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Antibacterial Activities of Zinc Oxide Nanostructures with Different
Structures
Rolen Brian P. Rivera1,2, Melchor J. Potestas1,2,
Ma. Reina Suzette B. Madamba2,3, Rey Y. Capangpangan4,
Bernabe L. Linog5,6 and Blessie A. Basilia7 and Arnold C. Alguno1,2,a
1Materials Science Laboratory, Department of Physics
2Premier Research Institute of Science and Mathematics (PRISM)
3Department of Biological Sciences MSU-Iligan Institute of Technology, Tibanga, Iligan City 9200,
Philippines
4Department of Chemistry, Caraga State University, Ampayon, Butuan City, Philippines
5Region XIII, Department of Education, Butuan City, Philippines
6College of Arts and Sciences, Saint Joseph Institute of Technology, Butuan City, Philippines
7Department of Science and Technology, Industrial Technology Development Institute
Bicutan, Taguig City, 1631, Philippines
aarnold.alguno@g.msuiit.edu.ph
Keywords: Antibacterial; nanostructures; zinc oxide; surface morphology.
Abstract. We report on antibacterial activities of Zinc oxide (ZnO) with different structures.
Fast furrier transform infrared spectroscopy ZnO nanostructures showed peaks in the range
between 450–600 cm-1 indicating the successful growth through the presence of Zn-O
stretching. On the other hand, impurities such as zinc complexes might be present due to the
appearance of peaks at 1110 cm-1, 1390 cm-1 and 1506 cm-1. Furthermore, SEM images
revealed that nanorods and sea-urchin like nanostructures are present in the produced ZnO
nanostructures. Nanorods exhibit a better antibacterial response than the sea-urchin like
structure. The change in structural morphology along with its purity has greatly influenced
the area of bacterial inhibition zone during antibacterial testing.
Introduction
Water is a fundamental necessity for life. The availability of fresh water is crucial for life
sustaining activities such as drinking, cooking, cleaning, bathing and etc. However, nowadays, due
to pollution, water can be infested by bacteria and other pathogenic microorganisms which can
cause diseases such as diarrhea, tuberculosis, skin diseases and other diseases which can lead to
death [1]. According to 2004 World Health Organization report, about 90% of all diseases
occurring in developing countries are related to the consumption of dirty water leading to nearly 4
billion reported cases of disease contracted from water in the world and these water borne disease
kill nearly 12 million people every year [2].
Some of the common bacteria that can be found on dirty water which is responsible for such
deadly diseases are the Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli). Moreover,
bacteria can be classified into two types according to their cell wall, the gram-negative (-) bacteria
and the gram-positive (+) bacteria. S.aureus is a gram-positive bacteria where its wall contains a
thick layer (20 - 50 nm) of peptidoglycan which is attached to techoic acids while E.coli is a gram-
negative bacteria where its cell wall comprises a thin peptidoglycan layer and contains an outer
membrane that confers resistance to hydrophobic compounds which covers the surface membrane
[3].
Solid State Phenomena Submitted: 2019-02-20
ISSN: 1662-9779, Vol. 294, pp 36-41 Accepted: 2019-03-06
doi:10.4028/www.scientific.net/SSP.294.36 Online: 2019-07-25
© 2019 Trans Tech Publications Ltd, Switzerland
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans
Tech Publications Ltd, www.scientific.net. (#505707952, Caraga State Univer-sity-08/07/19,01:14:55)
On the other hand, there are several ways of killing these bacteria in water. Disinfection of
drinking water is currently being carried out through physical and chemical techniques like
chlorination, ozonation and UV treatment. However, due to the materials and chemicals used, these
has some disadvantages such as, high toxicity due to the release of harmful disinfection by-products
and high cost due to high production and maintenance cost. Hence, we need to have an alternative
way of killing bacteria in water by having an antibacterial which is safe, biocompatible, low cost
and effective [4-5].
The most promising candidates as bactericidal agent are nanostructured material [5]. Among
many nanostructured material, zinc oxide (ZnO) is one of the most promising material because it
exhibits strong antibacterial activity, stable under harsh process condition, can be synthesized using
low cost techniques without compromising its effectiveness and bio-compatible [6]. Several studies
had revealed that ZnO is effective as an antibacterial material. Subhasree, et. Al. reported about the
antibacterial activity of nanorod and bulk ZnO against E.coli and S.aureus and they found out that
the percent reduction of these bacteria when introduced to ZnO is greater than 92%. Furthermore,
they observe that nanosize ZnO is more effective against bulk ZnO [7]. However, these studies
failed to consider the possible effect of different morphological structure on the antibacterial
activity of ZnO nanostructures. In this work, the antibacterial activity of ZnO nanostructures with
different morphological structures is studied. The effect of morphological change of ZnO
nanostructures on a gram positive and gram negative bacteria is revealed.
