International Journal of Pharma and Bio Sciences

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1 Research Article Nanotechnology International Journal of Pharma and Bio Sciences ISSN COMPARISON BETWEEN BIOLOGICAL AND CHEMICAL SYNTHESIS OF ZINC OXIDE NANOPARTICLES AND ITS INFLUENCE OVER SOME S A.B. ABEER MOHAMMED* Department of Microbial Biotechnology, Genetics Engineering and Biotechnology Research Institute, Universityof sadat city (usc), Egypt ABSTRACT This study reports comparison between bio and wet chemical synthesis of Zinc Oxide nanoparticles (ZnO NPS) biological synthesis by using cell free supernatant some isolate of bacillus cereus that have ability to convert zinc acetate to ZnO (NPS). On the other hand, wet chemical synthesis using zinc nitrate and sodium hydroxide as a surrogate. In this report we used two methods for improve antibiotics that effect against pathogenic bacteria. The present investigation describes the maximum zone of inhibition was observed in the bio ZnO NPs (5 µl) with antibiotics kanamycin (K).The effects of some antibiotics have more affected with ZnO nanoparticles about without and bacteria become more sensitive to some antibiotics in the same concentration. KEYWORDS: biosynthesis of ZnO(NPS), wet chemical synthesis ZnO(NPS), antibiotics A.B. ABEER MOHAMMED Department of Microbial Biotechnology, Genetics Engineering and Biotechnology Research Institute, University of sadat city, Egypt e.mail:- abeer.bayoume@gebri.usc.edu.eg or beromicro@gmail.com *Corresponding author B

2 INTRODUCTION Members of the Bacillus genus are generally found in soil and most of these bacteria have the ability to disintegrate proteins. In paddy field soil, most of the nitrogen source is stored as biomass protein and decomposes slowly to low molecular weight amino acids by the activity of soil protease. Soil protease is thought to be mainly supplied by soil microorganisms 1. Metal nanoparticles have been importance studied in the past decade. Nanosized materials have been an essential subject in basic and applied sciences. Biological systems have well planned and controlled physiological processes and their use in the nanoparticle synthesis is rapidly slowing importance. Different shapes and sizes of nanomaterials have their physicochemical properties compared to the bulk materials. There are opportunities to wholly utilize modern clinical technology NP research 15. Zinc oxide nanoparticles (ZnO NPs) have established considerable interest due to their unique antibacterial, antifungal, and UV filtering properties, high catalytic and photochemical activity 2. Metal nanoparticles have gained sizeable interest in various areas of science and technology. Numerous microorganisms have been synthesized metal nanoparticles such as bacteria 3, fungi and yeast. Bacteria favours the production of nanoparticles over eukaryotic microorganisms due to ease of management, easy genetic manipulation and the fact that studies on one bacterium can be easily work to others. Nanoparticles were synthesized using these airborne bacteria (Bacillus sp.). Bacillus subtilis can produce octahedral morphology inside the cell walls in the dimensions of 5 25 nm 4. Cell-free culture supernatants of five psychrophilic bacteria Pseudomonas antarctica, Pseudomonas proteolytica, Pseudomonas meridiana, Arthrobacter kerguelensis and Arthrobacter gangotriensis and two mesophilic bacteria Bacillus indicus and Bacillus cecembensis have been used to synthesize zno nanoparticles 5. The antibacterial activity of ZnO NPs was much stronger than ZnO powder. This could be simply explained as smaller particles having a larger surface to volume ratio and provides more efficient mean for antibacterial activity 6. The surface of ZnO is considered as an effective inhibition of bacterial growth 7. ZnO and TiO2 NPs were effected in a model bacterium, Salmonella typhimurium increase of nanoparticles in ecosystem 8. Antibacterial activity of ZnO was tested and the effect was more pronounced with the Gram-positive than the Gram-negative bacteria and also ZnO nanoparticles exhibited a preferential ability to kill cancerous HL60 cells as compared with normal peripheral blood mononuclear cells 9. MATERIALS AND METHODS MATERIALS Salmonella typhimurium ATCC 14028, Bacillus Subtilis ATCC 6633 and Micrococcus luteus ATCC 9341.The culture sample was lyophilized and resuspended in a nutrient broth with 1.0% NaCl at 37 C for 24 h into the viable culture source. All the chemicals were obtained from Sagmi Isolation of Bacillus cereus to synthesis ZnO nanoparticles Soil samples were collected from Sadat city, Egypt (University of Sadat city) and 20 g was suspended with 20 ml of sterile distilled water. Bacteria were isolated by serial dilution and streak plate methods. The aliquots (0.1 ml) were plated in triplicates on Nutrient Agar (NA) medium [(w/v) 0.5% peptone; 0.3% beef extract; 0.5% NaCl; 1.5% agar, ph 7] and incubated at 30 C for 48 h. The culture was maintained on NA slants at -20 C. Phenotypic characterization of the isolate was done by different tests referring to Bergey's Manual of Determinative Bacteriology and Agriculture handbook 10. Preparation of ZnO nano particles wetchemical method The zinc oxide (ZnO) nanoparticles were prepared by wet chemical method 17 using zinc nitrate and sodium hydroxide as precursors and B

