Biosynthesis and Production of Bacterial Cellulose: Enzymes, Precursors, and Optimization, Notas de estudo de Engenharia de Produção

Baixe Biosynthesis and Production of Bacterial Cellulose: Enzymes, Precursors, and Optimization e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! 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 struc- tural and mechanical properties and is highly pure as compared to plant cellulose. This ar- ticle presents a critical review of the available information on the bacterial cellulose with special emphasis on its fermentative production and applications. Information on the bio- synthetic pathway of bacterial cellulose, enzymes and precursors involved in bacterial cel- lulose 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, fer- mentation Introduction Polysaccharides are a structurally diverse group of biological macromolecules of widespread occurrence in nature. They can be divided according to their morpho- logical localization as: intracellular polysaccharides loca- ted inside, or as part of the cytoplasmic membrane; cell-wall polysaccharides forming a structural part of the cell wall; and extracellular polysaccharides located out- side 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 me- dium; 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 solu- tions. 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 su- gar derivatives, mainly glucose, galactose and rhamnose in different ratios. They are classified into two groups: homopolysaccharides (cellulose, dextran, mutan, pullu- lan, 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
2 or 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 micro- organisms. Exopolysaccharides find wide industrial ap- plications in food, pharmaceutical and other industries like textile, paper, cosmetics, gelling agents and medi- cines for wound dressing (3). Microbial cellulose is an exopolysaccharide produ- ced by various species of bacteria, such as those of the genera Gluconacetobacter (formerly Acetobacter), Agrobac- terium, 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 Acetobac- ter 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) *Corresponding author; Phone: ++91 22 24 145 616; Fax: ++91 22 24 145 614; E-mail: rekha@udct.org The molecular formula of bacterial cellulose (C6H10O5)n is the same as that of plant cellulose, but their physical and chemical features are different (6). Bacterial cellu- lose is preferred over the plant cellulose as it can be ob- tained 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 trans- fer 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 in- terlocked by hydrogen bonds so that a crystalline do- main is produced. This microfibrillar structure of bacte- rial 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 (ii) cellulose II, the thermodynamically more stable amorphous poly- mer (9). The differences in the assembly of cellulose I and cellulose II 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 perfor- mance speaker diaphragms, medical pads (10) and arti- ficial skin (11). 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. Consequent- ly, Acetobacter cellulose may always be more expensive to produce than conventional sources of cellulose (4). For this reason, successful commercialization of Aceto- bacter cellulose will depend on careful selection of appli- cations 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, xy- lan, or mannan may serve as structural cell wall poly- saccharides. Cellulose is found, although in small quan- tities, in all of the brown algae (Phaeophyta), most of the red algae (Rhodophyta), and most of the golden al- gae (Chrysophyta (Chrysophytes)) (13). It was also re- ported 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 cellu- lose in Oomycetes, accounting for about 15 % of the wall dry mass (14). Gram-negative species like Acetobac- ter, Agrobacterium, Achromobacter, Aerobacter, Sarcina, Azo- tobacter, Rhizobium, Pseudomonas, Salmonella and Alcali- genes produce cellulose. Cellulose is also synthesized by the Gram-positive bacterium Sarcina ventriculi, account- ing 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 (22). A. xylinum has been applied as model microorgan- ism 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 extra- cellular ribbons as part of primary metabolite. This bac- terium grows and produces cellulose from a wide vari- ety 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 sul- phoxide (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 suspen- sion 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 mor- phological 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 II LPS envelope Cellulose synthase Cellulose I Cytoplasmic membrane Fig. 1. Assembly of cellulose microfibrils by A. xylinum (36) protofibril of approx. 2–4 nm diameter, and the protofi- brils are bundled in the form of a ribbon-shaped micro- fibril of approx. 80´4 nm (36). Electron micrographs of the surface of the cell enve- lope indicate the presence of some 50 to 80 pore-like sites arranged in a regular row along the long axis of the cell and in evident combination with the extracellular cellu- losic ribbon (37,38). These discrete structures of the lipo- polysaccharide layer are presumed to be the sites of ex- trusion for precellulosic polymers. Aggregates of this size, rather than individual b-1
4 glucan chains, are pos- tulated to be the initial form of the cellulosic product (39). The existence of such tactoidal aggregates, and of analogous structures in algal preparations (39), suggests that the synthesis of many b-1
4 glucan chains simulta- neously at a spatially limited site is a common feature of the assembly of cellulose microfibrils in both higher and lower organisms (40). This process of assembly and crys- tallization of cellulose chains is usually described as cell directed because, although it occurs in the extracellular space, the mutual orientation and association of glucan chains, aggregates, microfibrils, bundles, and ribbon are apparently governed by the original pattern of extrusion sites (27). Stronger aeration or the presence of certain substan- ces that cannot penetrate inside the cells, but can form competitive hydrogen bonds with the b-1
4 glucan chains (carboxymethyl cellulose, fluorescent brightener, calcofluor white), bring significant changes in the supra- molecular organization of cellulose chains. Instead of the ribbon-like polymer, i.e. cellulose I, the thermody- namically more stable amorphous cellulose II is formed. The differences in the assembly of cellulose I and cellu- lose II outside the cytoplasmic membrane are well de- fined in Fig. 1. Typical chain elongation rate, which is 2 mm/min, corresponds to a polymerization of more than 108 glucose molecules in the b-1
