## 3 Results  
### Microbial Lipids Production in Two-Stage Batch Cultures  
The potential of _Y. lipolytica_ W29 to produce microbial lipids using VFAs as substrate was evaluated in two-stage batch cultures (TSC). _Yarrowia lipolytica_ W29 grew for 24 h in glucose or glycerol (growth phase) to obtain a higher cell density, followed by the addition of one pulse of 6 g\(\cdot\)L\({}^{-1}\) VFA mixture (lipogenic phase) to produce microbial lipids. During the first phase, yeast growth was similar in both substrates, no lag phase was observed, and the biomass concentration obtained after 24 h of cultivation was similar (Figure 1a,b). Glucose and glycerol were completely consumed after 24 h with a similar uptake rate (Table 1). In the lipogenic phase, _Y. lipolytica_ cells continued to grow due to the addition of VFAs that were also used for biomass proliferation, but with a lower specific growth rate. The stationary growth phase was reached at 48 h in both experiments--coinciding with the VFA consumption--and the final biomass concentration and the global biomass yield were statistically equal (\(p\geq 0.05\)). VFAs were consumed by the yeast cells with different uptake rates, depending on the substrate used in the growth phase (Table 1). However, in both conditions, a preference of the yeast for acetate was noted, the latter being completely consumed 12 h after the VFAs addition. Butyrate took 24 h to be assimilated in both experiments, whereas butyrate was consumed after 24 h and 30 h of VFAs addition to glucose (Figure 1a) and glycerol (Figure 1b) media, respectively. Figure 1: Time course of cellular growth (\(\Circinus\)), VFAs (\(\blacksquare\), acetate; \(\blacktriangle\), propionate; \(\blacklozenge\), butyrate), glucose (\(\bullet\)), and glycerol (x) concentration obtained in two-stage batch cultures of _Y. lipolytica_ W29. After 24 h of growth in glucose (**a**) or glycerol (**b**), a mixture of 6 g\(\cdot\)L\({}^{-1}\) VFAs (2 g\(\cdot\)L\({}^{-1}\) acetate, 2 g\(\cdot\)L\({}^{-1}\) propionate, and 2 g\(\cdot\)L\({}^{-1}\) butyrate) was added to the culture medium. Lipids content of _Y. lipolytica_ cells at 48 h (black bars) and 72 h (white bars) of cultivation (**c**). The error bars represent the standard deviation of two independent replicates. The lipids content of _Y. lipolytica_ W29 cells was not affected by the substrate used during the growth phase. Regardless of the substrate used in the growth phase, the yeast cells accumulated higher amounts of lipids at 48 h of cultivation (24 h after the VFAs addition), decreasing 1.5 times at 72 h (48 h after the VFAs addition) (Figure 1c). In all conditions, the maximum lipids production occurred at the end of the exponential growth phase, and utilization of intracellular lipids for metabolic activities was observed in the stationary phase owing to the complete depletion of the substrates (Figure 1a,b). As VFAs were completely consumed 30 h after the addition, and a lipid mobilization was observed in the lipogenic phase, two-stage batch cultures with 3 pulses of 6 g\(\cdot\)L\({}^{-1}\) VFA mixture (24 h, 48 h, 72 h) were carried out as an attempt to prevent substrate depletion, lipids turnover, and to boost lipids production. No enhancement in global biomass yield was attained in the experiments with 3 pulses compared to cultures with a single pulse (Table 1), but a higher biomass concentration was reached in the glycerol medium after 3 pulses of VFAs (Figure 2b). _Yarrowia lipolytica_ cells continued to grow after the addition of the first two pulses of VFAs, particularly in the experiments in which the growth phase was carried out with glycerol. However, yeast cells reached the stationary growth phase after the third pulse in both conditions, despite the almost total VFA consumption (Figure 2a,b). Moreover, with the addition of two further pulses, VFAs were consumed at a similar uptake rate to that observed after the first pulse.