3 soluble starch as stabilizing agent. Zinc nitrate, g (0.1mol), was added in the above solution. Then the solution was kept under constant stirring using magnetic stirrer to completely dissolve the zinc nitrate for one hour. After complete dissolution of zinc nitrate, 0.2 mol of sodium hydroxide solution was added under constant stirring, drop by drop touching the walls of the vessel. The reaction was allowed to proceed for 2 h after complete addition of sodium hydroxide. After the completion of reaction, the solution was allowed to settle for overnight and the supernatant solution was then discarded carefully. The remaining solution was centrifuged at 6,000 rpm for 10 min and the supernatant was discarded. Thus obtained nanoparticles were washed three times using distilled water. Washing was carried out to remove the byproducts and the excessive starch that were bound with the nanoparticles. After washing, the nanoparticles were dried at 80 C for overnight. During drying, complete conversion of zinc hydroxide into zinc oxide takes place 11. Biosynthesis of ZnO nanoparticles by Cell free supernatant ZnO nanoparticle was prepared by biosynthesis methods. 0.02M Zinc acetate dihydrate was added 50 ml of Cell free supernatant under constant stirring. The solution after 10min stirring at 1ml and 2.0M NaOH was added to make ph 12 resulted in a pale white aqueous solution. This was then placed in a magnetic stirrer for 2hrs. The pale white precipitate was then taken out and washed over and over again with distilled water followed by ethanol to get free of the impurities. Then a pale white powder of ZnO nanoparticles was obtained after drying at 60 C in oven over night (Gericke and Pinches, 2006). Antibiotic resistance and sensitivity The antibiotic discs, wet chemical and biosynthesis Zno nanoparticles for many pathogenic bacteria strains were used for the disc diffusion method 18 Broth cultures of the collected strains grown at 37 C and spread on both nutrient agar plate using sterilized glass spreader then, antibiotics [Ceftriaxone (30 µg / disc), kanamycin (30 µg/disc), Tetracycline (30 µg/disc), Neomycin (30 µg/disc)] and Zno nanoparticles were distributed on plate and kept at room temperature for 2 h, so that discs can diffuse on the agar media. The plates were then incubated at 37 C for 48 h and the growth of the bacteria was observed. The presence of a clear zone around the disc was the index of sensitivity to the antibiotic and Zno nanoparticles. The test results of antibiotic sensitivity were determined according to the inhibition zone diameter 13. The absence of clear zone or the presence of some colonies within the clear zone indicated the resistant to that antibiotic and Zno nanoparticles. The clear plate indicates that the strains were sensitive to this selective concentration and presence of colonies on the plate indicates that the strains were resistant to that selective concentration 14. B

