Accepted Manuscript Preparation, characterization and antibacterial properties of ZnO/kaoline nanocomposites Kateřina Dědková, Barbora Janíková, Kateřina Matějová, Pavlína Peikertová, Lucie Neuwirthová, Jan Holešinský, Jana Kukutschová PII: DOI: Reference:
S1011-1344(15)00119-0 http://dx.doi.org/10.1016/j.jphotobiol.2015.03.034 JPB 9999
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
28 January 2015 30 March 2015 31 March 2015
Please cite this article as: K. Dědková, B. Janíková, K. Matějová, P. Peikertová, L. Neuwirthová, J. Holešinský, J. Kukutschová, Preparation, characterization and antibacterial properties of ZnO/kaoline nanocomposites, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.03.034
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation, characterization nanocomposites
and
antibacterial
properties
of
ZnO/kaoline
Kateřina Dědkováa, Barbora Janíkováa, Kateřina Matějováb, Pavlína Peikertováa,c, Lucie Neuwirthováa, Jan Holešinskýd, Jana Kukutschováa a
Nanotechnology Centre, VŠB – Technical University of Ostrava, 17. listopadu 15, 708 33 Ostrava - Poruba, Czech Republic, [email protected] b
AGEL laboratory, Zalužanského 1192/15, 703 84 Ostrava–Vítkovice, Czech Republic
c
IT4Innovations Centre of Excellence, VŠB-Technical University of Ostrava, 17. listopadu 15, 70833 Ostrava - Poruba, Czech Republic d
Department of Material Engineering, VŠB – Technical University of Ostrava, 17. listopadu 15, 708 33 Ostrava - Poruba, Czech Republic
Abstract This paper describes laboratory preparation, characterization and antibacterial activity testing of ZnO/kaoline composites. ZnO/kaoline composites with 50 wt% of ZnO were laboratory prepared, dried at 105°C and calcined at 500 °C. XRPD analysis revealed that thermal treatment caused the phase transformation of Zn containing precursor into ZnO. Scanning and transmission electron microscopy techniques were used for characterization of morphology of the prepared samples. A standard microdilution test was used for evaluation of antibacterial activity using four common human pathogens (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aerigonosa). Daylight was used for induction photocatalytically based antibacterial activity. Second possible explanation of antibacterial activity of ZnO/kaoline could be the presence of biologically available forms of zinc. During the antibacterial activity assays the ZnO/kaoline composites exhibited antibacterial activity, where differences in an onset of the antibacterial activity and activity against bacterial strains were observed. The highest antibacterial activity was observed against S.aureus, where the lowest value of minimum inhibitory concentration was determined equal to 0.41mg/ml. Key words Kaoline/ZnO, nanocomposites, antibacterial activity, Staphylococcus aureus, XRD
1
Introduction
Nanomaterials are perspective materials which can give promising opportunities for enhanced properties in medical or environmental applications due to unique physical and chemical properties which are caused by their size in nano dimensions. They have large surface/volume ratio affecting their reactivity [1]. Nowadays the photocatalytic properties are being extensively investigated. The photooxidation of organic compounds to carbon dioxide is often used for decomposition of an organic pollutant in the air or for removal of wastewater impurities. Metal oxides or sulfides show photocatalytic properties. The most common photocatalysts in use are titanium dioxide, zinc oxide or cadmium sulfide [2]. Inorganic metal oxides may serve as effective disinfectants, due to their relatively non toxic profile, good chemical stability and efficient antibacterial activity. Numerous studies described strong antibacterial properties of different nanomaterials [1, 3-5]. Among metal oxide powders, ZnO demonstrates significant growth inhibition of broad spectrum of bacteria [6-8]. The suggested mechanism for the antibacterial activity of ZnO is based on catalysis due to formation of reactive oxygen species (ROS) [9]. Cell wall and cell membrane of bacteria are damaged upon the contact with nanoparticles of ZnO, which cause inhibition of bacterial growth [3]. Moreover, Zn2+ released from surface of ZnO nanoparticles can bind to the membranes of microorganisms and thus prolong the lag phase of the microbial growth cycle [10]. Since the catalysis of radical formation occurs on the particle surface, particles with larger surface area demonstrate stronger antibacterial activity. Therefore, as the size of the ZnO particles decreases their antibacterial activity increases [6]. Due to reactivity of ZnO nanoparticles, they may pose some environmental risks, given by their higher biological activity including potential penetration into cells and generation of reactive oxygen species [11-12]. If ZnO nanoparticles are anchored via chemical bond to a suitable matrix (e.g. clays) they still demonstrate photodegradable properties, however potential environmental risks are lowered due to the decreased mobility in the environmental media. Clay minerals, such as montmorillonite, kaolinite, vermiculite, etc. are natural materials from group of phyllosilicates. They are worldwide spread and have a broad scale of practical applications such as sorbents for metal cations [13]. Phyllosilicates have unique crystallochemical properties; therefore they may act as a suitable matrix for anchoring ZnO nanoparticles [12, 14-15]. Many published articles describe preparation of e.g. montmorillonite/TiO2 or vermiculite/TiO2 nanocomposites [16, 17], but only few studies deal with the preparation and antibacterial activity of nanocomposites with kaoline matrix [18], therefore kaoline was selected as a matrix in this study. The aim of the study was to prepare, and characterize kaoline/ZnO composite and evaluate its antibacterial activity for selected human pathogens in relation to the extent of daylight irradiation, and evaluate these prepared materials for their potential usage for antibacterial modification of surfaces in medical or sanitary applications.
