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The nutrient content of a commercial seawater culture medium for growing the microalga Isochrysis galbana was optimized using a stochastic strategy based on genetic algorithms. For this, 210 experiments spread over seven generations were carried out. This strategy reduced the number of assays by more than 90% compared to a factorial design involving the optimization of twelve nutrients simultaneously. The optimized medium outperformed the reference medium in all aspects. The genetic algorithm strategy achieved a polyunsaturated fatty acids (PUFAs) productivity of 7.8 mg L−1 day−1 in a continuous culture of I. galbana, corresponding to an increase of 15% compared to the commercial formulation. Carotenoids, on the other hand, increased by 50% d.w. In addition, PUFA yields were significantly improved, which allowed us to reduce the requirement of several nutrients, for instance, N (25%), Mo (20%), Mn (60%), Co (60%), and Cu (60%).

There is growing interest in finding microalgae species that efficiently convert dissolved nutrients contained in aquaculture effluents into highly valuable biomass. The different nitrogen forms that are present in aquaculture effluents are particularly concerning. This study demonstrated that the dinoflagellate Amphidinium carterae can acclimate to both combined and sole nitrogen sources such as nitrate, ammonium, and urea over a wide concentration range. As far as is known, it is the first time that a species of the genus Amphidinium has been successfully cultured with urea as the sole source of nitrogen. In the presence of 882 μM of nitrate, A. carterae tolerated urea concentrations up to 5000 μM. With respect to ammonium-N tolerance, it has been observed that it is lethal at concentrations higher than 441 μM. A robust laboratory experimental design was critical for accurately assessing this acclimation. Alternative N sources did not affect the production of high-value specific polyketide secondary metabolites from A. carterae, such as amphidinols, with an average concentration of 0.435 ± 0.038% biomass d.w. An analysis of the symbiotic microbial assemblages developed in a long-term A. carterae culture in an open raceway pond, and the fact that it is able to metabolize all three nitrogen sources simultaneously, supports the idea that this microalga has the potential to be successfully cultured with aquaculture effluents.

There is broad scientific evidence on the health benefits of omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially docosahexaenoic acid (DHA, 22:6n3) and eicosapentaenoic acid (EPA, 20:5n3). On the other hand, polar lipids (phospholipids, PLs, and glycolipids, GLs) are excellent emulsifying agents, making them useful both as food products and as excipients for drugs and cosmetics. The bioavailability of n-3 PUFAs in the form of PLs is greater than that of ethyl esters and triacylglycerols. This work has developed an easy method for the simultaneous extraction and fractionation of saponifiable lipids (SLs) from the microalga Nannochloropsis sp., using low-toxicity solvents. Firstly, a lipidic fraction very rich in neutral saponifiable lipids (NSLs) was obtained using hexane. Next, an EPA and polar lipids (PLs and GLs)-enriched fraction was obtained using ethanol (96%). Under optimal conditions, in the first extraction with hexane, an SL extract with 86.3% NSLs was obtained; this extract contained 88.9% of the biomass NSLs. Subsequently, in the second extraction step with ethanol (96%), an SL extract with 87.1% polar lipids and up to 35.2% EPA was obtained. This SL extract contained 87.1% of the polar lipids and 74.7% of the EPA from the Nannochloropsis sp. biomass.

The purpose of this study was to develop an economical agricultural fertilizer-based medium for the mass production of Isochrysis galbana Parke, a marine microalga producing docosahexaenoic acid (DHA) and considered an excellent feed for use in aquaculture. Different fertilizers were used to replace the main nutrients, nitrogen and phosphorus, and the micronutrients of the well-known commercial medium, ALGAL. The obtained results showed that I. galbana preferred nitrate, instead of ammonium, as the nitrogen source and phosphate as the phosphorus source. Nonetheless, a nitrate concentration above 4 mM inhibited cell growth. The alternative medium proposed achieved a biomass productivity 22% higher than that for the ALGAL medium, reducing the medium’s contribution to the overall biomass cost by over 95%, with protein, lipid, carbohydrate, and DHA contents at 44, 36, 25, and 1.47% of dry weight, respectively, thus providing a cheaper biomass with excellent nutritional value for use in aquaculture.

Sustainable dinoflagellate microalgae-based bioprocess designed to produce secondary metabolites (SMs) with interesting bioactivities are attracting increasing attention. However, dinoflagellates also produce other valuable bioproducts (e.g polyunsaturated fatty acids, carotenoids, etc.) that could be recovered and should therefore be taken into account in the bioprocess. In this study, biomass of the marine dinoflagellate microalga Amphidinium carterae was used to assess and optimise three different methods in order to obtain three families of high-value biochemical compounds present in the biomass. The existing processes encompassed a multi-step extraction process for carotenoids, fatty acids and APDs individually and are optimized for the integral valorization of raw A. carterae biomass, with SMs being the primary target compounds. Total process recovery yields were 97% for carotenoids, 80% for total fatty acids and 100% for an extract rich in APDs (not purified).

Microalgae are potential sources of high-value lipids and colorants for use in foods, cosmetics, and other applications. Biomass and metabolite productivities of photoautotrophic algae cultures are low because of limited availability of light. Therefore, mixotrophic cultures were investigated in parallel with photoautotrophic controls. In mixotrophy, some of the energy and carbon are supplied in the form of dissolved organic substrates in addition to inorganic carbon and light being available. The aim was to compare productivities of biomass, fatty acids, and carotenoid pigments in outdoor and indoor mixotrophic and photoautotrophic batch and continuous cultures. The edible and safe marine microalga Nannochloropsis gaditana was used in these studies. The alga could be grown mixotrophically using glucose and glycerol, but not acetate. Optimal concentrations of the organic carbon sources were 5 g L−1 for glucose and 1 g L−1 for glycerol. Mixotrophy substantially increased the biomass concentration and productivity relative to photoautotrophy. The maximum biomass productivity in mixotrophic batch cultures using glucose or glycerol was identical at 170 mg L−1 day−1, being 30% greater than control cultures. In continuous outdoor culture with glucose (5 g L−1) mixotrophy at 12 °C, the total carotenoids in the biomass were 83% higher compared to photoautotrophic control biomass, and the eicosapentaenoic acid (EPA) productivity was 2.2-fold higher relative to controls. The maximum EPA productivity was 11 mg L−1 day−1. Glucose mixotrophy increased the total lipids content in the biomass by 34% relative to photoautotrophic operation.

