Like the previous systems, 3 mL of concentrated microalgae suspension were measured in a precision balance in teflon tubes and further de-watered after centrifugation. The biomass pellet was re-suspended by adding 22.7 mL pure ethanol resulting in a ~ 96% ethanol extraction system at a ratio of 81 mL of solvent per 1 g dry biomass. Extraction took place in the dark, for 30 min at 60 °C in a water bath (SONOREX SUPER RK 510 H, Bandelin, Germany). Following this, the samples were centrifuged at 10,000 × g for 10 min. The supernatant was completely removed into a separate tube, where 6.81 mL hexane and 13.6 mL deionized water was added. After the formation of two distinct phases, 5 mL from the upper phase was removed into pre-weighted glass tubes and evaporated under N2. The above step was repeated with the addition of 5 mL fresh hexane. Gravimetric yields were measured gravimetrically.

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