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Silver supported on natural Mexican zeolite as an antibacterial material
Microporous and Mesoporous Materials 39 (2000) 431±444
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Silver supported on natural Mexican zeolite as an antibacterial material M. Rivera-Garza a,b, M.T. Olguõn a,*, I. Garcõa-Sosa a, D. Alc antara a, G. Rodrõguez-Fuentes c a
Departamento de Quõmica, Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, Col. Escand on, Delegaci on Miguel Hidalgo, C.P. 11801, M exico, D.F., Mexico b Facultad de Quõmica, Universidad Aut onoma del Estado de M exico, Tollocan esq. Col on s/n, Toluca Estado de M exico, Mexico c Laboratorio de Ingenierõa de Zeolitas, Universidad de la Habana, Zapata y calle 6, Vedado, Habana, Cuba Received 21 October 1999; received in revised form 31 March 2000; accepted 3 April 2000
Abstract The antimicrobial eect of the Mexican zeolitic mineral from Taxco, Guerrero exchanged with silver ions was investigated. The zeolitic mineral as well as sodium and silver zeolitic minerals were characterized by using X-ray diffraction, electron microscopy and IR spectroscopy techniques. The elementary composition of the zeolitic mineral was determined by atomic absorption and microanalyses (EDAX). Escherichia coli and Streptococcus faecalis as indicators of fecal contamination of water were chosen to achieve the antibacterial eect of the Mexican silver zeolitic mineral. The amount of silver in water after contact with the Mexican silver zeolitic mineral as a function of both time and initial sodium concentration in liquid media using 110m Ag and 22 Na as radiotracers was analyzed. It was found that the Mexican silver clinoptilolite±heulandite mineral eliminated the pathogenic microorganisms E. coli and S. faecalis from water with the highest amount of silver supported on the mineral after 2 h of contact time. Under these conditions, the silver level in water remained in 50 lg lÿ1 (NOM-041-SSA1-1993). Ó 2000 Elsevier Science B.V. All rights reserved.
1. Introduction Many pathogens ®nd their way into water, which is a common carriage vehicle for excreta. Also, number of diseases are indirectly associated with water and waste disposal. Stormwater runo is the natural route for surface wash and transport of fecal material to waters [1]. The use of bacteria as indicators of the sanitary quality of water dates back to the late 1800s, when Von Fristch described Klebsiella pneumoniae and *
Corresponding author.
K. rhinoscleromatis as microorganisms, characteristically found in feces [2], which are the major microbiological health hazards in natural waters. The coliform groups, fecal and total, have always been the most widely used indicator organisms. The fecal coliform subgroup of total coliforms is a much more speci®c indicator of fecal contamination. Among the coliforms in human feces, 96.4% are fecal [2]. Escherichia coli is the predominant member of the fecal coliform group. Fecal coliforms, and E. coli in particular, were originally chosen as fecal pollution indicators because of their relation to the typhoid±paratyphoid group and their occurrence in large numbers. Fecal
1387-1811/00/$ - see front matter Ó 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 7 - 1 8 1 1 ( 0 0 ) 0 0 2 1 7 - 1
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streptococci are also a primary indicator of fecal contamination. This group consists mainly of strains of Streptococcus bovis, S. durans, S. equinus, S. faecalis and S. faecium [3]. The World Health Organization (WHO) [4] recommended that any water intended for drinking should contain fecal and total coliform counts of 0, in any 100 ml sample. When either of these groups of bacteria are encountered in a sample, immediate investigative action should be taken. The removal or inactivation of pathogenic microorganisms is the last step in the treatment of wastewater. To achieve this, chemical and physical agents, such as chlorine and its derivatives, AgNO3 , ultraviolet light and radiation, are commonly used [1]. In the last 20 years, several investigations have been carried out concerning the use of synthetic and natural zeolites: A, X, Y, Z and clinoptilolite supporting metal ions (Ag, Cu, Zn, Hg, Sn, Pb, Bi, Cd, Cr, Ti) as bactericides for water disinfection [5±15]. The aim of this paper is to characterize and evaluate the antibacterial properties of natural Mexican clinoptilolite supporting Ag ions. E. coli and S. faecalis were chosen in this study as indicators of fecal contamination. 2. Experimental 2.1. Materials The Mexican zeolitic mineral clinoptilolite± heulandite from Taxco, Guerrero, Mexico was supplied by Lumogral company. The mineral was powdered and sieved. The diameter of the particle size selected to carry out the experimentation was 2 mm. The other reagents, of a commercial analytical grade, were used without further puri®cation. 2.2. Sodium Mexican zeolitic mineral A sample of the zeolitic mineral was treated with a concentrated NaCl solution. The solution was separated by centrifugation, and the solid was washed until the Clÿ1 test with AgNO3 was negative. The zeolitic mineral was dried at 80°C for 5 h.
2.3. Silver Mexican zeolitic mineral
1
Samples of the sodium Mexican zeolitic mineral were put into contact with AgNO3 solutions (from diluted to concentrated AgNO3 solutions in order to reach the maximum sorption, according to Breck [17]). The liquids were separated by centrifugation, and the solids were washed. The samples of the zeolitic mineral were then dried at 80°C for 5 h. 2.4. The chemical composition of the Mexican zeolitic mineral The amount of Na, K, Mg, Fe, Ca and Al in the clinoptilolite±heulandite (Mexican zeolitic mineral) was determined by absorption spectrophotometry. After the amount of Si was measured by gravimetric analysis, the water content in the zeolitic mineral was determined by thermogravimetric analysis. 2.5. Amount of Na and Ag in the silver Mexican zeolitic mineral The amounts of Na and Ag in natural sodium and silver zeolitic mineral, respectively, were determined by neutron activation analysis. Samples were irradiated in a TRIGA MARK III nuclear reactor for 40 s for Na and 3 h for Ag, with an approximate neutron ¯ux of 1013 neutron cmÿ2 sÿ1 . The photopeaks of 1368 keV from 24 Na and 658, 706, 764, 885, 937, 1384 and 1505 keV from 110m Ag were detected with a Ge/hyperpure solidstate detector coupled to a 4096 channel pulse height analyzer. 2.6. Characterization of zeolitic mineral 2.6.1. Thermogravimetric analysis The thermal analysis was carried out with a TGA 51 TA thermogravimetric analyzer, which was operated in an atmosphere of nitrogen and at a heating rate of 10 K minÿ1 from 293 to 573 K. 1
Mexican patent in progress.
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2.6.2. X-ray diraction Powder diractograms of the samples were obtained with a Siemens D500 diractometer coupled to a copper-anode X-ray tube. Conventional diractograms were used to identify the compounds. In order to determine the relation clinoptilolite/heulandite in the Mexican zeolitic mineral, a sample of this material was heated at 400°C for 24 h and its powder diractogram was then obtained. 2.6.3. Electron microscopy For scanning electron microscopy observations, the samples were mounted directly on the holders and covered with sputtered gold and then observed at 10 and 20 keV in a Phillips XL30 electron microscope. The microanalyses were carried out with a DX-4 sonde. 2.6.4. Infrared spectroscopy The IR spectra of each sample were obtained at room temperature by means of the FTIR Nicolet Magna IRTM 550 spectrometer at an interval of frequencies 400±4000 cmÿ1 . For this purpose, pellets with a mixture of the sample and KBr were prepared. For the evaluation of the data acquired, the ratio of the transmission 1053 cmÿ1 band (which is assigned to asymmetric stretching vibration of the external tetrahedra) to the transmission of the 455 cmÿ1 band (which is assigned to bending vibration of the internal tetrahedra) was used. 2.7. Microbiological experimentation 2.7.1. Microorganisms and growth conditions For microbiological experimentation, E. coli ATCC 8739 and S. faecalis ATCC 10741 were selected as indicators of fecal contamination of water. Luria Bertani (LB) was used as a growing medium for both the microorganisms, E. coli and S. faecalis. 2.7.2. Bacteria overnight cultures Bacteria were grown aerobically in LB broth at 37°C for 12 h. The cultures were centrifuged twice (10 000 rpm), and the cells were washed and sus-
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pended in distilled water, reaching a ®nal concentration of 103 cells mlÿ1 . 2.7.3. Dynamic aqueous microbiological system E. coli or S. faecalis suspended in 100 ml of distilled water were put into contact with 2.5, 5 and 10 mg of clinoptilolite±heulandite mineral samples exchanged with sodium or silver, and shaken at 37°C for 24 h. 2.7.4. Growth assay 0.1 ml were taken from samples of the above mixture (water bacteria Na or Ag zeolitic mineral) at 0, 2, 4, 6 and 24 h. These aliquots were diluted in distilled water. 0.1 ml of each diluted samples were spread on LB agar plates and incubated at 37°C for 24 h. Bacterial colonies were counted with in a cell counter C-110-76887 New Brunswick Scienti®c. Each growth assay was performed with three replicated samples. The values obtained were averaged to give the ®nal data with standard deviations. 2.8. Amount of Ag in water after contact with the silver zeolitic mineral 0.058 g of the sodium zeolitic mineral was put into contact with 5 ml of AgNO3 solution labeled with 110m Ag for 48 h. Then the phases were separated by centrifugation, and the solid phase was washed. The radioactivity was measured from 1 ml of the liquid phase. The zeolitic mineral labeled with 110m Ag was put into contact with 1.2 ml of distilled water. The mixture was shaken for 2 h. The solid was separated by centrifugation, and the radioactivity was measured from 1 ml of the liquid phase. This procedure was also repeated at 4, 28 and 76 h. 2.9. Amount of sodium in distilled water Two aliquot parts of 1 ml of distilled water used during the experimentation of this research were irradiated together with a Na standard in a Nuclear Reactor TRIGA MARK III in a similar neutron ¯ux and irradiation time conditions, as mentioned above.