Animal production inevitably causes the emission of greenhouse gases and the generation of large amounts of slurry, both representing a serious environmental problem. Photosynthetic microorganisms such as microalgae and cyanobacteria have been proposed as alternative strategies to bioremediate agricultural waste while consuming carbon dioxide and producing valuable biomass. The current study assessed the potential of the microalga Scenedesmus sp. to remove nutrients from piggery wastewater (PWW) and the influence of the microalga on the microbial consortia. Maximum N-NH4+ consumption was 55.3 &plusmn, 3.7 mg&middot, L&minus, 1&middot, day&minus, 1 while P-PO43&minus, removal rates were in the range 0.1&ndash, 1.9 mg&middot, 1. N-NH4+ removal was partially caused by the action of nitrifying bacteria, which led to the production of N-NO3&minus, N-NO3&minus, production values where lower when microalgae were more active. This work demonstrated that the photosynthetic activity of microalgae allows us to increase nutrient removal rates from PWW and to reduce the coliform bacterial load of the effluent, minimising both their environmental impact and health risks. Microalgae assimilated part of the N-NH4+ present in the media to produce biomass and did not to convert it into N-NO3&minus, as in traditional processes.

Microalgae-bacteria consortia have been proposed as alternatives to conventional biological processes to treat different types of wastewaters, including animal slurry. In this work, a microalgae-bacteria consortia (ABACO) model for wastewater treatment is proposed, it being calibrated and validated using pig slurry. The model includes the most relevant features of microalgae, such as light dependence, endogenous respiration, and growth and nutrient consumption as a function of nutrient availability (especially inorganic carbon), in addition to the already reported features of heterotrophic and nitrifying bacteria. The interrelation between the different populations is also included in the model, in addition to the simultaneous release and consumption of the most relevant compounds, such as oxygen and carbon dioxide. The implementation of the model has been performed in MATLAB software, the calibration of model parameters was carried out using genetic algorithms. The ABACO model allows one to simulate the dynamics of different components in the system, and the relative proportions of microalgae, heterotrophic bacteria, and nitrifying bacteria. The percentage of each microbial population obtained with the model was confirmed by respirometric techniques. The proposed model is a powerful tool for the development of microalgae-related wastewater treatment processes, both to maximize the production of microalgal biomass and to optimize the wastewater treatment capacity.

Primary urban wastewater was treated in outdoor raceways using microalgae-bacteria consortia dominated by Scenedesmus almeriensis. The current study aimed at assessing the effect of operational conditions, namely, culture depth and dilution rate, on the following: (i) biomass productivity, (ii) the nutrient removal capacity and (iii) the composition of the microalgae-bacteria consortium and the presence of unwanted microorganisms. Optimum dilution rates to process large quantities of wastewater during summer and achieve high biomass productivities were 0.3–0.5 day−1. Under the optimum operational conditions, nitrogen and phosphorus removal rates were higher than 90% while removal of chemical oxygen demand was 70%. Operating at different culture depths had a striking effect on the composition of the microalgae-bacteria consortium. The relative abundance of nitrifiers increased with culture depth and was minimised at 0.05 m: larger culture depths led to enhanced nitrifying activity and therefore to nitrate production and accumulation in the system. Results demonstrate the potential of microalgae-based wastewater treatment processes and the importance of selecting suitable operational conditions to maximise both, biomass production and nutrient removal by minimising the occurrence of nitrifying bacteria.

Photo-respirometric tecniques are applied for evaluating photosynthetic activity in phototrophic organisms. These methods allow to evaluate photosynthetic response under different conditions. In this work, the influence of nutrient availability (nitrate, ammonium, and phosphate) on the photosynthesis and respiration of Scenedesmus almeriensis was studied using short photo-respirometric measurements. Both photosynthesis and respiration increasing until saturation value and consecutively diminishing, presenting inhibition by high concentrations. Regarding the influence of phosphorus concentration in microalgae cells, a similar hyperbolic trend was observed but no inhibition was observed at high concentration. Based on these experimental data, the respiration, and the photosynthesis rate of S. almeriensis were modelled using Haldane equation for nitrate and ammonium data, and Monod equation for phosphate data. In addition, experiments were performed to determine the yield coefficients for both nitrogen and phosphorus in S. almeriensis cultures. The data showed that the nitrogen and phosphorous coefficient yields are not constant, being modified as a function of nutrients concentration, presenting the luxury uptake phenomena. Finally, the proposed models were incorporated into a simulation tool to evaluate the photosynthetic activity and the nutrient yield coefficients of S. almeriensis when different culture media and wastewaters are used as a nitrogen and phosphorous source for its growth.Key points• Microalgal photosynthesis/respiration vary as a function of nutrients availability.• Photosynthesis inhibition appears at high N-NO3-and N-NH4+concentrations.• Nutrient yield coefficients are influenced by luxury uptake phenomenon. Graphical abstract

Biofouling represents an important limitation in photobioreactor cultures. The biofouling propensity of different materials (polystyrene, borosilicate glass, polymethyl methacrylate and polyethylene terephthalate glycol-modified) and coatings (two spray-applied and nanoparticle-based superhydrophobic coatings and a hydrogel-based fouling release coating) was evaluated by means of a short-term protein test, using bovine serum albumin (BSA) as a model protein, and by the long-term culture of the marine microalga Nannochloropsis gaditana under practical conditions. The results from both methods were similar, confirming that the BSA test predicts microalgal biofouling on surfaces exposed to microalgae cultures; these secrete macromolecules, such as proteins, that have a high capacity for forming a conditioning film prior to cell adhesion. The hydrogel-based coating showed significantly reduced BSA and N. gaditana adhesion, whereas the other surfaces failed to control biofouling. Microalgal biofouling was associated with an increased concentration of sticky extracellular proteins at low N/P ratios (below 15).

In this study, the outdoor production of marine microalgae in raceway photobioreactors was investigated, modifying the centrate percentage in the culture medium (20, 30, 40 and 50%) and using two different dilution rates (0.2 day−1 and 0.3 day−1). The data obtained showed that microalgae are capable of producing biomass in addition to recovering the nutrients contained in the centrate. The best results for biomass productivity and light efficiency were obtained when the centrate was set at 20% with a dilution rate of 0.3 day−1. The biomass productivity was 32.42 g m−2·day−1 while the photosynthetic efficiency was 0.74 gbiomass·E−1 (3.66%). Regarding the nutrients, nitrogen (the majority being in the form of ammonium [NH4+]) and phosphorus were only fixed into biomass when optimal conditions were set; if this was not the case, they were lost to stripping or precipitation. The maximal nutrient removal capacities under the optimal conditions were 28.72 mgN·l−1·day−1 and 3.99 mgP·l−1·day−1. Population changes were determined by the dilution rate set whilst the centrate percentage had little effect. Four strains were present in the culture, Nannochloropsis g. being the main one. Biochemical changes did not vary greatly between the conditions set for the culture, with a composition rich in proteins and carbohydrates being observed. One can conclude that to produce marine microalgal biomass for a range of potential commodities such as feed, biofertilizers and biofuels, it is possible to use centrate from anaerobic digestion as the sole nutrient source, as a way of reducing costs.

