L. López-Rosales

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.

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.

New insights into shear-sensitivity in dinoflagellate microalgae

A modification of a flow contraction device was used to subject shear-sensitive microalgae to well-defined hydrodynamic forces. The aim of the study was to elucidate if the inhibition of shear-induced growth commonly observed in dinoflagellate microalgae is in effect due to cell fragility that results in cell breakage even at low levels of turbulence. The microalgae assayed did not show any cell breakage even at energy dissipation rates (EDR) around 10(12)Wm(-3), implausible in culture devices. Conversely, animal cells, tested for comparison purposes, showed high physical cell damage at average EDR levels of 10(7)Wm(-3). Besides, very short exposures to high levels of EDR promoted variations in the membrane fluidity of the microalgae assayed, which might trigger mechanosensory cellular mechanisms. Average EDR values of only about 4·10(5)Wm(-3) increased cell membrane fluidity in microalgae whereas, in animal cells, they did not.

Biofouling in photobioreactors for marine microalgae

Abstract 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.

Rapid method for the assessment of cell lysis in microalgae cultures

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.

Long-term culture of the marine dinoflagellate microalga Amphidinium carterae in an indoor LED-lighted raceway photobioreactor: Production of carotenoids and fatty acids

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.