ARTICLE IN PRESS
WATER RESEARCH 42 (2008) 3066 3074
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
The inhibitory effects of silver nanoparticles, silver ions,
and silver chloride colloids on microbial growth
Okkyoung Choia, Kathy Kanjun Dengb, Nam-Jung Kimc, Louis Ross Jr.d, Rao Y. Surampallie,
Zhiqiang Hua,
a
Department of Civil and Environmental Engineering, University of Missouri, E2509 Lafferre Hall, Columbia, MO 65211, USA
b
School of Engineering, Rice University, USA
c
Department of Mechanical and Aerospace Engineering, University of Missouri, USA
d
Electron Microscopy Core Facility, University of Missouri, USA
e
Region 7 Office, US Environmental Protection Agency, Kansas City, KS, USA
a r t i c l e i n f o a b s t r a c t
Emerging nanomaterials are of great concern to wastewater treatment utilities and the
Article history:
environment. The inhibitory effects of silver nanoparticles (Ag NPs) and other important Ag
Received 9 October 2007
species on microbial growth were evaluated using extant respirometry and an automatic
Received in revised form
microtiter fluorescence assay. Using autotrophic nitrifying organisms from a well-controlled
22 February 2008
continuously operated bioreactor, Ag NPs (average sizeź1476nm), Ag+ ions (AgNO3), and
Accepted 26 February 2008
AgCl colloids (average sizeź0.25 mm), all at 1 mg/L Ag, inhibited respiration by 8673%,
Available online 4 March 2008
4277%, and 4674%, respectively. Based on a prolonged microtiter assay, at about 0.5 mg/L
Keywords:
Ag, the inhibitions on the growth of Escherichia coli PHL628-gfp by Ag NPs, Ag+ ions, and AgCl
Silver nanoparticles
colloids were 5578%, 100%, and 6676%, respectively. Cell membrane integrity was not
Silver ion
compromised under the treatment of test Ag species by using a LIVE/DEAD BaclightTM
AgCl colloid
bacterial viability assay. However, electron micrographs demonstrated that Ag NPs attached
Microbial growth
to the microbial cells, probably causing cell wall pitting. The results suggest that nitrifying
Nitrification
bacteria are especially susceptible to inhibition by Ag NPs, and the accumulation of Ag NPs
Inhibition
could have detrimental effects on the microorganisms in wastewater treatment.
& 2008 Elsevier Ltd. All rights reserved.
(104 out of 502 nanoproducts surveyed) (Maynard and
1. Introduction
Michelson, 2006). These nanoparticles will likely enter the
Nanosilver (silver nanoparticle, Ag NP) materials have a wide sewage pipes and the wastewater treatment plants (WWTPs).
range of applications including spectrally selective coating for At present, little is known about the adverse effects of Ag NPs
solar energy absorption (Rand et al., 2004; Cole and Halas, on wastewater treatment and the environment.
2006), catalysis in chemical reactions (Zhai et al., 2006), It is known, however, that free silver ion (Ag+) is highly toxic
surface-enhanced Raman scattering for imaging (Yamamoto to a wide variety of organisms including bacteria. Metal
and Watarai, 2006), and antimicrobial sterilization (Savage toxicity to planktonic species such as algae (Lee et al., 2005)
and Diallo, 2005; Sambhy et al., 2006; Pal et al., 2007). Because and bacteria (Hu et al., 2002, 2003) is often governed by the
of their effective antimicrobial properties and low toxicity concentrations of aqueous free metal species (i.e., Ag+). The
toward mammalian cells, Ag NPs have become one of the inhibitory effect of Ag+ is believed to be due to its sorption to
most commonly used nanomaterials in consumer products the negatively charged bacterial cell wall, deactivating
Corresponding author. Tel.: +1 573 884 0497; fax: +1 573 882 4784.
E-mail address: huzh@missouri.edu (Z. Hu).