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2.10. Amount of Ag in water after interaction between silver zeolitic mineral and water with dierent concentrations of sodium 100 mg samples of the clinoptilolite±heulandite mineral exchanged with silver were put into contact with 1.2 ml of 1, 10, 50 and 100 mg mlÿ1 NaNO3 solutions labeled with 22 Na and shaken for 6 h. The phases were separated by centrifugation. Two aliquot parts of 1 ml from the liquid phase were irradiated in order to obtain the amount of silver in solution, under the same irradiation conditions, as described above. On the other hand, the 22 Na radioactivity from aliquot parts of 1 ml were measured. 2.11. Statistical analyses The signi®cative dierences among the antibacterial eects of the silver zeolitic mineral on E. coli and S. faecalis were explored by an analysis of variance and the Tuckey test. This was performed in order to determine the in¯uence of the mass and percentages of Ag in the zeolitic mineral on the antibacterial eects for both bacterial strains at the 95% con®dence level.
3. Results and discussion 3.1. The chemical composition of the Mexican clinoptilolite±heulandite mineral Table 1 shows the chemical composition of the Mexican clinoptilolite±heulandite mineral. The Table 1 Chemical composition of the Mexican clinoptilolite±heulandite mineral Components
(%)
SiO2 Al2 O3 Fe2 O3 MgO CaO Na2 O K2 O H2 O
66.2 9.7 1.5 4.5 2.9 0.5 3.7 11.0
ratio SiO2 /Al2 O3 obtained for this mineral was 6.8, which is similar to what was observed in clinoptilolite and heulandite rocks of various deposits, among which are Patagonia, CA/USA, Transcarpathia/Russia, Rhodopes/Bulgaria and Las Villas/ Cuba, whose values are between 5 and 7 [16]. The amount of potassium is higher than calcium and sodium in this Mexican zeolitic mineral, as can be seen in Table 1. The chemical composition of the heulandite±clinoptilolite series is characterized by remarkable changes in the SiO2 /Al2 O3 ratio as well as in the composition of exchangeable cations. According to the classi®cation of the clinoptilolite in relation to their chemical composition, the Mexican zeolitic mineral could correspond to a clinoptilolite with low silica and with alkali forms having K2 O Na2 O > CaO. 3.2. The sodium zeolitic mineral Table 2 shows the Na, Ca and K content of the Mexican clinoptilolite±heulandite mineral after treatment with sodium chloride. It can be observed that the sodium content in this mineral increased substantially, while the calcium content decreased at about a half in relation to its initial amount. No changes, however were observed in the K and Fe content after this treatment. This result suggests that Ca is the exchangeable ion in this zeolitic mineral. 3.3. Na /Ag ion exchange processes It was observed, a good linear correlation between the Na meq per 100 ml of the solutions in relation to the Ag meq gÿ1 of Mexican sodium zeolitic mineral after the ion exchange process. Table 2 Na, Ca, and K content in the Mexican zeolitic mineral before and after treatment with a NaCl solution Mineral
Content (%) Na
Ca
K
Clinoptilolite±heulandite Sodium clinoptilolite± heulandite
0.36 1.66
2.09 1.12
3.08 3.05
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This result suggests that the ion exchange in the zeolite takes place as follows: Na±Z AgNO3 solution ! Ag±Z NaNO3 ; where Z means Mexican clinoptilolite±heulandite mineral. For this reason, it is suggested that Na is preferentially exchangeable by Ag instead of by other ions, such as Ca2 and K , found in the treated Mexican sodium zeolitic mineral. 3.4. Characterization of zeolitic mineral 3.4.1. X-ray diraction In general, clinoptilolite tus contain 70±90% of clinoptilolite in association with authigenic species, such as montmorillonite, celadonite, chlorite, calcite, low cristobalite and sometimes, mordenite, as well as with high-temperature minerals such as quartz, plagioclase, biotite and potassium feldspar [16]. The principal components of the Mexican zeolitic mineral are shown in Table 3. Albite, moganite and biotite in the Mexican clinoptilolite± heulandite mineral were not found. It is well known that thermal behavior of both clinoptilolite and heulandite presents important dierences because of their thermal stability. Heulandite is completely destroyed after heating at 350°C for 3±4 h while clinoptilolite is stable at 750±800°C [15,17]. In order to distinguish these two principal components (clinoptilolite and heulandite) from the Mexican zeolitic mineral, a sample of this material was heated at 400°C for 24 h [18]. Fig. 1 shows the diraction patterns of the Mexican zeolitic mineral before and after thermal Table 3 Components of Mexican clinoptilolite±heulandite mineral Components
JCPDS reference cards
Clinoptilolite Heulandite Calcite Cristobalite Mordenite Quartz Stilbite
25-1349 25-0144 43-0697 11-0695 6-0239 33-1161 10-0433
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treatment. It is clear that the intensity of re¯ection observed at 10° decreases drastically after heating, which corresponds only to clinoptilolite. It is considered that before heating, this re¯ection corresponds to both clinoptilolite and heulandite. According to the results obtained, it is proposed that there is a heulandite±clinoptilolite relation given by heulandite=clinoptilolite 2:3. When the Mexican zeolitic mineral was treated with sodium or silver at a low concentration, no changes in the diraction patterns were observed. Only the Mexican zeolitic mineral with the highest Ag content showed dierences in its diraction pattern from 29° to 30°, in relation to the sodium zeolitic mineral diraction pattern (Fig. 2). These changes could be attributed to the Ag incorporation into the zeolite framework. 3.4.2. Electron microscopy The elemental composition of the Mexican zeolitic mineral obtained by chemical analyses and microanalyses by electron microscopy is similar. The size of the zeolitic mineral particles used in the microbiological experimentation was approximately of 2.0 mm. The microchemical elemental composition of Mexican sodium and silver zeolitic minerals obtained by EDAX changed in relation to the Mexican zeolitic mineral, as was observed by chemical analyses. In 1976, Mumpton and Ormsby reported the characterization of zeolites in sedimentary rocks of dierent regions [19]. The crystals of these materials have characteristic monoclinic symmetry of blades and laths, some of which are similar to the con shape of megascopic heulandite that occurs in vugs in basalts. They also observed ®bers of mordenite in some samples. The Mexican clinoptilolite±heulandite crystal morphology is similar to that observed by these authors, as can be seen in Fig. 3. No changes were observed in the morphology of the Mexican sodium zeolitic mineral in relation to the Mexican zeolitic mineral; however, in the Mexican silver zeolitic mineral, some small particles on the surface of the zeolitic material were observed, as can be seen in Fig. 4. When these small particles were microanalyzed, the results revealed a high concentration of Ag (Fig. 5). For this
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Fig. 1. X-ray diraction pattern (A) Mexican zeolitic mineral thermal treated at 400°C for 24 h and (B) Mexican zeolitic mineral.
Fig. 2. X-ray diraction pattern of (A) Mexican sodium zeolitic mineral (B) Mexican silver zeolitic mineral (24A Ag content).
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Fig. 3. SEM image of Mexican clinoptilolite±heulandite mineral. Note the characteristic monoclinic symmetry of blades and laths.
reason, it is proposed that the Ag exchanged in the zeolitic material reduces to Ag° in the presence of light [20]. This is a very important fact in the consideration of the eciency of the silver supported on Mexican natural zeolite as an antimicrobial material. 3.4.3. Infrared spectroscopy Flanigen and Khatami [21] have considered two groups of frequencies of vibration in all zeolites: internal vibrations of T±O (considered insensitive
437
Fig. 4. SEM image of the Mexican silver clinoptilolite±heulandite mineral (24A Ag content), 10 000.
to structure) and vibrations of external linkages between tetrahedra, due to topology and the mode of arrangement of the structure. No notable changes were observed in the vibration bands in the IR spectra of the Mexican zeolitic mineral treated with sodium as compared with the vibration bands of Mexican zeolitic mineral. When the sodium zeolitic mineral was treated with silver at the highest concentration, changes in some vibration bands (1050, 1400, 1700 and 3500 cmÿ1 ) were observed, as can be seen in Fig. 6.
Fig. 5. Elemental microanalyses of a silver crystal deposited on the Mexican zeolitic mineral.
