3.2. Biomass Extraction

Supercritical CO₂ extraction was performed in a bench scale apparatus (SFE-500, SEPAREX CHIMIE FINE, Champigneulles, France). The detailed apparatus description and extraction procedure is described in previously published work [55].

In this work, 80 g of C. vulgaris biomass were loaded in the extractor vessel. Dead space reduction and uniform flow distribution were achieved with the addition of glass bead (d = 4.5 mm) layers at the top and bottom of the vessel. The two separators operated at 60 and 10 bar respectively and 8 °C. Additionally, preliminary experiments showed that exhaustive extraction of C. vulgaris biomass was achieved with 94 kg_CO2/kg_biom, and therefore the solvent consumption was set at 100 kg_CO2/kg_biom for all of the performed experiments. The operational conditions of pressure, temperature and solvent flow rate were adjusted according to an experimental design and experimental error was determined through a quadruplicate repetition of the central point (see Table 1). The yield was determined by total biomass weight loss of the extraction vessel at the end of each experiment and all collected extracts were stored at −18 °C until further analysis.

In the case of the kinetic study, experiments were interrupted at regular periods of time for weight loss measurement. The experimental error was calculated from duplicate experiments.

Regarding the cosolvent addition, ethanol was inserted through a piston pump and ethanol content in CO₂ was set to 10% w/w.

Conventional extraction was performed with 1 g of C. vulgaris biomass and 37 mL of aq. ethanol 90% v/v. Sample and solvent were loaded into a jacketed vessel, stirred at 500 rpm and heated at 30 °C for 24 h in the dark by using a Carousel tech stirring hotplate (Radleys, Essex, UK). A condenser was connected to the top of the vessel for the minimization of solvent losses. The proposed solid-liquid extraction conditions have been optimized in a previously published study [34]. After extraction, the mixture was centrifuged for 8 min at 3000 rpm using a Hermle centrifuge Z206-A (Hermle AG, Baden-Württemberg, Germany). The supernatant was filtered using a ChromPure PTFE/L 0.45 μm filter (Membrane solutions, LLC, North Bend, OH, USA) and vacuum evaporated at 45 °C and 100 mbar using a Hei-VAP Advantage ML rotary evaporator (Heidolph Instruments GmbH & Co. KG, Bayern, Germany). SLE was performed in duplicate and the dry microalgal extracts were obtained after evaporation and stored at −18 °C until further analysis.