The effect of 1-ethyl-3-methylimidazolium acetate on the enzymatic degradation of cellulose

Highlights

• Cellulase action in concentrated ionic liquid.

• Novel approach to address cellulase activity directly using cellulose.

• Distinct degradation pattern for cellulose I and II.

Abstract

The effect of cellulose pre-treatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) on the enzymatic hydrolysis with a cellulase from Trichoderma reesei was studied. Enzymatic assays were performed with three different pulps – cotton linters, sulfite dissolving pulp, and eucalyptus Kraft pulp. The reaction kinetics were determined by two different methods: (i) the classical test based on the measurement of released reducing sugars from a water soluble cellulosic substrate (carboxy methyl cellulose (CMC)), and (ii) a novel approach where the enzymatic activity is determined as function of the molecular weight decrease of underivatized cellulose. Furthermore the impact of the pure ionic liquid on the stability of the enzyme was investigated. We found that enzymatic degradation of cellulose I followed a completely different degradation pattern in the molecular weight distribution and sugar solubilzation compared to the corresponding regenerated cellulose II.

Introduction

Fossil resources as the common raw materials for energy production and for chemical industries, are limited. Their energetic usage, i.e. burning, is the prime cause of carbon dioxide enrichment in the atmosphere. These negative aspects of the fossil starting materials, along with their prices steadily rising, make it understandable why renewable materials gain increasing interest. Due to its dominance with regard to both mass and available processing technologies, cellulose, the most abundant natural polymer [1], appears to be the logical alternative to successively replace fossil resources as raw material. Lignocellulosic biomass, which is not utilized for food production, is mainly composed of cellulose (35–50%), accompanied by hemicellulose (20–35%) and lignin (10–25%) [2]. These three biopolymers are assembled in the natural composite material “wood”, which has been thoroughly optimized by nature toward strength, endurance and permanence. Separation of the components is consequently rather difficult and laborious. On one side, cellulose is the basis of established industrial branches, such as the pulp, paper and fiber industries as well as more recent cellulose material applications. On the other side, many biorefinery approaches rely on cellulose to be degraded to glucose and small cellooligosaccharides (“saccharification”), which can be fermented to a variety of products, such as ethanol, propanol, acetic acid, biogas, or bioenergy, or be further converted to platform chemicals, e.g. furfural and furan. Cellulose can be degraded in the presence of hemicellulose and lignin with comparably low yields. Alternatively, the three wood constituents are at least partly separated from each other prior to cellulose degradation, which normally gives better outcomes. The saccharification of cellulose is said to be the key step [3], [4] in most biorefinery scenarios. Currently, two procedures are technically applied to hydrolyze cellulose. The first is the chemical cleavage of the 1,4-glycosidic bonds between the anhydroglucose units with the aid of mineral acids, usually sulfuric acid or hydrochloric acid, at elevated temperature and pressure, i.e. an acid-catalyzed hydrolytic cleavage. These processes suffer from relatively low yields and from the formation of significant amounts of byproducts that may interfere with further fermentation [5], [6]. Furthermore they are quite cost-intensive since they require input of considerable amounts of energy and the use of corrosion-resistant production lines. The second approach is the more environmentally friendly, less energy-demanding hydrolysis of cellulose with cellulolytic enzymes produced from fungi or bacteria. The enzymatic hydrolysis is usually carried out in aqueous media at low temperature and generates less undesirable byproducts [2]. However, also this strategy leads to low yields relative to the theoretical amount of glucose obtainable. The reason for this is the low accessibility of cellulose in many lignocellulosic materials. To tackle this problem, several methods of pretreatment have been developed. The aim of these pre-steps is to remove lignin and hemicellulose, reduce cellulose crystallinity and increase porosity and specific surface of the lignocellulosic material, while avoiding the formation of byproducts [7], which results in an overall improved accessibility of cellulose and thus higher glucose yields. The methods of pretreatment can be sub-classified into biological, chemical, physico-chemical and physical processes.

