Multi-scale characterization of calcium hydroxide mediated cell wall deconstruction during mild pretreatment

The ultrastructural and catalytic mechanisms underlying the efficient depolymerization of lignocellulosic biomass into high-quality sugar platforms under mild conditions remain poorly understood. A promising room temperature and high solid state pretreatment was called densifying lignocellulosic biomass with calcium hydroxide (ch) chemicals (DLC(ch)). It synergistically integrated mechanical densification and alkaline catalysis to overcome cell wall recalcitrance via compression-driven reagent penetration and expansion-induced structure loosening. Herein, we performed multi-modal visualization and characterization of corn stover (CS) cell walls to elucidate the underlying DLC(ch) mechanism. DLC pretreatment initially extruded chemical reagents into cell wall interior via mechanical compression. During post-treatment storage of DLC(ch)-CS, OH^- sustainably catalyzed lignin and carbohydrate bond cleavage, while Ca²⁺ chelated with soluble intermediates to form "cotton-like" calcium-organic deposits (~50% reduction in phenolics). Subsequent mild steam treatment induced cell wall swelling and the calcium-organic deposits filled the nanoscale interstices between cellulose microfibrils, forming a "sandwich-like" interlayer structure. This unique structure greatly enhanced cellulases accessibility to the otherwise occluded cellulose domains. Calcium-based DLC-CS pellets achieved a total sugar conversion of over 90% at a solid loading of 35% (w/w) via enzymatic hydrolysis, and the corresponding ethanol concentration reached 82.6 g/L. This study provided fundamental insights for the development of low-cost and high-efficiency lignocellulosic biorefinery technologies.