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[en] Highlights: • Chloroform:methanol 2:1 showed the highest lipid extraction efficiency. • Prolongation of extraction time over 2 h showed insignificant effect on EFAs yield and significantly increased FFAs. • Cell-disruption is not essential for lipid extraction from S. obliquus cells. • Hot-water treatment for 5 min showed significant increase in EFAs yield. - Abstract: During the transesterification of oil feedstock for biodiesel production, the reaction primarily happens at the ester bonds where the fatty acid chains meet the glycerol. Therefore, only esterified fatty acids (EFAs) are able to be turned directly into biodiesel by transesterification. In this study, an optimized procedure for EFAs recovery from the biodiesel promising microalga Scenedesmus obliquus was studied. The effect of different solvent mixtures (ratios), extraction times, pretreatments and cell-disruption methods on intracellular EFAs and free fatty acids (FFAs) yield was examined. Using of chloroform:methanol (C:M) 2:1 for 2 h was shown to be the best solvent mixture for lipid extraction which resulted in the highest EFAs yield. Furthermore, testing of different cell-disruption methods showed that cell-disruption is not essential for lipid extraction from S. obliquus cells. Although, microwave pretreatment showed significant increase in EFAs yield with respect to overnight oven drying at 80 °C, all showed insignificant differences to the control. Moreover, overnight cell freezing showed 7.7% significant reduction in EFAs yield with respect to the control, while hot-water treatment for 5 min showed significant increase by 13.7%. On the other hand, overnight cell incubation, in oven or freezing, resulted in significant increase in FFAs up to 7.44 and 12.47 mg g"−"1 of the dry weight, respectively. In addition, the present study showed that no pretreatment with isopropanol to inactivate the lipases is needed when hot-water pretreatment is performed. This study suggested that hot-water pretreatment not only deactivate lipases, but also enhances the EFAs recovery.