Structural features and properties of BC em meio agitado

Structural features and properties of BC em meio agitado

(Parte 1 de 3)

Structural features and properties of bacterial cellulose produced in agitated culture

Fime Chemicals Development Laboratories, Central Research Laboratories, Ajnomoto Co., Inc., 1-1 Susuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan

Received 14 November 1997; accepted for publication 6 February 1998

The structure and some properties of bacterial cellulose produced in agitated culture were studied. Scanning electron microscopy revealed that there was almost no difference between reticulated structures of bacterial cellulose ®brils produced in agitated culture and in static culture. Nevertheless, bacterial cellulose produced in agitated culture exhibited microstuctural changes, namely, a low degree of polymerization and a low crystallinity index. A CP=MAS 13C

NMR analysis revealed that the cellulose Iá content of the cellulose produced in agitated culture was lower than that of the cellulose produced in static culture. The bacterial cellulose produced in agitated culture had a lower Young's modulus of sheet, a higher water holding capacity and a higher suspension viscosity in the disintegrated form than that produced in static culture. 0969± 0239 # 1998 Blackie Academic & Professional

KEYWORDS: Bacterial cellulose, Acetobacter, structure, properties, agitated culture

Bacterial cellulose (BC) is produced by some kinds of acetic acid bacteria. Recently, bacterial cellulose has received much attention as a new functional material for industrial applications because of its characteristic features such as high Young's modulus and high sonic velocity, etc. These mechanical properties must be due to unique structural features of the bacterial cellulose (Yamanaka et al., 1989; Nishi et al., 1990).

It is well known that the macroscopic morphology of bacterial cellulose varies depending on the culture methods (Hestrin and Schramm, 1954; Dudman, 1959; Dudman, 1960; Marx-Figini and Pion, 1974). Bacterial cellulose can be produced by two kinds of culture method, namely static and agitated. Under static culture conditions a typical gelatinous membrane of bacterial cellulose is accumulated on the culture surface and under agitated culture conditions bacterial cellulose is accumulated on the culture surface and under agitated culutre conditons bacterial cellulose is produced in a well-dispersed slurry as irregular masses such as granule, stellate, and ®brous strand (Hestrin and Schramm, 1954). The agitated culture method seems to be suitable for the industrial production of bacterial cellulose and serves commercial application in various

0969±0239 # 1998 Blackie Academic & Professional To whom correspondence should be addressed.

®elds (Yoshinaga et al., 1997a, 1997b). We have investigated the agitation culture method and have established the production process using large-scale fermenters (Toyosaki et al., 1995; Kouda et al., 1997; Yoshinaga et al., 1997a, 1997b).

The static culture method has been adopted mainly for the investigation of cellulose production because cellulose de®cient mutants sometimes appears in agitated cultures; these mutants were believed to interfere with the fermentative production of bacterial cellulose (Hestrin and Schramm, 1954). Several researchers, however, have succeeded in producing bacterial cellulose using stable cellulose-producing strains in agitated culture (Dudman, 1960; Marx-Figini and Pion, 1974). Nevertheless, a patent claimed that a speci®c reticulated structure observed by scanning electron microscopy was generated in bacterial cellulose produced in agitated culture, but not in that produced in static culture (Ben-Bassat et al., 1986). Moreover, despite knowledge of the macroscopic morphology of cellulose (Yoshinaga et al. 1997a, 1997b) the inuences of cultural conditions on the microstucture such as crystallinity, crystalline polymorphism and degree of polymerization (DP) have not been clari®ed systematically.

Our recent study revealed that bacterial cellulose produced in agitated culture had a lower DP and crystallinity compared with that produced in static culture (Watanabe et al., 1994). It is important to claify the inuence of culture conditions on the structure and some properties of bacterial cellulose for applying the bacterial cellulose to various industrial applications as a new functional material.

In this paper, we have investigated differences in structures and properties of bacterial cellulose produced under agitated and static culture conditions.

