Cellulose: the structure slowly unravels

ANTOINETTE C. O'SULLIVAN

Department of Chemistry and School of Agriculture and Forestry Sciences, University of Wales,

Bangor, Gwynedd, LL57 2UW, UK

Received 13 May 1996; accepted for publication 3 December 1996

This article attempts to bring together basic and complex information which has been gathered

on cellulose structure, principally that of native cellulose, over the last few decades. Even though

advances have been made in the ®eld of crystallography, powder crystallography cannot yield a

de®nitive cellulose structure and single crystal diffraction is not possible due to the lack of

suitable crystals. Knowledge obtained on the biosynthesis of native cellulose and on the

polymorphy of cellulose and its derivatives help our understanding of ultrastructure. Many

inconsistencies between early crystallographic studies of native cellulose have been clari®ed by

the discovery that two polymorphs (á and â) of cellulose I exist. Models of the possible

ultrastructural arrangements within native cellulose have been put forward over the decades;

with advancement in technology, computer simulations of small and large systems are being

created to test the viability of these ultrastructural models. It is hoped that this review will aid

in the understanding of the complexity and uncertainties that still exist in this subject.

KEYWORDS: structure, computer, model, polymorphy

INTRODUCTION

Over 150 years ago, Anselme Payen discovered and isolated cellulose from green plants.Several reviews have been published on cellulose research (Preston, 1975, 1986; Sarko,1987; Chanzy, 1990; Okamura, 1991). These state that this compound is the mostabundant material on the earth: it is the main constituent of plants, serving to maintaintheir structure, and is also present in bacteria, fungi, algae and even in animals.Crystallographic research, yielding varying results, has also been extensively consideredin the above reviews. Despite the degree to which cellulose has been investigated, itsstructural features have not been identi®ed with absolute clarity and new information isconstantly being discovered by employing technological advances alongside conventionalanalytical tools. This review attempts to combine the facts and hypotheses of pastdecades with new eveidence on the polymorphy of native cellulose and the synthesis ofmicro®brils.

Cellulose research has been instrumental in advancing many analytical methods suchas crystallography and microscopy. To gain an understanding of all facets of thiscomplicated structure, the techniques used have been varied and the resultantterminology is vast. This latter point has frequently led to confusion especially ontopics such as the direction of cellulose chains in unit cells and the distinction betweenamorphous and crystalline cellulose.

Computational chemistry is one analytical technique which has developed extensivelyover the last three decades, initially being used to decipher complex crystallographic

CELLULOSE (1997) 4, 173±207

0969±0239 # 1997 Blackie Academic & Professional

data and now analysing the feasibility of cellulose models from an energeticperspective.

POLYMORPHY OF CELLULOSE

The polymorphy of cellulose and its derivatives has been well documented. Sixpolymorphs of cellulose (I, II, III1, III11, IV1 and IV11) can be interconverted, as shown inFig. 1 (Marchessault and Sarko, 1967; Walton and Blackwell, 1973; Marchessault andSundararajan, 1983). More recently, evidence for two polymorphs of cellulose I has beenoffered (VanderHart and Atalla, 1984; Sugiyama et al., 1991a); that is to say what waspreviously thought to be one polymorph (I) has now been found to be a mixture of twopolymorphs (Iá and Iâ). Proof of the polymorphy of cellulose comes from nuclearmagnetic resonance (NMR), infrared and diffraction studies (Blackwell and Marches-sault, 1971; Blackwell, 1982). Cellulose I, or native cellulose, is the form found innature. Cellulose II, the second most extensively studied form, may be obtained fromcellulose I by either of two processes: a) regeneration, which is the solubilization ofcellulose I in a solvent followed by reprecipitation by dilution in water to give celluloseII, or b) mercerization, which is the process of swelling native ®bres in concentratedsodium hydroxide, to yield cellulose II on removal of the swelling agent. Celluloses III1

and III11 (Marrinan and Mann, 1956; Hayashi et al., 1975) are formed, in a reversibleprocess, from celluloses I and II, respectively, by treatment with liquid ammonia or someamines, and the subsequent evaporation of excess ammonia (Davis et al., 1943; Sarko et

al., 1976; Sarko, 1987). Polymorphs IV1 and IV11 (Hess and Kissig, 1941; Gardiner andSarko, 1985) may be prepared by heating celluloses III1 and III11 respectively, to 206 8C,in glycerol.

BIOSYNTHESIS OF CELLULOSE

Cellulose is a ®brous, tough, water-insoluble substance, which is found in the protectivecell walls of plants, particularly in stalks, stems, trunks and all woody portions of plant

RegenerationMercerization

Cellulose I

Cellulose IIII

heat

Cellulose IVI

Cellulose II

NH3(I)

2NH3(g)

NH3(I)

2NH3(g)

Cellulose IIIII

heat

Cellulose IVII

FIGURE 1. Interconversion of the polymorphs of cellulose.

174 O'SULLIVAN

tissues. Before attempting to study the ultrastructure of this macromolecule, it is prudentto understand its molecular structure.

An unbranched homopolysaccharide, cellulose is composed of â-D-glucopyranoseunits linked by (1! 4) glycosidic bonds (Purves, 1954; Marchessault and Sundararajan,1983). These pyranose rings have been found to be in the chair conformation 4C1, withthe hydroxyl groups in an equatorial position (Fig. 2). In nature, cellulose chains have adegree of polymerization (DP) of approximately 10 000 glucopyranose units in woodcellulose and 15 000 in native cotton cellulose (SjostroÈm, 1981). There is someevidence for a lower degree of polymerization in primary cell walls as compared withsecondary cell walls. However, chain lengths of such large, insoluble molecules arerather dif®cult to measure, due to enzymic and mechanical degradation which mayoccur during analysis.

Available evidence suggests that cellulose is formed at, or outside, the plasmamembrane (Lamport, 1970; Brett and Waldron, 1990). Groups or rosettes of particles,or terminal complexes (TCs), are seen in the plasma membrane when viewed by freeze-fracture techniques (Okuda et al., 1994). These groups of TCs can be seen to beassociated with the ends of micro®brils (collections of cellulose chains), and arethought to be cellulose synthase complexes, involved in the elongation of wholecellulose micro®brils. Thus, all the chains in one micro®bril would have to be elongatedby the complex at the same rate. This requirement means that the complex would needto be comprised of many subunits, each elongating a single chain at a time. Since thereare between 30 and 200 chains in the cross-section of one micro®bril, this processwould be one of remarkable complexity, but nevertheless research has favoured it(Macchi and Palma, 1969).

Crystalline cellulose I is not the most stable form of cellulose. It is unlikely to besynthesized by crystallization of pre-formed cellulose chains, since such a processcarried out in vitro gives rise to the thermodynamically more favourable cellulose II.Hence, the most probable process is one in which crystallization accompanies, orfollows very closely, addition of glucose residues to the chains by the TCs. Such aprocess would permit the geometry of the enzyme complex to govern the crystallineform of the micro®bril. Crystallization of cellulose is thought to be brought about by aprotein, the top protein, which accompanies the TC. The synthesis of straight

OHO

HO

HOO

O

CH2OH

HOCH2

O

OH

O

1z

FIGURE 2. Fragment (repeating unit) of a cellulose chain.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 175

micro®brils by TCs was corroborated by Sugiyama et al. (1994), who found thattriclinic (Iá) micro®brils are unidirectional.

Matsuda et al. (1992) recently showed that cellulose crystallization in immaturecotton boll is directly related to the formation of intermolecular hydrogen bonds. Thismay be due to the fact that the cellulose molecules are arranged into a micro®bril asthey crystallize. A further discovery was that the synthesis of the cellulose micro®brilsof the tunicate Metondrocarpa uedia, was linked to vacuole like structures (Kimur andItoh, 1995). Thus, the area of native cellulose biosynthesis continues to unfold.

X-RAY CRYSTALLOGRAPHY OF CELLULOSE I

Cellulose has been under continuous investigation since its discovery, due to its structuralcomplexity, and has often been among the ®rst substances to be studied by new methods(Sarko, 1987). Among the techniques which have been used on cellulose are Fouriertransform infrared spectroscopy (FT±IR) (Marchessault and Liang, 1962), CrossPolarisation=Magic Angle Spinning (CP=MAS) NMR (Earl and VanderHart, 1981; Horiiet al., 1982, 1987a, 1987b; Dudley et al., 1983; Atalla and VanderHart, 1989; Yamamotoand Horii, 1993) neutron (Ahmed et al., 1976) and electron (Herbert and Muller, 1974)diffraction, Raman spectroscopy (Atalla and VanderHart, 1989), scanning electronmicroscopy (SEM), (Fengel and Stoll, 1989) atomic force microscopy (AFM), (Hanley et

al., 1992; Kuutti et al., 1995) transmission electron microscopy (TEM), (Purz et al.,1995) scanning tunnelling microscopy (STM), (Frommer, 1992) and computer basedstereochemical modelling (see section: Computer analysis of cellulose).

