In this study, the effects of bicarbonate addition on growth and pigment contents of the unicellular microalga Nannochloropsis oculata, were evaluated. N. oculata represents an interesting source of biomolecules widely used for food supplements and nutraceuticals. The bicarbonate was supplemented to microalgae cultures at concentrations of 0, 6, 18, 30, 42 and 60 mM. The cultures supplemented with salt at highest concentrations (42 and 60 mM) showed a significant increase in algal growth, demonstrated by the optical density spread. The intracellular content of pigments such as chlorophyll a and total carotenoids reached the highest values in cells from cultures supplied with bicarbonate. In fact, concentrations of bicarbonate from 30 to 60 mM strongly improved, for a short period of only 72 h, the cellular levels of chlorophylls and carotenoids. These are interesting pigments with commercial applications. The utilization of bicarbonate could represent an interesting sustainable opportunity to improve microalgae cultivation for cellular growth and pigment contents.

The implementation of gamete cryobanking in aquaculture may improve hatchery practices such as assisted reproduction and genetic selection of desired strains. This paper preliminarily investigated the technical feasibility of cryobanking semen of the common pandora Pagellus erythrinus, an important fishery resource in the Mediterranean area and a promising species for the diversification of productions. Post-thawing motility was evaluated in common pandora semen samples cryopreserved according to eight previously selected protocols and stored in liquid nitrogen for eight years. In order to mimic the practical use of long-term cryostored semen samples, the proportion of living sperm in which motility could be activated was evaluated, by computer-assisted analysis, on thawing and after 3 h of post-thawing incubation at 18 degrees C as biomarker of sperm viability. The assessed parameters were percentage of motile spermatozoa (TM), curvilinear velocity (VCL), lateral head displacement (ALH), beat-cross frequency (BCF) and dance (DNC). The cryostored sperm of the common pandora proved to have promising features. In fact, percentages of motile sperm higher than 40% were always obtained on thawing; moreover, the semen retained its activation ability in the times after thawing, showing no or slight alterations in the motility profile also when activated after 3 h of post-thawing incubation. The best post-thawing performances were recorded with the EG5%-based extender under the F1 curve (equilibration at 2 degrees C; freezing from +2 degrees C to -70 degrees C at 3.8 degrees C/min), that showed the highest percentages of motile cells (80 +/- 3) and no significant alterations in any of the evaluated motility parameters, followed by the EG10% based extender under the F2 curve (equilibration/cooling from 20 degrees C to 2 degrees C at 1 degrees C/min; freezing from +2 degrees C to -150 degrees C at 23.3 degrees C/min). Such a post-thawing resistance of long-term cryostored samples, encourages further studies for the inclusion of the semen of the common pandora in cryobanks aimed at use in reproductive biotechnologies for aquaculture applications and as a model in laboratory research. Therefore, the present paper contributes to the implementation of semen cryobanking as a technological tool in support to aquaculture, using as a model a species of potential aquaculture interest. In addition, the creation of germplasm cryobanks for aquatic species comply with the EU ARRIVE (Animal Research: Reporting of in Vivo Experiments) guidelines that promotes a coordinate use and sharing of biological samples from animal models in laboratory research.

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s−1 and kcat/Km of 3.5 × 107 M−1 ×s−1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

Botryococcus braunii strains are high-lipid producers. Despite this, their use as biodiesel feedstock is limited by their slow growth, which increases production costs. B. braunii wastewater bioremediation power, however, may be able to overcome this problem. This research evaluated the performance of two strains (UTEX-USA and IBL-Brazil) grown in pre-treated (influent) and treated (effluent) wastewater. The goal was to optimize algal-productivity and wastewater treatment in order to provide biomass rich in high-quality oil for biodiesel. The standard CHU-medium was used as control. Phosphorus was 100% removed in all treatments for both strains. Nitrogen removal was higher in wastewater (61-65%) than in CHU medium (48 and 61%), respectively for UTEX and IBL strains. Cultivation in the effluent generated higher biomass and lipid productivity for the UTEX strain, while the influent was the best for the IBL strain. The composition of the fatty-acids produced by Botryococcus was used to estimate the biodiesel quality. Hence, the biodiesels may have good fuel quality (high ignition and combustion efficiency) but limited application at low temperatures. The composition of fatty-acids, however, can be optimized by growing or mixing strategies. Therefore, Botryococcus braunii is a potential source for high quality biodiesel production.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 10(6) s(-1) for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (alpha-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 105 s−1 and kcat/Km of 3.6 × 107 M−1 × s−1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

The carbonic anhydrase (CA, EC 4.2.1.1) superfamily of metalloenzymes catalyzes the hydration of carbon dioxide to bicarbonate and protons. The catalytically active form of these enzymes incorporates a metal hydroxide derivative, the formation of which is the rate-determining step of catalytic reaction, being affected by the transfer of a proton from a metal-coordinated water molecule to the environment. Here, we report the cloning, expression, and purification of a particular CA, i.e., nacrein-like protein encoded in the genome of the Pacific oyster Magallana gigas (previously known as Crassostrea gigas). Furthermore, the amino acid sequence, kinetic constants, and anion inhibition profile of the recombinant enzyme were investigated for the first time. The new protein, CgiNAP2X1, is highly effective as catalyst for the CO2 hydration reaction, based on the measured kinetic parameters, i.e., kcat = 1.0 × 106 s−1 and kcat/KM = 1.2 × 108 M−1·s−1. CgiNAP2X1 has a putative signal peptide, which probably allows an extracellular localization of the protein. The inhibition data demonstrated that the best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with KIs in the range of 76–87 μM. These studies may add new information on the physiological role of the molluskan CAs in the biocalcification processes.

