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Research Article ISSN 2321 – 0494 Indexed in CAS, OPEN J-gate and GOOGLE SCHOLAR Received on: 18.10.2014., Revised and Accepted on: 27.10.2014
A Study on Antimicrobial Activity of Silica Supported Copper Oxide against Escherichia Coli Purvi B. Shukla,1 Manish Mishra,2,* Shailesh Dave,3 Monal shah,3 Mamta Purohit3 1 Department of Chemistry, Suresh Gyan Vihar University, Jaipur 302004, Rajasthan, India 2 Department of Chemical Engineering and Shah-Schulman Center for Surface Science and Nanotechnology, Faculty of Technology, Dharmsinh Desai University, College Road, Nadiad 387 001, Gujarat, India, 3 Department of Microbiology, Gujarat University, Ahmedabad 380 009, Gujarat, India ABSTRACT The antimicrobial property of silica supported copper oxide (Cu/SG) samples having varied amount of copper was studied to develop an efficient and cost effective antimicrobial material. The Cu/SG samples were prepared by wet impregnation of silica gel using aqueous solutions of copper salts (acetate and nitrate). The samples were characterized by various instrumental techniques viz. Xray diffraction study, elemental analysis, BET surface area measurement, zeta potential and thermogravimetric analysis. The copper loading in Cu/SG was found to be dependent of precursor and its concentration in aqueous solution used for impregnation. The impregnation using 0.5 M copper acetate aqueous solution resulted into highest loading of copper on silica (5.8 wt.% copper). The antimicrobial activity of Cu/SG samples was investigated against the gram negative bacteria Escherichia coli measuring optical density of the medium by UV-VIS spectrophotometer at 600 nm. The long lasting inhibitory effect (90-96%; 96 h) of Cu/SG sample (having 5.8 wt.% copper) against E. coli was observed at low loading of material, however the growth-inhibitory effect depends on the copper content. The investigation suggests that silica supported copper oxide can be useful as effective and long-lasting antimicrobial control systems.
Keywords: Antimicrobial materials; copper; copper oxide; Escherichia coli; silica supported copper. Corresponding author: Dr. M.K. Mishra
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*E-mail:
[email protected];
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INTRODUCTION In recent years, the development of new, cost effective and efficient antimicrobial materials has received great interest for removal of harmful microbial organisms resistant to antibiotics and other disinfectants, and also for protection from the microbial contamination in food production and storage, water purification, medical equipments and devices, etc. The noble metal nanoparticle based materials have been developed for purification of water from microbes [1]. The Nanoparticles of metals such as copper, silver and zinc are known for having excellent antibacterial activity [2]. In order to have enhanced antimicrobial activity, easy handling and reduced health risks of the metallic nanoparticles, these metal nanoparticles have also been studied in the supported form using silica, zeolites, polymers, carbon nanotube, etc. as support materials [3-7]. The metal nanoparticles in supported form possess low toxicity, high chemical and thermal stability, high and long lasting antibacterial activity, which make them suitable bactericidal agent for various applications like water purification, coating purpose in paint, plaster, etc. As compared to aqueous silver nitrate solution and supported silver materials such as zeolite and polymer, the higher effectiveness of silica nanocomposite in antimicrobial activity was found to be attributed to slow and controlled release of silver ions from silver nanoparticles embedded within the silica matrix, resulting into long-term antimicrobial activity [3,7]. Several mechanisms have been proposed to explain the inhibition effect of microbial growth by metal nanoparticles and metal nano-composites, which include substitution of essential ions, blocking of functional groups of proteins, denaturation of enzymes, production of free radicals (hydroperoxide), alterations of membrane integrity, etc. [8-10]. The copper, being inexpensive, is an appropriate metal to develop cost effective antimicrobial systems. Copper ions at high concentrations show significant