In 1858, Carl von NaÈgeli (von NaÈgeli, 1858) established the crystallinity of cellulosein the ®rst serious use of the polarizing microscope. This result was veri®ed 80 yearslater with the aid of powder X-ray crystallography (Meyer and Misch, 1937).Elucidation of the crystal structure of cellulose began with Sponsler and Dore (1926),who suggested a single chain unit cell with dimensions: a � 0.61; b (®bre axis) �1.034; c � 0.54 nm; and â � 888. This model assumed an alternation of (1! 1) and(4! 4) links along the chain. A more acceptable unit cell was deduced by Meyer andMark (1928) on the basis of different crystallographic parameters (a � 0:835; b � 1:03;c � 0:79 nm; and â � 848) and improved in a later model by Meyer and Misch (1937).The latter two models had a two chain unit cell, with uniform (1! 4) linkagesbetween successive residues, in agreement with chemical evidence published byHaworth (1925). Meyer and Misch explained the fact that the unit cell parameter alongthe ®bre axis direction is shorter than the fully extended length of a cellobiose residueby proposing a model with a kink, resulting from the formation of a hydrogen bondbetween the C(3) of one glucose residue and the ring oxygen of the next (Preston,1986).

It should be noted at this point that the convention for quoting unit cell parametershave been revised since the ®rst crystallography work on cellulose was carried out, andnow the ®bre axis of a cellulose unit cell is denoted by c and the unique angle isdenoted by ã. Furthermore, in early work the unique angle of the cellulose unit cell waswritten as an acute angle but in modern crystallography an obtuse angle is used. For thepurpose of clarity the modern conventions will be used throughout the remainder of thisliterature review.

176 O'SULLIVAN

Unit cell parameters for crystalline cellulose I are listed in Table 1. Variationsbetween the results of different research groups may be explained by several factors:

(1) Technical advances improved the ability to measure the parameters.(2) Parameters depend on the source of the cellulose and it has been suggested that

this may be linked to taxonomy (Okano et al., 1989).(3) Single-crystal diffraction is the only method that provides bond lengths, bond

angles and characteristic geometry. It is the ultimate structural method. On theother hand, polymer X-ray studies, as in the case of cellulose, use powdercrystallography. When carrying out powder X-ray crystallographic analysis theoperator has to input assumptions to obtain results. Thus, the structure ofcellulose cannot be determined with certainty by X-ray data alone, as the resultsobtained are dependent on the model input.

(4) Degree of crystallinity of samples may be a factor.(5) Sample purity, that is how many polymorphs of cellulose are present, is also

pertinent.

When studying some very highly crystalline celluloses (e.g. Valonia ventricosa), areasonably well resolved spectrum resulted, showing a slightly different diffractiondiagram which was taken to indicate an eight-chain unit cell (Honjo and Watanabe,1958). Dimensions given for this unit cell were quoted as twice those of a two chainunit cell; a � 1:576; b � 1:642; c (®bre axis) � 1:034 nm; and ã � 96:88 (Sarko andMuggli, 1974). Cotton, ramie, wood and other ®brous celluloses, of mediumcrystallinity, were found to be consistent with a two-chain monoclinic unit cell as

TABLE 1. X-ray crystallography data for native cellulose

Specimen c (nm) ã(8) Method Reference

NS 1.03 84 X-ray Meyer and Misch, 1937NS 1.034 88 X-ray Sponsler and Dore, 1926NS 1.03 84 X-ray Meyer and Mark, 1928Valonia 1.03 82 X-ray Fischer and Mann, 1960Bacteria 98.15 X-ray Wellard, 1954Valonia 1.033 96.65 X-ray Wellard, 1954Cladophora 1.034 96.45 X-ray Wellard, 1954Linen 1.034 97.23 X-ray Wellard, 1954Cotton 1.034 96.53 X-ray Wellard, 1954Ramie 1.034 96.38 X-ray Wellard, 1954Valonia 1.058 82 ED Honjo and Watanbe, 1958Ramie 1.039 95.90 X-ray Marchessault and Sarko, 1967Valonia 1.038 96.97 X-ray Nieduszynshi and Atkins, 1970Valonia 1.038 97.0 X-ray Gardner and Blackwell, 1974aNS 1.034 96.8 X-ray Sarko and Muggli, 1974Valonia 1.034 97.04 ED Claffey and Blackwell, 1976NS 1.034 96.38 X-ray French, 1978NS 1.034 96.5 X-ray Woodcock and Sarko, 1980Valonia 1.038 96.6 ED Okano and Sarko, 1984Ramie 1.037 97.6 X-ray Takahashi and Matsunaga, 1991

NS, not speci®ed; ED, electron diffraction.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 177

shown in Fig. 3, that is a unit cell with one non-908 angle (a 6� b 6� c andã 6� á � â � 908), with dimensions: a � 0:778; b � 0:820; c (®bre axis) � 1:034 nm;and angle ã � 96:58 (Woodcock and Sarko, 1980). It was accepted that the two-chainunit cells of other native celluloses could very nearly be considered as sub-cells of theeight-chain Valonia unit cell. The unit cell contains a two ring portion of the cellulosechain which is 1.034 nm in length, called the ®bre repeat.

Most workers agree that the symmetry of the unit cell is either close to, if notexactly, P21=c or belongs to a P1 space group (Fischer and Mann, 1960; Nyburg, 1961).The non 908 angle in the monoclinic unit cell was reported to tend towards a rightangle in the more advanced taxonomic groups (Okano et al., 1989). Two-fold screwaxes have been reported to exist for both of the symmetrically independent molecules inthe two-chain unit cell, but it has been proposed that such symmetry may exist for thebackbone of the cellulose chains, if not the pendant groups. An eight molecule unit cellhas been reported by Herbert and Muller (1974) as having triclinic symmetry, that is tohave a unit cell with no 908 angles (a 6� b 6� c and ã 6� á 6� â 6� 908).

FIGURE 3. Projections of a two-chain model of cellulose I (Valonia ventricosa)perpendicular to bc plane (Woodcock and Sarko, 1980).

178 O'SULLIVAN

Meyer and Misch (1937) postulated an antiparallel arrangement for the chains in theunit cell of native cellulose. This point has been contested many times and a parallelmodel is now more widely accepted for cellulose I (Gardner and Blackwell, 1974b;Sarko, 1973). There have been two possible types of parallel arrangement proposed forcellulose; parallel-up and parallel-down. This discussion was complicated by confusionover the actual de®nitions of parallel-up and parallel-down. According to Gardner andBlackwell (1974a) an `up' sheet consists of cellulose chains in which the z coordinateof O(5) is greater than that of C(5), as shown in Fig. 2, while a `down' sheet has all thecellulose chains with the z coordinate of C(5) greater than that of O(5). The z directionreferred to above is that along the z axis of the three-dimensional environment in whichthe unit cell is built and is parallel to the c or chain axis direction (Fig. 2). The positivedirection of the axis corresponds to increasing length of the c parameter of the unitcell. On this basis, Gardner and Blackwell declared that their model was `parallel-up'and differed considerably from the model of Sarko who stated that his model also was`parallel-up'. These two research teams were, however, considering different de®nitionsfor the a and b axes. For the work by Gardner and Blackwell the a axis correspondedto the intrasheet direction and the b axis corresponded to the intersheet direction. Thiswas the reverse in the models put forward by Sarko's group.

Models by both research groups were analysed by French and Howley (1989), who,using the Gardner and Blackwell de®nition for `up' but Sarko's usage of a and b axesfound that Sarko's models (Sarko and Muggli, 1974; Woodcock and Sarko, 1974) were`parallel-up' and that of Gardner and Blackwell (1974a) was parallel-down'. This articlewas intended to clear up ambiguity in the use of the terms `up', `down', parallel andantiparallel. It stated that a unit cell may be said to contain parallel chains if thedirection of the 1! 4 link is the same in both chains, and antiparallel if one chain hasthe 1! 4 link corresponding to the positive direction of the ®bre axis and the secondchain has the 1! 4 link in the negative direction of the ®bre axis. They representedthe chain arrangements in a unit cell as seen in Fig. 4. In this diagram each chain endis shown as a rectangular box of which two corners are indicated by the letters A and Bin a particular order. It can be seen that the difference between the `parallel-up' and`parallel-down' (Figs 4a and 4b, respectively) is in the direction of the glycosidic bondangle or, as stated by Gardner and Blackwell, in the order of the O5 and C5 atomsalong the chain axis. Takahashi and Matsunaga (1991) suggested that the crystalstructure differences among species (for example ramie, Valonia, cotton and wood) maybe brought about by the order or disorder of the stacking of two kinds of sheets, up anddown (Fig. 5).

