Elucidating the microstructural deconstruction and accessibility-driven enzymatic saccharification of reed biomass pretreated by p-toluenesulfonic acid-based deep eutectic solvent.

The recalcitrance of reed biomass, arising from the compact lignin-carbohydrate matrix and limited cellulose accessibility, substantially restricts its enzymatic conversion. In this study, a ternary p-toluenesulfonic acid (pTsOH)-based deep eutectic solvent (DES) was developed for selective deconstruction of reed and enhancement of enzymatic saccharification. ChCl:EG:pTsOH (1:2:0.4, molar ratio) operated at 80 °C for 30 min, achieving 58.8% delignification and 63.1% xylan removal while retaining 85.6% of the initial glucan. Structural characterization showed disruption of the lignin-hemicellulose matrix, increased cell-wall opening, and preservation of the cellulose I crystalline structure. Quantitative accessibility analysis revealed that Congo red adsorption was positively correlated with saccharification efficiency (R2 = 0.993), whereas the specific surface area of residual lignin determined by Azure B adsorption showed a strong negative correlation with saccharification efficiency (R2 = 0.992). These correlations indicate that cellulose accessibility and residual lignin surface properties are closely associated with enzymatic hydrolysis performance. Molecular dynamics simulations indicated strong hydrogen-bonding and van der Waals interactions between the DES components and representative lignocellulosic models, consistent with the experimentally observed fractionation trends. In addition, ChCl:EG:pTsOH exhibited good operational stability, with enzymatic saccharification remaining above 81% during five consecutive pretreatment runs. Mass-flow analysis further demonstrated effective redistribution of lignin and hemicellulose into the liquid fraction while preserving a cellulose-rich solid for subsequent hydrolysis. Overall, this work establishes quantitative relationships between DES-induced microstructural reconstruction, substrate accessibility, and enzymatic digestibility, providing mechanistic guidance for the rational design of acidic DES systems for lignocellulosic biorefining.