An important goal of this research was to identify the major ultra-structural and chemical modifications incorporated within lignocellulosic cell walls during AFEX using several microscopic, spectroscopic and spectrometric techniques. High resolution microscopic (SEM, TEM) and 3D-EM-Tomographic studies indicate an ultra-structural alteration of AFEX treated cell walls via formation of a nanoporous tunnel-like network. Closer analysis (via ESCA, AFM and confocal fluorescence microscopy) of outer cell wall surfaces shows heterogeneous deposits rich in AFEX cell wall extractives. Raman spectral data indicates conversion of cellulose I to III is intricately dependent on AFEX pretreatment conditions. More than 45 degradation products have been quantified using LC-MS/MS and GC-MS. Some of the major degradation products include organic acids, aromatics, phenolic acids and amides.

A fundamental understanding of physicochemical modifications incorporated within lignocellulosic cell walls during pretreatment and its effect on enzyme accessibility are critical to further advancements in reducing cell wall recalcitrance to bioprocessing. This understanding would be critical to re-engineer plant cell walls, hydrolytic enzymes and ethanologenic microbes amenable for cellulosic biorefineries.