Abstract:In the present study, Design of Experiment (DoE) as a statistical method was adopted for optimizing conditions for lipid extraction from scum sludge. Four different extraction variables were investigated: methanol to hexane ratio (%), solvent to sludge ratio (ml/g), temperature (°C), and extraction time (h). During the optimization process, saponifiable lipids (SLs) content of the extracted lipid was analyzed. Screening experiments revealed that methanol to hexane ratio (X1), solvents to sludge ratio (X2) and temperature (X3) showed a significant effect on Y lipid (p < 0.05). Lower methanol to hexane ratio and higher solvent to sludge ratio showed the highest positive effect on lipid yield (Y lipid ). No significant effect on extraction time on Y lipid was observed. The positive relationship between lower methanol to hexane ratio and the amount of lipid extracted can be attributed to the presence of higher amounts of neutral lipids in scum sludge. According to Box-Behnken design and Response surface method (RSM), the maximum lipid extraction yield (Y lipid ) predicted through numerical optimized conditions by the model for highest desirability (0.995) was 29.614% at methanol to hexane ratio (%) of 42%, solvent to sludge ratio (v/wt) of 51 ml/g, temperature at 87°C for extraction time of 6 hours. The FAMEs yield produced from ex-situ acid-catalyzed esterification/transesterification of the methanol-hexane co-solvent extracted lipid ranged between 7.9-9.3% (wt/wt) based on sludge weight. Fatty acid profile of FAMEs was found to be was found to be dominated by Oleic acid methyl ester (C18: 1) followed by methyl Palmitate (C16: 1) representing 39.4% and 24.3% of FAMEs composition respectively. The correlation analysis of extraction variables and FEMAs yield revealed that solvent to sludge ratio (ml/g) has the highest positive significant correlation with FAMEs yield (p-value < 0.05). However, methanol to hexane ratio (X1) and temperature (X3) were inversely correlated with FAMEs yield.