Journal of Industrial and Engineering Chemistry 19 (2013) 614–619

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Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec

Biofilm-inactivating activity of silver nanoparticles: A comparison with silver ions

Hee-Jin Park a, Soomin Park a, Jinkyu Roh b, Sujin Kim c, Kyunghee Choi c, Jongheop Yi a,
Younghun Kim b,*, Jeyong Yoon a,**
a
World Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Institute of
Chemical Process, Seoul National University (SNU), Daehak-dong, Gwanak-gu, Seoul 151-742, Republic of Korea
b
Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-dong, Nowon-gu, Seoul 139-701, Republic of Korea
c
National Institute of Environmental Research, Kyungseo-Dong, Seo-Gu, Incheon 404-708, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: The antimicrobial activity of silver nanoparticles (AgNPs) against Pseudomonas aeruginosa PA01
Received 9 August 2012 planktonic and biofilm bacteria was examined; their activity was compared with that of silver ions. The
Accepted 16 September 2012 inactivation of biofilms by AgNPs was greatly influenced by stirring, which caused an increased AgNP
Available online 24 September 2012
biosorption. Although the activity of AgNPs against planktonic cells was ca. 10% that of silver ions, their
activity against biofilm cells was comparable to the silver ions’ activity at the same concentration after
Keywords: 90 min under stirring (ca. 3.5 log inactivation). AgNPs inactivated biofilms in a biosorption-dependent
Silver nanoparticle (AgNP)
manner, whereas this was not the case for silver ions.
Biofilm
Pseudomonas aeruginosa
ß 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
Silver ion reserved.
Biosorption

1. Introduction can subsequently develop into three-dimensional biofilm struc-

Silver nanoparticles (AgNPs) are commonly used in commercial
products such as portable water filters [1], clothing [2,3], medical
devices [4], coatings for washing machines, and refrigerators [5]
because of their high antimicrobial activity. The Woodrow Wilson
International Center found that AgNPs were associated with the
greatest numbers of products in a survey of materials [6]. This
suggests that AgNPs are likely to have great exposure to the
environment and subsequent toxicity in ecosystems. Therefore,
studies of AgNPs should not only aim to advance their application,
but also establish and control their environmental impact.
Previous studies have evaluated the antimicrobial activity of
AgNPs [7,8] and the mechanisms of their activity have also been
investigated [9–11]. Suggested mechanisms for the antimicrobial
activity include induction of DNA damage [12], enzyme inhibition
[13], the generation of reactive oxygen species (ROS) [10,14], and
the disruption of cell membranes [7]. The aforementioned studies
were mostly investigated with planktonic cells, whereas the
impact of AgNPs on bacterial biofilms is not fully understood.
Biofilms are microbial populations enclosed in a matrix.
Planktonic bacterial cells can attach themselves to surfaces, and
produce adhesive extracellular polymeric substances (EPS). They
* Corresponding author. Tel.: +82 2 940 5768; fax: +82 2 941 5769.
** Corresponding author. Tel.: +82 2 880 8927; fax: +82 2 876 8911.
E-mail addresses: korea1@kw.ac.kr (Y. Kim), jeyong@snu.ac.kr (J. Yoon).
tures, a natural bacterial survival strategy, with a different
physiological state from planktonic cells [15]. Nutrients and
oxygen are limited in the bottom layers of biofilm cells, forming
dormant cells which have a low metabolic activity [16]. Bacterial
cells in biofilms are more tolerant to commonly used antimicrobial
agents than planktonic cells and are thus hard to control when they
develop in undesired areas [17]. As a result, they can cause serious
problems such as biofouling [18], biocorrosion [19], and infections
[20,21]; hence, they are also important for application aspects.
Several studies have examined the effects of AgNPs on bacterial
biofilms [22–25]. However, there are some limitations on
experimental designs such as the time point of AgNP exposure
to biofilms, exposure concentration, and evaluating condition and
method to accurately estimate the actual toxicity of AgNPs on
mature biofilms. Even though the pretreatment effect of AgNPs on
planktonic bacteria before biofilm development was reported to
inhibit subsequent biofilm formation, the results of such studies
cannot be used to determine the nature of the interactions that
occur between AgNPs and biofilms [24,25]. In addition, the effect of
AgNPs on mature biofilms was underestimated by the limited
experimental condition or inaccurate methods [22,23]. Fabrega
et al. [22] showed that AgNPs caused the sloughing of biofilms
without viability change. However, the maximum exposure
concentration of AgNPs was 2 mg/L, and the viability was verified
only by microscopic observation, suggesting that this issue is
worth of further investigation. Choi et al. [23] compared the
toxicity of AgNPs and silver ions on planktonic and biofilm

