Study of Nano-fiber Cellulose Production by Glucanacetobacter xylinum ATCC 10245

MATERIALS AND METHODS

Microorganism: Glucanacetobacter xylinium ATCC 10245 was obtained from Iranian Research Organization for Science and Technology (IROST). The ampoule containing the bacteria was opened aseptically at Laminar Air Flow cabinet as per supplier's instruction. The revived microorganism was propagated and stocks were prepared. Following media were used and inoculated with Glucanacetobacter xylinium to produce bacterial cellulose:

The flasks and plates were incubated at 28-30°C for 2-7 days at static condition. After the cultivation period, the formed gel at the liquid surface and between the pin array templates was removed, washed with deionized water and dried at 50°C. Subsequently, the cellulose membrane was treated with a 0.1 M NaOH solution at 90°C for 30 min to remove bacterial impurities and eventual cell debris. The membrane was again washed in deionized water to reach to neutral pH and then dehydrated by a freeze-dryer at -40°C.

Characterization: The prepared bacterial cellulose was characterized by Scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction (XRD) studies.

Scanning Electron Microscopy (SEM) analysis: SEM images of the samples were taken with a microscope (SEM, Philips, XL-30) to study the morphological changes. For the observations, BC membranes were freeze dried and placed over an aluminum support and sputtered with gold.

Fourier Transform-Infrared Spectrum (FTIS) analysis: FT-IR spectra were obtained using a BRUKER-Equinox 55 FT-IR spectrophotometer for the evaluation of chemical structures using a KBr pellet.

X-ray diffraction: XRD patterns were recorded on an X-ray diffractometer (Phlips PW 1140) by using Cu K_α (λ = 1.45 nm) radiation at 40 kV and 30 mA. The diffraction angle ranged from 5 deg to 40 deg. X-ray diffractometry was used to identify the phases of the cellulose membrane.

RESULTS AND DISCUSSION

Bacterial cellulose production: Table 1 reveals the production of bacterial cellulose in different cultivation media and type of vessels employed. As it can be seen from the Table 1, the surface area is an important factor and the production of bacterial cellulose occurs at the interface of air/liquid or solid surface. Bacterial cellulose has quite different structure from that of plant cellulose due to different synthesis procedure. During cultivation, the bacteria synthesize fine sub-elementary cellulose fibrils which are extruded from terminal enzyme complexes into the culture medium. Nascent cellulose extending from terminal enzyme complexes is initially amorphous and is gradually crystallized to cellulose. The obtained bacterial cellulose is dried by freeze-dryer. Figure 1 and 2 are typical images of bacterial cellulose synthesized by G. xylinum (ATCC 10245) under the static condition. Observation made by SEM indicates that the bacterial cellulose dried by freeze-drier contains more homogenous pores than those dried by hot air. Some isolated fibrils have less than 100 nm width. Pores with diameters lower than 100 μm are suitable for wound contracture (Czaja et al., 2006). They prolong the fibroblast migration through the porous template.

Table 1:	Production of cellulose in different medium

Fig. 1:	SEM image of produced BC with Gluconacetobacter xylinum ATCC 10245 in static culture, 2500x magnification

Fig. 2:	SEM image of produced BC with Gluconacetobacter xylinum ATCC 10245 in static culture, 40000x magnification

Beside the use of thin pore membranes in medical applications, membranes with pore sizes in the range of 10-100 μm are interesting materials to promote selective cell migration through the pores for specific cell cultures, with different cell sizes (Rambo et al., 2008). The sub-elementary fibrils are approximately 2-4 nm in diameter and are assembled into micro fibrils.

Table 2:	FT-IR spectra band assignments

These micro fibrils are then bundled to form ribbon-shaped fibrils of approximately 4 (thickness) x 80 (width) nm (Cai and Kim, 2010). The fibril length is in micro scale and some micro fibril-ends are not apparent in figures (Retegi et al., 2010). Cellulose biosynthesis is characterized by unidirectional growth and crystallization, where glucose molecules are linear bonded by β (1→4)- glycosidic bonds. The union of glycosidic chains forms oriented micro fibrils with intermolecular hydrogen bonds. The cellulose is crystallized outward the organisms, particularly in A. xylinum that synthesizes cellulose chains by introducing glucose units to the reducing ends of the polymer. The growth mechanism during bacterial activity determines the morphology of the final cellulose (Rambo et al., 2008).

Figure 3 depicts the FT-IR spectra of bacterial cellulose sample. FT-IR spectral band assignments of the sample are clearly listed in Table 2. In this case, a broad band at 3400 cm^-1 is attributed to O-H stretching vibration. The band at 2927 cm represents the aliphatic C-H stretching vibration. A sharp and steep band observed at 1065 cm^-1 is due to the presence of C-O-C stretching vibrations. The carbonyl amide group at 1652 cm^-1 in the FT-IR spectra of BC is due to the proteins and bacterial cells of BC’s suspension that is not easily wiped off after the NaOH treatment.

The morphological changes of ribbons are supposed to be related to the changes in microstructures such as crystallinity, I_α fraction and hydrogen bonding between the molecules (Sun et al., 2007). For pure BC, three main peaks located at 14.2, 16.6 and 22.4 deg can be identified in spectra for (110) (110) and (200) reflex ion planes of cellulose I (Kim et al., 2010). Like plant cellulose, BC has cellulose I structure. In the X-ray diffraction pattern of dry BC, peaks appear at 2θ = 14.5^o, 16.5^o and 22.5°for the (110) (110) and (200) reflex ion planes, respectively. However, during the cultivation, the (110) plane has a selective uniplanar orientation and the (110) peak becomes sharp and large. It was reported that, cellulose crystals become preferentially oriented in the (110) plane when water is removed from BC pellicle (Takai et al., 1975). Also, replacement of the water in BC pellicle with organic solvents prior to drying affects the orientation of the (110) plane.

Fig. 3:	FT- IR spectra of freeze- died BC synthesized under static culture condition

Fig. 4:	XRD pattern for produced BC

Polar solvents such as acetone and pyridine gave a higher selective uniplanar orientation, resulting in an increase in the intensity of the (110) peak, whereas non- polar solvents such as carbon tetrachloride and cyclohexane disturb the orientation and the intensity of the (110) peak decreases. Figure 4 shows the X-ray diffraction patterns of BC sample dried in the air. This produced BC is not pure and it should be purified and recrystallized.

CONCLUSION

BC synthesized by G. xylinum ATCC 10245 is in twisting network structure with many nano-fiber ribbons having the diameter of about 30 nm to 1 μm or more. Based on FT-IR spectrum produced BC has some impurities and it should be purified and recrystallized. The pore size is different with a range of more than 1 μm. Bacterial cellulose is proving to be a very versatile material. It can be used in a wide variety of biomedical applications, from topical wound dressings to the durable scaffolds required for tissue engineering. Many scientists are trying to develop novel biomaterials through biotechnological process. These new materials could be used in many biomedical and biotechnological applications, such as tissue engineering, drug delivery and, wound dressing medical implants. However, much interdisciplinary research is needed in order to bring microbial cellulose products to successful commercialization. For example, a wide variety of mammalian cells need to be seeded onto BC membranes in order to assess their viability and proliferation. A number of clinical studies are necessary to prove its usefulness and functionality. If bacterial cellulose proves to be effective in wound repair and tissue engineering, then it should be produced on an industrial scale. Due to its simple fermentation process, large scale bacterial cellulose production appears to be quite feasible.