Long-lasting biofouling formation on transparent fouling-release coatings for the construction of efficient closed photobioreactors.

In order to build an efficient closed-photobioreactor (PBR) in which biofouling formation is avoided, a non-toxic coating with high transparency is required, which can be applied to the interior surface of the PBR walls. Nowadays, amphiphilic copolymers are being used to inhibit microorganism adhesion, so poly(dimethylsiloxane)-based coatings mixed with poly(ethylene glycol)-based copolymers could be a good option. The 7 poly(dimethylsiloxane)-based coatings tested in this work contained 4% w/w of poly(ethylene glycol)-based copolymers. All were a good alternative to glass because they presented lower cell adhesion. However, the DBE-311 copolymer proved the best option due to its very low cell adhesion and high transmittance. Furthermore, XDLVO theory indicates that these coatings should have no cell adhesion at time 0 since they create a very high-energy barrier that microalgae cells cannot overcome. Nevertheless, this theory also shows that their surface properties change over time, making cell adhesion possible on all coatings after 8 months of immersion. The theory is useful in explaining the interaction forces between the surface and microalgae cells at any moment in time, but it should be complemented with models to predict the conditioning film formation and the contribution of the PBR's fluid dynamics over time.

Bioactives Overproduction through Operational Strategies in the Ichthyotoxic Microalga Heterosigma akashiwo Culture.

The red tide-forming microalga Heterosigma akashiwo has been associated with massive events of fish deaths, both wild and cultured. Culture conditions are responsible for the synthesis or accumulation of some metabolites with different interesting bioactivities. H. akashiwo LC269919 strain was grown in a 10 L bubble column photobioreactor artificially illuminated with multi-coloured LED lights. Growth and production of exopolysaccharides, polyunsaturated fatty acids (PUFAs), and carotenoids were evaluated under different culture modes (batch, fed-batch, semicontinuous, and continuous) at two irradiance levels (300 and 700 µE·s-1·m-2). Continuous mode at the dilution rate of 0.2·day-1 and 700 µE·s-1·m-2 provided the highest production of biomass, PUFAs (132.6 and 2.3 mg·L-1·day-1), and maximum fucoxanthin productivity (0.16 mg·L-1·day-1). The fed-batch mode accumulated exopolysaccharides in a concentration (1.02 g·L-1) 10-fold over the batch mode. An extraction process based on a sequential gradient partition with water and four water-immiscible organic solvents allowed the isolation of bioactive fucoxanthin from methanolic extracts of H. akashiwo. Metabolites present in H. akashiwo, fucoxanthin and polar lipids (i.e., eicosapentaenoic acid (EPA)), or probably such as phytosterol (ß-Sitosterol) from other microalgae, were responsible for the antitumor activity obtained.

In vitro evaluation of the potential allelopathic and ichthyotoxic effect of the raphidophyte Heterosigma akashiwo and the dinoflagellate Alexandriumcatenella.

Ichthyotoxic algal blooms cause economic losses throughout the world. However, the mechanisms and molecules proposed so far fail to explain the massiveness of these events. In this research, the allelopathic effect of two bloom-forming species (the raphidophyte Heterosigma akashiwo and dinoflagellate Alexandrium catenella) was evaluated between them and with Rhodomonas salina bioassay. Mono- and co-cultures were carried out with the aim of providing evidence of the relation between allelopathy and ichthyotoxicity. The allelopathic inhibitory effect of the A. catenella's supernatant was significantly enhanced when supernatants were obtained from co-cultures with direct contact between these species. We could not observe any allelopathic response provoked by H. akashiwo. On the other hand, A. catenella was able to decrease the cell concentration of H. akashiwo and R. salina. Besides, allelopathy and ichthyotoxicity were found for A. catenella's supernant, being the allelopathic effect not related to saxitoxin. These results reinforce the hypothesis that the allelopathic effect being regulated by the presence of other microalgae and could be responsible for ichthyotoxicity.

Sublethal effect of the toxic dinoflagellate Karlodinium veneficum on early life stages of zebrafish (Danio rerio).

Dinoflagellates of the genus Karlodinium are ichthyotoxic species that produce toxins including karlotoxins and karmitoxins. Karlotoxins show hemolytic and cytotoxic activities and have been associated with fish mortality. This study evaluated the effect of toxins released into the environment of Karlodinium veneficum strain K10 (Ebro Delta, NW Mediterranean) on the early stages of Danio rerio (zebrafish). Extracts of the supernatant of K10 contained the mono-sulfated KmTx-10, KmTx-11, KmTx-12, KmTx-13, and a di-sulfated form of KmTx-10. Total egg mortality was observed for karlotoxin concentration higher than 2.69 µg L-1. For 1.35 µg L-1, 87% of development anomalies were evidenced (all concentrations were expressed as KmTx-2 equivalent). Larvae of 8 days postfertilization exposed to 1.35 µg L-1 presented epithelial damage with 80% of cells in the early apoptotic stage. Our results indicate that supernatants with low concentration of KmTxs produce both lethal and sublethal effects in early fish stages. Moreover, apoptosis was induced at concentrations as low as 0.01 µg L-1. This is of great relevance since detrimental long-term effects due to exposure to low concentrations of these substances could affect wild and cultured fish.