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Fig. 6. IR spectra of (A) Mexican sodium zeolitic mineral and (B) Mexican silver zeolitic mineral (24A Ag content).
In the same ®gure, the vibration band of NOÿ 3 (1350±1400 cmÿ1 ) is not observed, for this reason, it is proposed that Ag is into the zeolite network occupied ion exchange sites. Rodriguez et al. [22] analyzed the natural clinoptilolite±heulandite by IR from Tasajeras deposit and assigned the vibration bands obtained. With this reference, the assignation of vibration bands shown in Table 4 can be proposed for the Mexican clinoptilolite±heulandite mineral. Table 4 Assignment of vibration bands for Mexican clinoptilolite±heulandite mineral from Taxco, Guerrero deposit Vibration modes Internal tetrahedra bending External tetrahedra double ring External tetrahedra linkage symmetric stretching External tetrahedra linkage asymmetric stretching Internal tetrahedra asymmetric stretching O±H bending
Frequency (cmÿ1 )
Intensity
463
Strong
604
Medium
795
Weak
1057
Strong
1203
Shoulder
1637
Wide
For a better evaluation of the data acquired, the ratio of transmission of the 1057 cmÿ1 band, which is assigned to asymmetric stretching vibration of the external tetrahedra, and the transmission of the analyzed band at 463 cmÿ1 , corresponding to the internal tetrahedral bending, were calculated (Table 5). Small dierences in the values were observed for the materials treated with sodium and silver; these can be attributed to the nature of the ions in the zeolite network, because the position of a cation in the clinoptilolite±heulandite structure is in¯uenced by its ionic potential [22]. 3.5. Mexican silver zeolitic mineral as an antimicrobial material 3.5.1. Mexican silver zeolitic mineral mass of 2.5 mg Table 6 shows the number of viable cells of E. coli or S. faecalis suspended in water after their contact with 2.5 mg of the Mexican sodium zeolitic mineral (represented in this work as 0 of Ag content) and silver zeolitic mineral, whose proportion of Ag in the silver Mexican zeolitic mineral was represented in this paper as A, 2A, 5A and 24A. This last Ag content means the maximum silver amount retained by the Mexican zeolitic mineral.
M. Rivera-Garza et al. / Microporous and Mesoporous Materials 39 (2000) 431±444
439
Table 5 Values of 1057/463 cmÿ1 determined for each cation forms of Mexican zeolitic mineral Absorbance ratio 1057/463 cmÿ1
Sample Mexican Mexican Mexican Mexican Mexican Mexican a
zeolitic mineral sodium zeolitic mineral silver zeolitic mineral A Ag content silver zeolitic mineral 2A Ag content silver zeolitic mineral 5A Ag content silver zeolitic mineral 24Aa Ag content
1.340 1.351 1.514 1.638 1.448 1.420
Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
Table 6 Antibacterial property of 2.5 mg of Mexican silver zeolitic mineral on E. coli (ATCC 8739) or S. faecalis (ATCC 10741) Contact time (h)
Proportion of Ag content in the Mexican zeolitic mineral
Number of viable cells of E. coli per ml
Number of viable cells of S. faecalis per ml
0
0a A 2A 5A 24Ab
2:73 0:35 103 2:70 0:40 103 2:83 0:37 103 2:56 0:73 103 2:50 0:43 103
1:33 0:05 103 1:16 0:05 103 1:16 0:05 103 1:20 103 1:20 0:10 103
2
0 A 2A 5A 24A
2:26 0:30 103 1:30 0:30 103 1:03 0:16 103 0 0
1:23 0:05 103 8:00 0:81 102 6:00 0:81 102 5:66 0:57 102 1:66 0:57 102
4
0 A 2A 5A 24A
2:36 0:58 103 6:33 0:51 102 3:33 0:51 102 0 0
1:20 103 5:33 0:52 102 3:66 0:52 102 1:66 0:52 102 1:00 102
6
0 A 2A 5A 24A
1:96 0:35 103 1:33 0:57 102 0 0 0
1:16 0:11 103 4:00 0:10 102 2:00 102 1:33 0:57 102 0
24
0 A 2A 5A 24A
1:83 0:40 103 0 0 0 0
1:13 0:05 103 2:66 0:57 102 1:33 0:57 102 0 0
a b
Refers to the sodium zeolitic mineral. Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
In this table, it is clearly observed that when E. coli suspended in water is in contact with the Mexican silver zeolitic mineral with 5A and 24A Ag content for 2 h, the number of viable cells of this microorganism goes down to zero. For the S. faecalis case, 6 h of contact with the Mexican
zeolitic mineral with 24A Ag content is needed to obtain the same result (viable-cell number equal to zero). These results show a dierence in the antibacterial eect of the Mexican silver zeolitic mineral on E. coli and S. faecalis. These results could be explained in relation to the microorganism
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structural dierences: S. faecalis has a thicker cellular wall than E. coli. For this reason, a higher contact time and a higher amount of Ag supported on the Mexican zeolitic mineral are needed to achieve the same eect as in E. coli [23,24]. It is observed in Table 6 that when the amount of Ag supported on the Mexican zeolitic mineral increases, the number of viable cells for both microorganisms, E. coli and S. faecalis decreases. Indeed, it is important to note that the contact time between the microorganisms suspended in water with the silver zeolitic mineral is an another important factor to consider, for as contact time increases, the viable-cell number of each studied microorganism decreases. For E. coli, a total bactericide eect is obtained after 24 hours at any Ag content of the zeolitic mineral, but for
S. faecalis only at the highest Ag content is a total eect observed. 3.5.2. Mexican silver zeolitic mineral mass of 5.0 mg Table 7 shows the number of viable cells of E. coli or S. faecalis suspended in water after being in contact with 5.0 mg of the Mexican sodium (0 Ag content) and silver zeolitic mineral (A, 2A, 5A and 24A Ag content) for dierent times. In this table, it is clear that when E. coli suspended in water is in contact with the Mexican silver zeolitic mineral with 2A, 5A and 24A Ag content for 2 h, the number of viable cells of this microorganism is equal to zero. S. faecalis also needs two hours of contact with the Mexican
Table 7 Antibacterial property of 5.0 mg of Mexican silver zeolitic mineral on E. coli (ATCC 8739) or S. faecalis (ATCC10741) Contact time (h)
Proportion of Ag content in the Mexican zeolitic mineral
Number of viable cells of E. coli per ml
Number of viable cells of S. faecalis per ml
0
0a A 2A 5A 24Ab
2:63 0:25 103 2:93 0:55 103 3:13 0:15 103 2:93 0:68 103 2:53 0:15 103
1:30 0:20 103 1:20 0:17 103 1:30 0:26 103 1:36 0:28 103 1:43 0:11 103
2
0 A 2A 5A 24A
2:70 0:20 103 3:00 0:73 102 0 0 0
1:20 0:10 103 9:00 0:10 102 7:33 0:52 102 6:00 0:63 102 0
4
0 A 2A 5A 24A
1:96 0:15 103 0 0 0 0
1:10 0:16 103 5:00 0:16 102 2:66 0:16 102 2:33 0:16 102 0
6
0 A 2A 5A 24A
1:90 0:26 103 0 0 0 0
1:00 0:87 103 3:33 0:52 102 0 0 0
24
0 A 2A 5A 24A
1:53 0:20 103 0 0 0 0
1:03 0:86 103 1:00 102 0 0 0
a b
Refers to the sodium zeolitic mineral. Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
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microorganism tested decreases as contact time increases. Although for E. coli a total bactericide eect is obtained after 4 hours with every Ag content, in S. faecalis the same eect was achieved only at the highest Ag content (24A Ag content).