CELLULOSE Iá AND Iâ

Evidence has been put forward for the existence of more than one polymorph ofcellulose in native samples. Simon et al. (1988a) postulated that a form of crystallinecellulose existed near the surface of a crystal which differed from the structure to befound at the centre of the crystal. These two crystalline forms were termed celluloses Iáand Iâ (Atalla and Vanderhart, 1989). Celluloses produced by primitive organisms weresaid to have the Iá component dominant, while those produced by the higher plants havethe Iâ form dominant. Iá and Iâ were found to have the same conformation of the heavyatom skeleton, but to differ in their hydrogen bonding patterns. Horii et al. (1987b)

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 179

suggested that the two 13C NMR spectra obtained for polymorphs Iá and Iâ correspondto the resonances for the two-chain and eight-chain unit cell regions of cellulose.

Looking at the crystallographic ®bre diagrams of two forms of cellulose, Halocynthia

and Microdictyon, a difference may be noted (Sugiyama et al., 1991a). The formergives a diffractogram which possesses a mirror of symmetry, while the latter does not.This was proposed to be due to the existence of two polymorphs of native cellulose; Iá,a meta-stable phase with a triclinic unit cell containing one chain and Iâ, having twochains in its monoclinic unit cell. The proportion of Iá=Iâ in Valonia was estimated tobe 65:35 (VanderHart and Atalla, 1984). All the re¯ections observed in the triclinicsample exist in the monoclinic sample but the reverse is not true. Thus, it was deducedthat two crystalline phases were being observed.

When the NMR spectral patterns of Valonia and Tunicin are examined (Belton et al.,1989; Sugiyama, 1992; Yamamoto and Horii, 1993), it may be seen that Tunicin has asimpler spectrum than Valonia. The latter contains two overlapping spectra, Iá and Iâ.It is possible to calculate the relative percentage of each of the polymorphs present.Tunicin possesses only the Iâ polymorph, and so gives the simpler spectrum wherehighly crystalline samples were used. Erata et al. (1995) found differences between thecorrelation peaks of Iá and Iâ cellulose in Cladophora which indicate essentialstructural differences.

The two distinct phases of native cellulose can be identi®ed by electron diffraction(Sugiyama et al., 1991b) and infrared spectroscopy (Sugiyama et al., 1991a). No pure

FIGURE 4. The x-y projection of the unit cells of cellulose packed with a) `parallel up', b)`parallel down' and c) antiparallel chains. The interactions between the AB points aredifferent for the three types of packings.

BA

BA

ABBA

AB

AB BA

AB

AB

BA

(a)

AB AB

AB

AB AB

BA

BA

BA

BA

BA

(b)

BA BA

BA BA BA

AB AB

AB

AB

AB

(c)

180 O'SULLIVAN

sample of Iá has been found in nature. It has been claimed by Lee et al. (1994) thatthis meta-stable polymorph can be synthesized. Also, several groups (Yamamoto et al.,1989; Debzi et al., 1991) have shown that the polymorph Iá can be converted to themore stable Iâ phase by annealing in various media. Annealing at 270 8C converts mostof the Iá to the Iâ form. Bacterial cellulose has the highest percentage of the Iápolymorph, that is 70%. The existence of Iá and Iâ polymorphs in cellulose samplesmay affect the reactivity of native cellulose as Iá is meta-stable, and thus more reactivethan Iâ. Yamamoto and Horii (1993) found that the proportion of Iá cellulose variesfrom 64% in Valonia and bacterial cellulose to 20% in ramie and cotton cellulose. Itwas also found (Yamamoto and Horii, 1994) that the presence of carboxymethylcellulose or xyloglucan decreases the content of Iá, and that Iá cellulose increases atlow temperatures.

Initial ambiguity in the interpretation of crystallographic data for native cellulosemay be largely attributed to the fact that the samples examined were not pure, in thatthey contained more than one polymorph. As the percentage of Iá=Iâ differs withsample type, the results obtained were not directly comparable. The co-existence of twopolymorphs of native cellulose, which have different stabilities, may lead to the Iápolymorph being a site of initial reaction in a micro®bril.

`AMORPHOUS' CELLULOSE

Wide-angle X-ray scattering has been used to study possible structures for amorphouscellulose. Diffraction studies show light and dark areas along a cellulose micro®bril,which have been attributed to crystalline and amorphous cellulose, respectively. Thesimplest conceivable model has `straight' cellulose chains isotropically distributed in thesample (Fink et al., 1987). However, a bent and twisted chain was also suggested(Paakkari et al., 1989). In later research (Chanzy, 1990), it was shown that a slightcurvature in the micro®bril brings speci®c domains in and out of the Bragg diffractionconditions, producing the successive bright and dark domains along the micro®bril axis.This implies that the cellulose micro®bril may be a continuous crystalline structure andnegates the use of light and dark areas observed in the diffraction patterns as evidencefor amorphous areas.

Valonia is a highly crystalline cellulose and, thus, frequently the object of study. Ofthe 600±1000 parallel chains which may exist in a micro®bril of Valonia, only 6±7%of the material is amorphous. Verlhac et al. (1990) suggested that the amorphousmaterial consists mostly of surface chains, which in the large Valonia micro®bril makesup a small percentage of the total. This may be linked to Valonia's low reactivity,(Debzi et al., 1991) denoted by a degree of substitution of only 1%, when subjected tochemical micro-structural analysis (Verlhac et al., 1990). The reactant involved in thistest was N,N-diethyl aziridinium chloride (DAC). For smaller micro®brils, like wood,the percentage surface area is greater and approximately equals the percentageamorphous content: 30% surface hydroxyls versus 33% amorphous material. In theprimary cell wall, the micro®bril is very small and 80% of the chains are on thesurface, and so according to this theory, there would be approximately 80% amorphousmaterial in these samples. Bacterial cellulose has a crystallinity of the order of 75%(Kulshreshta and Dweltz, 1973), and its crystals are around 5±6 nm in diameter.Assuming that these crystals have a nearly square cross-section, there are around 100

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 181

chains per crystal, with roughly 36 chains located at the crystal surface. Cotton is ratedas being 40±45% crystalline and as having a crystallite width of 4±5 nm (Morosoff,1974). A degree of substitution of 2.4% was measured for cotton (Verlhac et al., 1990).This may be compared with the result for Valonia outlined above, i.e. 1% degree ofsubstitution and more than 90% crystalline. This phenomenon is unique to cellulose I.The situation may be complicated, however, by the fact that triclinic cellulose I is meta-stable and may provide a more probable site of reaction.

The surface of crystalline cellulose or indeed areas of `amorphous' cellulose probablystill possess a degree of order. Thus `amorphous' cellulose cannot be considered trulyamorphous as, by de®nition, an amorphous material is one which is formless or lacks ade®nite shape.

CELLULOSE II

X-ray and electron diffraction work has been carried out to clarify the unit cells ofcellulose polymorphs I±IV. 13C CP=MAS NMR spectra (Horii et al., 1982; Dudley et al.,1983) show different signals for the C(4) and C(6) carbons of the polymorphs ofcellulose. It was found that the polymorphs may be divided into two groups; those with aunit cell similar to that of native cellulose (I, IIII and IVI) and those with a cellulose IItype arangement (II, IIIII and IVII). Thus, cellulose II has become an important focus ofstudy, being the second most investigated form of cellulose.

Preliminary work on cellulose II by Andress (1929) presented a two molecule unitcell (Fig. 6): a � 0:814; b � 0:914; c � 1:03 nm; and ã � 628. X-ray results forcellulose II are less reliable than those for cellulose I due to the large number ofdiffraction intensities which overlap each other (Buleon and Chanzy, 1978). Neutrondiffraction studies (Ahmed et al., 1976) directed interest toward a larger unit cellcontaining eight molecules, with parameters: a � 1:592; b � 1:822; c (®bre

FIGURE 6. Projections of cellulose II®bre unit cell (Sarko and Muggli, 1974).

UP UP

UP UP

DOWN DOWN

UP

UP UPUP

DOWN DOWN

FIGURE 5. Schematic representation for thestacking of the up- and down-sheet struc-tures.

182 O'SULLIVAN

repeat) � 1.031 nm; ã � 1178; and symmetry which was close to, but not exactly, P21.Table 2 illustrates other values obtained for cellulose II unit cells. It must beremembered that cellulose II may be arrived at from cellulose I by two distinct routes,i.e. mercerization (alkali treatment) and regeneration (solubilization and recrystalliza-tion), and that even though the unit cells resemble each other closely, there aredifferences. The a parameter for the regenerated product of ramie cellulose has beenquoted as 0.8662 nm and the mercerized ramie cellulose product as 0.8588 nm. Thevalue of ã is greater for mercerized specimens than for most regenerated samples(Wellard, 1954). Also, as the degree of purity of samples used has been shown to berelevant, it should be noted that regeneration gives a higher level of conversion ofcellulose I to cellulose II (Kolpak and Blackwell, 1976). Nyburg (1961) reported oneexample of naturally occurring cellulose II, in the genus Halicystis. Kuga et al. (1993)reported a mutant strain of Acetobacter xylinum as containing native folded-chaincellulose II.

