Bioethanol can be produced from lignocellulosic biomass feedstocks (wood, grasses, agricultural residues, and waste materials). The general steps for producing ethanol include pretreatment of substrates, saccharification to release the ferment­able sugars from polysaccharides, fermentation of the released sugars and, finally, a distillation step to separate the ethanol (Fig. 11.3).

Hydrolysis is usually catalyzed by cellulase enzymes and the fermentation is carried out by yeast or bacteria. Pretreatment of lignocellulosic materials is a prerequisite to facilitate the separation of cellulose, hemicellulose and lignin, so that complex carbohydrate molecules constituting the cellulose and hemicellulose can be broken down by enzyme-catalysed hydrolysis into their constituent simple sugars. Lignin consists of phenols and, for practical purposes, is not fermentable, while hemicellulose consists of 5-carbon sugars, and, although they are easily broken down into their constituent sugars such as xylose and pentose, the fermen­tation process is much more difficult, and requires efficient microorganisms that are able to ferment 5-carbon sugars to ethanol [2, 7, 48, 49]. Besides, in their natural state, cellulose fibers are highly crystalline and tightly packed, so pretreatment is necessary to increase the porosity and the accessible surface area for hydrolytic enzymes. Various biomass pretreatment methods have been used for the production of bioethanol, either simple such as steam explosion alone, or combined treatments such as steam explosion/ammonia, explosion/acid or alkali, fiber explosion/CO2, chemical hydrolysis/enzymatic processes [50—53]. The advantages of biological pretreatment include low energy requirement and mild environmental conditions, but the hydrolysis rate is very low.

In a pioneering study by Swatloski et al. [41], several ILs, in particular [BMIM] [Cl], were found to be capable of dissolving up to 25 % cellulose (by weight), forming highly viscous solutions. This prompted several other groups to test a variety of other ILs for their ability to dissolve cellulose [34, 43, 44, 54—61]. To optimize the use of lignocellulosic materials any pretreatment method should extract lignin and decrease the cellulose crystal structure. Lignocellulosic biomass can also be dissolved in ILs such as [AMIM][Cl], [BMIM][Cl] and [EMIM] [Ac] [62—64] and, for ionic liquids containing the same cation [BMIM], the ability to dissolve the residual lignin was dependent on the anion, as follows (in order): [MSO4] > [Cl] >> [Br] >>> [PF6] [65]. However, not all ILs have the capacity to dissolve cellulose. For example, it has been reported that, due to the presence of cationic hydroxy and allyl groups, alkanolammonium ILs cannot dissolve the crystal structure of cellulose [66]. In this context, ILs possessing coordinating anions (e. g., [Cl], [NO3], [Ac], [(MeO)2PO2]) which are strong hydrogen bond acceptors, have been found to be capable of dissolving cellulose in mild conditions by forming strong hydrogen-bonds with cellulose and other carbohydrates at high temperatures [22]. A good compromise between the solubility of lignin and cellu­lose is achieved with [EMIM][Ac] [67].

Liu et al. [68] provided an extensive review on the mechanism of cellulose dissolution in ionic liquids, demonstrating that the key parameters in the capacity of ILs, for cellulose dissolution are the cation and anion size and the ability to form hydrogen bonds with cellulose. Besides, the presence of water is disadvantageous to the solubility of carbohydrates, but is necessary for cellulose hydrolysis. However, high concentrations of water solvate the ions of the ionic liquid and thus prevent it from interacting with carbohydrates. A compromise must be reached between the water content and the cellulose hydrolysis rate in imidazolium ionic liquids [69].

Froschauer et al. [70] reported that a mixture of the cellulose dissolving IL EMIM OAc with 15-20 wt% of water is able to selectively extract hemicelluloses when mixed with a paper-grade kraft pulp for 3 h at 60 °C. This fractionation

method suggests the use of a cellulose solvent, which can serve repeatedly for complete dissolution of the purified cellulose fraction when applied undiluted.

It seems that although hydrophilic ILs are effective for the dissolution of cellulose, the activity of cellulases decreases significantly in their presence, which is consistent with what has been found for other enzymatic reactions. To overcome the negative effect of ILs on the enzymes, many research groups have regenerated cellulose from ILs prior to enzymatic saccharification, observing the faster hydrolysis of IL-regenerated cellulose compared to untreated cellulose [61, 67, 71]. Ionic liquid-treated cellulose was found to be essentially amorphous and more porous than native cellulose, both of which are effective parameters for enhancing the enzymatic action [72]. Figure 11.4 shows a scheme for regenerating cellulose from ILs prior to enzymatic saccharification for bioethanol synthesis.

After the pretreatment of cellulose with ionic liquids, the ILs must be removed; for this, methanol, ethanol and deionized water can be used as anti-solvents to regenerate cellulose from the cellulose/IL solutions. Enzymatic hydrolysis is affected by the anti-solvents used, the pretreatment temperature and the residual amount of ILs [73]. However, it has been shown that microwave irradiation (or sonication to a lesser degree) enhances the efficiency of dissolution compared with thermal heating, and, when used along with ionic liquid pretreatment, it increases the conversion of cellulose during the enzymatic reaction by making the external and internal surface area of cellulose more accessible. Similarly, Kamiya et al. [74] reported enzymatic in situ saccharification of cellulose in aqueous-ILs by adjusting the ratio of [EMIM] [DEP] to water, and Yang et al. [75] presented a new approach for enzymatic saccharification of cellulose in ionic liquids ([MMIM][DMP)-aqueous media, in which ultrasonic pretreatment is used to enhance the conversion of cellulose. Another strategy proposed to improve the stability of the cellulose in [BMIM][Cl] was to coat the immobilized enzyme particles with hydrophobic ILs. In this way, the stability of cellulase in hydrophobic IL/[BMIM][Cl] mixtures being greatly improved with respect to [BMIM] [Cl] alone [76].

Besides, it must be taken into account that cellulase activity is inhibited by cellobiose and, to a lesser extent, by glucose. Several methods have been developed to reduce such inhibition, including the use of high enzyme concentrations,
supplementation with в-glucosidases during hydrolysis, and removing sugars dur­ing hydrolysis. At this point, the importance of the purity should be mentioned, since water, halides, unreacted organic salts and organics easily accumulate in ionic liquids, thus influencing the solvent properties of the IL, and/or interfering with the biocatalyst.