2
Materials and methods 2.1. Studied nanocomposites
The entire preparation procedure consists of two main parts. The first part includes reaction between aqueous solution of sodium carbonate and zinc chloride in suspension of kaoline to obtain the precursor. After 3h stirring of the suspension, precursor was dried at 70 °C. The second part is based on thermal decomposition of the precursor to form zinc oxide. The thermal treatment of zinc oxide precursor is carried out in chamber furnace at 500 °C. The amount of reactants was chosen to achieve 50 wt. % ZnO in the final composites. The samples were assigned as ZinKa51 and ZinKa55, where the first number represents the amount of ZnO in composites (5 ≈ 50%) and the last number denoted the temperature of calcination (1 ≈ 100°C, 5 ≈ 500°C). 2.2. Microscopic and Phase Analysis Chemical composition of prepared composites was determined using energy dispersive X–ray fluorescence spectrometer (XRFS, SPECTRO XEPOS) equipped with 50W Pd X–ray tube. The samples were pressed into tablets for this measurement. X-ray powder diffraction (Bruker D8 Advance diffractometer equipped with detector VANTEC 1, rotational holder and CoKα lamp, λ=1.789 A˚) was used to obtain the phase composition of the samples. For the evaluation of phase composition database PDF 2 Release 2004 was used. Scanning electron microscope (Quanta FEG 450, FEI) and transmission electron microscope (JEM2100, JEOL) were used as microscopic methods for morphology characterization of the studied samples. The samples for SEM were coated with Au/Pd film. Mid-IR spectra were obtained by Fourier transform infrared spectrometer Nicolet 6700 (Thermo USA) using the single reflection ATR technique on a diamond crystal. Measurements were performed with a resolution of 4 cm-1 and 32 scans. Achieved data were processed using the OMNIC software. 2.3. Leaching test The leachates were prepared in accordance with European technical standard EN 12457-2. ZinKa samples were leached in deionised water (denoted as extraction agent DW) in continuous rotation container for 24 h to evaluate mobility of ZnO, resp. Zn2+ in water environment. After mixing for 24 h the mixture was centrifuged (3000 rpm) for 30 min and then filtered through the filter paper with density of 84 g/m2 in order to separate the solid phase. Zn was determined by flame AAS UNICAM 969.
2.4. Antibacterial Assessment Four different human pathogenic bacterial strains were used for the determination of antibacterial activity. Glucose broth (HiMedia) was used as a growth media for the purpose of antibacterial assay of the ZinKa composites. Turbidity of the inoculums was measured using Densi-La-Meter (LACHEMA). Incubation of bacteria was conducted in Biological thermostat BT 120M at 37°C. The antibacterial activity of the composite ZinKa was tested using standard microdilution method which enabled to determine the minimum inhibitory concentration (MIC) of tested substances. Disposable microtitration plates were used for the testing. Commercial solid blood agar plates for cultivation the bacteria without any additional modifications were used. Liquid growth media were prepared by instruction of supplier and after this sterilized in an autoclave. Suspension of ZinKa in growth media was diluted to achieve concentrations 100, 33.3, 11, 3.7, 1.2, 0.41, 0.014 mg/ml of ZinKa in growth media. Staphylococcus aureus 3953, Escherichia coli 3954, Enterococcus faecalis 4224 and Pseudomonas aerigonosa 1960 were achieved from the Czech Collection of Microorganisms (Czech Republic). Bacterial inoculums used had a cell concentration of 1.2x109 (S. aureus, E.coli), 1.3x109 (E. faecalis) 1.6x109 (P. aeruginosa) CFU/mL (colony-forming units per milliliter). Each cell at the micro titration plate was inoculated, and thus this plate is called the reaction plate. This plate was placed on the windowsill for 8 hours due to exposure to daylight irradiation. After defined time sections of growth inhibition presenting living bacterial cells were transferred from the reaction plate to pure growth media using the inoculation hedgehog. These re-inoculated plates were incubated at 37°C for 24h and then the MIC values were determined according to visible growth inhibition. This modification of the standard microdilution method eliminated issues with the determination of MIC caused by turbidity of death cells or caused by the presence of the solid ZinKa sample in a microtitration plate, because only living bacterial cells can be captured by needles of the inoculation hedgehog. Detailed description of antibacterial assessment was already published and could be seen in [5].
3
Results and discussion
Chemical composition of the prepared composites ZinKa51, ZinKa55 and pure kaoline is shown in table 1. Chemical composition analysis confirmed the presence of Zn in both composites. The content of ZnO in the composite dried at 100°C is lower than in the composite calcined at 500°C due to thermal processes, this fact is connected to higher value of LOI (loss of ignition) observed for dried composite (22.63%) in comparison to the composite calcined at 500 °C (5.72 %). The amount of ZnO in samples are 31.44 wt. % of ZnO for ZinKa51 and 41.00 wt. % of ZnO for ZinKa55. The ZinKa55 value shows the yield of ZnO approximately 82 %.
Table 1: Chemical composition and lost of ignition (wt. %) of pure kaoline (KA), ZinKa51, ZinKa55 composites estimated from XRFS results. sample
Na2O
MgO
Al2O3
SiO2
P2O5
SO3
K2O
CaO
TiO2
MnO
Fe2O3
ZnO
LOI
KA
0.071
0.140
34.80
49.70
0.048
0.056
1.193
0.061
0.847
0.003
0.559
-
11.52
ZinKa51
0.082
0.145
19.02
24.23
0.011
0.003
0.618
0.062
0.445
0.02
0.293
31.44
22.63
ZinKa55
0.110
0.180
21.66
28.41
0.028
0.018
0.800
0.094
0.573
0.025
0.375
41.00
5.72
LOI – lost of ignition, - means not present in pure kaoline The XRPD (X-ray powder diffraction) patterns of the pure kaoline, ZinKa51 and ZinKa55 are shown in Fig 1. The analysis verified muscovite and quartz as the typical admixtures of kaoline (KA). The XRPD pattern of composite ZinKa51 reveals the formation of Na2Zn3(CO3)4·(H2O)3 as the product of reaction of ZnCl2 and Na2CO3 in aqueous suspension. The presence of zincite (ZnO) was proved in the diffraction pattern of the composite ZinKa55. During the calcination of the ZinKa51 newly formed compound Na2Zn3(CO3)4·(H2O)3 were decomposed whereas ZnO is formed.
Fig. 1 XRPD pattern of pure kaoline (Ka), ZinKa51 and ZinKa55 - kaolinite (1), quartz (2), muscovite (3), Na2Zn3(CO3)4·(H2O)3 (4), ZnO (5). TEM image of the sample Ka (Fig. 2A) revealed smooth surface of kaolinite particles, whereas in the case of ZinKa55 (Fig. 2B) composite the surface of the kaolinite particles is covered by uniformly dispersed ZnO particles with dimension less than 100 nm. Analogical structure was observed for ZinKa51.