inhibition of bacterial growth [11-13], and have toxic effect on broad range of microorganisms. In recent year, there has been increased interest to develop copper based efficient and long lasting antimicrobial system for various applications. Copper nanoparticle with oxide layer formed on the surface was found to show excellent antimicrobial activity towards B. subtilis [14]. The metallic copper and copper alloys have also shown antibacterial activity over the pathogenic bacteria [15,16]. The antibacterial activity of copper oxide (CuO) nanoparticles generated by thermal plasma technology has been demonstrated against a range of bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli [17]. The studies of CuO nanoparticles incorporated into polymers suggested that release of copper ions into the local environment is required for optimal antimicrobial July-Sep 2014
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activity [17]. The Cu -exchanged montmorillonite (Cu-MMT) were studied for the antibacterial activity on Aeromonas hydrophila, which showed prolonged antibacterial activity of Cu-MMT as compared to copper sulfate [18]. The investigation on the mechanism of the antibacterial activity of Cu-MMT revealed that the antibacterial activity of Cu-MMT was mainly localized on the clay surface, and not due to the release of Cu2+ into solution. The excessive positive charge of Cu-MMT would make Cu-MMT attract A. hydrophila with negatively charged cellular wall and the copper cation would act directly on the bacteria adsorbed on the surface of Cu-MMT. Mingguang palygorskite clays modified with the silver and copper ion were studied for removing bacteria namely Escherichia coli and Staphylococcus aureus from aqueous solution showing elimination of the pathogenic microorganisms from water after 12 h of contact time [19]. The interesting features of the silica such as mesoporous nature, high surface area, easier attachment of metal ions on its surface as compared to polymeric support, high thermal resistant, irreversible binding of metal ions, etc. inspired to select it as support material for dispersion of copper oxide in the solid matrix. The desirable properties of silica and the beneficial antimicrobial property of copper oxide were combined to have enhanced activity of silica supported copper material for antimicrobial application. The wet impregnation of silica gel using copper acetate as precursor of copper with highest molar concentration (0.5 M) in aqueous solution was found to be appropriate method for highest loading of copper on silica (5.8 wt.% copper) giving better and fine dispersion of copper oxide onto the support material. The present study reveals that supported copper material possesses significant and long lasting inhibitory effect against E. coli at low loading of material indicating that this can be an effective antimicrobial control system for water purification, hygienic coating with polymers, plaster materials, paints, etc. 1. Experimental 1.1. Materials The chromatographic silica gel (100-200 mesh size), concentrated HCl (35%), copper acetate monohydrate (>98%), copper nitrate trihydrate (99.5%) were purchased from Merck, India. All the chemicals were used without any further purification. Gram negative bacteria (E. coli) culture (24 hours old) was obtained from Microbiology Department, Gujarat University, Ahmedabad. The related chemicals and other biological agents namely nutrient broth for growing and maintaining bacterial culture was obtained from Himedia.
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1.2. Synthesis of silica supported copper oxide samples The silica gel was washed with dil. HCl solution to remove any surface impurities followed by washing with distilled water and drying at 100°C for 14 h prior to use. The silica supported copper samples were synthesized by wet impregnation of silica (20 g) with aqueous solution of copper acetate or copper nitrate (200 mL) of different concentration under stirring for 24 h at room temperature. The copper impregnated materials were washed with distilled water to remove free copper and acetate/ nitrate ions and then dried at 120°C for 5 h in oven. The copper impregnated silica was calcined at 500°C for 5 h in muffle furnace to get silica supported copper sample (Cu/SG). The synthesis parameters of all the supported copper samples prepared and the sample names are summarized in Table 1. In the sample nomenclature used, Cu/SG stands for silica supported copper samples, A and N in parentheses denote the copper acetate and nitrate precursors respectively used in synthesis. The numbers 1 to 3 in parentheses represents the molar concentration of copper salt solution such as 0.5 M, 0.05 M and 0.025 M respectively. 