*2.6. Extraction of Lipids, Quantification and Evaluation of Fatty Acids Profile*

For the extraction of lipids, the Folch method was utilized [28] with some alterations [19]. Namely, 50 mg of dried cells were mixed with 10 mL of chloroform: MeOH 2:1 mixture and left overnight at room temperature. Then, distilled H2O was added at a volume equal to 20% of that of the organic mixture, and the mixture was centrifuged. After centrifugation, the lower phase was collected in glass tubes and washed with a MeOH: dH2O 1:1 mixture. The organic solvents used were evaporated in a vacuum oven for a maximum of 24 h in order to minimize the risk of sample oxidation. After the evaporation of the solvent, the total lipids were weighted. Following this, the collected fatty lipids had to be transformed into fatty acid methyl esters (FAME). For this reason, they were diluted in 1 mL pure chloroform and mixed with 2.5 mL solution of MeOH: HCl 92:8. The samples at that point were incubated at 60 ◦C for 15 min for the esterification to take place. The reaction was halted with the addition of 2.5 mL of CaCl<sup>2</sup> 5% (*v*/*v*). The methyl esters were extracted from the mixture using hexane, which proved to be a very good solvent, and analyzed by GC–MS, as described by Chalima et al. [19]. The quantification of fatty acids took place by employing a commercially accessible FAME combination (Supelco® 37, Sigma-Aldrich, St. Louis, MI, USA), whereas estimation of wt% DHA was considered out of the whole sum of five fatty acids that were primarily distinguished (C14:0, C16:0, C18:0, C18:1 and DHA).
