasive Pacific blue crab, Portunus segnis (Forskål, 1775) having potential biological activities.

The diversity of marine biomasses is a set of exploitable and renewable resources with application in several sectors. In this context, a co-culture based on three protease-producing bacterial isolates, namely Aeribacillus pallidus VP3, Lysinibacillus fusiformis C250R, and Anoxybacillus kamchatkensis M1V strains, was carried out in a medium based on the blue swimming crab Portunus segnis bio-waste. Proteases production was optimized using a central composite design (CCD). The highest level of proteases production obtained was 8,809 U/mL in a medium comprising 75 g/L of Portunus segnis by-product powder (Pspp). The biological value of Pspp and its obtained derivatives were evidenced via accredited protocols. The recovered protein hydrolysate (PHyd) was found to be active towards radical scavenging power and against angiotensin I-converting enzyme (ACE). The blue crab chitin (BC) extraction efficiency was achieved with a yield of 32%. Afterwards, chitosan was prepared through chitin N-deacetylation with a yield of 52%, leading to an acetylation degree (AD) of 19% and solubility of 90%. In addition, chitosan is found to be active against the growth of all pathogenic bacteria tested.

The Mediterranean mussel Mytilus galloprovincialis: responses to climate change scenarios as a function of the original habitat.

The impact of simulated seawater acidification and warming conditions on specimens of the mussel Mytilus galloprovincialis locally adapted to very distinct, widely separated sites in the Mediterranean Sea (Tunisia) and Atlantic Sea (Galicia, NW Spain) was evaluated in relation to key behavioural and eco-physiological parameters. Over the 2-month exposure to the experimental conditions, mussels were fed optimally to ensure that there are no synergistic interactions between climate change drivers and energetic status of the individuals. In general, regardless of origin (Atlantic or Mediterranean), the mussels were rather resilient to acidification for most of the parameters considered and they were able to grow in strongly acidified seawater through an increased feeding activity. However, shell strength decreased (40%) consistently in both mussel populations held in moderately and highly acidified seawater. The observed reduction in shell strength was not explained by slight alterations in organic matter, shell thickness or aragonite:calcite ratio. The combined effects of high acidification and warming on the key response of byssus strength caused a strong decline in mussel performance, although only in Galician mussels, in which the valve opening time decreased sharply as well as condition index (soft tissue state) and shell growth. By contrast, the observed negative effect of highly acidified scenario on the strength of Tunisian mussel shells was (partly but not totally) counterbalanced by the higher seawater temperature. Eco-physiological and behavioural interactions in mussels in relation to climate change are complex, and future scenarios for the ecology of the species and also the feasibility of cultivating them in Atlantic and Mediterranean zones are discussed.

Economic impact of human-induced shrinkage of Posidonia oceanica meadows on coastal fisheries in the Gabes Gulf (Tunisia, Southern Mediterranean Sea).

In early XXth century, the Gulf of Gabes (SE Tunisia) used to host the most extended Posidonia oceanica seagrass beds in the Mediterranean Sea, and a highly productive hotspot of benthic/demersal biodiversity. Sponge harvesting and seabed trawling provoked a first step of seagrass degradation. Subsequently, phosphogypsum releases from Gabes Industrial Complex, since mid-1970s, accelerated the decline of the remaining patches. A sharp reduction of coastal fisheries landings took place with the establishment of the last industrial plant units in 1985. The decrease in coastal commercial species landings was found to be directly correlated with P. oceanica decline. The trophic web system switched from a 'benthic-dominated' to a 'pelagic-dominated' system. The economic loss related to coastal fisheries was estimated at ~60 million € in 2014 and the 1990-2014 cumulated loss exceeded 750 million €. This first economic valuation of the only direct-use consumptive value of the coastal fishing service provided by P. oceanica in Gabes Gulf is a first step towards the assessment of the environmental cost of the negative externalities caused by the local phosphate industry. It may be used as a preliminary decision-making aid to consider alternative industrial solutions.

