Fatty Acid Profiling of Phytoplankton

Once all cultures of the same phytoplankton species reached stationary phase, phytoplankton cells were harvested by filtration through 3.0 μm cellulose nitrate membranes (Whatman, GE Healthcare), obtaining between 0.3 and 7.5 mg dry weight, depending on the species. Total lipids were extracted, and FA identified and analyzed as previously described (Calderini et al., 2022 (link)) without dividing the sample into different fractions. Shortly, total lipids were extracted with chloroform/methanol/water (4:2:1) using sonication (10 min). After evaporation of solvents under a nitrogen stream, 1 mL toluene was added, and fatty acids were transesterified overnight (50°C) using methanolic H₂SO₄ (1%, v/v). FA methyl esters were analyzed with a gas chromatograph equipped with a mass detector (GC–MS; Shimadzu Ultra) using a DB-23 column (30 m × 0.25 mm × 0.25 μm; Agilent). Quantification of FAs was based on peak integration using gcsolution software (version 2.41.00, Shimadzu). Peak areas of FAs were corrected by using two internal standards (phospholipid FA C19:0 and free FA C23:0; Larodan) added before lipid extraction.