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3

Bacterial Cellulose

Prof. Dr. Eng. Stanislaw Bielecki1, Dr. Eng. Alina Krystynowicz2, Prof. Dr. MariannaTurkiewicz3, Dr. Eng. Halina Kalinowska41 Institute of Technical Biochemistry, Technical University of LÛdz, Stefanowskiego4/10, 90-924 LÛdz, Poland; Tel: �48-4263-13442; Fax: �48-4263-402;E-mail: [email protected]

2 Institute of Technical Biochemistry, Technical University of LÛdz, Stefanowskiego4/10, 90-924 LÛdz, Poland; Tel: �48-4263-13442; Fax: �48-4263-402;E-mail: [email protected]



1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3 Structure of BC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4 Chemical Analysis and Detection . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

6 Physiological Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

7 Biosynthesis of BC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457.1 Synthesis of the Cellulose Precursor . . . . . . . . . . . . . . . . . . . . . . . . 457.2 Cellulose Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477.3 Mechanism of Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487.3.1 Mechanism of 1,4-�-Glucan Polymerization . . . . . . . . . . . . . . . . . . . 487.3.2 Assembly and Crystallization of Cellulose Chains . . . . . . . . . . . . . . . . 52

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7.4 Genetic Basis of Cellulose Biosynthesis . . . . . . . . . . . . . . . . . . . . . . 547.5 Regulation of Bacterial Cellulose Synthesis . . . . . . . . . . . . . . . . . . . . 557.6 Soluble Polysaccharides Synthesized by A. xylinum . . . . . . . . . . . . . . . 567.7 Role of Endo- and Exocellulases Synthesized by A. xylinum . . . . . . . . . . 57

8 Biodegradation of BC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

9 Biotechnological Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599.1 Isolation from Natural Sources and Improvement of BC-producing Strains . 609.1.1 Improvement of Cellulose-producing Strains by Genetic Engineering . . . . 609.2 Fermentation Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619.2.1 Carbon and Nitrogen Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619.2.2 Effect of pH and Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 639.2.3 Static and Agitated Cultures; Fermentor Types . . . . . . . . . . . . . . . . . 639.2.4 Continuous Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659.3 In vitro Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669.4 Chemo-enzymatic Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669.5 Production Processes Expected to be Applied in the Future . . . . . . . . . . 679.6 Recovery and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

10 Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

11 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6811.1 Technical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6911.2 Medical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7111.3 Food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7211.4 Miscellaneous Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

12 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

13 Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

14 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

A-BC bacterial cellulose from agitated cultureATP adenosine triphosphateBC bacterial celluloseCBH cellobiohydrolasec-di-GMP cyclic diguanosine monophosphateCel� cellulose-negative mutantCel6A, Cel7A cellobiohydrolases belonging to 6A and 7A families, respectivelyCM carboxymethyl-CS cellulose synthaseCSL corn steep liquorD aspartic acid

3 Bacterial Cellulose38

DMSO dimethyl sulfoxideDP degree of polymerizationE glutamic acidFBP fructose-1,6-biphosphate phosphataseFK fructokinaseFru-bi-P fructose-1,6-biphosphateFru-6-P fructose-6-phosphateG guanineGK glucokinaseGlc glucoseGlc-6(1)-P glucose-6(1)-phosphateG6PDH glucose-6-phosphate dehydrogenaseH-S medium Hestrin and Schramm medium (1954)IS insertion sequenceLip lipidLP UDPGPT: lipid pyrophosphate: UDPGlc- phosphotransferaseLPP lipid pyrophosphate phosphohydrolaseMan mannoseNMR nuclear magnetic resonancePC plant cellulosePDEA phosphodiesterase APDEB phosphodiesterase BPel� pellicle non-forming1PFK fructose-1-phosphate kinasePGA phosphogluconic acidPGI phosphoglucoisomerasePMG phosphoglucomutasePTS system of phosphotransferasesQ glutamineR arginineRha rhamnoseRib d-riboseS serineS-BC bacterial cellulose from static cultureTC terminal complexeU uridineUDP uridine diphosphateUDPGlc uridine diphosphoglucoseUGP pyrophosphorylase uridine diphosphoglucoseUMP uridine monophosphatev.v.m. volume per volume per minuteW tryptophan

Bacterial Cellulose 39

1

Introduction

Cellulose is the most abundant biopolymeron earth, recognized as the major compo-nent of plant biomass, but also a represen-tative of microbial extracellular polymers.Bacterial cellulose (BC) belongs to specificproducts of primary metabolism and ismainly a protective coating, whereas plantcellulose (PC) plays a structural role.