We present the data corresponding to the isolation and morphological and molecular characterization of a strain of Amphidinium carterae, isolated in Mallorca Island waters and now deposited in the microalgae culture collection of the Plant Biology and Ecology Department of the University of the Basque Country under the reference Dn241Ehu. The morphological characterization was made using two different techniques of microscopy and the molecular characterization by using the 28S rDNA sequences of D1 and D2 domains. This strain has been used for a culture study in an indoor LED-lighted pilot-scale raceway to determine its production of carotenoids and fatty acids, "Long-term culture of the marine dinoflagellate microalga Amphidinium carterae in an indoor LED-lighted raceway photobioreactor: Production of carotenoids and fatty acids." (Molina-Miras et al., 2018) [1]. (C) 2018 The Authors. Published by Elsevier Inc. This research was funded by the Spanish Ministry of Economy and Competitiveness (CTQ2014-55888-C3-02), the European Regional Development Fund Program, and the projects PPG17/67 from the UPV/EHU and IT1040-16 from the Basque Government.

A pilot-scale bioprocess was developed for the production of karlotoxin-enriched extracts of the marine algal dinoflagellate Karlodinium veneficum. A bubble column and a flat-panel photobioreactors (80–281 L) were used for comparative assessment of growth. Flow hydrodynamics and energy dissipation rates (EDR) in the bioreactors were characterized through robust computational fluid dynamic simulations. All cultures were conducted monoseptically outdoors. Bubble column (maximum cell productivity in semicontinuous operation of 58 × 103 cell mL−1 day−1) proved to be a better culture system for this alga. In both reactors, the local EDR near the headspace, and in the sparger zone, were more than one order of magnitude higher than the average value in the whole reactor (=4 × 10−3 W kg−1). Extraction of the culture and further purification resulted in the desired KTXs extracts. Apparently, the alga produced three congeners KTXs: KmTx-10 and its sulfated derivative (sulfo-KmTx-10) and KmTx-12. All congeners possessed hemolytic activity.

Marine dinoflagellate microalgae belonging to the genus Amphidinium are a key source of an interesting group of polyketide metabolites with potent bioactivities, wide-ranging functional diversity and stereochemical complexity, but low natural availabilities. The feasibility of a microalgae dinoflagellate-based sustainable bioprocess for producing amphidinols (APDNs) by photoautotrophic culture of Amphidinium carterae in a pilot-scale LED-illuminated bubble column photobioreactor (PBR) was therefore investigated. A fed-batch culture mode with pulse feeding strategy provided a growth pattern strongly limited by the availability of phosphate content in the culture medium that stimulated the production of cellular APDNs. Since A. carterae was found to be much more shear-sensitive than other shear-tolerant non-dinoflagellate microalgae, the culture height and air flow rate were established to ensure the absence of damaging levels of hydrodynamic stress. The biomass capacity yielded by the PBR at the end of the culture (0.540 g d.w. L−1 equivalent to 1.70 × 106 cell mL−1) corresponded to that estimated stoichiometrically from the experimentally determined biomass P-molar formula (C329 O126 H732 N69 S3 P1) and the total phosphorus and nitrogen balances. The downstream processing section was initially conceived to recover APDNs excreted by cells into the supernatant. A dry APDN-enriched extract concentration of 49 mg per liter of supernatant was obtained. This purification process led to partitioning of the extract into several fractions and sub-fractions thereof. Only two sub-fractions were studied, yielding thereof highly pure (>95% pure) luteophanol D and lingshuiol A, and a new, roughly purified (>80% pure) APDN, namely amphidinol 20. The percentages of luteophanol D, lingshuiol A and amphidinol 20 by dry weight of the APDN-enriched extract obtained were 1%, 0.39% and 0.31%, respectively, thus representing a concentration in the culture supernatant of 490, 191 and 152 μg L−1, respectively.

A study has been conducted to assess cell damage during centrifugation and to optimize this operation for the insect cell line Se301. Experiments were carried out in a discontinuous centrifuge using Falcon tubes of different sizes as containers. The cells were easily recovered from the cell suspension and the time required for complete sedimentation was found to be independent of tube size or design. Cell damage was observed once the cells were sedimented, therefore this process depends on both the residence time of the cells in the pellet and on the acceleration applied. The sensitivity of the cells to this operation was higher than for other naked microalgae or for very sensitive red blood cells. Indeed, excessive treatment produced cell damage that reduced the productivity of subsequent cultures. CFD simulations were carried out in 2D and a good agreement was found between the CFD and experimental values in terms of the time required for full sedimentation. In addition, this technique allowed the calculation of shear stress, a key variable in the study of cell sensitivity to flow.

Since the infection strategy in the baculovirus-insect cell system mostly affects production of the vector itself or the target product, and given that individual infection parameters interact with each other, the optimal combination must be established for each such specific system. In this work an artificial neural network was used to model infection strategy, including the cell concentration at infection, the multiplicity of infection, the medium recycle, and agitation intensity, and to evaluate the relative importance of each factor in the baculovirus production obtained. The results demonstrate that this model can be used to select an optimal infection strategy. For the baculovirus-insect cell system used in this study, this includes low multiplicity of infection and agitation intensity, along with high cell concentration at infection and medium recycle. Our model is superior to regression methods and predicts baculovirus production more precisely, thus meaning that it could be useful for the development of feasible processes, thereby improving process performance and economy.

The economic and/or energetic feasibility of processes based on using microalgae biomass requires an efficient cultivation system. In photobioreactors (PBRs), the adhesion of microalgae to the transparent PBR surfaces leads to biofouling and reduces the solar radiation penetrating the PBR. Light reduction within the PBR decreases biomass productivity and, therefore, the photosynthetic efficiency of the cultivation system. Additionally, PBR biofouling leads to a series of further undesirable events including changes in cell pigmentation, culture degradation, and contamination by invasive microorganisms; all of which can result in the cultivation process having to be stopped. Designing PBR surfaces with proper materials, functional groups or surface coatings, to prevent microalgal adhesion is essential for solving the biofouling problem. Such a significant advance in microalgal biotechnology would enable extended operational periods at high productivity and reduce maintenance costs. In this paper, we review the few systematic studies performed so far and applied the existing thermodynamic and colloidal theories for microbial biofouling formation in order to understand microalgal adhesion on PBR surfaces and the microalgae-microalgae cell interactions. Their relationship to the physicochemical properties of the solid PBR surface, the microalgae cell surfaces, and the ionic strength of the culture medium is discussed. The suitability and the applicability of such theories are reviewed. To this end, an example of biofouling formation on a commercial glass surface is presented for the marine microalgae Nannochloropsis gaditana. It highlights the adhesion dynamics and the inaccuracies of the process and the need for further refinement of previous theories so as to apply them to flowing systems, such as is the case for PBRs used to culture microalgae.