4 glucan (23). Fermentative Production of Microbial Cellulose In general, factors affecting cellulose production mainly include growth medium, environmental condi- tions and formation of byproducts. Generally, medium containing high carbon to limiting nutrient ratio (often nitrogen) is favourable for polysaccharide production. Conversion of 60–80 % of the utilized carbon source into crude polymer is commonly found in high yielding polysaccharide fermentations. The optimal design of the medium is very important for the growth of a microor- ganism and thus stimulating the formation of products. Nutrients required for the growth of a microorganism are carbon, nitrogen, phosphorus, sulphur, potassium and magnesium salts. Effect of medium components The fermentation medium contains carbon, nitrogen and other macro- and micronutrients required for the growth of organism. The changes in the medium com- ponents affect the growth and the product formation di- rectly or indirectly. Secretion of exopolysaccharides is usually most noticeable when the bacteria are supplied with an abundant carbon source and minimal nitrogen source (41). Sometimes a complex medium supplying amino acids and vitamins is also used to enhance the cell growth and production (42). Effects of various me- dium components on microbial cellulose production are described in the following sections. Carbon source Usually, glucose and sucrose are used as carbon sources for cellulose production, although other carbo- hydrates such as fructose, maltose, xylose, starch and glycerol have also been tried (43). G. hansenii PJK (KCTC 10505 BP) produced 1.72 g/L of cellulose when glucose was provided as carbon source (21). Acetobacter sp. V6 strain isolated from the traditionally fermented vinegar produced 4.16 g/L cellulose in a complex medium con- taining glucose as a carbon source (44). The effect of ini- tial glucose concentration on cellulose production is also important, since the formation of gluconic acid as a by- product in the medium decreases the pH of the culture and ultimately decreases the production of cellulose. Cellulose yields at initial glucose concentrations of 6, 12, 24 and 48 g/L were studied, and the consumption of glucose was found to be 100, 100, 68 and 28 % of the ini- tial concentration, respectively (43). Ishihara et al. (45) used xylose as a carbon source for the production of cellulose by A. xylinum IFO 15606 and obtained a yield of 3.0 g/L. Sucrose, mannitol and glu- cose were found to be the optimal carbon sources for cellulose production by A. xylinum NCIM 2526 (41). Glu- conacetobacter xylinus strain isolated from kombucha gave maximum cellulose production with mannitol as a car- bon source (46). Ethanol is used as additional carbon source and also to degenerate the cellulose non-producing cells of G. hansenii (Cel–), which can appear under submerged cul- ture conditions. Addition of ethanol increased cellulose production from 1.30 to 2.31 g/L in G. hansenii (21). Son et al. (47) studied the effect of the addition of ethanol on cellulose production by using newly isolated Acetobacter sp. A9 strain. It was observed that with the addition of 1.4 % (by volume) ethanol to the medium, cellulose pro- duction was 15.2 g/L, which was about four times higher than that without ethanol addition. Addition of ethanol was also found to eliminate spontaneous mutation of cellulose non-producing cells. The problem associated with the use of glucose as a carbon source for cellulose production is the formation of gluconic acid as byproduct in the medium which de- creases the pH of the culture and ultimately decreases the production of cellulose. Keshk and Sameshima (48) investigated the formation of gluconic acid and bacterial cellulose production in the presence of lignosulphonate. Gluconic acid production was decreased and bacterial cellulose production was increased when the medium was supplemented with lignosulphonate. This was at- tributed to the inhibition of gluconic acid formation in the presence of antioxidant and polyphenolic com- pounds in lignosulphonate. The effect of the addition of organic acids to GPY- -citric acid buffer medium was studied. Cellulose yield increased from 0.6 to 3.8 g/L in the presence of 20 g/L of acetic acid. The addition of organic acids other than acetic acid (succinic, lactic and gluconic acid) did not 111P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) increase the yield of cellulose. Acetic acid was consu- med as monomeric raw material and mass flow of glu- cose catabolism was partly substituted by another car- bon source (e.g. acetic acid) (49). Matsuoka et al. (42) observed that lactate had a stimulating effect on cellu- lose production when it was added with 4 % (by mass per volume) fructose containing corn steep liquor, yeast extract or peptone as a nitrogen source. However, time of initiation of bacterial cellulose production was differ- ent for different carbon sources. Naritomi et al. (50) also studied the effect of lactate on bacterial cellulose pro- duction from fructose in continuous culture by Acetobac- ter xylinum ssp. sucrofermentans BPR3001A. They report- ed that supplementing 12.5 g/L of lactate to the feed medium increased the cell concentration and fructose consumption at a steady state, resulting in a production rate of 0.90 g/(L·h) and a cellulose yield of 36 % at a di- lution rate of 0.1 h–1. The adenosine triphosphate (ATP) content of viable cells was maintained at a higher level by feeding with a lactate-supplemented medium rather than the unsupplemented corn steep liquor-fructose me- dium. They also indicated that lactate functioned as an energy source, not as a substrate for cellulose biosyn- thesis. Increased intracellular ATP resulting from lactate oxidation may have improved the fructose consumption and cellulose production in the continuous culture. Bae and Shoda (51) studied the production of bacte- rial cellulose by Acetobacter xylinum BPR2001 using mo- lasses medium in a jar fermentor. They reported that subjecting molasses to H2SO4-heat treatment gave a maximum cellulose concentration that was 76 % more than that achieved using untreated molasses, and also that the specific growth rate increased twofold. They also varied the initial sugar concentrations in the H2SO4-heat treated molasses from 23 to 72 g/L, and concluded that maintaining a lower concentration in the molasses is es- sential for efficient cellulose production in jar fermen- tors, the effect being attributed mainly to the complex nature of molasses. Bae and Shoda (52) studied the pro- duction of bacterial cellulose by intermittent and contin- uous fed-batch fermentation in molasses-based medium. They reported a production of 7.82 g/L when 200 mL of molasses medium was added five times by intermittent feeding, whereas maximum bacterial cellulose was ob- tained with a feeding rate of 6.3 g of sugar per h by con- tinuous feeding. Keshk and Sameshima (53) investigated the production of bacterial cellulose using sugarcane molasses in a Hestrin-Schramm medium, and indicated it to be a better carbon source than glucose for cellulose production. Premjet et al. (54) added components of sugarcane molasses such as sucrose, fructose, glucose, nitrogenous compounds, non-nitrogenous acids, nucleic acids, vita- mins, other carbohydrates, minerals and black colour substances individually or in combined forms into Hes- trin-Schramm medium, and investigated their effect on bacterial cellulose production by Acetobacter xylinum ATCC 10245. They concluded that the addition of vita- mins, amino acids, other carbohydrates, minerals and black colour substances to the molasses in the Hestrin- -Schramm medium with a mixture of sucrose and fruc- tose as the carbon source increased the bacterial cellu- lose yield. The black colour substance was the most effective in increasing the production of bacterial cellu- lose. Hornung et al. (55) studied the effect of the mass transfer rate of substrate on the microbial growth of the bacteria, cellulose formation, and the utilization of the substrate. A fundamental model for the diffusion of glu- cose through the growing cellulose layer was proposed. The model confirmed that the increase in diffusional re- sistance is indeed significant, but other factors should also be taken into account. Hornung et al. (56) noted that the growing cellulose is in contact with the wall of the box or beaker, and moves downwards into the nutrient broth with progress of time. They carried out experi- ments where this wall contact was eliminated and a con- stant rate of production over several weeks was found. This indicated the importance of understanding the role of the wall in the usual surface culture. Hornung et al. (57) reported the use of a novel aero- sol bioreactor working on a fed batch principle. This in- volved the