Another commonly studied aspect of cellulose II is the direction of the chains in theunit cell and the most prevalent view is that the two cellulose chains lie antiparallel toone another (Sarko and Muggli, 1974; Kolpak and Blackwell, 1976; Stipanovic andSarko, 1976).

CELLULOSE III

Celluloses IIII and IIIII may be made reversibly from celluloses I and II, respectively andthe polarity of the resultant cellulose chains has been stated to resemble that of thestarting material (Sarko, 1978; Sugiyama and Okano, 1989). A hexagonal unit cell isreported for cellulose III. Sarko et al. (1976) published cell parameters for cellulose IIII

as: a � 1:025; b � 0:778; c (®bre repeat) � 1:034 nm; and ã � 122:48. Their model didnot have strict P21 chain symmetry. Extensive research has been carried out on thereversible transformation of cellulose I into cellulose IIII by electron microscopy (Rocheand Chanzy, 1981), packing analysis (Chanzy et al., 1987), transmission electronmicroscopy (Chanzy et al., 1986), solid state 13C NMR (Sarko et al., 1976) and X-raydiffraction (Sugiyama and Okano, 1989).

The existence of liquid crystal type assembly of cellulose was used to investigatethe transformation of cellulose I to cellulose IIII through the cellulose I-EDAcomplex (Sarko et al., 1976; Roche and Chanzy, 1981; Reis et al., 1991). At thecrystalline level, the conversion from Valonia I to Valonia IIII involved an extensive

TABLE 2. Parameters for selected cellulose II samples�

Sample used a(nm) b(nm) ã

Mercerized bacterial 0.8014 0.9149 1178341

Mercerized ramie 70.970 0.9219 1178461

Mercerized linen 0.8059 0.9200 1188151

Viscose 0.7911 0.9134 117811

Viscose (Japanese) 0.7831 0.9186 1168491

Cuprammonium 0.7955 0.9167 1168571

�Meyer and Mark, 1928; Kolpak and Blackwell, 1976; Kolpak et al., 1978.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 183

decrystallization and fragmentation of the cellulose crystals. During the conversion backto cellulose I, partial recrystallization took place but the distortion and fragmentation ofthe crystals was irreversible. Electron diffraction analysis showed that the unique,uniplanar-axial orientation of the crystalline cellulose micro®brils (Fig. 7a) wasirreversibly lost during the swelling (Fig. 7b) and washing (Fig. 7c) steps leading tocellulose IIII. Following washing in methanol, the micro®bril adopted a convolutedcontour which contained small areas of crystallinity. The ®nal product cellulose I (Fig.7d), produced by hydrothermal treatment, had increased surface area and thus increasedactivity and larger domains of crystallinity than seen in the sample depicted in Fig. 7c.

Celluloses I and IIII were studied by 13C NMR and it was indicated that a reductionin the lateral dimensions of the crystallites occurred during the transformation from theformer to the latter polymorph (Sarko et al., 1976). Examination of the reversiblecomplexation of Valonia cellulose with ethylene-diamine was carried out. This spectralstudy indicated that a conformational change occurs at C(6), during the cellulose I tocomplex transition. The peak corresponding to the C(6)OH is tg in cellulose I(65.7 ppm) and possibly gt in the cellulose I-EDA complex (62.2 ppm). In cellulose IIII

the relevant peak occurs at 62.6 ppm. The regenerated cellulose I gave a spectrumwhich differed from that of the initial cellulose I. Electron diffraction studies show thatthe cellulose I-EDA complex has organized contours and non-uniform crystallinity,shown by a non-white area, whereas cellulose IIII has de®ned crystallinity (Fig. 7c).The variability of hydroxylmethyl conformation indicated above is interesting as it mayalso be used to study other transformations such as those occurring duringmercerization of cellulose I to cellulose II.

CELLULOSE IV

Celluloses IVI and IVII originate from celluloses I and II, respectively. In the latterinstance it is best to use regenerated cellulose (Zeronian and Ryu, 1987). Cellulose III isreported as being very nearly tetragonal but the conversion into cellulose IV is, in mostcases, only partial thus causing dif®culties in obtaining reliable X-ray data (Buleon andChanzy, 1980). One sample yielded the following parameters: a � 0:8068;b � 0:7946 nm; ã � 908. In Gardiner and Sarko's analysis of celluloses IVI and IVII,(Gardiner and Sarko, 1985) both polymorphs were said to crystallize in almost the sameorthogonal unit cell, with parameters for cellulose IVI: a � 0:803; b � 0:813; c (®brerepeat) � 1:034 nm and for cellulose IVII: a � 0:799; b � 0:810; and c (®bre repeat)� 1:034 nm. A P1 space group was suggested to be most probable for both polymorphs.

ALKALI-CELLULOSE

The majority view in the literature is that the polarity of cellulose chains differs betweencellulose I and cellulose II. That means that cellulose I is parallel but cellulose II isantiparallel. In the mercerization process (the treatment of cellulose I with alkali, toachieve cellulose II) no solubilization occurs, which seems to imply that the ®brousstructure of the cellulose would be maintained. In order to improve the understanding ofthis transformation, the changes that occur in cellulose during alkali treatment wereexamined (Okano and Sarko, 1985; Hayashi et al., 1989; Nishimura et al., 1991a,1991b). It was found, by Okano and Sarko (1985), that ®ve unique alkali-celluloses could

184 O'SULLIVAN

be generated reproducibly and that they could be divided into two types, based on theircrystallographic ®bre repeat (Okano and Sarko, 1984). Na-celluloses I, III and IV allexhibited a 1.0 nm repeat, while Na-celluloses IIA and IIB showed a 1.5 nm repeat and athree-fold helical chain conformation, not seen in crystalline celluloses. All the alkali-celluloses had a reasonable degree of crystallinity and a high degree of ®brousorientation. It is dif®cult to reconcile the claimed change in polarity with themercerization process. As Na-cellulose I cannot be reconverted to cellulose I, it wasdeduced that Na-cellulose I possesses an antiparallel arrangement: the same chainpolarity which is thought to exist in cellulose II. This may explain how cellulose I(parallel) can be converted to cellulose II (antiparallel) without solubilizing the cellulose.

Hayashi et al. (1989) discussed in excess of nine polymorphs of alkali-cellulose,which could be formed from cellulose I, II and=or III. It was deduced from theseresults that the hypothesis of the change in chain conformation, as the cause of theirreversibility, is more probable than that of the chain arrangement. The problem withthis argument is that the difference in the conformational energy between the two chainconformations is too small to bring about irreversibility.

It has been stated that there are two types of micro®brils in Valonia, that is thosewhich have cellulose chains with their 1! 4 glycosidic bonds uniformly in the positivez direction and those which have them uniformly in the negative z direction (Revol andGoring, 1983). This is in line with Figs 4a and 4b respectively. Cellulose micro®brilsare arranged in a criss-cross like arrangement, giving high strength to the material(Fig. 8). Diffraction studies of the cross-sections of micro®brils showed individual blackareas. These are supposed to correspond to micro®bril ends. The direction of themicro®bril can be determined by recording a diffraction diagram down the individualmicro®bril (Kolpak et al., 1978). In a small bundle of micro®brils various directions areobserved (Fig. 9). The �z=ÿ z directions of the micro®brils occur in a statistical

(a) (b)

(c) (d)

FIGURE 7. Schematic representation of aValonia micro®bril (MF) undergoing swel-ling in ethylene diamine (ETD): a) Initialsingle MF crystal (in black); b) Swollen MF,after uptake of one ETD molecule perglucose residue; c) Shrunken MF of ValoniaIII1, resulting from the washing in metha-nol of (b); d) MF as in (c) but afterthe hydrothermal treatment leading toregenerated Valonia I.

FIGURE 8. Criss-cross arrange-ment of native cellulose.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 185

distribution. This phenomenon may be used to explain solid-state conversion of nativecellulose II during mercerization (Sarko, 1987). Rearrangement of the chains in theoriginal unidirectional bundles of micro®brils of cellulose I give mixed directionmicro®brils in cellulose II.

Na-cellulose I was studied by X-ray crystallography (Nishimura et al., 1991a) andassigned a four chain unit cell with P2I space group (a � 0:883; b � 2:528; c (®brerepeat) � 1.029 nm; all angles 908). An antiparallel arrangement of chains was slightlyfavoured over a parallel one. Hydroxymethyl conformations were found to be tg in the`�z' chains and gt in the `ÿz' chains.

DENSITY AND DEGREE OF CRYSTALLINITY

The density of cellulose crystals, which may be determined by crystallography, is relatedto the structure of the substance. The density of crystalline cellulose, as found in a singlecrystal, is 1.59 g cmÿ3, whereas that of pure natural ®bre cellulose reaches only1.55 g cmÿ3 (Hermans, 1949). Hermans and Weidinger (1949) also found the so-calledcrystallinity derived from X-ray scattering was only 70% at most in native ®bres, whilethe remaining percentage of the cellulose was said to be `amorphous'. Degree ofcrystallinity may also be estimated by infrared spectroscopy on the basis of the relativeheight of certain bands (Fengel, 1992). Thus, the lower density found in natural ®bre maybe attributed to the presence of amorphous or less ordered cellulose.

