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Solubilization of activated sludge is a crucial process before its use as an appropriate renewable feedstock for biofuel generation which could be a legitimate alternative arrangement for contemporary concerns on fuel crisis, climate change and food security. The present study investigates the thermo-sonic assisted enzymatic pre-digestion of municipal waste activated sludge (MWAS) to cultivate oleaginous yeast Naganishia liquefaciens NITTS2 to produce lipids for biodiesel production. The maximum suspended solids reduction and sCOD observed were 36.42 +/- 0.7 and 41.35 +/- 0.5%, respectively at optimum conditions. The pre-digested sludge was used as a nutritional medium for yeast cultivation and the obtained maximum biomass and lipid content were 17.85 +/- 0.64 g/L and 65.43 +/- 1.60%, respectively. The consumption of nutrients present in the medium was analyzed before and after the batch cultivation. Lipid extraction was optimized using ultrasonication at different temperature and its characteristic profile was analyzed by GC-MS. Fatty Acid Methyl Esters (FAMES) was produced (88.45 +/- 1.2%) through enzymatic transesterification and further confirmed by H-1 NMR spectroscopy. Thus, the combined pre-digestion would help to improve the solids reduction in the MWAS and the solubilized sludge could be used as a renewable substrate for biodiesel production. (C) 2019 Elsevier Ltd. All rights reserved.