As described above, carboxylate salts have a good ability to dissolve cellulose and the dissolution mechanism has been analyzed. However these carboxylate type ILs also have a drawback in terms of thermal stability. These ILs still require heat to dissolve certain amounts of cellulose. To overcome these problems, methylphosphonate salts were proposed as stable CDILs. Fukaya and co-workers have synthesized 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][(CH3)(H)PO2]; IL5 in Scheme 2.6), and found that this IL5 had a good stability and dissolved cellulose without heating [28].

This IL5 has very low glass transition temperature (—86 °C), low viscosity (107 cP at 25 °C), and high Kamlet-Taft в value (1.00). The physicochemical properties of IL5 allow dissolution of 6 wt% cellulose within 1 h at 30 °C (Fig. 2.4), and allow it to dissolve 4 wt% cellulose without heating (at 25 °C) within 5 h.

Considering the above mentioned results, the structure of CDILs and cellulose solubility data are summarized in Table 2.5.

Our understanding of the mechanisms operating during fractionation of wood polymers using IL-based solvent systems is continuously improving. An increasing number of publications have appeared in the recent literature utilizing increasingly sophisticated and target-selective treatment systems. The fundamentals of action of the ILs during these treatments have been explored, and the importance of factors such as treatment conditions, solvent system reactivity, and structural features of plant cell walls have all been discussed to varying degrees and will be reviewed in this chapter. A large number of these citations have focused on the utility of ILs, as pretreatment method prior to biofuel production. Selective separation of the com­ponents in these pretreatments may not typically be the ultimate goal of the pretreatment, but frequently delignification takes place. Nevertheless, the publica­tions describing work oriented to increase enzyme activity on wood have offered useful insights into the action of ILs, which can also be utilized for the design of fractionation systems.

Sucrose is a disaccharide consisting of glucose and fructose moiety linked together by a glycosidic bond, and is easily hydrolyzed into fructose and glucose upon heating in ILs. Moreau et al. studied the dehydration of sucrose in 1-H-3-methyl imidazolium chloride ionic liquid, and found that it could be nearly quantitatively transformed into 5-HMF and unreacted glucose, thus 5-HMF was produced only from the fructose moiety and the glucose moiety was practically unused in the system [29]. Hu et al. investigated the conversion of sucrose in [EMIM][BF4] in the presence of SnCLr, and found that both the glucose and fructose moiety could be converted into 5-HMF (about 65 % yield) and most of the carbon in sucrose was converted [50].

Dehydration of sucrose (50 wt%) in choline chloride catalyzed by CrCl2 and CrCl3 resulted in 5-HMF yields of 62 and 43 %, respectively, in 1 h reaction time at 100 °C [37]. Outstanding results were reported by Lima et al. [53] who reported 5-HMF yield of 100 % when using a methyl iso-butyl ketone (MIBK) as co-solvent in the reaction system, [BMIM][Cl]/MIBK/CrCl3 system. These results are surpris­ing since yields for fructose and glucose alone in the system were only 88 and 79 %, respectively, under the same reaction conditions [53]. MIBK, however, is an undesirable additive, due to its volatility and its designation as a priority pollutant.

3.4.1 Synthesis of Fatty Acid Methyl Esters in the Presence ofDES

Biodiesel is a renewable biofuel made from oils or fats that can be used directly in the diesel engine [37]. Biodiesel is biodegradable, non-toxic and generates less pollutant emissions than “conventional” diesel [38] There are four main routes to produce biodiesel, direct use and blending of raw oils, micro-emulsions, thermal cracking and transesterification. The transesterification reaction is the most com­mon method to synthesize biodiesel. Much effort has been paid to decrease the total cost of biodiesel since it is significantly more expensive than fossil-derived diesel

[39] . Several studies have focused on the choice of the raw materials since it is the main cost contributing factor [40]. Thus waste cooking oils and industrial oils such as sludge palm oil (SPO) and acidic crude palm oil (ACPO) were used in the biodiesel production. During methanolysis of oils, glycerol is released. Due to high difference of polarity with biodiesel, released glycerol and excess of methanol can be removed by phase decantation. However, complete removal of glycerol from biodiesel is not an easy task and postpurification processes are necessary in order to reach the required ASTM specifications.

