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

Abstract

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, β-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 are 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.

Oscillating flow bioreactors: An enabling technology for sustainable biorefining operations?

“…Further studies would be necessary to give a sound explanation of the kinetic reaction behavior in the OFB. It has previously been suggested in that lower mixing speed leads to higher glucose yield, although the authors just worked in the range of 0 to 160 rpm. Interestingly, the lowest mixing showed fastest glucose production in the first hours, but almost no further conversion after 1000 minutes.…”

“…The improvements of initial good mixing become obvious only in the later course of the hydrolysis. The published modeled kinetic factors suggest a strong decay of rate constants for endo‐ and exoglucanase up to 100 rpm, and subsequent increase with highest rate constant >700 rpm. Earlier studies have suggested that initial mixing is crucial for hydrolysis performance to efficiently distribute the enzymes, while others highlighted that periods of vigorous mixing are sufficient .…”

“…It is known that enzyme‐substrate binding is a key in hydrolysis performance, as the enzyme activity has to unfold its potential on reactive sites . Insufficient binding can reduce the effectiveness of site interactions and lead to excess of enzyme input . To enhance enzyme activity, enzymatic engineering has been addressed by numerous researchers .…”

Influence of fluid dynamic conditions on enzymatic hydrolysis of lignocellulosic biomass: Effect of mass transfer rate

“…Using equations (2) to (8), mass transfer coefficient (k C ) values were estimated for runs 1 to 9 at different times. Results of these calculations are shown in Figure 3.…”

A multiscale three-zone reactive mixing model for engineering a scale separation in enzymatic hydrolysis of cellulose

“…(Gaikwad and Chakraborty, 2013); what is the optimally mixed reactor configuration for enzymatic hydrolysis in continuous systems? (Chakraborty et al, 2010);(c) what is the optimal reactor mixing strategy for batch hydrolysis of cellulose?…”