Changes in environmental stressors inevitably lead to an increasing need for innovative and more flexible monitoring tools. The aim of this work has been the characterization of the motility pattern of the cryopreserved sea bream semen after exposure to a dumpsite leachate sample, for the identification of the best representative parameters to be used as endpoints in an ecotoxicological bioassay. Sperm motility has been evaluated either by visual and by computer-assisted analysis; parameters concerning motility on activation and those describing it in the times after activation (duration parameters) have been assessed, discerning them in terms of sensitivity, reliability and methodology of assessment by means of multivariate analyses. The EC50 values of the evaluated endpoints ranged between 2.3 and 4.5ml/L, except for the total motile percentage (aTM, 7.0ml/L), which proved to be the less sensitive among all the tested parameters. According to the multivariate analyses, a difference in sensitivity among "activation" endpoints in respect of "duration" ones can be inferred; on the contrary, endpoints seem to be equally informative either describing total motile sperm or the rapid sub-population, as well as the assessment methodology seems to be not discriminating. In conclusion, the CRYO-Ecotest is a multi-endpoint bioassay that can be considered a promising innovative ecotoxicological tool, characterized by a high plasticity, as its endpoints can be easy tailored each time according to the different needs of the environmental quality assessment programs.

Cryopreservation is a valuable tool for aquaculture as it provides continuous seed production, regardless of the spawning season of the brood stock. The selection of a suitable cryoprotectant with low toxicity and high water solubility is important to avoid membrane injuries and intracellular ice crystallization. This study has been aimed at the assessment of the toxic effects of two usually applied cryoprotectants, 1-2 propylene glycol (PG) and methanol (MetOH), on spermatozoa of the of lion-paw scallop Nodipecten nodosus, by evaluating the sperm motility and the development of D larvae after fertilization procedure. Sperm was exposed at room temperature (22°C) for 10, 20 and 30 min to different concentration ranges of two cryoprotectants. Regarding the sperm motility, PG5%, PG7%, MetOH4% and MetOH6% did not show differences compared to control (semen incubated in seawater) (P < 0.05). The development of D larvae was not affected by the exposition to PG5%, MetOH 4% and MetOH 6%. These results indicate the potential use of both cryoprotectants for cryopreservation procedures.

Microalgae-oil yields and quality, associated with CO2-fixation rates, are able to enhance the feasibility of algal-biodiesel production and economics. Those issues were used as selective criteria applied to Trebouxiophyceae strains. Chlorella vulgaris and two Botryococcus strains were confirmed to grow and to produce high quality biodiesels at distinct levels (2.5 to 20%) of CO2-supplementation. Nevertheless, under nutrient-sufficient conditions, C. vulgaris showed the highest CO2-fixation rate (0.611gL−1d−1) and biomass production at 5% CO2- supplementation, while for Botryococcus terribilis and Botryococcus braunii, the maximum rates (0.614 and 0.555gL−1d−1 CO2) were obtained at 10%-supplementation. Under nutrient-deficient conditions lipids have increased to be above the contents found during the exponential growth-phase, by a maximum of 43%. The fatty-acid profiles varied according to strains and CO2-levels in cultures. Despite variation, palmitic, oleic and linoleic acids predominated. The higher percentage of oleic and palmitic acids over stearic acid, tended to balance the excess of the long chain-size and saturation effects on algal biodiesels' ignition and cold-flow properties. Thus, CO2-supplemented levels from 2.5 to 10.0% made biodiesels compliant with fuel-quality standards. Based on the obtained CO2-fixation rates biodiesels were projected to minimum yields of 42 to 46Lha−1d−1.

Gamete cryopreservation is a biotechnology that can guarantee a continuous supply of gametes, regardless of the seasonal reproductive cycle. In this study we developed a protocol for the cryopreservation of the sea urchin Paracentrotus lividus spermatozoa, with a view to the creation of cryobanks of semen to be used as a model system in laboratory research and ecotoxicological tests. All the key phases of the procedure were separately considered and the effect on sperm motility was evaluated by means of computer assisted analysis. The best results were obtained using 7% dimethylsulfoxide in 1% NaCl plus 0.04 M trehalose as the extender, at a freezing rate of −20 °C/min. On thawing, in semen samples cryopreserved in accordance with this protocol the velocity parameters of the sub-population of rapid sperm (best performing spermatozoa) did not significantly differ from semen on collection; in addition also the fertilization ability was restored, and about 50% of normal developed plutei larvae were obtained by thawed semen. The developed protocol is rapid and easy-to-perform; moreover, the use of gametes from reared urchins makes it unnecessary to continuously collect specimens from natural populations, making this procedure a promising starting point for the creation of alternative and more sustainable methodologies in laboratory research on sea urchin gametes and embryos.

