Keywords : Bacterial cellulose, Dielectric barrier discharge (DBD) plasma, Gelatin, Acetobacter xylinum ABSTRACT

Transcription

1 Surface modification of cotton fabric by DBD plasma treatment for preparation of cotton fabric-reinforced bacterial cellulose composites containing gelatin Kamonwan Thongthanoppakun a, Ratana Rujiravanit *,a,b,c a The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok, Thailand b NU-PPC Plasma Chemical Technology Laboratory, Chulalongkorn University, Bangkok, Thailand c Center of Excellence on Petrochemical and Materials Technology, Bangkok, Thailand Keywords : Bacterial cellulose, Dielectric barrier discharge (DBD) plasma, Gelatin, Acetobacter xylinum ABSTRACT The cotton fabric-reinforced bacterial cellulose composites (BC/cotton fabric composites) were fabricated by applying cell immobilization technique in order to reduce the production cost on the inoculum utilization. The surface of cotton fabric was pre-treated with dielectric barrier discharge (DBD) plasma before soaking into a gelatin solution in order to accomplish the deposition of gelatin onto the cotton fabric. The uniformity of gelatin deposited on the surface of cotton fabric was examined by color straining using amido black 10B. The gelatin content in the cotton fabric was calculated from the percent nitrogen content determined by elemental analyzer and X-ray photoelectron spectrophotometer (XPS). Moreover, the scanning electron microscope (SEM) images of BC/cotton fabric composites revealed that the presence of gelatin in the plasma-treated cotton fabric during cultivation led to denser network structure of BC, more inter-linked fibers between each layer of BC and more uniformity of pore-sized structure of BC. It might be concluded that DBD plasma treatment could enhance the interaction between cellulose in cotton fabric and gelatin, leading the higher amount of gelatin deposited in the cotton fabric and gelatin on the cotton fabric made the surface of cotton fabric preferable for the growth of the bacteria, resulting in more fiber production. * Ratana.r@chula.ac.th INTRODUCTION Nowadays, there are many types of wound dressing materials in markets. The selection of wound dressing materials depends on the type, cause and depth ofa wound. As dry dressings, i.e. cotton and gauze pad, they cannot provide moist wound environment. Moreover, when dressings are removed, they stick to the wound surface and disrupt the wound bed that can cause pain. Another type is advanced wound dressings. They have been developed for promoting healing through the creation of a moist wound environment. They not only create a moist wound environment but also reduce scarring. Cellulose is the most abundant organic compound on the earth. Cellulose from plant is normally surrounded with hemicellulose and lignin, leading to requirement of Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 1

2 purification process using hazardous alkali and acid treatment in order to obtain pure cellulose (Sun et al., 2008). Moreover, an growing demand for plant cellulose has increased wood consumption, causing deforestation and global environmental issue (Park et al., 2003). Although plant is the major source of cellulose, various kinds of bacteria are able to produce cellulose as well. Bacterial cellulose (BC) that is produced by bacterial cells (Acetobacter xylinum) has been used as biomaterial for medical field and food ingredient. Cellulose from Acetobacter xylinum has higher tensile strength and water holding capacity than that of plant cellulose. The molecular formula of BC (C 6 H 10 O 5 ) n is the same as that of plant cellulose, but their physical and chemical features are different. Gelatin is the most abundant protein and the main protein of connective tissue in animals. Gelatin is biopolymer due to its biocompatibility, biodegradability and nontoxicity. Moreover, it has anti-inflammatory, antioxidant, immunomodulatory and antiproliferative potentials. Nevertheless, in a large scale production of BC pellicles a damage from tearing of BC pellicle may occur during cultivation, sterilization, and packaging processes. Therefore, reinforcement by using cotton fabric was used to solve the problem. Cotton fabric was embedded into BC pellicle. However, the interaction between cotton fabric and BC pellicle needs to be improved. In this study, dielectric barrier discharge (DBD) plasma and gelatin was used to improve not only the interaction between cotton fabric and BC pellicle but also the production yield of cellulose. After cotton fabrics were treated by DBD plasma, followed by gelatin coating, BC cells were cultivated on the surface of cotton fabric by absorption immobilization method. The effect of gelatin coating on cotton fabric and DBD plasma treatment on production yield of BC, changing in chemical structure of the plasmatreated fabrics and morphology, of the BC composite were examined. EXPERIMENTAL A. Materials and chemicals Acetobacter xylinum strain TISTR 975 was purchased from Microbiological Resources Centre, Thailand Institute of Scientific and Technological Research (TISTR). D-glucose anhydrous (analytical grade) was purchased from Ajax Finechem. Yeast extract powder (bacteriological grade) was purchased from Biobasic. Sodium hydroxide anhydrous pellet (analytical grade) was purchased from Ajax Finechem. Glacial acetic acid (analytical grade) was purchased from RCI Labscan. Cotton fabric was purchased from Santext fabric, Thailand. Gelatin from cold water fish skin was purchased from Sigma-Aldrich. B. Methodology Preparation of gelatin solution Preparation 0%, 0.5%, 2% and 6% of gelatin solution by dissolving gelatin powder in distilled water and then heating at 30 degree Celsius. Surface treatment of cotton fabric The dielectric barrier made of glass has the thickness of 2 mm. The two parallel electrodes are made of stainless steel. The cotton fabric was put onto a glass plate inserted into the parallel electrodes of DBD plasma reactor for plasma treatment. The experiment was operated under the following condition: voltage of 50 kv, frequency of 325 Hz and the Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 2