CELLULOSE ULTRASTRUCTURE

In plants, polysaccharides exist in the primary, secondary and tertiary cell walls. Thetertiary cell wall contains a lower level of cellulose, being composed mainly of xylan.Primary and secondary walls differ in the arrangement of the cellulose chains. Theformer is less ordered and essentially composed of cellulose chains running in alldirections within the plane of the wall. In the secondary cell wall, the cellulose chains aregrouped in micro®brils which are parallel, giving a more densely packed arrangement,and are aligned more or less with the ®bre axis.

As technology has developed, the features of the cell wall have been observable inmore detail. High resolution electron microscopy lattice imaging shows the crystallineorganization of cellulose in wood ®brils. Negative staining (Heyn, 1966; Woodcock andSarko, 1980) used in conjunction with electron microscopy shows occasional pointsalong the ®bril at which the heavy metal stain seemed to penetrate: it was postulated

One Microfibril Second Microfibril A Bundle of Microfibrils

FIGURE 9. A schematic representation of the end of single micro®brils and of a bundleof micro®brils as seen by a diffraction pattern.

186 O'SULLIVAN

that non-glucose residues were located in these areas. This theory could be seen to besupported by the discovery, in 1961, that up to 50% of some sections of these nativecellulose ®brils were composed of non-glucose units. However, these early workers hadnot isolated pure cellulose micro®brils but were working with ®brils which were acombination of cellulose and hemicellulose (Dennis and Preston, 1961), and the non-glucose units are derived from the hemicelluloses. Breakdown of ®brils by chemicaltreatment (Dennis and Preston, 1961) yields colloidal celluloses which contain, at most,only traces of non-glucose sugars, even though these may be abundant in the parent®brillar material. This is because the material isolated by chemical means are cellulose®brils, almost free from hemicellulose contaminants. The resultant cellulose rods, whichwere measured as approximately 135 glucose residues in length, were thought torepresent crystallites of the micro®bril.

Despite development in microscopic techniques such as scanning electron microscopy(Fengel and Stoll, 1989), atomic force microscopy (Hanley et al., 1992) and scanningtunnelling microscopy (Kuutti et al., 1995) the chain length of such a large molecule ascellulose is still rather dif®cult to measure. The cellulose molecule in wood was foundto be approximately 3500±5000 nm in length when determined by degree ofpolymerization, as calculated by weight of cellulose molecules isolated by a varietyof methods (Fengel and Wegner, 1989), whilst the average size of each crystallite hasbeen determined by X-ray analysis to be 2±20 nm in width and 2±17 nm in thickness(depending on the cellulose sample used).

MODELS PROPOSED FOR CELLULOSE FIBRILLAR STRUCTURE

The supermolecular structure of cellulose can be characterized by the degree ofcrystallinity, crystallographic parameters, crystallite dimension and defectivity, structuralindices of amorphous domains, dimensions of ®brillar formation and other factors. Aseach of these has been studied in relation to celluloses, it is necessary to summarize themodels which have been proposed for the structure of cellulose to see how the presentlyheld views developed.

Frey-Wyssling (1953, 1954) put forward a model for a micro®bril with severalaggregated elementary ®brils (units of about 36 cellulose chains), also called micellarstrands, which were embedded in paracrystalline cellulose, an unordered crystallizedcellulose (Kratky and Mark, 1938; Nickerson, 1941). The existence of these elementary®brils within the cellulose micro®bril was questioned and the smallest ®bril wasreported to be where a micro®bril contains only one array of ordered cellulose chainsrather that a set of arrays. Another model is the fringe micellar model (Astbury, 1933)which has completely ordered or crystalline regions, which, without any distinctiveboundary, change into disordered or amorphous regions. In this model, a singlemolecule was thought to pass from one crystalline region to another through anamorphous area. Micro®brils 10±20 nm in width, visible by electron microscopy, weresaid to be combined in larger ®brils, or lamellae, to form ®bres. Disordered cellulosemolecules, as well as hemicellulose and lignin, were found to be located in the spacesbetween the micro®brils. These amorphous regions were oriented in the same directionas the cellulose micro®brils (Preston and Cronshaw, 1958). In his early work, Fengel(1971) suggested a model for the ultrastructural organization of the cell wallcomponents in wood which had several layers of hemicellulose molecules between

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 187

the ®brils (dimensions 12.0 nm) and a monomolecular layer of hemicellulose betweenthe elementary ®brils. Lignin was envisaged as surrounding the total micro®brillarsystem as shown in Fig. 10 (Fengel, 1971). Taylor and Wallace (1989) discussed theeffect of the hemicellulose xyloglucan binding to the ®brils. The extent of theassociation between cellulose and xyloglucan is dependent on the source of cellulose(Hayashi and Maclachlan, 1984). Binding of xyloglucan has been suggested as aregulator of cellulose ®brillar size (Sasaki and Taylor, 1984).

Elementary ®brils have been reported in recent literature (Lenz and Schurz, 1990), sothe controversy about the arrangement within ®brillar cellulose continues. Crystallites ofherbaceous plant cellulose and wood cellulose (Fink et al., 1990; Ioelovitch, 1992) havebeen determined to have widths in the order of 3.5±4.0 nm, while those for ¯axcellulose and cotton linters are 5.0±7.0 nm. Striations or marks on the surface ofmicro®brils have been observed by microscopy and have been attributed to the fact thatthe micro®bril originated from smaller units or elementary ®brils (Gardner andBlackwell, 1971; Okuda et al., 1994). Bourret et al. (1972) and Chanzy (1990) havedisputed this fact. Enzymic hydrolysis (Chanzy and Henrissat, 1983) of Valonia

cellulose divided the micro®brils into longitudinal crystalline sub-elements with widthsranging from below 2 nm to the full size of the initial Valonia micro®bril (14±18 nm)(Bourret et al., 1972). This span of sizes was taken as evidence against elementary®brils as no size was seen to be prevalent. The width of the Valonia micro®bril wasshown to correspond to the width of the crystallite, whose length was determined to beabove 100 nm, without any longitudinal periodicity. Tsekos et al. (1993) showed byelectron microscopy that, in a number of algae species, micro®brils may consist of two,

FIGURE 10. Cross-sectional model of the ultrastructural organization of the cellwall components in wood (Fengel, 1971).

188 O'SULLIVAN

three or four linear subcomponents (elementary ®brils). In some species, two or threemicro®brils were seen to be bundled together. Further electron microscopy studies byFujino and Itoh (1994) on algal cell walls suggested the existence of layers ofamorphous cellulose ®brils (8±10 nm) and of crystalline micro®brils (15±17 nm) andcross-bridges of approximately 2±4 nm in diameter. Thus, there appears to be noagreement as to the size and nature of micro®brils, perhaps due to a dependence ofcellulose structure on the sample source.

Terminal complexes (TCs) of various sizes have been observed by X-ray and electrondiffraction (Okuda et al., 1994) and by microscopy (Tsekos et al., 1993). It was thoughtthat the number of TC subunits could be linked to the size of the micro®bril produced.Membrane tetrads (Tsekos et al., 1993) have also been identi®ed, membrane-boundmulti-enzyme complexes which participate in the synthesis of matrix polysaccharides.

Looking at the micro®bril cross-sectional end shape, Valonia was found to be nearlysquare, as de®ned by DC TEM data (Chanzy, 1990). By this method, wood pulp(Chanzy, 1987) and ¯ax (NaÈslund et al., 1988) ®bres have also been studied anddistinct micro®brils revealed, but the image could not be suf®ciently resolved to displaytheir cross-sectional shape with certainty. Only in a few instances have nearly squaresections been seen. From this information it was found that the lateral widths ofValonia, Tunicin, ramie, wood and primary cell wall samples of cellulose were 20, 10,5, 3±4 and 1.8±2.0 nm respectively (Chanzy et al., 1979; Chanzy and Vuong, 1985;Chanzy, 1987, 1990; Kuga and Brown, 1987). However, Fink et al. (1995) found that,except for bacterial cellulose, crystallite sizes measured by wide angle X-ray scattering(WAXS) are distinctly smaller than those obtained from electron microscopy andsuggests that it is necessary to consider lattice distortions and the presence of twopolymorphs in native cellulose samples. Jacob et al. (1994) found that wood ®brils havedimensions which are remarkably constant: laterally 2:5� 0:14 nm.

Early workers expected the cellulose chains to have a coiled structure similar to otherpolymers. Bittiger and Husemann (1964a, 1964b) investigated the possible folding ofcellulose chains and found that samples with a degree of polymerization of 700, 420,162 and 120 formed rodlets upon precipitation, with a distinct length of 70 nm. Thislength corresponds to an extended chain length of about 135 glucose units and wastaken to indicate that the molecules would be folded at regular intervals, independent oftheir molecular weight. This theory presumes that the precipitation process causesbreaks in the chain at the folds.