zeolitic mineral, but a higher percentage (24A Ag content) of Ag is needed to obtain the same viablecell number (zero). These results are quite dierent from those obtained for 2.5 mg of Mexican silver zeolitic mineral because the mass increases from 2.5 to 5.0 mg, a better antibacterial eect is obtained for E. coli with a lower Ag content (2A Ag content); and for S. faecalis, the contact time necessary to kill all the bacteria decreases from 6 to 2 h at the same Ag content (24A Ag content). Also in Table 7, it is observed that as the amount of Ag supported on the Mexican zeolitic mineral increased, the amount of viable cells for both microorganisms, E. coli and S. faecalis, decreased in the same way as was observed previously for 2.5 mg of Mexican silver zeolitic mineral. In this case too, the viable-cell number of each
3.5.3. Mexican silver zeolitic mineral mass of 10.0 mg Table 8 shows the number of viable cells of E. coli and S. faecalis suspended in water after their contact with 10.0 mg of Mexican sodium (0 Ag content) and silver zeolitic mineral (A, 2A, 5A and 24A Ag content) at dierent contact times. In this table, it is clearly observed that when E. coli suspended in water is in contact with the Mexican silver zeolitic mineral with A, 2A, 5A and 24A Ag content for two hours, the number of
Table 8 Antibacterial property of 10.0 mg of Mexican silver zeolitic mineral on E. coli (ATCC 8739) or S. faecalis (ATCC10741) Contact time (h)
Proportion of Ag content in the Mexican zeolitic mineral
Number of viable cells of E. coli per ml
Number of viable cells of S. faecalis per ml
0
0a A 2A 5A 24Ab
2:13 0:75 103 2:23 0:63 103 2:26 0:30 103 2:00 0:86 103 2:06 0:20 103
1:23 0:11 103 1:26 0:11 103 1:30 0:10 103 1:20 0:10 103 1:26 0:11 103
2
0 A 2A 5A 24A
2:83 0:80 103 0 0 0 2:30 0:70 103
1:20 0:10 103 5:00 0:87 102 4:00 0:87 102 3:00 0:58 102 0
4
0 A 2A 5A 24A
0 0 0 0 0
1:23 0:11 103 2:66 0:15 102 1:33 0:57 102 0 0
6
0 A 2A 5A 24A
2:00 0:36 103 0 0 0 0
1:13 0:11 103 0 0 0 0
24
0 A 2A 5A 24A
2:40 0:55 103 0 0 0 0
1:06 0:15 103 0 0 0 0
a b
441
Refers to the sodium zeolitic mineral. Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
442
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Table 9 Optimal mass and contact time to obtain a total antibacterial eect with Mexican silver zeolitic mineral Bacteria
Mexican silver zeolitic mineral mass (mg)
Proportion of Ag content in the Mexican zeolitic mineral
Contact time (h)
E. coli
2.5 5.0 10.0
5A 2A A
2 2 2
S. faecalis
2.5 5.0 10.0
24Aa 24A 24A
6 2 2
a
Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
viable cells of this microorganism is equal to zero. For S. faecalis, two hours are also needed to reduce the number of viable cells to zero, but only with 24A Ag content. This latter result is similar to that obtained with 5.0 mg of the Mexican silver zeolitic mineral. In general, these results show a better antibacterial eect with less wt.% of Ag for both microorganisms. Moreover, the contact time necessary to reduce the number of viable cells to zero decreased. It is also important to note in Tables 6±8 that the Mexican sodium zeolitic mineral (0 Ag content) did not show any antibacterial eect. Table 9 shows the optimal masses of the Mexican silver zeolitic mineral with dierent Ag content and optimal contact times to achieve a total antibacterial eect on E. coli and S. faecalis, based on the results obtained. As can be seen, 5 mg of Mexican silver zeolitic mineral with 24A Ag content in contact for two hours with water is enough to remove both pathogenic microorganisms. 3.5.4. Amount of silver released to water after contact with the Mexican silver zeolitic mineral (24A Ag content) Table 10 shows the silver amount released into the aqueous media as a function of the contact time between phases. The solid phase corresponds to the Mexican silver zeolitic mineral (24A Ag content) and the liquid phase corresponds to water used in the microbiological experiments. It is important to note that there is a direct proportion between the silver amount in water and contact time. These results are important to be considered because Mexican normativeness establishes that
Table 10 Silver concentration in water at dierent contact times between the Mexican silver zeolitic mineral (10 mg, 24A Ag content) and the aqueous media Contact time (h)
Silver concentration in water (lg lÿ1 )
2 4 28 76
26.2 37.9 44.8 115.0
the silver level in water should be kept below 50 lg lÿ1 . As was mentioned before, 5 mg of Mexican silver zeolitic mineral with 24A Ag content in contact with water for two hours is suggested in order to remove both pathogenic microorganisms. Under these conditions, the silver level in water (26.2 lg Ag lÿ1 ) remains within the limit established by the Mexican normativeness [25]. 3.5.5. The amount of sodium in water The sodium concentration in water, present in all the microbiological experiments, was 281:5 54 lg lÿ1 . This indicates that release of silver in water after being in contact with Mexican silver zeolitic mineral (24A Ag content) is due to the ionic exchange Ag /Na (Table 10) and that these Ag ions diuse into the microorganism cells and kill them. 3.5.6. Silver concentration in water after contact between Mexican zeolitic mineral (24A Ag content) and water with dierent sodium concentrations Fig. 7 shows that as the sodium concentration in water increases, silver concentration in this liq-
M. Rivera-Garza et al. / Microporous and Mesoporous Materials 39 (2000) 431±444
Fig. 7. Silver amount found in water with dierent sodium concentrations after being in contact with Mexican silver zeolitic mineral.
uid media increases as well after 6 h of contact with the Mexican silver zeolitic mineral (24A Ag content). This result is important to consider because the ®nal silver concentration in the treated water with the Mexican silver zeolitic mineral depends on the initial sodium concentration in the wastewater. This amount of silver should not be higher than the maximum level established by the Mexican normativeness, as was mentioned before. Indeed, it is important to mention that water could contain other ions like Ca2 and Mg2 that can be exchanged by the Ag supported in the Mexican silver zeolitic mineral.
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For E. coli, there are several ratios between the Mexican silver zeolitic mineral mass and the content of Ag to obtain a total antibacterial eect. For instance, after 2 h of contact between the contaminated water with this microorganism and the silver zeolitic mineral, ratios of 2.5 mg-5A Ag content, 5.0 mg-2A Ag content and 10 mg-A Ag content, can be used. For S. faecalis, a total antibacterial eect of the silver zeolitic mineral is observed only with 24A Ag content. In this case, the total eect is not dependent on the mineral mass, as long as contact time is at least for 2 h. The Mexican sodium zeolitic mineral does not have a bactericide eect per se on E. coli and S. faecalis. Acknowledgements The authors thank the CONACyT for ®nancial support, project 26769-E. The technical support of C. Rodrõguez, M. Villa-Tomasa, E. Morales and F.A. Anastacio is gratefully acknowledged. The authors also thank L. Carapia, J. de la Torre and Thelma Falc on for their support in the characterization of the Mexican zeolitic mineral. The authors thank Lucia Hernandez Cruz for her help in editing the English. References
4. Conclusions The Mexican zeolitic mineral from Taxco, Guerrero has two main mineral components: clinoptilolite and heulandite. The mineral has a SiO2 /Al2 O3 ratio of 6.8 and presents the alkali form (K2 O Na2 O > CaO). The ratio between heulandite and clinoptilolite in the Mexican zeolitic mineral is 2.3:1. The Na plays a more important role in the Ag ion exchange process than that played by Ca2 and K , present also in the sodium zeolitic mineral. There are structural modi®cations in the Mexican silver zeolitic mineral because of the uptake of Ag ions into the zeolitic network.
[1] R.L. Droste, Theory and Practice of Water and Wastewater Treatment, Wiley, New York, 1997. [2] E.E. Geldreich, Bacterial populations and indicator concepts in feces, sewage, stormwater and solid wastes, in: R.L. Droste (Ed.), Theory and Practice of Water and Wastewater Treatment, Wiley, New York, 1997, p. 166. [3] B.A. Kenner, Fecal streptococcal indicators, in: R.L. Droste (Ed.), Theory and Practice of Water and Wastewater Treatment, Wiley, New York, 1997, p. 168. [4] World Health Organization (WHO), Guidelines for Drinking-Water Quality, vol. 2, Geneva, 1996. [5] H. Zenji, Manufacture of bactericidal zeolite, Jpn. Kokai Tokkyo Koho JP 01, 164, 721 [89, 164, 721] (CI.CO1B33/ 28), Appl. 87/324, 1987, p. 258. [6] M. Muneo, K. Hiroyuki, N. Masashi, H. Tsunenosuke, U. Keigo, Silver ion exchanged zeolites for desinfection of tap water, Jpn. Kokai Tokkyo Koho JP 04, 21, 517 [92, 21, 517] (C1. C01B33/34), Appl. 90/123, 1990, p. 661.
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[7] H. Junsuke, Manufacture of zeolite pellets, and their use in water treatment, Jpn. Kokai Tokkyo Koho JP 04, 275, 916 [92, 275, 916] (C1.C01B33/34). Appl. 91/36, 070, 1991. [8] H. Junsuke, M. Masaru, Zeolite-bound metal microbicides, Jpn. Kokai Tokkyo Koho JP 04, 83, 713 [92, 83, 713] (C1.C01B33/34), Appl. 90/197, 634, 1990. [9] O. Asao, T. Yoshiharu, H. Mitsuyoshi, O. Fumihiko, F. Yoshitaka, Antimicrobial Silicates, Jpn. Kokai Tokkyo Koho JP 04, 292, 410 [92, 292, 410] (C1.C01B33/26), Appl. 91/56, 888, 1991. [10] K. Shinichi, O. Kunie, N. Kayo, O. Masayuki, S. Takeshi, Suppression of microorganism growth by activated carbon and silver zeolite (Antimicrobial Granular Activated Carbon Column), Kogyo Yosui 436, 1995. [11] H. Toyoki, Manufacture of natural zeolite derived for improvement of water quality, Jpn. Kokai Tokkyo Koho JP 07, 148, 487 [95, 148, 487] (C1.C02F1/42), Appl. 93/299, 843, 1993. [12] T. Shunji, Manufacture of ceramic powder coated with antimicrobial agents, Jpn. Kokai Tokkyo Koho JP 07, 330, 527 [95, 330, 527] (C1.A01N59/04), Appl. 94/125, 005, 1994. [13] P.L. Barcelona, Actividad antimicrobiana del granulado zeolitico ZZ frente a bacterias de los generos, Escherichia, Vibrio, Salmonella y Shigella, Documento del Registro Sanitario de la Rep uplica de Cuba, Cuba, 1996. [14] P.M. Perez, Estudio del efecto de la concentraci on de metales pesados soportados en zeolitas naturales en la remoci on de microorganismos indicadores de contaminaci on, Tesis de Diploma, Universidad de la Habana, Cuba, 1998.
