**5. Cellulose allomorphs**

Two different ending groups are found in each cellulose chain edge. At one end of each of the chains, a non-reducing group is present where a closed ring structure is found. A reduc‐ ing group with both an aliphatic structure and a carbonyl group is found at the other end of the chains. The cellulose chain is thus a polarized molecule and the new glucose residues are

A wide variety of Gram-positive and Gram-negative bacterial species are reported to pro‐ duce cellulose, including *Clostridium thermocellum, Streptomyces* spp*., Ruminococcus* spp., *Pseudomonas* spp*., Cellulomonas* spp*., Bacillus* spp*., Serratia, Proteus, Staphylococcus* spp*.,* and

**Figure 2.** Crystalline and amorphous structure of cellulose. The crystalline structure is conserved by hydrogen bonds and Van der Waals forces, in amorphous structure exists twists and torsions that alter the ordered arrangement. Re‐

In plants, cellulose is synthesized by CESA proteins (Cellulose Synthase) embedded in plas‐

Cellulose crystallites are thought to be imperfect, the traditional two-phase cellulose model describes cellulose chains as containing both crystalline (ordered) and amorphous (less or‐ dered) regions. Crystalline structure of cellulose implies a structural arrangement in which all atoms are fixed in discrete position with respect to one another. An important feature of the crystalline array is that the component molecules of individual microfibrils are packed sufficiently tightly to prevent penetration not only by enzymes, but even by small molecules such as water. While its recalcitrance to enzymatic degradation may pose problems, one big

Highly ordered, crystalline regions are interspersed with regions containing disorganized or amorphous cellulose, which constitute 5 to 20% of the microfibril. Many studies have shown that completely disordered or amorphous cellulose is hydrolysed at a much faster rate than partially crystalline cellulose; this fact supports the idea that the initial degree of crystallinity

matic membrane arranged in hexameric groups called rosettes particles [25].

added at the non-reducing end allowing chain elongation (Figure 2) [23].

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

*Bacillus subtilis* [24].

ducing and non-reducing are shown.

**4. Cellulose crystallinity**

advantage of cellulose is its homogeneity [1, 26-27].

The crystalline structure of cellulose has been studied since its discovery in the 19th century, its structure was first established by Carl von Nageli in 1858, and the result was later veri‐ fied by X-ray crystallography [34-35].

In the past decades, many data on the polymorphism of cellulose were analysed, being the most reliable data published after 1984, when the results of NMR spectroscopic studies of cellulose were reported [36].

The repeating unit of the cellulose macromolecule includes six hydroxy groups and three oxygen atoms. Therefore, the presence of six hydrogen bond donors and nine hydrogen bond acceptors provides several possibilities for forming hydrogen bonds. Due to different arrangements of the pyranose rings and the possible conformational changes of the hydrox‐ ymethyl groups, cellulose chains can exhibit different crystal packings [37].

zation and subsequent recrystallization) [47]. Cellulose II, like cellulose Iβ, has the monoclin‐ ic unit cell (space group P21). The different arrangement of the chains (parallel in cellulose I<sup>β</sup> and antiparallel in cellulose II) is the most substantial difference between these two poly‐ morphs. The cellulose is a highly rigid macromolecule due to the presence of a three-dimen‐ sional hydrogen bond network in addition to the C-O-C bonds between the glucopyranose rings. In the absence of such hydrogen bond networks the chains are much more flexible. These hydrogen bonds are responsible for both the poor solubility of cellulose and the dif‐ ference in the reactivity of the hydroxy groups in esterification reactions (Figure 4) [37].

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**Figure 3.** Differences between the monoclinic and triclinic forms of cellulose I. a) In the monoclinic form, cellobiose units stagger with a shift of a quarter of the c-axis period (0.26 nm), whereas the triclinic form exhibits a diagonal shift of the same amount. The angles shown depend on which crystallographic face is being viewed. A glucose unit is rep‐ resented by rectangles (cellobiose, a dimer of glucose); image reproduced with publisher´s permission [23]. b) Mode of packing in the unit cell of cellulose I: mono and triclinic unit cell. Notice that the monoclinic angle γ is obtuse. Image

ammonia; in a reversible reaction. Besides producing the different allomorphs of cellulose, this chemical treatment can also alter other physical properties of cellulose, such as the de‐ gree of crystallinity and therefore enhanced cellulase accessibility and chemical reactivity. The degree of conversion of cellulose I to cellulose IIII depends on the reaction period and