1226-086X/$ – see front matter ß 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jiec.2012.09.013
 H.-J. Park et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 614–619
bacterial cells by means of a microrespirometric assay and found
higher resistance of biofilms to AgNPs than planktonic cells.
Nevertheless, the results should be re-evaluated from
the standpoint of the experimental method and condition of the
study, since bacterial cells were exposed to AgNPs in the presence
of medium and mineral oil that could induce particle aggregation
causing the underestimation of AgNP toxicity. Moreover, toxicity
was evaluated by means of a microrespirometric assay, which
measures oxygen consumption by the cells, despite the fact that
biofilms contain significant numbers of dormant bacterial cells
with low metabolic activity. Therefore, this assay is not suitable for
measuring the toxicity of AgNPs or silver ions on biofilm cells, and
it is impossible to accurately evaluate the actual toxicity of AgNPs
on biofilms based on these studies.
The goal of this study was to determine the extent of biofilm
inactivation by citrate-capped AgNPs in the absence of medium by a
plate counting method that can accurately evaluate the effect of
AgNPs on dormant cells in biofilms. Pseudomonas aeruginosa PA01,
which is widely used in biofilm studies, was used as a model
bacterial strain. The antimicrobial activity of AgNPs was compared
with that of silver ions as a reference, thus allowing the validity of the
exposure level of AgNPs to be established. In addition, the effects of
stirring on the inactivation of biofilms by AgNPs and biosorption of
AgNPs and silver ions by biofilms were also estimated to develop a
strategy for biofilm inactivation using AgNPs.
2. Materials and methods
2.1. Preparation of AgNP dispersion and silver ion solution
AgNP dispersions were purchased from ABC Nanotech Co.
(STU206011, Korea). This material has been selected as a reference
AgNP material by the Organization for Economic Co-operation and
Development (OECD) [26]. Particles were synthesized by aqueous
reduction of silver nitrate, dispersed by citric acid capping, and
suspended in deionized (DI) water. The dispersions contained
20 wt.% AgNPs serially diluted in DI water (Millipore, France) to the
desired concentration, which was confirmed by inductively
coupled plasma (ICP) spectrometry (ICPS-7500, Shimdzu, Japan).
A silver ion solution was prepared by dissolving silver nitrate
(Sigma–Aldrich) in DI water.
2.2. Physicochemical characterization of AgNPs
Electron light scattering spectrophotometry (ELS-8000, Otsuka,
Japan) was used to estimate the sizes and zeta potential (z, the
magnitude of the electrical charge at the double layer) of the
AgNPs. Transmission electron microscopy (TEM, JEM-3010, JEOL,
Japan) was also used to determined their sizes. The concentration
of silver ions in the AgNP dispersions was determined by
separation of the AgNPs and silver ions using a nanofiltration
membrane with 200 Da of cutoff size (NE-90, Woongjin, Korea) and
ICP measurements. UV/vis absorption spectra of the AgNPs were
obtained on an Agilent 8453 UV/vis spectrophotometer (Agilent,
Germany). A pH meter F-54BW (Horiba, Japan) was used to
determine pH.
2.3. Bacterial strain and growth
P. aeruginosa PA01 was obtained from the Center for Biofilm
Engineering at Montana State University. A single colony of P.
aeruginosa was inoculated in 40 mL of 1/10-strength tryptic soy
broth (TSB, BD, NJ) and the resulting suspension incubated at 37 8C
for 18 h. The bacterial cells were collected by three cycles of
centrifugation at 1000  g for 10 min and washed with 40 mL DI
water.
615
2.4. Biofilm formation
A Centers for Disease Control and Prevention (CDC) reactor
(Biosurface Technologies, MT) was used to produce biofilms [27].
The reactor contained 8 rods; each rod could hold 3 glass coupons
with a diameter of 1.1 cm, giving a surface area of both sides of ca.
1.9 cm2. P. aeruginosa PA01 cells were inoculated in 350 mL 1/100-
strength TSB, and incubated for 2 days in the sterile CDC reactor.
The initial population of P. aeruginosa PA01 was ca. 106 CFU/mL. For
the first day, biofilms were grown in the batch mode at 100 rpm
and room temperature (25  2 8C). During the second day, 1/300-
strength TSB was injected into the reactor at a flow rate of 8 mL/min.
2.5. Determination of antimicrobial activity and silver biosorption by
P. aeruginosa PA01 biofilms
Before investigating the interaction between the AgNPs and
biofilms, the antimicrobial activities of AgNPs and silver ions on
planktonic cells were evaluated. Approximately 106 CFU/mL of
planktonic P. aeruginosa PA01 was exposed to the desired
concentration of AgNPs or silver ions at 100 rpm and 25 8C. After
exposure, the bacterial cells were quenched by neutralizing
solution (10% sodium thioglycolate, 14.6% sodium thiosulfate)
[28]. Antimicrobial activity was determined by plate counting,
with the results expressed as log reductions (log N/N0).
Biofilm inactivation by AgNPs or silver ions was also evaluated
by plate counting. After biofilm formation, the rods were rinsed
with DI water and transferred to a new reactor containing AgNPs or
silver ions. The initial bacterial population of the biofilms was ca.
2  108 CFU/coupon (1.9 cm2). The biofilms were exposed to the
AgNPs or silver ions for similar durations under static condition or
with stirring (100 rpm) at room temperature. The coupons in the
rod were then rinsed with DI water and transferred to a falcon tube
containing phosphate buffered saline (PBS) and a neutralizing
solution. Bacterial cells in the biofilms were suspended in the
solution by sonication (1 min) and vortexing (2 min), and numbers
of surviving bacterial cells were determined by plate counting.
Antimicrobial activity was expressed as log reduction (log N/N0).
Silver concentrations of the solutions containing the post-exposure
biofilm cells were measured by ICP to evaluate the silver
biosorption by the biofilms. Silver biosorption was expressed as
mg Ag/1.9 cm2 (coupon surface area). The results are presented as
the means and standard deviations of triplicate experiments.
2.6. CLSM image analysis
Biofilms’ morphologies were analyzed by confocal laser
scanning microscopy (CLSM, Eclipse 90i, Nikon). The post-
exposure biofilm cells were stained using a BacLight Live/Dead
bacterial viability kit (Molecular Probes, USA), which includes
SYTO 9 and propidium iodide (PI). SYTO 9 and PI respectively stain
intact and damaged bacterial cells according to cell membrane
integrity [29]. The stained biofilms were sectionally imaged by
CLSM with a water immersion lens (60 object and numerical
aperture of 1.4). Images were then reconstructed by Imaris
(version 6.1.5, Bitplane AG) and presented as 3-dimensional
structures.
3. Results and discussion
3.1. Characterization of AgNPs
Average particle size of the AgNPs reported by the supplier was
7.9 nm and 12.2 nm, respectively, as measured by TEM and ELS.
However, using ELS, they were determined to be ca. 47 nm (Fig. S1,
Supplementary material). This was confirmed by TEM image
616 H.-J. Park et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 614–619

