The microbial cellulolytic enzymes (cellulases) can overcome the disadvantages of acid hydrolysis of cellulose. Being specific biological catalysts, secondary products of degradation are not formed and working under milder conditions (temperatures up to 60°C, pH of 2.5 to 5.5), exigencies to the enzymatic bioreac­tors are not very strict. Nevertheless, the reaction times are more prolonged than in the case of acid hydrolysis.

A significant number of microorganisms have the ability to biosynthesize cel — lulases. In general, the anaerobic bacteria degrade the cellulose through high — molecular-weight complex systems with cellulolytic activity named cellulosomes, as in the case of Clostridium thermocellum. The cellulosomes are extracellular structures consisting of spherical polypeptidic complexes that include an enzy­matic package with cellulase activity, which group together the enzymes through the action of one polypeptide with no hydrolytic activity involved in the adhesion of cellulosomes to the substrate. The cellulosomes increase the magnitude of the enzymatic action on the plant particles rich in cellulose due to the close contact of the bacterium with its enzymes and such particles (Fondevila, 1998). C. ther — mocellum cellulosomes are distributed not only in the culture medium, but also on the surface of bacterial cells, though several bacterial strains do not release appreciable amounts of these cellulolytic complexes to the medium. In this case, the cellulosomes are concentrated in the cell wall surface or inside its structure. These complexes have a high effectiveness in the degradation of crystalline cel­lulose (Lynd et al., 2002). In particular, anaerobic thermophilic bacteria exhibit high growth rates on cellulose and have enzymes with a high stability. Moreover, their culture requires less agitation energy. These bacteria could be potential can­didates for the direct conversion of cellulose to ethanol (Lee, 1997).

In contrast, aerobic cellulose-degrading microorganisms, including bacteria and fungi, break down this polysaccharide through the production of significant amounts of individual extracellular cellulases, although some enzymatic com­plexes can be occasionally found on the cell surface. Due to their individual nature, these cellulases exhibit a synergic action on the cellulose (Lynd et al., 2002). At an industrial level, a great variety of cellulases are produced and employed in the formulation of detergents, in the textile and food industries, and during the pro­duction of paper pulp and paper (Bhat, 2000). Most of the commercial cellulases are obtained from Trichoderma reesei, though a small portion is obtained from A. niger. Several reports can be found on the features of cellulase aerobic produc­tion by T. reesei. See, for example, the work of Marten et al. (1996). T. reesei releases a mixture of cellulases, among which at least two cellobiohydrolases, five endoglucanases, P-glucosidases, and hemicellulases can be found. The two cellobiohydrolases and one of the endoglucanases represent approximately 92% of total production of cellulases (Zhang and Lynd, 2004). Cellobiohydrolases break down P(1,4) linkages from nonreducing or reducing ends of the cellulose chain releasing cellobiose or even glucose, whereas endoglucanases hydrolyze these same linkages randomly inside the chain. The action of cellobiohydrolases causes a gradual decrease in the polymerization degree, while endoglucanases cause the rupture of cellulose into smaller chains reducing rapidly the polymer­ization degree. Endoglucanases especially act on amorphous cellulose, whereas cellobiohydrolases are capable of acting on crystalline cellulose as well (Lynd et al., 2002), as illustrated in Figure 5.1.

Although T. reesei produces some P-glucosidases, which is the result of hydrolyzing formed cellobiose into two molecules of glucose, their activities are not very high. Unfortunately, cellobiohydrolases are inhibited by cellobiose. Therefore, P-glucosidase from other sources needs to be added in order to com­plement the action of the cellulases from this fungus (Sanchez and Cardona, 2008). Dekker and Wallis (1983) have determined a ratio of FPU (filter paper

FIGURE 5.1 Schematic representation of cellulose hydrolysis using individual cel­lulolytic enzymes.

units) to P-glucosidase units of enzymatic activity as the minimum required to achieve more than 80% conversion of cellulose into glucose. The combined action of these enzymes is synergic leading to the conversion of cellulose into glucose (Beguin and Aubert, 1994; Walker et al. 1993). Factorial optimization techniques have been applied to the design of mixtures of cellulases from different sources along with P-glucosidase in order to maximize the yield of glucose produced (Kim et al., 1998). It has been suggested to develop a multicellulase plasmid in which the different cellulase genes could be expressed to produce cellulases with an optimum ratio from a single cultivation.

Cellulases should be adsorbed onto the surface of substrate particles before hydrolysis of insoluble cellulose takes place. The three-dimensional structure of

these particles in combination with their size and shape determines if P-glycosidic bonds are or are not accessible to enzymatic attack (Zhang and Lynd, 2004). This makes the hydrolysis process slower in relation to the enzymatic degradation of other biopolymers. For comparison, the hydrolysis rate of starch by amylases is 100 times faster than the hydrolysis rate of cellulose by cellulases under indus­trial processing conditions. Apparently, this difference in hydrolysis rates can be explained to a greater extent by the higher accessibility of the enzymes to the substrate, which is more limited in the case of the cellulose, than by the fact that the P(1,4) bond of cellulose is more difficult to hydrolyze than the a(1,4) bond of starch. In the case of the pretreated lignocellulosic complex, the cellulases can bind in a reversible way not only to the cellulose particles, but also to the lignin that reduces their effectiveness. In addition, the cellulases can bind in an irreversible way to the substrate provoking a progressive loss of enzymatic activity. It has been postulated that the addition of surfactants to the reaction mixture can improve the effectiveness of enzymatic cellulose hydrolysis due to the reduction of enzyme loss through irreversible binding to the substrate. The surfactant increases the rate of hydrolysis as well as prolongs the enzyme life. This allows the reduction of enzyme dosage to 50%. It has been reported that the usage of Tween-80 improved sugar production for any given particle size of cellulose when milled newsprint was used as a feedstock. Similarly, the use of sophorolipid increases by 67% the hydrolysis of steam-exploded wood (Duff et al., 1995; Helle et al., 1993).

One nonconventional approach for saccharification that demonstrates the diversity of trends for research and development of cellulose hydrolysis is the enzymatic hydrolysis in biphasic media, by which higher glucose concentrations can be attained. The goal of replacing part of the water with organic substances is explained by the need of ensuring the necessary rheological properties to accom­plish saccharification using higher substrate loads taking into account that glu­cose does not migrate to the organic phase. Cantarella et al. (2001) employed this approach for saccharification of steam-exploded wheat straw employing a medium with 25% (by volume) aqueous phase and 75% organic phase (acetates). Higher glucose concentrations (measured in the aqueous phase) were obtained compared to the case when only the aqueous phase was used. These concentrations reached about 150 g/L. However, when the aqueous phase was fermented using S. cer — evisiae, an increase in the lag-phase and a small reduction in ethanol yield were observed (nearly 86 to 92% of the theoretical maximum). Another approach to cellulose hydrolysis consists in the design of hydrolyzing agents, which are dif­ferent from mineral acids (that degrade the formed glucose) or enzymes. Mosier et al. (2002) have systematically studied several organic acids in order to assess their cellulose hydrolysis and glucose degradation characteristics in comparison to sulfuric acid. Their results indicate that maleic acid presented the best char­acteristics. In particular, this acid does not degrade the formed glucose. These studies are aimed at developing a nonprotein catalyst that mimics the action of the cellulases. In this way, maleic acid is a suitable catalytic domain for the synthesis of such enzymatic mimicry.