16-06: Evolution of cellulosic substrate during enzymatic hydrolysis: impact of substrate morphology and reactivity

The enzymatic hydrolysis of cellulose is still considered as a major limiting step in the biological production of biofuels from lignocellulosic biomass. This step involves the use of three types of cellulose degrading enzymes acting in synergy. Endoglucanases randomly cleave the b-1,4 glycosidic linkages of cellulose, cellobiohydrolases attack cellulose chain ends to produce cellobiose, and β-glucosidases hydrolyses cellobiose into glucose. The reaction rate decreases dramatically with the extent of conversion and this slowdown could be explained by enzyme related factors such as enzyme inactivation or product inhibition. Another important factor, investigated in this study, is the change in substrate reactivity.

 In our experiments, a cellulosic substrate (Avicel PH101) was partially hydrolysed (glucose conversion levels, 10%, 30%, 70%) by an enzymatic cocktail secreted by T. reesei. The evolution of crystallinity, degree of polymerization, particle size and adsorption capacity of pure cellulases from T. reesei were investigated. Substrate reactivity was also tested using the complete cocktail. Results showed the conservation of the crystallinity index and the degree of polymerization during enzymatic conversion associated with a decrease of the particle size. This reduction can be correlated to the diminution in the adsorption capacity of cellulases. However, this lower level of adsorption cannot exclusively explain the decrease in the reaction rate, suggesting also an impact on the substrate reactivity. These result will be included in a global kinetic model of hydrolysis of cellulose dissociating the action of each enzyme and integrating the inherent properties of a cellulosic substrate.