Understanding the fundamental mechanism behind accumulation of oligosaccharides during high solids loading enzymatic hydrolysis

During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by β-glucosidase, β-xylosidase and other enzymes to monomeric sugars. However, commercial enzyme mixtures do not hydrolyze all of these oligosaccharides and various oligomers may accumulate to represent up to 15-25% of the total polysaccharide solubilized during saccharification of biomass at high solid loading (>3% wt solids/volume). Oligomer accumulation represents a loss in yield of fermentable, monomeric sugars. Moreover, xylo-oligomers with a moderate degree of polymerization potentially act as inhibitors of enzymes during hydrolysis, and also may subsequently interfere with microbial fermentation. At present, little is understood about the structure of these oligomers and their specific impacts on the biofuels production pipeline. Here, we report fractionation, composition, and preliminary structural characterization of representative classes of oligosaccharides that accumulate during hydrolysis of Ammonia Fiber Expansion (AFEX^TM) pretreated corn stover. Oligosaccharides containing variations in the degree of polymerization, composition of monomeric sugar, and in extent of branching patterns have been identified. These different molecules were used as substrates with an array of enzymes including commercial sources, secretomes from novel highly cellulolytic microbes, and single enzymes obtained from phylogenetic analysis, gene synthesis and automated cell-free protein production. Correlations of physical characterizations of individual substrates with enzyme reactivity patterns indicate possible methods to more efficiently produce biomass hydrolysates with high titer of monomeric sugars.