ILs can also be used for the production of biodiesel since it can act both as catalyst and solvent. However, their cost and their complex synthesis are not competitive. In this context, few groups have attempted the use of DES for the removal of glycerol from biodiesel at the end of the methanolysis process.

Hayyan et al. have shown that DES can be used as a catalyst for esterification of free fatty acids (FFA) contained in vegetable oils. Hayyan et al. [40] They have used phosphonium-based DES (P-DES, made of p-toluenesulfonic acid mono­hydrate and alkyltriphenylphosphonium) in the pre-treatment of low grade oils. The authors have studied two catalyzed reactions (esterification and transesteri­fication) to produce biodiesel from Low grade crude palm oil (LGCPO), an agro­industrial raw material generated from oil palm mills. LGCPO was only considered for biodiesel production by very few studies. The esterification was performed in the presence of P-DES catalyst and methanol. Pre-treatment of LGCPO via ester­ification is necessary for conversion of high FFA to fatty acid methyl ester (FAME) since FFA causes a poisoning of basic sites used in biodiesel production. After treatment with the DES, only 0.88 % of FFA remained in LGPCO. Additionally, the DES can be recycled three times which represent a considerable advantage as compared to p-toluenesulfonic acid traditionally used in such case. The authors have also performed the transesterification of pre-treated LGPCO to produce FAME in the presence of P-DES. At the end of the reaction, it was shown that the P-DES was completely removed from the biodiesel since no P and K were detected.

Zhao et al. [41] have studied the enzymatic preparation of biodiesel from soybean oil using a ChCl/Glycerol DES (1:2 molar ratio). The transesterification reaction was performed in a mixture of DES and methanol. Different enzymes were tested. Reaction was heated at 50 °C in an oil bath. Authors have demonstrated that the highest triglycerides conversion was 88 %. This high conversion rate was obtained in the presence of Novozym 435/mL, 0.2 % (v/v) of water; 50 °C and 24 h with a volumic ratio of 7:3 of DES/methanol. DES is biocompatible with lipase. Moreover some authors have shown that DES can be used to reduce the purification cost of biodiesel through transesterification. This work has demon­strated that this DES is biocompatible with enzymes, which is of interest for the valorization of biomass.

Biodiesel, chemically defined as monoalkyl esters of long chain fatty acids, are derived from renewable feedstocks like vegetable oils and animal fats [16]. Recently, the production of biodiesel from lipid produced by oleaginous yeasts and algae has been obtained much attention [15—19]. Biodiesel has potentials

as an alternative to petroleum diesel. Emission of carbon dioxide into the atmosphere can be reduced by substituting diesel fuel with biodiesel [20]. The production of biodiesel from lipids include some key steps which are (1) lipids extraction from oily materials, such as soybean, sunflower seeds, and cell mass of oleaginous microorganisms, (2) esterification or transesterification of fatty acids or lipids, and (3) purification of fatty acid esters (Fig. 7.1). Biodiesel can be produced by either chemical or enzymatic conversion of lipids or fatty acids with a monohydric alcohol in the presence of acid or base catalysts, or lipases (Scheme 7.1). These procedures require a large quantity of organic solvents and corrosive acidic or basic catalysts. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have stimulated the search for alternative production methods and alternative substrates. In consideration of the unique prop­erties of ILs comparing to traditional solvents and acidic or basic catalysts, a lot of efforts by using ILs as solvents and catalysts have been devoted into developing a more clean and efficient process for biodiesel production.

Ngoc Lan Mai and Yoon-Mo Koo

Abstract The potential of ionic liquids as a green alternative to environmentally harmful volatile organic solvents has been well recognized. Being considered as “designer solvents”, ionic liquids have been used extensively in a wide range of applications including biotransformations. As compared to those in traditional organic solvents, enzyme performance in ionic liquids is showed enhance in their activity, enantioselectivity, stability, as well as their recoverability and recyclabil­ity. This chapter will cover the biocompatibility issue of ionic liquids with enzymes. The effects of ionic liquid properties on the enzymatic reactions and conformation of enzyme as well as methods for activation and stabilization of enzymes in ionic liquids will be described. In addition, the current attempts for rational design of biocompatible ionic liquids will be also discussed.

