Accepted Manuscript

Antimicrobial properties of ZSM-5 type zeolite functionalized with silver

M.J. Sánchez, J.E. Mauricio, A.R. Paredes, P. Gamero, D. Cortés

PII: S0167-577X(17)30039-3
DOI: http://dx.doi.org/10.1016/j.matlet.2017.01.039
Reference: MLBLUE 21992

To appear in: Materials Letters

Received Date: 7 May 2016

Revised Date: 27 December 2016
Accepted Date: 9 January 2017

Please cite this article as: M.J. Sánchez, J.E. Mauricio, A.R. Paredes, P. Gamero, D. Cortés, Antimicrobial properties
of ZSM-5 type zeolite functionalized with silver, Materials Letters (2017), doi: http://dx.doi.org/10.1016/j.matlet.
2017.01.039

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 Antimicrobial properties of ZSM-5 type zeolite functionalized with silver

M. J. Sánchez1, J. E. Mauricio2, A. R. Paredes2, P. Gamero1, D. Cortés1.

1
CINVESTAV IPN-Unidad Saltillo, Ave. Industria Metalúrgica No. 1062, Parque
Industrial Saltillo-Ramos Arizpe, Coah. C.P.25900, México. Tel. 52(844)4389600.
2
Facultad de Medicina, UA de C, Francisco Murguía Sur # 205, zona centro, Saltillo Coah.
C.P. 25000, México.
Corresponding autor: Miguel Sánchez; e-mail: miguelsanchez83@gmail.com

Abstract

Antimicrobial properties of high SiO2/Al2O3 ratio zeolites functionalized with silver have
been scarcely reported in the literature. The antimicrobial properties of the ZSM-5 type
zeolite functionalized with Ag+ ions were evaluated using culture media by placing in
contact the resultant zeolite with the Escherichia coli, Pseudomonas aeruginosa bacteria
and the Candida albicans fungus. The effect of the time on bactericidal activity was
examined with a kinetic study carried out to analyze the microorganism growth. The results
indicated that this functionalized zeolite inhibits the bacteria and fungus proliferation.

Keywords
Zeolite, ZSM-5, antibacterial, antifungal, silver

1. Introduction

The development of resistance to antibiotics and disinfectants, the occurrence of adverse

effects caused by the use of conventional antibacterial agents, has led scientists to look for
new antibacterial agents. Silver is one of the strongest antibacterial agents, and has been
known for its antibacterial activity since the times of ancient Greece[1].
The use of silver in medicine is growing, due to its broad spectrum of bacteria and fungi
inhibition[2]. It can be used to prevent wound colonization and for wound dressing[3], and to
prevent bacterial colonization on medical devices[4].
In recent years, there has been renewed interest by researchers to novel types of safe and
cost-effective antibacterial materials; as silver-based nanoparticles[5]. Zeolites are porous

1
crystalline aluminosilicate minerals with uniform molecular-sized pores. Contain metal
ions, such as sodium or calcium which are easily exchangeable with metals as silver,
copper, and zinc ions for their use as antibacterial agents.

Several materials using Ag+ ions have been reported, Ag-zeolites were the most successful
in elimination of bacteria[6]. Bioactivity, antimicrobial and fungal properties of high
SiO2/Al2O3 ratio zeolites, have been scarcely studied. It was reported the hydroxyapatite
growing over coatings made of silver ion-exchanged conventional ZSM-5 zeolite (ZSM-
5[Ag]) after incubation in simulated body fluid[7]. Metallic silver nanoparticles were
immobilized on silver ion doped ZSM-5 to obtain nanocomposites with anti-microfouling
properties[8].

In this paper, the functionalization of ZSM-5 type zeolite with ions Ag+ and the study of its
antibacterial and fungal properties are reported. It was demonstrated that ZSM-5[Ag] type
zeolite inhibits the growth of typically resistant microorganisms at sterile conditions.

2. Experimental section

2.1 Synthesis and functionalization of ZSM-5 type zeolite with silver ions

High SiO2/Al2O3 ratio ZSM-5 type zeolite was synthesized according with the method
reported by Cheng et al[9]. The obtained ZSM-5 type zeolite was exchanged by immersing 5
g of zeolite in its sodic form in 1000 mL of 0.1 N AgNO3 aqueous solution at 70 °C for 6 h
under magnetic stirring in absence of light. The solid product obtained by filtration was
washed 3 times with 500 mL of deionized water and dried at 100 °C for 24 h. A purple
powder (5 g) was obtained. Disk-shaped ZSM-5[Ag] zeolite samples (0.2 g in weight each)
were obtained by uniaxial pressing for 30 s at 499 MPa using a manual hydraulic press.

