**3. Microbial degradation of plant cell wall polysaccharides**

In order to survive, microorganisms developed, during the course of evolution, physiologi‐ cal mechanisms to cope with a variety of environmental factors. The acquirement of nu‐ trients represents a challenge for all living organisms, especially for microorganisms. Saprophytism, one of the most common lifestyle of microorganisms, involves living in dead or decaying organic matter, mainly composed by plant biomass. In this context, microorgan‐ isms developed cellular mechanisms in order to take energy from plant biomass, and one of this mechanisms involves the production and secretion of carbohydrate-active enzymes. These enzymes degrade the plant cell wall, releasing sugars monomers that can be used as substrates for the metabolism of the microorganism. The microbial use of plant biomass is pivotal for life on Earth, because it is responsible for large portions of carbon flux in the bio‐ sphere. In addition, plant cell wall-degrading enzymes (CWDEs) have a broad range of in‐ dustrial applications, such as within the food and feed industry and for sustainable production of many chemicals and fuels.

fers the complexity and rigidity of the plant cell wall, which is responsible for the protection of plant cell as a whole. In addition to offer protection against mechanical stress and osmotic lysis, the plant cell wall is an effective barrier against pathogens, including many microor‐ ganisms. However, during the course of evolution some microorganisms, in order to sur‐ vive, developed efficient strategies to degrade plant cell wall components, mainly the

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

Plant cell wall degradation mechanisms are pivotal for the lifestyle of many microorgan‐ isms, once they should be able to degrade the plant polymers to acquire nutrients from plants. For instance, saprophytic fungi inhabit dead organic materials like decaying wood and leaves. In order to take energy from these materials, these fungi need to produce en‐ zymes capable of degrading the majority of plant cell wall polysaccharides present in the bi‐ omass. The main mechanism through which fungi and other microorganisms degrade plant biomass consists of production and secretion of enzymes acting synergistically in the plant cell wall, releasing monomers that can be used by the microorganism as chemical energy. The next section will discuss mechanisms of cell wall degrading enzymes (CWDE) produc‐

tion by fungi, the most important producers of carbohydrate-active enzymes.

**3. Microbial degradation of plant cell wall polysaccharides**

In order to survive, microorganisms developed, during the course of evolution, physiologi‐ cal mechanisms to cope with a variety of environmental factors. The acquirement of nu‐

**Figure 2.** Schematic view of the hairy region of pectin.

polysaccharides [6].

The capacity to degrade lignocellulose is mainly distributed among fungi and bacteria. Cel‐ lulolytic bacteria can be found in different genus such as *Clostridium*, *Ruminococcus*, *Caldicel‐ lulosiruptor*, *Butyrivibrio, Acetivibrio*, *Cellulomonas, Erwinia, Thermobifida, Fibrobacter, Cytophaga,* and *Sporocytophaga*. Bacterial degradation of cellulolytic material is more restrict to biomass containing low amounts of lignin, once bacteria are poor producers of lignanas‐ es. Plant biomass produced in aquatic environment, containing little amounts of lignin, is typically degraded by bacteria, which are better adapted for an aquatic environment than fungi [7]. Cellulolytic bacteria can also be found in digestive tracts of herbivore animals [8]. Fungal cellulose utilization is distributed within the entire kingdom, from the protist-like *Chytridomycetes* to the advanced *Basidiomycetes*.

Concerning to lignin degradation, many white-rot basidiomycetes and some actinomycetes are able to produce lignin-degrading enzymes, especially peroxidases. For instance, *Phanero‐ chaete chrysosporium* and *Phlebia radiata* are well known producers of extracellular peroxidas‐ es [9], as well as *Coriolus tersicolor*, which was shown to produce the intracellular haem peroxidase upon the induction by phenolic compounds [10]. A white-rot basidiomycete, *Rigidoporous lignosus*, is known to secrete two oxidative enzymes, laccase and Mn peroxi‐ dase, responsible for solubilizing the lignin in a synergistic way [11].

The fungi *Hypocrea jecorina* (*Trichoderma reesei*) is the most important organism used in cellu‐ lase production [12, 13] and it has been the focus of cellulases research for over 50 years. Degradation of cellulose is performed by cellulases, a high specific class of enzymes able to degrade the cellulose glycosidic bonds. The filamentous fungi *Aspergillus niger* is known to produce a wide range of hemicellulose-degrading enzymes and it has been used for many industrial applications. As discussed above, hemicellulose is a complex class of polysacchar‐ ides composed by different units of sugars. In order to degrade hemicellulose, the organism should be able to produce a large set of enzymes (hemicellulases), acting in a synergistic way to hydrolyze such complex substrate. Therefore, the ascomycetes *T. reesei* and *A. niger* are considered the most important microorganisms for cellulase/hemicellulase production, and constitute the source of these enzymes for industrial applications, including the produc‐ tion of biofuels from plant biomass.

The plant cell wall-degrading machinery of aerobic and anaerobic microorganisms differs significantly, regarding to its macromolecular organization. The cellulase/hemicellulase 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‐ gesting a possible use of these nanostructures in cellulose hydrolysis as well [24].

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

Microbial Degradation of Lignocellulosic Biomass

http://dx.doi.org/10.5772/54325

213

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

Fungi play a central role in the degradation of plant biomass, producing an extensive ar‐ ray of carbohydrate-active enzymes responsible for polysaccharide degradation. The en‐ zyme sets for plant cell wall degradation differ between many fungal species, and our understanding about fungal diversity with respect to degradation of plant matter is es‐ sential for the improvement of new strains and the development of enzymatic cocktails

Carbohydrate-active enzymes are usually classified in different families, based on amino acid sequence of the related catalytic module. An extensive and detailed database present‐ ing these hydrolytic enzymes can be found at www.cazy.org (CAZymes, **C**arbohydrate-

cellulosic biofuel production in these yeast strains.

the gene expression regulation of these enzymes.

for industrial applications.

**5. Fungal enzyme sets for lignocellulosic degradation**
