Bacterial Celluloses (BC) are gaining importance in research and commerce due to numerous factors affecting the bacterial cellulose characteristics and application in different industries. The aim of the present study was to produce bacterial cellulose in different media using different cultivation vessels. Bacterial cellulose was produced by static cultivation of Glucanacetobacter xylinum ATCC 10245 in different culture media such as Brain Heart Agar, Luria Bertani Agar /Broth, Brain Heart Infusion, Hestrin-Schramm and medium no. 125. Cultivation of bacterium was conducted in various culture vessels with different surface area. The cellulose membrane was treated and purified with a 0.1 M NaOH solution at 90°C for 30 min and dried by a freeze- drier at -40°C to obtain BC. The prepared bacterial cellulose was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction (XRD). The amount of produced BC was related directly to the surface area of culture vessels.
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Cellulose is the most abundant natural polysaccharide found in nature with the chemical formula (C6H5O10)n (Chawla et al., 2009). The hydroxyl groups in cellulose are able to combine partially or completely with other chemicals to produce useful cellulosic derivatives. Cellulose biopolymer is formed by four methods: A isolation of cellulose from plants through lignin and hemicellulosic process which is a normal process (Tarchevsky et al., 1991; Klemm et al., 1998). biosynthetic (Vandamme et al., 1998; Jonas and Farah, 1998) enzymatic and (Kobayashi et al., 1991, 1992) chemical synthesis (Nakatsubo et al., 1996). Biological synthesis of cellulose is brought about by various microorganisms such as algae (Vallonia), fungi (Saprolegnia, Dictystelium and Discodium species), bacteria (Acetobacter, Achromobacter, Aerobacter, Agrobacterium, Pseudomonas, Rhizobium, Alcaligenes, Saecina and Zoogloea species). The above mentioned bacterial species are not able to synthesize fibrous cellulose extracellularly (Sun et al., 2007). Gluconobacter xylinum (formerly known as Acetobacter xylinus or Acetobacter xylinum) is Gram negative, rod shape, non pathogen, obligatory aerobic bacterium which belongs to the family of Acetobacteracea. A notable feature of this bacterium is its ability to produce extracellular cellulose as a pure, ultra fine fibrous network, possessing high crystallinity, water absorption and mechanical stability. Thus the produced cellulose network is known as pellicle (Klemm et al., 2001). The bacteria of genus Acetobacter are obligatory aerobic and are often found in fruits, vegetables, vinegar, fruit juice and alcoholic beverages. The mechanism of synthesizing bacterial cellulose helps the bacterium as follows: (a) provides oxygen at the surface of cultivation medium, (b) protect the bacteria against ultra violet rays and (c) maintains the humidity of cultivation medium. (Sun et al., 2007). Bacterial cellulose is gaining research interest due to properties like, fine fiber network, high water holding/absorption capacity, high mechanical strength (Hong et al., 2006; Putra et al., 2008) . Since the bacterial cellulose fiber's size is much smaller than that of plant cellulose, this makes the bacterial cellulose to be unique. High water maintenance capacity, high elasticity, high stability and compatibility of bacterial cellulose can be characteristics of cellulose produced by bacteria rather than isolated from plants (Czaja et al., 2006). Growth of bacteria under static or agitated conditions, media compositions will give rise to bacterial cellulose of different morphological characteristics (Ross et al., 1991). Bacterial cellulose produced under different cultural condition with various morphological characteristics and sizes, can be applied as stent coatings, for dura-mater substitution in tumor or trauma cases or as skin protection in cases of burning, deep wounds, periodontal tissue recovering (Rambo et al., 2008). In this study, attempts are made to produce bacterial cellulose in different media using different cultivation vessels.
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 100 mL Luria Bertani broth in 500 mL flask with the composition of peptone 10 g L-1, yeast extract 5 g L-1, NaCl 5 g L-1 with pH adjusted to 7|
|•||The 100 mL Brain Heart Infusion Broth in 500 mL flask with the pH of 7|
|•||The 100 mL Hestrin-Schramm broth in 500 mL Rough bottle (large surface area), with the following composition per liter: peptone 5 g, yeast extract 5 g, citric acid 1.15 g, disodium hydrogen phosphate 2.7 g and glucose 20 g with pH adjusted to 6 (Hestrin and Schramm, 1954)|
|•||Test tube (small surface area) and Petri plate (larger surface area than slant) containing Brain Heart infusion agar|
|•||Petri plate containing solid LB medium with composition as mentioned above. 2% agar was added to solidify the medium|
|•||Solid medium of Gluconobacter oxidans: The medium consisted of g L-1 yeast extracts 10 g, calcium carbonate 20 g, glucose 100 g, agar 15 g, pH of the medium was adjusted to 6.8 (recommended by the supplier of organism designated as medium No.125)|
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.
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 Kα (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 BCs 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, Iα 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 = 14.5o, 16.5o 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.
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.
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