This paper addresses a complete set of procedures to study and to better understand the emerging problem of the biofouling formation in photobioreactors (PBRs). Methodologies are described in detail for: (i) PBR and microalgae mat surface preparation, (ii) contact angles (CA) and zeta potential (ZP) measurements for both microalgal cells and PBR surfaces, and (iii) microscopic methods for studying the evolution of adhesion intensity. The impact that these methodologies may have on the photosynthetic apparatus of the cells, the biomass concentration and cell viability are also quantified. A lab-scale flat-plate PBR was used to perform a long-term batch culture of the marine microalgae Nannochloropsis gaditana , in which a devised rack of 25 PBR glass surfaces were submerged. To study the cell-to-cell and cell-to-PBR surface interactions, the existing surface thermodynamics and colloidal theories (XDLVO) were used. The major outcomes were: (1) that N. gaditana has a exposure time threshold to the electric field produced by the ZP meter; (2) a linear equation is provided for predicting the PBR surface potential as a function of the culture medium's ionic strength; (3) the biofouling growth curve on the PBR surface varied in line with the growth kinetic followed by the freely suspended culture cells; (4) the devised PBR slide rack system offers a versatile experimental platform for generating biofouling results, making it suitable for the in situ efficiency evaluation of antibiofouling coatings; (5) there was a significant variation in the surface free energy of the PBR surfaces and algal mats with respect to that present at the beginning of the culture, and, consequently, the application of thermodynamic theories failed to predict cell adhesion over long-term cultivation. However, the XDLVO model satisfactorily explained the dynamics of the adhesion studied. The reported results might be useful for research in the microalgal production and PBR engineering area.

Microalgae are becoming an important source of high-value products such as biostimulants, biopesticides, and other low-value products such as biofertilizers or aquafeed (fatty acids and carotenoids), among others. However, extracting the biomolecules contained in the microalgae cells is difficult due to the structure and composition of the microalgal cell wall. To overcome this difficulty, mechanical cell disruption methods, such as high-pressure homogenization (HPH), can be applied to facilitate the extraction of the compounds of interest. This work focuses on optimizing the extraction of biostimulants from microalgae biomass and obtaining amino acid concentrates that can be used in several applications. The research aims to study the effect of HPH pretreatment on the production of these different bioproducts using Scenedesmus sp. biomass grown in pig sludge as the raw material. For each product, the HPH pretreatment of the wet biomass must be optimized to select the optimal conditions and the number of passes. Soft treatment of the wet biomass at 200 bar is sufficient to observe a 10% increase in the germination index (GI) of watercress seeds, while greater treatment intensities reduce the GI with respect to the untreated biomass, possibly because this increases the concentration of the molecules responsible for the bioactivity, thus causing a toxic effect on the seeds. In the case of the amino acid concentrates, better results were obtained at 600 bar, achieving a hydrolysis level of 72.9%. Lastly, the concentrations of the phenolic compounds and the salicylic acid, related with enhanced biostimulant activity, were determined using spectrophotometric methods and High-Performance Liquid Chromatography- diode array (HPLC), respectively.

In this article, the influence of culture conditions (irradiance, temperature, pH, and dissolved oxygen) on the photosynthesis and the respiration rates of microalgae–bacteria consortia in wastewater treatment was analyzed. Specifically, some short photo-respirometric experiments, simulating outdoor raceway reactors, were performed to evaluate the response of microalgae, heterotrophic bacteria, and nitrifying bacteria to variations in environmental parameters. Results demonstrate that irradiance is the most dominant variable to determine microalgae photosynthesis rates. However, reduction in microalgae activity was not observed at higher irradiance, ruling out the existence of photoinhibition phenomena. Related to heterotrophic and nitrifying bacteria, their activities were strongly affected by the influence of temperature and pH. Moreover, the effect of dissolved oxygen concentrations on microalgae, and bacteria activities was studied, displaying a reduced photosynthetic rate at dissolved oxygen concentrations above 20 mg/L. Data have been used to develop an integrated model for each population (microalgae, heterotrophic bacteria, and nitrifying bacteria) based on considering the simultaneous influence of irradiance, temperature, pH, and dissolved oxygen. The models fit the experimental results in the range of culture conditions tested, and they were validated using data obtained by the simultaneous modifications of the variables. These individual models serve as a basis for developing a global biologic microalgae–bacteria model for wastewater treatment to improve the optimal design and management of microalgae-based processes, especially outdoors, where the cultures are subject to variable daily culture conditions.

The aim of this work was to optimize the production of fatty acid methyl ester (FAME, biodiesel) from wet Nannchloropsis gaditana microalgal biomass by direct enzymatic transesterification. This was done in order to avoid the high cost associated with the prior steps of drying and oil extraction. Saponifiable lipids (SLs) from microalgal biomass were transformed to FAME using the lipase Novozyme 435 (N435) from Candida antarctica as the catalyst, and finally the FAME were extracted with hexane. t-Butanol was used as the reaction medium so as to decrease lipase deactivation and increase mass transfer velocity. A FAME conversion of 99.5% was achieved using wet microalgal biomass homogenized at 140 MPa to enhance cell disruption, a N435:oil mass ratio of 0.32, methanol added in 3 stages to achieve a total of 4.6 cm 3 g −1 of oil and 7.1 cm 3 g −1 oil of added t-butanol, with a reaction time of 56 h. The FAME conversion decreased to 57% after catalyzing three reactions with the same lipase batch. This work shows the influence of the polar lipids contained in the microalgal biomass both on the reaction velocity and on lipase activity.

This work is the last of a series of three papers. The previous ones were performed in tubular photobioreactors and raceways. In the present work, the outdoor continuous production of Nannochloropsis gaditana in flat-panel reactors is studied over a two-year period, analysing the influence of the dilution rate imposed in addition to the nitrate and phosphate supplied. Maximum biomass and lipid yields of 0.19 g/l day and 38.0 mg/l day were obtained when operating at a dilution rate of 0.35 1/day and concentrations of 10.0 mM NO 3 − and 0.8 mM PO 4 3 − in the culture medium. By decreasing the specific nitrate input (below 2 mmol N/g day), and exposing the culture to moderate light availability and temperature, the volumetric productivity of saturated and monounsaturated fatty acids increased to 10.0 mg/l day. The specific nitrate input, together with other important parameters such as the average irradiance, temperature and dilution rate were used to develop a model describing this species' behaviour. The model is a powerful tool for further improvement and scaling-up of this technology.