analysis (Fig. S2). Particle diameter (D) was used to calculate the
number of silver atoms per AgNP (N) by the following equation
[25].
prD3
N¼ NA
6M
where r and M are the density (10.5 g/cm3) and the atomic mass
(107.87 g) of silver, respectively. NA is Avogadro’s constant
(6.023  1023). Each AgNP was calculated to contain ca. 3  106
silver atoms.
The zeta potential of the AgNPs was ca. 42 mV (data not
shown), allowing stable suspension in DI water. The AgNP
dispersion (10 mg/L total silver content) included ca. 0.08 mg/L
(<1%) ionic silver (data not shown), indicating that the effect of
free silver ions was negligible. The pH of the AgNP dispersion was
6.3 (data not shown), which would be unlikely to affect bacterial
viability per se. The UV–vis spectrum of the AgNPs (Fig. S3) shows
that they absorb light in the visible range. Therefore, all
experiments were conducted under dark conditions to eliminate
any effect by ambient light.
3.2. Antimicrobial activity of AgNPs on planktonic P. aeruginosa PA01
Fig. 1 shows the antimicrobial activities of AgNPs and silver ions
on planktonic P. aeruginosa PA01. The results for silver ions are
comparable with that of a previous study [29], though a slightly
higher antimicrobial activity was found in this work. This can be
attributed to the use of only DI water rather than a phosphate
buffered saline (PBS) solution. AgNPs at 10 mg/L showed similar
antimicrobial activity to a 1.0 mg/L solution of silver ions,
bacteria, thus lowering the antimicrobial activity found in this
study. The AgNP dispersion used in this study included a very low
proportion of silver ions (<1%), whereas AgNP dispersions in the
previous studies included higher proportions of residual silver
ions, which may have been responsible for their increased
antimicrobial activity [30]. In addition, the fraction of AgNPs
smaller than 5 nm has also been reported to have a significant
effect on antimicrobial activity. The very low amount of smaller
particles used herein may also have contributed to the particles
showing a lower antimicrobial activity than silver ions.
3.3. P. aeruginosa PA01 biofilm-inactivation by AgNPs
P. aeruginosa PA01 biofilms grown for two days on glass
coupons were exposed to 10 mg/L AgNPs or silver ions under static
conditions. The ions induced over a 2 log inactivation after
150 min, whereas a similar exposure to AgNPs only resulted in a
0.3 log inactivation of biofilm cells (Fig. 2a). This can be explained
by several mechanisms including differences in antimicrobial
activity and affinity between the materials and the biofilms, and
limited penetration by the AgNPs. The different antimicrobial
activities against planktonic bacteria could lead to different
extents of biofilm inactivation by the AgNPs and silver ions when
both materials are able to penetrate the biofilms. Silver ions also
have a good affinity for biological molecules such as amino acids in
0.0
(a)
-1.0
Log reduction (log N/N0)

