Bioethanol production from LCB includes basically the following steps: (1) hydrolysis of cellulose and hemicelluloses; (2) separation of released sugars from lignin residue (3) fermentation of sugars; (4) recovery and purification of ethanol to meet final specifications. The hydrolysis (saccharification) is one of the most important steps and is technically difficult to perform due to the poor accessibility of cellulose caused by many physical, chemical and structural factors mentioned above. It is an energy consuming task, contributing substantially to the economic costs of the process and is a subject of many research works (Mussatto et al. 2004; Sanchez et al. 2008; Alvira et al. 2010; Sannigrahi et al. 2010). Hydrolysis can be carried out using organic or strong inorganic acids or enzymes as cellulases and hemicellulases. Some characteristics of different conventional and prospective hydrolytic processes are summarised in Table 1.

Organic acids, mainly acetic and formic acids, are normally used in the autohydrolysis process and arisen upon hydrothermal treatment of LCB at high temperatures (170-220 °С) as the result of partial degradation of macromolecular components (acetylated xylan/ mannan and lignin). These relatively weak organic acids at low concentration are more effective in the hydrolysis of hemicelluloses to a significant extent than of cellulose. Consequently a pre-hydrolysis step is widely used in the production of dissolving pulps by kraft cooking when wood chips are processed prior to pulping by hydrothermal treatment to eliminate significant part of hemicelluloses (Sjostrom 1993). The pre-hydrolysis is also a part of pretreatment strategies aiming to hydrolyse selectively the hemicelluloses in LCB to obtain fermentable sugars and/or to improve cellulose accessibility towards hydrolytic enzymes. In this process, the monomeric sugars from hemicelluloses (xylose, galactose, glucose, mannose, and arabinose) and acetic acid are released in the medium (Lawford et al. 1993; Sanchez et al. 2008). Additionally, degradation of lignin/tannins and sugars originate biologically toxic compounds: gallic acid, syringic acid, pyrogallol, vanillic acid, furfural, 5- hydroxymethylfurfural, among others (Marques et al. 2009). Significant efforts were done to minimize the production of such highly toxic compounds, as well as acetic acid, for ethanol — producing microorganisms. The pretreatment should improve recovery of sugars from hemicelluloses, facilitate the cellulose hydrolysis step (when the main objective is the complete saccharafication of all polysccharides from LCB), and avoid the formation of inhibitors for subsequent fermentation processes (Mussatto et al. 2004; Alvira et al. 2010; Sannigrahi et al. 2010).

Inorganic acids (mainly H2SO4 and HCl) are effective hydrolysis catalysts and allow complete saccharification of LCB polysaccharides. There are some differences between the use of diluted (1-5%) and concentrated acids in the hydrolysis step. In the first case the complete saccharification takes place at high temperatures (160-180 °C) and leads to the formation of residual hydrolysis lignin (cellolignin) as a massive by-product (Sanchez et al. 2008). Due to drastic reaction conditions, sugars are readily degraded via intramolecular dehydration resulting in furfural from pentoses and 5-hydroxymethylfurfural from hexoses. All of these secondary products have a high inhibitory effect on the metabolism of microorganisms. In order to avoid sugars degradation, these compounds should be continuously removed from the reactor by continuous pumping of "fresh" acidic solution through the biomass bed (percolation hydrolysis). This process is used industrially since 1930th in former USSR and nowadays may be considered outdated due to its poor efficiency: low sugars recovery and production of high amounts of chemically inert hydrolysed lignin. The hydrolysis with concentrated acids (50-70% of H2SO4 or 30% HCl) allows for effective saccharification of LCB at moderate temperatures (30-80 °С) for short reaction time with high sugars yield. However, due to the technical difficulties and high consumption of acid, this hydrolysis method is not commercialized yet and is implemented only on pilot scale.