generation of an aerosol spray of glucose and its even distribution to the living bacteria on the me- dium-air interface. The apparatus was built and oper- ated up to eight weeks with a constant rate of cellulose production. The aerosol system provided the basis for an economic production of bacterial cellulose in surface culture. Bae and Shoda (51) and Sun et al. (58) optimized the media for the production of bacterial cellulose by using statistical techniques. Sun et al. (58) used the one-factor- -at-a-time and orthogonal array method for culture con- dition optimization. They reported the optimum medium composition of (in %): sucrose 5, protein peptone 1.5, ci- tric acid 0.2, Na2HPO4·12H2O 0.2, KH2PO4 0.2, MgSO4· ·7H2O 0.03, alcohol 1, pH=6.8, and cultivation at 28 °C for 6 days with maximum yield of 2 g/L of cellulose. Bae and Shoda (51) used Box-Behnken design to opti- mize the culture conditions and reported 14.3 g/L of bac- terial cellulose production with 4.99 % fructose, 2.85 % corn steep liquor, 28.33 % dissolved oxygen, and 0.38 % agar. Kim et al. (59) isolated Gluconacetobacter sp. RKY5 from persimmon vinegar and optimized the conditions for maximum cellulose production. The optimized me- dium composition for cellulose production was deter- mined to be (in %): glycerol 1.5, yeast extract 0.8, K2HPO4 0.3, and acetic acid 0.3. In this optimized culture me- dium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of cellulose after 144 h of shaken culture and 4.59 g/L were produced after 144 h of static culture. Seto et al. (60) studied the co-cultivation of Gluconacetobacter xylinus and Lactobacillus mali for maximum production of bacte- rial cellulose. A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bac- terial strains: a cellulose-producing acetic acid bacterium (st-60–12) and a lactic acid bacterium (st-20). Co-cultiva- tion of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield as compared to the st-60–12 monoculture. A simi- lar enhancement was observed in a co-culture with vari- ous L. mali strains, but not with other Lactobacillus sp. 112 P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) Nitrogen source Nitrogen is a main component of proteins necessary in cell metabolism, and comprises 8–14 % of the dry cell mass of bacteria. The effect of various nitrogen sources on the production of bacterial cellulose has been re- ported; casein hydrolyzate gave yield of 5 g/L, and peptone gave yield of 4.8 g/L of cellulose in A. xylinum (41). The addition of extra nitrogen favours the biomass production, but diminishes cellulose production (42). Matsuoka et al. (42) observed that corn steep liquor had a stimulating effect on cellulose production when it was added at 0.15 % (by volume) to the medium with 4 % (by mass per volume) fructose. This was attributed to the presence of lactate in corn steep liquor, which is ab- sent from other nitrogen sources. The effect of yeast extract concentrations on cell growth and cellulose production in different carbon me- dia has been studied. Yeast extract was added to the me- dium in the range from 5 to 60 g/L, while carbon sources comprised 20 g/L; the medium containing 40 g/L of yeast extract yielded maximum concentration (6.7 g/L) of cellulose (61). Effect of precursors The addition of precursor molecules is of consider- able importance in the polysaccharide synthesis in terms of metabolic driving force. Amino acids have been used by some researchers as a nitrogen source or as a stimu- lator for improving biopolymer yield (44). Methionine has an important effect on the cellulose production by Acetobacter xylinum ssp. sucrofermentans, accounting alone for 90 % of cell growth and cellulose production (42). Nicotinamide has also been found to be important for cellulose production. Evaluation of nicotinamide in the fraction range of 0.00001 to 0.00008 % showed that max- imal bacterial cellulose production was at 0.00005 % (44). Vitamins like pyridoxine, nicotinic acid, p-amino- benzoic acid and biotin were also found to be important for the cell growth and cellulose production, but vita- mins like pantothenate and riboflavin were found to have contradictory effects (42). The sugar nucleotides had an important effect on the cell growth and microbial cellulose production. UDPGlc has a stimulating effect on the production of microbial cellulose (44,62). Effect of the addition of water-soluble polysaccharides Bacterial cellulose production in a reactor is com- plex, mainly because the culture used consists of solid bacterial cellulose and cells, liquid medium and air gas. The heterogeneous coagulation of bacterial cellulose forms a large clump on the surface, with adhered cells and the subsequent deterioration of bacterial cellulose pro- duction. Therefore, a homogeneous and small bacterial cellulose suspension is essential for sufficient agitation and oxygen transfer. To minimize the clumps, several vis- cous water-soluble polysaccharides were supplemented to the medium. It is reported that the bacterial cellulose pellets became smaller in the medium supplemented with agar or acetan, which leads to improved bacterial cellulose production. Bacterial cellulose production in a 50-litre internal loop airlift reactor was increased from 6.3 to 8.7 g/L by the addition of 0.1 % agar (63). The ef- fect of agar fractions ranging from 0 to 1 % (by mass per volume) was studied on the Acetobacter xylinum BPR 2001 by Bae et al. (64). The yield of bacterial cellulose in- creased from 8 to 12.8 g/L at an agar fraction of 0.4 %. The culture broth containing agar was more viscous and the free cell number was higher than that of the broth without the agar, suggesting that agar may hinder the coagulation of bacterial cellulose in the broth. The spe- cific growth rate of the cellulose also increased in the presence of agar. Addition of agar was not associated with biochemical metabolism, but rather had a physico- chemical effect on the medium (64). Zhou et al. (65) studied the effect of the addition of sodium alginate on bacterial cellulose production by Acetobacter xylinum NUST4.1. It was observed that so- dium alginate hindered the formation of large clumps of bacterial cellulose, and enhanced cellulose yield. The yield of bacterial cellulose reached 6.0 g/L after the ad- dition of 0.04 % (by mass per volume) sodium alginate into the medium as compared to 3.7 g/L in the control. Miscellaneous The influence of lignosulphonate on bacterial cellu- lose productivity was studied by Keshk and Sameshima (48) using six strains of G. xylinus. The productivity of bacterial cellulose by all strains improved by almost 57 % in the presence of 1 % (by mass per volume) ligno- sulphonate. The stimulatory effect on the bacterial cellu- lose membrane production was attributed to the high molecular mass lignosulphonate fraction. Calcofluor white ST, an optical brightener for cellu- lose, increased the rate of glucose polymerization into cellulose. A. xylinum bacterium normally produces a rib- bon of cellulose that is a composite of crystalline micro- fibrils, but calcofluor white ST above 0.1 mM disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiomet- ric binding to glucose residues of newly polymerized cellulose polymer chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10–15 % (39). Addition of xylose isomerase to the me- dium containing D-xylose at the beginning of fermenta- tion increased the production of cellulose from 1.2 to 3 g/L (45). During continuous production of cellulose, A. xyli- num cells, which produce large amounts of cellulose, are rather difficult to transfer from one inoculum to the next as the cells frequently become entangled in the thick cel- lulosic pellicle. A large inoculum is necessary for the large scale production of cellulose. Cellulase enzyme preparations added at appropriate concentrations do not impede cell growth but dissolve cellulose around the cells. Using this technique, dense cultures of cellulose- -producing microorganisms may be produced and used for various purposes. Addition of cellulase at 0.000375 to 0.015 U/mL increased the rate of cellulose synthesis by A. xylinum. Brown (66) and Nakamura et al. (67) re- ported an increase in cellulose productivity by A. xyli- num even in the presence of a small amount of heat-de- natured cellulase. They also found that biosynthesis of cellulose in A. xylinum began at the earlier stage of fer- mentation as compared to the control. 