celulose bacteriana avancos 2009

celulose bacteriana avancos 2009

(Parte 1 de 8)

ISSN 1330-9862 review (FTB-2058)

Microbial Cellulose: Fermentative Production and Applications

Prashant R. Chawla, Ishwar B. Bajaj, Shrikant A. Survase and Rekha S. Singhal*

Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, IN-400019 Mumbai, India

Received: February 19, 2008 Accepted: December 3, 2008

Summary

Bacterial cellulose, an exopolysaccharide produced by some bacteria, has unique structural and mechanical properties and is highly pure as compared to plant cellulose. This article presents a critical review of the available information on the bacterial cellulose with special emphasis on its fermentative production and applications. Information on the biosynthetic pathway of bacterial cellulose, enzymes and precursors involved in bacterial cellulose synthesis has been specified. Characteristics of bacterial cellulose with respect to its structure and physicochemical properties are discussed. Current and potential applications of bacterial cellulose in food, pharmaceutical and other industries are also presented.

Key words: microbial cellulose, homopolymer, Acetobacter xylinum, Acetobacter hansenii, fermentation

Introduction

Polysaccharides are a structurally diverse group of biological macromolecules of widespread occurrence in nature. They can be divided according to their morphological localization as: intracellular polysaccharides located inside, or as part of the cytoplasmic membrane; cell-wall polysaccharides forming a structural part of the cell wall; and extracellular polysaccharides located outside the cell wall. Extracellular polysaccharides occur in two forms: loose slime, which is non-adherent to the cell and imparts a sticky consistency to bacterial growth on a solid medium or an increased viscosity in a liquid medium; and microcapsules or capsules, which adhere to the cell wall. They have a definite form and boundary, being only slowly extracted in the water or salt solutions. It is therefore possible to separate capsules and microcapsules from loose slime by centrifugation (1).

Exopolysaccharides are long chain polysaccharides consisting of branched, repeating units of sugars or sugar derivatives, mainly glucose, galactose and rhamnose in different ratios. They are classified into two groups:

homopolysaccharides (cellulose, dextran, mutan, pullulan, curdlan), and heteropolysaccharides(gellan, xanthan) (2). Homopolysaccharides consist of repeating units of only one type of monosaccharides (D-glucose or D-fruc- tose) joined by either a single linkage type (e.g. 1 2o r 1 4) or by a combination of a limited number of link- age types (e.g. 1 2 and 1 4). Heteropolysaccharides consist of multiple copies of oligosaccharides, containing three to eight residues, produced by a variety of microorganisms. Exopolysaccharides find wide industrial applications in food, pharmaceutical and other industries like textile, paper, cosmetics, gelling agents and medicines for wound dressing (3).

Microbial cellulose is an exopolysaccharide produced by various species of bacteria, such as those of the genera Gluconacetobacter (formerly Acetobacter), Agrobacterium, Aerobacter, Achromobacter, Azotobacter, Rhizobium, Sarcina, and Salmonella (4). Production of cellulose from Acetobacter xylinum was first reported in 1886 by A.J. Brown (5). He observed that the resting cells of Acetobacter produced cellulose in the presence of oxygen and glucose.

107P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009)

The molecularformulaof bacterialcellulose(C6H10O5)n is the same as that of plant cellulose, but their physical

and chemical features are different (6). Bacterial cellulose is preferred over the plant cellulose as it can be obtained in higher purity and exhibits a higher degree of polymerization and crystallinity index. It also has higher tensile strength and water holding capacity than that of plant cellulose, making it more suitable raw material for producing high fidelity acoustic speakers, high quality paper and dessert foods (4). Fibrils of bacterial cellulose are about 100 times thinner than that of plant cellulose, making it a highly porous material, which allows transfer of antibiotics or other medicines into the wound while at the same time serving as an efficient physical barrier against any external infection. It is therefore used extensively in wound healing (7).

Microbial cellulose exists as a basic structure known as microfibrils, which are composed of glucan chains interlocked by hydrogen bonds so that a crystalline domain is produced. This microfibrillar structure of bacterial cellulose was first described by Mühlethaler in 1949 (8). Electron microscopic observations showed that the cellulose produced by Acetobacter xylinum occurs in the form of fibres. The bacteria first secreted a structurally homogeneous slimy substance within which, after a short time, the cellulose fibers were formed.

