Antimicrobial Nanomaterial for Escherichia coli Inactivation

Under UVA Irradiation

Uv7

Sandra M. Mirandaa, b*, Vitor J.P. Vilara, Adrin M.T. Silvab, Joaquim L. Fariab, Rui Boaventuraa, Eugnia Pintoc
a
LSRE Laboratory of Separation and Reaction Engineering, bLCM Laboratory of Catalysis and Materials,
Associate Laboratory LSRE/LCM, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias,
4200-465 Porto, Portugal. cCEQUIMED-UP, Microbiology Service, Biological Sciences Department, Faculty of
Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, N 228, 4050-313 Porto. *smm@fe.up.pt

The photocatalytic inactivation of Escherichia coli combining

titanium dioxide (Degussa P25) or zinc oxide (ZnO) with UVA
light was evaluated under controlled conditions. The
photocatalyst concentration ranged from 0.125 to 5 mg mL-1. ZnO
showed a higher antimicrobial activity than P25, using the same
catalyst concentration. Inhibitor effects showed to be dependent
on photocatalyst concentration and UVA irradiation time. The
inhibitory effect was observed to be bactericidal. The microbial
inactivation by light without P25 or ZnO did not occur; but even in
the dark ZnO produced an inhibition of almost 50% after 20 min.

Introduction
Bacteria and fungi represent a very active group
of microbiological contaminants present in water
and air, responsible for millions of deaths and
diseases each year. The ability to destroy
microorganisms from drinking and waste waters is
of big concern and a challenge to many
organisations [1]. In this context, advanced
oxidation technologies (AOTs) proved to be very
promising as new disinfection methods [2, 3].
Research on heterogeneous photocatalysis, a
process that involve redox reaction induced by
radiation on the surface of a semiconductor, applied
to microorganisms inactivation has increased
substantially in the last years. Metal oxides such as
titanium dioxide (TiO2), zinc oxide (ZnO), tin oxide
(SnO2), iron (III) oxide (Fe3O4), and others have
been drawing increasing attention due to their
antibacterial activity [4]. However, the most widely
used is TiO2, still with growing interest, since in
1985 Matsunaga et al. reported for the first time the
microbicidal activity of TiO2/Pt [5]. Ever since, many
researches
have
reported
photocatalytic
inactivation of several microorganisms. Recently,
ZnO attracted special attention, due to its strong
antibacterial properties. Both materials are known
for their attractive properties of stability, low cost,
high catalytic activity, and strong antibacterial effect
[6-8].
In the present work, two commercial semiconductor powders, titanium dioxide (P25, EvonikDegussa GmbH) and zinc oxide (ZnO, Evonik),
were used as photocatalysts. E. coli, a well-known
Gram-negative bacteria model in laboratory
experiments, was used in the present work. The
effect of photocatalyst concentration and UVA
illumination time for E. coli inactivation is discussed.
Material and Methods
Each of the powders catalysts was added to
100 mL of deionized water (Milli-Q, 18.2 Mcm) to

obtain a nal concentration of 10 mgmL-1.

Afterwards, sonication was carried out for about 10
minutes to obtain a homogeneous dispersion of
nanoparticles. Several dilutions were carried out to
obtain a concentration range between 0.25 and
10 mgmL-1. Before each experiment E. coli was
inoculated into Mueller-Hinton agar (MH, Merck,
Germany) and incubated overnight at 37 C. E. coli
cells suspension was prepared and diluted in saline
solution to approximately 103-104 CFUmL-1. After
photocatalytic treatment, the cell concentration was
determined by the spread plate method which
consists in spreading 100 L of the treated
suspension into MH agar plates. After incubation at
37 C during 24 h, colonies were counted and the
number of CFUmL-1 calculated.
Photocatalytic experiments were performed in a 6
holes microtiter plate (see Figure 1).

Figure 1. Experimental set-up.

Briefly, 1 mL of photocatalyst suspension from

0.25 to 10 mgmL-1 was mixed with 1 mL of the
103-104 CFUmL-1 E. coli cells suspension. Reaction
plates were placed at a distance of 3.6 cm from the
UVA light (Sylvania Lynx.s 11W black light blue
lamp with a spectral range between 315-400 nm).
All experiments were carried out at room
temperature and magnetically stirred throughout the
experimental period to ensure adequate mixing and
contact between nanoparticles and E. coli cells. The

4x1 3
0

CFU.mL

-1

3x1 3
0

2x1 3
0
1x1 3
0

0
5
10

(min

15

20

2.5

0.2
5

0.5

1.2
5

0.1 0
25

-1

L
mg.m
Figure 2. Inhibitory effect of ZnO in the absence of UVA
irradiation over E. coli.
3

