2.2. Maximum fatty acid ethyl esters (FAEE) yield
To quantify the maximum conversion yield of FAEE, 0.3 g dry
microalgae biomass was added into a tube, mixed with 11.4 mL
mixture solvent (chloroform:methanol:water = 1:2:0.8; v/v/v),
and then the mixture was subjected to ultrasonic treatment for
30 min in order to allow lipids inside microalgae cells to be
extracted (Bligh and Dyer, 1959). Chloroform and water were
added to achieve the phase separation and the chloroform phase
containing the microalgae lipid. When the chloroform layer was
separated from the water layer, the chloroform layer was transferred
to a round-bottomed flask and evaporated in a rotary evaporator
to remove the solvent and obtain the crude lipids. The crude
lipids were mixed with 6 mL ethanol and 300 lL sulfuric acid in a
round-bottomed flask, which was heated to 90 C for 2.0 h. After
the transesterification reaction, the micro-reaction vessel was
cooled to room temperature. Afterwards, 6 mL deionized water
and 6 mL n-hexane was added into the micro-reaction vessel for
phase separation. The sample with 6 mg heptadecanoic acid ethyl
ester (C17:0) was added to the tube for GC analysis to determine
the maximum FAEE yield.