In Situ Rheological Method to Evaluate Feedstock Physical Properties Throughout Enzymatic Deconstruction

Abstract

Feedstock physical properties determine not only downstream flow behavior, but also downstream process yields. Enzymatic treatment of pretreated feedstocks is greatly dependent on upstream feedstock physical properties and choice of pre-processing Technologies. Currently available enzyme assays have been developed to study biomass slurries at low concentrations of ≤1% w/w. At commercially relevant biomass concentrations of ≥15% w/w, pretreated feedstocks have sludge-like properties, where low free water restricts movement of unattached enzymes. This work is an account of the various steps taken to develop a method that helps identify the time needed for solid-like biomass slurries transition into liquid-like states during enzymatic hydrolysis. A pre-processing technology that enables feedstocks in achieving this transition sooner will greatly benefit enzyme kinetics and thereby overall process economics. Through this in situ rheological properties determining method, we compared a model feedstock, Avicel ® PH101 cellulose, with acid pretreated corn stover. Novozymes Cellic ® CTec2 (80 mg protein/g glucan) can reduce 25% (w/w) Avicel from solid-like to liquid-like state in 5.5 h, as the phase angles rise beyond 45 • at this time. The same slurry needed 5.3 h to achieve liquid-like state with Megazyme endoglucanase (40 mg protein/g glucan). After 10.8 h, CTec2 slurry reached a phase angle of 89 • or complete liquid-like state but Megazyme slurry peaked only to 64.7 • , possibly due to inhibition by cello-oligomers. Acid pretreated corn stover at 30% (w/w) with a CTec2 protein loading of 80 mg/g glucan exhibited a solid-like to liquid-like transition at 37.8 h, which reflects the combined inhibition of low water activity and presence of lignin. The acid pretreated slurry also never achieved complete liquid-like state due to the presence of biomass residue. This method is applicable in several scenarios comparing varying combinations of pre-processing technologies, feedstock types, pretreatment chemistries, and enzymes. Using this method, we can generate a process chain with optimal flow behavior at commercially-relevant conditions.

Characterizing Variability in Lignocellulosic Biomass: A Review

“…When the phase angle is higher than 45°, materials present liquid-like behavior and vice versa. 132 Typically, a high solids loading (less use of water) reduces heating requirements and downstream product dilution and thereby reduces processing cost. 135−137 However, increasing solids concentration also leads to high yield stresses and viscosities, 138,139 which introduces challenges in catalyst mass transfer and biomass transfer from one treatment operation to another.…”

“…The study of viscoelastic properties of biomass before and after pretreatment and enzymatic hydrolysis provides an indication of flow behaviors. Rotational rheometers, torque rheometry, and oscillatory rheometry are commonly used to explore the viscoelastic properties, yield stress, viscosity curves, and compliance of pretreated biomass. − Capillary rheology is used to characterize important physical phenomena, such as liquid phase migration . Three interrelated parameterselastic modulus, viscous modulus, and phase angleare measured to study viscoelastic properties.…”

“…Three interrelated parameterselastic modulus, viscous modulus, and phase angleare measured to study viscoelastic properties. When the phase angle is higher than 45°, materials present liquid-like behavior and vice versa . Typically, a high solids loading (less use of water) reduces heating requirements and downstream product dilution and thereby reduces processing cost. − However, increasing solids concentration also leads to high yield stresses and viscosities, , which introduces challenges in catalyst mass transfer and biomass transfer from one treatment operation to another. − After pretreatment and enzymatic hydrolysis, apparent viscosity decreases due to the reduction of insoluble solids concentration. , …”

Constraints and advances in high-solids enzymatic hydrolysis of lignocellulosic biomass: a critical review

“…For instance, no effect was observed in the enzymatic hydrolysis of microcrystalline cellulose at 2% upon the addition of 10-35 mg of corn stover purified lignin per gram of cellulose [68] or of AFEX pretreated corn stover at increasing amounts of solids of 13.8, 19.3, and 24.9% [91]. Moreover, results from the enzymatic hydrolysis of 1% sugarcane bagasse treated by ball milling, a physical pretreatment that da Silva et al Biotechnol Biofuels (2020) 13:58 decreases cellulose crystallinity while keeping the native biomass composition, showed hydrolysis yields as high as 91% following 24 h of hydrolysis [92]; other researchers have also reported high biomass conversion yields in the presence of lignin [77,93,94]. Huang et al [94] observed that the hydrolysis yield was improved by 8-12% or decreased by 6-16% depending on the biomass source and pretreatment conditions that produced lignins with different characteristics.…”

“…These authors characterized each biomass and suggested that the lignin inhibition or stimulation effect is controlled by lignin hydrophobicity and the negative zeta potential, respectively. Still, Coffman et al [93] reported that the enzymatic hydrolysis of Avicel was faster in the presence of lignin. It is important to emphasize that these reports used an enzyme preparation containing LPMOs, which could be responsible for the boosting effect, as suggested by Cannella et al [77].…”