Statistical Experimental Design Optimization of Microbial Proteases Production under Co-Culture Conditions for Chitin Recovery from Speckled Shrimp Metapenaeus monoceros By-Product.

This study was designed with the aim to produce microbial proteases in presence of speckled shrimp by-product. For this reason, three strains belonging to Bacillus genus, namely, Aeribacillus pallidus VP3, Lysinibacillus fusiformis C250R, and Anoxybacillus kamchatkensis M1V were studied under co-culture procedure. A Taguchi L27 experimental design was applied to optimize the co-culture parameters. The experimental design was built with 9 factors (by-product powder concentration, the pH of the medium, the temperature, the sucrose concentration, the agitation speed, the inoculum sizes of VP3, M1V, and C250R strains, and the culture volume) at three different levels. The obtained results showed that a total protease activity of 8,182 U/mL could be achieved after 24 h of incubation in presence of 20 g/L shrimp by-product and 10 g/L sucrose, at an initial pH of 7, a 40°C temperature and absorbance, at 600 nm, of inoculum sizes of 0.1, 0.3, and 0.1 for VP3, M1V, and C250R strains, respectively. The agitation was set at 200 rpm, and the final volume was 25 mL. Taguchi's design allowed the identification of temperature, the inoculum size for strain VP3, the inoculum size for strain M1V, and the final culture volume as the most influencing variables. A Box-Behnken design with 27 experiments was carried out for the optimization of these four selected factors. Following such design, the highest protease production reached was 11,300 U/mL. This yield was obtained in a final culture volume of 15 mL containing 20 g/L shrimp by-product powder and 10 g/L sucrose and inoculated with VP3, C250R, and M1V strains at 0.05, 0.1, and 0.2, respectively. The flasks were incubated at 45°C for 24 h with shaking at 200 rpm. The efficiency of chitin extraction by co-cultivation was investigated under the latter conditions. The chitin yield from shells by-product was 16.7%. Fourier-Transform Infrared (FTIR) analysis of the obtained chitin displayed characteristic profiles similar to that of the commercial α-chitin.

Characterization of the role of phosphogypsum foam in the transport of metals and radionuclides in the Southern Mediterranean Sea.

The Gabes Gulf had received huge quantities of phosphogypsum discharged from fertilizer plants. Dumping phosphogypsum in coastal waters leads to the formation of foam layers which can float on the surface and be passively transported to distant areas. This is the first attempt at geochemical and mineralogical characterization of these industrial foams in order to understand their role in the dynamic and behavior of contaminants in marine environment. Chemically, phosphogypsum foams (PGFs) are heavily loaded with radiochemical contaminants. Their mineralogical composition showed a prevalence of synthetic gypsum followed by other secondary minerals including halite, quartz, dolomite, sphalerite-Cd and fluorapatite. PGFs are rich in organic matter (OM), precursor of their formation. Once released in gypseous water, the OM in solution undergoes agglomeration, cementing and flotation steps leading to the formation of floating foams. The foams' OM was found to control the mobility of industrial contaminants contributing then to the marine environment pollution. Consequently, PGFs are the main accumulating, transporting and dispersion agent of phosphogypsum radiochemical contaminants. Thus, PGFs removal has the potential to reduce enormously the dynamics of contaminants transferred from the fertilizer plants to the aquatic environment, reducing thus their impacts on the marine environment and health status in Gabes.

Characterization of phosphate rock and phosphogypsum from Gabes phosphate fertilizer factories (SE Tunisia): high mining potential and implications for environmental protection.