1.3. Characterization of silica supported copper samples The copper content in the copper containing samples was estimated by Atomic adsorption spectroscopy (AAS double beam Elico,SL-243,India). The material (1 g) was mixed with 20 mL aqua-regia (3:2, v/v). The mixture was heated till it dries up completely and then it was cooled. Concentrated H2SO4 (10 mL) was added to the dried material and heated for 15 min. The solution was cooled and diluted with double distilled water making up to 100 ml in volumetric flask, which was analyzed by AAS. The BET (Brunauer-Emmett-Teller) surface area (SBET) of the samples, degassed at 110ºC for 2 h under vacuum, was determined from N2 adsorption data (by using BET equation) measured at 77K using Quantachrome NOVA 1000e surface area analyzer. The X-ray diffraction (XRD) study of TiO2 and Ag-TiO2 samples was carried out using X-ray diffractometer (Bruker, Advanced D8) with Cu Kα radiation (λ = 1.5418 Å) and Lynx Eye detector to study the crystalline nature, type of phases and the crystallite size in the samples. The sample was scanned in 2θ range of 10–80º with a scanning rate of 0.02º s-1. The particles surface charge of the samples was analyzed by measuring zeta potential of the samples using Zetasizer (Malvern) by dispersing the samples in iso-propanol. Thermogravimetric analysis (TGA) of the samples was carried out using TGA/DSC thermal analyzer (Mettler Toledo, STARe SF1052) by heating the sample from 50°C to 800°C with the heating rate of 10°C/ min in the flow of nitrogen gas (50 mL/ min).
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1.4. Antimicrobial activity of supported copper samples The antibacterial activity of silica supported copper samples was studied against the gram negative E. coli bacterium. The young culture of E. coli was grown in a sterilized liquid nutrient broth medium at 37ºC and inoculated on a rotary shaker at 200 rpm for 24 h. The pH and bacterial inoculum size were 7.2 and 108 cells/ml, respectively. To assess the inhibition of E. coli growth in presence of supported copper samples, optical density of the medium was measured at different time interval using UV-VIS spectrophotometer (Systronic UV spectrophotometer-166) under ambient conditions. The optical density (OD) of the medium was measured at 600 nm; 0.5 mL E. coli culture was inoculated in a 4.5 mL sterilized Nutrient both medium along with supported copper samples. The % Inhibition of E.coli was calculated by considering positive control as 100% growth. The standard resazurin assay method was modified in the present study to determine the LD50 (lethal dose to kill 50% of total bacterial concentration) dose of the supported copper samples against E. coli micro-organism. The E. coli culture was grown overnight in 1:2 diluted nutrient broth for 24 h. The culture was diluted to an optical density at 600 nm of 0.1 (corresponding to 108 CFU/mL). A 25 μl culture was placed into different tubes and the supported copper samples ranging from 10-1200 µg/mL concentrations were added to the tubes. The total volume in all the tubes was made to 1.0 mL with normal saline. The resazurin dye solution (0.675%w/v; 20 µl) was then added into all the tubes. In the experiment, one tube was kept as positive control, which was without supported copper sample; while one tube was maintained as negative control (i.e., without E. coli and supported copper sample). All the tubes were then incubated at 37°C on a rotary shaker at 150 rpm. The change in color from purple to pink was noted after 24 h by visible examination and then LD50 was determined by analyzing the solution using spectrophotometer at 600 nm. All the conditions were optimized in pilot experiments to consistently be able to visually differentiate results of control and test. 2. RESULTS AND DISCUSSION 2.1. Characterization of supported copper samples Table 1: Synthetic parameters of silica supported copper samples prepared. S. No.
