10

Cellulose

Prof. Dr. Dieter Klemm1, Prof. Dr. Hans-Peter Schmauder2,Prof. Dr. Thomas Heinze31 Institute of Organic and Macromolecular Chemistry, Friedrich Schiller Universityof Jena, Humboldtstrasse 10, D-07743 Jena, Germany; Tel.: �49-3641-948-260;Fax: �49-3641-948-202; E-mail: c9kldi@uni-jena.de

2 Research Centre of Medical Technology and Biotechnology, Geranienweg 7,D-99947 Bad Langensalza, Germany; Tel.: �49-3603-833-140;Fax: �49-3603-833-150; E-mail: hpschmauder@fzmb.de

3 Bergische University of Wuppertal, FB 9, Chemistry, Gauss Strasse 20,D-42097 Wuppertal, Germany; Tel.: �49-202-439-2654; Fax: �49-202-439-2648;E-mail: theinze@uni-wuppertal.de

1 Introduction and Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . 277

2 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2782.1 Natural Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2782.2 Synthetic Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

3 Structure and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2803.1 Hydrogen Bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2803.2 Crystal Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2813.2.1 Cellulose I Polymorph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2813.2.2 Further Cellulose Polymorphs . . . . . . . . . . . . . . . . . . . . . . . . . . . 2823.3 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2833.4 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

4 Physiological Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

5 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2855.1 Synthesis of Substrates for the Polymerizing Enzyme . . . . . . . . . . . . . 2865.2 Polymerizing Enzyme System and Enzymology of Biosynthesis . . . . . . . 2875.3 Genetic Basis of Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2885.4 Regulation of Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

275

5.5 Summarizing Open Questions in Research on Plant Cellulose Synthesis . . 289

6 Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2906.1 Intracellular Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2906.2 Extracellular Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

7 Biotechnological Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

8 Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2938.1 Physical and Material Properties . . . . . . . . . . . . . . . . . . . . . . . . . . 2938.2 Chemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

9 Applications of Cellulose and Its Derivatives . . . . . . . . . . . . . . . . . . . 2989.1 Technical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2999.1.1 Regenerated Cellulose Products . . . . . . . . . . . . . . . . . . . . . . . . . . 2999.1.2 Microcrystalline Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3029.1.3 Cellulose Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3029.1.4 Cellulose Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3049.1.5 Oxidized Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3059.2 Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

10 Relevant Patents for Biosynthesis, Biodegradation, and Biological Applications 309

11 Current Problems and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . 310

12 Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

13 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

AGU anhydro glucopyranose unit(s)Bn benzylCA cellulose acetateCMC carboxymethyl celluloseCuam cuprammonium hydroxide [Cu(NH3)4]OHDMA N,N-dimethylacetamideDMF N,N-dimethylformamideDMSO dimethylsulfoxideDP degree of polymerizationDS degree of substitutionEHEC ethylhydroxyethyl celluloseHEC hydroxyethyl celluloseHPC hydroxypropyl celluloseMC methylcellulosemesylate methane sulfonateMHEC methylhydroxyethyl cellulose

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MHPC methylhydroxypropyl celluloseNMMO N-methylmorpholine-N-oxideSAXS small-angle X-ray scatteringSEC size-exclusion chromatographytosylate p-toluenesulfonateWAXS wide-angle X-ray scattering

1

Introduction and Historical Outline

Cellulose constitutes the most abundant,renewable polymer resource available todayworldwide. It has been estimated that byphotosynthesis, 1011 ±1012 tons are synthe-sized annually in a rather pure form, e.g., inthe seed hairs of the cotton plant, but mostlyare combined with lignin and other polysac-charides (so-called hemicelluloses) in thecell wall of woody plants (Kr‰ssig, 1993).Cellulose is a polymer raw material used

for two general purposes. For many centu-ries it has served mankind as a constructionmaterial, mainly in the form of intact woodand textile fibers such as cotton or flax, or inthe form of paper and board. On the otherhand, cellulose is a versatile startingmaterialfor chemical conversions, aiming at theproduction of artificial, cellulose-basedthreads and films as well as a variety ofstable cellulose derivatives used in manyareas of industry and domestic life (Klemmet al., 1998a). Empirical knowledge of dyingcellulose fibers, of burning wood, of prepar-ing charcoal, and of the biodegradation ofcellulose by rotting was acquired alreadythousands of years ago.Cellulose occupies a unique place in the