Cellulose is synthesized by bacteria be-longing to the genera Acetobacter, Rhizobi-um, Agrobacterium, and Sarcina (Jonas andFarah, 1998). Its most efficient producers areGram-negative, acetic acid bacteria Aceto-bacter xylinum (reclassified as Gluconaceto-bacter xylinus, Yamada et al., 1997; Yamada,2000), which have been applied as modelmicroorganisms for basic and applied stud-ies on cellulose (Cannon and Anderson,1991). Investigations have been focused onthe mechanism of biopolymer synthesis, aswell as on its structure and properties, whichdetermine practical use (Legge, 1990; Rosset al., 1991). One of the most importantfeatures of BC is its chemical purity, whichdistinguishes this cellulose from that fromplants, usually associated with hemicellulo-ses and lignin, removal of which is inher-ently difficult.

Because of the unique properties, result-ing from the ultrafine reticulated structure,BC has found a multitude of applications inpaper, textile, and food industries, and as abiomaterial in cosmetics and medicine(Ring et al., 1986). Wider application of thispolysaccharide is obviously dependent onthe scale of production and its cost. There-fore, basic studies run together with inten-sive research on strain improvement andproduction process development.

2

Historical Outline

Although synthesis of an extracellular gelat-inousmat by A. xylinumwas reported for thefirst time in 1886 by A. J. Brown, BCattracted more attention in the second halfof the 20th century. Intensive studies on BCsynthesis, using A. xylinum as a modelbacterium, were started by Hestrin et al.(1947, 1954), who proved that resting andlyophilized Acetobacter cells synthesized cel-lulose in the presence of glucose and oxygen.Next, Colvin (1957) detected cellulose syn-thesis in samples containing cell-free extractof A. xylinum, glucose, and ATP. Furthermilestones in studies on BC synthesis,presented in this review, contributed to theelucidation of mechanisms governing notonly the biogenesis of the bacterial polymer,but also that of plants, thus leading to theunderstanding of one of the most importantprocesses in nature. The true historicaloutline is presented throughout all theparagraphs below, including the references.

3

Structure of BC

Cellulose is an unbranched polymer of �-1,4-linked glucopyranose residues. Extensiveresearch on BC revealed that it is chemicallyidentical to PC, but its macromolecularstructure and properties differ from thelatter (Figure 1). Nascent chains of BCaggregate to form subfibrils, which have awidth of approximately 1.5 nm and belong tothe thinnest naturally occurring fibers, com-parable only to subelemental fibers of cellu-lose detected in the cambium of some plantsand in quinee mucous (Kudlicka, 1989). BCsubfibrils are crystallized into microfibrils(Jonas and Farah, 1998), these into bundles,and the latter into ribbons (Yamanaka et al.,


2000). Dimensions of the ribbons are 3±4(thickness) î70±80 nm (width), accordingto Zaar (1977), 3.2î133 nm, according toBrown et al. (1976), or 4.1î117 nm, accord-ing to Yamanaka et al. (2000), whereas thewidth of cellulose fibers produced by pulp-ing of birch or pine wood is two orders ofmagnitude larger (1.4±4.0î10�2 and 3.0±7.5î10�2 mm, respectively). The ultrafineribbons of microbial cellulose, the length ofwhich ranges from 1 to 9 �m, form a densereticulated structure (Figure 2), stabilized byextensive hydrogen bonding (Figure 3). BCis also distinguished from its plant counter-part by a high crystallinity index (above 60%)and different degree of polymerization (DP),usually between 2000 and 6000 (Jonas andFarah, 1998), but in some cases reachingeven 16,000 or 20,000 (Watanabe et al.,1998b), whereas the average DP of plantpolymer varies from 13,000 to 14,000 (Teeri,1997).

