Following pretreatment, hydrolysis and transformation of sugars into biofuels is carried out, generally by fermentation. As mentioned before, enzyme-based processes are preferred due to their high specificity under milder conditions with no formation of toxic compounds. The main enzymes involved in the hydrolysis of polysaccharides are cellulases and hemicellulases, which belong to the glycosyl hydrolase family (http://www. cazy. org/).

Cellulases, mainly derived from fungi such as Trichoderma reesei, consist of a mixture of several enzymes that act synergistically to facilitate cellulose degradation. The core enzymes of cellulases are endoglucanase (EG, endo-1,4-"- glucanohydrolase, or EC 3.2.1.4), exoglucanase (CBH, 1,4-"-glucan cellobiohydro — lase) and "-glucosidase (EC 3.2.1.21) and decompose the substrate in a stepwise manner. EG acts on amorphous regions of cellulose, creating free chain-ends that are further hydrolysed by CBH releasing cellobiose units, which are cleaved into glucose by the "-glucosidase.

Hemicelluloses are normally solubilised during pretreatment into monomers and oligomers with different degrees of polymerization. In other pretreatments such as AFEX, both celluloses and hemicelluloses remain in the pretreated material. Therefore, hemicellulases are necessary for conversion into monomeric sugars. Furthermore, these enzymes enhance cellulose hydrolysis by removing residual hemicelluloses from the fibres and reducing the inhibitory effect of the xylo — oligosaccharides (Qing et al. 2010). The complex structure of hemicellulose requires several enzymes to complete its hydrolysis, but they can be broadly classified as xylanases and mannanases (Gfrio et al. 2010).

The addition of other enzymes that affect lignin (peroxidases, oxidases, and laccases) have been also recommended as part of biochemical pretreatment to reduce lignin content (Wang et al. 2012) or to facilitate detoxification of pretreated materials prior to enzymatic hydrolysis and fermentation (Moreno et al. 2012).

Enzyme production and application has been estimated to be one of the main con­tributors to the cost of second generation ethanol production (Klein-Marcuschamer et al. 2012). Cellulases have lower specific activity than amylases used in first generation ethanol production, thus increasing enzyme dosage requirements. The enzyme loading for optimal conversion is determined by the type of feedstock used, pretreatment technology applied, pretreatment severity and solid concentration. Apart from optimization of pretreatment, different strategies are implemented to reduce enzyme loadings. These strategies include the development of robust enzyme producers, the improvement of existing enzyme systems and bioprospecting of new enzymes (Banerjee et al. 2010; Huang et al. 2011). Regarding the enzymatic process, the construction of tailor-made enzyme combinations adapted to feedstock and pretreatment and/or the addition of additives such as surfactants enhances the yield of enzymatic hydrolysis while reducing cellulase requirements. Moreover, surfactant addition could favour enzyme recycling (Tu and Saddler 2010).