annals of polymers. As early as 1838, Payenrecognized cellulose as a definitive sub-stance and coined the name ™cellulose∫(Payen, 1838). Cellulose as a precursor forchemical modifications has been used evenbefore its polymeric nature was recognizedand well understood. Milestones on this

pathway were the discovery of cellulosenitrate (commonly misnamed nitrocellu-lose) by Schˆnbein (1846), the preparationof Schweizer's reagent, i.e., a cuprammoni-um hydroxide solution representing the firstcellulose solvent (Schweizer, 1856, 1857,1859) in 1857, and the synthesis of anorgano-soluble cellulose acetate by Sch¸t-zenberger in 1865 (Sch¸tzenberger1865a,b). Partially functionalized cellulosenitrate mixed with camphor as softener wasone of the first polymeric materials used as a™plastic∫ and is well known under the tradename of Celluloid. Cellulose nitrates ofhigher N-content have been used extensivelyformilitary purposes. Today, cellulose nitrateis the only inorganic cellulose ester ofcommercial interest (Balser et al., 1986a).Regenerated cellulose filaments were ob-

tained by spinning cellulose dissolved incuprammonium hydroxide in an aqueousbath. By far the largest part of cellulose-basedartificial fibers have been manufactured forabout the last century by the so-called viscoseprocess, invented in 1892 by Cross et al.(1893). This process is practiced today withan output of about 3 million tons annuallyworldwide. It makes use of the formation ofcellulose xanthogenate, i.e., a water-soluble,less-stable anionic ester, prepared by reac-tion of cellulose with aqueous sodiumhydroxide and CS2 and its decompositionby spinning in an acid bath.The origin of cellulose chemistry as a

branch of polymer research can be tracedback to the fundamental experiments of H.Staudinger in the 1920s and 1930s on the

1 Introduction and Historical Outline 277

acetylation and deacetylation of cellulose;these experiments resulted in the concept ofpolymer-analogous reactions (Staudingerand Daumiller, 1937). According to thisconcept, functional groups of macromole-cules ± in the case of cellulose predomi-nantly hydroxyl groups ± can undergo thesame kind of reactions as the correspondinglow-molecular compounds. Further, it wasobserved that the supramolecular structureof the polymer may play an important role indetermining the rate and final degree ofconversion, as well as the distribution of thefunctional groups, which has been wellrecognized for cellulose.

2

Occurrence

The main source of cellulose is the occur-rence of this polysaccharide in different

types of plants often combined with otherbiopolymers. Of great scientific importanceis access to cellulose using enzymatic andchemical methods, respectively, developedduring the last decade.

2.1

Natural Sources

The primary occurrence of cellulose is theexisting lignocellulosic material in forests,with wood as the most important source.Other cellulose-containingmaterials includeagriculture residues, water plants, grasses,and other plant substances. Besides cellu-lose, they contain hemicelluloses, lignin,and a comparably small amount of extrac-tives (Hon, 1996). Commercial celluloseproduction concentrates on harvested sour-ces such as wood or on naturally highly puresources such as cotton (Table 1).

10 Cellulose278

Tab. 1 Chemical composition of some typical cellulose-containing materialsa

Source Composition (%)

Cellulose Hemicellulose Lignin Extract

Hardwood 43±47 25±35 16±24 2±8Softwood 40±44 25±29 25±31 1±5Bagasse 40 30 20 10Coir 32±43 10±20 43±49 4Corn cobs 45 35 15 5Corn stalks 35 25 35 5Cotton 95 2 1 0.4Flax (retted) 71 21 2 6Flax (unretted) 63 12 3 13Hemp 70 22 6 2Henequen 78 4±8 13 4Istle 73 4±8 17 2Jute 71 14 13 2Kenaf 36 21 18 2Ramie 76 17 1 6Sisal 73 14 11 2Sunn 80 10 6 3Wheat straw 30 50 15 5

a Adapted from Hon (1996).