0043-1354/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2008.02.021
ARTICLE IN PRESS
WATER RESEARCH 42 (2008) 3066 3074 3067
cellular enzymes, disrupting membrane permeability, and (PVA) (Aldrich) as the capping agent to control the growth of
ultimately leading to cell lysis and death (Ratte, 1999; Sambhy nanocrystals and agglomeration of nanoparticles. To dissolve
et al., 2006). The aqueous concentrations of Ag+ are typically PVA, a solution containing 0.06% (wt) PVA was heated to
low in wastewater treatment systems or in the natural 100 1C and cooled down to room temperature before use.
environment because of its strong complexation with various Silver particles were prepared by rapidly injecting 0.5 mL of
ligands such as chloride (Kspź10 9.75), sulfide (Kspź10 49), 10 mM NaBH4 into 20 mL PVA solution containing 0.25 mM
thiosulfate, and dissolved organic carbon (Shafer et al., 1998; silver nitrate at room temperature. After 5 min of stirring, the
Wang, 2003). As a result, silver toxicity to microorganisms is reaction mixture was stored at 4 1C before use.
generally not observed.
Nanosilver, a particle of Ag element, is a new class of
2.1.2. Silver ions
material with remarkably different physiochemical character-
A silver nitrate standard solution (14 mM, Fisher Scientific)
istics such as increased optical, electromagnetic and catalytic
was used as a source of Ag+ ions.
properties from the bulk materials (Wenseleers et al., 2002;
Kelly et al., 2003). Nanoparticles with at least one dimension of
2.1.3. Silver chloride colloids
100 nm or less have unique physicochemical properties, such
Aliquots of 100 mg/L AgCl colloids were prepared freshly by
as high catalytic capabilities and ability to generate reactive
vigorously mixing (700 rpm) 1 mL of 14 mM silver nitrate
oxygen species (ROS) (Limbach et al., 2007) (see recent review
standard solution and 1 mL of 28 mM sodium chloride with
by Nel et al., 2006). Silver in the form of nanoparticles could be
18 mL of distilled water. Twice as much sodium chloride as
therefore more reactive with its increased catalytic properties
silver nitrate was added to ensure complete complexation
and become more toxic than the bulk counterpart. Further-
with no residual Ag+ ions in the colloidal solution (confirmed
more, toxicity is presumed to be size- and shape-dependent
by Ag+ measurements with an ion-selective electrode).
(Pal et al., 2007), because small size nanoparticles (e.g.,
o10 nm) (Kloepfer et al., 2005; Morones et al., 2005) may pass
2.2. Microbial cultures
through cell membranes and the accumulation of intracellular
nanoparticles can lead to cell malfunction.
2.2.1. Autotrophic bacteria
Little work has been done to evaluate the inhibition of
The mixed and enriched nitrifying bacteria were cultivated in
microbial growth by different Ag species, especially Ag NPs in
a continuously stirred tank reactor (14 L) operated at solids
wastewater treatment systems where such information is
retention time of 20 d and hydraulic retention time of 1 d
valuable for operation planning and control. Both autotrophic
using seed from a local nitrifying activated sludge plant in
and heterotrophic microorganisms are important in waste-
Missouri, USA. The reactor was fed with an inorganic medium
water treatment. While heterotrophs are responsible for
containing ammonium (8.3 mM, NH4NO3) as the sole energy
organic and nutrient removal, autotrophs are responsible for
source and requisite macro- and micronutrients (Table 1). Low
nitrification that is considered as the controlling step in
concentrations of anions such as chloride and sulfate were
biological nitrogen removal because of the slow growth rate of
present in the reactor to minimize their complexation
nitrifying organisms and their sensitivity to temperature, pH,
potential with Ag+ ions. Sodium carbonate (0.5 M) was
dissolved oxygen (DO) concentration, and toxic chemicals
intermittently added to maintain the reactor pH at 7.570.1
(Blum and Speece, 1991; Hu et al., 2002). Consequently, the
and fulfilled both carbon and alkalinity requirements. After a
objective of this research was to evaluate the impact of
few months of operation, mixed liquor was periodically
important Ag species such as Ag NPs, Ag+ ions, and AgCl
withdrawn from the nitrifying reactor for batch respirometric
colloids on heterotrophic and autotrophic growth.
studies.