Keywords Enzyme • Biocompatible • Biotransformation • Ionic liquids • Molecular simulation • Rational design • QSAR

10.1 Introduction

Ionic liquids, which are composed entirely of ions and are liquid at room temper­ature, have been extensively used as a potential alternative to toxic, hazardous, flammable and highly volatile organic solvents due to their unusual and useful properties. Unlike traditional organic solvents, ionic liquids have many favorable properties such as negligible vapor pressure, wide liquid temperature range, non-flammability, high thermal and excellent chemical stability, high ionic con­ductivity, large electrochemical window, and ability to dissolve a variety of solutes. In addition, the physicochemical properties of ionic liquids such as melting point,

N. L. Mai • Y.-M. Koo (*)

Department of Marine Science and Biological Engineering, Inha University, Incheon 402-751, Republic of Korea e-mail: ymkoo@inha. ac. kr

Z. Fang et al. (eds.), Production of Biofuels and Chemicals with Ionic Liquids, Biofuels and Biorefineries 1, DOI 10.1007/978-94-007-7711-8_10,

© Springer Science+Business Media Dordrecht 2014 viscosity, density, hydrophobicity, polarity, and solubility can be finely tuned by simply selecting appropriate combination of cations and anion as well as attached substituents to customize ionic liquids for many specific demands, leading to the use of the terms “designer” and “task specific” ionic liquids [1]. In fact, these unique and tunable properties of ionic liquids make them as promising solvents, co-solvents, and reagents in wide range of applications including electrochemistry

[2] , analytical chemistry [3], organic and inorganic synthesis [4, 5], nanomaterial synthesis [6], polymerization [7], separation [8,9], and biotechnology [10] for more than a decade and their applications continue to expand.

In fields of biotechnology, ionic liquids have been widely used as solvents for biomolecule purification [11, 12], pretreatment of cellulosic biomass for biofuel production [13], and enzymatic reactions [14]. The extraction of biomolecules such as protein, enzymes and amino acids using ionic liquids as media or as ionic liquid — based aqueous two-phase systems (APTS) is gaining increasing attention in recent years which showed high extraction efficiency and recyclability. In addition, ionic liquids have been effectively used to pretreat cellulosic biomass for biofuel pro­duction. The pretreated cellulose showed an improvement in enzymatic hydrolysis compared to untreated cellulose. However, the most tremendous uses of ionic liquids in biotechnology are in biotransformation. Many excellent reviews have summarized the variety of enzymes used in ionic liquids [14—16]. However, this is not of focus in this chapter, but rather the issues regarding the compatibility of enzyme in ionic liquids are discussed.

After the ILs synthesis, the characterizations are followed to confirm the structures and purities. Nuclear magnetic resonance (NMR) spectrometry, Fourier transform infrared spectroscopy (FT-IR), the X-ray diffraction (XRD), elementary analysis (EA) and mass spectroscopy (MS) are common used techniques in ILs analysis. The

NMR included 1H NMR and 13C NMR, together with the FT-IR are widely to confirm the desired structures and functional groups of ILs. Meanwhile, the EA and MS are usually used to detect the ILs purities.

The common impurities are water, metathesis byproducts, sorbents and chemical drying agents, especially the water, which almost exit in all kinds of ILs, the water in ILs has various effects on the ILs applications, for the water sensitive reaction, the water removal is very necessary, while, sometimes a little amount of water in ILs may enhance the reaction, so the water content in ILs can be controlled by evaporated or vacuum drying according to the ILs applications. Some halide salts, alkali metals and heavy metal precipitation as byproducts in ILs, some of them are reduced by passing the ILs through silica gel [42], the precipitations are common filtered by millipore filters. In addition, some other methods, such as sorbents, distillation, zone melting and clean synthetic routes are also developed to obtain more pure ILs [39]. Sometimes the color of ILs would be dark when the reaction temperature is high, activated carbon is introduced to use in ILs discoloration. Meanwhile, the ILs those are solid at room temperature would be recrystallized to get better quality.

