Efficient lignin depolymerization is essential for biomass conversion into biofuels and biochemicals, yet the structural complexity of lignin presents significant challenges. Understanding hemicelluloses, their interactions with lignin, and the dynamic changes during biomass pretreatment is crucial for optimizing lignin removal and hemicellulose hydrolysis. This study utilizes solid-state Nuclear Magnetic Resonance (NMR) spectroscopy, combined with the quantitative MultiCP method, to investigate the structural dynamics change of lignin and hemicelluloses in unlabeled biomass samples under varying pretreatment conditions. Pretreatment experiments were performed using organosolv solvents with different acid catalysts, at temperatures ranging from 80 to 121°C and acid concentrations from 50 to 400 mM. Substrate samples with different lignin removal rates (0-92%) were analyzed using solid-state NMR (SS-NMR), along with Heteronuclear single quantum coherence spectroscopy (HSQC) analysis of isolated lignin, Well-resolved 13C NMR spectra revealed distinct peaks for lignin and hemicelluloses, with peak intensities decreasing as pretreatment severity increased. The SS-NMR results provide the first characterization of unlabeled dynamic pretreated biomass sample, showing changes in the aromatic S/G ratio in solid substrates under varying lignin removal rates. Notably, it is suggested that three-fold xylan (Xylan3f) and G-type aromatic units play a critical role in the linkage between hemicelluloses and lignin in biomass, offering new insights into the molecular interactions of lignin within the plant cell wall. These findings demonstrate the effectiveness of solid-state NMR in elucidating the spatial and compositional properties of plant cell walls, as well as its potential in modeling hydrolysis rates during biomass pretreatment.