In recent years several research groups have successfully addressed the refining of lignocellulosic biomass prior to an enzymatic degradation of cellulose by means of a pretreatment with ionic liquids (ILs) [4], [5], [8], [9], [10]. Ionic liquids are molten salts, consisting of an organic cation together with a usually inorganic anion, comprising unique characteristics, such as negligible vapor pressure, chemical and thermal stability, non-flammability and high dissolving power [11]. A number of ILs are reported to be suitable as cellulose solvents [12], [13], [14], some are even able to completely dissolve wood [9], [10], [15]. This makes ILs a promising tool for pretreating woody biomass. The IL 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), for example, was used to extract lignin from wood flour and decrease cellulose crystallinity – hence improving cellulose degradability to more than 90%[16]. The same IL was applied to dissolve wood and rye straw flour for subsequent precipitation of cellulose – which was this way separated from lignin – by addition of an antisolvent, such as methanol or water [9], [15]. In this context, dissolution of celluloses in ionic liquids was shown to causes a significant decrease in the molecular weight of the polymer. This degradation is caused by several factors, such as high temperature during dissolution, side reactions triggered by degradation products of ILs, and the direct reaction with imidazolium-type ILs with reducing ends and other carbonyls in celluloses [17]. However, a loss in the degree of polymerization of cellulose is rather advantageous for further conversion to mono- and oligosaccharides as long as it does not chemically alter the cellulose. But ILs also exhibit disadvantages, such as their enzyme-inactivating effect [3], [18], [19], [20], [21], [22], [23], [24], [25] the difficulty of purifying them, and their rather high price.

ILs have been studied with regard to their qualification as reaction media to host biotransformations [26], [27], especially in terms of hydrolase-catalyzed reactions (lipases and cellulases). In some cases ILs have been applied neat, in others they have been used in a mixture with aqueous buffer solutions. They have been shown to be an expedient alternative to conventional solvents, and to cause an enhancement in reaction rate, compared to aqueous buffer systems. The combination of ionic liquids and cellulase enzymes appeared to be an obvious and attractive option for cellulose processing in biorefineries. It would allow conducting the pretreatment of the cellulose containing biomass and its subsequent hydrolysis with enzymes in a one batch process and thus simplify saccharification of cellulose. Several accounts report about cellulose hydrolysis in a mixture of IL and aqueous buffer [3], [21], [28], [29]. Hydrophobic ionic liquids were better tolerated by cellulases than hydrophilic ones; however, incubation of cellulases as well as lipases in pure ILs resulted in a complete inactivation of the enzymes after a certain time [20]. While 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) turned out to cause irreversible inactivation and unfolding of cellulase from Trichoderma reesei [18], [EMIM]OAc, a powerful cellulose and wood solvent [30], was reported to cause slow inactivation of the enzyme. The enhancing effect of increased cellulose availability caused by IL pretreatment overcompensated the reduced enzyme activity in subsequent hydrolysis [3], [4], [20], [21], [28]. The same cellulase was immobilized on a resin and thus showed increased stability in pure 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM]NTf₂) compared to an aqueous buffer. Thereby it became possible to perform enzymatic saccharification of cellulose in ionic liquid solution [23]. Another successful attempt to degrade cellulose in 80% [EMIM]OAc at 90 °C was achieved by employing heterologously expressed cellulase from a hyperthermophilic origin [31]. With tris(2-hydroxyethyl)methylammonium methylsulfate (HEMA) as the solvent (99%), cellulase from Aspergillus niger was used to saccharify cellulose with 70% of enzymatic activity measured on aqueous solution [32].

In the present study we were looking at enzymatic degradation of cellulose in ILs, but from the viewpoint of the cellulose – which had not been considered so far – and not from the viewpoint of enzymes as in previous studies. We investigated the depolymerization of cellulose by cellulase after cellulose regeneration from [EMIM]OAc solution. This system was compared with the enzymatic reaction on cellulose I in aqueous suspension. For the first time the enzymatic activity was determined as a function of chain cleavage events, which can be calculated from the molar mass of the residual cellulose, instead of quantifying the soluble low-molecular mass carbohydrate fragments. We used this method to characterize the enzymatic activity, or in other words to measure the deactivating power of the deployed ionic liquid on the cellulase. It was also tested whether cellulase is able to function in highly concentrated [EMIM]OAc with catalytic amounts of water, and eventually the influence of cellulose purity and morphology on enzymatic degradation with different cellulose pulps. We hope that this study – with its focus on cellulose and its integrity under different degradation conditions – will be seen as a useful complementation of previous work that mainly focused on such cellulose degradation systems from the enzyme point of view.