Deciphering lignin heterogeneity in ball milled softwood: unravelling the synergy between the supramolecular cell wall structure and molecular events

Mechanical milling of lignocellulose has been used in several studies as a key pretreatment enabling the extraction of lignin from various sources for structural analysis. It is also applied as an alternative to wet chemical methods for lignin valorization. However, the changes caused to the plant cell walls at different hierarchical scales and how they relate to the molecular events are still poorly understood. In this context, we sought to gain deeper insights into molecular heterogeneity in milled cell walls, with a primary focus on lignin. A novel fractionation protocol was developed to enable the advanced analysis (1D and 2D NMR, SEC, XRD) of molecular populations in ball milled fiber walls. The methodology was applied to follow the emergence of such populations through the milling process, and in different milling environments. Lignin heterogeneity in the ball milled fibers was found to consist of distinct populations of small and large fractions of lignin carbohydrate complexes and pure lignin fractions, both with differences in lignin inter-unit abundances. Lignin-carbohydrate bonds of benzyl ester type were unequivocally demonstrated for the first time by combination of HSQC-HMBC NMR analysis. γ-Ester LCC and phenyl glycoside LCCs were also detected. Furthermore, an important branching point in lignin, previously controversial, namely the 4-O-etherified 5-5′ substructure, is unequivocally shown here by HSQC-HMBC analysis of the milled wood isolates, and supported by biomimetic lignin (DHP) to originate from the native structure. Based on the advanced characterization, the origin of lignin heterogeneity in ball milled fibers is proposed to result from the uneven distribution of the applied mechanical energy, where synergistic effects between crystalline and amorphous states play a central role. Accordingly, a plant cell wall model is proposed and a complete mechanism of its disintegration during the milling exercise is presented. The unveiled heterogeneity model of ball milled cell walls can serve as a useful guide for future studies on mechanical fractionation and valorization of lignocellulose based polymers.