The hydrolysis of polysaccharides by hydrolytic enzymes (cellulases and hemicellulases) is one of the most promising tools for the saccharification of LCB. Hydrolytic enzymes permit highly selective hydrolysis of polysaccharides at relatively low temperatures (30-60 °С), practically without emission of products from sugars degradation. Endo-cellulases break internal bonds to disrupt both the amorphous and the crystalline structures of cellulose, exposing its polysaccharide chains. Exocellulase cleaves two to four units from the ends of the exposed chains produced by endocellulase, while |3-glucosidase hydrolyses the exocellulase product into individual monosaccharides. Since no degradation of glucose occurs, more sugars could be available for a subsequent fermentation, which is the main advantage of this process. However, this process is slower when compared with acidic hydrolysis and hydrolytic enzymes have poor accessibility to polysaccharides of cell wall, especially cellulose. For these reasons this process is time consuming and results in low sugar yields. LCB enzymatic hydrolysis needs a preliminary treatment step to improve the accessibility of enzymatic attack. This preliminary step includes the application of physical methods (mechanical, hydrothermal, etc.) to disintegrate plant tissues and chemical/biochemical treatments to eliminate concomitant biopolymers, mainly lignin and hemicelluloses, hindering the cellulose accessibility. However, the enzymatic efficiency of cellulose conversion still needs to be improved.

The poor efficiency of mild acidic hydrolysis and, particularly, enzymatic biotreatment for direct saccharification of LCB, represents an obstacle for a successful production of second — generation biofuels. For this reason, the development of pretreatment techniques to improve cellulose accessibility and saccharification efficiency is a permanent challenge (Sanchez et al. 2008). A general perspective scheme for LCB conversion into ethanol is presented in Figure 6. The first step presumes LCB pretreatment invoked to degrade strong woody biomass matrix and thus blows away the integral tissues. Different lignocellulosic materials have different physic and chemical characteristics and consequently it is necessary to adopt a specific pretreatment suitable for each raw material. The selected pretreatment will have a determinant effect in the subsequent steps. The amount and type of simple sugars released, toxic compounds formed and their concentration, as well as the overall energy demand and wastewater required in the treatments, depend directly on the specific pretreatment applied (Mussatto et al. 2004; Alvira et al. 2010). Several methodologies for biomass pretreatments have been developed during the last decades. They can be classified into biological (using brown, white and soft-rot fungus or their lignolytic and cellulolytic enzymes to degrade lignin and hemicelluloses), physical (mechanical milling and extrusion), chemical (alkali or
acid pretreatments, ozonolysis, organosolv and pretreatment with ionic liquids) and physicochemical (steam explosion, hydrothermal treatment, ammonia fibre explosion, wet oxidation, microwaves, ultrasound and CO2 explosion) (Balat et al. 2008; Alvira et al. 2010; Sannigrahi et al. 2010). LCB pretreatment leads to partial or major removal of hemicelluloses in the form of mono — or oligosaccharides. Then, cellulose is prepared for the hydrolysis step, if the objective is fermentation of glucose from cellulose, or for further processing to obtain pulps for textile and paper products (Fig. 6). This extra step (dashed) can be catalysed by dilute or concentrated mineral acids or enzymes (cellulases).

Biomass

Fig. 6. Schematic steps for production of bioethanol from lignocellulosic biomass

Until now, fuel ethanol from LCB is not yet considered a viable alternative, mainly due to the high complexity involved on this process, compared with the cheaper oil derived fuels. However, in the last years, with the oil crisis, environmental concerns and the increased need for energy and fuels, bioethanol has become a realistic option in the energy market (Cardona et al. 2007; Sanchez et al. 2008). New research has been developed in order to overcome cellulosic to ethanol bioconversion problems and to make this process a cost — effective technology, with a process integration that combines different steps into one single unit (Lawford et al. 1993; Cardona et al. 2007). Furthermore, the process integration in other industrial plants, namely large scale industries, can be a good solution for reducing costs of bioethanol production, such as in pulp and paper mill industries, with the advantage of reduced release of subproducts.