Macroscopic morphology of BC strictlydepends on culture conditions (Watanabeet al., 1998a; Yamanaka et al., 2000). In staticconditions (Figure 4), bacteria accumulatecellulose mats (S-BC) on the surface ofnutrient broth, at the oxygen-rich air±liquidinterface. The subfibrils of cellulose arecontinuously extruded from linearly orderedpores at the surface of the bacterial cell,crystallized into microfibrils, and forced

deeper into the growth medium. Therefore,the leather-like pellicle, supporting the pop-ulation of A. xylinum cells, consists ofoverlapping and intertwisted cellulose rib-

3 Structure of BC 41

Fig. 1 Schematic model of BC micro-fibrils (right) drawn in comparison withthe `fringed micelles'; of PC fibrils(Iguchi et al., 2000; with kind permis-sion).

Fig. 2 Scanning electron microscopy images of BCmembrane from static culture of A. xylinum (a) andbacterial cell with attached cellulose ribbons (b).

bons, forming parallel but disorganizedplanes (Jonas and Farah, 1998). The adjacentS-BC strands branch and interconnect lessfrequently than these in BC produced inagitated culture (A-BC), in a form of irreg-ular granules, stellate and fibrous strands,well-dispersed in culture broth (Figure 5)(Vandamme et al., 1998). The strands ofreticulated A-BC interconnect to form a grid-like pattern, and have both roughly perpen-dicular and roughly parallel orientations(Watanabe et al., 1998a).

Differences in three-dimensional struc-ture of A-BC and S-BC are noticeable in theirscanning electron micrographs. The S-BCfibrils are more extended and piled aboveone another in a criss-crossing manner.Strands of A-BC are entangled and curved(Johnson and Neogi, 1989). Besides, they


Fig. 3 Interchain (a) and intrachain (b) hydrogenbonds in cellulose.

Fig. 4 BC pellicle formed in static culture.

have a larger cross-sectional width (0.1±0.2 �m) than S-BC fibrils (usually 0.05±0.10 �m). Morphological differences be-tween S-BC and A-BC contribute to varyingdegrees of crystallinity, different crystallitesize and I� cellulose content.

Two common crystalline forms of cellu-lose, designated as I and II, are distinguish-able by X-ray, nuclear magnetic resonance(NMR), Raman spectroscopy, and infraredanalysis (Johnson and Neogi, 1989). It isknown that in the metastable cellulose I,which is synthesized by the majority ofplants and also by A. xylinum in staticculture, parallel �-1,4-glucan chains arearranged uniaxially, whereas �-1,4-glucanchains of cellulose II are arranged in arandom manner. They are mostly antipar-allel and linked with a larger number ofhydrogen bonds that results in higherthermodynamic stability of the cellulose II.

A-BC has a lower crystallinity index and asmaller crystallite size than S-BC (Watanabeet al., 1998a). It was also observed that asignificant portion of cellulose II occurred in

BC synthesized in agitated culture. Innature, cellulose II is synthesized by only afew organisms (some algae, molds, andbacteria, such as Sarcina ventriculi) (Jonasand Farah, 1998); the industrial productionof this kind of cellulose is based on chemicalconversion of PC.

Using CP/MAS 13C-NMR it is possible toreveal the presence of cellulose I� and I� ±two distinct forms of cellulose (Watanabeet al., 1998a). These forms occur in algae-,bacteria-, and plant-derived cellulose. Thelatter one contains less I� cellulose than BC(Johnson and Neogi, 1989). The irreversiblecrystal transformation from cellulose I� to I�shifts the X-ray and CP/MAS 13C-NMRspectra because of the difference in the unitcell. S-BC contains more cellulose I� than A-BC. It was reported that the difference incellulose I� content between A-BC and S-BCexceeded that in crystallinity index (Wata-nabe et al., 1998a), and the mass fraction ofcellulose I� was closely related to the crys-tallite size.

4

Chemical Analysis and Detection

For the detection of either crystalline oramorphous cellulose, several direct dyes,specific for the linear �-1,4-glucan, are used(Mondal and Kai, 2000). All of them arefluorescent brightening agents and formdye±cellulose complexes, stabilized by vander Waals and/or hydrogen bonding. One ofthese dyes is the fluorescent brightenerCalcofluor. The direct dyes do not onlyenable visualization of cellulose chains, butalso have been intensively applied for studieson nascent cellulose chains association andcrystallization (see Section 7.3.2).