Bacterial strain

A strain of Acetobacter xylinum subsp. Sucrofermentans BPR2001 was used unless otherwise stated. This strain was isolated from a wild cherry fruit as one of the most potent cellulose-producers (Toyosaki et al., 1995).

Culture medium

The culture medium used was CSL-fru medium, which contained 20 ml=1 of corn-steep liquor and 40 g=1 of fructose. The detailed composition of this medium has been described previously (Toyosaki et al., 1995).

Culture methods

Culture methods have been described previously (Toyosaki et al., 1995). The agitated cultures were grown in a 1-liter jarfermenter (Able Co, Tokyo). The concentration of bacterial cellulose reached > 8g =1 in 40 h as reported previously (Toyosaki et al., 1995). The static cultures were grown in 30 ml of culture medium containing 3 ml of the same cell suspension in a 9 cm diameter petri dish at 28 8C for 10 days according to the method of Yamanaka et al. (1989).

Carboxymethyl cellulase (CMCase) activity

A mixture of 0.1 ml of culture supernatant and 15 ml of 0.6% (w=v) carboxymethyl cellulose (Nakarai Tesque Inc., Kyoto, Japan) was assayed for CMCase with a viscometer

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(Vibro Viscometer CVJ-5000, Chichibu Cement Co. Ltd., Tokyo, Japan) at 30 8C according to the method of Tahara et al. (1997). One unit of CMCase activity was de®ned as the amount of enzyme required to cause a 1% loss of viscosity in 2 h under these conditions.

Puri®cation of bacterial cellulose

The accumulated bacterial cellulose was washed in 0.1 NNaOH at 80 8C for 20 min to remove bacterial cells and other ingredients, and washed thoroughly with distilled water.

Scanning electron microscopy

The scanning electron microscope used for morphological observation was N-2250 from Hitachi Ltd., Tokyo Japan.

X-ray diffractometry

X-ray diffraction measurements were made with the symmetrical reection technique with collimated Ni ®ltered CuKá according to the method of Wada et al. (1993). The resolution of closely spaced reections was carried out by the least-squares method assuming a Gaussian reection pro®le for each reection preak, with the background scattering assumed to be composed of two Gaussian pro®les. The crystallinity index was calculated as the ratio of the area of the resolved crystalline peaks to the total area of a diffraction pro®le for 5±408. The crystallite size was estimated from the integrated width of the resolved crystalline peak by using Scherrer's equation (Nieduszynski and Preston, 1970).

CP=MAS 13C NMR analysis

CP=MAS 13C NMR measurements were conducted on the freeze-dried sample using a JEOL JMM-GSX200 spectrometer by the method of Yamamoto et al. (1993) and Yamamoto and Horii (1994). The spectra obtained were resolved into individual components using the curve-®tting program provided by JEOL to determine the mass fractions of cellulose Iá and cellulose Iâ (Yamamoto et al., 1993; Yamamoto and Horii, 1994).

Gel permeation chromatography

The weight-average degree of polymerization (DPw) and the DP distribution were determined for nitrated samples by high-performance gel permeation chromatography (GPC system, HLC-8020, Tosoh Co. Ltd. Tokyo, Japan) equiped with a refractive index (RI) detector according to the technique described by Kuga et al. (1989). Each cellulose sample was nitrated in a solution of fuming nitric acid=diphosphorous pentaoxide. Tetrahydrofuran was used as an eluent.

Disintegration of bacterial cellulose

The puri®ed bacterial cellulose was disintegrated in a homogenizer (Oster blender, Sunbeam-Oster Household Product Co., USA) at a concentation of 0.5% bacterial cellulose in water. The particle size of the disintegrated bacterial cellulose was analysed by the laser light scattering method with Microtrak FRA (Nikkiso Co. Ltd., Tokyo, Japan).