3.5x10

-1

5 mg.mL
-1
2.5 mg.mL
-1
1.25 mg.mL
-1
0.5 mg.mL

3.0x10

2.5x10

-1

2.0x10

CFU.mL

Results and Discussion

Control and disinfection experiments were
performed under different conditions: (a) in the dark
with no added semiconductor; (b) in the dark but in
the presence of the semiconductor photocatalyst;
(c) under UVA irradiation in the absence of
photocatalyst and (d) under UVA irradiation and in
the presence of the photocatalyst. Different
photocatalyst loads (0.125-5 mgmL-1) and different
irradiation times (0 to 20 min) were tested.
No visible effects on initial concentration of E. coli
were observed after 20 min in the dark or under
UVA irradiation, in the absence of any added
semiconductor.
In the dark with P25 no reduction in the E. coli
concentration was detected after 20 min (see Table
1), however, a significant inhibition was observed
for ZnO (see Figure 2). A complete inhibition was
observed after 20 min for the highest concentration.
For a 10-fold decrease of ZnO concentration, E. coli
inhibition drastically drops from 100 to 44%.
In the presence of catalysts and under UVA
irradiation, a significant inactivation was achieved.
With both catalysts, inhibitory effects showed to be
dependent on catalyst concentration and UVA
irradiation time. Loss of viable E. coli cells
increases with the catalyst concentration.
Considering P25, an almost complete inhibition was
observed for the highest concentrations after 5 min
of UVA irradiation (see Figure 3). For both
catalysts, after 10 min of UVA irradiation, there was
near complete inhibition of E. coli growth for

concentrations from 0.25 to 5 mgmL-1 (see Table 1

and 2). However, for the lowest concentration
studied, ZnO was more active, near 99.5%.

time

lamp was turned on (considered as t = 0) and

samples were periodically taken for cells counting
during 20 min. Samples without growth after
photocatalytic treatment were re-suspended in MH
broth for evaluation of the bactericidal effect. The
cultures were incubated at 37 C during 24 h and
100 L were spread into MH agar plates to evaluate
viable bacteria.
Control experiments were performed with or
without UVA light as so with or without
photocatalyst.
All experiments were repeated to confirm the
reproducibility of results, 5 times for P25 and 3
times for ZnO.
Material, photocatalyst suspensions and water
were sterilized by autoclaving (120 C for 15 min)
before use.

1.5x10

1.0x10

5.0x10

0.0
0

time (min)

Figure 3. Effect of P25 concentration on the inactivation of

103-104 CFUmL-1 E. coli suspension with UVA irradiation
for the first 5 min.

After 20 min of UVA irradiation, the residual

bacterial suspension of some experiments using
catalysts was re-suspended and incubated in the
dark. No re-growth of E. coli colonies was
detectable after 24 h at 37 C.

Table 1. Antimicrobial activity of P25 against E. coli during 20 min of UVA irradiation and after 20 min in the
absence of light.
CFUmL-1
0 min

5 min

10 min

15 min

20 min

20 min without
light

5 mgmL-1

2570620

510

2390800

2.5 mgmL-1

2340950

510

2490910

1.25 mgmL-1

3000780

3050

2450490

0.5 mgmL-1

3240870

100100

3070720

0.25 mgmL-1

2630700

930540

2010

2500670

0.125 mgmL-1

2970550

2110580

830480

210230

88

2460850

E. coli
(Control)

2450210

Table 2. Antimicrobial activity of ZnO against E. coli during 20 min of UVA irradiation
-1

CFUmL
E. coli
(Control)
-1

5 mgmL

-1

2.5 mgmL

1.25 mgmL-1
0.5 mgmL-1
-1

0.25 mgmL

-1

0.125 mgmL

3500510

0 min

5 min

10 min

15 min

20 min

2050550

3010

2360330

3010

2520480

5020

239050

11050

1870590

11040

3010

2070110

13040

2020

Conclusions
Photocatalytic inactivation of E. coli by UVA light in the presence of two commercial semiconductors, P25
and ZnO was obtained. Complete inhibition of E. coli growth by UVA light irradiation with both catalysts was
reached in 10 min. In both cases the antibacterial activity was significantly greater in the presence of light
than in the dark. ZnO exhibits better performance than P25 on the antimicrobial activity for E. coli.
Acknowledgements
Financial support for this work was mainly provided by FCT (Fundao para a Cincia e a Tecnologia) project PTDC/EQUEQU/100554/2008. This work was partially supported by project PEst-C/EQB/LA0020/2011 and PEst-OE/SAU/UI4040/2011,
financed by FEDER through COMPETE - Programa Operacional Factores de Competitividade, by FCT and by IJUPProjetos Pluridisciplinares 2011 U.Porto/Santander Totta.
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