The weight-average DP of cellulose andthe DP distribution are determined by high-performance gel-permeation chromatogra-

4 Chemical Analysis and Detection 43

Fig. 5 BC pellets formed in agitated culture.

phy (Watanabe et al., 1998a) of nitratedcellulose samples.

The differences between the reticulatedstructure of microbial cellulose, producedunder agitated culture conditions, and thedisorganized layered structure of cellulosepellicle, formed in static culture, are notice-able in scanning electronmicroscopy images(Johnson and Neogi, 1989).

To distinguish the parallel chain crystal-line lattice of cellulose I from the antiparallelone of cellulose II, X-ray diffraction, Ramanspectroscopy, infrared analysis, and NMRare applied (Johnson and Neogi, 1989). Thecrystallinity index and crystallite size arecalculated based on X-ray diffraction mea-surements (Watanabe et al., 1998a).

Two distinct forms of cellulose I, i.e.cellulose I� and I�, are not distinguishableby X-ray diffraction, and therefore CP/MAS13C-NMR analysis, carried out on freeze-dried cellulose samples, has to be performedto determine their mass fractions (Watanabeet al., 1998a).

The physicochemical properties of cellu-lose such as water holding capacity, viscosityof disintegrated cellulose suspension, andthe Young's modulus of dried sheets aredetermined using conventional methods(Watanabe et al., 1998a, Iguchi et al., 2000).

5

Occurrence

BC is synthesized by several bacterial genera,of which Acetobacter strains are best known.An overview of BC producers is presented inTable 1 (Jonas and Farah, 1998). The poly-mer structure depends on the organism,although the pathway of biosynthesis andmechanism of its regulation are probablycommon for the majority of BC-producingbacteria (Ross et al., 1991; Jonas and Farah,1998).

A. xylinum (synonyms A. aceti ssp. xyli-num, A. xylinus), which is the most efficientproducer of cellulose, has been recentlyreclassified and included within the novelgenus Gluconacetobacter, as G. xylinus (Ya-mada et al., 1998, 2000) together with someother species (G. hansenii, G. europaeus, G.oboediens, and G. intermedius).

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Physiological Function

In natural habitats, the majority of bacteriasynthesize extracellular polysaccharides,which form envelopes around the cells(Costeron, 1999). BC is an example of sucha substance. Cells of cellulose-producingbacteria are entrapped in the polymer net-work, frequently supporting the populationat the liquid±air interface (Wiliams andCannon, 1989). Therefore, BC-forming strainscan inhabit sewage (Jonas and Farah, 1998).The polymermatrix takes part in adhesion ofthe cells onto any accessible surface andfacilitates nutrient supply, since their con-centration in the polymer lattice is markedlyenhanced due to its adsorptive properties, incomparison to the surrounding aqueous


Tab. 1 BC producers (Jonas and Farah, 1998, modi-fied)

Genus Cellulose structure

Acetobacter extracellular pelliclecomposed of ribbons

Achromobacter fibrilsAerobacter fibrilsAgrobacterium short fibrilsAlcaligenes fibrilsPseudomonas no distinct fibrilsRhizobium short fibrilsSarcina amorphous celluloseZoogloea not well defined

environment (Jonas and Farah, 1998; Cos-teron, 1999). Some authors suppose thatcellulose synthesized by A. xylinum alsoplays a storage role and can be utilized by thestarving microorganisms. Its decompositionwould be then catalyzed by exo- and endo-glucanases, the co-presence of which wasdetected in the culture broth of somecellulose-producing A. xylinum strains (Oka-moto et al., 1994).

Because of the viscosity and hydrophilicproperties of the cellulose layer, the resist-ance of producing bacterial cells againstunfavorable changes (a decrease in watercontent, variations in pH, appearance oftoxic substances, pathogenic organisms,etc.) in an habitat is increased, and theycan further grow and develop inside theenvelope. It was also found that celluloseplaced over bacterial cells protects themfrom ultraviolet radiation. As much as 23%of the acetic acid bacteria cells covered withBC survived a 1h treatment with ultravioletirradiation. Removal of the protective poly-saccharide brought about a drastic decreasein their viability (3% only) (Ross et al., 1991).

