Effects of extractive ammonia pretreatment on the ultrastructure and glycan composition of plant cell walls

Extractive Ammonia (EA) is a novel pretreatment for lignocellulosic biomass that uses anhydrous liquid ammonia to convert the native cellulose I_β into cellulose III.  This modified cellulose allomorph can double enzymatic hydrolysis rates, allowing significant enzyme savings during biomass conversion to fuels and chemicals.  Simultaneously, this pretreatment can selectively extract lignin from the plant cell wall, leaving 95 percent of the carbohydrates in the ammonia-insoluble phase.  Depending on the processing conditions, EA can extract up to ~50 percent of the lignin present in corn stover, which can potentially be used for conversion to fuels and chemicals. Lignin removal by EA also helps improving enzymatic hydrolysis yields due to reduced enzyme inhibition.  The ammonia-insoluble lignin remains intertwined with the structural carbohydrates that compose the plant cell wall and can be recovered in the unhydrolyzed solids that result from enzymatic hydrolysis.  In order to better understand the effects of EA on the ultrastructure and components of plant cell walls, we used Glycome Profiling and immunohistochemistry by employing suite of monoclonal antibodies that recognize various plant cell wall glycans.  We also captured high resolution electron microscopy images (both transmission and scanning).  Here, we report the changes in extractability, cell wall sugars and lignin upon EA treatment along with ultrastructural differences as a function of EA pretreatment conditions.  These are further correlated with enzymatic hydrolysis yields in order to define the key structural modifications that impact cell wall recalcitrance.