B
A
Fig. 2 TEM images of Ka (A) and ZinKa55 (B) The SEM images of the studied samples (Fig. 4) proved the matrix consisting of micro-sized kaolinite particles having a layered structure with sub-micron particles of ZnO attached onto the surface of the clay matrix. ZnO particles cover the surface of kaoline matrix almost homogeneously and are not attached on edges of kaoline sheets. A
B
Fig. 3 SEM images of ZinKa51 (A) and ZinKa55 (B) FTIR studies of the composites were carried out in the mid-IR region 4000-400 cm-1, whereas the absorption region of the diamond crystal was removed from the obtained spectra. The obtained spectra are shown in Fig. 4. Kaoline spectrum shows all its characteristic absorbtion bands. The bands are assigned to the particular bonds as follows: Al-O-H (3684, 3651, 3619, 937, and 911 cm-1), Si-O (1114, 788, 641, and 456 cm-1), Si-O-Al (997, 750, 692, and 522 cm-1) and Si-O-Si (1024 cm-1). In the spectrum of ZinKa51 are all characteristic bands of kaoline are also clearly visible. However, some new bands appear in the spectrum. The band at 3394 cm-1 corresponds to the absorbed water in the sample; this band vanished after calcination (ZinKa55). Other bands in the lower wavenumbers (1506, 1457, 1394, 1094,
and 836 cm-1) corresponding to the carbonates in the sample, which is decayed after calcination, because these bands are missing in the spectrum of ZinKa55. The presence of carbonate (Na2Zn3(CO3)4·(H2O)3) was also proved by XRPD. In the composite ZinKa55 is also observable the transformation of the kaoline to the metakaoline, which is not yet terminated. This transformation is indicated by broader shape of the bands about 1000 cm -1 and vanishing of the inner octahedral structure of the Al-O-H bonds (3700-3600 cm-1).
Fig. 4 Measured mid-IR spectra of pure kaoline (KA) and composites ZinKa51 and ZinKa55. The concentrations of leached Zn after material interaction with DW are listed in Table 2. Table 2: Concentration of leached Zn from ZinKa samples in extracts after interaction with extraction agent DW
Zn Sample [mg/l] ZinKa51
0.031± 0.002
ZinKa55
0.57± 0.03
Leached amount of Zn is aprox. 0.5 wt. % from ZinKa55 and even lower from Zinka51. Taking into account that ZnO content in the photoactive composite materials is 41 wt. % and 31 wt% respectively, it can be concluded that ZnO nanoparticles are tightly anchored on the clay matrix in both photoactive composites (ZinKa51 and ZinKa55). Antibacterial activity expressed as the MIC values of the ZinKa samples was evaluated using four bacterial strains. Values of MIC for ZinKa samples against all bacteria are summarized in tables (Tab. 3). Pure kaoline did not exhibit antibacterial activity therefore MIC could not be determined and are not included in the tables. The highest antibacterial activity was obtained against S.aureus (0.41 mg/ml for both ZinKa samples). Faster onset of antibacterial activity of the ZinKa samples against S.aureus and E.coli was observed in comparison to E.faecalis and P.aeruginosa. It could be caused by different requirements for living condition e.g. pH, etc. of E.faecalis and P.aeruginosa, when S.aureus and E.coli are more sensitive to changes in living conditions so it is not that difficult to cause growth inhibition of the cells comparing to other two strains. Generally, P. aeruginosa is the most resistant from bacteria used in experiments. Bacterial cells have also some defense mechanism which can help them to resist the treatment of ZinKa samples or other chemicals. The difference between antibacterial activity of ZinKa51 and ZinKa55 was observed in the case of E. faecalis and P. aeruginosa while ZinKa51 had better activity and does not contain any ZnO as proved by XRPD method. This fact imply that the effect of Zn2+ ions on the antibacterial activity of the ZinKa51 composites is higher than visible light induced photocatalytical activity of ZnO in the case of ZinKa55. Our results are in accordance to results of the study describing proposed mechanism of antibacterial activity based on Zn 2+ ions and consequent diffusion of these ions into the cytoplasm [19]. Decrease of the MIC values during the reaction time was observed for the ZinKa samples against S. aureus and E. coli. The MIC values >100 mg/ml means that, the MIC were not determined, which can be caused by the MIC value being higher than 100 mg/ml, and thus above highest concentration of the composite applied in the growth media. In comparison with previous work [5] where daylight induced antibacterial activity of kaolinite/nanoTiO2 (KATI) composites was studied, we can say that the onset of antibacterial activity of ZinKa composites is faster than KATI, especially against S. aureus. Generally lower values of MIC were determined for KATI. The reason of the difference in the obtained MIC values could be caused by using the lamp with wide spectrum bulb in the case of KATI experiments instead of using natural daylight in ZinKa experiments. As it has been mentioned above, there might be also difference in the proposed mechanism of daylight induced antibacterial activity and additional role of Zn2+ ions.