The separation and purification methods of ILs can be decided by different water solubility of ILs. The cation and anion composed of ILs both influence the water solubility of ILs, of course, the increased alkyl chain (n) length of the cation decreases the water solubility of ILs, and n = 10 is a boundary of liquid and solid phase of ILs. With a short alkyl chain cation, the ILs containing halide, acetate, sulfate, or phosphate are generally liquids insoluble with water, while the ILs with BF4 or PF6 are mostly water-immiscible [39].

The first description of the use of ILs as reaction media for etherification of cellulose is a patent related to the carboxymethylation of the polysaccharide [70]. Carboxymethyl cellulose as well as alkyl — and hydroxyalkyl ethers of cellu­lose are of huge commercial interest but reports on etherification of cellulose in ILs are rare compared to the vast number of publications related to the esterifications in these media. ILs applied for cellulose dissolution are rather hydrophilic and homo­geneous etherification of cellulose is more difficult to realize because the reagents/ bases applied and/or the derivatives formed are not completely soluble in the reaction mixtures.

Solid NaOH has been utilized as base for carboxymethylation of cellulose in ILs,

i. e., this derivatization occurs heterogeneously despite the fact that cellulose is dissolved in the ILs prior to the reaction. Moreover, the polysaccharide solution forms a gel-like system shortly after the addition of the reagent [13]. As a conse­quence, DS values obtained are rather low (<0.5). Cellulose dissolving ILs are also immiscible with hexamethyldisilazane (HMDS), a rather hydrophobic reagent utilized for the conversion of cellulose into the corresponding silyl ethers. A biphasic silylation procedure has been reported, in which cellulose is dissolved in an IL and treated with HMDS, dissolved in IL-immiscible toluene [42]. The reaction involves transition of the reagent into the polar IL phase and of the cellulose derivative, which becomes increasingly hydrophobic upon silylation, into the non-polar toluene phase. These phase transitions influence the course of the derivatization reaction, e. g., in terms of overall DS, distribution of silyl groups along the polymer chain, and product homogeneity. These reactions are difficult to control because they strongly depend on multiple reaction conditions, such as temperature, stirring speed, viscosity, and liquid/liquid interface area. Completely homogeneous preparation of trimethylsilyl cellulose (TMSC) with a broad range of DS values from 0.4 to 2.9 has been achieved in EMIMAc/chloroform mixtures [41]. The co-solvent efficiently solubilizes HMDS as well as TMSC formed, which otherwise precipitate from the reaction mixtures at DS > 2.

Hydroxyethyl- and hydroxypropyl celluloses, which are among the most impor­tant cellulose derivatives applied commercially, e. g., as additives in paint, cement, and household products, could be prepared in ILs by conversion of cellulose with gaseous ethylene — or propylene oxide [37, 38]. The reaction proceeds heteroge­neous and the degree of molecular substitution (MS) could be increased by addition of DMF or DMSO. The increased reactivity is most likely a result of the improved solubilization of the hydroxyalkylation reagents. EMIMAc was found to be the most suitable solvent for this etherification because the acetate anion can catalyze ring opening of the oxiranes, which results in increased MS. By addition of catalytic amounts of sodium/magnesium acetate, cellulose hydroxyalkyl ethers could also be obtained in imidazolium chloride based ILs. In addition, low-melting quaternary ammonium chlorides and formates could be utilized [71].

Triphenylmethyl (trityl) substituents have been exploited in polysaccharide research as protecting group for the primary hydroxyl group that is more accessible for bulky moieties [72, 73]. Ethers with DS values around 1 possessing a pro­nounced 6-O-functionalization, could be obtained by homogeneous conversion of cellulose dissolved in BMIMCl with trityl chloride [40]. By using the more reactive p-methoxytrityl chloride, a maximum DS of 1.8 could be achieved in AMIMCl but complete protection of position 6 and 2 was not possible.

