Rc membrane syringe filter

Ten mg of lyophilized biomass was vigorously mixed with a chloroform–methanol mixture (2:1, v/v) for lipid extraction. Then, 0.5 mg of heptadecanoic acid (C17:0) was added as an internal standard. For transesterification, 1 mL of methanol and 300 μL of sulfuric acid were added to the samples, and incubated at 100 °C for 20 min. Each sample was then mixed with 1 mL of deionized water, followed by centrifugation to separate the organic (lower) phase, which was obtained by filtration using 0.20 μm RC-membrane syringe filters (Sartorius Stedim Biotech, Germany). FAMEs were analyzed using a gas chromatograph (GC) (HP 6890, Agilent, USA) equipped with a flame ionized detector (FID) and an HP-INNOWax polyethylene glycol column (HP 19091N-213, Agilent, USA). The oven temperature of the GC was increased from 50 to 250 °C at a rate of 15 °C per min. The FAME contents in these samples were determined by reference to a 37-component mix of FAME standards (F.A.M.E. MIX C8-C24, Supelco, USA).

All sample solutions were dissolved in methanol. Standard stock solutions of compounds were prepared at a concentration of 1 mg/mL and were then serially diluted with methanol to obtain a calibration curve of standard solutions at various concentration levels ranging from 12.5–200 μg/mL for all three compounds. ARAE extract was prepared at a concentration of 5 mg/mL. All solutions for analysis were filtered through 0.45 μm regenerated cellulose (RC) membrane syringe filters (Sartorius, Germany).

Microalgal cells were harvested by centrifugation at 5035×g for 10 min. (Supra-22K, Hanil Science Industrial, Republic of Korea). The pellets were washed twice with deionized water, lyophilized at − 80 °C for 3 days and then used for lipid extraction. For the lipid extraction, a chloroform–methanol mixture (2:1, v/v) was added to 10 mg of lyophilized cells and then vigorously mixed in a Teflon-sealed screw-capped Pyrex tube (Pyrex, USA) for 10 min. 0.5 mg of heptadecanoic acid (C17:0) were added as an internal standard for gas chromatography (GC). For transesterification which converts extracted lipid into fatty acid methyl esters (FAMEs), 1 mL of methanol and 300 μL of sulfuric acid were added and then incubated at 100 °C for 20 min. After cooling down, 0.3 N concentration of sodium hydroxide was added to rinse the remaining methanol and sulfuric acid. After centrifugation at 4000 rpm for 10 min at room temperature, the organic phase (lower layer) was filtered using a 20 μm RC-membrane syringe filter (Sartorius Stedim Biotech, Germany). FAMEs were analyzed by GC (HP5890, Agilent, USA) equipped with a flame ionized detector (FID) and an HP-INNOWAX polyethylene glycol column (30 m × 0.32 mm × 0.5 μm; H19091 N-213, Agilent, USA). The FAME composition and content were determined with a 37-component mix of FAME standards (F.A.M.E. Mix C8–C24, Supelco, USA).