113P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) The average thickness (or the grammage) of the fi- nal dried pellicle strongly affects its performance and hence must be carefully controlled. The fermentation process must be interrupted at a certain stage in order to obtain, after the final treatments, a dried pellicle pre- senting a grammage within a defined interval. Due to the relatively high complexity of the process, it is diffi- cult to determine the time to stop the fermentation, leading to industrial losses that are frequently higher than 60 %. Borzani and De Souza (85) invented a simple method to control the bacterial cellulose production in static culture in order to obtain dried pellicles of desired average thickness. An equation was proposed to evalu- ate the volume of inoculated medium to be placed in a given tray, so that a completely dried film of a desired average thickness could be produced: V G A R A t= × + × + ×( )1 a s r r /2/ where G is the grammage of the dried film (g/m2), A is the area of air/medium interface (m2), a is a relative increase in film area due to pressing, r is the density of inoculated medium (g/mL), s is dried content of wet film (g/g), t is time (day), and R is evaporation rate (L/(day·m2). Continuous production The microbial cellulose can be produced under sta- tic conditions. Hestrin-Schramm medium was added to the smaller or larger culture pans. The depth of the cul- ture medium in each pan was 3–4 mm. The media were inoculated with the subcultured A. xylinum at 28 °C un- der static conditions. After two days of incubation, the edge of the pellicle produced on the surface was picked up, passed through the sodium dodecyl sulphate (SDS) bath to denature the bacterial cell wall. After 2 or 3 days of static cultivation, thin bacterial cellulose gel was formed and the harvest was started on the roller system. The tensile strength of the filament was found to be sig- nificantly stronger than the ordinary cellulose fibres (86). Genetic Modification for Production of Bacterial Cellulose The Gram-negative bacterium A. xylinum has been studied as a model organism for cellulose biosynthesis. This organism has a cellulose synthase operon (AxCes operon) consisting of three or four genes (87–90). In ad- dition, two genes, cmcax and ccpax, are located in the upstream region of the operon (91). De Wulf et al. (92) attempted to improve cellulose productivity genetically by generating a mutant with re- stricted ketogluconate synthesis. They used UV muta- genesis to obtain a ketogluconate-nonproducing mutant from a parent strain, and its cellulose production in- creased from 1.8 g/L by the parent strain to 3.3 g/L af- ter 10 days of shaking culture. This was supported by the fact that, when glucose or sucrose is used as the car- bon source by G. xylinus, the main product is not cellu- lose, but ketogluconate, which decreases the pH, cell growth and cellulose production. G. xylinus secretes not only water-insoluble cellulose, but also acetan, a viscous water-soluble polysaccharide which decreases fluidity of the culture broth adversely affecting the cellulose production. Acetan consumes UDPGlc for self-synthesis, which is also the starting ma- terial for cellulose synthesis. If acetan is not synthesized, the amount of UDPGlc used for cellulose synthesis is ex- pected to increase, resulting in an increase in cellulose production. Ishida et al. (93) generated the acetan-non- producing mutant strain, EP1, from the parental strain G. xylinus BPR2001. However, the cellulose production by EP1 decreased in the shake flask culture, although the productivity was maintained at the same level as that of the parent strain BPR2001 in a static culture. They found that the culture broth of EP1 became a het- erogeneous suspension, containing large flocks formed by the aggregation of cells and cellulose, compared to that of the parent strain in the shake flask. This might be because acetan increases the viscosity of the culture, pre- venting the coagulation of cells and cellulose, resulting in an increased production. They also reported that when agar was added to the medium, similar effect to that of acetan was shown towards cellulose production. When agar was added at the start of cultivation, cellu- lose production was observed from the beginning of the experiments, and the cultivation time was reduced to two thirds as compared to that without the addition of agar. The gene dgc1 is known to be important for activat- ing bacterial cellulose synthesis. Bae et al. (62) reported on cloning and sequencing the dgc gene associated with the regulation of c-di-GMP produced by A. xylinum BPR 2001 and determined the relationship between the struc- tural characteristics and production of cellulose formed by the dgc1 gene-disrupted mutant cultured using differ- ent cultivation methods. They unexpectedly found that the cellulose production by a mutant was almost the same as that of the parent strain in static and shake flask cultivations. Moreover, when the mutant was cultivated in the stirred tank reactor, the cellulose production in- creased by 36 % compared to that of the parent strain. They speculated that although dgc1 was disrupted, cel- lulose production increased because other genes, dgc2 and dgc3, which are functionally similar to those of dgc1, worked complementarily or were more stimulatory for cellulose synthesis. Therefore, they concluded that dgc1 disruption is not critical for total bacterial cellulose pro- duction. However, Tal et al. (94) reported that cellulose production decreased with dgc1 disruption. Although these two results seem to be inconsistent, the cultivation time used by Tal et al. (94) was too short to evaluate the final cellulose production, as the growth rate of the mu- tant was slower than that of the parental strain. Kawano et al. (95) reported on cloning of cellulose synthesis related genes from A. xylinum ATCC23769 and ATCC53582. They cloned about 14.5 kb of DNA frag- ments from A. xylinum ATCC23769 and ATCC53582, and determined their nucleotide sequences. The sequenced DNA regions contained endo-b-1,4-glucanase, cellulose complementing protein, cellulose synthase subunits AB, C and D, and b-glucosidase genes. They found that cel- lulose production by ATCC53582 was 5 times greater than that of ATCC23769 during a 7-day incubation. In 116 P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) A. xylinum ATCC53582, the synthesis of cellulose contin- ued after glucose was consumed, suggesting that a me- tabolite of glucose, or a component of the medium other than glucose, may be a substrate for the production of cellulose. They concluded that the synthesis of cellulose and the growth of bacterial cells are contradictory. To understand the structure–function relationships of endoglucanase gene (CMCax), which is involved in both cellulose hydrolysis and synthesis, Kawano et al. (96) studied crystallization and preliminary crystallo- graphic analysis of the cellulose biosynthesis-related protein CMCax (EC 3.2.1.4) from A. xylinum. They over- expressed CMCax in Escherichia coli, then purified and crystallized it. CMCax, a protein coded by the CMCax