In this research work, Ag NPs and AgCl colloids with larger
sizes were synthesized and characterized by UV vis spectro-
2.2.2. Heterotrophic bacteria
scopy and electron microscopy. The inhibitory effects on the
The test heterotrophic bacterium was Escherichia coli PHL628-
autotrophic and heterotrophic growth were determined by a
gfp, a gift from Dr. Anthony Hay at Cornell University. This
short-term extant respirometric assay and an automatic
strain tagged with a green fluorescence protein (GFP) was a
microtiter assay, respectively. Environmental scanning elec-
derivative of the E. coli K12 that forms biofilms as a
tron microscopy (ESEM) was applied as a complementary
consequence of the over-expression of curli (Junker et al.,
technique to examine the microbial/nanoparticle interac-
2006). The test strain was grown overnight on a mechanical
tions. The mode of action of nanosilver toxicity was finally
shaker (200 rpm) at room temperature in a nutrient-rich
discussed based on the results of membrane integrity using a
medium (BBLTM containing 5 g/L GelysateTM peptone and
LIVE/DEAD BaclightTM bacterial viability kit.
3 g/L beef extract, pH 6.970.2).
2. Materials and methods 2.3. Silver species characterization
2.1. Silver materials Aliquots of the prepared Ag NP suspensions were periodically
scanned from 250 to 700 nm to obtain absorption spectra
2.1.1. Silver nanoparticles using a UV vis spectrophotometer (Cary 50, Varian, CA).
Ag NPs were synthesized by reducing silver nitrate with Additional aliquots were used to determine the stability of
sodium borohydrate (NaBH4) and adding polyvinyl alcohol the Ag NPs by measuring the concentrations of Ag+ ions in the
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3068 WATER RESEARCH 42 (2008) 3066 3074
Table 1  Composition of the growth nutrients in reactor 2.5. Heterotrophic growth determined by an automated
influent
microtiter assay
To evaluate the inhibitory effects of Ag species on hetero-
Compound Concentrations in reactor influent
trophic growth, E. coli PHL628-gfp was grown in nutrient broth
mg/L Cations Anions
(BBL) at room temperature overnight. For the microtiter
(mM) (mM)
fluorescence assay, aliquots of the fresh medium (190 mL)
were pipetted into eight parallel wells of a 96-well microplate
Mg(NO3)2 61 0.41Mg2+ 0.82NO 3
(i.e., 8 replicates), and aliquots (10 mL) of overnight E. coli
Ca(NO3)2 41 0.25Ca2+ 0.25NO 3
NaNO3 879 10.34Na+ 10.34NO 3
cells were inoculated in each well. Aliquots of the Ag NP
NH4NO3 667 8.33NH+a 8.33NO 3
4
suspension, Ag+ or AgCl colloidal solution were added
K2HPO4 3.9 0.04K+ 0.02HPO 4
individually to each well to reach predetermined Ag concen-
FeCl2 4H2O 2 0.01Fe2+ 0.02Cl b
trations. The cells were exposed to ambient air and mixed
MnSO4 H2O 3.4 0.02 n2+ 0.02SO2 c
4
intermittently to support their growth on the plate. A program
(NH4)6Mo7O24 4H2O 1.2 0.006NH+a
4
was made to incubate the samples with vigorous mixing for
0.001Mo7O6
24
CuSO4 0.8 0.01Cu2+ 0.01SO2 c 10 s per hour before the fluorescence intensities (535 nm)
4
Zn(NO3)2 6H2O 1.8 0.01Zn2+ 0.02NO 3
excited at 485 nm were recorded automatically every hour for
Ni(NO3)2 6H2O 0.3 0.001Ni2+ 0.002NO 3
24 h. The plate was pre-equilibrated at room temperature
(2572 1C) for 0.5 h and the fluorescence (in relative fluores-
a
Total NH+ź8.336 mM.
4
cence unit, RFU) of microbial suspensions was measured with
b
Total Cl ź0.02 mM.
a fluorescence microreader (VICTOR3, PerkinElmer, Shelton,
c
Total SO2 ź0.03 mM.
4
USA).
The time-dependent microbial growth associated with
organic substrate oxidation in the 96-microwells was simu-
Ag NP suspensions using a silver ion/sulfide selective lated using an exponential growth model:
electrode (Denver instrument, Denver, CO).