Since the establishment of the coastal industrial complex in Gabes city (Gulf of Gabes, SE Tunisia), hundred million tons of untreated phosphogypsum have been discharged in the open sea causing serious environmental problems. To better understand the dynamic and behavior of phosphate/phosphogypsum contaminants from raw ores to marine environment, a chemical, organic, mineralogical, and morphological characterization of phosphate rock and phosphogypsum was conducted using several sophisticated techniques. The chemical analysis showed that phosphate and phosphogypsum contain high loads of trace elements and that the transfer factors of pollutants varied from 5.83% (U) to 140% (Hg). Estimated annual flows of phosphogypsum contaminants into the marine environment ranged between 0.05 (Re) and 87,249.60 (F) tons. The phosphate rock was found to be formed by carbonate fluorapatite, calcite, dolomite, natural gypsum, quartz, calcite-Mg, apatite, pyrite, fluorite, and sphalerite-Cd and phosphogypsum by synthetic gypsum and sphalerite-Cd. The phosphate was found to be richer in organic compounds compared to phosphogypsum. Based on this work, the Tunisian phosphogypsum has a high mining potential and encourages the development of an economically beneficial and environmentally friendly phosphogypsum-treating industry.

Seawater quality assessment and identification of pollution sources along the central coastal area of Gabes Gulf (SE Tunisia): Evidence of industrial impact and implications for marine environment protection.

Temperature, pH and trace elements (F, P, Cr, Cu, Zn, Cd, and Pb) contents were determined in 16 stations as well as in 2 industrial and 2 domestic discharge sources, in the central coastal area of the Gulf of Gabes. Compared to the northern and southern areas of the study area, the highest contents of contaminants were reached in the central area which hosts the coastal industrial complex. The seawater in this central area was also found to be acid and of higher temperature. Based on the Water Pollution Index results, an increasing degradation gradient of the seawater quality was revealed from northern and/or southern stations to central ones, categorized as 'strongly to seriously affected'. Phosphogypsum wastes dumped by the Tunisian Chemical Group (GCT) seem to have continuously degraded the seawater quality in the study area. A rapid intervention is needed to stop the effects on the marine environment.

Biomonitoring of coastal pollution in the Gulf of Gabes (SE, Tunisia): use of Posidonia oceanica seagrass as a bioindicator and its mat as an archive of coastal metallic contamination.

Within the framework of a study on the extent and history of marine pollution in the central area of Gabes Gulf, the concentrations of four trace metals (Cd, Cu, Pb, and Zn) were assessed in three different tissues of the seagrass species, Posidonia oceanica (leaves, rhizomes, and roots), in the mat of P. oceanica meadows at different depths from the sea floor (- 30 to - 150 cm) and in sediments. The results showed that P. oceanica leaves accumulate more Cd, Cu, and Zn, whereas Pb was found to be more concentrated in roots. The analysis of P. oceanica mat highlighted a clear decreasing gradient of trace metal concentrations from lower (- 30 cm) to higher (- 150 cm) depths. Considering that P. oceanica mat continuously rises above the initial level, with a rate of 1 m per century (~1 cm year-1; Molinier and Picard Ann Inst Océanogr Fr 27:157-234, 1952), the latter observation suggested that the pollution level in Gabes Gulf increased continuously during the last few decades. The results of the P. oceanica mapping in the study area showed a continuous regression of its meadows as well as its local disappearance from various areas in the central part of Gabes Gulf. The current status of P. oceanica is most likely due to the cumulative effects of the discharges from the coastal industrial complex of Gabes-Ghannouche represented mainly by the phosphogypsum discharges. This study confirms the usefulness of P. oceanica not only as a bioindicator of the "health status" of coastal ecosystems but also as a record register tracing back the history and temporal evolution of coastal contamination level.

Heavy metal contamination and ecological risk assessment in the surface sediments of the coastal area surrounding the industrial complex of Gabes city, Gulf of Gabes, SE Tunisia.

