Assessment of the marine microalga Chrysochromulina rotalis as bioactive feedstock cultured in an easy-to-deploy light-emitting-diode-based tubular photobioreactor.

Marine microalgae have potential to be low-cost raw materials. This depends on the exploitation of different biomass fractions for high-value products, including unique compounds. Chrysochromulina rotalis, an under-explored haptophyte with promising properties, was the focus of this study. For the first time, C. rotalis was successfully cultivated in an 80 L tubular photobioreactor, illuminated by an easy-to-use light-emitting-diode-based system. C. rotalis grew without certain trace elements and showed adaptability to different phosphorus sources, allowing a significant reduction in the N:P ratio without compromising biomass yield and productivity. The design features of the photobioreactor provided a protective environment that ensured consistent biomass production from this shear-sensitive microalgae. Carotenoid analysis showed fucoxanthin and its derivatives as major components, with essential fatty acids making up a significant proportion of the total. The study emphasizes the tubular photobioreactor's role in sustainable biomass production for biorefineries, with C. rotalis as a valuable bioactive feedstock.

A step forward in sustainable pesticide production from Amphidinium carterae biomass via photobioreactor cultivation with urea as a nitrogen source.

This study addresses the problem of replacing nitrate and ammonium with urea as a greener nitrogen source in the mass cultivation of the microalga Amphidinium carterae for the development of amphidinol-based phytosanitary products. To solve this problem, a nuclear magnetic resonance assisted investigation evaluated the effect of nitrogen sources on growth and metabolic profiles in photobioreactors. Urea-fed cultures exhibited growth kinetics comparable to nitrate-fed cultures (µmax = 0.30 day-1, Pbmax = 43 mgL-1day-1). Urea-fed cultures had protein, lipid, and carbohydrate contents of 39.5%, 14.5%, and 42.4%, respectively, while nitrate-fed cultures had 27.9 %, 17.5% and 48.1%, respectively. Metabolomics revealed nitrogen source-dependent metabotypes and a correlation between amphidinols and polyunsaturated fatty acids. The amphidinol-to-nitrogen yield coefficient in urea-fed cultures (135 mg/g) was approximately 2.5 times higher than in nitrate-fed cultures. The potent antiphytopathogenic activity exhibited by extracts from urea-fed cultures underscores the potential of urea as a sustainable nitrogen source in microalgae-based biorefineries.

Life-cycle assessment of a microalgae-based fungicide under a biorefinery approach.

The aim of this work was to perform a life-cycle analysis of the production process of a fungicide based on amphidinols. Two scenarios were evaluated: (1) biorefinery process -biofungicide, fatty acids and carotenoids were considered as co-products-, and (2) biofungicide as only product. Inventory data were taken and scaled-up from previous work on pilot-scale reactors, as well as lab-scale downstream equipment. A yearly production of 22,000 L of fungicide, was selected as the production objective. Despite, photosynthetic biomass is a sink of anthropogenic CO2, harvesting and downstream processing have large carbon footprints that exceed the biomass fixed carbon. Producing the biofungicide resulted in 34.61 and 271.33 ton of CO2e (15 years) for the Scenarios 1 and 2, respectively. Different commercial agricultural fungicides were compared with the microalgal fungicide. A lower impact of the microalgal product for most of the indicators, including carbon footprint, was shown.

Treatment of secondary urban wastewater with a low ammonium-tolerant marine microalga using zeolite-based adsorption.

The low tolerance of marine microalgae to ammonium and hyposalinity limits their use in urban wastewater (UWW) treatments. In this study, using the marine microalga Amphidinium carterae, it is demonstrated for the first time that this obstacle can be overcome by introducing a zeolite-based adsorption step to obtain a tolerable UWW stream. The maximum ammonium adsorption capacities measured in the natural zeolite used are among the highest reported. The microalga grows satisfactorily in mixtures of zeolite-treated UWW and seawater at a wide range of proportions, both with and without adjusting the salinity, as long as the ammonium concentration is below the threshold tolerated by the microalgae (6.3 mg L-1). A proof of concept performed in 10-L bubble column photobioreactors with different culture strategies, including medium recycling, showed an enhanced biomass yield relative to a control with no UWW. No noticeable effect was observed on the production of specialty metabolites.

An integrated approach for the efficient separation of specialty compounds from biomass of the marine microalgae Amphidinium carterae.

An amphidinol-prioritized fractioning approach was for the first time developed to isolate multiple specialty metabolites such as amphidinols, carotenoids and fatty acids using the biomass of the marine microalgae Amphidinium carterae. The biomass was produced in a raceway photobioreactor and the exhausted culture media were reused, thus fulfilling sustainability criteria employing a circular economy concept. The integrated bioactive compounds-targeted approach presented here consisted of four steps with which recovery percentages of carotenoids, fatty acids and amphidinols of 97%, 82% and 99 %, respectively, were achieved. The proposed process was proved to be a better extraction system for this microalga than another based on a sequential gradient partition with water and four water-immiscible organic solvents (hexane, carbon tetrachloride, dichloromethane and n-butanol). The proposed process could be scaled-up as a commercial solid-phase extraction technology well-established for industrial bioprocesses.