indicating a 10 greater antimicrobial activity of the ions

compared to the nanoparticles under the test conditions used
here. However, the AgNPs showed much greater antimicrobial -2.0
activity in terms of particle number or atomic number (ca. 3  105).
This may be indicative of unique antimicrobial mechanisms for the
AgNPs and silver ions. -3.0
The antimicrobial activity of AgNPs has been compared with
that of silver ions in several previous studies [30,31]. They have
been reported to exhibit a higher toxicity to bacteria than silver -4.0 AgNP 10 mg/L
ions at similar silver concentrations, whereas the opposite was Ag+ 10 mg/L
observed here. This difference may be due to surface coatings [32],
ionic ratios [31], or different particle sizes of the AgNPs [30]. The -5.0
0 30 60 90 120 150
citrate coating on the particles could inhibit direct contact with the
Time (min)

0.0
(b) 3.0
AgNP 10 mg/L
AgNP 10 mg/L
-1.0 Ag+ 1 mg/L
2.5 Ag+ 10 mg/L
Log reduction (log N/N0)

Biosorption (µg/1.9 cm )
2

-2.0 2.0

-3.0 1.5

-4.0 1.0

-5.0 0.5

-6.0 0.0
0 10 20 30 40 0 30 60 90 120 150
Time (min) Time (min)

Fig. 1. Inactivation of planktonic P. aeruginosa PA01 by AgNPs and silver ions (25 8C, Fig. 2. (a) Biofilm inactivation and (b) silver biosorption by biofilms under static
pH 6.3, 100 rpm, N0 = 106 CFU/mL). condition (25 8C, pH 6.3, N0 = 2  108 CFU/1.9 cm2).
 H.-J. Park et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 614–619 617