In [45] solved the crystal structure of cellulose IIII by synchrotron X-ray and neutron fiber diffraction analyses, and showed that it has a lower packing density than cellulose Iα or I<sup>β</sup>

*Cellulose IV* can be most easily prepared by heating cellulose III, and therefore, two poly‐ morphs of it also exist -celluloses IVI and IVll obtained respectively, from celluloses IIII and IIIII. In general, cellulose IV could be prepared by treatment in glycerol at 260 °C after trans‐ formation into cellulose II or III. Cellulose I cannot be transformed directly into cellulose IV

Fibrillation makes cellulose IVI less suitable for crystallographic analysis: that is, it makes it more difficult to interpret cellulose IVI as a crystal. For these reasons, it is unclear whether is a crystal with an orthogonal unit cell or a less crystalline form of cellulose I [49]. A thorough

review of cellulose crystalline allomorphs can be found elsewhere [46-47].

and *IIIII* can be formed from cellulose I and II, respectively, by treatment with

reproduced with permission from PNAS Copyright (2012).

the temperature used in the final stage of the treatment [47-48].

*Cellulose IIII*

(Figure 4).

[46, 49].

Four different crystalline allomorphs of cellulose have been identified by their characteristic X-ray diffraction patterns and solid-state 13C nuclear magnetic resonance (NMR) spectra: cel‐ luloses I, II, III (IIII, IIIII) and IV (IVI, IVII). The most important allomorphs are cellulose I and II [22].

Some difference in symmetry and chain geometry have been found in unit cell dimensions of various allomorphs and some parameters have been established: a, interchain distance, b unit chain length and c, intersheet distance, as well as the angles α, β and γ which are the angles between b and c, a and c, and a and b, respectively, (Table 3) [38-40].


I, Fresh water algae *Glaucocystis nostochinearum;* Iβ*,* Tunicate *Halocynthia roretzi;* II, Ramie cellulose (mercerized); II, Regenerated cellulose (Fortisan); IIII , Marine algae *Cladophora.* All crystal structures have been determined at 293ºK, except allomorph II (Fortisan) that was also determined at 100ºK (italics).

**Table 3.** Unit cell parameters of different cellulose allomorphs obtained by X-ray diffractions.

*Cellulose I* is the most abundant form found in nature, is a mixture of two distinct crystalline forms: cellulose Iα, the predominant form isolated from bacteria (*Acetobacter xylinum*) and fresh water algae (*Glaucosystis nostochinearum*); and cellulose Iβ is the major form in higher plants such as cotton and wood celluloses, ramie and animal celluloses, for example in the edible ascidian *Halocynthia roretzi* [4]. Cellulose from the marine algae *Claudophora* sp. and *Valonia ventricosa* is a mixture of both forms, predominating I<sup>α</sup> [37]. Currently, cellulose I is receiving increased attention due to its potential use in bioenergy production.

*Cellulose Iα* has a triclinic one-chain unit cell where parallel cellulose chains stack through van der Waals interactions, with progressive shear parallel to the chain axis. *Cellulose Iβ* has a monoclinic two-chain unit cell, which means parallel cellulose chains stacked with alternat‐ ing shear (Figure 3) [46].

*Cellulose II* is the most crystalline thermodynamic stable form, it can also be obtained from cellulose I by two distinct routes: mercerization (alkali treatment) and regeneration (solubili‐ zation and subsequent recrystallization) [47]. Cellulose II, like cellulose Iβ, has the monoclin‐ ic unit cell (space group P21). The different arrangement of the chains (parallel in cellulose I<sup>β</sup> and antiparallel in cellulose II) is the most substantial difference between these two poly‐ morphs. The cellulose is a highly rigid macromolecule due to the presence of a three-dimen‐ sional hydrogen bond network in addition to the C-O-C bonds between the glucopyranose rings. In the absence of such hydrogen bond networks the chains are much more flexible. These hydrogen bonds are responsible for both the poor solubility of cellulose and the dif‐ ference in the reactivity of the hydroxy groups in esterification reactions (Figure 4) [37].

arrangements of the pyranose rings and the possible conformational changes of the hydrox‐

Four different crystalline allomorphs of cellulose have been identified by their characteristic X-ray diffraction patterns and solid-state 13C nuclear magnetic resonance (NMR) spectra: cel‐ luloses I, II, III (IIII, IIIII) and IV (IVI, IVII). The most important allomorphs are cellulose I and

