**4. Ethanol production from the fermentable feedstock from lignocellulosic biomass**

Fermentative production of ethanol is largely performed nowadays through the use of starch or sucrose provided by agricultural crops such as wheat, corn or sugarcane. In Bra‐ zil, for instance, the ethanol production through yeast fermentation of substrates from sugarcane is a well-known and consolidated process. However, the improvement of fer‐ mentative processes towards utilization of lower-value substrates such as lignocellulosic residues is emerging as a valuable approach for reducing the production cost and conse‐ quently increasing the use of ethanol as biofuel. In sugarcane mills, for instance, a large quantity of sugarcane bagasse, which is a great source of lignocellulosic residue, is pro‐ duced as a by-product of the industrial process. The sugarcane bagasse can be used as a lower-value substrate to produce the so-called second generation ethanol, in other words the ethanol generated from lignocellulosic material. The conversion of lignocellulose to ethanol requires challenging biological processes that includes: (i) delignification in order to release free cellulose and hemicellulose from the lignocellulosic material; (ii) depolyme‐ rization of the carbohydrates polymers from the cellulose and hemicellulose to generate free sugars; and (iii) fermentation of mixed hexose and pentose sugars to finally produce ethanol [25]. Glucose presents approximately 60% of the total sugars available in cellulosic biomass. The yeast *Saccharomices cerevisiae* is the most important microorganism able to ferment glucose (hexose), generating ethanol [26]. However, the presence of pentose sug‐ ars such as xylose and arabinose represents a challenge for the fermentation of these sug‐ ars in lignocellulosic biomass, once *S. cerevisiae* is not able to efficiently ferment C5 sugars. The naturally occurring microorganisms able to ferment C5 sugars include *Pichia stipitis*, *Candida shehatae*, and *Pachysolen tannophilus* [27]. From these microorganisms, the yeast *P. stipitis* has the highest ability to perform xylose fermentation, producing ethanol under low aeration rates. It appears that ethanol yields and productivity from xylose fermenta‐ tion by *P. stipitis* are significantly lower than glucose fermentation by *S. cerevisiae* [28]. Therefore, genetic improvement of yeasts is a valuable tool to obtain strains able to fer‐ ment pentoses, hexoses and, in addition, produce ethanol with a high yield and a high ethanol tolerance as well. Genetically engineered organisms with C5 fermenting capabili‐ ties already include *S. cerevisiae*, *Escherichia coli*, *Zymomonas mobilis* and *Candida utilis* [28-31]. Studies on fungi degradation of lignocellulosic material could yield promising candidate genes that could be subsequently used in engineering strategies for improved cellulosic biofuel production in these yeast strains.

apparatus of anaerobic bacteria is frequently assembled into a large multienzyme com‐ plex, named cellulosomes [14, 15]. This complex contains enzymes with a variety of ac‐ tivities such as polysaccharide lyases, carbohydrate esterases and glycoside hydrolases [16-18]. Basically, the catalytic components of the cellulosomes include a structure named dockerins, which are noncatalytic modules that bind to cohesin modules, located in a large noncatalytic protein acting as scaffold [15]. The protein-protein interaction between dockerins and cohesins allows the integration of the hydrolytic enzymes into the com‐ plex [19, 20]. It has been demostrated that scaffoldins are also responsible for the anchor‐ ing of the whole complex onto crystalline cellulose, through a noncatalytic carbohydratebinding module (CBM) [21]. The main studies concerning cellulosomes are being focused on anaerobic bacteria, especially from *Clostridium* species, but a range of other anaerobic bacteria and fungi were shown to produce cellulosomal systems. These include anaerobic bacteria such as *Acetivibrio cellulolyticus*, *Bacteroides cellulosolvens*, *Ruminococcus albus*, *Ru‐ minococcus flavefaciens*, and the anaerobic fungi of the genera *Neocalimastix*, *Pyromices* and *Orpinomyces* [14, 15]. Cellulosome-based complexes design and construction is a promis‐ ing approach for the improvement of hydrolytic activity systems. Cellulosomes able to integrate fungal and bacterial enzymes from nonaggregating systems could be generated to increase hydrolytic activities and consequently the biomass saccharification [22]. In ad‐ dition, genetic manipulations could be used in order to introduce genes responsible for the synthesis of cellulosome into microorganisms able to ferment simple sugars but that do not have a functional plant cell wall-degrading machinery [23]. Alternatively, micro‐ organisms naturally synthesizing cellulosomes could be engineered to increase their ca‐ pacity to produce ethanol from lignocellulose [15]. Recently, using the architeture of cellulosomes as template, self-assembling protein complexes were successfully designed and constructed. These protein complexes were termed xylanosomes, and were designed specifically for hemicellulose hydrolysis, but demonstrated synergy with cellulases, sug‐

212 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

gesting a possible use of these nanostructures in cellulose hydrolysis as well [24].

Fermentative production of ethanol is largely performed nowadays through the use of starch or sucrose provided by agricultural crops such as wheat, corn or sugarcane. In Bra‐ zil, for instance, the ethanol production through yeast fermentation of substrates from sugarcane is a well-known and consolidated process. However, the improvement of fer‐ mentative processes towards utilization of lower-value substrates such as lignocellulosic residues is emerging as a valuable approach for reducing the production cost and conse‐ quently increasing the use of ethanol as biofuel. In sugarcane mills, for instance, a large quantity of sugarcane bagasse, which is a great source of lignocellulosic residue, is pro‐ duced as a by-product of the industrial process. The sugarcane bagasse can be used as a lower-value substrate to produce the so-called second generation ethanol, in other words the ethanol generated from lignocellulosic material. The conversion of lignocellulose to

**4. Ethanol production from the fermentable feedstock from**

**lignocellulosic biomass**

In summary, many microorganisms are able to produce and secrete hemicellulolytic en‐ zymes, but fungi are pointed as the most important microorganisms concerning the biomass degradation. The significance of secreted enzymes in the life of these organisms and the bio‐ technological importance of filamentous fungi and their enzymes prompted an interest to‐ wards understanding the mechanisms of expression and regulation of the extracellular enzymes, as well as the characterization of the transcription factors involved. The next sec‐ tions of this chapter will discuss the fungal enzyme sets for lignocellulosic degradation and the gene expression regulation of these enzymes.