Tab. Experimental MIC values (mg/ml)
Exposure time Sample
180min
240min
300min
1 day
2 day
3 day
Staphylococcus aureus
ZinKa51
100
11.1
11.1
1.2
0.41
0.41
ZinKa55
100
11.1
11.1
1.2
0.41
0.41
Escherichia coli
ZinKa51
>100
>100
100
100
33.3
33.3
ZinKa55
>100
>100
100
100
33.3
33.3
Enterococcus faecalis
ZinKa51
>100
>100
>100
33.3
33.3
33.3
ZinKa55
>100
>100
>100
>100
>100
>100
Pseudomonas aeruginosa
ZinKa51
>100
>100
>100
100
100
100
ZinKa55
>100
>100
>100
>100
>100
>100
4
Conclusions
ZnO/kaoline composites were laboratory prepared by thermal decomposition of Na2Zn3(CO3)4·(H2O)3 which is the product of the reaction of zinc chloride and Na2CO3 in aqueous suspension with kaolinite. Transmission and scanning electron microscopy confirmed the presence of ZnO particles and proved that ZnO particles are bound onto the surface of kaoline. XRPD proved formation of zincite during the thermal decomposition of Na2Zn3(CO3)4·(H2O)3 at 500°C. FTIR analysis revealed also presence of the carbonate in the composite ZinKa51 and the partial transformation of the kaoline to the metakaoline in the ZinKa55. XRFS revealed approximately 84 % yield of ZnO during the proposed synthesis procedure. Antibacterial assays using four common human pathogen bacterial strains showed that ZinKa samples have antibacterial potency. Both ZinKa samples exhibited faster onset of antibacterial activity against S. aureus and E.coli in comparison to E. faecalis and P. aeruginosa. The lowest MIC was achieved against S. aureus for both ZinKa samples. Composites ZinKa could find potential applications e.g. in the field of antibacterial modification of surfaces of various materials, when its activity was observed under daylight irradiation thus UV light is not necessary to induce the antibacterial activity of ZinKa composites. Moreover, these composites may pose lower environmental risks due to the bonding of ZnO nanaoparticles to kaoline matrix. Acknowledgement The work was supported by the IT4Innovations Centre of Excellence (Project reg. no. cz.1.05/1.1.00/02.0070) and by the project SP2015/54 (Ministry of Education Youth and Sport). References [1] A. Fujishima, N.R. Tata, A.T. Donald, Titanium dioxide photocatalysis,J. Photochem. Photobiol. C(2000) 1-21. [2] A. Wold, Photocatalytic Properties of TiO2, Chem.Mater. 5(3) (1993) 280-283. [3] Z. Huang, X. Zheng, D. Yan, G. Yin, X.Liao, Y.Kang,Y. Yao, D. Huang, B. Hao, Toxicological effect of ZnO Nanoparticles Based on Bacteria, Langmuir 24 (2008) 41404144. [4] K. Dědková, V.A. Fernandéz, L. Kvítek, R. Prucek, A. Panáček, Study of antibacterial activity of silver NPs against animal pathogens. Adv. Sci. Eng. Med. 3 (2011) 93 – 96. [5] K. Dědková, K. Matějová, J. Lang, P. Peikertová, K. MamulováKutláková, L. Neuwirthová, K. Frydrýšek, J. Kukutschová, Antibacterial activity of kaolinite/TiO2 composites in relation to irradiation time, J.Photochem.Photobiol. B 135 (2014) 17-22. [6] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, Antibacterial activity of ZnO suspensions on a broad spectrum of microorganisms, FEMS Microbiol. Lett.279 (2008) 71-76.
[7] N. Talebian, S.M. Amininezhad, M. Doudi, Controllable synthesis of ZnO nanoparticles and their morphology-dependent antibacterial and optical properties, J. Photochem. Photobiol. B 120 (2013) 66-73. [8] K. Kairyte, A. Kadys, Z. Luksiene, Antibacterial and antifungal activity of photoactivatedZnO nanoparticles in suspension, J. Photochem. Photobiol. B 128 (2013)78-84. [9] O. Yamamoto, J. Sawai, T. Sasamoto, Activated Carbon Sphere with Antibacterial Characteristics,Mater. Trans.43 (2002) 1069-1073. [10] S. Atmaca, K. Gul, R. Clcek, The Effect of Zinc on Microbial Growth, Turk. J. Med. Sci. 28 (1998) 595-597. [11] N.L. Stock, J. Séller, K. Vinodgal, P.V. Kamat, Combinative sonolysis and photocatalysis for textile dye degradation, Environ. Sci. Technol. 34 (2000) 1747-1750. [12] L. Korösi, J. Németh, I. Dékány, Structural and photooxidation properties of SnO2/layer silicate nanocomposites, Appl. Clay Sci. 27 (2004) 29-40. [13]M.G. Fonseca, M.M. de Oliveira, L.N.H. Arakaki, Removal of cadmium, zinc, manganese and chromium cations from aqueous solutions by a clay mineral, J. Hazard. Mater. 137 (2006) 288-292. [14] T. Szabó, J. Németh, I. Dékány, Zinc oxide nanoparticles incorporated in ultrathin layer silicate films and their photocatalytic properties, Colloids Surf A: Physicochem. Eng. Aspects 230 (2003) 23-35. [15] V. Matějka, M. Šupová, V. Klemm, D. Rafaja, M. Valášková, J. Tokarský, J. Lešková, E. Plevová, Vermiculite interlayer as a reactor for CdS ultrafine particles preparation, Micropor. Mesopor.Mater. 129 (2010) 118-125. [16] Y. Kameshima, Y. Tamura, A. Nakajima, K. Okada, Preparation and properties of TiO2/montmorillonite composites, Appl. Clay Sci. 45 (2009) 20-23. [17] L.C.R. Machado, C.B. Torchia, R.M. Lago, Floating photocatalysts based on TiO2 supported on high surface area exfoliated vermiculite for water decontamination, Catal. Commun. 7 (2006) 538-541. [18] M.N. Chong, V. Vimonses, S. Lei, B. Jin, C. Chow, C. Saint, Synthesis and characterization of novel titania impregnated kaolinite nano-photocatalyst, Micropor. Mesopor.Mater. 117 (2009) 233-242. [19] J. Panigrahi, D.Behera, I.Mohanty, U. Subudhi, B.B. Nayak, Radio frequency plasma enhanced chemical vapor based ZnO thin film deposition on glass substrate: A novel approach towards antibacterial agent. Appl. Surf. Sci. 258 (2011) 304-311.
Composites ZnO/kaoline (ZinKa) crystallite size less than 100nm were prepared. Analytical methods revealed ZnO tightly anchored on the kaoline matrix.
Antibacterial properties induced by daylight were observed due to photocatalysis. Antibacterial assays found the ZinKa samples to have antibacterial potency. ZinKa exhibited antibacterial properties to human pathogens.