As a naturally occurring material, cellu-lose may contain byproducts leading toapplication problems and difficulties inchemical modification reactions. However,up-to-date cellulose isolation and purifica-tion yield materials of high purity andvariability. Table 2 gives some examples ofsuch cellulose materials. The values of thedegree of polymerization (DP) (molecularweight�DPî162 g mol�1), included in Ta-ble 2, manifest the huge variety inmolecularweight available.A rather new approach to pure cellulose

gaining increasing interest is the lab-scaleproduction of the polymer by acetic acid-producing bacteria, such as Gluconacetobact-er xylinum and Acanthamoeba castellani(Tarchevsky and Marchenko, 1991). Algaeform another origin of cellulose, e.g.,Valoniaventricosa and Chaetamorpha melagonicum.The cellulose obtained is highly crystallineand is useful for studying polymorphs of thepolymer (see Section 3.2.1) (Vander Hartand Atalla, 1984; Isogai et al., 1989; Sugiya-ma et al., 1990; Yamamoto et al., 1989).Cellulose of the Valonia type is found infungal cell walls as well. In addition, thereare several celluloses of animal origin, ofwhich tunicin, a cell wall component ofascidians, has been extensively studied.

2.2

Synthetic Cellulose

The chemosynthesis of functionalized cellu-lose by ring-opening polymerization of 3,6-di-O-benzyl-�-d-glucopyranose 1,2,4-ortho-pivalate (Nakatsubo et al., 1996), or by step-wise reactions of selectively protected �-d-glucose as, e.g., 1-allyl-2,6-di-O-acetyl-3-benzyl-4-O-(p-methoxybenzyl)-�-d-glucopyranoside(Nishimura et al., 1993) has been experi-mentally realized. Up to now, completedeprotection of the polymers may yieldcellulose with a rather low DP in the rangefrom 9 to 55, dependent upon the appliedprotecting groups. The non-biosyntheticpreparation of cellulose of a molecularweight of 6300 g mol�1 was described in-volving an enzymatic polymerization using�-d-cellobiosyl fluoride as a substrate forpurified cellulase, in amixture of acetonitrileand acetate buffer at pH 5 (Kobayashi et al.,1991, 1994). Whether the cellulose I orcellulose II polymorph (see Section 3.2.1 and3.2.2) is formed depends on the solventcomposition and the purity of the cellulase(Lee et al., 1994). This approach has inter-esting potential for the control of molecularweight and dispersity of the celluloseformed. Further, the enzyme-controlled ster-eospecifity and regioselectivity may mini-mize or even avoid the laborious, multi-step

2 Occurrence 279

Tab. 2 Carbohydrate composition and degree of polymerization (DP) of some cellulose samplesa

Sample Producer Carbohydrate composition (%) DP

Glucose Mannose Xylose

Avicel Fluka 100 ± ± 280Sulfate pulp V-60 Buckeyeb 95.3 1.6 3.1 800Sulfate pulp A-6 Buckeye 96.0 1.8 2.2 2000Sulfite pulp 5-V-5 Borregaardc 95.5 2.0 2.5 800Linters Buckeye 100 ± ± 1470Linters Buckeye 100 ± ± 2000

a Adapted from Heinze (1998a) b Buckeye Cellulose Corp., 1001 Tillman Street, Memphis/Tennessee38108±0407, USA. c Borregaard ChemCell, P.O. Box 162, N-1701 Sarpsborg, Norway.

chemical protection-deprotection proce-dures that generally are required in polysac-charide chemistry.

3

Structure and Analysis

Cellulose is a polydisperse linear homopol-ymer, consisting of regio- and enantioselec-tively �-1,4-glycosidic linked d-glucopyra-nose units (so-called anhydroglucose units[AGU]) (Figure 1). It has been shown by 1H-NMR spectroscopy that the �-d-glucopyra-nose adopts the 4C1 chain conformation, thelowest free energy conformation of themolecule (Kr‰ssig, 1993). As a consequence,the hydroxyl groups are positioned in thering plane (equatorial), while the hydrogenatoms are in the vertical position (axial). Thepolymer contains free hydroxyl groups at theC-2, C-3, and C-6 atoms. Based on the OHgroups and the oxygen atoms of both thepyranose ring and the glycosidic bond,ordered hydrogen bond systems form vari-ous types of supramolecular semi-crystallinestructures.