2.2 Antibacterial assays

A clinically isolate Gram negative E. coli, P. aeruginosa bacteria and C. albicans fungus
were selected as testing strain to evaluate the antimicrobial activity and were cultivated in a
nutrient broth (BD Bioxon). The glassware used in the study were sterilized in an autoclave
at 121 °C and 20 atm during 15 min and the test samples by UV irradiation during 15 min
before culturing.

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For the inhibition halo test, samples were placed in contact with an agar plate uniformly
covered with the bacteria and incubated at 36 °C during 24 h. For the kinetics testing,
ZSM-5[Ag] zeolite was immersed in 100 mL of nutrient broth containing 2 µL of
inoculums. The flasks were covered and placed in a wrist-action shaker at 100 rpm at 36
°C, a sample of 5 mL of the solution was removed at different times and the bacteria and
fungus proliferation was evaluated by turbidimetric readings with a wavelength 490 nm.
The bacteria proliferation in the nutrient broth without ZSM-5[Ag] zeolite was used as a
control.

3. Results

3.1 Properties of the ZSM-5[Ag] zeolite.

The SEM image (Fig. 1a) indicates the material is constituted by agglomerates particles
with different shapes in circular form obtained during crystallization reaction. The
corresponding EDS spectrum demonstrates that the material contains mainly Si, Al, Cl and
Ag, which it was obtained by exchanging sodium for silver ions (Fig. 1b). The XRD
pattern shows the ZSM-5[Ag] zeolite as a unique crystalline phase (Fig. 1c).

a) Si b)

C Ag

Cl
Al
Na

20 µm
Intensidad

1.00 2.00 3.00 4.00

KeV

50 1
c) b)

ASG JCPDS 079-1638

Intensity (a.u)

13 3
15 1
01 3
13 1

40 0
02 221 2

0 10 0
05 3
12 1

21 1

55 2
30 1

43 2
42 1
13 2

10
10 20
20 30
30 40
40 50
50
22Θ
Θ (°)
(°)

Fig. 1. ZSM-5[Ag] zeolite. (a) zeolitic material micrograph; (b) punctual chemical composition; (c)
XRD pattern.

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3.2 Inhibitory effect of bacteria growing around ZSM-5[Ag]
The antibacterial activity of the zeolite ZSM-5 was tested against E. coli and P. aeruginosa.
Figure 2a the control sample where E. coli grows around the unfunctionalized ZSM-5 is
shown. In Figure 2b is observed a significant inhibition halo about 1cm diameter around
the ZSM-5[Ag] zeolite formed after 24 h in contact with the bacteria cultivated in agar. A
similar situation occurs in the P. aeruginosa in presence of ZSM-5[Ag]. Figure 2d shows
an inhibition halo of 0.5 cm in diameter around the ZSM-5[Ag]. In the sample treated at the
same conditions (control) it is observed the growth of the bacteria around the
unfunctionalized ZSM-5, Figure 2c.
The standard SNV 1959-1992, mentions that the inhibition zone higher than 1 mm is
considered good for antibacterial activity[10]. The results of inhibition halo test indicate that
zeolite ZSM-5[Ag] has an excellent ability to inhibit the bacterial growth and be considered
of clinical interest in bacteria resistant to antibiotics.

a) ZSM-5 b) ZSM-5 [Ag]

c) ZSM-5 d) ZSM-5 [Ag]

Fig. 2. Inhibition halo a) ZSM-5-E.coli, b) ZSM-5[Ag]-E. coli, c) ZSM-5-P. aeruginosa, d) ZSM-

5[Ag]-P. aeruginosa.

The effect of time on bactericidal activity was examined with the kinetic study to analyze
bacteria growth after 24 h. Figure 3a illustrates the kinetic bacteria growth, E. coli nutrient
broth (control), and the bacteria growth E. coli in presence of zeolite, ZSM-5[Ag]-E. coli.

