1.1 Cellulolytic capability of organisms: Difference in the cellulose-degrading strategy

Different strategies for the cellulose degradation are used by the cellulase-producing microorganisms: aerobic bacteria and fungi secrete soluble extracellular enzymes known as non complexed cellulase system; anaerobic cellulolytic microorganisms produce complexed cellulase systems, called cellulosomes (Sun et al., 2002). A third strategy was proposed to explain the cellulose-degrading action of two recently discovered bacteria: the aerobic Cytophaga hutchinsonii and the anaerobic Fibrobacter succinogenes (Ilmen et al., 1997).

• Non-complexed cellulase system. One of the most fully investigated non-complexed cellulase system is the Trichoderma reesei model. T. reesei (teleomorph Hypocrea jecorina) is a saprobic fungus, known as an efficient producer of extracellular enzymes (Bayer et al., 1998). Its non-complexed cellulase system includes two cellobiohydrolases, at least seven endoglucanases, and several P-glucosidases. However, in T. reesei cellulases, the amount of fi-glucosidase is lower than that needed for the efficient hydrolysis of cellulose into glucose. As a result, the major product of hydrolysis is cellobiose. This is a dimer of glucose with strong inhibition toward endo — and exoglucanases so that the accumulation of cellobiose significantly slows down the hydrolysis process (Gilkes et al., 1991). By adding fi-glucosidase to cellulases from either external sources, or by using co-culture systems, the inhibitory effect of cellobiose can be significantly reduced (Ting et al., 2009).

It has been observed that the mechanism of cellulose enzymatic hydrolysis by T. reesei involves three simultaneous processes (Ting et al., 2009):

1. Chemical and physical changes in the cellulose solid phase. The chemical stage includes changes in the degree of polymerization, while the physical changes regard all the modifications in the accessible surface area. The enzymes specific function involved in this step is the endoglucanase.

2. Primary hydrolysis. This process is slow and involves the release of soluble intermediates from the cellulose surface. The activity involved in this step is the cellobiohydrolase.

3. Secondary hydrolysis. This process involves the further hydrolysis of the soluble fractions to lower molecular weight intermediates, and ultimately to glucose. This step is much faster than the primary hydrolysis and ft-glucosidases play a role for the secondary hydrolysis.

• Complexed cellulase system. Cellulosomes are produced mainly by anaerobic bacteria, but their presence have also been described in a few anaerobic fungi from species such as Neocallimastix, Piromyces, and Orpinomyces (Tatsumi et al., 2006; Watanabe & Tokuda, 2010). In the domain Bacteria, organisms possessing cellulosomes are only found in the phylum Firmicutes, class Clostridia, order Clostridiales and in the Lachnospiraceae and Clostridiaceae families. In this latter family, bacteria with cellulosomes are found in various clusters of the genus Clostridium (McCarter & Whiters, 1994; Wilson, 2008). Cellulosomes are protuberances produced on the cell wall of the cellulolytic bacteria grown on cellulosic materials. These protuberances are stable enzyme complexes tightly bound to the bacteria cell wall but flexible enough to bind strongly to cellulose (Lentig & Warmoeskerken, 2001). A cellulosome contains two types of subunits: non-catalytic subunits, called scaffoldins, and enzymatic subunits. The scaffoldin is a functional unit of cellusome, which contain multiple copies of cohesins that interact selectively with domains of the enzymatic subunits, CBD (cellulose binding domains) and CBM (carbohydrates binding modules). These have complementary cohesins, called dockerins, which are specific for each bacterial species (Fig. 4) (Gilligan & Reese, 1954; Lynd et al., 2002; Arai et al., 2006;).

For the bacterial cell, the biosynthesis of a cellulosome enables a specific adhesion to the substrate of interest without competition with other microorganisms. The cellulosome allows several advantages: (1) synergism of the cellulases; (2) absence of unspecific adsorption (McCarter & Whiters, 1994; Zhang & Lynd, 2004). Thanks to its intrinsic Lego-like architecture, cellulosomes may provide great potential in the biofuel industry.

The concept of cellulosome was firstly discovered in the thermophilic cellulolytic and anaerobic bacterium, Clostridium thermocellum (Wyman, 1996). It consists of a large number of proteins, including several cellulases and hemicellulases. Other enzymes that can be included in the cellulosome are lichenases.

• Third cellulose-degrading strategy. The third strategy was recently proposed to explain the

cellulose-degrading behavior of two recently sequenced bacteria: Cytophaga hutchinsonii and Fibrobacter succinogenes (Ilmen, 1997). C. hutchinsonii is an abundant aerobic cellulolytic soil bacterium (Fagerstam & Petterson, 1984), while F. succinogenes is an anaerobic rumen bacterium which was isolated by the Rockville, (Maryland), and San

Diego (California) Institute of Genomic Research (TIGR) (Mansfield et al., 1998). In the aerobic C. hutchinsonii no genes were found to code for CBM and in the anaerobic F. succinogenes no genes were identified to encode dockerin and scaffoldin. Thus, a third cellulose degrading mechanism was proposed. It includes the binding of individual cellulose molecules by outer membrane proteins of the microrganisms followed by the transport into the periplasmic space where they are degraded by endoglucanases (Ilmen, 1997).