Microalgal Lipid Profiling via Modified Folch's Method

A modified Folch’s method was used for FAME analysis [15 (link), 25 (link)]. The 10 mg of lyophilized microalgal cells were mixed with chloroform–methanol solvent mixture (2:1, v/v) and vortexed vigorously for 10 min. As an internal standard, 0.5 mg of heptadecanoic acid (C17:0) was added to vortexed samples and 1 mL of methanol and 300 μL of sulfuric acid were added to sample in order. The samples were incubated in hot plate for 20 min at 100 °C for transesterification. After cooling the samples in room temperature, 1 mL of deionized water was added to each of samples and samples were vortexed for 5 min. After centrifugation (4000 rpm for 10 min, at 25 °C), the separated lower layer for organic phase was taken and filtered by using a 0.20 μm RC-membrane syringe filter (Sartorius Stedim Biotech, Germany). FAMEs in organic phase were detected via gas chromatograph (GC) (HP 6890, Agilent, Wilmington, DE, USA) with an HP-INNOWax polyethylene glycol column (HP 19,091 N-213, Agilent) and a flame ionization detector (FID). The temperature of GC oven was increased from 50 to 250 at 15 °C per min. Composition and contents of FAME in the sample were determined by comparison with a 37-component mix of FAME standards (F.A.M.E. MIX C8-C24, Supelco, USA).