In the current study, the potentiality and optimization of lipid extraction from secondary sludge for biodiesel production were investigated. Four lipid extraction parameters were examined and used for process optimization and model development using Design of Experiment (DoE) method (namely methanol to hexane ratio-%, solvent to sludge ratio-ml/g, temperature-oC and extraction time-h). During the optimization process, free fatty acid (FFA) and saponifiable lipids (SLs) content of the extracted lipid were analyzed. The results revealed that, the maximum lipid extraction yield (
Ylipid) predicted through numerically optimized conditions by the model for highest desirability (0.99) was 16.5% at methanol to hexane ratio (%) of 84%, solvent to sludge ratio (v/wt) of 45 ml/g, temperature at 90oC for 6 hours extraction time. The extracted lipid contained a maximum amount of 31% (wt/wt) FFA, where palmatic acid was predominant. The FAMEs yield produced from ex-situ acid-catalyzed esterification/transesterification of the methanol-hexane co-solvent extracted lipid ranged between 4.5-5% (wt/wt) based on sludge weight. Fatty acid profile of FAMEs was found to be dominated by methyl palmitate (C16:0) representing 36% of FAMEs composition, followed by palmitoleic acid methyl ester (C16:1), oleic acid methyl ester (C18:1) and stearic acid methyl ester (C18:1) representing 24%, 18% and 10% of the FFA composition respectively. PCA analysis showed that solvent to sludge ratio (ml/g) has the highest significant positive effect on FAMEs yield (p-value <0.05) where methanol to hexane ratio (X1), temperature (X3) and extraction time (X4) were inversely correlated with FAMEs yield. The results indicated the feasibility of using secondary sludge as an alternative feedstock for biodiesel production. However, the optimized conditions for maximizing extracted lipid content should not be considered suitable for FAMEs yield as well.