(793g) Inline Rheometry to Identify Mass Transfer Issues in Enzymatic Hydrolysis of Biomass At High Solids Concentration

A limiting factor in enzymatic hydrolysis of lignocellulosic biomass at high solids concentration (>15% w/w), critical for industrial biofuel applications, is the high yield stress and viscosity of wet biomass that causes mass transfer limitations. Due to hygroscopicity of pretreated biomass, very little free water is available at high solid concentrations leading to uneven enzyme application and thereby impeded enzymatic hydrolysis. In-line rheometry can be a valuable analytical tool that can unveil mass transfer issues relevant to the kinetics of biomass hydrolysis. At the ABPDU, we developed a method to correlate varying rheological properties of biomass-enzyme system to the chemical composition (monomeric and oligomeric sugars) by conducting enzymatic hydrolysis in-situ a stress controlled rheometer and obtain a rheokinetic profile.

Ionic liquid ([C₂mim][OAc]) pretreated switchgrass (18.5% w/w) was hydrolyzed with a commercial cellulase mixture (54 mg protein/ g glucan of CTec 2, Novozymes, CA) and tested in a vane-in-cup attachment on a rheometer (Malvern Kinexus, Boston, MA) maintained throughout the study at 50°C. Real-time rheological properties of biomass-enzyme mixture were studies through an oscillatory experiment conducted at a single frequency (1 Hz) and stress (1 Pa), which lied within the linear viscoelastic region of the pretreated solids. The time required for the biomass-enzyme mixture to convert to “liquid-like” slurry (δ > 45 rad) was 2.53 hours, where the glucose and oligomer yields were 12.16% and 42.89% (w/w glucan in pretreated biomass). Initial application of endoglucanase alone reduced the viscosity of biomass improved the action of the other two cellulase enzymes. Even at low viscosity, rheological properties provided insights into interactions at the molecular level. Beyond 3 hours, δ and G* along with elastic (G’) and viscous (G”) moduli exhibited oscillating profiles, with slight increase and a drastic decrease in every 3 to 4 minutes between 3 and 6 hours of hydrolysis. These results indicated (i) product and substrate inhibition effect on enzymes (decrease in G* and glucose yields at 4.67 hours) and (ii) changes in water retention capabilities of the biomass itself (increase in G* with constant glucose yields at 4.83 hours). The state-of-the art techniques, which are typically off-line chemical methods, cannot detect biomass behavior elucidated in this study as they rely on samples obtained from pre-determined time intervals. Obtaining real-time biophysical information correlated with changing chemical composition of biomass –enzyme mixtures will enable us to obtain a comprehensive understanding of enzyme-substrate interactions and thereby allow in improving mass transfer and the overall hydrolysis process at high solids concentration.