Green pretreatment strategies for enhanced microbial lipid fermentation and synergistic high-quality lignin recovery for next-generation integrated biorefineries

• Pretreated bioenergy grass yields microbial lipids and preserves uncondensed lignin. • NADES pretreatment resulted in higher sugar and yeast lipid yields than HT or refined sugars. • Enzymatic hydrolysis of ChCl:LA pretreated biomass resulted in 83.5% of glucose yield. • Two-stage R. toruloides cultures of NADES sugars produced 19.36 g/L lipids. • ChCl:LA pretreatment gave >89% pure lignin with high phenolic OH and low S/G. Miscanthus × giganteus (Mxg) is a warm-season perennial grass being commercialized as a biomass feedstock for temperate farms. Three process strategies were compared for converting Mxg into single-cell oil and extracted lignin intermediates, as hydrothermal (HT) processing and two natural deep eutectic solvents (NADES), ChCl:LA (choline chloride:lactic acid) and ChCl:Gly (choline chloride:glycerol). Pretreatments were performed at 10% and 50% solids loading at 140°C, 2 h; HT: 190°C, 10 min. Enzymatic hydrolysates, generated at 10% solids using washed and unwashed biomass, were evaluated for microbial lipid production. Maximum glucose conversions from washed biomass reached 83.5% (ChCl:LA), 52.7% (ChCl:Gly), and 74.0% (HT) . Remarkably, NADES-derived hydrolysates effectively replaced refined sugars for the cultivation of the oleaginous yeast Rhodotorula toruloides, achieving ∼51% higher biomass (OD 90.7) and lipid titers of 19.36 g/L within 45 h using a two-stage fermentation strategy. Lipid contents ranged from 34.5–44.5% dry weight, demonstrating reduced reliance on purified sugars. Beyond carbohydrate valorization, ChCl:LA pretreatment enabled high-purity lignin recovery (>89%) in a lignin-first strategy. Structural analyses (2D-HSQC and ³¹P NMR) showed syringyl (78.15%), guaiacyl (15.15%), and p-hydroxyphenyl (6.41%) units, with higher phenolic hydroxyl content (0.91 mmol/g) and a lower S/G ratio (0.19) than HT lignin (0.87 mmol/g, S/G 0.22). These attributes favor downstream lignin depolymerization into low-molecular-weight aromatics. Overall, NADES pretreatment simultaneously enhances microbial lipid yields and recovers high-quality lignin , advancing the economic feasibility of renewable diesel and sustainable aviation fuel (SAF) production from bioenergy crops within an integrated biorefinery framework.