gene, is an endoglucanase that has cellulose-hydrolyzing activity, while the CCPax product is suggested to be in- volved in cellulose crystallization (97). Kawano et al. (90) reported that over-expression of the CMCax gene in A. xylinum enhances cellulose production, so does the addi- tion of the CMCax protein into the culture medium. Kawano et al. (98) revealed a regulation mechanism of CMCax expression in a cellulose non-producing mutant of A. xylinum using an enzyme assay and real-time quan- titative reverse transcriptase polymerase chain reaction (qRT-PCR). They also studied condensation activity in A. xylinum, which produces b-glucodisaccharides from glucose. Furthermore, they investigated the CMCax gene expression in a wild-type strain using real-time qRT-PCR and concluded that gentiobiose induced CMCax expres- sion and enhanced CMCax activity, suggesting that the concentrations of gentiobiose in the culture regulate cel- lulose production in A. xylinum. Shigematsu et al. (99) cloned a gene fragment encod- ing a putative pyrroloquinoline quinone glucose dehy- drogenase from Gluconacetobacter xylinus strain BPR2001, which was isolated as a high bacterial cellulose pro- ducer when using fructose as the carbon source. A glu- tamate dehydrogenase (GDH)-deficient mutant of strain BPR2001, namely GD-I, was generated via gene disrup- tion using the cloned gene fragment. Strain GD-I pro- duced no gluconic acid but it produced 4.1 g/L of cellu- lose aerobically in a medium containing glucose as the carbon source. The ability of strain GD-I to convert glu- cose to bacterial cellulose was approx. 1.7-fold higher than that of the wild type. Nobles and Brown (100) reported on the functional expression of a partial cellulose synthase operon (acs - ABDC) of G. xylinus in the unicellular cyanobacterium, Synechococcus leopoliensis strain UTCC 100 (synonym, Synechococcus elongatus strain PCC 7942), resulting in the production of non-crystalline cellulose. They sought to combine the prodigious cellulose biosynthetic capacity of G. xylinus with the photosynthetic ability of cyano- bacteria. Properties Microbial cellulose possesses high crystallinity, high tensile strength, extreme insolubility in most of the sol- vents, moldability and high degree of polymerization (101–103). The thickness of cellulose fibrils is generally 0.1–10 mm, one hundred times thinner than that of cellu- lose fibrils obtained from plants with good shape re- tention. Its water holding capacity is over 100 times (by mass) higher. Microbial cellulose is far stronger than plant cellulose (82,104). Macroscopic morphology of cel- lulose strictly depends on the culture conditions, which can easily be tailored for the physicochemical properties. Wanichapichart et al. (105) demonstrated that cellulose fibre had the degree of polymerization of 793, with a cor- responding molecular mass of approx. 142.73 kDa. Cellulose is soluble in concentrated acids like sul- phuric, hydrochloric or nitric acid. It is also soluble in 8.5 % NaOH solution. The solubility of cellulose in the alkali can be increased by adding 1 % of urea to the so- lution (106). At higher temperatures (>300 °C) the biopolymer degrades, although the alkali-treated cellulose membra- ne is more stable (between 343 and 370 °C). Composites prepared by adding bacterial cellulose and microfibril- lated cellulose (MFC) processed through fibrillation of kraft pulp were compared for mechanical properties and it was found that the bending strength increased up to 425 MPa, while the Young’s modulus increased from 19 to 28 GPa, nearly retaining the modulus of the bacterial cellulose sheets (107,108). The mechanical properties of cellulose are due to the uniqueness of uniform nano-sca- lar network structure, which is oriented bi-dimensiona- lly when compressed. George et al. (101) studied the swelling property of cellulose under different conditions. NaOH at lower concentration caused greater swelling in fibres as com- pared to other alkalis at the same concentrations. The percentage mass gain by the cellulose membranes after immersion in different alkali solutions was found to be in the order of NaOH>KOH>Na2CO3>K2CO3. The per- vaporation characteristics of deproteinated microbial cel- lulose membrane were investigated over a wide range of water-ethanol feed composition and it was found to be promising for dehydration of azeotropes of ethanol. It has a high selectivity towards water at a reasonable flux (109). The basic characteristics of cellulose membra- ne as a molecular separation medium in aqueous condi- tions, and with modification of the structure by chemi- cal treatments for controlling its molecular permeation characteristics are well described (110). The most attrac- tive feature of bacterial cellulose production is the abil- ity to control and modify not only the physical characte- ristics, but also the chemical composition of the cellulose fibre (72). The structure of the cellulose assembly can be altered by using direct dyes (amide black, trypan red), fluorescent brightening agents (congo red) or derivatives like carboxymethyl cellulose (111–113). A. xylinum was cultured in Hestrin-Schramm medium (control medium) and Hestrin-Schramm medium containing acetyl gluco- mannan. The presence of acetyl glucomannan in the me- dium prevents the assembly of cellulose microfibrils and changes the crystal structure of cellulose (86). Cultiva- tion of A. xylinum in Hestrin-Schramm medium contain- ing glucuronoxylan (xylan medium) showed loose bun- dles of cellulose microfibrils in the medium. In contrast, cellulose ribbons were formed in the pectin medium. Glucuronoxylan in the medium prevented the assembly 117P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) of cellulose microfibrils and changed the crystal struc- ture of cellulose, whereas pectin in the medium scarcely had an effect (114). Chemical Structure Cellulose is a homopolymer consisting of glucose glycosidically linked in a b-1
4 conformation (Fig. 3). The repeating unit of the polymer synthesis consists of two glucose molecules bonded together in such a way that one molecule is rotated 180 degrees with respect to the other. The chemical structure of bacterial cellulose is simi- lar to that of plant cellulose but the degree of polymer- ization differs from about 13 000 to 14 000 for plant and 2000–6000 for bacterial cellulose (115). The glucose units in cellulose are bound together to produce a long straight unbranched polymer chain and the capacity to form intermolecular hydrogen bonds between adjacent glucan chains is extremely high. A. xylinum cellulose consists of ribbons of microfibrils generated at the surface of the bacterial cell. The dimensions of the ribbons are 3–4 nm thick and 70–80 nm wide. The shape of microbial cellu- lose sheet seems to be maintained by hydrophobic bonds. It is reported that in the course of time inter- and intra- molecular hydrogen bonds initially occur in each cellu- lose sheet, and then the cellulose crystalline structure is formed with the development of hydrogen bonds between cellulose sheets (23). The microfibrilar structure of microbial and plant cellulose under the scanning electron microscope (SEM) is shown in Fig. 4. The structural differences of cellulose produced by stationary and agitated culture were stud- ied by using nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) (81,82). The existence of tunnels as observed by scanning elec- tron microscope (SEM) argues for some kind of coordi- nation during the pellicle formation and a random for- mation of cellulose microfibrils (116). Two common crystalline forms of cellulose designated as I and II are distinguishable by X-ray diffraction (117). The microbial cellulose observed under SEM showed a significant difference in appearance of the external