Tab.3 Experimental MIC values (mg/ml)
Exposure time Sample
180min
240min
300min
1 day
2 day
3 day
Staphylococcus aureus
ZinKa51
100
11.1
11.1
1.2
0.41
0.41
ZinKa55
100
11.1
11.1
1.2
0.41
0.41
Escherichia coli
ZinKa51
>100
>100
100
100
33.3
33.3
ZinKa55
>100
>100
100
100
33.3
33.3
Enterococcus faecalis
ZinKa51
>100
>100
>100
33.3
33.3
33.3
ZinKa55
>100
>100
>100
>100
>100
>100
Pseudomonas aeruginosa
ZinKa51
>100
>100
>100
100
100
100
ZinKa55
>100
>100
>100
>100
>100
>100
Fig. 1
S1011-1344(15)00119-0 http://dx.doi.org/10.1016/j.jphotobiol.2015.03.034 JPB 9999
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
28 January 2015 30 March 2015 31 March 2015
Please cite this article as: K. Dědková, B. Janíková, K. Matějová, P. Peikertová, L. Neuwirthová, J. Holešinský, J. Kukutschová, Preparation, characterization and antibacterial properties of ZnO/kaoline nanocomposites, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.03.034
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation, characterization nanocomposites
and
antibacterial
properties
of
ZnO/kaoline
Kateřina Dědkováa, Barbora Janíkováa, Kateřina Matějováb, Pavlína Peikertováa,c, Lucie Neuwirthováa, Jan Holešinskýd, Jana Kukutschováa a
Nanotechnology Centre, VŠB – Technical University of Ostrava, 17. listopadu 15, 708 33 Ostrava - Poruba, Czech Republic, [email protected] b
AGEL laboratory, Zalužanského 1192/15, 703 84 Ostrava–Vítkovice, Czech Republic
c
IT4Innovations Centre of Excellence, VŠB-Technical University of Ostrava, 17. listopadu 15, 70833 Ostrava - Poruba, Czech Republic d
Department of Material Engineering, VŠB – Technical University of Ostrava, 17. listopadu 15, 708 33 Ostrava - Poruba, Czech Republic
Abstract This paper describes laboratory preparation, characterization and antibacterial activity testing of ZnO/kaoline composites. ZnO/kaoline composites with 50 wt% of ZnO were laboratory prepared, dried at 105°C and calcined at 500 °C. XRPD analysis revealed that thermal treatment caused the phase transformation of Zn containing precursor into ZnO. Scanning and transmission electron microscopy techniques were used for characterization of morphology of the prepared samples. A standard microdilution test was used for evaluation of antibacterial activity using four common human pathogens (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aerigonosa). Daylight was used for induction photocatalytically based antibacterial activity. Second possible explanation of antibacterial activity of ZnO/kaoline could be the presence of biologically available forms of zinc. During the antibacterial activity assays the ZnO/kaoline composites exhibited antibacterial activity, where differences in an onset of the antibacterial activity and activity against bacterial strains were observed. The highest antibacterial activity was observed against S.aureus, where the lowest value of minimum inhibitory concentration was determined equal to 0.41mg/ml. Key words Kaoline/ZnO, nanocomposites, antibacterial activity, Staphylococcus aureus, XRD
1
Introduction
Nanomaterials are perspective materials which can give promising opportunities for enhanced properties in medical or environmental applications due to unique physical and chemical properties which are caused by their size in nano dimensions. They have large surface/volume ratio affecting their reactivity [1]. Nowadays the photocatalytic properties are being extensively investigated. The photooxidation of organic compounds to carbon dioxide is often used for decomposition of an organic pollutant in the air or for removal of wastewater impurities. Metal oxides or sulfides show photocatalytic properties. The most common photocatalysts in use are titanium dioxide, zinc oxide or cadmium sulfide [2]. Inorganic metal oxides may serve as effective disinfectants, due to their relatively non toxic profile, good chemical stability and efficient antibacterial activity. Numerous studies described strong antibacterial properties of different nanomaterials [1, 3-5]. Among metal oxide powders, ZnO demonstrates significant growth inhibition of broad spectrum of bacteria [6-8]. The suggested mechanism for the antibacterial activity of ZnO is based on catalysis due to formation of reactive oxygen species (ROS) [9]. Cell wall and cell membrane of bacteria are damaged upon the contact with nanoparticles of ZnO, which cause inhibition of bacterial growth [3]. Moreover, Zn2+ released from surface of ZnO nanoparticles can bind to the membranes of microorganisms and thus prolong the lag phase of the microbial growth cycle [10]. Since the catalysis of radical formation occurs on the particle surface, particles with larger surface area demonstrate stronger antibacterial activity. Therefore, as the size of the ZnO particles decreases their antibacterial activity increases [6]. Due to reactivity of ZnO nanoparticles, they may pose some environmental risks, given by their higher biological activity including potential penetration into cells and generation of reactive oxygen species [11-12]. If ZnO nanoparticles are anchored via chemical bond to a suitable matrix (e.g. clays) they still demonstrate photodegradable properties, however potential environmental risks are lowered due to the decreased mobility in the environmental media. Clay minerals, such as montmorillonite, kaolinite, vermiculite, etc. are natural materials from group of phyllosilicates. They are worldwide spread and have a broad scale of practical applications such as sorbents for metal cations [13]. Phyllosilicates have unique crystallochemical properties; therefore they may act as a suitable matrix for anchoring ZnO nanoparticles [12, 14-15]. Many published articles describe preparation of e.g. montmorillonite/TiO2 or vermiculite/TiO2 nanocomposites [16, 17], but only few studies deal with the preparation and antibacterial activity of nanocomposites with kaoline matrix [18], therefore kaoline was selected as a matrix in this study. The aim of the study was to prepare, and characterize kaoline/ZnO composite and evaluate its antibacterial activity for selected human pathogens in relation to the extent of daylight irradiation, and evaluate these prepared materials for their potential usage for antibacterial modification of surfaces in medical or sanitary applications.
2
Materials and methods 2.1. Studied nanocomposites
The entire preparation procedure consists of two main parts. The first part includes reaction between aqueous solution of sodium carbonate and zinc chloride in suspension of kaoline to obtain the precursor. After 3h stirring of the suspension, precursor was dried at 70 °C. The second part is based on thermal decomposition of the precursor to form zinc oxide. The thermal treatment of zinc oxide precursor is carried out in chamber furnace at 500 °C. The amount of reactants was chosen to achieve 50 wt. % ZnO in the final composites. The samples were assigned as ZinKa51 and ZinKa55, where the first number represents the amount of ZnO in composites (5 ≈ 50%) and the last number denoted the temperature of calcination (1 ≈ 100°C, 5 ≈ 500°C). 2.2. Microscopic and Phase Analysis Chemical composition of prepared composites was determined using energy dispersive X–ray fluorescence spectrometer (XRFS, SPECTRO XEPOS) equipped with 50W Pd X–ray tube. The samples were pressed into tablets for this measurement. X-ray powder diffraction (Bruker D8 Advance diffractometer equipped with detector VANTEC 1, rotational holder and CoKα lamp, λ=1.789 A˚) was used to obtain the phase composition of the samples. For the evaluation of phase composition database PDF 2 Release 2004 was used. Scanning electron microscope (Quanta FEG 450, FEI) and transmission electron microscope (JEM2100, JEOL) were used as microscopic methods for morphology characterization of the studied samples. The samples for SEM were coated with Au/Pd film. Mid-IR spectra were obtained by Fourier transform infrared spectrometer Nicolet 6700 (Thermo USA) using the single reflection ATR technique on a diamond crystal. Measurements were performed with a resolution of 4 cm-1 and 32 scans. Achieved data were processed using the OMNIC software. 2.3. Leaching test The leachates were prepared in accordance with European technical standard EN 12457-2. ZinKa samples were leached in deionised water (denoted as extraction agent DW) in continuous rotation container for 24 h to evaluate mobility of ZnO, resp. Zn2+ in water environment. After mixing for 24 h the mixture was centrifuged (3000 rpm) for 30 min and then filtered through the filter paper with density of 84 g/m2 in order to separate the solid phase. Zn was determined by flame AAS UNICAM 969.
2.4. Antibacterial Assessment Four different human pathogenic bacterial strains were used for the determination of antibacterial activity. Glucose broth (HiMedia) was used as a growth media for the purpose of antibacterial assay of the ZinKa composites. Turbidity of the inoculums was measured using Densi-La-Meter (LACHEMA). Incubation of bacteria was conducted in Biological thermostat BT 120M at 37°C. The antibacterial activity of the composite ZinKa was tested using standard microdilution method which enabled to determine the minimum inhibitory concentration (MIC) of tested substances. Disposable microtitration plates were used for the testing. Commercial solid blood agar plates for cultivation the bacteria without any additional modifications were used. Liquid growth media were prepared by instruction of supplier and after this sterilized in an autoclave. Suspension of ZinKa in growth media was diluted to achieve concentrations 100, 33.3, 11, 3.7, 1.2, 0.41, 0.014 mg/ml of ZinKa in growth media. Staphylococcus aureus 3953, Escherichia coli 3954, Enterococcus faecalis 4224 and Pseudomonas aerigonosa 1960 were achieved from the Czech Collection of Microorganisms (Czech Republic). Bacterial inoculums used had a cell concentration of 1.2x109 (S. aureus, E.coli), 1.3x109 (E. faecalis) 1.6x109 (P. aeruginosa) CFU/mL (colony-forming units per milliliter). Each cell at the micro titration plate was inoculated, and thus this plate is called the reaction plate. This plate was placed on the windowsill for 8 hours due to exposure to daylight irradiation. After defined time sections of growth inhibition presenting living bacterial cells were transferred from the reaction plate to pure growth media using the inoculation hedgehog. These re-inoculated plates were incubated at 37°C for 24h and then the MIC values were determined according to visible growth inhibition. This modification of the standard microdilution method eliminated issues with the determination of MIC caused by turbidity of death cells or caused by the presence of the solid ZinKa sample in a microtitration plate, because only living bacterial cells can be captured by needles of the inoculation hedgehog. Detailed description of antibacterial assessment was already published and could be seen in [5].
3
Results and discussion
Chemical composition of the prepared composites ZinKa51, ZinKa55 and pure kaoline is shown in table 1. Chemical composition analysis confirmed the presence of Zn in both composites. The content of ZnO in the composite dried at 100°C is lower than in the composite calcined at 500°C due to thermal processes, this fact is connected to higher value of LOI (loss of ignition) observed for dried composite (22.63%) in comparison to the composite calcined at 500 °C (5.72 %). The amount of ZnO in samples are 31.44 wt. % of ZnO for ZinKa51 and 41.00 wt. % of ZnO for ZinKa55. The ZinKa55 value shows the yield of ZnO approximately 82 %.
Table 1: Chemical composition and lost of ignition (wt. %) of pure kaoline (KA), ZinKa51, ZinKa55 composites estimated from XRFS results. sample
Na2O
MgO
Al2O3
SiO2
P2O5
SO3
K2O
CaO
TiO2
MnO
Fe2O3
ZnO
LOI
KA
0.071
0.140
34.80
49.70
0.048
0.056
1.193
0.061
0.847
0.003
0.559
-
11.52
ZinKa51
0.082
0.145
19.02
24.23
0.011
0.003
0.618
0.062
0.445
0.02
0.293
31.44
22.63
ZinKa55
0.110
0.180
21.66
28.41
0.028
0.018
0.800
0.094
0.573
0.025
0.375
41.00
5.72
LOI – lost of ignition, - means not present in pure kaoline The XRPD (X-ray powder diffraction) patterns of the pure kaoline, ZinKa51 and ZinKa55 are shown in Fig 1. The analysis verified muscovite and quartz as the typical admixtures of kaoline (KA). The XRPD pattern of composite ZinKa51 reveals the formation of Na2Zn3(CO3)4·(H2O)3 as the product of reaction of ZnCl2 and Na2CO3 in aqueous suspension. The presence of zincite (ZnO) was proved in the diffraction pattern of the composite ZinKa55. During the calcination of the ZinKa51 newly formed compound Na2Zn3(CO3)4·(H2O)3 were decomposed whereas ZnO is formed.
Fig. 1 XRPD pattern of pure kaoline (Ka), ZinKa51 and ZinKa55 - kaolinite (1), quartz (2), muscovite (3), Na2Zn3(CO3)4·(H2O)3 (4), ZnO (5). TEM image of the sample Ka (Fig. 2A) revealed smooth surface of kaolinite particles, whereas in the case of ZinKa55 (Fig. 2B) composite the surface of the kaolinite particles is covered by uniformly dispersed ZnO particles with dimension less than 100 nm. Analogical structure was observed for ZinKa51.