3.1

Hydrogen Bonding

Both intra- and intermolecular hydrogenbonding occurs in cellulose. The detailedstructure of this hydrogen-bond network isstill a subject of discussion. The presence ofintramolecular hydrogen bonds is of high

relevance with regard to the single-chainconformation and stiffness. The existence ofhydrogen bonds between O-3-H and O-5�(2.75 ä, means of neighboring AGU) of theadjacent glucopyranose unit and O-2-H andO-6� (2.87 ä) in native crystalline cellulose(cellulose I, Figure 2) can be concluded fromX-ray diffraction and NMR- and IR spectro-scopical data (Liang andMarchessault, 1959;Marchessault and Liang, 1960; Gardner andBlackwell, 1974; Sarko and Muggli, 1974).In cellulose II crystallites, the hydrogen

bonds are essentially the same as thoseproposed for cellulose I, considering theO-3-H and O-5� (2.69 ä) hydrogen bond. Theconformation of the C-6 hydroxymethylgroup differs in each chain since the chainsare oriented anti-parallel in the unit cell, i.e.,the CH2OH groups of the respective chainsare not equivalent (Kolpak and Blackwell,1976). Because one of the chains has oneintramolecular hydrogen bond per AGUwhile the other chain has two, a morecomplex hydrogen-bonding network occurs(Stipanovic and Sarko, 1976).

10 Cellulose280

Fig. 1 Molecular structure of cellulose.

Fig. 2 Hydrogen bond system of cellulose I.

The intermolecular hydrogen bonding incellulose is responsible for the sheet-likenature of the native polymer. Today, inter-molecular hydrogen bonding between onlytheOHgroup at the C-6� andC-3� (��means ofthe neighboring chain) positions of cellulosemolecules adjacently located in the samelattice plane (020 planes) is assumed (Black-well et al., 1977).The intermolecular hydrogen bonding in

cellulose II is significantly more complexcompared to that of cellulose I. The anti-parallel chain model enables the formationof not only interchain but also of interplanehydrogen bonds. The most widely acceptedrepresentation of the bonding situation hasbeen given by Kolpak and Blackwell (1976,1978), as shown in Figure 3. It should bepointed out again that the hydrogen bondingof cellulose has been interpreted in manyways, as nicely discussed by Gilbert andKadla (1998).

3.2

Crystal Structure

The order of the macromolecules in acellulose fiber is not uniform throughoutthe whole structure. There exist regions oflow order (so-called amorphous regions) aswell as of very high crystalline order. Theexperimental evidence available today isadequately interpreted by a two-phase mod-el, the fringed fibril model, assuming low-order (amorphous) and high-order (crystal-line) regions and neglecting the rather smallamount of matter with an intermediate stateof order (Hearle, 1958).The relative amount of polymer within the

highly ordered regions is usually assessedfrom wide-angle X-ray scattering (WAXS)patterns or from the evaluation of a 13C CP-MAS NMR spectrum. The degree of crystal-linity of cellulose (usually in the range of40% to 60%) covers a wide range and

depends on the origin and pretreatment ofthe sample (Fink and Walenta, 1994).

3.2.1

Cellulose I PolymorphCellulose exists in several crystal modifica-tions, differing in unit cell dimensions and,possibly, in chain polarity. For crystallinenative cellulose, i.e., cellulose I, Meyer,Mark, and Misch (Meyer and Mark, 1929;Meyer and Misch, 1937) proposed a unit cellof the crystal lattice, already 60 years ago,that is still applicable for practical purposestoday (Figure 4). This model assumes amonoclinic unit cell with the space group

3 Structure and Analysis 281

Fig. 3 Most probable bond pattern of cellulose I(Kolpak and Blackwell, 1976, 1978).

