## 4 Discussion  
The amount of microbial lipids produced in batch cultures is usually low owing to the difficulties of optimizing the culture and operational conditions for simultaneous cellular growth (nitrogen-rich media) and lipids synthesis (nitrogen-limited media) [17]. Two-stage batch cultures, an approach that encompasses two distinct phases--the growth phase (1 st phase), followed by the lipid synthesis in the lipogenic phase (2 nd phase)--was proved to be advantageous for increasing lipids production by _Y. lipolytica_ NCYC 2904 [9], _Y. lipolytica_ MUCL 28849 [18], _Cryptococcus curvatus_[19], and _Apiotrichum porosum_[20] (Table 3). Thus, in an attempt to achieve high lipids concentration in _Y. lipolytica_ W29 cultures, several experiments were carried out in which the cell proliferation phase in glucose or glycerol was followed by the lipogenic phase in VFAs. Regardless of the substrate (glucose or glycerol) used in the growth phase, and the two-stage batch strategy (single pulse of 6 g:L\({}^{-1}\) VFAs or three pulses of 6 g:L\({}^{-1}\) VFAs each), _Y. lipolytica_ W29 was able to accumulate lipids using VFAs as a sole carbon source (Table 1). However, two distinct patterns of lipids production were observed according to the addition strategy: (1) in experiments with a single VFAs pulse, lipids content decreased from 48 h to 72 h of cultivation; and (2) in experiments with 3 pulses of VFAs, a slight increase of lipids content occurred from 48 h to 72 h, without a further significant decrease. In the first strategy, VFAs were completely consumed after 24 h of addition (48 h of cultivation) and no other carbon source remained in the culture medium until the end of the experiments (Figure 1). Thus, lipids synthesized by _Y. lipolytica_ W29 up to 48 h were mobilized for cellular metabolic activities during the stationary growth phase. By contrast, the addition of 3 pulses made it possible to maintain the VFA concentration above zero during 96 h of cultivation (Figure 2), which contributed to preventing lipid turnover. Moreover, the maximum lipids content was reached in the stationary growth phase, indicating that the VFAs added were channeled towards lipids biosynthesis instead of biomass production. Furthermore, the maximum lipids content obtained in experiments with 3 pulses was 1.6 times higher than that attained with a single pulse. The excess of a carbon source and depletion of nitrogen are the principal factors that trigger the onset of lipogenesis in _Y. lipolytica_ growing in hydrophilic substrates [21]. In fact, in experiments with a single pulse of 18 g:L\({}^{-1}\) VFAs, a higher synthesis of lipids was obtained compared to the other two-stage batch strategies, particularly 24 h after the addition of VFAs. By contrast, this was the experiment where lower biomass production was reached. In this condition, there was a residual amount of glucose in the medium at the moment of VFAs addition, and a higher amount of VFAs was supplemented at once, resulting in a higher carbon/nitrogen (C/N) ratio, and favoring lipids production. According to the literature, in a de novo lipid biosynthetic pathway--as occurs from glucose and acetate--high C/N ratios favor lipids synthesis instead of yeast cell proliferation. This metabolic route is generally induced by the limitation of nitrogen, leading to a decrease in yeast growth, since this is an essential nutrient for nucleic acid and protein synthesis. Yet, the carbon sources continue to be assimilated and are channeled for lipids biosynthesis [17; 21]. It has been reported that the biosynthesis of microbial lipids by _Y. lipolytica_ from butyrate and propionate is an ex novo metabolic pathway [22], which is independent of the C/N ratio [21]. However, in this study, a clear preference for acetate--which is uptaken via the de novo pathway--was observed. Similar results were obtained by [PERSON] et al. [9], demonstrating that the C/N ratio is an important factor affecting lipids production by _Y. lipolytica_ strains during the lipogenic phase.