BACKGROUND Waste oils are gaining interest as an alternative carbon source in fermentation processes as an option for the development of cost-effective and renewable processes. In this study, the production of the biosurfactant surfactin by three different Bacillus subtilis strains was investigated using waste frying oils as carbon source in two different forms: (i) emulsified waste oil (EWO), and (ii) emulsified and enzymatically hydrolyzed waste oil (HWO). Thus, the objective of this work is to analyze the effect of a hydrolytic pretreatment on the increase in the bioavailability of the waste oil over the cell metabolism and surfactin yields. RESULTS Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) analyses confirmed that the biosurfactant produced by all three strains is surfactin, the congener distribution being dependent on the strain and medium composition. Although the emulsification of the waste oil led to an increase in biomass production, surfactin concentration did not improve significantly, whatever the strain used. However, HWO shifted the cell metabolism towards the production of the surfactin congener C15 (with 15 carbon atoms on its hydrophobic chain) almost exclusively. CONCLUSIONS The reported behavior of B. subtilis strains growing on hydrolyzed frying oil could be of interest for the production of pure surfactin congeners from waste materials. (c) 2019 Society of Chemical Industry