proteins [33], whereas negatively charged AgNPs can be electro-
statically repulsed from the negatively charged (20 mV) surfaces
of bacterial cells [34]. The AgNPs could therefore be prevented
from being taken up by the biofilms without any external force,
resulting in a reduced biofilm inactivation. The size of the AgNPs
may also hinder their penetration into biofilms compared with
smaller silver ions, possibly also contributing to the different
extents of biofilm inactivation.
These proposed mechanisms could be tested by measuring the
biosorption of silver by the biofilms. Biosorption includes AgNPs or
silver ions present in biofilm cells, the EPS matrix, and on the
surface of a biofilm. AgNPs were taken up to a lesser extent than
silver ions under static conditions (Fig. 2b), suggesting that
biosorption may be responsible for the different extents of biofilm
inactivation. It is also possible that stirring could aid silver
biosorption by biofilms. To examine this issue, similar tests were
conducted under stirring at 100 rpm, the same speed used for
biofilm formation. Before the tests, it was confirmed that stirring at
100 rpm for 150 min did not significantly induce a detachment of
biofilm cells (data not shown) and a change of the AgNP particle
size (Fig. S4).
As shown in Fig. 3a, P. aeruginosa PA01 biofilm inactivation was
increased by both the AgNPs and silver ions under stirring. After
30 min, stirring enhanced the inactivation of biofilms by silver ions
by almost 2 log, a more than 2 log inactivation greater than the
corresponding value for AgNPs. After 60 min, biofilm inactivation
(a) 0.0
-1.0
Log reduction (log N/N0)
by AgNPs was similar to that for silver ions. As expected, AgNP
biosorption was significantly increased by stirring (Fig. 3b), while
only a slight increase in the biosorption of silver ions was observed.
The following hypothesis explains this observation: The
penetration of AgNPs is limited by their size or by electrostatic
repulsion, and this restricted biosorption is overcome by the
external force of stirring. As the biosorption of silver ions was not
increased by stirring, the high affinity of silver ions for biological
molecules may prevent an increased penetration by stirring. The
relationship between biofilm inactivation and silver biosorption
was examined using the results shown in Figs. 2 and 3. The AgNPs
showed a good correlation (Fig. 4a), indicating that they inactivate
biofilm cells in a biosorption-dependent manner. Silver ions
showed a lower correlation between biofilm inactivation and silver
biosorption compared with the AgNPs (Fig. 4b). Biofilm-inactiva-
tion by silver ions was affected more by exposure time than by
silver biosorption (Figs. 2 and 3).
Table 1, calculated from the results of Figs. 3 and 4, shows the
inactivation efficiency of AgNPs for a 3 log inactivation of P.
aeruginosa PA01 biofilm cells in comparison with silver ions, as
expressed by exposure time, biosorption, and effective biosorption
number. Silver ions inactivated P. aeruginosa PA01 biofilms more
efficiently than AgNPs in terms of exposure time. However, the
amount of AgNPs taken up by the biofilms was lower than
the amount of silver ions; consequently, the effective number of
AgNPs taken up was lower than that of silver ions. This suggests
that AgNPs and silver ions have different patterns of P. aeruginosa
PA01 biofilm inactivation: AgNPs were more toxic to P. aeruginosa
PA01 biofilms than silver ions at similar levels of silver biosorption,
AgNP 10 mg/L
Ag+ 10 mg/L

-2.0

-3.0

-4.0

-5.0
0 30 60 90 120 150
Time (min)

(b) 3.0

2.5
Biosorption (µg/1.9 cm2 )

2.0

1.5

1.0

0.5 AgNP 10 mg/L

+
Ag 10 mg/L

0.0
0 30 60 90 120 150
Time (min)

Fig. 3. (a) Biofilm inactivation and (b) silver biosorption by biofilms under stirring Fig. 4. Correlation between biofilm inactivation by (a) AgNPs (b) silver ions and
condition (25 8C, pH 6.3, 100 rpm, N0 = 2  108 CFU/1.9 cm2). silver biosorption (replotted data from Figs. 2 and 3).
618 H.-J. Park et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 614–619