Experimental Details
ZnO powders have been synthesized from aqueous solutions of ammonium hydroxide (NH4OH)
and zinc sulfate (ZnSO4) where the total volume is 200ml by addition of deionized water. The
solution was stirred at 360 rpm for 30 minutes. Then the solution is introduced into a bath and wait
until the temperature of the bath is at 70℃. When the temperature of the bath is at 70℃, maintain
this for 5 hours. Finally, collect the powders by filtering it using the filter paper then wash it with
deionize water and dry at room temperature. A 0.03M ZnSO4 is mixed with varying amount of
NH4OH and mix using magnetic stirrer with a temperature of 70℃. After 5 hours, the ZnO powder
was collected and filtered. The resulting powered was dried at room temperature.
The ZnO powders are characterized using the JEOL JSM-6510LA Analytical scanning
electron microscope. Fast fourier transform spectroscopy was carried out using Perkin
Elmer Spectrum 100 FT-IR Spectrometer in the wavenumber range of 4000-500 cm-1. The
antibacterial response of the synthesized ZnO powders is tested for antibacterial activities using
Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The bacterial was grown on
nutrient agar slants at 35⁰C for 24 hours. The test is conducted using agar well-diffusion assay.
Muller Hilton Agar (MHA) was used for well-diffusion susceptibility test of each bacterial species.
Eight mm wells were punched onto the agar after inoculating 100µL of 1.5x106 CFU/ml each of the
bacteria cultures. Sealing of each wells using the molten agar followed prior to the application of
ZnO nanoparticle treatments to prevent leakage from the bottom of each wells. A standard
concentration of 25mg/ml of ZnO nanostructures were used and introduced to the wells. The ZnO
powders are then exposed to S.aureus and E.coli and obtain the quantitative and qualitative data of
its antibacterial activity. Measurements of zones of inhibition were done using the carbon dial
caliper. Triplicates were carried out and correlated against the controls.
Results and Discussion
Inhibition zones of the antimicrobial assay using ZnO with 1M and 3M of NH4OH against
S.aureus are shown on Figure 1(a). It is observed that the inhibition zone of S.aureus using 1M of
NH4OH (23mm) is bigger as compared to the inhibition zone of S.aureus using 3M of NH4OH
(9mm). On the other hand, the inhibition zones of antimicrobial assay of sample 1M and 3M of
NH4OH against E.coli is shown in Figure 1(b). It shows that a 14mm diameter inhibition zone can
be observed for sample 1M while sample 3M shows no inhibition zone. This implies that sample
Solid State Phenomena Vol. 294 37
1M is a more potent antibacterial than sample 3M for both gram positive and gram negative
bacteria. ZnO nanostructures grown using 1M and 3M NH4OH are more potent to S.aureus than
E.coli, similar to previously reported work published elsewhere [7]. This might be due to the reason
that gram positive bacteria has cell wall consist of one cytoplasmic membrane while gram negative
bacteria has cell wall consist of another outer membrane covering its cytoplasmic membrane [3].
Consequently, it is apparent that the 1M NH4OH ZnO nanostructures has a greater
antibacterial activity than the 3M NH4OH ZnO nanostructures. This might be attributed to
the morphological structure and the purity of the ZnO nanostructures.
Figure 1. Antimicrobial assay of 25mg/ml concentration ZnO against (a) S. aureus (b) E. coli with
1M and 3M NH4OH concentrations.
Figure 2 shows the SEM micrographs of ZnO nanostructures using 1M and 3M concentration.
The ZnO nantrucrures using 1M NH4OH concentration has a hexagonal rod structure with sizes
around 149nm – 410nm as shown in Figure 2(a,b). The reason for this might be due to the shapes of
ZnO nanostructures are closely related to the nucleation rate, growth rate and the ion concentration
of the solution [8]. On the other hand, 3M concentration produced sea urchin-like structures with
pointed tips with sizes around 100nm – 200nm as shown in Figure 2(c,d). The pointed tips might be
due to the crystal etching of ZnO hexagonal nanorods caused by the basic environment created by
the excess OH- ions due to high concentration of NH4OH for 3M NH4OH concentration.