Mark and coworkers (Mark, 1967; Mark et al., 1969) came to the conclusion that thestructural carbohydrate must be aggregated into continuous extended-chain ®laments ofnearly perfect crystallinity. Such a conformation was said to be the mostthermodynamically stable, and for this reason the molecules would tend to assumethis form (Lindenmeyer, 1965). The folded-chain crystal represents a meta-stableconformation and the same polymer was said to be able to exist in the extended orfolded forms, depending on the conditions of crystallization. These facts seem toindicate the possibility of a folded conformation in cellulose derivatives but not innative cellulose. On the basis of experimental evidence, straight cellulose moleculeswere identi®ed in ramie ®bres (Muggli et al., 1969).

Mark (1967, 1971) reviewed various cellulose chain models from a mechanicalstand-point and found that extended-chain conformations yielded axial stiffnessconstants that are compatible with the experimental observations. However, a variety

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 189

of folded-chain conformations yielded maximum stiffnesses incompatible with ex-perimental results.

Sarko and Marchessault (1969) offered a further opinion on the presence of foldedchains. They suggested that the folded chain occurs during the nucleation mechanism ofcrystallization, which is kinetically controlled. The suggestion was that a non-foldedcellulose chain model could be one which excluded the possibility of crystallization.This theory was disproved in later studies, when computer based energy analysis wasadded to the techniques used to examine cellulose. A single straight, extended chainwas found to be thermodynamically less stable, but giving rise to a crystal structure oflower energy. More recently, it has been reported (Wolters-Arts et al., 1993; Emons,1994; Emons and Kieft, 1994) that cellulose in plant cell walls exhibited a helicoidalstructure.

Hermans and Weidinger (1949a) suggested that discontinuities in the ®brillarstructure could be explained by the presence of dislocations (Fig. 11), i.e. disorders in anearly perfectly crystalline lattice but this model has not yet been substantiated. For thisto be true breaks in cellulose chains would be required or chains within a micro®brilwould need to be of non-uniform length. Rowland and Roberts (1972) proposed thatcellulose micro®brils consist of completely crystalline areas with various types ofsurface imperfections, such as distorted surfaces and twisted or strained regions of thecrystalline elementary ®brils.

Cellulose chain orientation relative to the micro®bril surface and to the cell wall hasbeen studied by diffraction studies (Frey-Wyssling and MuÈhlethaler, 1951; Wellard,1954; Tsekos et al., 1993) over many decades. These various studies lead to similarconclusions. All the cellulose chains lie parallel to the 020 plane but it is the 110lattice plane which is parallel to the micro®bril surface and to the plasma membranesurface of the cell (Fig. 12). When reading literature on this subject it is important, yetagain, to recall the change in the convention of labelling the unit cell parameters, asdiscussed above.

HYDROGEN BONDING

Whether native cellulose consists of elementary ®brils or not, its ultrastructure is largelydue to the presence of covalent bonds, hydrogen bonds and van der Waals forces.Hydrogen bonding within cellulose chains may act to determine the `straightness' of thechain. Interchain hydrogen bonds might introduce order or disorder into the system,depending on its regularity.

When considering hydrogen bonding, it is essential to note the conformation of theC(6) hydroxymethyl group. There are three possible minimum energy orientations forthis substituent to the pyranose ring; trans-gauche (tg), gauche-trans (gt) and gauche-gauche (gg), as illustrated in Fig. 13 (Shefter and Trublood, 1965). The reason for thedifference in stability between these three staggered conformers is the relative proximityof the oxygen and carbon substituents. Both cellulose I and cellulose II have beenstudied in depth to decipher any pattern in their hydroxymethyl groups which may exist.

Sundaralingam (1968) reported that the trans-gauche (tg) conformation had not beenobserved in crystal structures as this conformation had a higher energy than the gt orgg orientations. He disputed its inclusion in models for cellulose I proposed by Petitpasand Mering (1956). Of the two remaining possibilities, it was found that the gt

190 O'SULLIVAN

O<6>

H<622>

C<4>H<621>

O<5>

H<5>

H<621>

C<4>

H<622>

O<5>

O<6>

(a)

H<621>

H<5>

H<622>

O<5>

O<6>

C<4>

(b) (c)

H<5>

O<6>

C<4>

H<622>

O<5>

H<621>

FIGURE 13. The three most probable rotational positions of the hydroxymethyl group:(a) gt, (b) gg, (c) tg.

3.5 nm

FIGURE 11. The structural modelof the elementary ®bril of cellulosewith chain dislocations (Hermansand Weidinger, 1949a).

110

020

FIGURE 12. Lamination of crystallized cellulose Ialong plane 110.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 191

conformation was much more likely to occur than the gg. Although the gt conformationis favoured in mono- and disaccharides such as cellobiose (Brown, 1966), this cannotbe taken to preclude the occurrence of the tg orientation in the cellulose polymer, if itallows additional stability through hydrogen bonding.

Models of cellulose with each of the three orientations (tg, gt and gg) were comparedto X-ray data to obtain the best ®t. This ®t may be described as the model with thelowest reliability factor (R). For cellulose I, a tg orientation gave the best ®t (Gardnerand Blackwell, 1974a) with the R value for tg, gt and gg models being 0.242, 0.292and 0.349, respectively. For cellulose II, both regenerated (Buleon and Chanzy, 1978)and mercerized (Kolpak and Blackwell, 1976) forms were reported to have a gtconformation throughout the cellulose chain at the corner of the unit cell, but a tgarrangement for the centre chain. However, some rotation about the suggested minimumorientations (tg, gt and gg) was accepted as being possible. The majority view,expressed in the literature, is that cellulose I has a tg conformation and cellulose II hasa gt conformation throughout the chains (Woodcock and Sarko, 1980; Okamura, 1991).

Derived from the study of hydroxymethyl orientations, intra- and inter-chainhydrogen bond patterns were suggested for cellulose I and II. The former has twointramolecular hydrogen bonds at (O)5-(OH)31 and (OH)2-(O)61 and an interchainhydrogen bond between (O)6-(O)311 as shown in Fig. 14 (Tsuboi, 1957; Mann andMarrinan, 1958; Liang and Marchessault, 1959; Marchessault and Liang, 1960).Cellulose II is reported as having intrachain hydrogen bonding at (OH)3-(O)51 and anintermolecular hydrogen bond at (OH)6-(O)211 for corner chains and (OH)6-(O)311 forcentre chains. An extra dimension was also added to the hydrogen bonding in celluloseII over cellulose I, in the form of an inter-sheet interaction between (OH)2 (cornerchain) ± (O)211 (centre chain), absent in native cellulose. The lengths of the hydrogenbonds in cellulose I are reported to be 0.275 nm (Tasker et al., 1994). Fengel et al.(1995) found that the transformation from cellulose I to II is determined by the splittingand new formation of inter- and intramoleculear hydrogen bonds.

A further consideration, in view of recent evidence, is the conformation which existsin each of the native cellulose polymorphs Iá and Iâ. Molecular dynamics simulationscarried out by Heiner et al. (1995) indicate that the Iâ has a tg hydroxy-methyl

(a)

O

O

OO

O

O

O

O

O

O

O

O

O

O

O

O<3>

O<5>

O<6>O<3>

O<5>

O<3>O<6>

cb

(b)

OO

O

O

O

OO

OO

O

O

O

O

O

O<3>

O<5>O<2>

O<6>O<5>

O<3>

O<2>O<6>

FIGURE 14. Hydrogen bonding pattern for (a) cellulose I and (b) cellulose II.

192 O'SULLIVAN

conformation in more than 90% of cases (92.0 and 99.8 for planes of a cellulosemodel). For the Iá polymorph, 81.0% of hydroxymethyl groups were found to be tg. Ineach situation the remainder of the percentage is represented by a mixture of the ggand gt conformations. This agrees with carbon NMR studies carried out by Yamamotoand Horii (1993).

Cellulose IIII has intramolecular hydrogen bonds between (OH)3-(O)5I and (O)2-(OH)61 and intermolecular associations between (O)3-(O)611 as in cellulose I. Forcelluloses IVI and IVII, in addition to the usual two intramolecular hydrogen bondspresent in most of the other crystalline celluloses, both polymorphs seem to be wellestablished by the intermolecular hydrogen bonding along the 020 plane.

Differences in the hydrogen bonding patterns reported for models of cellulose I andII are not solely derived from deviations in hydroxymethyl conformation but also fromthe fact that the polarity of the chains are popularly thought to differ; a parallelarrangement (Gardner and Blackwell, 1974a) is attributed to cellulose I and anantiparallel arrangement is attributed to cellulose II.

Intersheet associations are further affected by the fact that a relative shift of thecentre and corner chains, along the ®bre axis, has been observed (Gardner andBlackwell, 1974a). This amounts to c=4, or half a ring in length, for cellulose I or II.Cellulose III is reported to show a smaller shift of 0.09 nm (Sarko et al., 1976), ascompared with 0.26 nm (c=4).

