Characterizing the Effect of AFEX Pretreatment Severity On Corn Stover Cell Wall Ultra-Structural Modifications and Formation of Ammonolysis by-Products

Ammonia fiber expansion (AFEX) pretreatment is a low severity thermochemical pretreatment that enhances the enzymatic digestibility of lignocellulosic biomass through several ultra-structural changes within the cell wall that are poorly understood. The effect of varying pretreatment severity (e.g. moisture/ammonia loading, temperature, residence time) on corn stover cell walls was examined through 3D-TEM based tomography. The lower pretreatment severities were shown to target the middle lamella creating large pores (100-500 nm) within it. While, at higher severities the middle lamella collapsed and there was significant delamination within the secondary cell walls leading to the formation of lignin-rich globules originating from the S3-secondary cell wall layer. For certain ammonia pretreatment severities, cellulosic microfibril bundles (10-20 nm thickness) were exposed from within the outer cell walls facing the cell lumen. Closer analysis (via ESCA, AFM and confocal fluorescence microscopy) of outer cell wall surfaces shows heterogeneous deposits rich in AFEX cell wall extractives. EM based immunolabeling helped locate arabinoxylans deposited on the outer cell wall surfaces after AFEX. Raman spectral data indicates conversion of cellulose I to III is intricately dependent on AFEX pretreatment conditions. The surface area and pore volume created within the different regions of the cell wall was estimated through 3D-image analysis using MATLAB. The extent of ammonolysis vs. hydrolysis of the various lignin-carbohydrate complex linkages were estimated using LC-MS/MS and GC-MS based methodologies. The effect of cell wall porosity on enzyme diffusion was explored using simple model simulations.