Sequential Supercritical Extraction of Carotenoid- and Chlorophyll a-Enriched Fractions from Nannochloropsis oculata

Carotenoids and chlorophylls are lipophilic microalgal pigments with antioxidant and other bioactive properties relevant to food, cosmetic and pharmaceutical applications. Because most solvents, including green solvents, co-extract both pigment classes, carotenoid-rich extracts often retain substantial chlorophyll and require chromatographic purification. Sequential extraction of the same biomass with solvents of increasing polarity may instead fractionate pigments during extraction, reducing purification demands and supporting integrated biomass use in microalgal biorefineries. This study evaluated the effects of temperature and pressure on a two-stage supercritical extraction of carotenoids and chlorophyll a from Nannochloropsis oculata. Freeze-dried biomass (0.5 g) was extracted with supercritical CO₂ (30 min static; 45 min dynamic, 8 mL min⁻¹), and the residual biomass was then extracted with supercritical CO₂ (5 mL min⁻¹) and ethanol as cosolvent (20 mL; 30 min static; 30 min dynamic), added directly to the extraction cell. Temperature (35–70 °C) and pressure (10–25 MPa) were investigated using a central composite rotatable design (2²), comprising 11 experimental runs. Total carotenoids and chlorophyll a were quantified by visible spectrophotometry with correction for spectral overlap. Pigment content was expressed per gram of dry biomass, and purity as the proportion of the target pigment in the summed pigment content of each extract. Effects were considered significant at p < 0.05. In the CO₂ stage, pressure was the only significant factor for carotenoid content, whereas temperature was the only significant factor for carotenoid purity. The highest carotenoid content (143 µg g⁻¹) was obtained at the upper pressure level (25.0 MPa; 52.5 °C), and the highest purity (88%) at the upper temperature level (70 °C; 17.5 MPa). Carotenoid purity in all CO₂ extracts (26–88%) exceeded the carotenoid proportion in the biomass (15%), showing that pure CO₂ preferentially extracted carotenoids over chlorophyll a. In the CO₂ + ethanol stage, neither factor significantly affected chlorophyll a content or purity within the studied domain. Chlorophyll a purity varied narrowly (79–83%), and content reached 4.1 mg g⁻¹ (52.5 °C; 25.0 MPa). The higher polarity conferred by ethanol likely favours co-solubilization of both pigment classes, which may explain the narrow purity range and the limited influence of temperature and pressure in this stage. Sequential extraction yielded fractions with distinct pigment compositions: a carotenoid-enriched fraction obtained with pure CO₂, whose content and purity were influenced by different operating variables, and a chlorophyll a-enriched fraction recovered with ethanol-modified CO₂. These findings highlight the potential of sequential supercritical extraction using solvents of different polarities to obtain differentiated pigment fractions for microalgal biorefineries.

Acknowledgments: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul.