Antimicrobial Properties of Zeolite-X and Zeolite-A Ion ...

Antimicrobial Properties of Zeolite-X and Zeolite-A Ion-Exchanged with Silver, Copper, and Zinc Against a Broad Range of Microorganisms Selami Demirci, Zeynep Ustaoğlu, Gonca Altın Yılmazer, Fikrettin Sahin & Nurcan Baç Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-013-0647-7

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Author's personal copy Appl Biochem Biotechnol DOI 10.1007/s12010-013-0647-7

Antimicrobial Properties of Zeolite-X and Zeolite-A Ion-Exchanged with Silver, Copper, and Zinc Against a Broad Range of Microorganisms Selami Demirci & Zeynep Ustaoğlu & Gonca Altın Yılmazer & Fikrettin Sahin & Nurcan Baç

Received: 2 September 2013 / Accepted: 4 November 2013 # Springer Science+Business Media New York 2013

Abstract Zeolites are nanoporous alumina silicates composed of silicon, aluminum, and oxygen in a framework with cations, water within pores. Their cation contents can be exchanged with monovalent or divalent ions. In the present study, the antimicrobial (antibacterial, anticandidal, and antifungal) properties of zeolite type X and A, with different Al/Si ratio, ion exchanged with Ag+, Zn2+, and Cu2+ ions were investigated individually. The study presents the synthesis and manufacture of four different zeolite types characterized by scanning electron microscopy and X-ray diffraction. The ion loading capacity of the zeolites was examined and compared with the antimicrobial characteristics against a broad range of microorganisms including bacteria, yeast, and mold. It was observed that Ag+ ion-loaded zeolites exhibited more antibacterial activity with respect to other metal ion-embedded zeolite samples. The results clearly support that various synthetic zeolites can be ion exchanged with Ag+, Zn2+, and Cu2+ ions to acquire antimicrobial properties or ion-releasing characteristics to provide prolonged or stronger activity. The current study suggested that zeolite formulations could be combined with various materials used in manufacturing medical devices, surfaces, textiles, or household items where antimicrobial properties are required. Keywords Zeolite-X . Zeolite-A . Antimicrobial . Silver . Copper . Zinc

Introduction Zeolites composed of silicon, aluminum, and oxygen in a framework with cations, water within pores, are basically nanoporous alumina silicates [1]. Silica is a neutral regular tetrahedronin in which positive charge of silicon ion is balanced by oxygen. However, there S. Demirci : Z. Ustaoğlu : G. A. Yılmazer : F. Sahin (*) Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, 34755 Kayisdagi, Istanbul, Turkey e-mail: [email protected] N. Baç Department of Chemical Engineering, Faculty of Engineering and Architecture, Yeditepe University, Kayisdagi 34755 Istanbul, Turkey

Author's personal copy Appl Biochem Biotechnol

is an unbalanced negative charge in the alumina structure. Therefore, the total structure of the zeolite has a negative charge, and this charge is balanced by cations (mainly by Na+ or K+). These cations can be exchanged in a solution by a desired positive ions such as heavy metals or ammonium ions [2]. The capacity of cation exchange depends on the ratio of silica/alumina in the structure. Generally, zeolites with a low silica/alumina (Si/Al) ratio have higher ion exchange capacity [3]. According to Si/Al ratio, there are several types of natural and synthetic zeolites including zeolite-β, zeolite-A, zeolite-X, and zeolite-Y, which are the most common commercial adsorbents. The general formula of zeolite is shown in the equation as Mx/n [(AlO2)x(SiO2)y]·zH2O where M stands for cations [4]. Although research on biological applications of zeolites is a new field, it is becoming increasingly an area of interest. It can be proposed that zeolites will be used widely in the near future because of their well-defined structure, reversible binding to small molecules, selectivity of shape and size, and their ability of behaving like metalloenzymes and regulating immune system properties [5, 6]. In addition to flavorless, odorless, and harmless properties of zeolites, antimicrobial characteristics can be obtained by ion-exchange process. The most common ion used in the exchange process is silver due to its stability and broad spectrum of antibacterial effect [7]. It has been reported that silver-embedded zeolite type A was found to be antibacterial against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus [8]. Furthermore, zeolite type X has also been impregnated with silver to have bactericidal effect against E. coli, Pseudomonas aeruginosa, and S. aureus [9]. Apart from synthetic zeolites, natural zeolites have also been made antibacterial by cation exchange process. Faujasite zeolite doped with silver has been manufactured, and its antimicrobial properties have been examined against bacteria and yeast [10]. Moreover, other heavy metals, mainly zinc, copper, nickel, mercury, tin, lead, bismuth, cadmium, chromium, and thallium have been used in the ion-exchange procedure to make natural and synthetic zeolites antimicrobial [11–13]. Although silver exhibited superior antibacterial activity, zinc and copper exchanged Na-clinoptilolite have shown inhibitory effect against P. aeruginosa and E. coli [14]. The current study aimed to investigate the antimicrobial (antibacterial, anticandidal, and antifungal) properties of zeolite type X and A, with different Al/Si ratio, ion exchanged with Ag+, Zn2+, and Cu2+ individually. The study presents the preparation and manufacture of four different zeolite types characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The ion loading and releasing capacity of the zeolites was examined and compared with the antimicrobial characteristics against bacteria, yeast, and mold.

Materials and Methods Materials and Reagents Sodium aluminate (Al 2 O 3 /1.4Na 2 O), sodium hydroxide (NaOH/0.07H 2 O), sodium metasilicatepentahydrate (Na2O/SiO2:5H2O), and ludox (SiO2/5H2O), used for zeolite synthesis, were obtained from Sigma-Aldrich. Silver nitrate (AgNO3), zinc chloride (ZnCl2), copper sulfate pentahydrate (CuSO4·5 H2O) and anhydrous copper sulfate (CuSO4), used for ionexchange process, were obtained from Sigma-Aldrich. Potato dextrose agar, sabouraud dextrose agar, tryptic soy agar, sabouraud dextrose broth (SDB), and tryptic soy broth (TSB), used in antimicrobial activity tests, were purchased from Merck (Darmstadt, Germany). Six-branch manifold filtration system and incubator shaking cabinet CERTOMAT BS-T (Sartorius, Göttingen, Germany) were used during zeolite synthesis and ion exchange process. SEM Zeiss EVO 40 (Jena, Germany), Sputter Coater BAL-TEC SCD 005 (Balzers, Switzerland)


and X-ray diffraction D8 FOCUS ASX Bruker (MA, USA) were used for zeolite characterization. Inductively coupled plasma-mass spectrometry (ICP-MS) measurements were conducted by X Series 2 ICP-MS device (Thermo Scientific, MA, USA) and Cetac ASX-520 (CETAC Technologies, NE, USA) auto-sampler. Zeolites samples were solved in acidic solutions by means of using CEM Mars microwave (CEM Corporation, Mathews, USA). Zeolite Synthesis Different types of zeolites were synthesized using sodium metasilicate pentahydrate (Na2O·SiO2·5H2O) and ludox (SiO2·5H2O) used as a silica source, sodium aluminate (Al2O3/1.4 Na2O) as an alumina source, and additional sodium hydroxide (NaOH/0.07 H2O). Required amount of water was divided into two plastic 1-L flasks. Sodium metasilicate pentahydrate and water were mixed in a flask, and sodium aluminate, sodium hydroxide, and the remaining amount of water were mixed in another flask concurrently. These two separate solutions were mixed after both solutions were dissolved completely. The flasks were placed into a shaker for 5 min at 200 rpm. Afterward, the solutions were put in the oven at 90 °C for crystallization about 72 h. At the end of the crystallization period, samples were filtered by using membrane filtration setup, and filtered zeolites were dried in the oven at 90 °C for 24 h [15]. Zeolites were taken from oven and ground up with a mortar and pestle. An additional grinding step was applied with the aid of household-type coffee grinder. Different types of zeolites, which have various Si/Al ratios, were synthesized. Zeolite X (Z1, and Z2) with high synthesis gel Si/Al ratio, 3.2 and 8, respectively, and Zeolite A (Z3, and Z4) with low synthesis gel Si/Al ratio, 0.84 and 1.6, respectively, were synthesized. The synthesis gel formulas of the zeolite types were listed in Table 1. Ion-Exchange with Metal Ions (Ag+, Zn2+, Cu2+) For the cation exchange process, different metal ions were used and prepared in same molarities. 1 M AgNO3, 1 M ZnCl2, and 1 M CuSO4·5 H2O ion exchange reaction solutions were prepared. The solutions were mixed at 200 rpm for 3 days in dark environment. Ionexchange process for silver, copper, and zinc zeolites can be denoted as in Eqs. 1, 2, and 3. Ionloaded zeolites were vacuum-filtered using vacuum filtration system and dried at 90 °C for approximately 24 h. Zeolites were ground using a mortar and pestle, and a conventional coffee grinder.

Table 1 Gel formulas of zeolite samples

Na−Z þ AgNO3 ↔Ag−Z þ NaNO3

ð1Þ

Na−Z þ CuSO4 ↔Cu−Z þ Na2 SO4

ð2Þ

Na−Z þ ZnCl2 ↔Zn−Z þ NaCl

ð3Þ

Z1

17Na2O/Al2O3/8SiO2/666H2O

Z2 Z3

4.64Na2O/Al2O3/3.2SiO2/400H2O 2Na2O/Al2O3/1.6SiO2/200H2O

Z4

2.5Na2O/Al2O3/0.84SiO2/194H2O


SEM, XRD, and ICP-MS Analysis Scanning Electron Microscopy (SEM) SEM was used to obtain the size and morphology of zeolite crystallites. In order to prevent evaporation during the imaging, zeolites were oven dried at 90 °C for 24 h. For scanning electron microscopy analysis, the zeolite samples were placed on double-sided sticky tape and coated with sputtered gold with BAL-TEC SCD (Balzers, Switzerland) 005 Sputter Coater. SEM images were obtained by using Zeiss EVO 40 (Jena, Germany). Energy dispersive spectroscopy was at beam voltage of 10.00 kV. XRD Analysis X-ray powder diffraction was used to determine the structural characterization of zeolites. The X-ray powder diffraction patterns were obtained using a Bruker D8 Advance Powder X-ray diffractometer (MA, USA) with Cu K-radiation, operated at 20 mA and 40 kV. The 2θ values were between 5° and 38°, and the scan speed was 3 s, and degree step was 0.02°. ICP-MS Analysis ICP-MS was used to indicate metal contents of ion-exchanged zeolites. ICP-MS results were obtained by using X Series 2 ICP-MS Thermo Scientific (MA, USA). Certain amount of the zeolite samples (about 15 mg) were mixed with 15 mL acid mixture (40%HNO3 +30%HCl+ 30%HF) and poured into microwave sample holders. Microwave was operated at 1,600 Watt, 210 °C, and 600 psi. Samples were filtered through 0.2-μm membrane filters and diluted 200 μL/10 mL (zeolite solution/2 % HNO3 solution). The procedure was repeated for copper and zinc zeolites as described. However, silver zeolite was dissolved in 100 % HNO3 due to the insufficient solubility of AgCl in water. Dissolved zeolite solutions were diluted to 50 mL. One hundred microliters of the each solution was added into 10 mL water, and metal ion contents were measured. Release of Metal Ions from Zeolites The release of metal ions from Ag+-, Zn2+-, and Cu2+-loaded zeolites into tryptic soy broth was examined using ICP-MS. Thirty-six centrifuge tubes (three tubes for each sample) were filled with 10 mL TSB, and 20.480 mg metal-loaded zeolites were added into each tube individually. Centrifuge tubes were put into a shaker and rotated at 180 rpm. Samples were centrifuged at 4,000 rpm for 15 min at each time point (0.5, 1, 2, 6, 12, and 24 h), and metal ion content of the supernatants was measured using ICP-MS. Each sample was measured three times. Antimicrobial Activity Test Microbial Strains The zeolite formulations were individually tested against a range of seven microorganisms, which consisted of four bacteria, one fungus, and two yeast species. The list of microorganisms used in the present study is given in Table 2. The Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University (Istanbul, Turkey), provided microorganisms.


Table 2 List of microorganisms tested for antimicrobial activity

Bacteria

S. aureus E. coli P. aeruginosa Bacillus cereus

Yeast

Candida albicans Candida glabrata

Fungi

Aspergillus niger Penicillium vinaceum

Minimum Inhibition Concentration (MIC) Assay Antimicrobial properties of metal ion embedded zeolite were evaluated via determining MIC values by broth dilution method as described earlier with slight modifications [16]. Ag+-, Zn2+-, and Cu2+-loaded zeolites were suspended at a concentration of 2,048 μg/ml in 10 ml TSB and SDB for bacteria and fungi, respectively. Twofold dilutions were conducted using appropriate broth media. Suspensions of 1×107 CFU/mL bacteria, 1×105 CFU/mL yeast and 1×103 spore/mL fungi were prepared from fresh cultures. The 0.5 mL of inocula was added to 4.5 mL of serially diluted zeolite suspensions. Inoculated suspensions were incubated 24 h for bacteria, 48 h for yeast at 36±1 °C, and 72 h at 27±1 °C for fungi stirring at 150 rpm. One hour before MIC incubation, stirring was stopped. Minimum inhibition concentration was determined as the lowest concentration in which microbial growth was inhibited.

Results SEM Analysis Z1 and Z2, which are faujausite zeolite X (NaX), have an octahedral shape. The ideal crystal system of the FAU-X is cubic, but crystal habit could be octahedral. Crystal sizes of samples Z1 and Z2 are approximately 9 μm. Samples Z3 and Z4 are types of zeolite A (LTA-Na) that have cubic structure, but they have beveled edges which is very normal due to the lack of shaking during synthesis. All crystals grew in a hydrothermal batch, and they were somewhat agglomerated. Nevertheless, there are some perfect cubic structures that emerged during the synthesis. The crystal size of the sample Z3 is between 2 and 5 μm, while Z4 is between 1 and 3 μm (Fig. 1). X-ray Diffraction According to the XRD route, Z1 has a phase formula of Na2Al2Si2.5O9·6.2H2O. Figure 2a shows the X-ray pattern for Z2. The peaks in the XRD patterns match the lines of aluminum silicate hydrate with composition of Na2Al2Si2.5O9·6.2H2O which belongs to Zeolite NaX [17]. Figure 2b shows the X-ray pattern for zeolite 3, and red lines represent sodium aluminum silicate hydrates that have Na96Al96Si96O384·216H2O which is an exact match with the chemical formula of LTA zeolite. Also, Z4 has the phase formula of LTA, which is Na96Al96Si96O384·216H2O. Table 3 indicates the X-ray diffraction results for zeolite samples.


Fig. 1 SEM images of zeolite formulations; a zeolite 1, b zeolite 2, c zeolite 3, d zeolite 4

ICP-MS Analysis of Metal-Loaded Zeolites The ICP-MS analysis showed the metal ion contents of the all ion-exchanged zeolite samples. Table 4 shows the amount of metal ions (%w/w) loaded into four different zeolite samples. According to the ICP-MS analysis, Z1 has the highest silver content, which is equal to 42.745± 0.649 %. Z10 follows it with 37.325±0.660 %, and Z1 has the lowest silver content with 31.435±0.987. For zinc ion-exchanged zeolites, Z1 has a zinc content of 18.090±0.166 % which is the highest among samples tested, and Z2 has a zinc content of 10.840±0.063 % which is the lowest. For copper ion-exchanged zeolites, the highest copper content was observed at Z1 which is equal to 14.940±0.253 %, and the lowest copper content was observed at Z2 with 9.865±0.050 %. Release Assay The concentrations of the Ag+, Zn2+, and Cu2+ released from metal ion-loaded zeolites into the TSB were measured with ICP-MS after 0.5-, 1-, 2-, 6-, 12-, and 24-h time intervals. Metal ion content for Ag+, Zn2+, and Cu2+ ion-exchanged zeolites are given at Table 5 in parts per million (ppm) values. For all types of zeolites, as time progresses, metal content in the TSB increases. Results clearly showed that, among all three types of ion-loaded zeolites, copper ion has the highest release ability. After 24 h, Cu-Z2 released 184.700±1.996 ppm copper ion, which has the lowest value among copper zeolites, which is even higher than all other type of zinc- and silver-loaded zeolites. However, after 6 h incubation period, there is no significant concentration change in the TSB.


Fig. 2 X-ray patterns for; a zeolite 2, b zeolite 3

Antimicrobial Activity The antimicrobial effects of ion-exchanged zeolites were investigated by determining MICs against eight different microorganisms (Table 6). According to the MIC results, different metal ion-embedded zeolites exhibited varying antimicrobial activity against bacteria, yeast, and fungi. Among them, silver zeolites were found to be the most effective ions with respect to


Table 3 Crystal compositions of zeolites obtained from XRD

Sample Number

Composition

Z1

Na2Al2Si2.5O9·6.2H2O

Z2

Na8(AlSiO4)6(OH)2-2H2O (sod cage) Na2Al2Si2.5O9.6·2H2O

Z3

Na96Al96Si96O384·216H2O

Z4

Na96Al96Si96O384·216H2O

copper and zinc ion-exchanged zeolites. As a group of microbial species tested, bacteria were found to be the most sensitive to silver zeolites. Bacterial growth was inhibited by silver zeolite with as low concentration as 16 μg/mL, though the concentration required for the inhibition of candidal and fungal growth was found to be as much as 1024 μg/mL. On the other hand, copper and zinc ion-loaded zeolite samples were found to display more antifungal and anticandidal characteristics, relatively. As another subject, different zeolite types with varying Al/Si ration exhibited diverse antimicrobial effect against bacteria, yeast, and fungi. Generally, metal ion-doped Z1 was found to be superior to Z2 in zeolite X group, and Z4 exhibited greater antimicrobial effect with respect to Z3 for all three metal ions.

Discussion Metal ions have been used as antimicrobial agents for centuries without exact knowledge about the mechanism of action. Silver, zinc, and copper are the most important metal ions used for their efficient antimicrobial activity. In several studies, they have been reported to have biocidal activity on both gram+ and gram- bacteria, yeast, and molds [18–21]. However, they can easily be oxidized and lose their antimicrobial characteristics due to change in their oxidation state from X+ to X0. Therefore, they need a specific host such as nanoporous natural or synthetic zeolites that have been ion exchanged with various metal ions including Ag, Cu, Zn, Hg, Sn, Pb, Bi, Cd, Cr, and Ti for water disinfection [22]. In the current study, structural, antimicrobial, and ion exchange properties of Ag+-, Zn2+-, and Cu2+-embedded synthetic zeolites types X and A were investigated. Z3 and Z4 formulations were found to have the same crystal structure according to the XRD measurements. However, ICP-MS, metal ion release, and MIC experiments show difference in results. The reason for these differences could be varying size distribution of zeolites. In addition, metal ions could have been trapped in the crystal water and were not able to be released during the MIC experiments. Generally, low Si/Al ratio increases the capacity of ion exchange. Therefore, it is easy to explain why Z4 and Z3 with low Si/Al ratio have been found to exchange more ions than Z2. On the other hand, although Z1 has higher Si/Al ratio than Z3 and Z4, the Table 4 Ag+, Zn2+, and Cu2+ ion content (% w/w) of zeolite samples Metal ion/zeolite type

Z1

Z2

Z3

Z4

Silver

42.745±0.649

31.435±0.987

33.240±0.660

37.325±0.660

Zinc

18.090±0.166

10.840±0.063

10.955±0.122

13.305±0.159

Copper

14.940±0.253

9.865±0.050

10.245±0.097

13.440±0.211

Author's personal copy Appl Biochem Biotechnol Table 5 Release of metal ions from zeolite samples Zeolite/ 0.5 time (h)

1

2

6

12

24

Ag-Z1

45.730±0.507a 51.070±0.710

54.490±0.275

64.250±0.550

65.640±0.827

66.750±0.317

Ag-Z2

42.107±0.940

44.460±0.498

47.540±0.443

61.430±0.572

64.340±0.606

65.630±0.293

Ag-Z3

39.340±0.327

40.020±0.324

45.320±0.378

69.940±0.798

70.800±0.153

70.830±0.554

Ag-Z4

22.940±0.138

25.110±0.270

35.280±0.450

55.270±0.814

57.460±0.672

58.070±0.322

Zn-Z1

52.320±0.433

56.460±0.452

57.560±0.641

59.470±1.293

61.870±0.472

63.540±1.214

Zn-Z2

75.230±0.719

79.220±1.518

79.680±1.095

81.270±1.008

82.630±0.828

82.880±1.742

Zn-Z3

26.840±0.347

30.430±0.194

31.590±0.797

39.430±0.423

39.900±0.318

40.690±0.219

Zn-Z4 Cu-Z1

35.550±0.547 40.430±0.224 45.040±0.324 50.510±0.559 50.570±1.108 51.280±0.762 179.200±2.186 225.700±1.638 260.700±3.629 290.200±0.507 295.300±2.684 300.100±5.409

Cu-Z2

70.050±1.141

Cu-Z3

161.500±1.132 202.300±1.147 230.100±3.404 253.200±0.92

Cu-Z4

214.300±0.276 266.700±1.420 291.900±2.513 315.300±1.657 317.500±0.255 325.000±2.335

a

77.080±0.262

107.000±0.763 159.700±0.358 175.500±0.875 184.700±1.996 260.700±1.104 265.000±0.591