Cellulase enzymes are not the only catalysts that are effective for the saccharifica­tion of polysaccharides. In a number of reaction schemes for the utilization of biomass, lignocellulose must be hydrolyzed into monosaccharides. This is impor­tant not just in fermentation of biomass into ethanol, but also if the saccharides are to undergo processing directly into commodity chemicals or fuels. Without the use of ILs, the most common methods to hydrolyze polysaccharides into monosaccha­rides is through enzymatic hydrolysis or acid catalyzed hydrolysis [19]. This process can be slow and expensive, in part due to the necessity of heterogeneous reactions due to the insolubility of cellulose in conventional solvents. The ability of ILs to solubilize biomass has led to a considerable research effort in the hydrolysis of cellulose. These studies have focused on a wide range of catalysts, from conventional acid catalysts, both solid and homogeneous, to novel metal catalysts and ILs that are designed to be both solvent and catalyst.

Acid hydrolysis of lignocellulose is well understood and has been used to quantitatively saccharify biomass for decades [24, 86]. The specific reaction occur­ring in the hydrolysis of cellulose begins with the formation of a conjugate acid leading to the cleavage of the glycosidic linkage as a water molecule is added and a H+ ion is released [87]. Because many ILs are stable under acidic conditions, coupling the saccharification ability of acids with the dissolution ability of ILs is a natural choice. The acidity in any IL can only be as high as the conjugate acid of the anion of the IL. If HCl is added to an acetate based ILs, for example, acetic acid will be formed and the acetate anion from the IL will be effectively replaced by a chloride anion. The pH scale is not an appropriate measure of the acidity of an IL, as pH is defined in dilute aqueous solutions. For this reason, the Hammett acidity, as measured by nitroanilines with known pKa values, is used to determine the acidity in ILs [88, 89].

A number of studies have been conducted in which biomass, acid, and an IL have been mixed to hydrolyze the polysaccharides into mono — or oligosaccharides. Proof of this concept was demonstrated by dissolving various cellulose sources, including spruce wood, in BMIMCl and adding HCl, sulfuric acid, nitric acid, or phosphoric acid with heat and stirring. With this method, glucose yields as high as 43 % and total reducing sugars as high as 77 % could be obtained after 9 h [90]. Sievers et al. were able to depolymerize pure cellulose and the cellulose and hemicellulose in loblolly pine in 1-butyl-3-methylimidazolium chloride (BMIMCl) using 0.2 wt% trifluoroacetic acid as a catalyst. In the case of pure cellulose, 97 % could be transformed into soluble mono — or oligosaccharides after 2 h at 120 °C, while 62 % of the pine wood could be converted into soluble products (representing 97 % of the carbohydrate content of the wood) [91]. Other experiments have been done with AMIMCl and added HCl to hydrolyze eucalyptus, pine, and spruce thermomechanical pulps. Higher HCl loadings and longer reaction times resulted in higher degrees of hydrolysis and yielded products consistent with lignin depolymerization [92].

A study by Li et al. demonstrated the hydrolysis of corn stalk, rice straw, pine wood, and sugarcane bagasse using combinations of the ILs 1-butyl-3-methylimi — dazolium bromide (BMIMBr), AMIMCl, 1-hexyl-3-methylimidazolium chloride (C6MIMCl), 1-butyl-3-methylimidazolium hydrogensulfate (BMIMHSO4), and 1-(4-sulfobutyl)-3-methylimidazolium hydrogensulfate (SBMIMHSO4) with HCl, sulfuric acid, nitric acid, phosphoric acid, and maleic acid. BMIMCl coupled with HCl was found to be the most effective system. Interestingly, sulfuric acid was less effective than HCl, possibly due to interaction between the sulfuric acid and lignin [93]. While this study successfully hydrolyzed cellulose in ILs as other researchers had demonstrated, it also worked with the naturally acidic ILs BMIMHSO4 and SBMIMHSO4. Other groups have worked with acidic ILs as catalysts in a number of situations [53, 58, 61]. Amarasekara et al. also utilized this novel acidic IL to depolymerize cellulose and found that 1-(1-propylsulfonic)-3-methylimidazolium chloride and 1-(1-butylsulfonic)-3-methylimidazolium chloride could dissolve cel­lulose up to a loading of 20 g per 100 g of IL at room temperature. The cellulose could then be hydrolyzed with the addition of water and mild heating (70 °C) to produce up to 62 % yield of reducing sugars and 14 % yield of glucose [62]. In one interesting study by Zhang et al., a simple EMIMCl/water system without added acid was used to hydrolyze cellulose at temperatures between 90 and 140 °C with a total reducing sugar yield of up to 97 %. This study of EMIMCl/water systems also demonstrated the ability of “neutral” ILs such dialkyl — and trialkylimidazolium chlorides to lower the pH of an aqueous solution, enhancing cellulose hydrolysis [94].

