For each sample, approximately 3 mL of suspension were measured in Teflon tubes (Nalgene® Oak Ridge Centrifuge Tubes, Teflon® FEP, 50 mL Thermo Scientific), which at 100 g·L−1 concentration corresponds to 0.3 g biomass. The probes were then centrifuged (Heraeustrade; Megafugetrade 8R, ThermoFischer Scientific, Germany) at 10,000 × g for 10 min and the supernatant was removed and measured to evaluate the remaining water in the system which was equal to approx. 1.5 mL. The biomass pellet was then re-suspended by adding 16.1 mL ethanol and 6.6 mL hexane. The composition of the system at the beginning of the extraction was thus 1:0.41:0.09 ethanol:hexane:water with a ratio of 81 mL of solvent per 1 g dry weight. It should be noted, that the water present is the leftover in the biomass pellet from the above centrifugation step, without any extra water addition at this stage.

After rigorous vortexing, the samples were left to agitate on an agitator for 24 h, in the dark and at room temperature. Once extraction was completed, the probes were centrifuged at 10,000 × g for 10 min, to separate the solvent from the residual biomass. From the supernatant, 6.1 mL were removed into a separate tube, where an additional 18.2 mL hexane and 2.9 mL distilled water were added. From the two distinct phases formed, 15 mL from the upper, hexane lipid-rich phase was removed into pre-weighted glass tubes and evaporated under N2. The lipid yields were then calculated gravimetrically. Incubated samples were treated in an identical manner.

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