Mixing Effects on the Kinetics of Enzymatic Hydrolysis of Avicel for Batch Production of Cellulosic Ethanol

This article presents a tightly coupled experimental and theoretical study to explore the effects of mixing and mass transfer on the kinetics and dynamics of cellulase-mediated cellulose (Avicel) hydrolysis for bioethanol production in batch reactors. The kinetic parameters (K-M and V-Max) for the three enzymes (endoglucanase, exoglucanase, beta-glucosidase) that constitute cellulase are determined at various mixing speeds: 0 (no mixing), 40, 80, and 150 rpm (high mixing). The experimental values of K-M and V-Max fitted to algebraic expressions that quantify them as functions of mixing speed, and that, in the asymptotic limit of complete mixing, give their purely kinetic values (without any mass transfer disguise). The glucose and reducing sugar yields as well as the degree of polymerization (DP) for Avicel are measured at all four mixing speeds for 45 h of incubation. Maximum yields of 76% for glucose and 87% for reducing sugar are obtained at no mixing condition (i.e., mass transfer controlled regime), and the DP was found to reduce from 300 to 41. An unsteady-state multistep three-enzyme kinetic model incorporating competitive and noncompetitive inhibition caused by the products glucose (monomer) and cellobiose (dimer) is simulated using the experimentally obtained K-M and V-Max values at various mixing speeds, and our model simulations are validated with our experiments. Our analysis shows that the K-M and V-Max for the three cellulase enzymes remain mass-transfer disguised kinetic parameters even at high mixing speeds, and we quantify the purely kinetic values they attain in the limit of perfect mixing. Our experiments and simulations show that lower mixing speeds increase glucose and reducing sugar yields and decrease DP by preventing the products glucose and cellobiose from coming in contact with the active sites of the cellulase, thus reducing product inhibition, an observation that may significantly reduce the energy costs for bioethanol production.