The specific effects of chemical and physical factors on lipid synthesis of isolated Aspergillus awamori (MTCC11639) was investigated with the final aim of producing biodiesel. Design of experimental methodology (DOE) was employed to understand the fungal lipid synthesis with response to the selected factors. An orthogonal array (OA) was designed to evaluate the influence of eight factors using three levels of factor (2(1) x 3(7)) variation with a L18 experimental matrix. Among the selected factors, pH influenced the lipid production substantially (41.6%), followed by glucose (16.3%) and incubation temperature (4.3%). Interaction of proteins with nitrates showed highest severity index (SI, 85.1%), while NaCl with incubation time documented lowest SI (20.0%). Validation experiments with the obtained optimum conditions showed improved lipid productivity of 31%, along with 90% substrate degradation. Composition of fatty acid methyl esters (FAME) of fungal lipids showed higher saturated fatty acid (SFA) than unsaturated fatty acids (USFA). (C) 2013 Elsevier Ltd. All rights reserved.