[15] G. Rodrõguez-Fuentes, Propiedades ®sico-quõmicas y aplicaciones industriales de la clinoptilolita natural, Tesis de Doctorado, Centro Nacional de Investigaciones Cientõ®cas, Cuba, 1987. [16] G.V. Tsitsishvili, T.G. Andronikashvili, G.N. Kirov, L.D. Filizova, Natural Zeolites, Ellis Horwood, UK, 1992. [17] W. Breck, Zeolite Molecular Sieve, Wiley/Interscience, New York, 1974. [18] F.A. Mumpton, Clinoptilolite rede®ned, Am. Miner. 45 (1960) 351. [19] F.A. Mumpton, W.C. Ormsby, Morphology of zeolites in sedimentary rocks by scanning electron microscopy, Clays and Clay Miner. 241 (1976) 1. [20] P. Borell, Fotoquõmica, Editorial El Manual Moderno, Mexico, DF, 1980. [21] E.M. Flanigen, H. Khatami, Molecular Sieve Zeolites, vol. 1, American Chemical Society, Washington, 1971. [22] G. Rodrõguez-Fuentes, A.R. Ruiz-Salvador, M. Mir, O. Picazo, G. Quintana, M. Delgado, Thermal and cation in¯uence on IR vibrations of modi®ed natural clinoptilolite, Micropor. Mesopor. Mater. 20 (1998) 269. [23] S. Silver, in: Bacterial Heavy Metal Detoxi®cation and Resistance Approaches, Mongkolsuk (Ed.), Plenum Press, New York, 1992. [24] A. Viarengo, Biochemical eects of trace metals, Marine Pollut. Bull. 16 (1985) 153. [25] Norma O®cial Mexicana NOM-127-SSA1-1994, Salud ambiental, agua para uso y consumo humano, lõmites permisibles de calidad y tratamiento a que debe someterse el agua para su potabilizaci on. Publicada en el Diario O®cial de la Federaci on el 18 de enero de 1996.
www.elsevier.nl/locate/micromeso
Silver supported on natural Mexican zeolite as an antibacterial material M. Rivera-Garza a,b, M.T. Olguõn a,*, I. Garcõa-Sosa a, D. Alc antara a, G. Rodrõguez-Fuentes c a
Departamento de Quõmica, Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, Col. Escand on, Delegaci on Miguel Hidalgo, C.P. 11801, M exico, D.F., Mexico b Facultad de Quõmica, Universidad Aut onoma del Estado de M exico, Tollocan esq. Col on s/n, Toluca Estado de M exico, Mexico c Laboratorio de Ingenierõa de Zeolitas, Universidad de la Habana, Zapata y calle 6, Vedado, Habana, Cuba Received 21 October 1999; received in revised form 31 March 2000; accepted 3 April 2000
Abstract The antimicrobial eect of the Mexican zeolitic mineral from Taxco, Guerrero exchanged with silver ions was investigated. The zeolitic mineral as well as sodium and silver zeolitic minerals were characterized by using X-ray diffraction, electron microscopy and IR spectroscopy techniques. The elementary composition of the zeolitic mineral was determined by atomic absorption and microanalyses (EDAX). Escherichia coli and Streptococcus faecalis as indicators of fecal contamination of water were chosen to achieve the antibacterial eect of the Mexican silver zeolitic mineral. The amount of silver in water after contact with the Mexican silver zeolitic mineral as a function of both time and initial sodium concentration in liquid media using 110m Ag and 22 Na as radiotracers was analyzed. It was found that the Mexican silver clinoptilolite±heulandite mineral eliminated the pathogenic microorganisms E. coli and S. faecalis from water with the highest amount of silver supported on the mineral after 2 h of contact time. Under these conditions, the silver level in water remained in 50 lg lÿ1 (NOM-041-SSA1-1993). Ó 2000 Elsevier Science B.V. All rights reserved.
1. Introduction Many pathogens ®nd their way into water, which is a common carriage vehicle for excreta. Also, number of diseases are indirectly associated with water and waste disposal. Stormwater runo is the natural route for surface wash and transport of fecal material to waters [1]. The use of bacteria as indicators of the sanitary quality of water dates back to the late 1800s, when Von Fristch described Klebsiella pneumoniae and *
Corresponding author.
K. rhinoscleromatis as microorganisms, characteristically found in feces [2], which are the major microbiological health hazards in natural waters. The coliform groups, fecal and total, have always been the most widely used indicator organisms. The fecal coliform subgroup of total coliforms is a much more speci®c indicator of fecal contamination. Among the coliforms in human feces, 96.4% are fecal [2]. Escherichia coli is the predominant member of the fecal coliform group. Fecal coliforms, and E. coli in particular, were originally chosen as fecal pollution indicators because of their relation to the typhoid±paratyphoid group and their occurrence in large numbers. Fecal
1387-1811/00/$ - see front matter Ó 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 7 - 1 8 1 1 ( 0 0 ) 0 0 2 1 7 - 1
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streptococci are also a primary indicator of fecal contamination. This group consists mainly of strains of Streptococcus bovis, S. durans, S. equinus, S. faecalis and S. faecium [3]. The World Health Organization (WHO) [4] recommended that any water intended for drinking should contain fecal and total coliform counts of 0, in any 100 ml sample. When either of these groups of bacteria are encountered in a sample, immediate investigative action should be taken. The removal or inactivation of pathogenic microorganisms is the last step in the treatment of wastewater. To achieve this, chemical and physical agents, such as chlorine and its derivatives, AgNO3 , ultraviolet light and radiation, are commonly used [1]. In the last 20 years, several investigations have been carried out concerning the use of synthetic and natural zeolites: A, X, Y, Z and clinoptilolite supporting metal ions (Ag, Cu, Zn, Hg, Sn, Pb, Bi, Cd, Cr, Ti) as bactericides for water disinfection [5±15]. The aim of this paper is to characterize and evaluate the antibacterial properties of natural Mexican clinoptilolite supporting Ag ions. E. coli and S. faecalis were chosen in this study as indicators of fecal contamination. 2. Experimental 2.1. Materials The Mexican zeolitic mineral clinoptilolite± heulandite from Taxco, Guerrero, Mexico was supplied by Lumogral company. The mineral was powdered and sieved. The diameter of the particle size selected to carry out the experimentation was 2 mm. The other reagents, of a commercial analytical grade, were used without further puri®cation. 2.2. Sodium Mexican zeolitic mineral A sample of the zeolitic mineral was treated with a concentrated NaCl solution. The solution was separated by centrifugation, and the solid was washed until the Clÿ1 test with AgNO3 was negative. The zeolitic mineral was dried at 80°C for 5 h.
2.3. Silver Mexican zeolitic mineral
1
Samples of the sodium Mexican zeolitic mineral were put into contact with AgNO3 solutions (from diluted to concentrated AgNO3 solutions in order to reach the maximum sorption, according to Breck [17]). The liquids were separated by centrifugation, and the solids were washed. The samples of the zeolitic mineral were then dried at 80°C for 5 h. 2.4. The chemical composition of the Mexican zeolitic mineral The amount of Na, K, Mg, Fe, Ca and Al in the clinoptilolite±heulandite (Mexican zeolitic mineral) was determined by absorption spectrophotometry. After the amount of Si was measured by gravimetric analysis, the water content in the zeolitic mineral was determined by thermogravimetric analysis. 2.5. Amount of Na and Ag in the silver Mexican zeolitic mineral The amounts of Na and Ag in natural sodium and silver zeolitic mineral, respectively, were determined by neutron activation analysis. Samples were irradiated in a TRIGA MARK III nuclear reactor for 40 s for Na and 3 h for Ag, with an approximate neutron ¯ux of 1013 neutron cmÿ2 sÿ1 . The photopeaks of 1368 keV from 24 Na and 658, 706, 764, 885, 937, 1384 and 1505 keV from 110m Ag were detected with a Ge/hyperpure solidstate detector coupled to a 4096 channel pulse height analyzer. 2.6. Characterization of zeolitic mineral 2.6.1. Thermogravimetric analysis The thermal analysis was carried out with a TGA 51 TA thermogravimetric analyzer, which was operated in an atmosphere of nitrogen and at a heating rate of 10 K minÿ1 from 293 to 573 K. 1
Mexican patent in progress.