Table 1 The extent of inactivation of the suspended biofilm cells by AgNPs

Requirement of exposure time, biosorption, and effective biosorption number of
and silver ions was comparable, though slightly higher inactivation
AgNPs or silver ions for a 3 log inactivation of biofilm cells (data from Figs. 3 and 4).
was observed for the AgNPs at similar concentrations, despite the
Chemical Exposure Biosorption Effective biosorption fact that bacterial cells from the biofilms would be expected to be
exposure time (min) (mg) number for biofilm
more tolerant than planktonic bacteria [15]. This indicates that
inactivation (atom or
particle no./cell no.
biofilm cells are easily inactivated by AgNPs, even if they are
in biofilms) detached from the biofilms.
AgNPs were found to have a similar activity as silver ions
AgNP 60 1.5a 14 particles/cella
Silver ion 30a 2.1 6  107 atoms/cell against biofilms in the presence of an external force such as
a stirring. This new finding suggests that they are potentially
Better efficiency on biofilm inactivation.
important both in the environment and in specific types of
applications. When AgNPs are exposed to the environment, they
could show considerable toxicity on mature biofilms in natural
whereas silver ions showed a higher toxicity in terms of exposure environments or wastewater treatment systems. They could also
time. Their mechanisms of biofilm inactivation may also be be used in the control of biofilms, for example to prevent biofouling
different, and this will require further investigation. in membrane processes and infections.
CLSM images were recorded to examine the morphologies of P. As previously reported, AgNPs can spontaneously aggregate in
aeruginosa PA01 biofilms exposed to AgNPs or silver ions under the presence of ionic components (such as Ca2+) in the
stirring. The green and red colors in Fig. 5 indicate live and dead environment [35]. This phenomenon could be used in controlling
cells, respectively. Almost all of the biofilm cells were inactivated both their environmental impact and their potential applications,
by both AgNPs (Fig. 5b) and silver ions (Fig. 5c) without significant since aggregation results in a reduction of toxicity and their
disruption of the biofilms. However, a slight amount of biofilm efficiency to control biofilms. To diminish the toxicity of AgNPs on
cells could have been detached from the biofilms; a similar biofilms in the environment, AgNPs could be coated with a
disruption of biofilm cells can also occur in the environment. material that is unstable in the presence of ionic components in the
Therefore, the inactivation of cells detached from the biofilms by environment. The effective control of biofilms using AgNPs
AgNPs and silver ions was investigated. requires that the particles be stable during the treatment process.
P. aeruginosa PA01 biofilm cells on the coupon surface were Furthermore, the treatment should be conducted under conditions
suspended in DI water and then exposed to AgNPs or silver ions. of stirring rather than static conditions to obtain higher efficiency.
The circles in Fig. 6 show the log reduction of biofilm cells Strategies for reducing the environmental impact of AgNPs and
suspended in DI water. The log reductions of planktonic bacteria by enhancing their applications are opposite objectives, so an
the AgNPs and silver ions from Fig. 1 are included for comparison. effective method for recovering AgNPs should be considered to