B
A
Fig. 2 TEM images of Ka (A) and ZinKa55 (B) The SEM images of the studied samples (Fig. 4) proved the matrix consisting of micro-sized kaolinite particles having a layered structure with sub-micron particles of ZnO attached onto the surface of the clay matrix. ZnO particles cover the surface of kaoline matrix almost homogeneously and are not attached on edges of kaoline sheets. A
B
Fig. 3 SEM images of ZinKa51 (A) and ZinKa55 (B) FTIR studies of the composites were carried out in the mid-IR region 4000-400 cm-1, whereas the absorption region of the diamond crystal was removed from the obtained spectra. The obtained spectra are shown in Fig. 4. Kaoline spectrum shows all its characteristic absorbtion bands. The bands are assigned to the particular bonds as follows: Al-O-H (3684, 3651, 3619, 937, and 911 cm-1), Si-O (1114, 788, 641, and 456 cm-1), Si-O-Al (997, 750, 692, and 522 cm-1) and Si-O-Si (1024 cm-1). In the spectrum of ZinKa51 are all characteristic bands of kaoline are also clearly visible. However, some new bands appear in the spectrum. The band at 3394 cm-1 corresponds to the absorbed water in the sample; this band vanished after calcination (ZinKa55). Other bands in the lower wavenumbers (1506, 1457, 1394, 1094,
and 836 cm-1) corresponding to the carbonates in the sample, which is decayed after calcination, because these bands are missing in the spectrum of ZinKa55. The presence of carbonate (Na2Zn3(CO3)4·(H2O)3) was also proved by XRPD. In the composite ZinKa55 is also observable the transformation of the kaoline to the metakaoline, which is not yet terminated. This transformation is indicated by broader shape of the bands about 1000 cm -1 and vanishing of the inner octahedral structure of the Al-O-H bonds (3700-3600 cm-1).
Fig. 4 Measured mid-IR spectra of pure kaoline (KA) and composites ZinKa51 and ZinKa55. The concentrations of leached Zn after material interaction with DW are listed in Table 2. Table 2: Concentration of leached Zn from ZinKa samples in extracts after interaction with extraction agent DW
Zn Sample [mg/l] ZinKa51
0.031± 0.002
ZinKa55
0.57± 0.03
Leached amount of Zn is aprox. 0.5 wt. % from ZinKa55 and even lower from Zinka51. Taking into account that ZnO content in the photoactive composite materials is 41 wt. % and 31 wt% respectively, it can be concluded that ZnO nanoparticles are tightly anchored on the clay matrix in both photoactive composites (ZinKa51 and ZinKa55). Antibacterial activity expressed as the MIC values of the ZinKa samples was evaluated using four bacterial strains. Values of MIC for ZinKa samples against all bacteria are summarized in tables (Tab. 3). Pure kaoline did not exhibit antibacterial activity therefore MIC could not be determined and are not included in the tables. The highest antibacterial activity was obtained against S.aureus (0.41 mg/ml for both ZinKa samples). Faster onset of antibacterial activity of the ZinKa samples against S.aureus and E.coli was observed in comparison to E.faecalis and P.aeruginosa. It could be caused by different requirements for living condition e.g. pH, etc. of E.faecalis and P.aeruginosa, when S.aureus and E.coli are more sensitive to changes in living conditions so it is not that difficult to cause growth inhibition of the cells comparing to other two strains. Generally, P. aeruginosa is the most resistant from bacteria used in experiments. Bacterial cells have also some defense mechanism which can help them to resist the treatment of ZinKa samples or other chemicals. The difference between antibacterial activity of ZinKa51 and ZinKa55 was observed in the case of E. faecalis and P. aeruginosa while ZinKa51 had better activity and does not contain any ZnO as proved by XRPD method. This fact imply that the effect of Zn2+ ions on the antibacterial activity of the ZinKa51 composites is higher than visible light induced photocatalytical activity of ZnO in the case of ZinKa55. Our results are in accordance to results of the study describing proposed mechanism of antibacterial activity based on Zn 2+ ions and consequent diffusion of these ions into the cytoplasm [19]. Decrease of the MIC values during the reaction time was observed for the ZinKa samples against S. aureus and E. coli. The MIC values >100 mg/ml means that, the MIC were not determined, which can be caused by the MIC value being higher than 100 mg/ml, and thus above highest concentration of the composite applied in the growth media. In comparison with previous work [5] where daylight induced antibacterial activity of kaolinite/nanoTiO2 (KATI) composites was studied, we can say that the onset of antibacterial activity of ZinKa composites is faster than KATI, especially against S. aureus. Generally lower values of MIC were determined for KATI. The reason of the difference in the obtained MIC values could be caused by using the lamp with wide spectrum bulb in the case of KATI experiments instead of using natural daylight in ZinKa experiments. As it has been mentioned above, there might be also difference in the proposed mechanism of daylight induced antibacterial activity and additional role of Zn2+ ions.
