Lignin barrier reconstruction outweighs delignification in determining enzymatic hydrolysis of pretreated poplar

Abstract

Efficient lignin removal is widely considered essential for improving enzymatic hydrolysis of lignocellulose. However, lignin redistribution and structural changes during pretreatment may reconstruct the lignin barrier, impacting enzymatic hydrolysis. In this study, poplar was treated with a sequential hydrothermal–deep eutectic solvent pretreatment (HP–DES) and compared with standalone hydrothermal and DES pretreatments. Despite higher delignification efficiencies, HP‐DES‐pretreated substrates exhibited substantially lower enzymatic hydrolysis yields (33.5%–55.3%) than single DES‐pretreated substrates (41.2%–74.8%). To elucidate this counterintuitive behavior, lignin redistribution, structural evolution, and enzyme–lignin interactions were investigated using microscopic imaging, spectroscopic characterization, and reconstructed lignocellulose experiments. The results revealed that HP‐DES pretreatment promoted lignin migration toward the substrate surface, increased the number of phenolic hydroxyl groups, and enhanced lignin condensation, thereby intensifying enzyme non‐productive binding. These findings unraveled that lignin barrier reconstruction outweighs delignification in determining enzymatic hydrolysis, giving new insights into effective pretreatment process design.