and internal surfaces of the pellicles. The external surfaces had irregular clusters of fibrils, whereas internal sur- faces were organized in fractured sections. At higher magnification, layers of tunnels in the bacterial cellulose of about 7 mm in diameter were found (118). The solid- -phase nitration and acetylation processes for bacterial cellulose were studied using CP/MAS 13C NMR spec- troscopy together with wide-angle X-ray diffractometry and transmission electron microscopy (TEM). The rela- tive reactivity of the OH groups in the glucose residues was found to decrease in the order of 6'OH>2'OH>3'OH. Moreover, the nitration rate greatly depends on the con- centration of nitric acid in the reaction media. At lower concentrations, the 6'OH groups in the crystalline and disordered components were subjected to nitration at nearly the same rate, indicating that these two compo- nents were distributed almost randomly in the entire region of each microfibril. In contrast, all OH groups un- derwent nitration very rapidly at the higher concentra- tion, although nitration levelled off to a certain extent for 3'OH groups. In solid-phase acetylation, no regio- selective reactivity was observed among the three kinds of OH groups, which may be due to the characteristic reaction that precedes in a very thin layer between the acetylated and nonacetylated regions in each microfibril (119). Recovery and Purification The microbial cellulose obtained after fermentation is not pure; it contains some impurities like cells and/or the medium components. Care must be taken in the in- terpretation of such yields, as crude products will often contain cells, which are bound to the polymer when it is recovered from fermentation broth (23). The fermented broth has to be purified to obtain pure cellulose. The process of isolation and purification of microbial cellu- lose is described in Fig. 5. The most widely used process of purification of bac- terial cellulose in the culture medium is the treatment with alkali (sodium hydroxide or potassium hydroxide), organic acids like acetic acid or repeated washing of the mixtures with the reverse osmosis water or hot tap wa- 118 P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) Non-reducing end Reducing end n-2 OH OH OH OH O OH OH OH O OH OH OH O OH OH Fig. 3. Repeating units of cellulose a) b) Fig. 4. Microfibrillar organization between (a) Acetobacter cellu- lose, and (b) wood pulp (5000´) (7) of stirred tank fermentations. The isolation of a geneti- cally stable strain with a substantially reduced ability to form gluconic acid has been achieved by relatively stan- dard techniques of mutagenesis and selection; this strain is reported to allow rapid and reliable culture on a glu- cose substrate, from which the major product is fibrillar cellulose. Another approach to increase the bacterial cellulose production is to genetically modify the bacteria. G. xylinus has a long doubling time compared to most other bacteria, such as E. coli and B. subtilis. Since their growth rates are relatively faster than that of G. xylinus, genetic modification of these bacteria will also be one possible means of increasing bacterial cellulose produc- tion. A very promising line of advance toward obtaining industrially valuable strains is in the direct genetic ma- nipulation of the genes coding for the catalysts of cellu- lose synthesis, their adjunctive regulatory enzymes, and the relevant associated membrane structures such as the postulated extrusion pores. However, bacterial cellulose obtained from genetically modified organisms may face regulatory restrictions in medical and food industry. References 1. J.F. Wilkinson, The extracellular polysaccharides of bacte- ria, Bacteriol. Rev. 22 (1958) 46–73. 2. I.W. Sutherland, Structure-function relationship in micro- bial exopolysaccharides, Biotechnol. Adv. 12 (1994) 393–448. 3. I.W. Sutherland, Novel and established applications of mi- crobial polysaccharides, Trends Biotechnol. 16 (1998) 41–46. 4. M. Shoda, Y. Sugano, Recent advances in bacterial cellulo- se production, Biotechnol. Bioprocess Eng. 10 (2005) 1–8. 5. A.J. Brown, On an acetic ferment which forms cellulose, J. Chem. Soc. Trans. 49 (1886) 432–439. 6. F. Yoshinaga, N. Tonouchi, K. Watanabe, Research pro- gress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material, Biosci. Biotechnol. Biochem. 61 (1997) 219–224. 7. W. Czaja, A. Krystynowicz, S. Bielecki, R.M. Brown Jr., Mi- crobial cellulose – The natural power to heal wounds, Bio- materials, 27 (2006) 145–151. 8. K. Mühlethaler, The structure of bacterial cellulose, Bio- chim. Biophys. Acta, 3 (1949) 527–535. 9. X. Yu, R.H. Atalla, Production of cellulose II by Acetobacter xylinum in the presence of 2,6-dichlorobenzonitrile, Int. J. Biol. Macromol. 19 (1996) 145–146. 10. E.J. Vandamme, S. De Baets, A. Vanbaelen, K. Joris, P. De Wulf, Improved production of bacterial cellulose and its application potential, Polym. Degrad. Stabil. 59 (1998) 93– 99. 11. Y. Nishi, M. Uryu, S. Yamanaka, K. Watanabe, N. Kitamu- ra, M. Iguchi, S. Mitsuhashi, The structure and mechanical properties of sheets prepared from bacterial cellulose, J. Mater. Sci. 25 (1990) 2997–3001. 12. R.L. Legge, Microbial cellulose as a specialty chemical, Biotechnol. Adv. 8 (1990) 303–319. 13. P.A. Richmond: Occurrence and Functions of Native Cellu- lose. In: Biosynthesis and Biodegradation of Cellulose, C.H. Haigler, P.J. Weimer (Eds.), Marcel Dekker, Inc. New York, USA (1991) pp. 5–23. 14. S. Isizawa, M. Araragi: Chromogenicity of Actinomycetes. In: Actinomycetes: The Boundary Microorganisms, T. Arai (Ed.), Toppan Co., Tokyo, Japan (1976) pp. 43–65. 15. W.D. Bellamy, Single cell proteins from cellulosic wastes, Biotechnol. Bioeng. 16 (1974) 869–880. 16. R.M. Brown Jr., The biosynthesis of cellulose, Food Hydro- colloids, 1 (1987) 345–351. 17. Z. Gromet-Elhanan, S. Hestrin, Synthesis of cellulose by Acetobacter xylinum. VI. Growth on citric acid-cycle inter- mediates, J. Bacteriol. 85 (1963) 284–292. 18. U. Geyer, D. Klemm, H.P. Schmauder, Kinetics of the utili- zation of different C sources and the cellulose formation by Acetobacter xylinum, Acta. Biotechnol. 14 (1994) 261–266. 19. U. Geyer, T. Heinze, A. Stein, D. Klemm, S. Marsch, D. Schumann, H.P. Schmauder, Formation, derivatization and applications of bacterial cellulose, Int. J. Biol. Macromol. 16 (1994) 343–347. 20. J.Y. Jung, J.K. Park, H.N. Chang, Bacterial cellulose pro- duction by Gluconoacetobacter hansenii in an agitated cultu- re without living non-cellulose producing cells, Enzyme Microb. Technol. 37 (2005) 347–354. 21. J.K. Park, J.Y. Jung, Y.H. Park, Cellulose production by Gluconacetobacter hansenii in a medium containing ethanol, Biotechnol. Lett. 25 (2003) 2055–2059. 22. T. Yoshino, T. Asakura, K. Toda, Cellulose production by Acetobacter pasteurianus on silicone membrane, J. Ferment. Bioeng. 81 (1996) 32–36. 23. S. Bielecki, A. Krystynowicz, M. Turkiewicz, H. Kalinow- ska: Bacterial Cellulose. In: Polysaccharides and Polyamides in the Food Industry, A. Steinbüchel, S.K. Rhee (Eds.), Wi- ley-VCH Verlag, Weinheim, Germany (2005) pp. 31–85. 24. V.P. Puri, Effect of crystallinity and degree of polymeriza- tion of cellulose on enzymatic saccharification, Biotechnol. Bioeng. 26 (1984) 1219–1222. 25. C. Wiegand, D. Klemm, Influence of protective agents for preservation of Gluconacetobacter xylinus on its cellulose production, Cellulose, 13 (2006) 485–492. 26. D.P. Delmer, Y. Amor, Cellulose biosynthesis, Plant Cell, 7 (1995) 987–1000. 27. P. Ross, R. Mayer, M. Benziman, Cellulose biosynthesis and function in bacteria, Microbiol. Rev. 55 (1991) 35–58. 28. N. Tonouchi, T. Tsuchida, F. Yoshinaga, T. Beppu, S. Hori- nouchi, Characterization of the biosynthetic pathway of cellulose from glucose and fructose in Acetobacter xylinum, Biosci. Biotechnol. Biochem. 