Some difference in symmetry and chain geometry have been found in unit cell dimensions of various allomorphs and some parameters have been established: a, interchain distance, b unit chain length and c, intersheet distance, as well as the angles α, β and γ which are the

**Unit cell parameters**

*a b c* **Α β γ** I 6.717(7) 5.962(6) 10.400(6) 118.08(5) 114.80(5) 80.37(5) (41) I<sup>β</sup> 7.784(8) 8.201(8) 10.380(10) 90 90 96.5 (42) II 8.10(1) 9.03(1) 10.31(1) 90 90 117.10(5) (43)

IIII 4.450(4) 7.850(8) 10.310(10) 90 90 105.10(5) (45)

I, Fresh water algae *Glaucocystis nostochinearum;* Iβ*,* Tunicate *Halocynthia roretzi;* II, Ramie cellulose (mercerized); II,

*Cellulose I* is the most abundant form found in nature, is a mixture of two distinct crystalline forms: cellulose Iα, the predominant form isolated from bacteria (*Acetobacter xylinum*) and fresh water algae (*Glaucosystis nostochinearum*); and cellulose Iβ is the major form in higher plants such as cotton and wood celluloses, ramie and animal celluloses, for example in the edible ascidian *Halocynthia roretzi* [4]. Cellulose from the marine algae *Claudophora* sp. and *Valonia ventricosa* is a mixture of both forms, predominating I<sup>α</sup> [37]. Currently, cellulose I is

*Cellulose Iα* has a triclinic one-chain unit cell where parallel cellulose chains stack through van der Waals interactions, with progressive shear parallel to the chain axis. *Cellulose Iβ* has a monoclinic two-chain unit cell, which means parallel cellulose chains stacked with alternat‐

*Cellulose II* is the most crystalline thermodynamic stable form, it can also be obtained from cellulose I by two distinct routes: mercerization (alkali treatment) and regeneration (solubili‐

10.35(1) *10.34(1)*

**Bond lengths (Å) Angles (º) Ref.**

90 *90*

, Marine algae *Cladophora.* All crystal structures have been determined at 293ºK,

90 *90* 117.11(2) *117.11(2)* (44)

ymethyl groups, cellulose chains can exhibit different crystal packings [37].

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

angles between b and c, a and c, and a and b, respectively, (Table 3) [38-40].

II [22].

**Allo morph**

II

8.03(1) *8.03(1)(*

Regenerated cellulose (Fortisan); IIII

ing shear (Figure 3) [46].

9.04(1) *9.02(1)*

except allomorph II (Fortisan) that was also determined at 100ºK (italics).

**Table 3.** Unit cell parameters of different cellulose allomorphs obtained by X-ray diffractions.

receiving increased attention due to its potential use in bioenergy production.

**Figure 3.** Differences between the monoclinic and triclinic forms of cellulose I. a) In the monoclinic form, cellobiose units stagger with a shift of a quarter of the c-axis period (0.26 nm), whereas the triclinic form exhibits a diagonal shift of the same amount. The angles shown depend on which crystallographic face is being viewed. A glucose unit is rep‐ resented by rectangles (cellobiose, a dimer of glucose); image reproduced with publisher´s permission [23]. b) Mode of packing in the unit cell of cellulose I: mono and triclinic unit cell. Notice that the monoclinic angle γ is obtuse. Image reproduced with permission from PNAS Copyright (2012).

*Cellulose IIII* and *IIIII* can be formed from cellulose I and II, respectively, by treatment with ammonia; in a reversible reaction. Besides producing the different allomorphs of cellulose, this chemical treatment can also alter other physical properties of cellulose, such as the de‐ gree of crystallinity and therefore enhanced cellulase accessibility and chemical reactivity. The degree of conversion of cellulose I to cellulose IIII depends on the reaction period and the temperature used in the final stage of the treatment [47-48].

In [45] solved the crystal structure of cellulose IIII by synchrotron X-ray and neutron fiber diffraction analyses, and showed that it has a lower packing density than cellulose Iα or I<sup>β</sup> (Figure 4).

*Cellulose IV* can be most easily prepared by heating cellulose III, and therefore, two poly‐ morphs of it also exist -celluloses IVI and IVll obtained respectively, from celluloses IIII and IIIII. In general, cellulose IV could be prepared by treatment in glycerol at 260 °C after trans‐ formation into cellulose II or III. Cellulose I cannot be transformed directly into cellulose IV [46, 49].

