2-02: What are the key substrate factors which limit the hydrolysis of biomass by cellulases?

Typically, the multi-component “cellulase mixture” encounters a lignocellulosic substrate in which both the chemical nature of components such as the lignin and hemicellulose and physical factors such as the degree of polymerization and crystallinity of the cellulose and the overall surface area/porosity of the cellulose all limit enzyme accessibility. Over the past several years, while focusing on applying pretreatment processes to produce substrates amenable to subsequent hydrolysis by cellulases and maximizing carbohydrate recovery, we have shown that pretreated substrates exhibit various characteristics whose interplay governs the accessibility of cellulases to the substrate. For example, the long fibers of softwoods generally result in larger substrate particles after pretreatment via the organosolv process compared to small particles generated by steam pretreatment, suggesting that the likely greater surface area of the smaller particles would result in more effective hydrolysis. However, it was apparent that the presence of varying amounts of lignin and hemicelluloses, which limits direct accessibility to the cellulose, also plays a significant role, indicating that many physicochemical characteristics influence accessibility. Our recent work on organosolv and steam pretreated feedstocks such as corn stover, poplar and lodgepole pine have shown that substrate characteristics such as swelling (which can occur to varying degrees, depending on the substrate and pretreatment method used) can have a tremendous effect on the subsequent ease of enzyme hydrolysis at both high and low solids loadings and at enzyme loadings varying from 5-60 FPU/g cellulose. Methods such as Simons’ staining, fiber quality analysis, pulp viscosity, water retention value and zero span measurements derived from the pulp and paper industry were used to better understand the relative ease or recalcitrance of a given pretreated substrate to enzymatic hydrolysis. We will suggest what substrate characteristics have to be modified to ensure, fast, effective enzymatic hydrolysis.