P21 and two anti-parallel cellobiose chainsegments running in opposite directionsalong the fiber axis. The dimensions of thiscell are given in Table 3.Numerous authors have suggested that

the unit cell of native cellulose may dependon the source. Honjo and Watanabe (1958)concluded from low temperature electrondiffractograms a doubling of the size of theunit cell. Sarko and Muggli (1974) agree tothis eight-chain unit cell but state that theMeyer-Misch model also adequately repre-sents most of the crystallographic evidenceof native crystalline cellulose. Based on themore refinedWAXS technique, Gardner andBlackwell (1974) proposed for cellulose

(from Valonia alga) a monoclinic lattice withtwo parallel running chains and assumedthis to be valid for cellulose I in general.Sarko and Muggli (1974) proposed from acombined packing and X-ray intensity anal-ysis a triclinic lattice cell with two cellulosechain segments running parallel along thefiber axis.Atalla and Vanderhart (1984) showed that

native cellulose consists of two differentcrystal structures, cellulose I� and I�, usinghigh-resolution, solid-state 13C NMR spec-troscopical studies. There are differences inthe resonances of the C-1 atoms. A singletfor cellulose I� and a doublet for cellulose I�appears at about 106 ppm. This rather smalldifference indicates a different hydrogen-bonding pattern of the glycosidic linkages.Bacterial cellulose and Valonia cellulose(from alga) contain a large amount of I�modification, while in ramie, cotton, andwood cellulose, the I� phase is the dominat-ing modification. The I� modification isdescribed as a triclinic P-1 structure, withone cellulose chain per unit cell, whereas theI� phase is assumed to be a monoclinic unitcell of the Meyer-Misch type (space group P-21 with two chains per unit cell) as concludedfrom electron diffraction experiments (Su-gijama et al., 1991). According to Yamamotoand Horii (1993), the I� phase is metastabileand can be transformed (not completely,however) into the thermodynamically morestable I� phase by annealing at 260�C to280�C.

3.2.2

Further Cellulose PolymorphsBesides cellulose I, cellulose II is the mostimportant crystalline form of cellulose froma technical and commercial point of view.Cellulose II can be prepared by precipitatingdissolved cellulose into an aqueous medi-um; this is the typical process for thetechnical spinning of man-made cellulose

10 Cellulose282

Fig. 4 Unit cell of cellulose I according to theMeyer-Misch model.

Tab. 3 Unit cell dimensions of various celluloseallomorphsa

a-axis (ä) b-axis (ä) c-axis (ä) �-axis (ä) Polymorph

7.85 8.17 10.34 96.4 Cellulose I9.08 7.92 10.34 117.3 Cellulose II9.9 7.74 10.3 122 Cellulose III7.9 8.11 10.3 90 Cellulose IV

a As accomplished by Kr‰ssig (1993).

fibers. It is also obtained by the so-calledmercerization process, i.e., by soaking cellu-lose in aqueous NaOH (17% to 20%, w/v)followed by decomposition of the intermedi-ate by neutralization or washing out theNaOH. Mercerization is used to activate thepolymer prior to the production of technicalcellulose ether. The process of transforma-tion of cellulose I to cellulose II is generallyconsidered to be irreversible.Cellulose II is formed naturally by a

mutant strain of Gluconacetobacter xylinum(Kuga et al., 1993) and occurs in the algaHalicystis (Sisson, 1938), which were bothvery useful to provide an insight into thecrystal structure of cellulose II.The crystalline modification of cellulose

III is obtained by treating native cellulosewith liquid ammonia (below ±30�C) or anorganic amine such as ethylene diamine,followed by washing with alcohol. Smalldifferences in lattice dimensions exist be-tween the two submodifications celluloseIIII and IIIII. As the fourth modificationreported so far, cellulose IV is formed upontreatment of the other modification ofcellulose in a suitable liquid at high temper-ature and under tension (Figure 5, seeTable 3).

3.3

Morphology

Cellulose morphology represents a well-organized architecture of fibrillar elements.It has been considered that the elementaryfibril of native cellulose is the smallestmorphological unit with a diameter of about3.5 nm (M¸hlethaler, 1965; Heyn, 1966;Fengel and Wegener, 1989). Recent electronmicroscopic and WAXS data indicate thatthe diameter may differ in the range of 3 to35 nm depending on the cellulose source(Table 4, Chanzy et al., 1986; Fink et al.,1990). The so-called microfibril is describedas the lowest well-defined morphologicalentity, although it consists of non-uniformsubunits (Fink et al., 1990). The length ofthe microfibril can reach micrometers,which in turn forms the macrofibrils witha diameter in the range of micrometers(Fengel and Wegner, 1989; Kr‰ssig, 1993).Micro- and macrofibrils represent the

construction units of the cellulose fiber

3 Structure and Analysis 283

Fig. 5 Transformation of cellulose into its various polymorphs.