In this paper, a new photo-respirometry method for determining the rates of the main metabolic processes of microalgae-bacteria consortia in microalgae-based wastewater treatment processes has been developed and tested. The proposed protocol consists on applying dark and light periods to a microalgae-bacteria consortium in the presence of different substrates and measuring the rate of oxygen production. This allows determining the activity of microalgae, heterotrophic bacteria and nitrifying bacteria separately. The method has been optimized in terms of the operation strategy, including the starvation period required, the biomass concentration and the irradiance during the measurements. Results show that a starvation period of one to three days is necessary depending on the nutrient concentration. The optimal experimental conditions determined were a biomass concentration of 0.5 g/L and an irradiance of 200 μmol photons/m2·s. Furthermore, microalgae-bacteria samples from seven photobioreactors (indoor and outdoor) with different nutrient sources have been evaluated applying the methodology proposed. Regardless of the wastewater type, the microalgae activity is the main metabolic process, with heterotrophic activity increasing along with the chemical oxygen demand (COD) in the wastewater. Nitrifying activity was only observed when high ammonium concentrations were present. The developed method is a powerful tool to adequately manage and operate wastewater treatment processes using microalgae/bacteria consortia, providing valuable information to model wastewater treatment systems with microalgae and determine kinetic parameters.

Amphidinium carterae is a marine dinoflagellate microalga that produces high-value bioactive polyketides such as amphidinols and amphidinolides, which have remarkable cytotoxic, antifungal and antitumoral activities. This species also produces docosahexaenoic acid and the carotenoid peridinin as high-value byproducts. The development of any sustainable microalgae-based bioprocess must comprise complete valorization of the biomass and reuse of the exhausted culture medium as much as possible to comply with the circular economy concept. In this work we have assessed the effect of recycling the cell-free supernatant on the growth kinetics and production of amphidinols by A. carterae photoautotrophically cultured in a pilot-scale raceway-type photobioreactor in semi-continuous mode. Acclimation to the recycled medium was first studied on a laboratory scale and was accomplished in the fourth sub-culture, except for pigment content, which decreased throughout the process. No measurably negative effect was detected in the cells. Maximum growth rates and cell productivities increased by 60% with use of 75% spent medium, as a result of mixotrophic consumption of the organic carbon present in that medium, remaining constant thereafter. In the photobioreactor, acclimation took 77 days and was also accomplished in the fourth sub-culture, with no negative effects on cell health. Biomass and cell productivities were higher with the recycled medium than with the fresh medium. Pigments decreased throughout the acclimation process, probably due to organic carbon-based mixotrophic growth. Amphidinols and docosahexaenoic acid biomass contents were not affected by medium recycling, which will significantly improve the economics of amphidinols production from A. carterae cultures on a circular economy basis.

The feasibility of the long-term (>170 days) culture of a dinoflagellate microalga in a raceway photobioreactor is demonstrated for the first time. Amphidinium carterae was chosen for this study as it is producer of interesting high-value compounds. Repeated semicontinuous culture provided to be a robust operational mode. Different concentration levels of the f/2 medium nutrients (i.e. f/2×1–3) were assayed. The composition f/2×3 (N:P = 5), combined with a sinusoidal irradiance pattern (L/D = 24:0) with a 570 µE m−2 s−1 daily mean irradiance, maximized the biomass productivity (2.5 g m−2 day−1) and production rate of the valuable carotenoid peridinin (19.4 ± 1.35 mg m−2 L−1 with nearly 1% of the biomass d.w.). Several carotenoids and polyunsaturated fatty acids were also present in significant percentages in the harvested biomass (EPA, 1.69 ± 0.31% d.w.; DHA, 3.47 ± 0.24% d.w.), which had an average P-molar formulate of C40.7O21.2H73.9N3.9S0.3P1.

A feed-forward back-propagation neural network (FBN) was used as a tool for predicting the growth dynamics of the microalga Karlodinium veneficum in a culture medium with any specified concentrations of the key nutrients. A 3-layered FBN configuration of 27-25-1 nodes was used. This FBN satisfactorily represented the nonlinear interactions among all the nutrients of a culture medium containing up to 25 different components. The FBN model was trained using the growth dynamics data from more than 420 batch culture experiments involving different media compositions. The relative impact of individual nutrients, the initial cell concentration and the culture duration on growth profiles were determined through a systematic analysis of the partitioning of the FBN connection weights. Microelements and vitamins together had a higher relative impact on growth compared to the impact of the macronutrients. The trained FBN successfully predicted the cell concentration dynamics in cultures with previously untested initial conditions. The FBN proved to be an excellent tool for predicting the growth curves in the range of culture conditions that were relevant to this study.

The dinoflagellate Karlodinium veneficum was grown in bubble column photobioreactors and a genetic algorithm-based stochastic search strategy used to find optimal values for the culture parameters gas flow rate, culture height, and nozzle sparger diameter. Cell production, concentration of reactive oxygen species (ROS), membrane fluidity and photosynthetic efficiency were studied throughout the culture period. Gas-flow rates below 0.26Lmin(-1), culture heights over 1.25m and a nozzle diameter of 1.5mm were found to provide the optimal conditions for cell growth, with an increase of 60% in cell production with respect to the control culture. Non-optimal conditions produced a sufficiently high shear stress to negatively affect cell growth and even produce cell death. Cell physiology was also severely affected in stressed cultures. The production of ROS increased by up to 200%, whereas cell membrane fluidity decreased by 60% relative to control cultures. Photosynthetic efficiency decreased concomitantly with membrane fluidity.

In this paper, we have studied the outdoor production of the freshwater microalgae Scenedesmus sp. in two open reactors (32 m2): thin-layer (1.2 m3) and raceway (4.4 m3), using centrate from anaerobic digestion as the sole nutrient source. The aim was to recover valuable nutrients (nitrogen and phosphorus) from effluents in order to maximize biomass productivity. Experiments were performed in semicontinuous mode, modifying the centrate percentage within the culture medium. The optimal centrate percentage was 30% — above this value the culture's performance reduced, probably due to ammonium excess (above 122 mg l− 1). Using the raceway reactor, biomass productivity was 24 g m− 2 day− 1, whereas using the thin-layer reactor, it increased up to 42 g m− 2 day− 1. Nitrogen and phosphorus removal was demonstrated to be proportional to biomass productivity, with maximal values up to 38 mgN l− 1 day− 1 and 3.9 mgP l− 1 day− 1 being removed, respectively. Ammonium stripping was only relevant in the raceway reactor, due to its lower biomass productivity, with more than 40% of the inlet nitrogen being lost to the air. The thin-layer reactor also proved to be more photosynthetically efficient, with maximal values of 4.7% being measured. An economic analysis demonstrated that the thin-layer reactor allowed a reduction in the biomass production cost, in addition to utilizing waste as the nutrient source, with a minimum production cost of 0.9 €/kgbiomass being estimated. In conclusion, it is possible to use centrate from anaerobic digestion as the sole nutrient source for the large-scale production of Scenedesmus sp. biomass thus reducing the biomass production cost by avoiding the use of expensive and non-sustainable fertilizers; while also obtaining returns from the treatment of this type of residue. Such a combination helps to increase the possibility of producing commodities, or biofuels, from microalgae by coupling their production to treatment processes.