60 (1996) 1377–1379. 29. S. Valla, D. H. Coucheron, E. Fjaervik, J. Kjosbakken, H. Weinhouse, P. Ross, D. Amikam, M. Benziman, Cloning of a gene involved in cellulose biosynthesis in Acetobacter xy- linum: Complementation of cellulose-negative mutant by the UDPG pyrophosphorylase structural gene, Mol. Gen. Genet. 217 (1989) 26–30. 30. I.M. Saxena, R. M. Brown Jr.: Cellulose Biosynthesis in Acetobacter xylinum: A Genetic Approach. In: Cellulose and Wood – Chemistry and Technology, C. Schuerch (Ed.), John Wiley & Sons, Inc., New York, USA (1989) pp. 537–557. 31. F.C. Lin, R.M. Brown Jr., J.B. Cooper, D.P. Delmer, Synthe- sis of fibrils in vitro by a solubilized cellulose synthase from Acetobacter xylinum, Science, 230 (1985) 822–825. 32. R.M. Brown Jr., I.M. Saxena, Cellulose biosynthesis: A mo- del for understanding the assembly of biopolymers, Plant Physiol. Biochem. 38 (2000) 57–67. 33. L. Einfeldt, D. Klemm, H.P. Schmauder, Acetylated car- bohydrate derivatives as C-sources for Acetobacter xylinum, Nat. Prod. Res. 2 (1993) 263–269. 34. N.I. De Iannino, R.O. Couso, M.A. Dankert, Lipid-linked intermediates and the synthesis of acetan in Acetobacter xylinum, J. Gen. Microbiol. 134 (1988) 1731–1736. 35. F.C. Lin, R.M. Brown Jr.: Purification of Cellulose Synthase from Acetobacter xylinum. In: Cellulose and Wood: Chemistry and Technology, C. Schuerch (Ed.), John Wiley & Sons, Inc., New York, USA (1989) pp. 473–492. 121P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) 36. M. Iguchi, S. Yamanaka, A. Budhiono, Bacterial cellulose – A masterpiece of nature’s arts, J. Mater. Sci. 35 (2000) 261– 270. 37. R.M. Brown Jr., J.H.M. Willison, C.L. Richardson, Cellulose biosynthesis in Acetobacter xylinum: Visualization of the site of synthesis and direct measurement of the in vivo process, Proc. Natl. Acad. Sci. USA, 73 (1976) 4565–4569. 38. K. Zaar, Visualization of pores (export sites) correlated with cellulose production in the envelope of the Gram-negative bacterium Acetobacter xylinum, J. Cell Biol. 80 (1979) 773–777. 39. M. Benziman, C.H. Haigler, R.M. Brown Jr., A.R. White, K.M. Cooper, Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum, Proc. Natl. Acad. Sci. USA, 77 (1980) 6678–6682. 40. D.P. Delmer, Cellulose biosynthesis, Ann. Rev. Plant Phy- siol. 38 (1987) 259–290. 41. K.V. Ramana, A. Tomar, L. Singh, Effect of various carbon and nitrogen sources on cellulose synthesis by Acetobacter xylinum, World J. Microbiol. Biotechnol. 16 (2000) 245–248. 42. M. Matsuoka, T. Tsuchida, K. Matsushita, O. Adachi, F. Yoshinaga, A synthetic medium for bacterial cellulose pro- duction by Acetobacter xylinum subsp. sucrofermentans, Biosci. Biotechnol. Biochem. 60 (1996) 575–579. 43. S. Masaoka, T. Ohe, N. Sakota, Production of cellulose from glucose by Acetobacter xylinum, J. Ferment. Bioeng. 75 (1993) 18–22. 44. H.J. Son, H.G. Kim, K.K. Kim, H.S. Kim, Y.G. Kim, S.J. Lee, Increased production of bacterial cellulose by Acetobacter sp. V6 in synthetic media under shaking culture condi- tions, Bioresour. Technol. 86 (2003) 215–219. 45. M. Ishihara, M. Matsunaga, N. Hayashi, V. Ti{ler, Utiliza- tion of D-xylose as carbon source for production of bacte- rial cellulose, Enzyme Microb. Technol. 31 (2002) 986–991. 46. V.Y. Nguyen, B. Flanagan, M.J. Gidley, G.A. Dykes, Char- acterization of cellulose production by a Gluconacetobacter xylinus strain from kombucha, Curr. Microbiol. 57 (2008) 449–453. 47. H.J. Son, M.S. Heo, Y.G. Kim, S.J. Lee, Optimization of fer- mentation conditions for the production of bacterial cellu- lose by a newly isolated Acetobacter sp. A9 in shaking cul- tures, Biotechnol. Appl. Biochem. 33 (2001) 1–5. 48. S. Keshk, K. Sameshima, Influence of lignosulfonate on crystal structure and productivity of bacterial cellulose in a static culture, Enzyme Microb. Technol. 40 (2006) 4–8. 49. K. Toda, T. Asakura, M. Fukaya, E. Entani, Y. Kawamura, Cellulose production by acetic acid-resistant Acetobacter xylinum, J. Ferment. Bioeng. 84 (1997) 228–231. 50. T. Naritomi, T. Kouda, H. Yano, F. Yoshinaga, Effect of lac- tate on bacterial cellulose production from fructose in con- tinuous culture, J. Ferment. Bioeng. 85 (1998) 89–95. 51. S. Bae, M. Shoda, Statistical optimization of culture condi- tions for bacterial cellulose production using Box-Behnken design, Biotechnol. Bioeng. 90 (2005) 20–28. 52. S. Bae, M. Shoda, Bacterial cellulose production by fed- -batch fermentation in molasses medium, Biotechnol. Progr. 20 (2004) 1366–1371. 53. S. Keshk, K. Sameshima, The utilization of sugar cane mo- lasses with/without the presence of lignosulfonate for the production of bacterial cellulose, Appl. Microbiol. Biotechnol. 72 (2006) 291–296. 54. S. Premjet, D. Premjet, Y. Ohtani, The effect of ingredients of sugar cane molasses on bacterial cellulose production by Acetobacter xylinum ATCC 10245, Sen-i Gakkaishi, 63 (2007) 193–199. 55. M. Hornung, M. Ludwig, A.M. Gerrard, H.P. Schmauder, Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction (Part 1), Eng. Life Sci. 6 (2006) 537–545. 56. M. Hornung, M. Ludwig, A.M. Gerrard, H.P. Schmauder, Optimizing the production of bacterial cellulose in surface culture: Evaluation of product movement influences on the bioreaction (Part 2), Eng. Life Sci. 6 (2006) 546–551. 57. M. Hornung, M. Ludwig, H.P. Schmauder, Optimizing the production of bacterial cellulose in surface culture: A no- vel aerosol bioreactor working on a fed batch principle (Part 3), Eng. Life Sci. 7 (2007) 35–41. 58. D.P. Sun, J.D. Zhang, L.L. Zhou, M.Y. Zhu, Q.H. Wu, C.Y. Xu, Production of bacterial cellulose with Acetobacter xyli- num 1.1812 fermentation, J. Nanjing Univ. Sci. Technol. 29 (2005) 601–604. 59. S.Y. Kim, J.N. Kim, Y.J. Wee, D.H. Park, H.W. Ryu, Produc- tion of bacterial cellulose by Gluconacetobacter sp. RKY5 iso- lated from persimmon vinegar, Appl. Biochem. Biotechnol. 131 (2006) 705–715. 60. A. Seto, Y. Saito, M. Matsushige, H. Kobayashi, Y. Sasaki, N. Tonouchi, T. Tsuchida, F. Yoshinaga, K. Ueda, T. Beppu, Effective cellulose production by a coculture of Gluconace- tobacter xylinus and Lactobacillus mali, Appl. Microbiol. Bio- technol. 73 (2006) 915–921. 61. Y.K. Yang, S.H. Park, J.W. Hwang, Y.R. Pyun, Y.S. Kim, Cellulose production by Acetobacter xylinum BRC5 under agitated condition, J. Ferment. Bioeng. 85 (1998) 312–317. 62. S.O. Bae, Y. Sugano, K. Ohi, M. Shoda, Features of bacte- rial cellulose synthesis in a mutant generated by disrup- tion of the diguanylate cyclase 1 gene of Acetobacter xyli- num BPR 2001, Appl. Microbiol. Biotechnol. 65 (2004) 315– 322. 63. Y. Chao, M. Mitarai, Y. Sugano, M. Shoda, Effect of addi- tion of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor, Biotechnol. Progr. 17 (2001) 781–785. 64. S. Bae, Y. Sugano, M. Shoda, Improvement of bacterial cel- lulose production by addition of agar in a jar fermentor, J. Biosci. Bioeng. 97 (2004) 33–38. 65. L.L. Zhou, D.P. Sun, L.Y. Hu, Y.W. Li, J.Z. Yang, Effect of addition of sodium alginate on bacterial cellulose produc- tion by Acetobacter xylinum, J. Ind. Microbiol. Biotechnol. 34 (2007) 483–489. 66. M.R. Brown, Use of cellulase preparations in the cultiva- tion and use of cellulose-producing microorganisms. Euro- pean patent 19870307513 (1993). 67. T. Nakamura, K. Tajima, M. Fujiwara, M. Takai, J. Hayashi, Cellulose production by Acetobacter xylinum in the presen- ce of cellulose, Use of Minerals in Papermaking (1998) 3–8. 68. Y. Chao, T. Ishida, Y. Sugano, M. Shoda, Bacterial cellulose production by Acetobacter xylinum in a 50L internal-loop airlift reactor, Biotechnol. Bioeng. 68 (2000) 345–352. 69. S. Hestrin, M. Schramm, Synthesis of cellulose by Aceto- bacter xylinum: II. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose, Biochem. J. 58 (1954) 345–352. 