Tab. 4 Range of microfibril diameter of variouscellulose samplesa

Sample Microfibril diameter (nm)

Bacterial cellulose 4±7Cotton linters 7±9Ramie 10±15Dissolving pulp 10±30Valonia cellulose 10±35

a Source: Fink et al. (1990).

cell-wall architecture, which is characterizedby layers differing in fibril texture (Figure 6).The fibers consist of different layers, with thefibril position giving different densities andtextures, as shown for a cotton fiber and adelignified spruce pulp fiber. The primarywall (P) fibrils have a diameter of about10 nmand are positioned crosswise to a layerwith a thickness of about 50 nm. Thesecondary cell wall (S) consists of two layers,S1 and S2, with a thickness of about 100 nm(cotton) to 300 nm (spruce pulp). The S1 andS2 layers contain most of the cellulose mass.The fibrils are aligned parallelly and packeddensely in a flat helix. The inner layer closestto the fiber lumen (W) is the tertiary layer(T), which is rather thin and has fibrilsaligned in a flat helix as well (Kr‰ssig, 1993).

3.4

Analysis

Qualitatively, cellulose can be determined byX-ray spectroscopy, by color reactions withKI or ZnCl2, by reacting with a solution of

iodine in sulfuric acid or phosphoric acid,and with Congo red and others (Blazej et al.,1979). However, these color reactions are notspecific.For a quantitative determination, the

Cross-Bevan method and the K¸rschner-Hoffer method are quite appropriate (Blazejet al., 1979). For these methods, the acces-sory components are removed by a treatmentwith gaseous hydrogen chloride and subse-quent heating with aqueous sodium sulfiteor with nitric acid/ethanol after pretreat-ment with aqueous potassium hydroxide,respectively (K¸rschner and Popik, 1962).In the analysis of cellulose and its deriv-

atives, instrumental methods are employedfor assessing the size and chemical structureof the macromolecules within the entity of agiven sample. Moreover, instrumental de-tection methods also are required for themonitoring of structurally relevant parame-ters during continuous fractionation of thepolymer or chromatographic separation ofits fragments. The spectroscopic methodspreferentially used are UV/visible- (Fengeland Wegener, 1989), IR- (Grˆbe, 1989), andNMR-spectroscopy (Nehls et al., 1994). To-day, predominately HPLC and gas chroma-tography are used to analyze the fragmentsobtained by acid or enzyme degradation(Mischnick, 1995; Heinze et al., 1994;Heinze, 1998b; Saake et al., 2000).

4

Physiological Function

Cellulose is the main component of plantcell walls. Protection of cells and formationof structures are as such the main functionsof cellulose during plant life. The definedcell shapes and position of cells to each otherare the basis for plant morphology. There-fore, cellulose is essential for plant life as weknow it. Even structures based on old cell

10 Cellulose284

Fig. 6 Scheme of the ™morphological architecture∫of a cotton fiber (a) and a delignified spruce woodfiber (b) according to Kr‰ssig (1993). C�cuticle(rich in pectins andwaxes); L� lumen;ML�middlelamella (mainly lignin); P�primary wall; R� rever-sal of the fibril spiral; S1�secondary wall (™windinglayer∫); S2�secondary wall (main body); T� terti-ary wall; W�wart layer.

