Identification and characterisation of the zooplankton genus Tigriopus as a natural source of astaxanthin and high-value fatty acids (EPA and DHA)

Carotenoids are molecules involved in essential physiological processes in humans. For this reason, the biotech industry constantly seeks to develop new methods to produce these molecules. This thesis is part of the LEAF (LutEin Algae Feasibility) project, whose initial objective was to cultivate lutein-rich microalgae and collect them using zooplankton as a more cost-effective alternative to the marigold flower. However, our results suggest that this method, using the microalgae species Dunaliella salina and the zooplankton species Artemia franciscana, produces limited amounts of lutein and with low purity. Therefore, we reoriented our work to characterize the marine harpacticoid copepod Tigriopus californicus. This species does not limit itself to accumulating metabolites of interest such as Artemia, but produces them from ingested or selfmanufactured precursors. Specifically, Tigriopus synthetizes the carotenoid astaxanthin and the omega-3 fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Our feeding experiments confirmed that T. californicus assimilated carotenoids from all the feed sources used and converted them into astaxanthin. In addition, T. californicus used short-chain fatty acids to produce and accumulate EPA and DHA. Specifically, T. californicus produced the highest amounts of astaxanthin, EPA and DHA (1.53 mg g-1, 0.139 μg g-1 and 0.204 μg g-1 of dry mass, respectively), when Nannochloropsis oceanica was used as a food source (p <0.05 when compared with the other diets). Of note, T. californicus produced 0.098 μg of DHA per g of dry biomass when fed with baker's yeast (which does not have alpha-linolenic acid, essential in animals to synthesize DHA), suggesting that Tigriopus can synthesize DHA de novo. Exposure of T. californicus to abiotic stimuli suggests, as has been reported by other authors, that light and temperature affects the production of these molecules in Tigriopus. We found that the exposure of Tigriopus to actinic light significantly increased the production of astaxanthin (0.65 mg g-1 of dry biomass) and total fatty acids (2.786 μg g-1 of dry biomass). On the other hand, moderate temperatures (21 °C) favoured the production of these molecules. This study suggests that T. californicus can produce a series of high-value molecules from more common molecules, making this species of zooplankton a promising candidate for producing krill-like oil, rich in EPA and DHA and with higher concentrations of astaxanthin. These results warrant further research to investigate the adaptation of T. californicus to mass production in the reactor developed in the LEAF project.