The Isolation of Specialty Compounds from Amphidinium carterae Biomass by Two-Step Solid-Phase and Liquid-Liquid Extraction.

The two main methods for partitioning crude methanolic extract from Amphidinium carterae biomass were compared. The objective was to obtain three enriched fractions containing amphidinols (APDs), carotenoids, and fatty acids. Since the most valuable bioproducts are APDs, their recovery was the principal goal. The first method consisted of a solid-phase extraction (SPE) in reverse phase that, for the first time, was optimized to fractionate organic methanolic extracts from Amphidinium carterae biomass using reverse-phase C18 as the adsorbent. The second method consisted of a two-step liquid-liquid extraction coupled with SPE and, alternatively, with solvent partitioning. The SPE method allowed the recovery of the biologically-active fraction (containing the APDs) by eluting with methanol (MeOH): water (H2O) (80:20 v/v). Alternatively, an APD purification strategy using solvent partitioning proved to be a better approach for providing APDs in a clear-cut way. When using n-butanol, APDs were obtained at a 70% concentration (w/w), whereas for the SPE method, the most concentrated fraction was only 18% (w/w). For the other fractions (carotenoids and fatty acids), a two-step liquid-liquid extraction (LLE) method coupled with the solvent partitioning method presented the best results.

Physiological Response of Atlantic Salmon (Salmo salar) to Long-Term Exposure to an Anesthetic Obtained from Heterosigma akashiwo.

Despite the invaluable role of anesthetics as a tool for ensuring animal welfare in stressful situations, there is currently a lack of anesthetic drugs that meet the requirements of intensive aquaculture. In response to the growing interest in anesthetic substances of natural origin, this study evaluated the physiological and health impact of an anesthetic based on an extract of the microalga Heterosigma akashiwo on juvenile salmon (Salmo salar) exposed for a period of 72 h. To simulate a condition closer to reality where fish are subjected to stimuli (e.g., transport), the animals were exposed to 50 mg L-1 of algal extract and to physical stress. Functional, physiological, and histological parameters were evaluated in blood and tissues at different sampling periods (0, 24, and 72 h). There was no mortality and the induction and recovery times observed were within the established criteria for anesthetic efficacy. The anesthetic extract did not induce any side effects, such as stress or metabolic damage, indicating that this extract is a viable option for supporting fish welfare during deleterious events. This study provides information to support that the anesthetic extract tested, derived from H. akashiwo, is a promising candidate drug for operations requiring sedation (e.g., Salmonid transport).

Neuro-2a cell-based assay for toxicity equivalency factor - proposal and evaluation in Chilean contaminated shellfish samples.

There are two official PSP detection methods (mouse bioassay and HLPC-FLD) and a number of alternative methods. Ethical considerations have led to regulations being adopted in some countries that limit or prohibit the application of mouse bioassay. Analytical methodologies (e.g. HPLC-FLD or LC-MSMS) have the disadvantages of not being able to detect new toxins or analogues or reflecting the overall toxicity of the sample. In addition, they require highly trained personnel and expensive equipment, which are not always available. In this work, we have evaluated a method based on the Neuro-2a cell-based assay to detect substances that inhibit voltage-dependent sodium channels (Manger's method). We tested PSP standards and natural samples contaminated with PSP. Here we demonstrate that the adapted Manger's method is suitable for calculating Toxicity Equivalency Factors (TEF) for STX-analogues. The method was shown to be useful for screening contaminated natural samples in concentrations above the regulatory limit for these toxins (80 µg STX equivalents/100 g shellfish). We were able to detect PSP in 19 natural mollusc samples from South Chile despite the presence of other marine toxins. These preliminary results suggest that the method could be used as a first step in screening programmes.

Simultaneous effect of temperature and irradiance on growth and okadaic acid production from the marine dinoflagellate Prorocentrum belizeanum.

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I0). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·(m-2)·s(-1)), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ(m)) was 0.204 day(-1) at 25 °C and 40 µE·(m-2)·s(-1). Photo-inhibition was observed at I0 > 40 µEm(-2)s(-1), leading to culture death at 120 µE·m(-2)·s(-1) and 28 °C. Cells at I0 ≥ 80 µE·m(-2)·s(-1) were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ(m)-I0 curves and another empirical model for relating µ(m)-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I0-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.