walls from already dead cells are crucialfunctional units of higher plants (xylem).The main building blocks of the primary

cell wall of plants consist of differentcomponents, e.g., pectins, hemicelluloses,celluloses, and proteins. In the primary cellwall, the non-cellulosic components domi-nate; on this basis, the mechanical stabilityof the primary cell wall is low. While in lateron formed structures of the cell wall andunits important for plantmorphogenesis thefibrillar structure of cellulose stabilizes theplant organism. The later on formed struc-tures of the cell wall and cell units importantfor plant morphogenesis were stabilizedby the fibrillar character of cellulose Theprimary cell wall swells and forms jelly-likestructures. (To the groups of hemicellulosesbelong glucanes of the (1�3)-� as well as(1�4)-�-, gluco-, and galactomannanes andmainly xylanes; Sitte et al., 1991) The cellu-lose molecules in the primary cell walls havehigh degrees of polymerization between2000 and 15,000 anhydroglucose units inlong, non-branchedmolecules. The cellulosechains are twisted along the axis of theglucan chains (180�) and stabilized by hydro-gen bonds between the chains. As a result,the rings of the pyranoses lie approximatelyin the same level, forming ligaments. Thesmaller chains have lengths around 8 �m.These chains associate to elementary fibrilshaving a diameter of about 5±30 nm. In thesecondary cell walls, the fibrils associate tomicrofibrils with diameters of about 5±30 nm. These microfibrils have an organ-ization in crystal lattices, bringing a highstability into the cell walls of plants. Theseassociated cellulose fibrils bring the maincontribution to the high mechanicalstrength of the plant cell walls. The tensilestrength of plant cell walls has a basis notonly in the association of the chains byhydrogen bonds but also in sticking togetherwith other components of the primary cell

wall, such as proteins, pectines, and hemi-celluloses. For further increasing the stabil-ity of plants, lignin is incorporated into theplant architecture. The portion and distribu-tion of the different components of cell wallsdefine the final properties of the plant partsand tissues. Examples of the role andstructure of hemicelluloses in the plant cellwall system are given by Henriksson et al.(2000).Rose et al. (1997) have found that specific

proteins, so-called expansins, are able toinduce the extension of isolated plant cellwalls in vitro and to disrupt the non-covalentinteractions between hemicelluloses andcellulose microfibrils. Because the primarycell wall acts as the main factor for cellenlargement, this processmay be an integralpart to plant cell expansion. Using expan-sins, the role of the different componentswithin this primary cell wall can be studied.Some of these reactions and substanceformations will be regulated by ethyleneand other phytohormones.The microtubule arrays are of high im-

portance because of their involvement in theorientation of cellulose microfibrils. Theplant interphase tubulins play an importantrole in these processes and have influenceson structuring the microfibrils within thecell-wall system (Moore et al., 1997).

5

Biosynthesis

The biosynthesis of cellulose is not yetcompletely elucidated. Moreover, contraryresults have been described and discussed inmany papers. During the last few years, thenumbers of patents in this field has in-creased because of the interesting possibilityto increase the cellulose content of plantsand to construct new and more efficientplants. Because of the ability of some

5 Biosynthesis 285

bacterial strains to form cellulose and be-cause of the similarity of the biosyntheticapparatus in some aspects, much of theresearch was done using these bacteria.Results obtained with the bacterium Gluco-nacetobacter xylinum as a model organismare discussed in Volume 5 of Biopolymers,Chapter 3. Table 5 contains a summarizationof different relevant publications, which willnot be discussed in detail, while Table 6shows a selection of interesting patents.

5.1

Synthesis of Substrates for the PolymerizingEnzyme

The only substrate for cellulose biosynthesisis UDP-glucose. The biosynthesis of thisenergy-rich compound follows the normalbiosynthetic pathways in the cells, startingfrom glucose (Figure 7). The enzyme cellu-lose synthase accepts only UDP-glucose as asubstrate; moreover, it was noticed that by

feeding modified glucoses to bacteria (Glu-conacetobacter xylinum), as well as to plantcells or cell extracts, no significant formationof modified celluloses could be detected(Schmauder, unpublished; Brown, personalcommunication).Another possible source for UDP-glucose

could be sucrose synthase, an enzymeassociated with the plasma membrane,e.g., of cotton fibers. Because of this location,a direct channeling of the substrate UDP-glucose to the polymerizing enzyme ispossible. But the regulation, control, andtargeting of this process is unknown in wideareas. Other possible sources for the stabi-lizing and transport of the substrate areannexin-like molecules, which are able tobind UDP-glucose, e.g., a 170-kDa polypep-tide was co-purified with the cellulosesynthase. This protein shows some homol-ogies to the yeast �-1,3-glucan synthase (seealso Brown, 1999; Delmer, 1997, 1999a,b,2000a,b).