In the present study, the concentrations of 6 trace metals (Hg, Cd, Cu, Pb, Cr and Zn) were assessed in the surface sediments of the central coastal area of Gabes Gulf to determine their contamination status, source, spatial distribution and ecological risks. The ranking of metal contents was found to be Zn>Cd>Cr>Pb>Cu>Hg. Correlation analysis indicated that Cd and Zn derived mainly from the Tunisian Chemical Group phosphogypsum. The other pollutants may originate from other industrial wastes. Metallic contamination was detected in the south of chemical complex, especially in the inter-harbor zone, where the ecological risk of surface sediments is the highest, implying potential negative impacts of industrial pollutants. The spatial distribution of pollutants seems to be due to the effect of harbor installations and coastal currents. The metallic pollution status of surface sediments of Gabes Gulf is obvious, very worrying and requires rapid intervention.

Dynamic energy budget model: a monitoring tool for growth and reproduction performance of Mytilus galloprovincialis in Bizerte Lagoon (Southwestern Mediterranean Sea).

Mussel farming is the main economic activity in Bizerte Lagoon, with a production that fluctuates depending on environmental factors. In the present study, we apply a bioenergetic growth model to the mussel Mytilus galloprovincialis, based on dynamic energy budget (DEB) theory which describes energy flux variation through the different compartments of the mussel body. Thus, the present model simulates both mussel growth and sexual cycle steps according to food availability and water temperature and also the effect of climate change on mussel behavior and reproduction. The results point to good concordance between simulations and growth parameters (metric length and weight) for mussels in the lagoon. A heat wave scenario was also simulated using the DEB model, which highlighted mussel mortality periods during a period of high temperature.

Characterization of the protozoan parasite Marteilia refringens infecting the dwarf oyster Ostrea stentina in Tunisia.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. The life cycle of this species is still poorly known, although there is evidence of the need for intermediate host(s). In the present study, we have used molecular approaches to identify this parasite in samples of the dwarf oyster Ostrea stentina after reports of massive mortality along the Tunisian coasts. In 2009 we evaluated the status of O. stentina from Monastir and checked if there was an infection with M. refringens, using polymerase chain reaction assays. Of the 103 tested O. stentina, 85 were PCR-positive using a Marteilia genus-specific assay. Additional assays were subsequently carried out on some samples collected in 2010 in Monastir and processed for histology, transmission electron microscopy and complementary molecular analyses. PCR was carried out to amplify the IGS and ITS regions. Histological and transmission electron microscopy analyses allowed us to confirm the presence of this parasite in the digestive gland tissue of O. stentina and to characterize it at the ultrastructural level. This is the first record of the occurrence of M. refringens in the oyster O. stentina along the Tunisian coasts.

Observation of a Bonamia sp. infecting the oyster Ostrea stentina in Tunisia, and a consideration of its phylogenetic affinities.

The small non-commercial oyster Ostrea stentina co-occurs with commercially important Ostrea edulis in the Mediterranean Sea, yet its disposition with respect to the destructive pathogens Bonamia ostreae and Marteilia refringens is unknown. We began an evaluation of the Bonamia spp. infection status of O. stentina from Hammamet, Tunisia, in June 2007 using polymerase chain reaction diagnostics followed by histology and in situ hybridization. Of 85 O. stentina sampled, nine were PCR-positive for a Bonamia sp. using a Bonamia genus-specific assay; of these nine, one displayed the uninucleate microcells associated with oyster hemocytes characteristic of Bonamia spp. There was no associated pathology. DNA sequencing of the parasite from this one infected individual revealed it to be of a member of the Bonamia exitiosa/Bonamia roughleyi clade, an identification supported by positive in situ hybridization results with probes specific for members of this clade, and by the morphology of the parasite cells: nuclei were central, as in B. exitiosa, not eccentric, as in B. ostreae. There is no basis for identifying the Tunisian parasite as either B. exitiosa or B. roughleyi, however, as these species are genetically indistinguishable. Likewise, there is no basis for identifying any of the other Bonamia spp. with affinities to the B. exitiosa/B. roughleyi clade, from Argentina, Australia, Spain, and the eastern USA, as one or the other of these named species. Though they are clearly distinct from Bonamia perspora and B. ostreae, justification for drawing species boundaries among the primarily austral microcells with affinities to B. exitiosa and B. roughleyi remains elusive.