Fibrillation makes cellulose IVI less suitable for crystallographic analysis: that is, it makes it more difficult to interpret cellulose IVI as a crystal. For these reasons, it is unclear whether is a crystal with an orthogonal unit cell or a less crystalline form of cellulose I [49]. A thorough review of cellulose crystalline allomorphs can be found elsewhere [46-47].

Although considerable progress has been made in elucidating the crystal structures of cellu‐ lose in microfibrils, they are still not well understood, and a deeper understanding of cellu‐ lose structure is required [50-51].

Aerobic bacteria with cellulolytic activities of the order *Actinomycetales* (phylum *Actinobacte‐ ria*) have been found on soils, water, humus, agricultural waste (sugar cane) and decaying leaves, these bacteria excretes enzymes capable of degrading cellulose (cellulases) [52]. In aerobic bacteria *Pseudomonas fluorescens* subsp. cellulosa, *Streptomyces lividans* and *Cellulomo‐*

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Some anaerobic bacteria with cellulolytic activity are Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Ruminococcus flavefaciens, Clostridium cellulovorans, C. cellulolyticum and

Due to the significant diversity in the physiology of cellulolytic bacteria, sometimes is diffi‐ cult to classify bacteria as mentioned above, therefore, on this basis, they can be placed into three diverse physiological groups: (1) fermentative anaerobes, typically Gram-positive, (*Clostridium* and *Ruminococcus)*, but with a few Gram-negative species (*Butyvibrio* and *Aceti‐ vibrio*) that are phylogenetically related to the *Clostridium* assemblage (*Fibrobacter*); (2) aero‐ bic Gram-positive bacteria (*Cellulomonas* and *Thermobifida)* and (3) aerobic gliding bacteria,

The ability to utilize lignocellulosic material is widely distributed among fungi, from chytri‐ diomycetes to basidiomycetes. Among fungi, the most efficient at using wood as substrate are the basidiomycetes, considered the principal taxonomic group involved in the aerobic degradation of wood with all its components, they are the main organic material decompo‐ sition agents. These aerobic fungi produce extracellular enzymes allowing lignocellulose degradation (lacasses, hemicellulases, and cellulases), although some Ascomycetes are able to degrade cellulosic compounds as well. Unlike aerobic fungi, some of the Chytridiomy‐ cetes anaerobic fungi, have multienzymatic complexes similar to cellulosomes of bacteria [1, 3, 62-63] some members are anaerobic species living in the gastrointestinal tract of rumi‐

Examining the taxonomic composition of cellulolytic fungi inhabiting the decaying leaves and rotting woods of forest soils, zygomycetes are represented by a single genus, *Mucor,* while ascomycetes and basidiomycetes are represented by genera such as *Chaetomium, Tri‐ choderma, Aspergillus, Penicillium*, *Fusarium, Coriolus, Phanerochaete, Schizophyllum, Volvariella, Pycnoporus* and *Bjerkandera.* Two of the most studied fungi, due to their industrial relevance,

Nowadays, more than 14,000 fungi, which are active against cellulose and other insoluble fibres, are known [1, 24, 64-66]. A more detailed list of cellulose degrading bacteria and fun‐

Selective pressure of evolution is the force driving microorganisms to adapt a new environ‐ ment, in anaerobic conditions is necessary a machinery for the extracellular degradation of substrates, such as the recalcitrant crystalline components of the plant cell wall. Due to this, the anaerobes tend to adopt different strategies for degrading plant components, being the

nants such as *Anaeromyces, Caecomyces, Neocallimastix, Orpinomyces* and *Piromyces.*

are *Trichoderma reesei* and *Phanerochaete chrysosporium*.

cellulosomes the most remarkable feature.

**7. Cellulose degradation mediated by cellulosome**

*nas fimi* cellulolytic systems of degradation have been reported [56-58].

C. thermocellum [59-61].

gi is listed in Table 5.

(*Cytophaga* and *Sporocytophaga)* [1, 53].

**Figure 4.** Projections of the crystal structures of cellulose I (α,and β) II and III down the chain axes directions. C, O, and H atoms are represented as gray, red, and white balls, respectively. Covalent and hydrogen bonds are represented as full and dashed sticks, respectively. H atoms involved in hydrogen bonding are explicitly represented for only cellulose IIII. Only the major components of hydrogen bonds are represented. Adapted with permission from [45]. Copyright (2012) American Chemical Society.
