F. García-Camacho

A mechanistic model of photosynthesis in microalgae including photoacclimation dynamics

In this study, an extension and actualization of a dynamic model of photosynthesis, previously published (Camacho Rubio et al., 2003), is presented. This model uses the concept of Photosynthetic Unit (PSU). In it, the processes of excited PSU net disappearance rate, photoinhibition and damaged PSU repair have been redefined in the context of photochemical and non-photochemical quenching. The phenomenon of photoacclimation in microalgae has been extensively incorporated into this model, significantly improving its prediction capabilities. A significant number of previously reported experimental results are successfully interpreted by the novel formulation: 1. Photosynthetic response of cells photoacclimated to different constant continuous irradiances (photoacclimated cells); 2. Short-term photosynthetic response of cells non-photoacclimated to different constant continuous irradiances (non-photoacclimated cells); 3. Kinetics of the photoacclimation response; 4. Photosynthetic response under intermittent light. It is expected that this model will contribute notably to the simulation of industrial algal mass cultures.

Protein production using the baculovirus‐insect cell expression system

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.

Bioactives from microalgal dinoflagellates

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.

Mixotrophic growth of Phaeodactylum tricornutum on fructose and glycerol in fed-batch and semi-continuous modes.

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.

Cytotoxicity of yessotoxin and okadaic acid in mouse T lymphocyte cell line EL-4

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.

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 (µm) was 0.204 day−1 at 25 °C and 40 µE·m−2·s−1. Photo-inhibition was observed at I0 > 40 μEm−2s−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 µm-I0 curves and another empirical model for relating µm-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.

An optimisation approach for culturing shear-sensitive dinoflagellate microalgae in bench-scale bubble column photobioreactors.

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.

Shear-induced changes in membrane fluidity during culture of a fragile dinoflagellate microalga

The commonly used shear protective agent Pluronic F68 (PF68) was toxic to the marine dinoflagellate microalga Protoceratium reticulatum, but had a shear‐protective effect on it at concentrations of ≤0.5 g L−1. Supplementation of P. reticulatum cultures with PF68 actually increased the fluidity of the cell membrane; therefore, the shear protective effect of PF68 could not be ascribed to reduced membrane fluidity, an explanation that has been commonly used in relation to its shear protective effect on animal cells. Data are reported on the membrane fluidity of P. reticulatum and its response to the presence of PF68 under sublethal and lethal turbulence regimens. The membrane fluidity was found to depend strongly on the level of lipoperoxides in the cells produced under lethal agitation.

Modelling of multi-nutrient interactions in growth of the dinoflagellate microalga Protoceratium reticulatum using artificial neural networks

This study examines the use of artificial neural networks as predictive tools for the growth of the dinoflagellate microalga Protoceratium reticulatum. Feed-forward back-propagation neural networks (FBN), using Levenberg-Marquardt back-propagation or Bayesian regularization as training functions, offered the best results in terms of representing the nonlinear interactions among all nutrients in a culture medium containing 26 different components. A FBN configuration of 26-14-1 layers was selected. The FBN model was trained using more than 500 culture experiments on a shake flask scale. Garsons algorithm provided a valuable means of evaluating the relative importance of nutrients in terms of microalgal growth. Microelements and vitamins had a significant importance (approximately 70%) in relation to macronutrients (nearly 25%), despite their concentrations in the culture medium being various orders of magnitude smaller. The approach presented here may be useful for modelling multi-nutrient interactions in photobioreactors.

Pilot-scale bubble column photobioreactor culture of a marine dinoflagellate microalga illuminated with light emission diodes

Production of biomass of the shear-sensitive marine algal dinoflagellate Karlodinium veneficum was successfully scaled-up to 80L using a bubble column photobioreactor. The scale factor exceeded 28,500. Light-emission diodes were used as the light source. The diel irradiance profile mimicked the outdoor profile of natural sunlight. The final cell concentration in the absence of nutrient limitation in the scaled-up photobioreactor was nearly 12×10(5)cellsmL(-1), or the same as in laboratory culture systems. The pH-controlled culture (pH=8.5) was always carbon-sufficient. The culture was mixed pneumatically by using a superficial air velocity of 1.9×10(-3)ms(-1) and the temperature was controlled at 21±1°C.