Ammonia Fiber Expansion (AFEX) is a leading biomass pretreatment process that reduces the recalcitrance of lignocellulosics to further biological processing (i.e. enzymatic hydrolysis and fermentation). Lignocellulosic biomass is AFEX treated using concentrated aqueous and/or essentially anhydrous liquid ammonia (30-95% w/w) at moderate temperatures (70-120°C). After a few minutes treatment the ammonia is explosively released and recovered. AFEX treated biomass is easily hydrolyzed by enzymes to monomeric sugars and the sugars further fermented to ethanol. However, we still lack a complete fundamental understanding of the plant cell wall morphological and ultra-structural changes and degradation compounds produced during AFEX. Improved fundamental understanding of the pretreatment may enhance both the pretreatment itself as well as downstream biological processing.

We examine the morphological changes in the plant cell wall of lignocellulosic biomass upon AFEX pretreatment, using scanning electron (SEM) and laser scanning confocal microscopic (LSCM) imaging. The degradation compounds (e.g., organic acids and phenolics) produced during AFEX are identified by liquid and gas chromatography followed by mass spectroscopy (LC-MS/MS and GC-MS). Xylo-oligosaccharides produced during AFEX are identified using size exclusion chromatography (HPSEC) and quantified using acid hydrolysis. Supporting insights on the ligno-phenolics and organic acids deposited on the biomass surface by AFEX are obtained through Prussian blue analysis (total phenolics) and electron spectroscopy for chemical analysis (ESCA). The potential inhibitory role of the compounds produced during AFEX on subsequent enzymatic hydrolysis and microbial fermentation and their implications for high solid loadings is also explored.