Solid acid catalysts have also been successfully employed to hydrolyze the polysaccharides of biomass. A pair of studies by Rinaldi and coworkers demon­strates the ability of H-type ion exchange resins and zeolites to catalyze the depolymerization of cellulose. This process showed continuously decreasing degree of polymerization of cellulose along with a continuously increasing yield of reducing sugars, reaching 13 % reducing sugar yield after 5 h using Amberlyst 15 to depolymerize cellulose [95]. Further study examined the mech­anisms of Amberlist 15dry as an acid catalyst and determined the effects of catalyst concentration, substrate concentration, temperature, and impurities have on the reaction. Additionally, they found that the catalyst releases H+ ions into solution, which subsequently catalyze cellulose depolymerization instead of acting as a true heterogeneous catalyst [96]. Zhang and Zhao also studied H-form zeolites and H-type ion exchange resins in ILs as a method for depolymerizing cellulose but with the addition of microwave irradiation to effect the reaction. The combination of high surface area H-form zeolites and microwave irradiation produced a much quicker reaction, yielding 37 % glucose after only 8 min [97], as compared to the 13 % reducing sugar yield obtained by Rinaldi et al. after 5 h. In other work, silica was modified with tethered sulfonic acid functionalized ionic liquids and subse­quently used to hydrolyze cellulose in BMIMCl [98]. When using solid catalysts, especially ion exchange resins, with ILs, it is important to note that ion exchange between the catalyst and IL will most likely occur. This makes the catalysis homo­geneous and alters the composition of the IL.

Ionic liquids are biocompatible with enzymes if they allow enzymes to operate efficiently in them. These ILs must maintain an appropriate balance between the activity and stability of enzymes or even exalt their properties. It is well known that the catalytic activity of an enzyme strongly depends on this 3D structure or native conformation, which is maintained by a high number of weak internal interactions (e. g. hydrogen bonds, van der Waals, hydrophobic interactions, etc.), as well as interactions with other molecules, mainly water, as a natural solvent of living systems. Thus, water is the key component of all non-conventional media, because of the importance that enzyme-water interactions have in maintaining the active conformation of the enzyme. Few clusters of water molecules are required for the catalytic function, in which hydrophobic solvents typically permit higher enzymatic activity than hydrophilic ones due to their tendency to strip some of these essential water molecules [77].

Generally, there are three ways to use ionic liquids in a biocatalytic process:

1. As a pure solvent in nearly anhydrous conditions;

2. In aqueous solutions as a co-solvent;

3. In biphasic systems.

When an ionic liquid is used as a pure solvent, proper control of the water content, or, better, of water activity, is of crucial importance as since minimum amount of water is necessary to maintain the enzyme’s activity. But, when aqueous solutions of ILs are used as reaction media, the IL and the assayed concentration are key criteria because of the high ability of water-miscible ILs to deactivate enzymes. Therefore, the water content is one of the key factors for enzymatic transester­ification reactions for biodiesel production because excess water causes reverse hydrolysis reaction. So, the amount of water required to provide optimal enzyme activity differs according to the type of enzyme and composition of the reaction medium.

The most important properties of ionic liquids when they are used with biocatalysts are their polarity, hydrophilicity, viscosity and purity. The significance of these factors depends on the system used. Recently, Yang [78] has reported a good review on the effect of ionic liquids on enzymes for biotransformations.