## 2 Materials and Methods  
### Microorganism and Media Preparation  
_R. toruloides_ ATCC 204091 was used to produce lipids and carotenoids. Yeast cells were maintained in 20% glycerol stock at \(-80\)\({}^{\circ}\)C. They were activated overnight in 50 mL. YPD liquid medium (20 g/L glucose, 10 g/L yeast extract, and 20 g/L peptone) at 180 rpm and 28 \({}^{\circ}\)C, using 250-mL baffled conical Erlemmeyer flasks.  
Due to their differences in nutrient composition, agro-food waste and lignocellulosic urban residues were used to promote cell growth and trigger intracellular lipid accumulation. The agri-food waste used came from the Spanish horticulture sector, specifically from a mixture of discarded tomatoes, peppers, and cucumbers. These wastes were collected and provided by Fundacion Cajmar (Almeria, Spain). The soluble fraction obtained after homogenization, crushing, and centrifugation of the waste was used as a culture medium for yeast growth. The discarded vegetables were subjected to crushing using an industrial blender (Dianamix TR/bM 330, SAMMIC, Azkoitia, Spain) and then subjected to centrifugation (3000\(\times\)\(g\), 15 min) using a basket centrifuge RTL2 BD (Comfeifa, Barcelona, Spain). The soluble fraction was then collected and filter sterilized using a 0.22 \(\upmu\)m Nalgene(tm) Rapid-Flow(tm) Sterile Disposable Filter Units (ThermoFisher Scientific, Waltham, MA, USA). With the aim of increasing the initial sugar content, the resulting liquid was also subjected to concentration using a rotavapor (Buchi, Uster, Switzerland) until the initial sugar concentration was doubled. Both non-concentrated and concentrated liquid fractions were analyzed, as described below.  
On the other hand, the hydrolysate resulting from subjecting a lignocellulosic urban tree pruning residue to pretreatment and enzymatic hydrolysis was used as a medium for lipid accumulation. This residue was collected and supplied by CEDER-CIEMAT (Soria, Spain). This residue was mainly composed of collected pruning mixtures from elms, plane, and acacia trees. This municipal forestry waste was chopped and milled at 8-10 mm before pretreatment. The residue, whose composition in main components was as follows (% dry weight basis): Cellulose 34.7; hemicellulose 18.7; and Lignin 26.4, was pretreated at CIEMAT-Moncloa (Madrid, Spain) using a 2-L steam explosion reactor designed based on Mansonite technology. The pretreatment conditions (190 \({}^{\circ}\)C, 10 min, and 40 mg H\({}_{2}\)SO\({}_{4}\)/g lignocellulosic biomass) were selected based on previous results, considering solid recovery after pretreatment, its chemical composition, and the enzymatic hydrolysis yields [19]. After pretreatment, the water-insoluble solids were separated by filtration and subjected to 72 h of enzymatic hydrolysis at 15% (_w/w_) solids concentration, an enzyme loading of 20 mg of protein/g glucan (SAE0020, 220 mg protein/mL; Merck, Germany), 50 \({}^{\circ}\)C temperature, and pH 5.0. The resulting hydrolysate was collected by filtration, concentrated by vacuum evaporation using a rotavapor until a glucose concentration of about 250 g/L (to prevent dilution during the pulse-feeding cultivation strategy), and then filter-sterilized through a 0.22 \(\upmu\)m filter. The concentrated hydrolysate was analyzed prior to its use as described below.