Isolation and Structural Elucidation of New Amphidinol Analogues from Amphidinium carterae Cultivated in a Pilot-Scale Photobioreactor.

The demand for valuable products from dinoflagellate biotechnology has increased remarkably in recent years due to their many prospective applications. However, there remain many challenges that need to be addressed in order to make dinoflagellate bioactives a commercial reality. In this article, we describe the technical feasibility of producing and recovering amphidinol analogues (AMs) excreted into a culture broth of Amphidinium carterae ACRN03, successfully cultured in an LED-illuminated pilot-scale (80 L) bubble column photobioreactor operated in fed-batch mode with a pulse feeding strategy. We report on the isolation of new structurally related AMs, amphidinol 24 (1, AM24), amphidinol 25 (2, AM25) and amphidinol 26 (3, AM26), from a singular fraction resulting from the downstream processing. Their planar structures were elucidated by extensive NMR and HRMS analysis, whereas the relative configuration of the C-32→C-47 bis-tetrahydropyran core was confirmed to be antipodal in accord with the recently revised configuration of AM3. The hemolytic activities of the new metabolites and other related derivatives were evaluated, and structure-activity conclusions were established. Their isolation was based on a straightforward and high-performance bioprocess that could be suitable for the commercial development of AMs or other high-value compounds from shear sensitive dinoflagellates.

Biofouling in photobioreactors for marine microalgae.

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.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean).

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

Salinity as an Abiotic Stressor for Eliciting Bioactive Compounds in Marine Microalgae.

This study investigated the impact of culture medium salinity (5-50 PSU) on the growth and maximum photochemical yield of photosystem II (Fv/Fm) and the composition of carotenoids, fatty acids, and bioactive substances in three marine microalgae (Chrysochromulina rotalis, Amphidinium carterae, and Heterosigma akashiwo). The microalgae were photoautotrophically cultured in discontinuous mode in a single stage (S1) and a two-stage culture with salt shock (S2). A growth model was developed to link biomass productivity with salinity for each species. C. rotalis achieved a maximum biomass productivity (Pmax) of 15.85 ± 0.32 mg·L-1·day-1 in S1 and 16.12 ± 0.13 mg·L-1·day-1 in S2. The salt shock in S2 notably enhanced carotenoid production, particularly in C. rotalis and H. akashiwo, where fucoxanthin was the main carotenoid, while peridinin dominated in A. carterae. H. akashiwo also exhibited increased fatty acid productivity in S2. Salinity changes affected the proportions of saturated, monounsaturated, and polyunsaturated fatty acids in all three species. Additionally, hyposaline conditions boosted the production of haemolytic substances in A. carterae and C. rotalis.

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.

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.

Pathogens and predators impacting commercial production of microalgae and cyanobacteria.

Production of phytoplankton (microalgae and cyanobacteria) in commercial raceway ponds and other systems is adversely impacted by phytoplankton pathogens, including bacteria, fungi and viruses. In addition, cultures are susceptible to productivity loss, or crash, through grazing by contaminating zooplankton such as protozoa, rotifers and copepods. Productivity loss and product contamination are also caused by otherwise innocuous invading phytoplankton that consume resources in competition with the species being cultured. This review is focused on phytoplankton competitors, pathogens and grazers of significance in commercial culture of microalgae and cyanobacteria. Detection and identification of these biological contaminants are discussed. Operational protocols for minimizing contamination, and methods of managing it, are discussed.

Effect of Nitrogen, Phosphorous, and Light Colimitation on Amphidinol Production and Growth in the Marine Dinoflagellate Microalga Amphidinium carterae.

The marine dinoflagellate microalga Amphidinium carterae is a source of amphidinols, a fascinating group of polyketide metabolites potentially useful in drug design. However, Amphidinium carterae grows slowly and produces these toxins in tiny amounts, representing a hurdle for large-scale production. Understanding dinoflagellate growth kinetics under different photobioreactor conditions is imperative for promoting the successful implementation of a full-scale integrated bioproduct production system. This study evaluates the feasibility of growing Amphidinium carterae under different ranges of nitrogen concentration (NO3- = 882-2646 µM), phosphorus concentration (PO33- = 181-529 µM), and light intensity (Y0 = 286-573 µE m-2 s-1) to produce amphidinols. A mathematical colimitation kinetic model based on the "cell quota" concept is developed to predict both algal growth and nutrient drawdown, assuming that all three variables (nitrogen, phosphorous and light) can simultaneously colimit microalgal growth. The model was applied to the semicontinuous culture of the marine microalgae Amphidinium carterae in an indoor LED-lit raceway photobioreactor. The results show that both growth and amphidinol production strongly depend on nutrient concentrations and light intensity. Nonetheless, it was possible to increase Amphidinium carterae growth while simultaneously promoting the overproduction of amphidinols. The proposed model adequately describes Amphidinium carterae growth, nitrate and phosphate concentrations, and intracellular nitrogen and phosphorus storage, and has therefore the potential to be extended to other systems used in dinoflagellate cultivation and the production of bioproducts obtained therein.

Characterization of bubble column photobioreactors for shear-sensitive microalgae culture.

The shear-sensitive marine algal dinoflagellate Karlodinium veneficum was grown in a cylindrical bubble column photobioreactor with an internal diameter of 0.044 m. Initial liquid height varied from 0.5 to 1.75 m, superficial gas velocities from 0.0014 to 0.0057 ms-1, and nozzle diameter from 1 to 2.5 mm. Computational fluid dynamics was used to characterize the flow hydrodynamics and energy dissipation rates. Experimental gas holdup and volumetric mass transfer coefficient strongly depended on the liquid height and correlated well with the Froude number. Energy dissipation near the head space (EDtop) was one order of magnitude higher than the average energy dissipation in the whole reactor (EDwhole), and the value in the sparger zone (EDspar) was one order of magnitude higher than EDtop. Cultures of K. veneficum were limited by CO2 transfer at low EDwhole and severely stressed above a critical value of EDwhole.