The effect of culture conditions on the productivity of biomass and high value products by microalgae has been studied in order to maximise these parameters and meet the nutritional requirements for aquaculture. To this end, a range of temperatures (15–30 °C), incident photon flux densities (PFD) (250–1600 μmol·s − 1 ·m − 2 ) and dilution rates (0.1–0.5 d − 1 ) have been tested in indoor Nannochloropsis gaditana cultures. Predictive models for biomass and high value product productivities are presented. Photoinhibition was observed in cultures at both high incident PFD and temperature. The maximum biomass, EPA, vaucheriaxanthin and β-carotene productivities (0.429 g·L − 1 ·d − 1 and 17.2, 1.05 and 1.31 mg·L − 1 ·d − 1 , respectively) were achieved at 25 °C, with an average irradiance of 170 μmol·s − 1 ·m − 2 and a dilution rate of 0.3 d − 1 .

The aim of this work was to obtain biodiesel (methyl esters) from the saponifiable lipids (SLs) fraction of the microalga Nannochloropsis gaditana, whose biomass dry weight contains 12.1 wt% of these lipids. SLs were extracted from the microalga as free fatty acids (FFAs) for subsequent transformation to methyl esters (biodiesel) by enzymatic esterification. Extraction as FFAs rather than as SLs allows them to be obtained with higher purity. Microalgal FFAs were obtained by direct saponification of lipids in the biomass and subsequent extraction-purification with hexane. Esterification of FFAs with methanol was catalysed by lipase Novozym 435 from Candida antarctica. Stability studies of this lipase in the operational conditions showed that the esterification degree (ED) attained with the same batch of lipase remained constant over six reaction cycles (36 h total reaction time). The optimal conditions attained for 4 g of FFAs were 25°C, 200 rpm, methanol/FFA molar ratio of 1.5:1, Novozym 435/FFA ratio of 0.025:1 w/w and 4 h reaction time. In these conditions the ED attained was 92.6%, producing a biodiesel with 83 wt% purity from microalgal FFAs. Several experimental scales were tested (from 4 to 40 g FFAs), and in all cases similar EDs were obtained.

In this paper freshwater microalgae production is studied using centrate from anaerobic digestion as the only nutrient source. The objective is not only to maximize biomass productivity but also to eliminate contaminants in order to release water which is clean enough for reuse in compliance with regulations. Experiments were performed indoors using two microalgae strains, Muriellopsis sp. and Pseudokirchneriella subcapitata, to establish the optimal centrate percentages within the culture medium. Results demonstrated that with 40–50% of centrate in the culture medium, productivity reached values of up to 1.13 and 1.02 g·l− 1·day− 1 for Muriellopsis sp. and P. subcapitata, respectively. In addition, the removal of nitrogen and phosphorous contained in the culture medium exceeded 90%, the COD at the outlet being lower than 100 mg·l− 1. Above 50% of centrate, toxicity exists and culture performance decreases. No nitrification was observed as the ammonium was completely consumed at centrate percentages lower than 20%. Muriellopsis sp. demonstrated itself to be the more robust strain tolerating higher ammonium concentrations as well as achieving a higher quantum yield, of up to 0.50 g·E− 1, and a higher nitrogen removal rate, of up to 47.5 mgN·l− 1·day− 1 and 3.8 mgP·l− 1·day− 1. Consequently, it is possible to use centrate from anaerobic digestion as the sole nutrient source for the production of freshwater microalgae, thus reducing the biomass production cost by avoiding the use of expensive and non-sustainable fertilizers; not to mention the returns obtained from treating this type of residue. This combination can help to increase the possibilities of producing biofuels from microalgae by means of coupling their production to other treatment processes.

A solid understanding of the effect of hydrodynamic forces encountered by microalgae in bioprocesses would benefit existing bioprocesses, eventually allowing an increase in their productivity. For this purpose, a sensitive method able to quantify cell lysis is crucial. Most of the available protocols and methods intended for this purpose were developed for animal or insect cells. In the case of microalgae, the commercial kits tested were unable to determine the cell lysis extension. The method proposed here was developed to relate the release of a cytoplasmic component (enzyme lactate dehydrogenase (LDH)) with cell lysis by measuring the NADH (reduced form of nicotinamide cofactor adenine dinucleotide) produced by LDH. Although different commercial kits based on similar processes are available, they are more complicated to use and not applicable to microalgae nor when longer-term tests are to be performed.

The aim of this paper is to solve problems that may arise in microalgae cultivation facilities at a hatchery in such a way that its activities are not disrupted. Thus, the effect of storage conditions (time, initial biomass concentration, presence or absence of light, addition of preservatives and conservation atmospheres) on cell viability, evolution of sample concentration, contamination, fatty acid and pigment content for long-term preservation of Nannochloropsis gaditana biomass has been studied. The polyunsaturated fatty acid content was maintained and the eicosapentaenoic acid (EPA, 20:5n3) content was kept stable at 3.6 % (d.w.). The total pigments content was also preserved at 0.85 % (d.w.), what allowed maintaining the antioxidant activity of samples. The results show that samples preserved both as concentrated inocula ready to run a photobioreactor, at an initial biomass (N. gaditana) concentration of 5 g L−1, and as pastes to be used as live food if any incident happens in a hatchery, at 150 g L−1, can be stored for up to 4 months at 4 °C under nitrogen or air atmosphere of conservation maintaining the cell viability and the sample concentration stable and at low incident irradiance (35 μmol photons m−2 s−1). This allows the photosynthetic activity of cells to be preserved in the case of samples at 5 g L−1. The bacterial load was kept at low level by the regular addition of preservative agents.