Acetobacter xylinum produces two forms of cellulose: (i) cellulose I, the ribbon-like polymer, and (i) cellulose I, the thermodynamically more stable amorphous polymer (9). The differences in the assembly of cellulose I and cellulose I outside the cytoplasmic membrane are described in Fig. 1. Microfibrillar structure of bacterial cellulose is responsible for most of its properties such as high tensile strength, higher degree of polymerization and crystallinity index. Bacterial cellulose is used as a diet food and to produce new materials for high performance speaker diaphragms, medical pads (10) and artificial skin (1). Relatively high cost of the production of cellulose may limit its application to high value-added products as well as speciality chemicals (12). Significant cost reductions are possible with improvements in fer- mentation efficiency and economics of scale, the lower limit of the cost of microbial cellulose being determined by the price of the raw material substrates. Consequently, Acetobacter cellulose may always be more expensive to produce than conventional sources of cellulose (4). For this reason, successful commercialization of Acetobacter cellulose will depend on careful selection of applications where its superior performance can justify its higher cost.

Strains Producing Cellulose

Cellulose is found in groups of microorganisms like fungi, bacteria, and algae. In green algae, cellulose, xylan, or mannan may serve as structural cell wall polysaccharides. Cellulose is found, although in small quantities, in all of the brown algae (Phaeophyta), most of the red algae (Rhodophyta), and most of the golden algae (Chrysophyta (Chrysophytes)) (13). It was also reported to be present in some fungi, forming inner cell wall layer, usually in association with b-1 3/b-1 6- -linked D-glucan. Chitin is completely replaced by cellulose in Oomycetes, accounting for about 15 % of the wall dry mass (14). Gram-negative species like Acetobacter, Agrobacterium, Achromobacter, Aerobacter,S arcina, Azotobacter, Rhizobium, Pseudomonas, Salmonella and Alcaligenes produce cellulose. Cellulose is also synthesized by the Gram-positive bacterium Sarcina ventriculi, accounting for about 15 % of the total dry cell mass (15). The most effective producers of cellulose are A. xylinum (16– 19), A. hansenii (20,21) and A. pasteurianus (2).

A. xylinum has been applied as model microorganism for basic and applied studies on cellulose. It is most commonly studied source of bacterial cellulose because of its ability to produce relatively high levels of polymer from a wide range of carbon and nitrogen sources (23). It is a rod-shaped, aerobic, Gram-negative bacterium that produces cellulose in the form of interwoven extracellular ribbons as part of primary metabolite. This bacterium grows and produces cellulose from a wide variety of substrates and is devoid of cellulase activity (24). Various strains producing cellulose and their yields are depicted systematically in Table 1.

It is important to preserve the chosen bacterial strain to guarantee reproducibility of the work as well as to shorten the preparation time. Various techniques like freezing in a suspension using glycerol, dimethyl sulphoxide (DMSO) or skimmed milk as protective agents and drying in gelatin drops have been studied for the preservation of the strain. A useful preservation method should provide high survival rates of A. xylinum, and should have no influence on the cellulose formation. The use of glycerol and skimmed milk as protective agents for freezing is not recommended, since they alter the structure of cellulose produced by A. xylinum and influence the bacterial metabolism. Freezing in a suspension with DMSO has proven to be more efficient with high survival rates and no determinable influence on the structure of the formed bacterial cellulose. Drying the bacterial cells in gelatin drops had no effect on the morphological structure and kinetic parameters, but showed very low survival rate (25).

108 P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009)

Cellulose

ProtofibrilCellulose export component

Cellulose I

LPS envelope Cellulose synthase

Cellulose I

Cytoplasmic membrane Fig. 1. Assembly of cellulose microfibrils by A. xylinum (36)

Biosynthetic Pathway

Synthesis of bacterial cellulose is a precisely and specifically regulated multi-step process, involving a large number of both individual enzymes and complexes of catalytic and regulatory proteins, whose supramolecular structure has not yet been well defined. Pathways and mechanisms of uridine diphosphoglucose (UDPGlc) synthesis are relatively well known, whereas molecular mechanisms of glucose polymerization into long and unbranched chains still need exploring.

