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The most common microorganisms used in fermentation for bioethanol production are not capable of metabolising oligosaccharides (Carvalheiro et al., 2008). Therefore, after cellulose is purified, there is the need to hydrolyse cellulose fibres into its monomers, glucose.

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Hydrolysis is a common reaction in biochemistry in which water reacts to break linkages as those found in polymers, nucleic acids, etc. (Nelson and Cox, 2004). Lignocellulosic biomass hydrolysis is usually performed after an adequate pretreatment, which increases significantly hydrolysis efficiency (Hamelinck et al., 2005).

The conversion of cellulose into glucose can be catalysed by acid or enzymes and the most common and studied processes are enzymatic hydrolysis and acid (concentrated or diluted) hydrolysis (Kumar et al., 2009). More recently, subcritical water hydrolysis has been gaining attention as an alternative process (Yu et al., 2007).

1.7.1.1. Enzymatic hydrolysis

Enzymatic hydrolysis is the most common process to convert cellulose into glucose (Van Dyk and Pletschke, 2012). During enzymatic hydrolysis, the enzymes act as catalyst in the hydrolysis process and they are very specific, which leads to high efficiency and no generation of degradation products from glucose. Moreover, due to moderate operating conditions, the utility costs in this process are usually low (Duff and Murray, 1996).

The enzyme used in this process, cellulase, is a mixture formed by three enzyme types: endoglucanase, which breaks -1,4-glycosidic linkages randomly; exoglucanase, which acts in the termini -1,4-glycosidic linkages liberating cellobiose or glucose; and -D-glucosidase which breaks -1,4-glycosidic of small oligosaccharides (cellobiose, cellotriose) (Wyman et al., 2004). These enzymes can be obtained from fungi (Orpinomyces sp., Piromyces sp.) or bacteria sources (Thermobifida fusca) (Wyman et al., 2004). Under optimal conditions, enzymatic hydrolysis was reported to yield up to 95% of glucose (Hamelinck et al., 2005).

The cost of the enzymes used to be a significant drawback for large-scale process. However, with the recent increase in the interest of a cost-effective cellulosic bioethanol

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process, significant progress has been achieved in terms of both efficiency and cost of enzymes (Maris et al., 2006). Moreover, the two already mentioned large-scaled existent technologies, Proesa and Iogen, use enzymatic hydrolysis for cellulose conversion into glucose (Damaso et al., 2014).

Challenges faced during enzymatic hydrolysis process are the high sensitivity of enzymes to pH and temperatures, thus, control of these parameters is mandatory in order to obtain high efficiency and prevent enzyme damage (Van Dyk and Pletschke, 2012). Moreover, enzyme efficiency can be severely compromised by inhibitory substances generated during previous pretreatments such as organic acids and furans (Van Dyk and Pletschke, 2012) and products of enzymatic hydrolysis such as glucose and cellobiose have also an inhibitory effect in the enzymes (García-Aparicio et al., 2006). Finally, although the process is usually performed in mild conditions such as pH 5 and 45-50oC, it takes a long period, usually in the range of hours to few days (Yu et al., 2007).

1.7.1.2. Acid hydrolysis

Acid hydrolysis is largely used in lignocellulosic biomass pretreatment in order to make cellulose more accessible. Nevertheless, it can also be used to hydrolyse cellulose into glucose.

During acid hydrolysis, the addition of H+ acts as catalyst in the cleavage of glycosidic bonds. In general, 50-60% sugars yield can be obtained using dilute acid hydrolysis at temperatures about 220oC (Wyman et al., 2004) and yields as high as 90% have been reported using concentrated acid (30-70%) at mild temperatures (40o_{C) (Hamelinck et al., 2005).} Furthermore, this method is effective in a wide range of different feedstocks, particularly residues such as municipal waste that presents large variability in the composition (Harmsen et al., 2010).

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The advantages of acid hydrolysis, compared to enzymatic process, is the faster rate and non-requirement for a previous treatment (Lenihan et al., 2010). However, due to the drawbacks (mentioned in acid pretreatments), large scale acid hydrolysis has not been evaluated.

1.7.1.3. Subcritical water (SBW) hydrolysis

Subcritical water is referred to water at high temperatures and under sufficiently pressure to maintain a liquid state, however, below the critical point (Tc=374oC, Pc=22.1MPa) (Kruse and Dinjus, 2007).

SBW water is commonly investigated as an environment-friendly pretreatment in which hemicellulose is removed. Moreover, the common approach after the SBW treatment is to submit the solid fraction, composed of cellulose and lignin, to enzymatic hydrolysis for glucose production (Ingram et al., 2011). Nevertheless, at increased temperatures, SBW can also hydrolyse the cellulose fraction into glucose.

Water properties such as dielectric constant (), density and ionic product (Kw) change according to temperature and pressure. Figure 1-9 shows these water properties changing according to the temperature.

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An increase in temperature causes an increase in Kw due to auto-ionization, which creates hydronium ions (H3O+_{), depending on water temperature and pressure conditions (Ruiz} et al., 2013). This leads to increase in autohydrolysis as the produced H3O+ ions catalyse hydrolysis of bonds in a similar way as diluted acid hydrolysis, by attacking glycosidic linkages in both cellulose and hemicellulose and also acetyl groups in hemicellulose (Carvalheiro et al., 2008).

The dielectric constant is a property of solvents relateing the electrical fields around particles and it affects reactions and equilibrium rates (Mohsen-Nia et al., 2010). The decrease in due to the increase in temperature is related to the degree of hydrogen bonds (Kruse and Dinjus, 2007). The decrease of bothand densitychange water properties as a solvent, improving solubility of non-polar substances (Bröll et al., 1999).

There have been many studies of SBW hydrolysis of pure cellulose (Abdullah et al., 2014, Kumar and Gupta, 2008). On the other hand, there are only few studies using SBW for hydrolysis of the complex lignocellulosic biomass. Cheng et al. (2008) used SBW to hydrolyse switchgrass in a batch reactor at temperatures from 250-350oC and residence times from 0- 5min. (Cheng et al., 2009). Prado et al. (2013) hydrolysed sugarcane bagasse using SBW at temperatures from 213-290oC in a flow reactor with flow rates ranging between 11-55mL/min (Prado et al., 2014). Nevertheless, these studies focus on cellulose hydrolysis, not in the recovery of hemicellulose and lignin, which results in degradation of theses fractions.

There are also studies using supercritical water for biomass hydrolysis (Lü and Saka, 2010, Zhao et al., 2009c), however, at these harsh conditions, corrosion problems are more significant and equipment costs are considerably higher (Bröll et al., 1999).

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Comparing SBW hydrolysis with acid and enzymatic hydrolysis, SBW is non-toxic reagent, has a much faster reaction rate and also it is not inhibited by intermediate and end- products formed during the hydrolysis (Zhao et al., 2009c). On the other hand, monosaccharides yield achieved by SBW from lignocellulosic biomass is low when compared to acid and enzymatic hydrolysis (Yu et al., 2007). Moreover, one of the key factors of SBW hydrolysis is the balance between the severity needed for cellulose hydrolysis and the formation of fermentation inhibitors generated to great extents at harsh conditions (Rogalinski et al., 2008).