Innowax capillary column

Algal lipid was stained with Nile red as described previously [7] . Subsequently, after adjusting the microalgal concentration to the same levels across treatments, relative quantifications of Nile red staining were performed by measuring the absorbance spectrophotometrically.
In addition, lipid extraction and subsequent GC analysis were performed by following Folch's method [11, 20] . Briefly, microalgal biomass was harvested by centrifugation (5,000 ×g, 10 min) and then washed twice with deionized water. The remaining cell pellet was freeze-dried and then subsequently used for lipid extraction and analysis. The total lipids were extracted from the 10 mg of lyophilized biomass with a chloroform-methanol (2:1 (v/v)) solvent mixture. Fatty acid methyl esters (FAMEs) were produced from the extracted lipid by transesterification reaction according to previous studies [11, 20] . The FAMEs in the organic phase were analyzed by gas chromatography (HP5890; Agilent, USA) with a flame-ionized detector (FID) and INNOWAX capillary column (Agilent, USA; 30 m × 0.32 mm × 0.5 µm). Each FAME component was identified and quantified by comparing the retention times and peak areas with those of the FAME standard solutions.

The procedure for the quantification of fatty acid methyl esters (FAMEs) was based on the modified Folch method [23] . 10 mg of freeze-dried and powdered biomass was mixed to a solvent mixture of chloroform: methanol (2 mL, 2:1, v/v) in duplicate. After vortexing for 20 minutes, FAMEs were formed by addition 1 mL of methanol and 300 µL of H 2 SO 4 and incubation at 100°C for 20 minutes. After cooling down, 1 mL of distilled water was added to the sample, which was then vortexed for 5 minutes and centrifuged at 4,000 rpm for 10 minutes. The lower layer including the organic solvent was analysed with gas chromatography (HP 5890, Agilent, USA) with a flame ionized detector (FID) and INNOWAX capillary column (Agilent, USA). The GC column temperature was programmed as follows: (1) initial column temperature at 50 °C, hold for 1 min, (2) increase to 200 °C at a rate of 15 °C min -1 , hold for 9 min, and (3) increase to 250 °C at a rate of 2 °C min -1 , maintain for 2 min. Individual FAME component was identified and quantified by comparing the retention times and peak areas with those of the FAMEs standard solutions, respectively. The internal standard was Supelco 37 Component FAME Mix, item no. 47885-U, Sigma-Aldrich.