This work investigated the production of Scenedesmus sp. in semi-continuous mode in three pilot-scale outdoor raceways (7.2 m2) using flue gas for CO2 supply and centrate from the anaerobic digestion of urban wastewater as the sole nutrient source. Experiments were performed at different culture depths, 5, 10 and 15 cm, while evaluating two centrate concentrations (30% and 45%) at dilution rates of 0.2 and 0.3 d−1. Under optimal conditions of 30% centrate, 0.3 d−1 dilution rate and a 15 cm culture depth, a maximum biomass productivity of 22.9 g m−2 d−1 was obtained. The optical properties of the cultures were studied and the results showed a photosynthetic efficiency of up to 2.0% and a quantum yield of 0.3 g biomass E−1. Nitrogen and phosphorus removal rates of 3 g N m−2 d−1 and 0.6 g P m−2 d−1 were recorded, respectively. Lipid productivity of 2.3 g m−2 d−1 was determined possessing a suitable fatty acids profile for biofuel production.

This work investigates the production of the native microalgae strain Scenedesmus sp. in semi-continuous mode at lab scale in open raceway-simulating reactors and using centrate as the culture medium. The biomass productivity and nutrient removal capacity of Scenedesmus sp. at different dilution rates were investigated indoors as well as its tolerance to centrate as the culture medium at different concentrations. A biomass productivity of 7.80 g/m2 day was obtained at 200 μE/m2 s, 5 cm culture depth, 0.30 1/day of dilution rate and 60% centrate while nitrogen and phosphorus removal rates were 1.50 g/m2 day and 0.15 g/m2 day, respectively. The produced biomass characterization under these conditions showed a lipid content of 12.60% d wt. along with a favorable fatty acids profile with 57.70% of total fatty acids composed of saturated and monounsaturated fatty acids. Subsequently, the effect of light intensity and culture depth on biomass productivity and nutrient uptake as well as the biochemical composition and fatty acids profile was studied using two irradiance levels (200 and 1000 μE/m2 s) and four culture depths (5 cm, 10 cm, 15 cm and 20 cm). Under optimal conditions of 1000 μE/m2 s, 60% centrate, 0.30 1/day dilution rate and 15 cm culture depth, a maximum biomass productivity of 22.20 g/m2 day was obtained. Nitrogen and phosphorus removal rates of 2.00 gN/m2 day and 0.40 gP/m2 day, respectively, were recorded. An amount of 11.70% d wt. of lipids was determined along with a suitable fatty acids profile for biofuel production.

In the present work, a study on the performance, culture parameters and modeling of the growth and lipid productivity of Nannochloropsis gaditana in outdoor pilot-scale raceway ponds is made. During the experiments, modifications to the dilution rate and nutrient supply were performed, with maximum productivities of 0.19 gBIOMASS/l day and 30.4 mgLIPIDS/l day obtained at 0.30–0.36 1/day and 10.5 mM NO3−. The impact of average irradiance within the culture (in the range of 280–350 μE/m2 s) and the specific nitrate input (SNI, below 0.4 mmol N/g day) was correlated with an enhancement of saturated and monounsaturated fatty acid contents, which attained maximum values of 29.5% and 33.0% of total FAs, respectively. The modeling of growth, lipid and fatty acid production rates was developed taking into account the irradiance, dilution rate and SNI, which might prove useful in the scaling-up of raceway ponds with this species.

A genetic algorithm has been used to optimize the composition of the culture medium for growing the microalga Nannochloropsis gaditana, based on the nutrients composition of the commercial medium ALGAL. This strategy was carried out through the implementation of 270 experiments spread over nine generations, which allowed achieving an eicosapentaenoic acid (EPA) productivity of 17.8 m gL(-1) d(-1) in a continuous culture of N. gaditana, with an increase of 23% compared to the commercial medium. The EPA yield on nitrogen and phosphorous, 0.042 and 1.146 g(EPA) g(s)(-1), respectively, were 40% and 5-fold higher, respectively, than the values obtained with the nitrogen-optimized ALGAL medium. This improvement was obtained with the medium G-8, which also allowed reducing the requirement of several nutrients such as P, Mo, Mn in 74%, 69% and 66%, respectively, as well as the thiamine content a 46%.

The shear-sensitive dinoflagellate microalga Karlodinium veneficum was grown in a sparged bubble column photobioreactor. The influence of mass transfer and shear stress on cell growth and physiology (concentration of reactive oxygen species, membrane fluidity and photosynthetic efficiency) was studied, and a model describing cell growth in term of mass transfer and culture parameters (nozzle sparger diameter, air flow rate, and culture height) was developed. The results show that mass transfer limits cell growth at low air-flow rates, whereas the shear stress produced by the presence of bubbles is critically detrimental for air flow rates above 0.1 vvm. The model developed in this paper adequately represents the growth of K. veneficum. Moreover, the parameters of the model indicate that bubble rupture is much more harmful for cells than bubble formation.

This work studied outdoor pilot scale production of Nannochloropsis gaditana in tubular photobioreactors. The growth and biomass composition of the strain were studied under different culture strategies: continuous-mode (varying nutrient supply and dilution rate) and two-stage cultures aiming lipid enhancement. Besides, parameters such as irradiance, specific nitrate input and dilution rate were used to obtain models predicting growth, lipid and fatty acids production rates. The range of optimum dilution rate was 0.31–0.35 1/day with maximum biomass, lipid and fatty acids productivities of 590, 110 and 66.8 mg/l day, respectively. Nitrate limitation led to an increase in lipid and fatty acids contents (from 20.5% to 38.0% and from 16.9% to 23.5%, respectively). Two-stage culture strategy provided similar fatty acids productivities (56.4 mg/l day) but the neutral lipids content was doubled.

The baculovirus-insect cell expression system is widely used in producing recombinant proteins. This review is focused on the use of this expression system in developing bioprocesses for producing proteins of interest. The issues addressed include: the baculovirus biology and genetic manipulation to improve protein expression and quality; the suppression of proteolysis associated with the viral enzymes; the engineering of the insect cell lines for improved capability in glycosylation and folding of the expressed proteins; the impact of baculovirus on the host cell and its implications for protein production; the effects of the growth medium on metabolism of the host cell; the bioreactors and the associated operational aspects; and downstream processing of the product. All these factors strongly affect the production of recombinant proteins. The current state of knowledge is reviewed.