XźX0emt (1)
The sizes of Ag NPs and AgCl colloids were characterized by
where X and X0 are final and initial biomass concentrations,
an FEI Quanta 600F ESEM (resolution: 3 nm at 30 kV, FEI
respectively, as reflected by the fluorescence intensity. The
Company, OR) equipped with a scanning transmission
parameters of the specific microbial growth rate, m, were
electron microscopy (STEM) detector. The Ag NP suspension
determined via least-squares error analysis using the SOLVER
was added to standard carbon-coated TEM grid. Images of the
routine in Microsoft Excel.
samples were taken at an accelerating voltage of 30 keV.
2.6. Microbial/nanoparticle interaction
2.4. Autotrophic growth determined by extant
respirometry
The microbial/nanoparticle interaction was visualized using
the FEI Quanta 600F SEM in the environmental (ESEM) mode
Autotrophic growth inferred from oxygen uptake rates due to
that allows organic samples to be examined without applying
ammonia oxidation was measured in triplicate using a batch
a conductive coating prior to imaging. The enriched nitrifying
extant respirometric assay (Hu et al., 2002). Aliquots (60 mL) of
culture amended with commercially available Ag NPs (10 nm,
nitrifying bacteria were collected from the nitrifying reactor.
Nanostructured & Amorphous Materials, Inc., Houston, TX)
MOPS[3-(N-morpholino) propanesulfonic acid, pH adjusted to
was placed in an Al cup on a cold stage (10 1C) and imaged at
7.5] at a final concentration of 20 mM was added to maintain
ca. 7 Torr and 80% relative humidity. In order to obtain higher-
relatively constant pH of 7.5 during ammonium oxidation.
resolution images, bacteria amended with our own Ag NPs
The nitrifying bacterial suspensions were amended with Ag
were examined under high-vacuum conditions utilizing a
NPs, Ag+ ions and AgCl colloids individually at the final
concentration range of 0.1 1 mg/L Ag, filled into the respiro- back-scattered electron (BSE) detector. The nanoparticle
samples synthesized in our laboratory were prepared using
metric bottles with no headspace, and then tightly capped.
a standard protocol described above, critically point dried,
Every batch respirometric test was accompanied by a positive
and coated with 10 nm of Pt.
control (e.g., untreated nitrifying bacteria only) at room
temperature (2572 1C). The nitrifying bacterial suspensions
were aerated with pure oxygen gas before aliquots of NH+-N 2.7. LIVE/DEAD bacterial viability assays
4
(10 mg/L N as NH4NO3) were injected. Magnetic stirring at ca.
100 rpm was provided in the bottles to ensure complete Experiments were carried out in the presence and absence of
mixing. A decrease in the DO level in the respirometric vessel nanoparticles to determine the cell viability of heterotrophic
was measured by a DO probe (YSI model 5300A, Yellow (E. coli PHL628 without GFP tagged) and autotrophic cultures
Springs, OH) and continuously monitored at 4 Hz by an by using a LIVE/DEAD BaclightTM bacterial viability kit
interfaced personal computer. The degree of inhibition of (Molecular Probes, Eugene, OR) (Hu et al., 2003). Viable and
autotrophic microbial growth was inferred from the differ- dead cells were detected by differential staining with a
ence between the measured specific oxygen uptake rate in mixture of a green fluorochrome, SYTO 9 (stains all cells, live
the absence and presence of the Ag species (Hu et al., 2002). or dead), and a red fluorochrome, propidium iodide (stains
ARTICLE IN PRESS
WATER RESEARCH 42 (2008) 3066 3074 3069
only bacteria with damaged membranes). A reduction in the absorption band with a maximum of about 400 nm, a
SYTO 9 fluorescent emission results when both dyes are characteristic peak of Ag NPs (Petit et al., 1993; Kong
present in the cell. Dead cells subject to 75% ethanol killing and Jang, 2006), indicating the presence of Ag NPs in the
for 1 h were provided as a positive control. To reduce solution. Due to the excitation of plasma resonances
background fluorescence, the microbial suspension was or interband transitions, some metallic nanoparticle disper-
washed with 0.85% NaNO3 after centrifuging at 10,000g for sions exhibit unique bands/peaks (Creighton and Eadont,
15 min. After adding aliquots of microbial suspensions and 1991). The broadness of the peak is a good indicator of
stain solution to each well of a 96-well microplate, the plate the size of nanoparticles. As the particle size increases,
was incubated at room temperature in the dark for 15 min, the peak becomes narrower with a decreased bandwidth
and the relative fluorescence intensity was measured by the and an increased band intensity (Petit et al., 1993; Kong
VICTOR fluorescence microreader. Enumeration of stained and Jang, 2006). Furthermore, there is an inverse linear
cells was facilitated by excitation at 485 nm and detection at relationship between the full-width at half-maxi-
642 nm (red) and 535 nm (green), for propidium iodide and mum (FWHM) and the diameter of particles (Petit et al.,
SYTO 9, respectively. 1993):
230
FWHMź50þ (2)
D
3. Results and discussion
where both FWHM and the particle diameter (D) are in
nanometers. The size of the Ag NPs was estimated as
3.1. Characterization of Ag NPs and AgCl colloids
approximately 16 nm based on Eq. (2). This result is consistent
with the STEM results, which showed a size distribution
The absorption spectrum (Fig. 1) of dark brown Ag NPs
between 10 and 40 nm (Fig. 2) of the Ag NPs with an average of
prepared by chemical reduction showed a surface plasmon
1476nm.