Long-term biofouling formation mediated by extracellular proteins in Nannochloropsis gaditana microalga cultures at different medium N/P ratios.

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 whose cells secrete macromolecules, such as proteins, with a high capacity for forming a conditioning film before 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).

Improved extraction of bioactive compounds from biomass of the marine dinoflagellate microalga Amphidinium carterae.

The extraction of three families of compounds (carotenoids, fatty acids and amphidinols) from the biomass of two strains of Amphidinium carterae (ACRN03 and Dn241EHU) was improved by tuning cell disruption and solvent extraction operations. The extraction of carotenoids was evaluated using alkaline saponification (0%-60% KOH d.w.) at different temperatures (25-80 °C). High levels of carotenoids were obtained at 60 °C using freeze-dried biomass, not subjected to cell disruption methods. The ACRN03 strain required 20% KOH whereas the Dn241EHU strain did not require saponification since carotenoid degradation was observed. The extraction efficiencies were determined with a wide range of pure solvents and mixtures thereof. Two empirical non-linear equations were used to correlate extraction percentages for each family of compounds with the Hildebrand solubility parameter (δT) and the polarity index of the solvents (PI). Thresholds of δT and PI of around 20 MPa1/2 and 6, respectively, were determined for the extraction of amphidinols, consistent with antiproliferative activity measurements.

Assessment of multi-step processes for an integral use of the biomass of the marine microalga Amphidinium carterae.

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).

Data on the Amphidinium carterae Dn241EHU isolation and morphological and molecular characterization.

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].

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 ±â€¯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 ±â€¯0.31% d.w.; DHA, 3.47 ±â€¯0.24% d.w.), which had an average P-molar formulate of C40.7O21.2H73.9N3.9S0.3P1.

Maximizing carotenoid extraction from microalgae used as food additives and determined by liquid chromatography (HPLC).

Microalgae are an interesting source of natural pigments that have valuable applications. However, further research is necessary to develop processes that allow us to achieve high levels of carotenoid recovery while avoiding degradation. This work presents a comprehensive study on the recovery of carotenoids from several microalgae genera, optimizing carotenoid extraction using alkaline saponification at various temperatures and KOH concentrations. Results show that I. galbana requires a temperature of 60 °C and <10% KOH, N. gaditana and K. veneficum require 60 °C and no saponification, P. reticulatum requires 40 °C and 10% KOH, T. suecica and H. pluvialis require 25 °C and 40% KOH while C. sp. and S. almeriensis require 80 °C and 40% KOH. The influence of the solvent on carotenoid recovery was also studied. In general terms, an ethanol:hexane:water (77:17:6 v/v/v) mixture results in good yields.

Pilot-scale outdoor photobioreactor culture of the marine dinoflagellate Karlodinium veneficum: Production of a karlotoxins-rich extract.

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.

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.

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.

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. Garson's 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.

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.

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.

Differential secretion of chicken growth hormone variants after growth hormone-releasing hormone stimulation in vitro.

Variants of growth hormone (GH) are present in most vertebrates. Chicken GH (cGH) undergoes posttranslational modifications that contribute to its structural diversity. Although the 22-kDa form of GH is the most abundant, some other variants have discrete bioactivities that may not be shared by others. The proportion of cGH variants changes during ontogeny, suggesting that they are regulated differentially. The effect of growth hormone-releasing hormone (GHRH) on the release of cGH variants was studied in both pituitary gland and primary cell cultures, employing sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and densitometry. GHRH (2 nM, 2 h) stimulated the secretion of most of the size variants of cGH although the amplitude of increase was not equal for each one. A differential effect on the secretion of GH size variants, particularly on the 22- (monomer) and 26-kDa (putatively glycosylated) cGH isoforms was found in both systems. In the whole pituitary culture, the proportion of the 26-kDa immunoreactive cGH increased 35% while the 22 kDa decreased 31% after GHRH treatment in comparison with the controls. In the primary cell culture system, the proportion of the glycosylated variant increased 43% whereas the monomer and the dimer decreased 22.26 and 29%, respectively, after GHRH stimulation. Activators of intracellular signals such as 1 mM 8-bromo-cAMP and 1 microM phorbol myristate acetate had a similar effect to that obtained with GHRH. The data support the hypothesis that GH variants may be under differential control and that GHRH promotes the release of a glycosylated cGH variant that has an extended half-life in circulation.