STRUCTURE AND PROPERTIES OF BACTERIAL CELLULOSE 189

Water holding capacity

Water holding capacity (WHC) was determined by the partially modi®ed centrifuging method of Robertson and Eastwood (1981) as follows: 10 ml suspension containing about 200 mg of disintegrated bacterial cellulose was transferred to a 15 ml polypropylene centrifuge tube. The tube was centrifuged for 15 min at various centrifugal forces. The volume of the resulting sediment was measured. The dried weight of bacterial cellulose contained in the sediment was measured after drying at 105 8C. Water holding capacity was expressed as water volume per dried weight of bacterial cellulose.

Viscosity of suspension

Viscosity of suspension of disintegrated bacterial cellulose was measured using a Brook®eld viscometer (DVL-B, Tokimec Co., Tokyo, Japan) equipped with No. 4 rotor at 60 rev=min after 30 s of rotation.

Young's modulus

The suspension containing disintegrated bacterial cellulose was cast in a plastic petri dish and dried at 50 8C to make a sheet. The dried sheet was peeled off and cut into a ribbon of 5 3 30 m. The thickness of the specimen ranged from 50 to 100 ìm. The Young's modulus of the dried ®lm was measured by a longitudinal oscillation tester (DMS 210, Seiko Denshi Co. Ltd., Tokyo, Japan). The initial tension was 250 g=mm2, the displacement length was 20 ìm, and the frequency was 10 Hz.

Structural features of bacterial cellulose produced in the agitated culture

The appearance of the two types of bacterial cellulose, agitated and static, are quite different. In the static culture, it is well known that the bacterial cellulose is accumulated at the surface of the culture medium as a gelatinous membrane. In contrast, many particles of various sizes (10 ìm to 1 m) and various shapes (spherical, ellipsoidal, stellate or ®brous) were accumulated in well-dispersed suspension in the agitated culture as reported previously (Hestrin and Schramm, 1954; Dudman, 1960; Yoshinaga et al., 1997a, 1997b).

Fig. 1 shows the scanning electron micrographs of bacterial cellulose prepared in static and agitated cultures. Both samples show the reticulated structure consisting of ultra®ne cellulose ®brils. The ®ne spherical particles of bacterial cellulose produced in the agitated culture have a reticulated structure similar to that of the gelatinous membrane produced in the static culture. The microscopic morphology of the reticulated structure is similar for the two specimens as examined by scanning electron microscopy. However, careful observations of the photographs revealed that there were some morphological differences in the ®brils and the reticulated structures between the two samples. The ®brils of bacterial cellulose produced statically (St-BC) were more highly extended (Fig. 1b). In contrast, the ®brils of bacterial cellulose produced under agitated conditions (Ag-BC) were curved and entangled with each other resulting in a denser reticulated structure than those of St-BC (Fig. 1a). Moreover, the width of the Ag-BC ®brils appears to be slightly smaller than that of St-BC, although it is dif®cult to estimate the size of each ®bril accurately in the limited view area after coating with

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palladium and gold. Such morphology of the ®brils may be formed by the turbulent ow in the agitated culture. These morphological changes are supposed to relate to the changes in microstructures, namely, DPw, crystallinity and content of cellulose Iá described below.

FIGURE 1. Scanning electron micrographs of bacterial celluloses produced in agitated and static cultures. (a) Bacterial cellulose reticulated structure in granules accumulated in the agitated culture. (b) Bacterial cellulose reticulated structure in gelatinous membrane produced in the static culture.

STRUCTURE AND PROPERTIES OF BACTERIAL CELLULOSE 191

Fig. 2 shows X-ray diffraction patterns of the two types of bacterial celluloses. These patterns are apparently similar to each other. However, a deconvolution analysis revealed the difference in the structural features between bacterial celluloses in the agitated and static cultures (Table 1); Ag-BC has lower crystallinity index and smaller crystallite size of a crystallographic plane (110) than St-BC.

(Parte 1 de 3)

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