Values were expressed in parts per million values

capacity of ion exchange was found to be superior. It contains the highest amount of metal ions in the zeolite framework. The reason for this could be the presence of sodalite cages that could not combine via double six-membered rings during the synthesis. Na+ ions bounded to those sodalite cages ion-exchanged with metal ions and increased the metal amount in the powder. In the literature, the metal content loaded into various zeolites has generally been lower in comparison with the current study. It has been found the Ag+ content of ion-exchanged zeolite type A was 16.16 % (w/w) [8] and zeolite type X was 5.8 % (w/w) [9]. However, in the present study, silver ion content was found between 31.435±0.987 and 42.745±0.649, zinc ion content was found between 10.840±0.063 and 18.090±0.166, and copper ion content was found between 9.865±0.050and 14.940±0.253 %. The reason for the difference might be the metal ionic species could be absorbed on the surface as well as ion exchanged within the cages. As metallic ions attached to the surface are not stable, concentration of metal ion exchange solution should be decreased and further optimized for enhanced and prolonged antimicrobial Table 6 MIC values of Ag+-, Cu2+-, and Zn2+-loaded zeolites against microorganisms tested Silver (Ag+) Zeolite type

Copper (Cu2+) 2

Zinc (Zn2+)

1

2

3

4

1

3

4

1

2

3

4

E. coli

64

64

32

64

256

P. aeruginosa Bacillus cereus

16 64

32 64

32 16

128 512 32 512

512

512

2,048 2,048

S. aureus

32

64

32

64

Candida albicans

512

1,024 256 128 512

1,024 512

1,024 2,048 2,048 2,048 2,048

Candida glabrata

512

1,024 256 128 256

1,024 512

1,024 2,048 2,048 2,048 2,048

Aspergillus niger

512

1,024 512 128 512

2,048 1,024 1,024 512

Microorganism,μg/mL

Penicillium vinaceum 1,024 512

1,024 1,024 256

2,048 1,024 1,024 2,048 1,024 2,048 2,048 2,048 1,024 1,024 2,048 2,048 2,048 2,048

1,024 2,048 1,024 1,024 512

512 128 512

2,048 2,048 2,048

1,024 1,024 1,024

2,048 1,024 1,024 2,048 2,048 2,048 2,048


activity. Other than that, it was observed that the selectivity of both types of zeolites towards silver is higher with respect to copper and zinc. These results are consistent with the research reported by Top and Ülkü [14]. They have found that free energies for exchange of silver ions has been superior to copper and zinc, Ag+ >Zn2+ >Cu2+. Similar to absorption capacity, Agzeolites were found to be the most effective against bacteria tested. Ag-zeolites exhibited the most powerful antibacterial effect with the MIC values ranging between 16 and 128 μg/ml. On the other hand, copper- and zinc-loaded zeolites displayed moderate antibacterial efficacy with the MIC value ranging between 256 and 2,048 μg/ml. The low antibacterial effect of Znzeolite and Cu-zeolite is in agreement with the study reported by Kim et al. [23]. They have observed that, while Ag+-incorporated ceramics based on hydroxyapatite exerted remarkable antibacterial effect, Zn2+- and Cu2+-incorporated ceramics displayed little or no antibacterial effect against E. coli. Antifungal activities of metal ion-exchanged zeolite types A and X were also investigated by broth dilution method. In contrast to the antibacterial effect, Zn2+- and Cu2+-embedded zeolites displayed considerable inhibitory effect of fungal growth with respect to Ag-zeolites. Similar results have been reported in the literature earlier [12, 24]. These results can be explained by the fact that zinc and copper, but not silver, belong to essential elements for fungi. Although fungal species require some heavy metals such as zinc for their metabolic activities, those metals can also be toxic at a concentration a little over that is required. Therefore, copper and zinc ions might have been taken by ion transporter of fungal systems in which metal ions accumulate and display fungicidal activity at high concentrations. In conclusion, the results clearly support that various synthetic zeolites can be ionexchanged with Ag+, Zn2+, and Cu2+ ions to acquire antimicrobial properties (antibacterial, anticandidal, or antifungal), or ion releasing characteristics to provide prolonged or stronger activity. The study also proposed that proper metallic ion could be loaded into zeolite formulation with an optimized Si/Al ratio to obtain required biocidal activity. According to the desired property, zeolite formulations can be combined with different materials such as polypropylene or polyethylene used in manufacturing medical devices, surfaces, textiles, or household items where antimicrobial properties are required. Acknowledgments This research was supported by Yeditepe University. The authors deny any conflicts of interest.

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