Multi-faceted studies on the effects of extractive ammonia pretreatment on cell wall ultrastructure and glycan composition of plant biomass

Extractive Ammonia (EA) pretreatment is a novel and effective process for biomass pretreatment for cellulosic biofuel production. Compared to AFEX^TM pretreatment, EA requires a lower moisture content and employs higher ammonia loading. These factors enable formation of cellulose III (more easily digestible than cellulose II and native cellulose) and selective lignin solubilization in liquid ammonia followed by lignin extraction, leading to a very effective biomass pretreatment process. In-depth understanding of cell wall ultrastructural modifications induced by EA pretreatment remains unknown. In this study, we monitored cell wall ultrastructural modifications induced by EA pretreatment in corn stover stem using a complementary set of immunological tools namely, glycome profiling and immunolocalization employing a comprehensive suite of cell wall glycan-directed monoclonal antibodies (mAbs). In addition, we employed scanning electron microscopy and electron microscopic tomography to monitor and visualize corn stover stem cell wall ultrastructural modifications caused by EA pretreatment. Glycome profiling of EA pretreated biomass demonstrated increased extractability of non-cellulosic wall glycans including xyloglucan, xylan, and pectic backbone. In-situ localization studies with specific mAbs against the above glycans further elucidated cell wall modifications induced by EA pretreatments. SEM studies show that EA pretreatment induces significant disruption of primary cell walls rather than secondary cell walls. EA pretreatment also caused delamination and kinking of primary walls with deposited lignin residues locally on the walls. The information gathered provides in-depth details of the pretreatment mechanism and a better understanding of factors affecting biomass recalcitrance.