A shoulder at approximately 425 nm was noticed in UV vis
absorption spectra, indicating a broad distribution of particle
2
sizes and shapes in the solution because of crystallization, as
was confirmed by STEM imaging. The position and the
number of peaks in the absorption spectra are dependent
1.5
on the shape of the particles: for an ellipsoidal particle there
are two peaks whereas for spherical silver particles there is
only one peak centered at about 400 nm (Creighton and
1 Eadont, 1991; Petit et al., 1993).
The concentrations of Ag+ ions were measured simulta-
neously to evaluate the stability of Ag NPs in the suspension.
The beginning Ag+ concentration to make the Ag NP suspen-
0.5
sions was 27 mg/L (0.25 mM). At the completion of the
reaction, the residual Ag+ concentration was 0.670.1 mg/L.
The Ag+ concentration remained largely unchanged at the
0
end of 1 day of resting at room temperature. Afterward, the
250 350 450 550 650
Ag+ concentrations increased gradually (data not shown), as
Wavelength, (nm)
also indicated from the changes of solution color.
Fig. 1  UV vis absorption spectra of an Ag NP suspension
During a week of Ag+ monitoring at room temperature, the
recorded immediately after chemical reduction (solid line)
color of Ag NP suspensions changed from dark brown to
and after 1 week (dash line) at room temperature.
ab
35
30
25
20
15
10
5
0
5 10 15 20 25 30 35
Diameter, (nm)
Fig. 2  STEM image of Ag NPs prepared by chemical reduction (a) and the particle size distribution (b). The average particle
size was 14 nm. Bar size: 500 nm.
Absorbance
Relative Abundance, (%)
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3070 WATER RESEARCH 42 (2008) 3066 3074
100
yellow, presumably due to oxidative dissolution of the Ag NPs
4AgþO2þ2H2Oź4Agþþ4OH (3)
Ag NPs
80
The color change associated with particle dissolution and
Ag+
the presence of multiple UV vis absorption bands indicate
AgCl
60
the existence of Ag NPs of various shapes and sizes, as was
confirmed by STEM imaging (Fig. 2). To minimize the
interference of dynamic changes of Ag NPs, we used the
40
freshly prepared Ag NP suspensions that were stored shortly
(a few days) at 4 1C before use, during which no significant
20
changes of Ag+ concentrations were observed in the suspen-
sion (data not shown).
Silver chloride colloids (100 mg/L Ag) were prepared with an
0
average size of ca. 0.25 mm. The particle sizes ranged from 0.1
0 0.2 0.4 0.6 0.8 1
to 2 mm. A constant low Ag+ concentration was detected Silver concentration, (mg/L)
because of the overdose of chloride. The fraction of Ag+ was
Fig. 4  Nitrification inhibition as a function of the
measured to be less than 0.1% of the total Ag in the AgCl
concentrations of silver in the form of Ag NPs, Ag+ ions,
colloidal solution.
and AgCl colloids. Error bars indicate one standard
deviation.
