2.4.4. Fatty acid methyl esters
The fatty acid compositions of the lipid produced by C. curvatus were determined by analysis of fatty acid methyl esters (FAMEs). The FAMEs were produced by transesterification: methanol was added to the extracted lipid with sulfuric acid (2.5% V/V H2SO4/CH3OH) as a catalyst; the reaction was allowed to proceed for 45 min at 90°C. Then 1 ml H2O and 2 ml n-hexane were added. The FAMEs dissolved into the n-hexane. The solution was centri-fuged at 2000 rpm for 15 min, to separate the water from the FAME/hexane phase which was then transferred into glass vials using Pasteur pipettes.
The FAMEs in n-hexane were analyzed using a gas chromatog-raphy (6890N, Agilent, USA) equipped with a flame ionized detec-tor (FID) and an INNOWAX capillary column (Agilent, USA, 30 m x 0.32 mm x 0.5 µm). The column temperature was pro-grammed as follows: (1) initial column temperature 100°C, hold for 5 min, (2) increase to 250°Cat 10°C/min, hold for 30 min.The split ratio was 1:10 (v/v). The injector and the detector tem-
peratures were both set at 250°C. FAME components were identi-fied and quantified by comparing the retention times and peak areas with those of standard solutions.