COMPUTER ANALYSIS OF CELLULOSE

Computer modelling has been used to study cellulose for many decades and some of theresults are discussed below. Many of the calculations carried out on cellulose have usedvarying levels of constraint to reduce the computing power and time required to obtain aresult. This method of analysis has been used in combination with more conventionalmethods of analysis to test the viability of proposed models.

Due to the small amount of diffraction data available for cellulose in comparison tothe number of atoms that must be located in each of three dimensions, it has been thepractise to construct a stereochemically reasonable polymer model. The diffractionintensity information is then used to locate the chain within the unit cell and todetermine the actual position of the hydroxymethyl groups (French and Murphy, 1977).Several chain arrangements were generated in an effort to reduce the reliability factor(R) for X-ray data. Attempts were made to produce a result which ®tted in withexpectations for cellulose, based on information possessed for related structures. X-raydiffraction of single crystals is the only method that can provide, de®nitively, bondlength, bond angles and characteristic geometry for compounds (Henrissat et al., 1987).However, in the study of polymers by diffraction methods a proposed model has to becreated and hence the result is not absolute. Therefore, it is important to refer to singlecrystal studies that provide data on which to base cellulose models.

By adding a mathematical approach to the quest for the `best ®t', proposed modelswere analysed by computer based stereochemical modelling (Tasker et al., 1994).Statistics were applied to determine the probabilities of the chain conformations andarrangements occurring. As with all methods used for the structural determination ofcellulose, the results of computer analysis cannot be termed absolute, as they depend onthe level of sophistication of the instrumentation and the quality of the algorithms used.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 193

CONSTRAINTS AND ASSUMPTIONS USED DURING MODEL BUILDING

Despite the signi®cant progress that is being made in broadening the scope of molecularmodelling and reducing the approximations that are included in calculations and modelsused, there are still limits to what can be achieved. Polysaccharides are multi-atomsystems, even when disaccharides or oligosaccharides are considered. A cellulosemicro®bril comprises more atoms than can be dealt with by the commercial packagesavailable at present. Thus, only sections of the sytem can be modelled and constraints areoften placed on the amount of freedom allowed in the variation of bond lengths, bondangles and torsion angles.

Stereochemical modelling of cellulose was limited, initially, by the power of thecomputers available and by the lack of suitable force ®elds. Glucose and cellobiosehave both been considered monomers for the polymer, cellulose. Structural aspectswhich were considered in the computer generation of cellulose chains included thestraightness of chains, puckering of rings, glycosidic bond angles, orientation of chains,hydroxymethyl conformation and unit cell parameters. A major assumption in theconstruction of polysaccharide models was that the geometry of sugar monomers isvery similar to that which exists in saccharide oligomers and polymers. French andMurphy (1977) found that the bond lengths and bond angles of â-glucose residues arerelatively constant but that the conformation angles show a variation of as much as 128.Glucose has been found to be an unsuitable monomer for cellulose as it does notcomprise the â(1±4) linkage that exists in cellulose. Thus, cellobiose and derivativesthereof have been more commonly employed.

Using cellobiose crystallographic data as a basis for cellulose models addeduncertainty. Cellobiose has its hydroxymethyl group in a gt conformation (Bittiger andHusemann, 1964b). Thus, if the calculation does not have the ability to consider severaldifferent conformations and arrangements, the results will be inherently inaccurate,unless the structure of the cellulose polymorph to be studied has the same conformationas cellobiose. Early workers did not consider the possibility of differences in the basicring structure occurring during polymerization of cellobiose. Thus, the bond angles andbond lengths of their models were ®xed at those of cellobiose throughout thecalculation, not allowing for any puckering of the pyranose ring.

DEVELOPMENT OF FORCE FIELDS AND ENERGY MINIMIZATION

To survey the development of stereochemical analysis of cellulose, one may begin withthe work of the 1960s to improve minimization functions (Powell, 1964). This allowedresults to be produced faster and with greater reliability for less constrained models. Itwas necessary to direct the mathematics towards measuring speci®c chemical energieswhich determine stabilities and predict the relative probabilities of different conforma-tions occurring. Scott and Scheraga (1965) developed a method of calculating internalrotation barriers by considering exchange interaction of electrons and non-bonded or vander Waals interactions by a semi-empirical method. Allinger (1977) introduced animproved force ®eld for molecular mechanics calculations of the structure and energiesof hydrocarbons. This is known as Molecular Mechanics 2 (MM2). Further force ®elds,such as MMP2 (Sprague et al., 1987), MM3 and UFF (Rappe et al., 1991) have sincebeen devised. The Universal Force Field (UFF) can deal with complex systems which

194 O'SULLIVAN

cannot be handled with the Molecular Mechanics (MM) packages but it is not as accurateat dealing with simple hetero-atomic molecules.

As, initially, the functions developed were for less complex molecules, it was thoughtprudent to attempt a prediction of cellulose structure by working out models for smallermolecules. One example of this was the study of â-D-cellotetraose (Poppleton andMathieson, 1968).

Arnott and Scott (1972) applied the linked-atom constrained least-squares approach tothe analysis of the structure of polysaccharides. In this approach, it was found to beacceptable to keep the bond lengths and bond angles ®xed. The parameters used wereobtained from X-ray studies of the polysaccharides or the monomers involved. It wasconcluded that the variability of the glycosidic bond angles was not greater than that ofthe ring bond angles.

Rees (1970) stated that the torsional angles at each glycosidic linkage incarbohydrate oligomers and polymers may be used to predict their overall shape.There are two of these variables in cellulose: the dihedral angles Ö and Ø. It was alsofound that the ¯exibility of the glycosidic system is determined by the equatorialsubstituents on both residues which are next to the glycosidic oxygen (Rees and Scott,1971). The use of oligomer analysis has continued to be important in trying to elucidatethe macromolecular cellulose structures. An example of this is the work of Gessler etal. (1995) which drew parallels between â-D-cellotetraose hemihydrate and cellulose IIstructures.

CHAIN POLARITY AND HYDROXYMETHYL CONFORMATION

In examining the deductions obtained from computer based stereochemical analysis onX-ray data of cellulose, it must be remembered that the models contained constraints andassumptions. Rees and Skerrett (1968) examined the models previously proposed byMeyer and Misch (1937) and Hermans (1946b): a `straight' and a `bent' chain model. Itwas concluded that the latter was the most likely, on the basis of van der Waals energy.In arriving at this conclusion, the pyranose ring conformation was constrained and thehydroxymethyl group was treated as equivalent to a methyl group in its van der Waalsinteractions. Model-building calculations carried out on cellodextrin (Rees and Skerrett,1970) showed that the only important steric contacts were with adjacent residues, andthese favoured folding of crystalline cellulose chains. Straight-chain cellulose is howevera more acceptable model (Mark, 1967, 1971; Mark et al., 1969; Muggli et al., 1969;Sarko and Marchessault, 1969). By analysing models for cellulose produced by earlierworkers, many discrepancies in these models were noted. For example, Pizzi and Eaton(1987) noted that the model by Kolpak et al. (1978) was much more stable than thatproposed by Sarko. French and Murphy (1977) studied the Gardner and Blackwellmodels (1974a, 1974b) for Valonia, which were originally stated to be `parallel-up' and`parallel-down' (French and Howley, 1989).

Speci®c conformations and orientations (Stipanovic and Sarko, 1976) were proposedfor cellulose I and cellulose II, one example being triclinic unit cells with all thehydroxymethyl groups in or near the tg position. Native cellulose was found to possessparallel chain polarity, and antiparallel was found to be consistent with cellulose II.Gardner and Blackwell (1974a, 1974b) agreed with the chain polarity and conformationassigned to cellulose I, and speci®ed a P21 space group which is a monoclinic unit cell.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 195

Cellulose II (Andress, 1929; Sarko and Muggli, 1974; Buleon and Chanzy, 1978) wasstated by other workers to have antiparallel chain polarity, with the two chains havingdifferent hydroxymethyl conformations (centre chain tg and corner chain gt). Mostresearch groups came to accept that parallel and antiparallel chain polaritiescorresponded to cellulose I and cellulose II respectively and went on to look at otheraspects of cellulose. However, as the chain sense and conformations of cellulose havenot been conclusively proven, there is still scope for investigation. Kroon-Batenburg et

al. (1996) published a study which indicated that it is not possible to disregard thepossibility of a parallel cellulose II or to presume that there is only one feasibleconformational pattern for celluloses I and II.

D-Glucose (Brady, 1986), native cellulose and regenerated cellulose (Pertsin et al.,1986) have been studied by molecular mechanics. In dealing with cellulose theparameters for glucose, as obtained from X-ray crystallography, were used to create amonomer. This was polymerized and packed in accordance with the symmetry assignedto cellulose in previous work (Nyburg, 1961; Gardner and Blackwell, 1974b). Suchconsiderations included the investigation of two chains, with a two-fold screw axis,arranged in a unit cell of P21 symmetry; one chain was positioned at the centre andone at the corner. Various arrangements, for example parallel or antiparallel chainorientations, were considered and the minimum energy calculated. The best crystalmodels of cellulose II were approximately 1.5 kcal molÿ1 lower in energy than those forcellulose I.