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Biosynthesis of BC

Synthesis of BC is a precisely and specificallyregulated multi-step process, involving alarge number of both individual enzymesand complexes of catalytic and regulatoryproteins, whose supramolecular structurehas not yet been well defined. The processincludes the synthesis of uridine diphospho-glucose (UDPGlc), which is the celluloseprecursor, followed by glucose polymeri-zation into the �-1,4-glucan chain, andnascent chain association into characteristicribbon-like structure, formed by hundreds oreven thousands of individual cellulosechains. Pathways and mechanisms of

UDPGlc synthesis are relatively well known,whereas molecular mechanisms of glucosepolymerization into long and unbranchedchains, their extrusion outside the cell, andself-assembly into fibrils require furtherelucidation.

Moreover, studies on BC synthesis maycontribute to better understanding of PCbiogenesis.

7.1

Synthesis of the Cellulose Precursor

Cellulose synthesized byA. xylinum is a finalproduct of carbon metabolism, which de-pending on the physiological state of the cellinvolves either the pentose phosphate cycleor the Krebs cycle, coupled with gluconeo-genesis (Figure 6) (Ross et al., 1991; Tonou-chi et al., 1996). Glycolysis does not operatein acetic acid bacteria since they do notsynthesize the crucial enzyme of this path-way ± phosphofructose kinase (EC 2.7.1.56)(Ross et al., 1991). In A. xylinum, cellulosesynthesis is tightly associated with catabolicprocesses of oxidation and consumes asmuch as 10% of energy derived fromcatabolic reactions (Weinhouse, 1977). BCproduction does not interfere with otheranabolic processes, including protein syn-thesis (Ross et al., 1991).A. xylinum converts various carbon com-

pounds, such as hexoses, glycerol, dihydroxy-acetone, pyruvate, and dicarboxylic acids, intocellulose, usually with about 50% efficiency.The latter compounds enter the Krebs cycleand due to oxalacetate decarboxylation topyruvate undergo conversion to hexoses viagluconeogenesis, similarly to glycerol, dihy-droxyacetone, and intermediates of the pen-tose phosphate cycle (Figure 6).

The direct cellulose precursor is UDPGlc,which is a product of a conventional path-way, common ofmany organisms, includingplants, and involving glucose phosphoryla-

7 Biosynthesis of BC 45

tion to glucose-6-phosphate (Glc-6-P), cata-lyzed by glucokinase, followed by isomer-ization of this intermediate to Glc-�-1-P,catalyzed by phosphoglucomutase, and con-version of the latter metabolite to UDPGlc byUDPGlc pyrophosphorylase. This last en-zyme seems to be the crucial one involved incellulose synthesis, since some phenotypiccellulose-negative mutants (Cel�) are specif-ically deficient in this enzyme (Valla et al.,1989), though they display cellulose syn-thase (CS) activity, this was confirmed invitro by means of observation of cellulosesynthesis, catalyzed by cell-free extracts of

Cel� strains (Saxena et al., 1989). Further-more, the pyrophosphorylase activity variesbetween different A. xylinum strains and thehighest activity was detected in the mosteffective cellulose producers, such as A.xylinum ssp. sucrofermentans BPR2001.The latter strain prefers fructose as a carbonsource, displays high activity of phosphoglu-coisomerase, and possesses a system ofphosphotransferases, dependent on phos-phoenolpyruvate. The system catalyzes con-version of fructose to fructose-1-phosphateand further to fructose-1,6-biphosphate (Fig-ure 6).


Fig. 6 Pathways of carbon metabolism in A. xylinum. CS, cellulose synthase (EC 2.4.2.12); FBP, fructose-1,6-biphosphate phosphatase (EC 3.1.3.11); FK, glucokinase (EC 2.7.1.2); G6PDH, glucose-6-phosphatedehydrogenase (EC 1.1.1.49); 1PFK, fructose-1-phosphate kinase (EC 2.7.1.56); PGI, phosphoglucoisomerase;PMG, phosphoglucomutase (EC 5.3.1.9); PTS, system of phosphotransferases; UGP, pyrophosphorylaseUDPGlc (EC 2.7.7.9); Fru-bi-P, fructose-1,6-bi-phosphate; Fru-6-P, fructose-6-phosphate; Glc-6(1)-P, glucose-6(1)-phosphate; PGA, phosphogluconic acid; UDPGlc, uridine diphosphoglucose.

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