Clearly, from these results, two types of structures of ZnO - nanorods and sea urchin-like
nanotructures. Moreover, the surface area of these structures are investigated and found out that the
sea urchin-like structure has a higher surface to volume ratio due to higher density of ZnO than the
nanorods which means that sample 3M might be the reason to exhibit more potent antibacterial than
sample 1M since the higher surface area will have a greater interaction to the bacteria due to the
increase in contact with the bacterial effluent. In addition, higher surface area also has greater
potential reactive surface sites.
9 mm
+
_
23 mm
(a)
1M
3M
+
_
+
No inhibition
14 mm
+
-
(b)
1M
3M
-
38 Functional Materials and Metallurgy II
Figure 2. Scanning electron images of ZnO nanostructures with different NH4OH concentrations:
(a,b) 1M (c,d) 3M.
The broad peaks around 3380 cm-1 for samples 1M and 3M corresponds to the O-H stretching
which may be due to the water moisture on the samples. Zn-O stretching can be seen from the peaks
around 500-601 cm-1 for both samples which indicates the presence of ZnO nanostructures.
However, it is evident that the Zn-O stretching peaks for sample 1M (nanorods) is more emphasized
than on sample 3M (sea urchin). This means that the nanorods have more ZnO purity than the sea
urchin-like structures.
Figure 3. FTIR spectra of ZnO nanostructures with different NH4OH concentrations.
(a)
(d)
(b)
(c)
Solid State Phenomena Vol. 294 39
The ZnO nanostructures using 1M (nanorods) have better antibacterial activity than sample 3M
(sea urchin-like) might be due to greater surface to volume ratio as indicated in the higher density as
seen from the SEM images. It is believed that 1M concentration has higher purity than sample 3M
concentration. It is suggested that there is a competition between the total surface area and the
purity of the ZnO nanostructures.
There are many mechanisms proposed by previous reports on the antibacterial activity of
ZnO nanostructure, such as ROS generation, release of Zn2+ ions and electrostatic
interaction [9]. However, some of this is still controversial and not yet established. One of
this is the ROS generation such as H2O2 which is due to the photo catalysis of ZnO
nanostructure caused by UV activation under light exposure. These H2O2 molecules are able
to pass through the bacterial cell wall subsequently leading to injuries and destroy cell parts,
finally triggering cell death. However, a problem exists because antibacterial activity is also
observed under dark condition when there is no light to cause photo catalysis as reported by
Adams et .al [10]. It was suggested that release zinc ions (Zn2+) in medium containing ZnO
and bacteria which has significant effect in the active transport inhibition as well as in the
amino acid metabolism and enzyme system disruption which causes bacterial death [11].
The ZnO nanostructures toxicity is referred to the solubility of Zn2+ in the medium
including the bacteria. However, the insolubility of ZnO impedes the distribution of zinc
ions into the medium and thus limits this antimicrobial effect.
Summary
ZnO nanostructures of different surface morphologies were successfully synthesized. The
antibacterial activity of ZnO nanostructure has been greatly affected by varying the NH4OH
concentration. The reason for this might be due to the surface morphology and the purity of ZnO
nanostructures. ZnO with 3M NH4OH concentration has higher surface area than ZnO with 1M
NH4OH concentration. Consequently, a higher surface area would most likely result to a greater
antibacterial activity due to a higher interaction on the reactive surface. The antibacterial activity
of ZnO nanostructure has been greatly affected by varying the NH4OH concentration. Larger
inhibition zones are observed for ZnO nanostructure with 1M NH4OH concentration than with 3M
NH4OH concentration which indicate that 1M has a better antibacterial activity than 3M. This
effective inhibition of bacteria might be due to the surface morphology of ZnO with 3M
NH4OH concentration which provide higher surface area than ZnO with 1M NH4OH
concentration. Consequently, a higher surface area would most likely result to a greater
antibacterial activity due to a higher interaction on the reactive surface. However, the ZnO
purity of the sample has also significant effect on its antibacterial activity because the
electrostatic interaction and the ROS generation. It is believed that higher purity of ZnO will
have greater antibacterial capability. It might be a competition between purity and surface
area of ZnO nanostructure that will determine its antibacterial capability.
Acknowledgments
Scholarship grants of R.B.P. Rivera from DOST- ASTHRDP is acknowledged.
40 Functional Materials and Metallurgy II
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