Several pretreatment strategies focus on the removal of a significant part of lignin from plant cell walls, as it is considered the main barrier to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The ability to chemically differentiate and spatially locate lignins across cell walls provides an important contribution to the effort to improve these processes. Here, we present a novel approach using synchrotron phase-contrast tomography to probe the physicochemical features of plant cell walls. In addition to the 3D cellular architecture, the distribution of dense packed lignin–carbohydrate complexes (or, simply dense lignin) over larger regions and within cell walls has been successfully provided. We examined in particular the effect of the sequential H2SO4 and NaOH pretreatment on sugacane bagasse. Our results revealed that aggregates of dense lignin form elongated 3D structures in cell corners (CC) following the orientation of the sclerenchyma fibers; a remarkable result in the light of the conventional perception that lignin particles are discrete and roughly spherical. After the acid pretreatment, a considerable fraction of the dense lignin primarily located in CCs was removed; and it was further dissolved with the subsequent alkali treatment. Unexpectedly, the two-step pretreatment did not contribute to the cellulose accessibility in terms of available surface area and porosity. This study provides new insights into the underlying mechanisms of biomass deconstruction by pretreatments; and the approach established here can be extended to other systems relevant to the bioenergy and biotechnology arenas.
