Lipid Extraction and Quantification

Wet cells were directly methylated to determine fatty acid content and composition according to the following procedure. 1 mL of lipid sample was transferred into 16 mm × 125 mm screw-cap pyrex culture tubes. 1 mL 0.1 mg/mL of C13:0 (Tridecanoic acid) was added as the internal standard. The mixture was incubated at 85°C for 15 min, followed by the addition of 5.3 mL methanol and 0.7 mL 10 M NaOH solutions. Then the solution was neutralized with 0.58 mL H₂SO₄; the resulting solution was vortexed and incubated at 85°C for 15 min. 2 mL H₂O and 2 mL hexane were added, and the mixture was vortexed at room temperature for 15 min to extract the fatty acid methyl esters (FAMEs) for Gas Chromatography-Mass Spectrometer (GC-MS) analysis. After extraction, the mixture is centrifuged at 8000 × g for 1 min, and the top hydrophobic phase was transferred to a vial for GC-MS analysis.

Samples were analyzed in a GC-MS (QP2010 Ultra) with a capillary column RTX-5MS (30 m × 0.25 mm × 0.25 mm). The following settings were used: 120°C (1 min), increased at 7°C/min to 250°C, increased at 8°C/min to 295°C, hold at 295°C for 7 min. Helium was used as carrier gas. Mass spectrometer operating conditions were as follows: electron impact energy 70 eV; emission current 250 μA, transfer line 310°C; source temperature 230°C; scan rate 0.8 scans/-s and mass range 40–650 Da. Fatty acids were identified and quantified by comparison with commercial FAME standards normalized to methyl tridecanoate (C13:0). Total lipid content was calculated as the sum of total fatty acid contents for five FAMEs: methyl palmitate (C16:0), methyl palmitoleate (C16:1), methyl stearate (C18:0), methyl oleate (C18:1), and methyl linoleate (C18:2). The addition of tridecanoic acid was used as the internal standard, which was carried through the entire analysis procedure and transesterified into its methyl ester.