## 3 Results and Discussion
### Effects of Media Composition on the Intracellular Accumulation of Lipids and Carotenoids  
The oleaginous yeast _R. toruloides_ is a promising microorganism that can co-produce both lipids and carotenoids from acetyl-CoA. As depicted in Figure 3, the designed pulse-feeding strategy allowed the accumulation of a total lipid content of 42-45% (\(w/w\)), independently of using the non-concentrated and the concentrated agro-food waste streams for the growth phase. The total amount of FAMEs showed, however, different accumulation percentages, accounting for up to 43.5% and 35.7%, respectively. In addition, lower sugar-to-lipid conversion yields were also estimated when using the concentrated agro-food waste stream (0.25 g/g vs. 0.20 g/g) (Table 3). These differences in the accumulation of fatty acids (FAs) and the conversion yields can be attributed to the lower stress exerted on yeast cells when using the concentrated growth medium, thus supporting the differences observed for the cultivation parameters. It is important to note that the higher cell biomass concentration reached allowed the final lipid concentration to increase from 16.4 g/L to 23.3 g/L. Moreover, despite the differences in the content of FAMEs, both the distribution of major groups and the individual percentage of FAs were similar when using the non-concentrated and the concentrated agro-food waste media (Figure 3, Table 3). Saturated and monounsaturated FAs represent more than 85% of the total FA content, with palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) major contributors. Figure 3: Total lipid production and fatty acid distribution after cultivating _R. toruloides_ ATCC 204091 in the non-concentrated and concentrated agro-food waste streams as growth media following a pulse-feeding strategy with the enzymatic hydrolysate of a lignocellulosic urban pruning residue to trigger lipid accumulation. The content in FAMEs is directly correlated with storage lipids. The mechanisms to increase storage lipids depend on the microbial strain. Under nitrogen limitation, the model yeast _Yarrovia lipolytica_ increases its lipid pool by mainly driving the carbon source into the lipid biosynthetic pathways with minor transcriptional regulation [35]. In contrast, [PERSON] et al. [33] have reported a significant lipid regulation in _R. toruloides_ during nitrogen starvation, supporting the hypothesis of an active lipid remodeling process in this yeast species. These authors observed a shift in the distribution of lipids from mostly phospholipids towards mostly storage lipids. Similarly, the severe nutrient-limiting conditions when using the non-concentrated agro-food waste during the growth phase resulted both in a higher content of storage lipids and lower differences between the storage lipids and the total lipids (Figure 3). In addition to lipids, a total intracellular content of 665 and 736 \(\upmu\)g/g DCW of carotenoids was observed during the pulse-feeding strategy using the non-concentrated and the concentrated agro-food waste, respectively (Figure 4). The highest concentrations were reached at the end of the cultivation process, which might be indicative of the benefit of having nutrient-limiting conditions to favor carotenoid accumulation. Among the different carotenoids produced by _R. toruloides_, major components considering the distribution profile are torulene \(>\gamma\)-carotene \(>\) torulardhoin \(>\beta\)-carotene (Table 4). During the cultivation process, \(\gamma\)-carotene and \(\beta\)-carotene remained constant or slightly decreased, while the concentration of torulene and torularhodin increased after each pulse addition, almost doubling their concentration between the growth phase and at the end of the overall process. Microbial carotenoid production is considered a promising bioprocessing alternative to current practices. Different yeast species from the _Rhodotorula_, _Rhodosporidium_, and _Sporobolomyces_ genera have been investigated for carotenoid production due to their natural capacity to produce these components.