(657f) Effects of High Frequency Ultrasound On Cellulose Morphology and Enzymatic Hydrolysis

Lignocellulosic biomass is a complex three structural biopolymers containing cellulose (about 50%), hemicellulose (25%) and lignin (25%). Conversion of biomass to ethanol consists of three basic steps: (1) pretreatment of biomass; (2) hydrolysis of cellulose; and (3) fermentation of glucose. Cellulose is a linear polymer of anhydroglucose units (-C₆H₁₂O₅-) with the cellobiose repeating unit linked in the β-1,4 positions. Strong inter-chain hydrogen bonding causes the cellulose polymer chains in plant cell walls to bundle together ~ 10² at a time into a mixed crystalline and amorphous “fibrils” with diameter of 1-10 nm, and the fibrils aggregate into larger, interconnected fibers with diameters of 10-100 nm. Only a small fraction of the cellulose is accessible for cellulose binding at the beginning of hydrolysis, and accessibility changes as hydrolysis consumes and disintegrates the fibrils, making new binding sites available. The hydrolysis of cellulose can be carried out using dilute acids, concentrated acids or enzymes. Enzymatic hydrolysis is preferred over acid hydrolysis as it avoids the formation of fermentation inhibition products and does not require drastic operating conditions and material of construction. However, enzymatic hydrolysis of cellulose is a very slow process which requires, in general, about 70-120 hrs for achieving appreciable conversions (~60-80%). In this work, we have investigated the intensification of enzymatic cellulose hydrolysis using high-frequency, low-intensity ultrasound generated by novel multi-frequency reactor and quantify the effects of ultrasound frequency, ultrasound power, and concentrations of cellulose, cellobiase and cellulase on the rate of cellulose hydrolysis by determining glucose yields and the cellulose morphology at different ultrasonication conditions. The morphology of cellulose before and after ultrasonication was investigated with nitrogen adsorption-desorption (BET), X-ray diffraction (XRD), scanning electron microscopy (SEM) and helium ion microscopy (HIM). It was observed that the reducing sugar (glucose) yield obtained in the presence of ultrasound was double that in the absence of it using Avicel microcrystalline cellulose and cellulase+cellobiase enzymes at 50°C and 5.2 pH. It was also observed that the surface area of the native cellulose (Avicel PH 101) used in this study increased from 2.18 m²/gm after hydrolysis to 2.68 m²/gm in the presence of enzyme only, and to 4.32 m²/gm in the presence of ultrasound + enzyme, representing an increase of 23% and 100% (i.e, the surface area nearly doubled), respectively. HPLC chromatographic peaks for both enzymes indicate they are stable even after 44 hrs of ultrasonication and not degrading due to the mechanical and chemical effects of ultrasound. Also, the optimization of the enzymatic cellulose hydrolysis carried out using Taguchi statistical design of experiment methodology, indicated that cellulose concentration of 2% (w/v), cellulase loading of 11.25 FPU cellulase/ gm cellulose, cellobiase loading of 19.25 CBU cellobiase/ gm cellulose and ultrasonic power of 104 W constituted the optimal conditions for optimum glucose yield. The results suggest that the improved cellulose hydrolysis using ultrasound and enzymes could be attributed to a number of factors such as, increase in the interfacial area of the cellulose due to its fragmentation in the presence of ultrasonic cavitation; increase in the activity of the enzyme in the presence of ultrasound; increase in the activity of the cellulose surface due to its continuous cleaning in the presence of the ultrasound which improves the binding of the enzyme to the cellulose; and increased transport of enzymes toward the cellulose surface.

References

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