Computer modelling of cellulose by molecular and brownian dynamics methods wasdiscussed by Khalatur et al. (1986). The chemical bond lengths and bond angles wereassumed to be the same as for the cellobiose molecules in the crystal state, and theformer were not allowed to vary beyond certain constraints along the eight ringfragments (168 atoms) in the model being studied. The energies of certainconformations, and the possible interchanging of conformations, were investigated.The conclusion was that conformational changes in the macromolecule as a wholeoccur very slowly.

Khalatur et al. (1986) favoured eight ring chain models for regenerated andmercerized cellulose: the hydrogen bonding patterns showed no signi®cant differencesand these models had the cellulose chains arranged in an antiparallel sense. However,for cellulose I the best models (`parallel-down' and antiparallel) were very close inenergy and possessed statistically equivalent RII factors. Pertsin et al. (1986) favouredthe antiparallel model for cellulose I, in view of its similarities to the cellulose IImodel, thus facilitating conversion of cellulose I to cellulose II without solubilization.

Studying the possible conformations for a segment of cellulose chain, six glucoseunits in length, it was found that four slightly twisted chain minima had very similarconformational energies (Simon et al., 1988b), upon minimization, and each facilitatedintramolecular hydrogen bonding. However, the possibility of an in®nite chain foldingback on itself was not ruled out. Two straight-chain segments of ten disaccharide unitseach, in an antiparallel arrangement, provided enough attractive interactions to induce asharp fold in cellulose II (Fig. 15) which could allow the possibility for transition froma parallel cellulose I to an antiparallel cellulose II.

The controversy about the hydroxymethyl conformation and orientation of the chainscontinued. Miller and Li (1989) investigated a two molecule unit cell for cellulose I anddeduced that the O(6) atoms have a gt conformation on the corner chains and a tg

196 O'SULLIVAN

orientation on the centre chains. Work published at the same time (Millane andNarusiah, 1989), supported a crystal structure consisting of a parallel arrangement withall primary hydroxyl groups in the tg conformation. Other workers (Takahashi andMatsunaga, 1991) suggested that the crystalline structure of cellulose was not uniformly`parallel-up' but a mixture of `parallel-up' and `parallel-down'. Kroon-Batenburg et al.(1996) found that micro®bril models with various tg and gt conformations patterns werestable. This variety of possible cellulose micro®bril systems would aid therationalization of the mercerization process, that is, cellulose I to cellulose II withoutsolubilization.

Pizzi and Eaton (1985a, 1985b) identi®ed a cause and mechanism for termination ofthe crystalline zone of cellulose I from their study of ®ve chain crystals ofcellotetraosides. They attributed this feature to localized variation in the hydrogenbond pattern, and also proposed a helicoidal conformation for `free' or amorphouscellulose. The merit of cellobioside over cellobiose as a monomer for cellulose I wasalso commented on.

MOLECULAR DYNAMICS AND ITS APPLICATION

With the advent of molecular dynamics (Khalatur et al., 1986) a greater level of freedomwithin the model could be allowed but bond length and certain bond angle constraintswere retained by some workers to speed up the calculations involved. It has been shownthat the geometry of pyranose rings varies with linkage conformation (Henrissat andChanzy, 1985; Simon et al., 1988a; Millane and Narusiah, 1989; Miller and Li, 1989).Thus, forcing the rings in modelled cellulose to retain the dimensions they hold incellobiose is erroneous (Frey-Wyssling and MuÈhlethaler, 1951) and would make surfacesimulation dif®cult due to the disregard to end-effect implicit in this practise.

Cellulose I

Cellulose II

FIGURE 15. Schematic representation of successive stages of the transformationof antiparallel cellulose II from a parallel cellulose I.

CELLULOSE: THE STRUCTURE SLOWLY UNRAVELS 197

Molecular dynamics simulations carried out by Kroon-Batenburg and Kroon (1992)on cellulose II models worked on the premise that all the primary hydroxyl groups arein the gt conformation. Kroon-Batenburg and Kroon (1990) utilized a computermodelling package GROMOS (van Gunsteren) to perform molecular dynamics (MD)simulations of methyl-â-D-glucoside in water. This study encompassed the stability ofthe tg conformation of the hydroxymethyl group in isolated molecules of methyl-â-D-glucoside and methyl-â-D-cellubioside and the decrease in probability of observing thisconformation in polar solvents. In the GROMOS force ®eld, CH and CH2 groups aretreated as united atoms and no special hydrogen-bond potential is included, in order tosimplify the calculations. The parameters used were taken from work by Koehler et al.(1987) and in addition, van der Waals parameters were applied throughout thecalculations. Only the 4C1 chair conformation was considered, and during simulation notransitions to other shapes occurred. For both the tg and gt conformations the globalminima were obtained by varying the conformations of the hydroxy groups and the exo-anomeric torsional angle. A box was constructed containing the molecule of interest, ina particular conformation, with 242 water molecules. The water molecules were keptrigid by using the SHAKE method (van Gunsteren and Berendsen, 1977). Theconclusions drawn from this work were that cellulose I had hydroxymethyl groups in atg conformation, allowing it to form two intramolecular hydrogen bonds and thatcellulose II had a gt conformation, allowing only one intramolecular hydrogen bond.

Molecular dynamics is now used commonly in most simulation studies but resultsindicate that, even for the simplest of systems, many picoseconds are needed to obtainacceptably low estimated standard deviations (Hooft et al., 1992). For large systemssuch as cellulose micro®brils, it may be necessary to allow molecular dynamicscalculations to run for several weeks of computing time to obtain satisfactory results.Thus, the use of approximations and constraints in all or parts of a structure are stillcommon. The merit of molecular dynamics on an unconstrained system is that it allowsthe system to relax.

ADVANCED MODELS OF CELLULOSE

A structure for the single-chain Iá cellulose unit cell was built by French et al. (1993).The existence of two polymorphs of native cellulose i.e. Iá and Iâ was also investigatedby Heiner et al. (1995). They reported the monoclinic (Iâ) phase was 8.7 kJ molÿ1

cellobioseÿ1 more stable than the triclinic phase. This agrees with the ®ndings of Aablooet al. (1994), which were obtained by using both the rigid-ring method PLMR and thefull-optimization molecular mechanics program MM3(90). Reisling and Brickmann(1995) devised a model for a cellulose chain consisting of â-glucose hexamers.

Models have been built for sections of native crystalline cellulose, for example thatof French et al. (1993) which contained 24 to 32 monosaccharide residues and that ofO'Sullivan (1995) which was a 16 cellulose chain model of a cellulose micro®bril.Non-bond inter- and intramolecular interactions have been calculated for cellulosemolecules and similar systems to determine what the stabilizing forces are in acellulose micro®bril. It has been stated by Kooijman et al. (1992) that crystal packingof a compound is determined solely by intermolecular interactions. While Pizzi andEaton (1984, 1987) found that hydrogen bonding and van der Waals forces are thepredominant factors in the stabilization of the minimum energy conformations of

198 O'SULLIVAN

cellobiose and methyl-â-cellobioside and of the crystalline zone for cellulose II andcellulose I. They also found that cellulose II was more stable than cellulose I. Due tothe importance of non-bond interactions in determining minimum energy conformationsit is vital that they are included in the force ®eld that is used for modelling cellulose.However, as the magnitude of such forces decrease greatly with increases in interatomicdistances, the interaction energy between two atoms may be calculated when theinteracting atoms lie within a speci®ed cut-off range, thereby reducing computationtime signi®cantly. Heiner et al. (1995) produced models of Iá and Iâ cellulose usingthe Gromos 87 force ®eld on which hydroxymethyl conformations were carried out.

Molecular modelling may be used to study physical properties of cellulose as shownin the work of Lee¯ang et al. (1992), who studied the elastic modulus of cellulose Iand II by molecular mechanics. Computer simulation is now used in conjunction withmany other methods of analysis such as diffraction studies as discussed above, NMRspectroscopy (Henrissat et al., 1987; Heiner et al., 1995) and surface analysis(O'Sullivan, 1995; Woodcock et al., 1995).

CONCLUSION

From the above discussion it is obvious that the structure of cellulose is complex and itsstudy requires the consideration of many factors. Investigation into the many aspects ofcellulose structure is being continued by research groups worldwide and new resultsappear in the literature constantly. This trend shall continue as more is learned aboutcellulose by the application of an increasing number of techniques. All knowledge gainedabout cellulose structure is vital to life today as cellulose and its derivatives are used inso many industrial applications.

ACKNOWLEDGEMENTS

I would like to thank the BioComposites Centre, Bangor for funding this work and ProfsM. S. Baird and W. B. Banks for many helpful discussions.

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