3 electrode gap of 5 mm. The flowing air gas was introduced directly through the gap of electrodes. Preparation of culture medium The culture medium used for bacterial cellulose production by Acetobacter xylinum contained 4.0 % w/v D-glucose and 2.0 % w/v yeast extract powder in distilled water. Then, the culture medium was sterilized by autoclaving at 121ºC for 30 minutes. Preparation of inoculum Freeze-dried Acetobacter xylinum TISTR 975 inoculum was activated by adding Acetobacter xylinum TISTR 975 in a 50 ml Erlenmeyer flask containing 200 ml of culture medium. After a static incubation at 30 ºC for 2 days, a bacterial cellulose pellicle appeared on the surface of culture medium. After that, 10 ml of stock culture medium was transferred to a 500 ml Erlenmeyer flask containing 100 ml of culture medium, followed by incubation at 30 ºC to obtain the inoculum for production of BC. Preparation of pure BC An inoculum of Acetobacter xylinum TISTR 975 (3 ml) was added into a 600 ml beaker containing 100 ml of culture medium. After that the beaker was kept in a static incubator at 30 ºC at a specific cultivation time. a bacterial cellulose pellicle was produced on the surface of culture medium. Preparation of BC composite Before immersing cotton fabric in culture medium to produce bacterial cellulose composite, the cotton fabric was immersed in a gelatin solution at concentrations of 0, 0.5, 2 and 6%. After that 3mL of Acetobacter xylinum TISTR 975 inoculum was dropped on cotton fabrics after that the cotton fabric was immersed at the surface of 100 ml of culture medium. After cultivation in a static incubator at 30 ºC for 3 days, bacterial cellulose pellicle was formed on the surface of cotton fabric. Purification of BC After incubation, bacterial cellulose pellicles produced on the surface of culture medium were harvested and purified by boiling them in 4.0% w/v sodium hydroxide solution at 90 ºC for 24 hours (repeated for 3 times) in order to remove bacterial cells and remaining culture medium, followed by neutralizing with 1.5 % w/v acetic acid solution at room temperature for 30 minutes and then immersed in distilled water until ph become neutral. The bacterial cellulose pellicles were kept in distilled water until use. RESULTS AND DISCUSSION A. Effect of DBD Plasma on Cotton Fabric coated with gelatin The comparison between DBD plasma-untreated and DBD plasma-treated cotton fabric with gelatin coating was done. The results from CHN analyzer showed that both DBD plasma-untreated and DBD plasma-treated cotton fabric with the immersion in 0% gelatin solution had 0% nitrogen content. In the cases of DBD plasma-untreated and DBD plasmatreated cotton fabric with the immersion in 0.5%, 2% and 6% gelatin solution, The results from CHN analyzer revealed that DBD plasma-treated cotton fabric had higher gelatin content than DBD plasma-untreated cotton fabric for every gelatin concentrations as shown in figure 1. Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 3

4 Figure 1. Comparison on nitrogen content in DBD plasma-untreated cotton fabric and DBD plasma-treated cotton fabric as a function of gelatin concentration. B. Morphology of Cotton fabric coated with gelatin The morphology of cotton fabric coated with gelatin was examined by using FESEM technique. Figure 2 showed the comparison of SEM images of DBD plasma-treated cotton fabrics coated with different gelatin concentrations. The results showed that gelatin not only deposited on the surface of cellulose fiber but also filled in the space between the fibers at higher gelatin concentration. The high concentration of gelatin solution would provide high amount of gelatin deposited on the cotton fabric. Accordingly, the polar functional groups of gelatin could extensively interact with the polar functional groups of the cotton fabric by hydrogen bonding interaction. (a) (b) Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 4

5 (c) (d) Figure 2. SEM images of DBD plasma-treated cotton fabrics after coating with gelatin in (a) 0%,(b) 0.5%, (c) 2% and (d) 6% gelatin solution at the magnification of 4,000 times. C. Morphology of composite The cross-sectional morphology of BC composite was investigated by SEM. The SEM images showed that the gelatin coating on cotton fabric resulted in denser structure, more inter-linked fibers between each layer of cellulose and more uniformity of pore size of cellulose as shown in figure 3. (a) (b) (c) (d) Figure 3. SEM images of cross-sectional morphology of bacteria cellulose composites produced by using cotton fabric coating with (a) 0%, (b) 0.5%, (c) 2% and (d) 6% of gelatin solution at the magnifications of 1,500 times. Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 5

6 CONCLUSIONS Cell immobilization technique was used to produce cotton fabric-reinforced BC composites (BC/cotton fabric composites) in order to reduce production cost on inoculum, generate better interaction between BC and cotton fabric, and reduce cultivation time. Moreover, it was found that production yield of BC became higher when gelatin was coated on cotton fabric. The deposition of gelatin on cotton fabric was accomplished by surface pretreatment using DBD plasma. By the combination of the above mentioned methods, BC/cotton fabric composites were successfully prepared for wound dressing application. ACKNOWLEDGEMENTS The authors would like to acknowledge the financial support from the PTT Public Company Limited. REFERENCES Brown, A.J. (1886). On an acetic ferment which forms cellulose. Journal of the Chemical Society, Transactions, 49, Harkins, A.L., Duri, S., Kloth, L.C., and Tran, C.D. (2014). Chitosan cellulose composite for wound dressing material. Part 2. Antimicrobial activity, blood absorption ability, and biocompatibility. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 102(6), Li, Y., Jiang, H., Zheng, W., Gong, N., Chen, L., Jiang, X., and Yang, G. (2015). Bacterial cellulose-hyaluronan nanocomposite biomaterials as wound dressings for severe skin injury repair. Journal of Materials Chemistry B, 3(17), Liu, C.Z., Wu, J.Q., Ren, L.Q., Tong, J., Li, J.Q., Cui, N., Brown, N.M.D., and Meenan, B.J. (2004). Comparative study on the effect of RF and DBD plasma treatment on PTFE surface modification. Materials Chemistry and Physics, 85(3), Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 6