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.

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.

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.