Sample code
Copper precursor
Molar conc. of
Copper content (wt.%)a
copper solution 1
Cu/SG(A1)
Cu(CH3COO)2
0.5
5.8
2
Cu/SG(A2)
Cu(CH3COO)2
0.05
0.5
3
Cu/SG(A3)
Cu(CH3COO)2
0.025
0.46
4
Cu/SG(N1)
Cu(NO3)2
0.5
1.8
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Calculated from ICP analysis. From the elemental analysis of supported copper samples by AAS (Table 1), the copper
content in the samples was found to be highest with copper acetate precursor showing its highest loading efficiency as compared to copper nitrate. However, with decreasing the concentration of copper acetate, loading of copper also decreases and highest copper loading (5.8 wt.%) was obtained with 0.5 M copper acetate solution. The BET surface area (SBET) of silica (515 m2/ g) was slightly decreased in the supported copper sample containing highest copper content (i.e., Cu/SG(A1); 511 m2/ g) indicating the fine dispersion of copper species in the samples. The XRD pattern (Figure 1) of Cu/SG(A1) did not show any diffraction peaks corresponding to crystallites of copper species, which also indicates that the copper species are highly dispersed on the surface of the support and they are
Counts/ s
too less as well as small to be detected.
10
20
30
40 50 2 Theta
60
70
80
Figure 1: XRD patterns of silica and supported copper (Cu/SG(A1)) sample.
The zeta potential of the sample with highest copper content was measured to examine the surface charge of the particles. The particles of silica were negatively charged (-10.6 mV), which further increased in the supported copper particles (for Cu/SG(A1)) to -20.9 mV. The increased negative surface charge may be due to presence of copper oxide species on silica surface. TGA-DTG profiles (Figure 2 a and 2b) clearly indicates significant loading of copper acetate in the sample prepared by using 0.5 M copper acetate solution as compared to 0.05 M copper acetate, and 0.5 M and 0.05 M copper nitrate solutions showing significant weight loss for adsorbed copper acetate. The TGA graph shows a weight loss near 100°C for loss of water present in the samples and in the temperature range of 150°C to 350°C can be attributed to the decomposition of acetate and nitrate ions indicating the loading of copper ions along with their counter anions (acetate and nitrate groups) on the silica surface (Figure 2 a). The weight loss from 400°C to 600°C may be representing the dehydroxylation reaction of copper hydroxide species (which might have formed from thermal July-Sep 2014
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decomposition of copper acetate/ nitrate) with silanols. This indicates that the calcination of supported copper material in the temperature range of 400°C to 600°C may stabilize the adsorbed copper species on silica surface forming silica supported copper oxide. Therefore, all the supported copper materials were calcined at 500°C. The DSC graph of Cu/SG (A1) confirms that the supported copper oxide species are thermally stable showing no heat change during thermal treatment in the range of 50°C to 600°C (Figure 3). From characterization results, it can be concluded that the supported copper sample prepared by using 0.5 M concentration of copper acetate and calcined at 500°C possesses significant amount of finely dispersed copper (5.8 wt.%) as thermally stable copper oxide species on the surface of silica.
(ii) TGA
(i) DTG
(i) TGA
(ii) TGA
Figure 2a: TGA-DTG profiles of uncalcined copper samples prepared by using (i) 0.5 M and (ii) 0.05 M solutions of copper acetate (i.e., Cu/SG(A1)-D and Cu/SG(A2)-D).