70. S. Kongruang, Bacterial cellulose production by Acetobacter xylinum strains from agricultural waste products, Appl. Biochem. Biotechnol. 148 (2008) 245–256. 71. N. Noro, Y. Sugano, M. Shoda, Utilization of the buffering capacity of corn steep liquor in bacterial cellulose produc- tion by Acetobacter xylinum, Appl. Microbiol. Biotechnol. 64 (2004) 199–205. 72. A. Shirai, M. Takahashi, H. Kaneko, S. Nishimura, M. Ogawa, N. Nishi, S. Tokura, Biosynthesis of a novel poly- saccharide by Acetobacter xylinum, Int. J. Biol. Macromol. 16 (1994) 297–300. 73. T. Kouda, T. Naritomi, H. Yano, F. Yoshinaga, Effects of oxygen and carbon dioxide pressures on bacterial cellulose 122 P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009) production by Acetobacter in aerated and agitated culture, J. Ferment. Bioeng. 84 (1997) 124–127. 74. S. Tantratian, P. Tammarate, W. Krusong, P. Bhattarakosol, A. Phunsri, Effect of dissolved oxygen on cellulose pro- duction by Acetobacter sp., J. Sci. Res. Chula Univ. 30 (2005) 179–186. 75. J.W. Hwang, Y.K. Yang, J.K. Hwang, Y.R. Pyun, Y.S. Kim, Effects of pH and dissolved oxygen on cellulose produc- tion by Acetobacter xylinum BRC5 in agitated culture, J. Biosci. Bioeng. 88 (1999) 183–188. 76. Y. Chao, Y. Sugano, M. Shoda, Bacterial cellulose produc- tion under oxygen-enriched air at different fructose con- centrations in a 50-liter, internal-loop airlift reactor, Appl. Microbiol. Biotechnol. 55 (2001) 673–679. 77. J.Y. Kim, J.N. Kim, Y.J. Wee, D.H. Park, H.W. Ryu, Bacte- rial cellulose production by Gluconacetobacter sp. RKY5 in a rotary biofilm contactor, Appl. Biochem. Biotechnol. 137 (2007) 529–537. 78. A. Krystynowicz, W. Czaja, A. Wiktorowska-Jezierska, M. Gonçalves-Miœkiewicz, M. Turkiewicz, S. Bielecki, Factors affecting the yield and properties of bacterial cellulose, J. Ind. Microbiol. Biotechnol. 29 (2002) 189–195. 79. J.Y. Jung, T. Khan, J.K. Park, H.N. Chang, Production of bacterial cellulose by Gluconacetobacter hansenii using a novel bioreactor equipped with a spin filter, Korean J. Chem. Eng. 24 (2007) 265–271. 80. W. Czaja, D. Romanovicz, R.M. Brown, Structural investi- gations of microbial cellulose produced in stationary and agitated culture, Cellulose, 11 (2004) 403–411. 81. C. Tokoh, K. Takabe, M. Fujita, H. Saiki, Cellulose synthe- sized by Acetobacter xylinum in the presence of acetyl glucomannan, Cellulose, 5 (1998) 249–261. 82. K. Watanabe, M. Tabuchi, Y. Morinaga, F. Yoshinaga, Struc- tural features and properties of bacterial cellulose pro- duced in agitated culture, Cellulose, 5 (1998) 187–200. 83. T. Kouda, H. Yano, F. Yoshinaga, Effect of agitator configu- ration on bacterial cellulose productivity in aerated and agitated culture, J. Ferment. Bioeng. 83 (1997) 371–376. 84. D. Klemm, B. Heublein, H.P. Fink, A. Bohn, Cellulose: Fas- cinating biopolymer and sustainable raw material, Angew. Chem. Int. Edit. 44 (2005) 3358–3393. 85. W. Borzani, S.J. De Souza, A simple method to control the bacterial production of cellulosic films in order to obtain dried pellicles presenting a desired average thickness, World J. Microbiol. Biotechnol. 14 (1998) 59–61. 86. N. Sakairi, H. Asano, M. Ogawa, N. Nishi, S. Tokura, A method for direct harvest of bacterial cellulose filaments during continuous cultivation of Acetobacter xylinum, Carbohydr. Polym. 35 (1998) 233–237. 87. H.C. Wong, A.L. Fear, R.D. Calhoon, G.H. Eichinger, R. Mayer, D. Amikam, M. Benziman, D.H. Gelfand, J.H. Meade, A.W. Emerick, R. Bruner, A. Ben-Bassat, R. Tal, Ge- netic organization of the cellulose synthase operon in Acetobacter xylinum, Proc. Natl. Acad. Sci. USA, 87 (1990) 8130–8134. 88. I.M. Saxena, K. Kudlicka, K. Okuda, R.M. Brown Jr., Char- acterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: Implications for cellu- lose crystallization, J. Bacteriol. 176 (1994) 5735–5752. 89. T. Nakai, A. Moriya, N.Tonouchi, T. Tsuchida, F. Yoshinaga, S. Horinouchi, Y. Sone, H. Mori, F. Sakai, T. Hayashi, Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001, Gene, 213 (1998) 93–100. 90. S. Kawano, K. Tajima, H. Kono, T. Erata, M. Munekata, M. Takai, Effects of endogenous endo-b-1,4-glucanase on cel- lulose biosynthesis in Acetobacter xylinum ATCC23769, J. Biosci. Bioeng. 94 (2002) 275–281. 91. R. Standal, T.G. Iversen, D.H. Coucheron, E. Fjaervik, J.M. Blatny, S. Valla, A new gene required for cellulose produc- tion and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon, J. Bacteriol. 176 (1994) 665–672. 92. P. De Wulf, K. Joris, E.J. Vandamme, Improved cellulose formation by an Acetobacter xylinum mutant limited in (keto)gluconate synthesis, J. Chem. Technol. Biotechnol. 67 (1996) 376–380. 93. T. Ishida, Y. Sugano, T. Nakai, M. Shoda, Effects of acetan on production of bacterial cellulose by Acetobacter xylinum, Biosci. Biotechnol. Biochem. 66 (2002) 1677–1681. 94. R. Tal, H.C. Wong, R. Calhoon, D. Gelfand, A.L. Fear, G. Volman, R. Mayer, P. Ross, D. Amikam, H. Weinhouse, A. Cohen, S. Sapir, P. Ohana, M. Benziman, Three cdg ope- rons control cellular turnover of cyclic di-GMP in Acetobac- ter xylinum: Genetic organization and occurrence of con- served domains in isoenzymes, J. Bacteriol. 180 (1998) 4416–4425. 95. S. Kawano, K. Tajima, Y. Uemori, H. Yamashita, T. Erata, M. Munekata, M. Takai, Cloning of cellulose synthesis re- lated genes from Acetobacter xylinum ATCC23769 and ATCC53582: Comparison of cellulose synthetic ability bet- ween strains, DNA Res. 9 (2002) 149–156. 96. S. Kawano, Y. Yasutake, K. Tajima, Y. Satoh, M. Yao, I. Ta- naka, M. Munekata, Crystallization and preliminary crys- tallographic analysis of the cellulose biosynthesis- related protein CMCax from Acetobacter xylinum, Acta Crystallogr. F, Struct. Biol. Cryst. Commun. 61 (2005) 252–254. 97. T. Nakai, Y. Nishiyama, S. Kuga, Y. Sugano, M. Shoda, ORF2 gene involves in the construction of high-order structure of bacterial cellulose, Biochem. Biophys. Res. Commun. 295 (2002) 458–462. 98. S. Kawano, K. Tajima, H. Kono, Y. Numata, H. Yamashita, Y. Satoh, M. Munekata, Regulation of endoglucanase gene (cmcax) expression in Acetobacter xylinum, J. Biosci. Bioeng. 106 (2008) 88–94. 99. T. Shigematsu, K. Takamine, M. Kitazato, T. Morita, T. Na- ritomi, S. Morimura, K. Kida, Cellulose production from glucose using a glucose dehydrogenase gene (gdh)-defi- cient mutant of Gluconacetobacter xylinus and its use for bioconversion of sweet potato pulp, J. Biosci. Bioeng. 99 (2005) 415–422. 100. D.R. Nobles Jr., R.M. Brown Jr., Transgenic expression of Gluconacetobactor xylinus strain ATCC 53582 cellulose syn- thase genes in the cyanobacterium Synechococcus leopolien- sis strain UTCC 100, Cellulose, 15 (2008) 691–701. 101. J. George, K.V. Ramana, S.N. Sabapathy, A.S. Bawa, Physi- co-mechanical properties of chemically treated bacterial (Acetobacter xylinum) cellulose membrane, World J. Micro- biol. Biotechnol. 21 (2005) 1323–1327. 102. J. George, K.V. Ramana, S.N. Sabapathy, H.J. Jambur, A.S. Bawa, Characterization of chemically treated bacterial (Acetobacter xylinum) biopolymer: Some thermo-mechani- cal properties, Int. J. Biol. Macromol. 37 (2005) 189–194. 103. A.R. White, R.M. Brown Jr., Enzymatic hydrolysis of cel- lulose: Visual characterization of the process, Proc. Natl. Acad. Sci. USA, 78 (1981) 1047–1051. 104. S. Schrecker, P. Gostomski, Determining the water holding capacity of microbial cellulose, Biotechnol. Lett. 27 (2005) 1435–1438. 105. P. Wanichapichart, S. Kaewnopparat, K. Buaking, W. Put- hai, Characterization of cellulose membranes produced by Acetobacter xylinum, J. Sci. Technol. 24 (2002) 855–862. 106. B. £aszkiewicz, Solubility of bacterial cellulose and its structural properties, J. Appl. Polym. Sci. 67 (1998) 1871– 1876. 123P.R. CHAWLA et al.: Fermentative Production of Microbial Cellulose, Food Technol. Biotechnol. 47 (2) 107–124 (2009)