*4.4. Fatty Acid Extraction*

Fatty acid (FA) analysis was performed following the modified method of Bligh and Dyer [27] as modified by [28,29]. Before extraction, an internal standard (23:0) was added to every sample for quantification purpose (0.5 mg mL<sup>−</sup>1). Lipids were extracted with a 20 min ultrasonication (sonication bath, 80 kHz, Fisherbrand™) in a mixture of distilled water, chloroform and methanol in ratio 1:1:2 (v:v:v, in mL). Lipids were concentrated under N2 flux, and saponified, in order to separate FA, with a mixture of NaOH (2 mol L<sup>−</sup>1) and methanol (1:2, v:v, in mL) at 90 °C during 90 min. Saponification was stopped with 500 μL hydrochloric acid. Samples were then incubated with BF3-methanol at 90 °C during 10 min to transform free fatty acids into fatty acids methyl esters (FAME), which were isolated and kept frozen in chloroform. Just before analysis, samples were dried under N2 flux and transferred to hexane. One μL of the mixture was injected in a gas chromatograph (GC, Varian CP-3800 equipped with flame ionization detector), which allowed separation and quantification of FAME. Separation was performed with a Supelco® OMEGAWAX 320 column (30 m × 0.32 mm i.d., 0.25 μm film thickness) with He as carrier gas. The following temperature program was used: 60 °C for 1 min, then raise to 150 °C at 40 °C·min−<sup>1</sup> (held 3 min), then raise to 240 °C at 3 °C·min−<sup>1</sup> (held 7 min) at 1 mL min<sup>−</sup>1. FAME Peaks were identified by comparison of the retention time with analytical standards. Additional identification of the samples was performed using a gas chromatograph coupled to mass spectrometer (GC-MS, Varian 450GC with Varian 220-MS). Compounds annotation was performed by comparing mass spectra with NIST 2017 library. Fatty acids were quantified using the FID detector and the internal standard (C23). Corresponding fatty acids are designated as X:Y*n*-Z, where X is the number of carbons, Y the number of double bonds and Z the position of the ultimate double bond from the terminal methyl (see [30] for additional information about naming convention).