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2.6.2. X-ray diraction Powder diractograms of the samples were obtained with a Siemens D500 diractometer coupled to a copper-anode X-ray tube. Conventional diractograms were used to identify the compounds. In order to determine the relation clinoptilolite/heulandite in the Mexican zeolitic mineral, a sample of this material was heated at 400°C for 24 h and its powder diractogram was then obtained. 2.6.3. Electron microscopy For scanning electron microscopy observations, the samples were mounted directly on the holders and covered with sputtered gold and then observed at 10 and 20 keV in a Phillips XL30 electron microscope. The microanalyses were carried out with a DX-4 sonde. 2.6.4. Infrared spectroscopy The IR spectra of each sample were obtained at room temperature by means of the FTIR Nicolet Magna IRTM 550 spectrometer at an interval of frequencies 400±4000 cmÿ1 . For this purpose, pellets with a mixture of the sample and KBr were prepared. For the evaluation of the data acquired, the ratio of the transmission 1053 cmÿ1 band (which is assigned to asymmetric stretching vibration of the external tetrahedra) to the transmission of the 455 cmÿ1 band (which is assigned to bending vibration of the internal tetrahedra) was used. 2.7. Microbiological experimentation 2.7.1. Microorganisms and growth conditions For microbiological experimentation, E. coli ATCC 8739 and S. faecalis ATCC 10741 were selected as indicators of fecal contamination of water. Luria Bertani (LB) was used as a growing medium for both the microorganisms, E. coli and S. faecalis. 2.7.2. Bacteria overnight cultures Bacteria were grown aerobically in LB broth at 37°C for 12 h. The cultures were centrifuged twice (10 000 rpm), and the cells were washed and sus-
433
pended in distilled water, reaching a ®nal concentration of 103 cells mlÿ1 . 2.7.3. Dynamic aqueous microbiological system E. coli or S. faecalis suspended in 100 ml of distilled water were put into contact with 2.5, 5 and 10 mg of clinoptilolite±heulandite mineral samples exchanged with sodium or silver, and shaken at 37°C for 24 h. 2.7.4. Growth assay 0.1 ml were taken from samples of the above mixture (water bacteria Na or Ag zeolitic mineral) at 0, 2, 4, 6 and 24 h. These aliquots were diluted in distilled water. 0.1 ml of each diluted samples were spread on LB agar plates and incubated at 37°C for 24 h. Bacterial colonies were counted with in a cell counter C-110-76887 New Brunswick Scienti®c. Each growth assay was performed with three replicated samples. The values obtained were averaged to give the ®nal data with standard deviations. 2.8. Amount of Ag in water after contact with the silver zeolitic mineral 0.058 g of the sodium zeolitic mineral was put into contact with 5 ml of AgNO3 solution labeled with 110m Ag for 48 h. Then the phases were separated by centrifugation, and the solid phase was washed. The radioactivity was measured from 1 ml of the liquid phase. The zeolitic mineral labeled with 110m Ag was put into contact with 1.2 ml of distilled water. The mixture was shaken for 2 h. The solid was separated by centrifugation, and the radioactivity was measured from 1 ml of the liquid phase. This procedure was also repeated at 4, 28 and 76 h. 2.9. Amount of sodium in distilled water Two aliquot parts of 1 ml of distilled water used during the experimentation of this research were irradiated together with a Na standard in a Nuclear Reactor TRIGA MARK III in a similar neutron ¯ux and irradiation time conditions, as mentioned above.
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2.10. Amount of Ag in water after interaction between silver zeolitic mineral and water with dierent concentrations of sodium 100 mg samples of the clinoptilolite±heulandite mineral exchanged with silver were put into contact with 1.2 ml of 1, 10, 50 and 100 mg mlÿ1 NaNO3 solutions labeled with 22 Na and shaken for 6 h. The phases were separated by centrifugation. Two aliquot parts of 1 ml from the liquid phase were irradiated in order to obtain the amount of silver in solution, under the same irradiation conditions, as described above. On the other hand, the 22 Na radioactivity from aliquot parts of 1 ml were measured. 2.11. Statistical analyses The signi®cative dierences among the antibacterial eects of the silver zeolitic mineral on E. coli and S. faecalis were explored by an analysis of variance and the Tuckey test. This was performed in order to determine the in¯uence of the mass and percentages of Ag in the zeolitic mineral on the antibacterial eects for both bacterial strains at the 95% con®dence level.
3. Results and discussion 3.1. The chemical composition of the Mexican clinoptilolite±heulandite mineral Table 1 shows the chemical composition of the Mexican clinoptilolite±heulandite mineral. The Table 1 Chemical composition of the Mexican clinoptilolite±heulandite mineral Components
(%)
SiO2 Al2 O3 Fe2 O3 MgO CaO Na2 O K2 O H2 O
66.2 9.7 1.5 4.5 2.9 0.5 3.7 11.0
ratio SiO2 /Al2 O3 obtained for this mineral was 6.8, which is similar to what was observed in clinoptilolite and heulandite rocks of various deposits, among which are Patagonia, CA/USA, Transcarpathia/Russia, Rhodopes/Bulgaria and Las Villas/ Cuba, whose values are between 5 and 7 [16]. The amount of potassium is higher than calcium and sodium in this Mexican zeolitic mineral, as can be seen in Table 1. The chemical composition of the heulandite±clinoptilolite series is characterized by remarkable changes in the SiO2 /Al2 O3 ratio as well as in the composition of exchangeable cations. According to the classi®cation of the clinoptilolite in relation to their chemical composition, the Mexican zeolitic mineral could correspond to a clinoptilolite with low silica and with alkali forms having K2 O Na2 O > CaO. 3.2. The sodium zeolitic mineral Table 2 shows the Na, Ca and K content of the Mexican clinoptilolite±heulandite mineral after treatment with sodium chloride. It can be observed that the sodium content in this mineral increased substantially, while the calcium content decreased at about a half in relation to its initial amount. No changes, however were observed in the K and Fe content after this treatment. This result suggests that Ca is the exchangeable ion in this zeolitic mineral. 3.3. Na /Ag ion exchange processes It was observed, a good linear correlation between the Na meq per 100 ml of the solutions in relation to the Ag meq gÿ1 of Mexican sodium zeolitic mineral after the ion exchange process. Table 2 Na, Ca, and K content in the Mexican zeolitic mineral before and after treatment with a NaCl solution Mineral
Content (%) Na
Ca
K
Clinoptilolite±heulandite Sodium clinoptilolite± heulandite
0.36 1.66
2.09 1.12
3.08 3.05
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This result suggests that the ion exchange in the zeolite takes place as follows: Na±Z AgNO3 solution ! Ag±Z NaNO3 ; where Z means Mexican clinoptilolite±heulandite mineral. For this reason, it is suggested that Na is preferentially exchangeable by Ag instead of by other ions, such as Ca2 and K , found in the treated Mexican sodium zeolitic mineral. 3.4. Characterization of zeolitic mineral 3.4.1. X-ray diraction In general, clinoptilolite tus contain 70±90% of clinoptilolite in association with authigenic species, such as montmorillonite, celadonite, chlorite, calcite, low cristobalite and sometimes, mordenite, as well as with high-temperature minerals such as quartz, plagioclase, biotite and potassium feldspar [16]. The principal components of the Mexican zeolitic mineral are shown in Table 3. Albite, moganite and biotite in the Mexican clinoptilolite± heulandite mineral were not found. It is well known that thermal behavior of both clinoptilolite and heulandite presents important dierences because of their thermal stability. Heulandite is completely destroyed after heating at 350°C for 3±4 h while clinoptilolite is stable at 750±800°C [15,17]. In order to distinguish these two principal components (clinoptilolite and heulandite) from the Mexican zeolitic mineral, a sample of this material was heated at 400°C for 24 h [18]. Fig. 1 shows the diraction patterns of the Mexican zeolitic mineral before and after thermal Table 3 Components of Mexican clinoptilolite±heulandite mineral Components
JCPDS reference cards
Clinoptilolite Heulandite Calcite Cristobalite Mordenite Quartz Stilbite
25-1349 25-0144 43-0697 11-0695 6-0239 33-1161 10-0433
435
treatment. It is clear that the intensity of re¯ection observed at 10° decreases drastically after heating, which corresponds only to clinoptilolite. It is considered that before heating, this re¯ection corresponds to both clinoptilolite and heulandite. According to the results obtained, it is proposed that there is a heulandite±clinoptilolite relation given by heulandite=clinoptilolite 2:3. When the Mexican zeolitic mineral was treated with sodium or silver at a low concentration, no changes in the diraction patterns were observed. Only the Mexican zeolitic mineral with the highest Ag content showed dierences in its diraction pattern from 29° to 30°, in relation to the sodium zeolitic mineral diraction pattern (Fig. 2). These changes could be attributed to the Ag incorporation into the zeolite framework. 3.4.2. Electron microscopy The elemental composition of the Mexican zeolitic mineral obtained by chemical analyses and microanalyses by electron microscopy is similar. The size of the zeolitic mineral particles used in the microbiological experimentation was approximately of 2.0 mm. The microchemical elemental composition of Mexican sodium and silver zeolitic minerals obtained by EDAX changed in relation to the Mexican zeolitic mineral, as was observed by chemical analyses. In 1976, Mumpton and Ormsby reported the characterization of zeolites in sedimentary rocks of dierent regions [19]. The crystals of these materials have characteristic monoclinic symmetry of blades and laths, some of which are similar to the con shape of megascopic heulandite that occurs in vugs in basalts. They also observed ®bers of mordenite in some samples. The Mexican clinoptilolite±heulandite crystal morphology is similar to that observed by these authors, as can be seen in Fig. 3. No changes were observed in the morphology of the Mexican sodium zeolitic mineral in relation to the Mexican zeolitic mineral; however, in the Mexican silver zeolitic mineral, some small particles on the surface of the zeolitic material were observed, as can be seen in Fig. 4. When these small particles were microanalyzed, the results revealed a high concentration of Ag (Fig. 5). For this
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Fig. 1. X-ray diraction pattern (A) Mexican zeolitic mineral thermal treated at 400°C for 24 h and (B) Mexican zeolitic mineral.