4 RESULTS AND DISCUSSION Int J Pharm Bio Sci 2015 July; 6(3): (B) Antibacterial activity Table (1): Characteristics of Bacillus cereus Identification and Characterization of Bacillus cereus The bacterium grew aerobically and formed white colonies. The morphological and biochemical characteristics are presented in Table 1 Characteristics Morphological identification Gram s staining Results Small, white, dried colonies Gram positive, rod Indole production - Methyl red test - Voges - Proskauer test + Citrate utilization test + Starch hydrolysis + a) glucose + b) Mannitol - c) Lactose - d) Sucrose + D-glucose + b) Mannitol - +, Positive results; -, negative results. Based on Bergey's manual of determinative bacteriology the phenotypic characteristics of isolate, belongs to the genus Bacillus. Table 1 Zone of inhibition for Salmonella typhimurium ATCC S.NO. Concentration Of without ZNO (NPS) With ZNO (NPS) Biosynthesis Wet Chemical 1 30µg /disc Ceftriaxone (CRO) µg/disc kanamycin (K) µg/disc Tetracycline (TE) µg/disc Neomycin (N) B

5 Table 2 Zone of inhibition for Bacillus Subtilis ATCC 6633 S.NO. Concentratio n Of without ZNO (NPS) With ZNO (NPS) Biosynthesis Wet Chemical 1 30µg /disc Ceftriaxone (CRO) µg/disc kanamycin (K) µg/disc Tetracycline (TE) µg/disc Neomycin (N) Table 3 Zone of inhibition for Micrococcus luteus ATCC 9341 S. N O. Concentration Of without ZNO (NPS) With ZNO (NPS) Biosynthesi Wet s Chemical 1 30µg /disc Ceftriaxone (CRO) µg/disc kanamycin (K) µg/disc Tetracycline (TE) µg/disc Neomycin (N) The well diffusion experiments were performed by antibiotic discs [Ceftriaxone (30 µg /disc), kanamycin (30 µg/disc), Tetracycline (30 µg/disc) and Neomycin (30 µg/disc)], wet chemical and biosynthesis ZnO nanoparticles against the Salmonella typhimurium ATCC 14028, Bacillus Subtilis ATCC 6633 and Micrococcus luteus ATCC As shown in (Table,1) the combination of bio ZnO NPs with antibiotics kanamycin (K) against Salmonella typhimurium ATCC showed maximum inhibition zone. Ceftriaxone (CRO) without ZnO nanoparticles showed no effect inhibitory. Tetracycline (TE) with Zno(NPs) also showed the same effect. The maximum zone of inhibition was observed in the bio ZnO NPs with antibiotics kanamycin (K) against Bacillus Subtilis ATCC 6633 (Table, 2 ). After that, it showed no inhibitory effect for Ceftriaxone (CRO) without ZnO nanoparticles (Table,2). Finally, the highest zone of inhibition was found in the bio ZnO NPs with kanamycin (K) and Neomycin (N) against Micrococcus luteus ATCC 9341 (Table, 3 ). Later it, showed the minimum zone of inhibitory Ceftriaxone (CRO) without ZnO nanoparticles and Ceftriaxone (CRO) with chemical ZnO nanoparticles (Table,3). Similar results for Tetracycline (TE) with Zno(NPs) and improved effect for Tetracycline (TE) without Zno(NPs) according to 15 reported Bacillus cereus as a biotemplating agent for the formation of zinc oxide B

6 Int J Pharm Bio Sci 2015 July; 6(3): (B) nanoparticles with simple thermal decomposition of zinc acetate by maintaining the original ph of the reaction mixtures. In this study, bacterial strains were isolated from Egyptain soil, which can be used as supernatant to biosynthesis ZnO nanoparticles and the composition of nutrient media plays a fundamental role in the biosynthesis of metallic and/or oxide nanoparticles. Figure 1 TEM results of chemical synthesized zinc oxide nanoparticles. Figure 2 TEM results of biosynthesized zinc oxide nanoparticles The sample was dissolved in ethanol and then a drop on the surface of copper grid for TEM analtsis. Figure (1) showed TEM image of chemical ZnO nanoparticles showing average size of 5-20 nm. Nanoparticles has a larger grain size, uniform shape and polycrystalline in nature. Figure (2) showed TEM image biosynthesis ZnO nanoparticles with average size of 2-5 nm. Nanoparticles have a smaller size, uniform shape and polycrystalline in chemical synthesis16. CONCLUSION The present biosynthesis method was a low cost approach, and can produce Zinc oxide ( NPs) nearby room temperature so that improved effectiveness of almost drug against pathogenic bacteria can be achieved. On the B

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