Production of Amphidinols and Other Bioproducts of Interest by the Marine Microalga Amphidinium carterae Unraveled by Nuclear Magnetic Resonance Metabolomics Approach Coupled to Multivariate Data Analysis.

This study assessed the feasibility of an NMR metabolomics approach coupled to multivariate data analysis to monitor the naturally present or stresses-elicited metabolites from a long-term (>170 days) culture of the dinoflagellate marine microalgae Amphidinium carterae grown in a fiberglass paddlewheel-driven raceway photobioreactor. Metabolic contents, in particular, in two members of the amphidinol family, amphidinol A and its 7-sulfate derivative amphidinol B (referred as APDs), and other compounds of interest (fatty acids, carotenoids, oxylipins, etc.) were evaluated by altering concentration levels of the f/2 medium nutrients and daily mean irradiance. Operating with a 24 h sinusoidal light cycle allowed a 3-fold increase in APD production, which was also detected by an increase in hemolytic activity of the methanolic extract of A. carterae biomass. The presence of APDs was consistent with the antitumoral activity measured in the methanolic extracts of the biomass. Increased daily irradiance was accompanied by a general decrease in pigments and an increase in SFAs (saturated fatty acids), MUFAs (monounsaturated fatty acids), and DHA (docosahexaenoic acid), while increased nutrient availability lead to an increase in sugar, amino acid, and PUFA ω-3 contents and pigments and a decrease in SFAs and MUFAs. NMR-based metabolomics is shown to be a fast and suitable method to accompany the production of APD and bioactive compounds without the need of tedious isolation methods and bioassays. The two APD compounds were chemically identified by spectroscopic NMR and spectrometric ESI-IT MS (electrospray ionization ion trap mass spectrometry) and ESI-TOF MS (ESI time-of-flight mass spectrometry) methods.

The use of an artificial neural network to model the infection strategy for baculovirus production in suspended insect cell cultures.

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.

Modeling shear-sensitive dinoflagellate microalgae growth in bubble column photobioreactors.

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 0.1vvm. 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.

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.

The effect of spent medium recycle on cell proliferation, metabolism and baculovirus production by the lepidopteran Se301 cell line infected at very low MOI.

The aim of this paper was to study the effect of spent medium recycle on Spodoptera exigua Se301 cell line proliferation, metabolism, and baculovirus production when grown in batch suspension cultures in Ex-Cell 420 serum-free medium. The results showed that the recycle of 20% of spent medium from a culture in mid-exponential growth phase improved growth relative to a control culture grown in fresh medium. Although both glucose and glutamine were still present at the end of the growth phase, glutamate was always completely exhausted. The pattern of the specific glucose and lactate consumption and production rates, as well as the specific glutamine and glutamate consumption rates, suggests a metabolic shift at spent medium recycle values of over 60%, with a decrease in the efficiency of glucose utilization and an increase in glutamate consumption to fuel energy metabolism. Baculovirus infection provoked a change in the metabolic pattern of Se301 cells, although a beneficial effect of spent medium recycle was also observed. Both growth rate and maximum viable cell density decreased relative to uninfected cultures. The efficiency of glucose utilization was dramatically reduced in those cultures containing the lowest percentages of spent medium, whereas glutamine and glutamate consumption was modulated, thereby suggesting that infected cells were devoted to virus replication, retaining their ability to incorporate the nutrients required to support viral replication. Recycle of 20% of spent medium increased baculovirus production by around 90%, thus showing the link between cell growth and baculovirus production.

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.

Adaptation of the Se301 insect cell line to suspension culture. Effect of turbulence on growth and on production of nucleopolyhedrovius (SeMNPV).

As chemical pesticides are being banned as control agents for agricultural pests, the use of the highly specific, safe to non-target organisms baculoviruses has been proposed. These viruses can be produced either in vivo or in vitro. In vitro production requires appropriated host insect cell lines with the ability for growing as freely-suspended cells. In this work, the Spodoptera exigua Se301 cell line was used to produce the commercially available S. exigua nucleopolyhedrovirus (SeMNPV) in suspension. Se301 cells showed to be very sensitive to the hydrodynamic shear rates developed in bioreactors. A process of progressive adaptation to freely-suspended cultures using protective additives against shear stress and disaggregant was proposed. The best combinations were polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) with the disaggregant dextran sulfate (DS). Both static and freely-suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Production of occluded baculovirus (OB) increased with the multiplicity of infection (MOI > 0.1).

Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas.

Chemical treatment with hydroxyurea (HU) has been selected as a simple and low cost strategy to generate a cell population enriched for the G1 phase. After the chemical treatment with HU, cells were stimulated with anti-mIgG to test if the positive effects of anti-mIgG on CD40 expression and specific IgG2a production rate were improved upon a cell population with a higher percentage of cells in G1 phase at the beginning of the cell culture. In addition, other treatments assayed in this work were the cell stimulation with Lipopolysaccharide (LPS) both before and after the HU treatment. It has been observed that the use of HU under conditions able to maintain the cells in viable state (0.1 mM for 20 h), has a negative effect on CD40 expression and specific IgG2a production rate induced by anti-mIgG. The positive effect of LPS on cell stimulation induced by anti-mIgG is reduced on cells treated with HU.