Fig. 2. X-ray diraction pattern of (A) Mexican sodium zeolitic mineral (B) Mexican silver zeolitic mineral (24A Ag content).
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Fig. 3. SEM image of Mexican clinoptilolite±heulandite mineral. Note the characteristic monoclinic symmetry of blades and laths.
reason, it is proposed that the Ag exchanged in the zeolitic material reduces to Ag° in the presence of light [20]. This is a very important fact in the consideration of the eciency of the silver supported on Mexican natural zeolite as an antimicrobial material. 3.4.3. Infrared spectroscopy Flanigen and Khatami [21] have considered two groups of frequencies of vibration in all zeolites: internal vibrations of T±O (considered insensitive
437
Fig. 4. SEM image of the Mexican silver clinoptilolite±heulandite mineral (24A Ag content), 10 000.
to structure) and vibrations of external linkages between tetrahedra, due to topology and the mode of arrangement of the structure. No notable changes were observed in the vibration bands in the IR spectra of the Mexican zeolitic mineral treated with sodium as compared with the vibration bands of Mexican zeolitic mineral. When the sodium zeolitic mineral was treated with silver at the highest concentration, changes in some vibration bands (1050, 1400, 1700 and 3500 cmÿ1 ) were observed, as can be seen in Fig. 6.
Fig. 5. Elemental microanalyses of a silver crystal deposited on the Mexican zeolitic mineral.
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Fig. 6. IR spectra of (A) Mexican sodium zeolitic mineral and (B) Mexican silver zeolitic mineral (24A Ag content).
In the same ®gure, the vibration band of NOÿ 3 (1350±1400 cmÿ1 ) is not observed, for this reason, it is proposed that Ag is into the zeolite network occupied ion exchange sites. Rodriguez et al. [22] analyzed the natural clinoptilolite±heulandite by IR from Tasajeras deposit and assigned the vibration bands obtained. With this reference, the assignation of vibration bands shown in Table 4 can be proposed for the Mexican clinoptilolite±heulandite mineral. Table 4 Assignment of vibration bands for Mexican clinoptilolite±heulandite mineral from Taxco, Guerrero deposit Vibration modes Internal tetrahedra bending External tetrahedra double ring External tetrahedra linkage symmetric stretching External tetrahedra linkage asymmetric stretching Internal tetrahedra asymmetric stretching O±H bending
Frequency (cmÿ1 )
Intensity
463
Strong
604
Medium
795
Weak
1057
Strong
1203
Shoulder
1637
Wide
For a better evaluation of the data acquired, the ratio of transmission of the 1057 cmÿ1 band, which is assigned to asymmetric stretching vibration of the external tetrahedra, and the transmission of the analyzed band at 463 cmÿ1 , corresponding to the internal tetrahedral bending, were calculated (Table 5). Small dierences in the values were observed for the materials treated with sodium and silver; these can be attributed to the nature of the ions in the zeolite network, because the position of a cation in the clinoptilolite±heulandite structure is in¯uenced by its ionic potential [22]. 3.5. Mexican silver zeolitic mineral as an antimicrobial material 3.5.1. Mexican silver zeolitic mineral mass of 2.5 mg Table 6 shows the number of viable cells of E. coli or S. faecalis suspended in water after their contact with 2.5 mg of the Mexican sodium zeolitic mineral (represented in this work as 0 of Ag content) and silver zeolitic mineral, whose proportion of Ag in the silver Mexican zeolitic mineral was represented in this paper as A, 2A, 5A and 24A. This last Ag content means the maximum silver amount retained by the Mexican zeolitic mineral.
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Table 5 Values of 1057/463 cmÿ1 determined for each cation forms of Mexican zeolitic mineral Absorbance ratio 1057/463 cmÿ1
Sample Mexican Mexican Mexican Mexican Mexican Mexican a
zeolitic mineral sodium zeolitic mineral silver zeolitic mineral A Ag content silver zeolitic mineral 2A Ag content silver zeolitic mineral 5A Ag content silver zeolitic mineral 24Aa Ag content
1.340 1.351 1.514 1.638 1.448 1.420
Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
Table 6 Antibacterial property of 2.5 mg of Mexican silver zeolitic mineral on E. coli (ATCC 8739) or S. faecalis (ATCC 10741) Contact time (h)
Proportion of Ag content in the Mexican zeolitic mineral
Number of viable cells of E. coli per ml
Number of viable cells of S. faecalis per ml
0
0a A 2A 5A 24Ab
2:73 0:35 103 2:70 0:40 103 2:83 0:37 103 2:56 0:73 103 2:50 0:43 103
1:33 0:05 103 1:16 0:05 103 1:16 0:05 103 1:20 103 1:20 0:10 103
2
0 A 2A 5A 24A
2:26 0:30 103 1:30 0:30 103 1:03 0:16 103 0 0
1:23 0:05 103 8:00 0:81 102 6:00 0:81 102 5:66 0:57 102 1:66 0:57 102
4
0 A 2A 5A 24A
2:36 0:58 103 6:33 0:51 102 3:33 0:51 102 0 0
1:20 103 5:33 0:52 102 3:66 0:52 102 1:66 0:52 102 1:00 102
6
0 A 2A 5A 24A
1:96 0:35 103 1:33 0:57 102 0 0 0
1:16 0:11 103 4:00 0:10 102 2:00 102 1:33 0:57 102 0
24
0 A 2A 5A 24A
1:83 0:40 103 0 0 0 0
1:13 0:05 103 2:66 0:57 102 1:33 0:57 102 0 0
a b
Refers to the sodium zeolitic mineral. Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
In this table, it is clearly observed that when E. coli suspended in water is in contact with the Mexican silver zeolitic mineral with 5A and 24A Ag content for 2 h, the number of viable cells of this microorganism goes down to zero. For the S. faecalis case, 6 h of contact with the Mexican
zeolitic mineral with 24A Ag content is needed to obtain the same result (viable-cell number equal to zero). These results show a dierence in the antibacterial eect of the Mexican silver zeolitic mineral on E. coli and S. faecalis. These results could be explained in relation to the microorganism
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structural dierences: S. faecalis has a thicker cellular wall than E. coli. For this reason, a higher contact time and a higher amount of Ag supported on the Mexican zeolitic mineral are needed to achieve the same eect as in E. coli [23,24]. It is observed in Table 6 that when the amount of Ag supported on the Mexican zeolitic mineral increases, the number of viable cells for both microorganisms, E. coli and S. faecalis decreases. Indeed, it is important to note that the contact time between the microorganisms suspended in water with the silver zeolitic mineral is an another important factor to consider, for as contact time increases, the viable-cell number of each studied microorganism decreases. For E. coli, a total bactericide eect is obtained after 24 hours at any Ag content of the zeolitic mineral, but for
S. faecalis only at the highest Ag content is a total eect observed. 3.5.2. Mexican silver zeolitic mineral mass of 5.0 mg Table 7 shows the number of viable cells of E. coli or S. faecalis suspended in water after being in contact with 5.0 mg of the Mexican sodium (0 Ag content) and silver zeolitic mineral (A, 2A, 5A and 24A Ag content) for dierent times. In this table, it is clear that when E. coli suspended in water is in contact with the Mexican silver zeolitic mineral with 2A, 5A and 24A Ag content for 2 h, the number of viable cells of this microorganism is equal to zero. S. faecalis also needs two hours of contact with the Mexican
Table 7 Antibacterial property of 5.0 mg of Mexican silver zeolitic mineral on E. coli (ATCC 8739) or S. faecalis (ATCC10741) Contact time (h)
Proportion of Ag content in the Mexican zeolitic mineral
Number of viable cells of E. coli per ml
Number of viable cells of S. faecalis per ml
0
0a A 2A 5A 24Ab
2:63 0:25 103 2:93 0:55 103 3:13 0:15 103 2:93 0:68 103 2:53 0:15 103
1:30 0:20 103 1:20 0:17 103 1:30 0:26 103 1:36 0:28 103 1:43 0:11 103
2
0 A 2A 5A 24A
2:70 0:20 103 3:00 0:73 102 0 0 0
1:20 0:10 103 9:00 0:10 102 7:33 0:52 102 6:00 0:63 102 0
4
0 A 2A 5A 24A
1:96 0:15 103 0 0 0 0
1:10 0:16 103 5:00 0:16 102 2:66 0:16 102 2:33 0:16 102 0
6
0 A 2A 5A 24A
1:90 0:26 103 0 0 0 0
1:00 0:87 103 3:33 0:52 102 0 0 0
24
0 A 2A 5A 24A
1:53 0:20 103 0 0 0 0
1:03 0:86 103 1:00 102 0 0 0
a b
Refers to the sodium zeolitic mineral. Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
M. Rivera-Garza et al. / Microporous and Mesoporous Materials 39 (2000) 431±444
microorganism tested decreases as contact time increases. Although for E. coli a total bactericide eect is obtained after 4 hours with every Ag content, in S. faecalis the same eect was achieved only at the highest Ag content (24A Ag content).
