## 3 Results and Discussion
### Potential of Biowastes as Culture Media
Due to its high glucose content, the potential of using the lignocellulosic urban pruning residues as a carbon source for SCO production, which may contribute to the development of biorefining processes both from the economic and from the environmental point of view. Optimization of the Pulse-Feeding Strategy for the Cultivation of R. toruloides Using Biowastes with Different Nature  
Different strategies have been investigated for SCO production using oleaginous yeasts, including batch operation, fed-batch, and continuous approaches. Among them, fed-batch and continuous cultivation modes usually exhibit higher conversion yields than the batch operational mode due to improved substrate utilization [17]. A pulse-feeding strategy was designed to optimize the sugar-rich streams obtained from the agro-food waste and the lignocellulosic urban pruning residue. In addition, the yeast _R. toruloides_ was selected as a microbial cell factory due to its potential to obtain carotenoids simultaneously during SCO production. _R. toruloides_ was cultivated in the agro-food stream to foster cell growth. Subsequently, after sugar depletion, two pulses of the concentrated lignocellulosic hydrolysate were added to the culture to trigger lipid accumulation. Figure 1 shows the time-course cultivation process for _R. toruloides_ following this pulse-feeding strategy. _R. toruloides_ cells could consume both glucose and fructose within the first 24 h, reaching a cell concentration of 21.0 g/L. The sugar consumption rates decreased significantly after the first and second pulse additions. The second pulse was added after 72 h from inoculation, thus requiring 48 h for sugar depletion after the first pulse addition. In addition, a total sugar concentration of 28.3 g/L still remained in the medium after 120 h of cultivation. A final cell biomass concentration of 36.4 g/L was observed at this point. It is important to note that although the total number of viable cells remained constant after 24 h of the cultivation process, cell viability decreased from 92.8% at 24 h to 51.9% and 35.7% after 72 h (before the second pulse addition) and 120 h, respectively. The lower sugar consumption rates and the reduction in cell viability might be indicative of cells suffering from nutrient limitation. With the aim of increasing the initial nutrient content in the system and avoiding external nutrient addition, the agro-food residue was concentrated by vacuum evaporation before inoculation until the sugar content was doubled. As can be observed in Figure 2, the use of the two-time concentrated agro-food stream improved both sugar consumption rates and cell viability within the cultivation processes. In this case, pulses were added after 24 h and 48 h, and complete sugar depletion was observed after 96 h of cultivation. Due to the higher initial sugar concentration, the cell biomass content also increased up to 32.1 g/L and 55.5 g/L after 24 h and 96 h, respectively. On the other hand, the cell viability remained above 75% until the second pulse addition (48 h) and 3-3.5 times higher number of total viable cells (2.8-3.9\(\cdot\)10\({}^{9}\) vs. 0.9-1.2\(\cdot\)10\({}^{9}\) viable cells/mL) were observed, being indicative of the lower stress pressure \begin{table}
\begin{tabular}{l c} \hline \hline
**Component** & **g/100 g of Pretreated Biomass** \\ \hline Glucan (Cellulose) & 43.96 \(\pm\) 0.44 \\ Hemicellulose & 3.74 \\ Xylan & 2.84 \(\pm\) 0.14 \\ Galactan & 0.69 \(\pm\) 0.05 \\ Arabinan & 0.06 \(\pm\) 0.01 \\ Mannan & 0.16 \(\pm\) 0.01 \\ Acid-insoluble lignin & 42.17 \(\pm\) 0.81 \\ Ash & 2.41 \(\pm\) 0.31 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Chemical composition of the water-insoluble solid fraction resulting from steam explosion pretreatment (190 \({}^{\circ}\)C, 10 min, 40 mg H\({}_{2}\)SO\({}_{4}\)/g raw biomass) of the lignocellulosic urban pruning residue. SCO production, cell biomass concentrations are directly related to the concentrations of the final product.