In-situ Rheological Measurements to assess Cellulases for application on Biomass at High Solids Concentrations

Inhibitions on cellulase activities caused by cell-wall constituents including lignin and xylan have been extensively studied, but at low solids concentrations (<10% w/w). Enzymatic hydrolysis of pretreated lignocellulosic biomasses at high solids concentrations or HSC (>15% w/w), critical for industrial biofuel applications, requires cellulases to perform effectively in spite of insufficient free water. The presence of lignin coupled with rheology of HSC biomass can further aggravate cellulase activity. At the AB-PDU, we successfully developed an in-situ rheological property determining method that displays the transition of "solid-like" HSC lignocellulosic biomass to "liquid-like" slurry in real-time. A faster transition led to higher availability of free water, reduced mass transfer limitations, and improved kinetics. Through our method, we determined that Novozymes CTec2 and Megazyme Endoglucanase (80 mg protein/g glucan) can reduce a 25% (w/w) Avicel from “solid-like” to “liquid-like” state in 5.5 and 5.25 hours, respectively as the phase angle or PA rises beyond 45° at these times. After 10.75 hours, the CTec2-carrying slurry behaved completely “liquid-like” with PA reaching 88° while the Megazyme endoglucanase could not induce a similar state in the slurry with the PA peaking at 64.7°. This phenomenon was observed due to the inhibition from cello-oligomers. When 30% (w/w) acid pretreated solids was treated with Novozymes CTec2, the transition occurred at 37.75 hours reflecting the combined influence of presence of lignin and lack of free water. Through this study, we compare enzymes by assessing the influence of lignin and free water on enzyme activities at HSC.