Synergistic Sequential Phosphoric Acid-Deep Eutectic Solvent Pretreatment-Based Biorefinery: Selective Fractionation of Sugarcane Bagasse and Isolation of Structurally Preserved Lignin

The development of selective and sustainable fractionation strategies is critical for advancing lignocellulosic biorefineries. In this study, a synergistic sequential pretreatment approach combining dilute phosphoric acid (PA) and a choline chloride-lactic acid deep eutectic solvent ( ChCl-LA DES) was developed for efficient fractionation of sugarcane bagasse (SCB). The initial PA treatment selectively removed ~ 93% of hemicellulose while enhancing cellulose accessibility and lignin extractability. Subsequent DES treatment enabled over 93% lignin recovery and yielded more than 45 wt% cellulose-rich pulp ( DES-cellulose ) containing ~ 84% α-cellulose. The ChCl-LA DES system was characterized using physicochemical and thermal analyses, confirming its suitability as a stable and effective lignin-solubilizing medium. The recovered DES-lig nin exhibited nanoscale features, as evidenced by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Detailed structural characterization using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) spectroscopy revealed the preservation of key native lignin linkages (e.g., β-O-4) and the presence of derivatized structures, indicating minimal condensation during pretreatment. This structural integrity highlights the advantage of the sequential PA-DES strategy over conventional approaches that often degrade lignin. X-ray diffraction (XRD) analysis of the cellulose-rich fraction demonstrated disruption of hydrogen bonding and reduced crystallinity, supporting improved downstream processability. Overall, the proposed sequential pretreatment enables controlled and selective deconstruction of lignocellulosic biomass using environmentally benign solvents, achieving high component recovery while preserving lignin functionality. This work provides a promising and scalable pathway for integrated biorefinery applications and lignin valorization.