zeolitic mineral, but a higher percentage (24A Ag content) of Ag is needed to obtain the same viablecell number (zero). These results are quite dierent from those obtained for 2.5 mg of Mexican silver zeolitic mineral because the mass increases from 2.5 to 5.0 mg, a better antibacterial eect is obtained for E. coli with a lower Ag content (2A Ag content); and for S. faecalis, the contact time necessary to kill all the bacteria decreases from 6 to 2 h at the same Ag content (24A Ag content). Also in Table 7, it is observed that as the amount of Ag supported on the Mexican zeolitic mineral increased, the amount of viable cells for both microorganisms, E. coli and S. faecalis, decreased in the same way as was observed previously for 2.5 mg of Mexican silver zeolitic mineral. In this case too, the viable-cell number of each
3.5.3. Mexican silver zeolitic mineral mass of 10.0 mg Table 8 shows the number of viable cells of E. coli and S. faecalis suspended in water after their contact with 10.0 mg of Mexican sodium (0 Ag content) and silver zeolitic mineral (A, 2A, 5A and 24A Ag content) at dierent contact times. In this table, it is clearly observed that when E. coli suspended in water is in contact with the Mexican silver zeolitic mineral with A, 2A, 5A and 24A Ag content for two hours, the number of
Table 8 Antibacterial property of 10.0 mg of Mexican silver zeolitic mineral on E. coli (ATCC 8739) or S. faecalis (ATCC10741) Contact time (h)
Proportion of Ag content in the Mexican zeolitic mineral
Number of viable cells of E. coli per ml
Number of viable cells of S. faecalis per ml
0
0a A 2A 5A 24Ab
2:13 0:75 103 2:23 0:63 103 2:26 0:30 103 2:00 0:86 103 2:06 0:20 103
1:23 0:11 103 1:26 0:11 103 1:30 0:10 103 1:20 0:10 103 1:26 0:11 103
2
0 A 2A 5A 24A
2:83 0:80 103 0 0 0 2:30 0:70 103
1:20 0:10 103 5:00 0:87 102 4:00 0:87 102 3:00 0:58 102 0
4
0 A 2A 5A 24A
0 0 0 0 0
1:23 0:11 103 2:66 0:15 102 1:33 0:57 102 0 0
6
0 A 2A 5A 24A
2:00 0:36 103 0 0 0 0
1:13 0:11 103 0 0 0 0
24
0 A 2A 5A 24A
2:40 0:55 103 0 0 0 0
1:06 0:15 103 0 0 0 0
a b
441
Refers to the sodium zeolitic mineral. Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
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M. Rivera-Garza et al. / Microporous and Mesoporous Materials 39 (2000) 431±444
Table 9 Optimal mass and contact time to obtain a total antibacterial eect with Mexican silver zeolitic mineral Bacteria
Mexican silver zeolitic mineral mass (mg)
Proportion of Ag content in the Mexican zeolitic mineral
Contact time (h)
E. coli
2.5 5.0 10.0
5A 2A A
2 2 2
S. faecalis
2.5 5.0 10.0
24Aa 24A 24A
6 2 2
a
Represents the maximum Ag amount retained by the Mexican zeolitic mineral.
viable cells of this microorganism is equal to zero. For S. faecalis, two hours are also needed to reduce the number of viable cells to zero, but only with 24A Ag content. This latter result is similar to that obtained with 5.0 mg of the Mexican silver zeolitic mineral. In general, these results show a better antibacterial eect with less wt.% of Ag for both microorganisms. Moreover, the contact time necessary to reduce the number of viable cells to zero decreased. It is also important to note in Tables 6±8 that the Mexican sodium zeolitic mineral (0 Ag content) did not show any antibacterial eect. Table 9 shows the optimal masses of the Mexican silver zeolitic mineral with dierent Ag content and optimal contact times to achieve a total antibacterial eect on E. coli and S. faecalis, based on the results obtained. As can be seen, 5 mg of Mexican silver zeolitic mineral with 24A Ag content in contact for two hours with water is enough to remove both pathogenic microorganisms. 3.5.4. Amount of silver released to water after contact with the Mexican silver zeolitic mineral (24A Ag content) Table 10 shows the silver amount released into the aqueous media as a function of the contact time between phases. The solid phase corresponds to the Mexican silver zeolitic mineral (24A Ag content) and the liquid phase corresponds to water used in the microbiological experiments. It is important to note that there is a direct proportion between the silver amount in water and contact time. These results are important to be considered because Mexican normativeness establishes that
Table 10 Silver concentration in water at dierent contact times between the Mexican silver zeolitic mineral (10 mg, 24A Ag content) and the aqueous media Contact time (h)
Silver concentration in water (lg lÿ1 )
2 4 28 76
26.2 37.9 44.8 115.0
the silver level in water should be kept below 50 lg lÿ1 . As was mentioned before, 5 mg of Mexican silver zeolitic mineral with 24A Ag content in contact with water for two hours is suggested in order to remove both pathogenic microorganisms. Under these conditions, the silver level in water (26.2 lg Ag lÿ1 ) remains within the limit established by the Mexican normativeness [25]. 3.5.5. The amount of sodium in water The sodium concentration in water, present in all the microbiological experiments, was 281:5 54 lg lÿ1 . This indicates that release of silver in water after being in contact with Mexican silver zeolitic mineral (24A Ag content) is due to the ionic exchange Ag /Na (Table 10) and that these Ag ions diuse into the microorganism cells and kill them. 3.5.6. Silver concentration in water after contact between Mexican zeolitic mineral (24A Ag content) and water with dierent sodium concentrations Fig. 7 shows that as the sodium concentration in water increases, silver concentration in this liq-
M. Rivera-Garza et al. / Microporous and Mesoporous Materials 39 (2000) 431±444
Fig. 7. Silver amount found in water with dierent sodium concentrations after being in contact with Mexican silver zeolitic mineral.
uid media increases as well after 6 h of contact with the Mexican silver zeolitic mineral (24A Ag content). This result is important to consider because the ®nal silver concentration in the treated water with the Mexican silver zeolitic mineral depends on the initial sodium concentration in the wastewater. This amount of silver should not be higher than the maximum level established by the Mexican normativeness, as was mentioned before. Indeed, it is important to mention that water could contain other ions like Ca2 and Mg2 that can be exchanged by the Ag supported in the Mexican silver zeolitic mineral.
443
For E. coli, there are several ratios between the Mexican silver zeolitic mineral mass and the content of Ag to obtain a total antibacterial eect. For instance, after 2 h of contact between the contaminated water with this microorganism and the silver zeolitic mineral, ratios of 2.5 mg-5A Ag content, 5.0 mg-2A Ag content and 10 mg-A Ag content, can be used. For S. faecalis, a total antibacterial eect of the silver zeolitic mineral is observed only with 24A Ag content. In this case, the total eect is not dependent on the mineral mass, as long as contact time is at least for 2 h. The Mexican sodium zeolitic mineral does not have a bactericide eect per se on E. coli and S. faecalis. Acknowledgements The authors thank the CONACyT for ®nancial support, project 26769-E. The technical support of C. Rodrõguez, M. Villa-Tomasa, E. Morales and F.A. Anastacio is gratefully acknowledged. The authors also thank L. Carapia, J. de la Torre and Thelma Falc on for their support in the characterization of the Mexican zeolitic mineral. The authors thank Lucia Hernandez Cruz for her help in editing the English. References
4. Conclusions The Mexican zeolitic mineral from Taxco, Guerrero has two main mineral components: clinoptilolite and heulandite. The mineral has a SiO2 /Al2 O3 ratio of 6.8 and presents the alkali form (K2 O Na2 O > CaO). The ratio between heulandite and clinoptilolite in the Mexican zeolitic mineral is 2.3:1. The Na plays a more important role in the Ag ion exchange process than that played by Ca2 and K , present also in the sodium zeolitic mineral. There are structural modi®cations in the Mexican silver zeolitic mineral because of the uptake of Ag ions into the zeolitic network.
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