10 Cellulose286

Tab. 5 Selected papers for biosynthesis and structure of cellulose

Topic of the paper Authors/Applicants

Role of callose synthase and other (1,3)-�-glucan synthase in cellulosebiosynthesis; enhancing of cellulose synthesis by cellobioses

Him et al., 2000

Effect of retardants on cellulose biosynthesis in cotton; effects on fibers andseedlings

Akhunov et al., 2000

Review about genes and proteins involved in cellulose synthesis in plants; roleof the sucrose synthase for substrate formation; orientation of the microfibrildeposition; role of membrane-associated cellulase in biosynthesis process

Delmer et al., 2000

Cellulose structure elucidation using atomic force microscopy Baker et al., 2000Estimation of the relations between Cellulose I� and I� in wood; application of13C-NMR

Newman, 1999

Supramolecular architecture; fibril formation and its regulation Kataoka and Kondo,1999a,b

Cellulose biosynthesis as a binding factor for CO2 Hayashi et al., 1998bReviews on cellulose biosynthesis; comparison of synthesis by microorganismsand by plants

Brown, 1996; Brownet al., 1996; Kudlickaand Brown, 1996

General reviews concerning cellulose biosynthesis by bacteria, fungi, and plants Blanton and Haigler,1996

Summarizing those effects, Brett (2000)states that UDP-glucose on the one hand orsucrose on the other, as well as furthersoluble intermediates from these pathways,could serve as possible precursors.

5.2

Polymerizing Enzyme System and Enzymologyof Biosynthesis

Different data are described for the cellulosesynthase as the active enzyme system incellulose formation. Carpita and Vergara(1998) discuss the polypeptide formed as aresult of the CelA gene family (cotton), witha size of about 110 kD, and the existence ofeight transmembrane domains.

Brown and Saxena (2000) and Delmer(1999b) describe that the cellulose-synthasecomplex has a rosette structure character-ized by ultrastructural investigations. Thisstructure is highly symmetrically arranged(about six-fold) and contains transmem-brane particle subunits. From these sub-units, the crystalline cellulose I will begenerated. In this review, the historical datafor this finding are discussed in detail. Thecatalytic subunit is a transmembrane proteinwith some transmembrane regions.Brown and Saxena (2000) discuss four

different models for cellulose synthesis:

1) The most acceptable model, model 1,works with the assumption that the

5 Biosynthesis 287

Tab. 6 Selected patents for biosynthetic pathways

Topic of the patent Authors/Applicants

Overexpression of cellulose synthase genes for modulating expression ofenzymes involved in synthesis of plant cell walls

Taylor and Turner, 2000

Polynucleotides encoding cellulose synthase for acceleration of plant growthand up-regulation of cellulose synthase level; modifying of lignin biosynthesis

Carraway et al., 2000

Cellulose synthase gene from poplar; application for altering cellulose andlignin composition

Chiang et al., 2000

New genes encodes maize cellulose synthase polypeptides; modulation ofexpression of cellulose synthase in plants; production of transgenic plantsexpressing the new protein

Bowen et al., 2000

Genes for cellulose synthase; application of these genes for improving plantstalk quality; increase of cellulose in stalks etc.

Dhugga et al., 2000

Transgenic plant expressing cell-wall modulating proteins as a basis for, e.g.,altered morphology, increased growth, modified fiber lengths, increasedcellulose and starch content

Shani et al., 1999

Isolated genes encoding polypeptides involved in cellulose biosynthesis,transgenic plants, expressed in sense or anti-sense orientation, ribozxymes, co-suppression, gene-targeting molecules

Arioli et al., 1999

Fig. 7 Intracellular activation of glucose as the precursor for cellulose biosynthesis. 1: Glucokinase; 2:Phosphoglucomutase; 3: UDP-glucose-pyrophosphorylase; UDP�uridine 5�-diphopsphate; glc�glucose;glc-6-P�glucose-6-phosphate; glc-1-P�glucose-1-phosphate; Pi� inorganic phosphate.