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, -1°C/min chilling rate (18°C to 5°C) and -13°C/min freezing rate (5°C to -80°C), followed by immersion in liquid nitrogen.

A new approach to environmental studies was investigated by the authors, who propose the use of cryopreserved biological systems in ecotoxicological bioassays. The feasibility of spermiotoxicity tests using cryopreserved semen of the sea bream Sparus aurata, with sperm motility parameters as the endpoint, was evaluated. Thawed sperm was incubated in environmental samples (sediment elutriate and dumpsite leachate) and in a reference toxicant (cadmium) at scaled concentrations. Motility was then evaluated by video-microscopy using both visual and computer-assisted analyses. Activation time, sperm motility and velocity and motility duration were assessed on thawing and at the end of the incubation time, and the difference with respect to the control was statistically evaluated. All the endpoints of the bioassay proved to have good sensitivity even at the highest dilutions of the tested matrices. Observed differences in the sensitivity thresholds of the endpoints were considered to be representative of different aspects of sperm physiology. Therefore the proposed bioassay is a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies for aquatic environments.

The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays.Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination.The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L-1). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen.The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies. (C) 2012 Elsevier Inc. All rights reserved.

A pilot re-circulating system was used for the cultivation of two Mediterranean calanoid copepods: Temora stylifera and Centropages typicus. The system automatically concentrated the naupliar and copepodite stages. Temora stylifera was fed the flagellate Rhodomonas baltica or Prorocentrum minimum, whereas C. typicus was fed with a mixture of R. baltica or P. minimum and Tetraselmis suecica. Both copepods also received Isochrysis galbana. After 21 days, the T. stylifera population increased 26-fold, reaching a density of 38 000 individuals, mostly represented by nauplii (88%). The maximum density recorded was 380 ind. L-1, with a production of 370 nauplii L-1. On average, the egg hatching success for this copepod during the rearing period was 54%, with the highest viability in April and May (>75%). The C. typicus population increased more than 10-fold after 7 weeks of rearing, reaching a density of 123 000 individuals, mainly represented by nauplii (>90%). The highest naupliar production was 100 ind. L-1, with a mean egg hatching success of 68%. This system may be useful to produce nauplii and copepodite stages to be used as live, alternative or complementary food for fish larvae or to provide a ready source of organisms for physiological and bioassay studies.

The application of many current-use pesticides has increased after the disuse of persistent, bioaccumulative or toxic ones as DDT or chlordane. Many of the used pesticides are considered less dangerous towards the environment for their physico-chemical properties. This study investigated the toxic effects of three current-use pesticides, pentachlorophenol (PCP), azinphos-methyl (AZM), and chlorpyrifos, on Mediterranean sea urchin Paracentrotus lividus early development and offspring quality. The experimental results showed that the most toxic pesticides were PCP and AZM at EC50 level. Nevertheless at low concentration PCP resulted the less toxic compound and showed EC1 value more protective than NOEC. PCP at high concentration seemed to modify cytoskeleton assembly, while at low concentrations, it could alter the deposition of the larval skeleton. OPs at low concentrations until 300 μg/l showed a similar toxicological behaviour with a trend corresponding to the pesticide concentrations. At high concentration (500 μg/l) the effect mainly observed was the embryos pre-larval arrest. This investigation highlighted the relevance to evaluate, in coastal seawaters, the levels of the used pesticides to understand the real impact on benthic populations mainly in sites characterized by intensive agriculture or floriculture activities, such as the coastal areas of the Mediterranean Sea.

The possibility of using the sperm of teleosts as a model system for ecotoxicological assessments has been explored by evaluating sperm motility parameters: (1) time to reach the maximum motility value (activation time), (2) maximum motility value, (3) duration of maximum motility value, and (4) total time of motility (until class 0). Sperm of Dicentrarchus labrax, Sparus aurata, Diplodus puntazzo and Pagellus erythrinus were analysed and compared. The effects of dimethylsulfoxide, ethylene glycol, propylene glycol, glycerol and methanol on sperm motility in these marine species were investigated. Among the systems tested, sperms of S. aurata and D. labrax were the most sensitive to the tested xenobiotics and S. aurata spermatozoa were shown to be easier to manage for ecotoxicological assays.

This article evaluates the sensitivity of two endpoints in ecotoxicological tests using nauplii of Tigriopus fulvus: the classical index of mortality and the variation in the number of moults. The experiment was conducted by exposing the nauplii to three different types of chemical compounds: copper (heavy metal), pentachlorophenol (pesticide) and phenanthrene (polycyclic aromatic hydrocarbon). For each substance, 50% effect concentration, no observed effect concentration and lowest observed effect concentration were evaluated for both endpoints. The system showed good sensitivity for pentachlorophenol and copper, although no relevant effects were found for phenanthrene. The endpoint ‘number of moults’ during larval development showed higher sensitivity than the mortality endpoint.