Tab. Experimental MIC values (mg/ml)
Exposure time Sample
180min
240min
300min
1 day
2 day
3 day
Staphylococcus aureus
ZinKa51
100
11.1
11.1
1.2
0.41
0.41
ZinKa55
100
11.1
11.1
1.2
0.41
0.41
Escherichia coli
ZinKa51
>100
>100
100
100
33.3
33.3
ZinKa55
>100
>100
100
100
33.3
33.3
Enterococcus faecalis
ZinKa51
>100
>100
>100
33.3
33.3
33.3
ZinKa55
>100
>100
>100
>100
>100
>100
Pseudomonas aeruginosa
ZinKa51
>100
>100
>100
100
100
100
ZinKa55
>100
>100
>100
>100
>100
>100
4
Conclusions
ZnO/kaoline composites were laboratory prepared by thermal decomposition of Na2Zn3(CO3)4·(H2O)3 which is the product of the reaction of zinc chloride and Na2CO3 in aqueous suspension with kaolinite. Transmission and scanning electron microscopy confirmed the presence of ZnO particles and proved that ZnO particles are bound onto the surface of kaoline. XRPD proved formation of zincite during the thermal decomposition of Na2Zn3(CO3)4·(H2O)3 at 500°C. FTIR analysis revealed also presence of the carbonate in the composite ZinKa51 and the partial transformation of the kaoline to the metakaoline in the ZinKa55. XRFS revealed approximately 84 % yield of ZnO during the proposed synthesis procedure. Antibacterial assays using four common human pathogen bacterial strains showed that ZinKa samples have antibacterial potency. Both ZinKa samples exhibited faster onset of antibacterial activity against S. aureus and E.coli in comparison to E. faecalis and P. aeruginosa. The lowest MIC was achieved against S. aureus for both ZinKa samples. Composites ZinKa could find potential applications e.g. in the field of antibacterial modification of surfaces of various materials, when its activity was observed under daylight irradiation thus UV light is not necessary to induce the antibacterial activity of ZinKa composites. Moreover, these composites may pose lower environmental risks due to the bonding of ZnO nanaoparticles to kaoline matrix. Acknowledgement The work was supported by the IT4Innovations Centre of Excellence (Project reg. no. cz.1.05/1.1.00/02.0070) and by the project SP2015/54 (Ministry of Education Youth and Sport). References [1] A. Fujishima, N.R. Tata, A.T. Donald, Titanium dioxide photocatalysis,J. Photochem. Photobiol. C(2000) 1-21. [2] A. Wold, Photocatalytic Properties of TiO2, Chem.Mater. 5(3) (1993) 280-283. [3] Z. Huang, X. Zheng, D. Yan, G. Yin, X.Liao, Y.Kang,Y. Yao, D. Huang, B. Hao, Toxicological effect of ZnO Nanoparticles Based on Bacteria, Langmuir 24 (2008) 41404144. [4] K. Dědková, V.A. Fernandéz, L. Kvítek, R. Prucek, A. Panáček, Study of antibacterial activity of silver NPs against animal pathogens. Adv. Sci. Eng. Med. 3 (2011) 93 – 96. [5] K. Dědková, K. Matějová, J. Lang, P. Peikertová, K. MamulováKutláková, L. Neuwirthová, K. Frydrýšek, J. Kukutschová, Antibacterial activity of kaolinite/TiO2 composites in relation to irradiation time, J.Photochem.Photobiol. B 135 (2014) 17-22. [6] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, Antibacterial activity of ZnO suspensions on a broad spectrum of microorganisms, FEMS Microbiol. Lett.279 (2008) 71-76.
[7] N. Talebian, S.M. Amininezhad, M. Doudi, Controllable synthesis of ZnO nanoparticles and their morphology-dependent antibacterial and optical properties, J. Photochem. Photobiol. B 120 (2013) 66-73. [8] K. Kairyte, A. Kadys, Z. Luksiene, Antibacterial and antifungal activity of photoactivatedZnO nanoparticles in suspension, J. Photochem. Photobiol. B 128 (2013)78-84. [9] O. Yamamoto, J. Sawai, T. Sasamoto, Activated Carbon Sphere with Antibacterial Characteristics,Mater. Trans.43 (2002) 1069-1073. [10] S. Atmaca, K. Gul, R. Clcek, The Effect of Zinc on Microbial Growth, Turk. J. Med. Sci. 28 (1998) 595-597. [11] N.L. Stock, J. Séller, K. Vinodgal, P.V. Kamat, Combinative sonolysis and photocatalysis for textile dye degradation, Environ. Sci. Technol. 34 (2000) 1747-1750. [12] L. Korösi, J. Németh, I. Dékány, Structural and photooxidation properties of SnO2/layer silicate nanocomposites, Appl. Clay Sci. 27 (2004) 29-40. [13]M.G. Fonseca, M.M. de Oliveira, L.N.H. Arakaki, Removal of cadmium, zinc, manganese and chromium cations from aqueous solutions by a clay mineral, J. Hazard. Mater. 137 (2006) 288-292. [14] T. Szabó, J. Németh, I. Dékány, Zinc oxide nanoparticles incorporated in ultrathin layer silicate films and their photocatalytic properties, Colloids Surf A: Physicochem. Eng. Aspects 230 (2003) 23-35. [15] V. Matějka, M. Šupová, V. Klemm, D. Rafaja, M. Valášková, J. Tokarský, J. Lešková, E. Plevová, Vermiculite interlayer as a reactor for CdS ultrafine particles preparation, Micropor. Mesopor.Mater. 129 (2010) 118-125. [16] Y. Kameshima, Y. Tamura, A. Nakajima, K. Okada, Preparation and properties of TiO2/montmorillonite composites, Appl. Clay Sci. 45 (2009) 20-23. [17] L.C.R. Machado, C.B. Torchia, R.M. Lago, Floating photocatalysts based on TiO2 supported on high surface area exfoliated vermiculite for water decontamination, Catal. Commun. 7 (2006) 538-541. [18] M.N. Chong, V. Vimonses, S. Lei, B. Jin, C. Chow, C. Saint, Synthesis and characterization of novel titania impregnated kaolinite nano-photocatalyst, Micropor. Mesopor.Mater. 117 (2009) 233-242. [19] J. Panigrahi, D.Behera, I.Mohanty, U. Subudhi, B.B. Nayak, Radio frequency plasma enhanced chemical vapor based ZnO thin film deposition on glass substrate: A novel approach towards antibacterial agent. Appl. Surf. Sci. 258 (2011) 304-311.
Composites ZnO/kaoline (ZinKa) crystallite size less than 100nm were prepared. Analytical methods revealed ZnO tightly anchored on the kaoline matrix.
Antibacterial properties induced by daylight were observed due to photocatalysis. Antibacterial assays found the ZinKa samples to have antibacterial potency. ZinKa exhibited antibacterial properties to human pathogens.
Tab.3 Experimental MIC values (mg/ml)
Exposure time Sample
180min
240min
300min
1 day
2 day
3 day
Staphylococcus aureus
ZinKa51
100
11.1
11.1
1.2
0.41
0.41
ZinKa55
100
11.1
11.1
1.2
0.41
0.41
Escherichia coli
ZinKa51
>100
>100
100
100
33.3
33.3
ZinKa55
>100
>100
100
100
33.3
33.3
Enterococcus faecalis
ZinKa51
>100
>100
>100
33.3
33.3
33.3
ZinKa55
>100
>100
>100
>100
>100
>100
Pseudomonas aeruginosa
ZinKa51
>100
>100
>100
100
100
100
ZinKa55
>100
>100
>100
>100
>100
>100
Fig. 1
