Canola materials including processed flake and cake were investigated as both of carbon and nitrogen sources for the production of new oil containing polyunsaturated fatty acids (PUFAs). With conversion of the fungi Mortierella alpina, modified lipids enhanced the oil profile with the addition of three PUFAs, gamma-linolenic acid (GLA, gamma-CI8:3n6), arachidonic acid (ARA, C20:4n6) and eicosapentaenoic acid (EPA, C20:5n3). The ratio of polyunsaturated to saturated fatty acid (P/S) increased 50% compared to the original canola oil. ARA yield of 20.3 mg/g flake was comparable to 20.1 mg/g glucose, and higher than 12.5 mg/g cake. EPA yield of 3.3 mg/g flake was greater than 2.7 mg/g cake, and much greater than 0.3 mg/g glucose. The mixed culture of two strains, M. alpina and Pythium irregulare, resulted in higher production of ARA and EPA than their single cultures. Mixed culture achieved ARA yields of 26 mg/g flake, 23 mg/g cake and 38 mg/g glucose, and had favorable lipid profiles with P/S of 6.5 and 8.7 for growth on flake and cake, respectively. in addition, a method was developed to estimate the bioconversion rate from digested canola oil to fungal oil, which was nearly 50% when canola flake and cake were utilized as substrate. (C) 2008 Elsevier Inc. All rights reserved.