4
As observed, the beginning of the growth phase and duplication is the same for both
systems. At a time of 180 min, the presence of ZSM-5 [Ag] in contact with the bacteria, has an
inhibitory effect on the cellular duplication, against E. coli in nutritive broth (control). The kinetic
of the P. aeruginosa (Figure 3b), it can be seen a decrease at 60 min compared with the
curve of the bacteria (control) that shows antibacterial behavior the ZSM-5[Ag]-P.
aeruginosa. The functionalized zeolite inhibits by up 50% the bacterial growth, doing more
effective the antibacterial effect in comparison with ZSM-5[Ag]-E. coli.

0.10 0.20
a) b)
0.09 0.18 P. aeuroginosa
E. coli
P. aeuroginosa + ZSM-5[Ag]
0.08 E. coli + ZSM-5 [Ag] 0.16

0.07 0.14
Optical Density (%A)

Optical Density (%A)

0.06 0.12

0.05 0.10

0.08
0.04

0.06
0.03
0.04
0.02
0.02
0.01
0.00
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450
Time (min) Time (min)

Fig. 3. Bacterial growth curves a) E. coli. b) P. aeruginosa exposed to the zeolite ZSM-5
functionalized with silver.

3.3 Antifungal properties of ZSM-5 type zeolite functionalized with silver

To evaluate the fungal properties of the zeolite ZSM-5 and ZSM-5[Ag], both materials
were put in contact with fungus C. albicans. In Figure 4a an effective fungal growth, even
at the limits of the unfunctionalized zeolite particles, can be observed. However, an
inhibition halo of fungal growth about 0.3 cm of diameter around the ZSM-5[Ag] particles
can be seen in Figure 4b. Taking into account the standard SNV 195920-1992, the
inhibition halo showed by ZSM-5[Ag] is considered appropriate for antifungal activity.
The influence of the ZSM-5[Ag] on the antifungal properties was analyzed by exposure to
C. albicans and a kinetic study was carried out to analyze fungus growth during 420 min.
Figure 4c shows the system C. albicans in nutrient broth begins the exponential phase at
60 min and it is kept up to 420 min. The curve of ZSM-5[Ag]-C. albicans tends to decrease
at 120 min (exponential phase) being noticed that at this phase the slopes of the two
systems are different being the smallest slope the resulting of the ZSM-5[Ag]-C. albicans.

5
This means that the zeolite has an inhibitory effect of growth and proliferation of the
fungus.

The way in which ZSM-5[Ag] acts as antifungal, is due to the fact that membrane-active
antibacterial and antifungal drugs (in this case silver ions bonded to the zeolite) can disrupt
the phospholipid bilayer that constitutes the fungus membrane, causing the cell death[11].

c) 0.7
C. albicans
0.6 C. albicans + ZSM-5[Ag]

0.5
Optical Density (%A)

0.4

0.3

0.2

0.1

0.0

0 50 100 150 200 250 300 350 400 450

Time (min)

Fig. 4. ZSM-5 zeolite a) Inhibition halo ZSM-5-C.albicans, b) Inhibition halo ZSM-5[Ag]-C.

albicans, c) Growth curve of C. albicans exposed to the zeolite ZSM-5[Ag].

4. Conclusions
Silver ions confer antifungal and antibacterial properties to the zeolite ZSM-5 surface. The
functionalized zeolite inhibits the growth of the bacteria and fungus. This was demonstrated
with the study in which the functionalized zeolite was placed in contact with highly
infectious pathogens microorganisms that represent a problem of great clinical and
epidemiological importance, such as E. coli, P. aeruginosa, bacteria and C. albicans
fungus. In all cases the presence of a halo of inhibition of microorganism growth and

6
kinetic studies confirmed that the ZSM-5 zeolite functionalized with Ag+ inhibits the
growth of microorganisms.

Acknowledgments
M. J. Sánchez gratefully acknowledge the support of CONACyT-México for the
scholarship No. 34796. It is important to mention that this work was supported by SENER-
CONACyT-Hidrocarburos project No. 144453.

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Highlights

The functionalization of ZSM-5 type zeolite with ions Ag+ and the study of its antibacterial
and fungal properties are proposed.

ZSM-5[Ag] type zeolite inhibits the growth of typically resistant microorganisms at septic
conditions.

Silver ions confer antifungal and antibacterial properties to the zeolite ZSM-5 surface.

The functionalized zeolite inhibits the growth of the bacteria and fungus