(ii) TGA
(i) DTG
(i) TGA
(ii) TGA
Figure 2b: TGA-DTG profiles of uncalcined copper samples prepared by using (i) 0.5 M and (ii) 0.05 M solutions of copper nitrate (i.e., Cu/SG(N1)-D and Cu/SG(N2)-D). July-Sep 2014
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Figure 3: DSC profile of Cu/SG(A1)-C sample. 2.2. Antibacterial activity of samples 100
Inhibition (%) after 24 h
80
60
40
20
0 Cu/SG(A1)
Cu/SG(A1)-Dried
Cu/SG(A2)
Cu/SG(A3)
Cu/SG(N1)
Supported copper sample
Figure 4: Antimicrobial activity of supported copper samples. The silica gel without copper did not show antibacterial activity as no inhibition of E. coli growth was observed with pure silica. The supported copper sample (Cu/SG(A1)) after calcination has significantly higher inhibition activity against E. coli as compared to uncalcined material (Cu/SG(A1)-Dried) (Figure 4). The calcination of the material decomposes supported copper acetate
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and form copper oxide species over silica surface, which seem to be active for antibacterial activity [17]. Furthermore, the sample prepared using acetate precursor with 0.5 M concentration showed highest growth inhibition, which is attributed to high loading of copper oxide on silica. All the supported copper samples were found to be having antibacterial activity showing inhibition effect against E. coli (Figure 4), however, the inhibition decreases with decreasing copper content in the samples. The Cu/SG(A1) having 5.8% copper showed highest inhibition effect (96%) for 24 h. Copper-silica composite calcined at 500°C possesses highly dispersed copper oxide species over silica surface exposing most of copper resulting into high antibacterial activity [3]. The antibacterial activity of Cu/SG samples with 5.8% and 0.5% copper i.e., Cu/SG(A1) and Cu/SG(A2), respectively was examined for 96 h, which revealed the long-lasting antibacterial performance of the silica supported copper materials showing 90 to 96% inhibition of E. coli growth from 24 to 96 h (Figure 5). The results showed very high antimicrobial activity since beginning (4 h) of inoculation with Cu/SG(A1), which indicates that the supported copper species are easily supplying copper ions for killing the bacteria. Thus, the study demonstrates the long-term antimicrobial applicability of this material. The bactericidal mechanism of silica supported copper materials is exactly not known, but it is expected that copper ions released from Cu/SG sample enters the bacterial cells and interrupts the biochemical processes inhibiting the bacterial growth. The additional probable mechanism may be the formation of active oxygen species (e.g., hydrogen peroxide) due to redox reactions between Cu2+ and Cu+, which may damage the cytoplasmic membrane of bacteria [20]. The Resazurin assay method was used to determine the lethal dose (LD50) of supported copper materials against E. coli to kill 50% of the total bacterial concentration. The LD50 for Cu/SG(A1) was found to be in the range of 80 to 100 µg/mL (Figure 6), which revealed the highest bactericidal activity of Cu/SG(A1) at very low concentration. The significant inhibitory effect was also observed with Cu/SG(A2) giving LD50 at 600 µg/mL and with Cu/SG(N1), LD50 of was found in the range of 1000 to 1200 µg/mL.
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Cu/SG(A1)
Inhibition (%)
80
60
40 Cu/SG(A2)
20
0 0
20
40
Time (h)
60
80
100
% Inhibition
Figure 5: Growth inhibition of E. coli with silica supported copper samples with time. 100 90 80 70 60 50 40 30 20 10 0 10
20
40
60
80
100
200
400
600
800
1000 1200
Concentration (ppm) Cu/SG(A1)
Cu/SG(N1),
Cu/SG(A2)
Figure 6: LD50 of supported copper materials against E. coli.
3. CONCLUSIONS The antimicrobial property of silica supported copper (Cu/SG) samples was studied against the gram negative bacterium Escherichia coli. The significant and long lasting inhibitory effect (9096%; 24 h to 96 h) of Cu/SG sample (having 5.8 wt.% copper) against E. coli was observed at low loading of material. The study suggest that silica supported copper can be promising material as antimicrobial agent against E.coli contaminated system.
