## 1 Introduction  
Microbial lipids, also known as microbial oils, bio- oils, or single-cell oils (SCO), have been under the spotlight during the last decades owing to their potential application as food supplements and nutraceuticalals--polyunsaturated fatty acids (PUFA), such as those belonging to the omega-3 and omega-6 lipids--or as a renewable energy source for the biodiesel industry--saturated (SFA) and monounsaturated (MUFA) fatty acids [1]. Oleaginous yeasts are able to accumulate more than 20% (\(w/w\)) of their dry weight as intracellular lipids, mainly in the form of triacylglycerglycerols (TAG). Comparatively to other oleaginous microorganisms (e.g., filamentous fungi or microalgae) or vegetable crops, yeasts have many attractive characteristics including high biomass and lipids productivity, utilization of several substrates, less seasonal or climate dependence, short life cycle, higher metal ion tolerance and the relative ease of culturing [2, 3]. In particular, the non-conventional yeast _Yarrowia lipolytica_ has been considered a model yeast for microbial lipids production, owing to its superior capacity for lipids accumulation and ability to use various and cheap raw materials, such as wastes and by-products of the agro-industrial sector [4]. Lipids accumulated by _Y. lipolytica_ have, in general, a similar fatty acid composition and energy value to common vegetable oils, making them a promising feedstock for biodiesel production [1]. Biodiesel is a non-toxic, sulfur-free, renewable, and biodegradable fuel, and its combustion emits fewer greenhouse gases than petroleum diesel [5]. Biodiesel is mainly obtained from vegetable oils, but the limited supply and high cost of these feedstocks make its production not economically viable [6]. Biodiesel derived from _Y. lipolytica_ lipids is a promising technology and does not compete with arable land for food production as in the case of crop-based oil resources used as raw material for biodiesel production. However, the high costs of carbon and nitrogen sources, the amount of lipids accumulated per unit of cellular mass, and the post-processing costs associated with oil extraction from cells are still challenges for the large-scale production of microbial lipids [7]. In this regard, the use of abundant, renewable, and low-value raw materials as substrates for microbial lipids production decreases the overall costs and enables the development of a more sustainable and affordable process within the circular bioeconomy assumptions. Volatile fatty acids (VFAs), generated as intermediates during anaerobic fermentation of organic wastes, have been selected as a renewable and low-cost feedstock for microbial lipids production by _Yarrougia_ species [8; 9; 10; 11]. Regardless of the substrate and anaerobic fermentation conditions, VFAs comprise short-chain organic acids (C2-C6), specifically, acetic (30-80%), propionic (5-32%), and butyric (11-30%) acids [9; 12]. Compared to sugar-based substrates, these organic acids have higher theoretical conversion yields and shorter metabolic pathways to intracellular lipids [13]. The main strategies reported so far have studied the conversion of VFAs to microbial lipids in batch cultures [9; 10; 11; 12]. Previous works have demonstrated that the two-stage batch strategy (growth phase in glucose, followed by the addition of VFAs for lipid synthesis) enhances microbial lipids production by _Y. lipolytica_ NCYC 2904 [9]. Thus, this study aims to evaluate the ability of _Y. lipolytica_ W29 to synthesize intracellular lipids from a VFA mixture (acetate, propionate, and butyrate) during the lipogenic phase after yeast growth in glucose or glycerol. For the first time, a two-stage batch strategy with 3 pulses of 6 g\(\cdot\)L\({}^{-1}\) VFA mixture was tested. _Yarrougia lipolytica_ W29 was able to assimilate 18 g\(\cdot\)L\({}^{-1}\) VFAs without the inhibition of VFA uptake rate and cellular growth, producing higher amounts of microbial lipids. The estimation of the properties of biodiesel produced from _Y.