Fig. 5. CLSM images of (a) control biofilms, (b) biofilm exposed to AgNP 10 mg/L, (c) biofilms exposed to silver ion 10 mg/L (25 8C, pH 6.3, 100 rpm, N0 = 2  108 CFU/1.9 cm2).
 H.-J. Park et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 614–619
Fig. 6. Suspended biofilm cell inactivation by AgNPs (dashed and dotted lines from
Fig. 1, 25 8C, 100 rpm, N0 = 106 CFU/mL).
minimize their negative environmental impact when they have
been stabilized for enhanced performance.
4. Conclusions
This study estimated the antimicrobial activity of citrate-
capped AgNPs on P. aeruginosa PA01 biofilms, and their activity
was compared with the silver ions’ activity. P. aeruginosa PA01
biofilm-inactivating activity of AgNPs was significantly enhanced
by stirring, which caused an increase of AgNP biosorption by
biofilms. The antimicrobial activity of AgNPs against biofilm cells
was comparable to that of silver ions at the same concentration
after 90 min under stirring (ca. 3.5 log inactivation), although their
activity against planktonic bacteria was ca. 10% that of silver ions.
AgNPs inactivated biofilm cells in a biosorption dependent
manner, whereas silver ions were not. In addition, less amount
of AgNP biosorption by biofilms was needed to achieve the same
log inactivation as compared with silver ions. AgNPs showed
comparable antimicrobial activity against both suspended biofilm
cells and planktonic bacterial cells, indicating that bacterial cells
detached from the biofilms can be also easily inactivated by AgNPs.
Acknowledgments
This research was supported by WCU (World Class University)
program through the National Research Foundation of Korea
funded by the Ministry of Education, Science and Technology (R31-
10013) and by National Institute of Environmental Research,
Korea. These contributions are greatly appreciated.
619
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jiec.2012.09.013.
References
[1] P. Jain, T. Pradeep, Biotechnology and Bioengineering 90 (2005) 59.
[2] S. Dubas, P. Kumlangdudsana, P. Potiyaraj, Colloids and Surface A 289 (2006)
289.
[3] T. Benn, P. Westerhoff, Environmental Science and Technology 42 (2008)
4133.
[4] F. Furno, K. Morley, B. Wong, B. Sharp, P. Arnold, S. Howdle, R. Bayston, P. Brown, P.
Winship, H. Reid, Journal of Antimicrobial Chemotherapy 54 (2004) 1019.
[5] Y. Kim, J. Kim, H. Cho, D. Rha, J. Kim, J. Park, B. Choi, R. Lim, H. Chang, Y. Chung,
Inhalation Toxicology 20 (2008) 575.
[6] Project on Emerging Nanotechnologies, Consumer Products Analysis, 2009.
[7] I. Sondi, B. Salopek-Sondi, Journal of Colloid and Interface Science 275 (2004) 177.
[8] A. Panacek, L. Kvitek, R. Prucek, M. Kola, R. Vec eova, N. Pizurova, V. Sharma, T.
Nevcna, R. Zboil, Journal of Physical Chemistry B 110 (2006) 16248.
[9] C. Lok, C. Ho, R. Chen, Q. He, W. Yu, H. Sun, P. Tam, J. Chiu, C. Che, Journal of
Proteome Research 5 (2006) 916.
[10] E. Hwang, J. Lee, Y. Chae, Y. Kim, B. Kim, B. Sang, M. Gu, Small 4 (2008) 746.
[11] M. Rai, A. Yadav, A. Gade, Biotechnology Advances 27 (2009) 76.
[12] J. Morones, J. Elechiguerra, A. Camacho, K. Holt, J. Kouri, J. Ramirez, M. Yacaman,
Nanotechnology 16 (2005) 2346.
[13] N.S. Wigginton, A.D. Titta, F. Piccapietra, J. Dobias, V.J. Nesatyy, Environmental
Science and Technology 44 (2010) 2163.
[14] A. Ivask, O. Bondarenko, N. Jepihhina, A. Kahru, Analytical and Bioanalytical
Chemistry 398 (2010) 701.
[15] L.P. John, E.R. Mark, G.F. Roger, Biofilm, Infection, and Antimicrobial Therapy, CRS
Press, Boca Raton, 2006.
[16] T. Mah, G. O’Toole, Trends in Microbiology 9 (2001) 34.
[17] J. Nickel, I. Ruseska, J. Wright, J. Costerton, Antimicrobial Agents and Chemother-
apy 27 (1985) 619.
[18] P. Piriou, S. Dukan, Y. Levi, P. Jarrige, Water Science and Technology 35 (1997) 283.
[19] H.A. Videla, International Biodeterioration and Biodegradation 49 (2002) 259.
[20] J. Costerton, P. Stewart, E. Greenberg, Science 284 (1999) 1318.
[21] L. Hall-Stoodley, J. Costerton, P. Stoodley, Nature Reviews Microbiology 2
(2004) 95.
[22] J. Fabrega, J. Renshaw, J. Lead, Environmental Science and Technology 43 (2009)
9004.
[23] O. Choi, C.-P. Yu, G. Esteban Fernández, Z. Hu, Water Research 44 (2010) 6095.
[24] A. Dror-Ehre, A. Adin, G. Markovich, H. Mamane, Water Research 44 (2010) 2601.
[25] K. Kalishwaralal, S. BarathManiKanth, S. Pandian, V. Deepak, S. Gurunathan,
Colloid Surface B 79 (2010) 340.
[26] OECD, Guidance Manual for the Testing of Manufactured Nanomaterials, 2010.
[27] Center for Biofilm Engineering, Standardized Biofilm Methods Team, CDC Biofilm
Reactor Operator’s Manual, 2003.
[28] R. Tilton, B. Rosenberg, Applied and Environmental Microbiology 35 (1978) 1116.
[29] J. Kim, B. Pitts, P. Stewart, A. Camper, J. Yoon, Antimicrobial Agents and Chemo-
therapy 52 (2008) 1446.
[30] O. Choi, Z. Hu, Environmental Science and Technology 42 (2008) 4583.
[31] E. Bae, H.-J. Park, J. Lee, Y. Kim, J. Yoon, K. Park, K. Choi, J. Yi, Environmental
Toxicology and Chemistry 29 (2010) 2154.
[32] A.M. El Badawy, R.G. Silva, B. Morris, K.G. Scheckel, M.T. Suidan, T.M. Tolaymat,
Environmental Science and Technology 45 (2011) 283.
[33] V. Lee, H. Li, T. Lau, R. Guevremont, K. Siu, Journal of the American Society for Mass
Spectrometry 9 (1998) 760.
[34] S.H. Hong, J. Jeong, S. Shim, H. Kang, S. Kwon, K.H. Ahn, J. Yoon, Biotechnology and
Bioengineering 100 (2008) 379.
[35] A.M. El Badawy, T.P. Luxton, R.G. Silva, K.G. Scheckel, M.T. Suidan, T.M. Tolaymat,
Environmental Science and Technology 44 (2010) 1260.