Biochemical reactions of cellulose synthesis by A. xylinum are extensively documented (16,26). It is a precisely and specifically regulated multi-step process, involving a large number of individual enzymes and complex of catalytic and regulatory proteins (Fig. 2). The process includes the formation of UDPGlc, which is the precursor in the formation of cellulose, followed by glu- cose polymerization into the b-1 4 glucan chain and a nascent chain which forms ribbon-like structure of cellulose chains formed by hundreds or even thousands of individual cellulose chains, their extrusion outside the

109P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009)

Table 1. Different strains producing microbial cellulose

Microorganism Carbon source Supplement Culture time Yield/(g/L) Reference

A. xylinum BRC 5 glucose ethanol, oxygen 50 h 15.30 (75) G. hansenii PJK (KCTC 10505 BP) glucose oxygen 48 h 1.72 (20) G. hansenii PJK (KCTC 10505 BP) glucose ethanol 72 h 2.50 (21) Acetobacter sp. V6 glucose ethanol 8 day 4.16 (4) Acetobacter sp. A9 glucose ethanol 8 day 15.20 (47) A. xylinum BPR2001 molasses none 72 h 7.820 (52) A. xylinum BPR2001 fructose agar oxygen 72 h 14.10 (64) A. xylinum BPR201 fructose agar 56 h 12.0 (64) Acetobacter xylinum ssp. sucrofermentans BPR2001 fructose oxygen 52 h 10.40 (68) Acetobacter xylinum ssp. sucrofermentans BPR2001 fructose agar oxygen 4 h 8.70 (68) Acetobacter xylinum E25 glucose no 7 day 3.50 (78) G. xylinus strain (K3) mannitol green tea 7 day 3.34 (46) Gluconacetobacter xylinus IFO 13773 glucose lignosulphonate 7 day 10.10 (48) Acetobacter xylinum NUST4.1 glucose sodium alginate 5 day 6.0 (65) Gluconacetobacter xylinus IFO 13773 sugar cane molasses no 7 day 5.76 (53) Gluconacetobacter sp. RKY5 glycerol no 144 h 5.63 (59)

Co-culture of Gluconacetobacter sp. st-60–12

GK Glucose-6-phospate

Fructose-6-phospate Glucose-1-phospate

Fructose -1,6-biphosphateFructose-1-phospate Fructose

UDPGlc Glucose

Cellulose

Phosphogluconic acid

Pentose phosphate cycle

G6PDH NAD, NADP

FK 1FPk

Krebs cycle Gluconeogenesis

Fig. 2. Biochemical pathway for cellulose synthesis by A. xylinum. CS cellulose synthase, GK glucokinase, FBP fructose-1,6-biphosphate phosphatase, FK fructokinase, 1FPk fructose-1-phosphatekinase, PGI phosphoglucoisomerase,PMG phosphoglucomutase, PTS system of phosphotransferases, UGP pyrophosphorylase uridine diphosphoglucose, UDPGlc uridine diphosphoglucose, G6PDH glucose-6-phosphate dehydrogenase, NAD nicotinamide adenine dinucleotide, NADP nicotinamide adenine dinucleotide phosphate cell, and self-assembly into fibrils (23). In A. xylinum, cellulose synthesis is tightly associated with catabolic processes of oxidation and consumes as much as 10 % of energy derived from catabolic reactions. Production of bacterial cellulose does not interfere with other anabolic processes, including protein synthesis. A. xylinum follows either pentose phosphate cycle or the Krebs cycle coupled with gluconeogenesis (27,28).

Synthesis of the Cellulose Precursor

A. xylinum converts various carbon compounds, such as hexoses, glycerol, dihydroxyacetone, pyruvate, and dicarboxylic acids, into cellulose, usually with about 50 % efficiency. Pyruvate and dicarboxylic acids enter the Krebs cycle and due to oxalacetate decarboxylation to pyruvate undergo a conversion to hexoses via gluconeogenesis, similarly to glycerol, dihydroxyacetone, and intermediates of the pentose phosphate cycle (Fig. 2). The direct cellulose precursor is UDPGlc, which is a product of a conventional pathway, common in many organisms, including plants, and involving glucose phosphorylation to glucose-6-phosphate (Glc-6-P), catalyzed by glucokinase, followed by isomerization of this inter- mediate to Glc-a-1-P, catalyzed by phosphoglucomutase, and conversion of the latter metabolite to UDPGlc by UDPGlc pyrophosphorylase. This enzyme seems to be the crucial one involved in cellulose synthesis, since some phenotypic cellulose-negative mutants (Cel–)a re specifically deficient in this enzyme (29), although they display cellulose synthase (CS) activity, which was confirmed in vitro by means of observation of cellulose synthesis, catalyzed by cell-free extracts of Cel– strains (30). Furthermore, the pyrophosphorylaseactivityvariesamong different A. xylinum strains and the highest activity was detected in the most effective cellulose producers, such as A. xylinum ssp. sucrofermentans BPR2001 (23).

(Parte 1 de 8)

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