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ACKNOWLEDGEMENT Authors are thankful Dr. M.B. Dholakia, Principal, L.D. College of Engineering and Dr. Shailesh Dave, Head of Department, Department of Microbiology, Gujarat University for providing necessary facilities to pursue the research work. Authors are thankful to D.D. University for providing necessary facilities. REFERENCES 1. Pradeep T, Anshup. Noble metal nanoparticles for water purification: A critical review. Thin Solid Films 2009; 517: 6441–6478. 2. Horiguchi H. Chemistry of antibacterial and anti-mildew. Sankyo Press: Tokyo; 1980, p. 46– 59. 3. Egger S, Lehmann RP, Height MJ, Loessner MJ, Schuppler M. Antimicrobial Properties of a Novel Silver-Silica Nanocomposite Material, Applied and Environmental Microbiology 2009; 75: 2973–2976. 4. Mohan R, Shanmugharaj AM, Hun RS. An efficient growth of silver and copper nanoparticles on multiwalled carbon nanotube with enhanced antimicrobial activity. Journal of Biomedical Materials Research B: Applied Biomaterials 2011; 96B: 119-126. 5. Valodkar M, Rathore PS, Jadeja RN, Thounaojam M, Devkar RV, Thakore S. Cytotoxicity evaluation and antimicrobial studies of starch capped water soluble copper Nanoparticles. Journal of Hazardous Materials 2012; 201– 202: 244– 249. 6. Appendini P, Hotchkiss JH. Review of antimicrobial food packaging. Innovative Food Science & Emerging Technologies 2002; 3: 113–126. 7. Kumar R, Howdle S, Munstedt H. Polyamide/silver antimicrobials: effect of filler types on the silver ion release. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2005; 75: 311–319. 8. Nies DH. Microbial heavy-metal resistance. Applied Microbiology and Biotechnology 1999; 51: 730-750. 9. Ohsumi Y, Kitamoto K, Anraku Y. Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion. Journal of Bacteriology 1988; 170: 2676-2682. 10. Rodriguez-Montelongo L, de la Cruz-Rodriguez LC, Farías RN, Massa EM. Membraneassociated redox cycling of copper mediates hydroproxide toxicity in Escherichia coli. Biochimica et Biophysica Acta 1993; 1144: 77-84.
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11. Gordon AS, Howell LD, Harwood V. Responses of diverse heterotrophic bacteria to elevated copper concentrations. Canadian Journal of Microbiology 1994; 40: 408-411. 12. Sani RK, Peyton BM, Brown LT. Copper–induced inhibition of growth of Desulfovibrio desulfuricans G20: assessment of its toxicity and correlation with those of zinc and lead. Applied and Environmental Microbiology 2001; 67: 4765-4772. 13. Cervantes C, Gutierrez-Corona F. Copper resistance mechanisms in bacteria and fungi. FEMS Microbiology Reviews 1994; 14: 121-137. 14. Ruparelia JL, Chatterjee AK , Duttagupta SP, Mukherji S. Strain specificity in antimicrobial activity of silver and copper nanoparticles. Acta Biomaterialia 2008; 4: 707–716. 15. Espírito Santo C, Lam EW, Elowsky CG, Quaranta D, Domaille DW, Chang CJ, Grass G. Bacterial killing by dry metallic copper surfaces. Applied and Environmental Microbiology 2011; 77: 794-802. 16. Kielemoes J, Verstraete W. Influence of copper alloying of austenic stainless steel on multispecies biofilm development. Letters in Applied Microbiology 2001; 33: 148-152. 17. Ren G, Hu D, Cheng EWC, Vargas-Reus MA, Reip P, Allaker RP. Characterisation of copper oxide nanoparticles for antimicrobial applications. International Journal of Antimicrobial Agents 2009; 33: 587–590. 18. Hu CH, Xu ZR, Xia MS. Antibacterial effect of Cu2+-exchanged montmorillonite on Aeromonas hydrophila and discussion on its mechanism. Veterinary Microbiology 2005; 109: 83–88. 19. Zhao D, Zhou J, Liu N. Preparation and characterization of Mingguang palygorskite supported with silver and copper for antibacterial behavior. Applied Clay Science 2006; 33: 161–170. 20. Raffi M, Mehrwan S, Bhatti TM, Akhter JI, Hameed A, Yawar W, Hasan M. Investigations into the antibacterial behavior of copper nanoparticles against Escherichia coli. Annals of Microbiology 2010; 60: 75–80.
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Graphical abstract A study on antimicrobial activity of silica supported copper oxide against Escherichia coli The silica supported copper oxide prepared by wet impregnation method was found to be efficient, long lasting and cost effective antimicrobial material.
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