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Enzymatic hydrolysis of ionic liquid-pretreated lignocellulose

Abstract

Cellulose is the most abundant organic compound on Earth, and is found in lignocellulosic biomass. In order to access this carbon source for biofuel production, a process must be developed that breaks down the natural barriers the plant has in place to protect itself from degradation. The natural breakdown of biomass by organisms involves a variety of enzymes that work together to make the carbon source available. Cellulose can be hydrolyzed in an acid-catalyzed process by cellulase enzymes to form glucose.

The enzymatic hydrolysis of cellulose relies on the initial adsorption of cellulases to the solid lignocellulose surface, and thus the reaction can be limited by surface area. Additionally, hemicellulose and lignin combine to act as barriers to adsorption, both physically and through competitive binding that inactivates the enzymes. Cellulose crystallinity also inhibits enzymatic degradation by limiting surface area and decreasing the rate of enzymatic hydrolysis. All of these factors make enzymatic hydrolysis of cellulose slow for untreated biomass, and make pretreatment a necessary step for biofuel production. Several pretreatment methods have been developed to make biomass more amenable to enzymatic hydrolysis. A promising approach is biomass dissolution in ionic liquids (ILs) followed by the addition of an anti-solvent to precipitate the cellulosic fraction of the biomass prior to hydrolysis. A cellulase mixture can then quickly and efficiently hydrolyze the precipitated biomass.

The enzymatic hydrolysis by a cellulase cocktail of Miscanthus x giganteus, a lignocellulosic biomass, pretreated with the IL 1-ethyl,3-methylimidazolium acetate ([Emim][OAc]) was studied. The IL pretreatment time and temperature parameters have been studied, and a kinetic model was developed to optimize the pretreatment conditions for improved cellulose and hemicellulose enzymatic conversion. This kinetic model indicated a wide range of optimal pretreatment conditions, from high temperatures / short times to lower temperatures / long times. Variables obtained from the kinetic model are within reported ranges of experimentally obtained values for other pretreatments, indicating that the model may be broadly applicable to a variety of lignocellulosic pretreatment processes.

Since ionic liquid pretreatment provides a readily-hydrolysable substrate, other factors, such as enzyme loading, product inhibition, and solids loading, become important. For industrially-relevant processes, the enzymatic hydrolysis must produce and tolerate high levels of glucose. We have investigated the effects of glucose concentration on the enzymatic hydrolysis of Avicel, [Emim][OAc]-pretreated Avicel, and [Emim][OAc]-pretreated Miscanthus. Both cellobiose and glucose production were monitored over time, and cellobiose was found to be present at appreciable concentrations when high levels of glucose were present. This effect is more pronounced with [Emim][OAc]-pretreated substrates. A competitive inhibition model was fit to the hydrolysis data and found to fit moderately well. However, this model was unable to capture both the fast initial glucose production and prolonged cellobiose presence, highlighting the need for a more mechanistic kinetic model.

By studying the enzymatic hydrolysis of pretreated lignocellulose, we will be able to better understand and direct the engineering of substrates, enzymes, and processes for more effective hydrolysis under industrially relevant conditions.

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