AbstractThe prerequisites for a commercial fermentation process of biosurfactants include the use of low‐ or negative‐cost substrates and maximum conversion yields. Under competitive market conditions, the price of canola oil is expected to decrease in response to its increased supply. Lactose, obtained from cheese whey, is a by‐product of the dairy industry. In this work, canola oil with glucose or lactose as carbon sources was used as substrates to produce sophorose lipids (SLs) by means of the yeastCandida bombicola. Fermentations were conducted in either shaker flasks or 1‐L Bellco (Vineland, NJ) stirred reactors for 5–7 d at 450 rpm and 30°C. The production of SLs reached 150–160 g/L in a medium consisting of 10% glucose, 10.5% canola oil, 0.1% urea and 0.4% yeast extract. When lactose was substituted for glucose, 90–110 g/L SL was obtained. The apolar SL 17‐l‐([2′‐O‐β‐d‐glucopyranosyl‐β‐d‐glucopyransoyl]oxy)‐octadecanoic acid 1′‐4″‐lactone 6′,6″‐diacetate (SL‐1) was the major one (73%) when canola oil was used instead of safflower oil (SL‐1, 50%). Use of canola oil generally resulted in increased yields of SLs comparable to the yields obtained when safflower oil was used in the medium. Other literature reports present yields of 70 g/L and 120 g/L SLs, respectively, with these substrates.