Mixotrophic cultures of Phaeodactylum tricornutum were carried out in bubble columns using fructose and glycerol in indoor fed-batch and semi-continuous modes. In the fed-batch cultures, different nutrient-addition strategies, combined with stepwise increments in the light intensity, were assayed. It was found that glycerol promoted significantly higher biomass productivity than fructose. A glycerol-induced photoinhibition that arrested the growth of P. tricornutun was also observed. As this was considered a limitation as regards transferring the fed-batch mode to outdoor conditions, this information was used to culture P. tricornutum in semi-continuous mode. Similar glycerol-induced photoinhibition was not observed in these cultures, even at highest dilution rates. Although the highest biomass (1.5 g L(-1) d(-1)) and EPA (40 mg L(-1) d(-1)) productivities found in the semi-continuous cultures were lower than those obtained photoautotrophically in outdoor photobioreactors, the findings showed that semi-continuous mode was an excellent candidate for transferring mixotrophic culture to an outdoor setting.

Nannochloropsis gaditana is a microalga with a high nutritional value and a protein and polyunsaturated fatty acid (PUFA) content that makes it interesting as a feed in aquaculture. To maximize its productivity and nutritional value in large-scale culture, a well-known commercial medium was optimized to the most favorable nutrient level using commercial fertilizers. Optimal growth conditions were obtained in the alternative fertilizer-based medium at a nitrogen concentration of 11.3 mM, a phosphorus concentration of 0.16 mM, and a micronutrient concentration of 30 μL L(-1). This alternative medium allowed to obtain a biomass concentration similar to that achieved when using the commercial formula but with a reduction in Cu, Fe, and Mo content of 71%, 89%, and 99%, respectively. A maximum biomass productivity of 0.51 g L(-1) d(-1) was obtained. The eicosapentaenoic acid and protein contents of the biomass were 2.84% and 44% of dry weight, respectively.

Continuous cultures of Nannochloropsis gaditana, Tetraselmis chuii, Tetraselmis suecica and Phaeodactylum tricornutum were carried out at different dilution rates and culture media in order to check their influence on biomass productivity. N. gaditana attained maximum biomass productivity of 0.49 g/l day at a dilution rate of 0.42 1/day. The influence of nitrate concentration on biomass productivity was tested by continuous cultures of N. gaditana. At 8.0 mM nitrate and dilution rates ranging between 0.30 and 0.40 1/day, maximum biomass productivities were achieved. To enhance lipid accumulation, a two-stage culture strategy consisting in a first stage of nitrate-replete conditions followed by a nitrate-depleted phase was performed. The accumulated productivity was 51 mgFATTY ACIDS/l day. Results showed an important change in the fatty acids profile and an increase in the neutral lipids content, representing 73.1% of total lipids. Additionally, the combination of nitrogen depletion and light stress was proved to contribute to lipid enhancement.

Nannochloropsis gaditana is a good producer of proteins and valuable fatty acids for aquaculture. Recycling of culture medium is interesting for microalgae commercial production as it cuts costs and prevents environmental contamination. The recycled medium must be sterilized to prevent the buildup of unwanted metabolites and microorganisms. We tested several sterilization methods: filtration, ozonation, chlorination, addition of hydrogen peroxide and heating. Results showed that the most successful method is ozonation lowering the bacterial load to 1.910(3)CFUs/mL, which is 1000-fold and 10-fold lower than the supernatant obtained after harvesting and the initial filtered medium, respectively. Continuous cultures of N. gaditana were grown using this recirculated supernatant. A maximum biomass productivity of 0.8 g/L/d composed of ∼50% proteins and 40% lipids with more than 3%d.w. EPA was obtained making this biomass very interesting for aquaculture.

In order to obtain microalgal saponifiable lipids (SLs) fractions containing different polar lipid (glycolipids and phospholipids) contents, SLs were extracted from wet Nannochloropsis gaditana microalgal biomass using seven extraction systems, and the polar lipid contents of some fractions were reduced by low temperature acetone crystallization. We observed that the polar lipid content in the extracted lipids depended on the polarity of the first solvent used in the extraction system. Lipid fractions with polar lipid contents between 75.1% and 15.3% were obtained. Some of these fractions were transformed into fatty acid methyl esters (FAMEs, biodiesel) by methanolysis, catalyzed by the lipases Novozym 435 and Rhizopus oryzae in tert-butanol medium. We observed that the reaction velocity was higher the lower the polar lipid content, and that the final FAME conversions achieved after using the same lipase batch to catalyze consecutive reactions decreased in relation to an increase in the polar lipid content.

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).

Dinoflagellate microalgae are an important source of marine biotoxins. Bioactives from dinoflagellates are attracting increasing attention because of their impact on the safety of seafood and potential uses in biomedical, toxicological and pharmacological research. Here we review the potential applications of dinoflagellate toxins and the methods for producing them. Only sparing quantities of dinoflagellate toxins are generally available and this hinders bioactivity characterization and evaluation in possible applications. Approaches to production of increased quantities of dinoflagellate bioactives are discussed. Although many dinoflagellates are fragile and grow slowly, controlled culture in bioreactors appears to be generally suitable for producing many of the metabolites of interest.

Yessotoxin (YTX) and okadaic acid (OA), algal toxins accumulated in edible shellfish, were previously shown to induce a specific and reversible T Cell Receptor (TCR) down-regulation in T lymphocyte EL-4 cells, in a time and concentration-dependent manner, via protein kinase C (PKC) and serine/threonine protein phosphatase 2A (PP2A) activities. In this study we have evaluated the development of other signs of toxicity induced by low concentrations of YTX or OA for 3 days of treatment. Concentrations of YTX as low as 1 nM decreased a 35% the concentration of viable cells after 48 h exposure to the toxin, while concentrations as little as 5 nM YTX or OA were sufficient to induce membrane blebbing. The concentration of YTX that produced after 24 h of incubation a 50% reduction in maximum cell viability (EC50₂₄) was approximately 46 nM, whereas with OA over 75% of the cells were still viable after exposure to 100 nM OA. According to our results, the cytoskeleton of EL-4 cells seems to be a cell component particularly sensitive to YTX and OA with disruption of F-actin cytoskeleton in these cells treated with concentrations of YTX or OA as low as 5 nM at 48 h incubation. Toxicity by YTX or OA involved typical hallmarks of apoptosis and an increase of reactive oxygen species (ROS) production. The cytotoxic effects of YTX and OA reported here, and the previously demonstrated potential of these toxins to regulate the activity of EL-4 cells through the regulation of TCR expression, rise reasonable concern about possible risks for human health associated to the chronic exposure to low amounts of YTX or OA itself or enhanced by the presence of other shellfish toxins specially by a population potentially at risk such as immunocompromised patients.

A genetic algorithm stochastic search strategy was used to optimize the culture medium for producing the toxic marine dinoflagellate microalga Protoceratium reticulatum. The optimized medium contained 26 different components (macronutrients, trace elements, vitamins). The use of this medium allowed a 60% enhancement in the final cell concentration relative to the control culture based on L1 medium. The final titer of yessotoxins was improved by 40% relative to the control medium.