A pretreatment step is necessary for the enzymatic hydrolysis process. It is able to remove the lignin layer and to decristallize cellullose so that the hydrolytic enzymes can easily access the biopolymers. The pretreatment is a critical step in the cellulosic bioethanol technology because it affects the quality and the cost of the carbohydrates containing streams (Balat et al., 2008). Pretreatments methods can be classified into different categories: physical, physiochemical, chemical, biological, electrical, or a combination of these (kumar et al., 2009), (Table 3).

On the whole, the final yield of the enzymatic process depends on the combination of several factors: biomass composition, type of pretreatment, dosage and efficiency of the hydrolytic enzymes (Alvira et al., 2010).

The use of enzymes in the hydrolysis of cellulose is more advantageous than use of chemicals, because enzymes are highly specific and can work at mild process conditions. Despite these advantages, the use of enzymes in industrial applications is still limited by several factors: the costs of enzymes isolation and purification are high; the specific activity of enzyme is low compared to the corresponding starch degrading enzymes. As consequence, the process yields increase at raising the enzymatic proteins dosage and the hydrolysis time ( up to 4 days) while, on the contrary, decrease at raising the solids loadings. One typical index used to evaluate the performances of the cellulase preparations during the enzymatic hydrolysis is the conversion rate to say the obtained glucose concentration per time required to achieve it (g glucose/L/h/). Some authors reported conversion rates of softwoods substrates (5%w/ v solids loading) in the range 0.3-1.2 g/L/h (Berlin et al., 2007). In general, compromise conditions are necessary between enzymes dosages and process time to contain the process costs.

In 2001, the cost to produce cellulase enzymes was 3-5$ per gallon of ethanol (0.8-1.32$/liter ethanol), (Novozymes and NREL)[1]. In order to reduce the cost of cellulases for bioethanol production, in 2000 the National Renewable Laboratory (NREL) of USA has started collaborations with Genencor Corporation and Novozymes. In particular, in 2004, Genencor has achieved an estimated cellulase cost in the range $0.10-0.20 per gallon of ethanol (0.03-

0. 05$/liter ethanol) in NREL’s cost model (Genencor, 2004)[2]. Similarly, collaboration between Novozymes and NREL has yielded a cost reduction in the range $0.10-0.18 per gallon of ethanol (0.03-0.047$/liter ethanol), a 30-fold reduction since 2001 (Mathew et al., 2008).

Unlike the acid hydrolysis, the enzymatic hydrolysis, still has not reached the industrial scale. Only few plants are available worldwide to investigate the process (pretreatment and bioconversion) at demo scale. More recently, the steam explosion pretreatment, investigated for several years in Italy at the ENEA research Center of Trisaia (De Bari et al., 2002, 2007), is now going to be developed at industrial scale thanks to investments from the Italian Mossi & Ghisolfi Group.