3.2. Effect of Ag species on autotrophic growth
An extant respirometric technique was developed to deter-
mine biokinetic parameters from small pulses of substrate statistical difference (p40.05) of inhibition between AgCl
(e.g., NH+) while minimizing changes in the microbial colloids and Ag+ ions. At this small size, silver chloride
4
physiological state (Chandran and Smets, 2000; Hu et al., colloids appeared to reduce the bacterial growth as effectively
2002). Fig. 3 shows a representative respirograph of ammonia
as Ag+.
oxidation after an aliquot of ammonium was injected at
100 s in the enriched nitrifying microbial suspension. The 3.3. Effect of Ag species on heterotrophic growth
lack of change in DO illustrates nitrification inhibition in the
presence of Ag NPs. There was no significant pH change Consistent with the results from autotrophic growth study, Ag
before and after the test because of the addition of MOPS. NPs inhibited E. Coli growth. While no inhibition was observed
As shown in Fig. 4, at 1 mg/L Ag in the nitrifying suspension, at Ag NP concentrations below 1.0 mM, the heterotrophic
the inhibitions by Ag NPs, Ag+ ions, and AgCl colloids were growth rate was reduced significantly by 55% as the Ag NP
8673%, 4277%, and 4674%, respectively. Of all the Ag species concentrations increased to 4.2 mM (Table 2). The IC 50
tested, Ag NPs presented the highest inhibition on nitrifying inhibition by the Ag NP suspension, or the half maximal
bacterial growth. Interestingly, the freshly prepared AgCl inhibitory concentration, was estimated to be 4.0 mM(nź8).
colloids with an average size of 0.25 mm also inhibited Surprisingly, silver ion was the most toxic species to inhibit
nitrification. At the same level of Ag dose, there was no heterotrophic growth. At 4.2 mM ( 0.5 mg/L Ag), the inhibi-
tions on the growth of E. coli PHL628-gfp were 5578%, 100%,
and 6676% by Ag NPs, Ag+ ions, and AgCl colloids, respec-
tively. E. Coli treated with 1 mg/L Ag (or 9.3 mM) in the forms of
Ag NPs, Ag+ ions, or AgCl did not exhibit signs of growth (data
25
not shown). The inhibition on heterotrophic growth appeared
to be more severe from the long-term microtiter fluorescence
20
A assays, as we reported earlier that inhibition on microbial
growth with longer period of metal exposure tends to be more
15
significant (Hu et al., 2004).
B A slight lag phase of E. coli growth ( 1.5 h) was observed
10
during the automatic microtiter assays (Fig. 5). Stationary
phase was reached after incubation of the heterotrophic
5
strain for approximately 12 h at room temperature (2571 1C).
Upon the addition of Ag NPs in the microbial suspension, a
0
slight decrease of fluorescence efficiency (i.e., fluorescence
0 200 400 600 800
quenching) with increasing Ag NP concentrations was
Time, (s)
observed. In the case of AgCl colloids, the quenching effect
Fig. 3  Nitrification inhibition inferred from the decrease of was less significant (data not shown). The results are
specific oxygen uptake rate (slope of curve A) in the consistent with the existing experimental data (Sabatini
presence of Ag NPs, as compared with control (curve B) after et al., 2007; Yamaguchi et al., 2007), indicating that the
an aliquot of ammonium nitrate was injected individually at overlap between the GFP-tagged microbial fluorescence and
approximately 100 s. the plasmon absorption of Ag nanoparticles may slightly
Inhibition, (%)
2
O , (mg/L)
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WATER RESEARCH 42 (2008) 3066 3074 3071
Table 2  Specific growth rates (l) of E. coli PHL628-gfp and the inhibitions by silver species at different concentrations
Concentration (mM) Ag NP Ag+ AgCl colloid
m (d 1) Inhibition (%) m (d 1) Inhibition (%) m (d 1) Inhibition (%)
1.4 0.40 (70.03) 17 (75) 0.41 (70.02) 11 (74) 0.43 (70.01) 7 (74)
2.8 0.34 (70.03) 30 (76) 0.14 (70.03) 69 (76) 0.35 (70.02) 24 (75)
4.2 0.22 (70.04) 55 (78) 0 100 0.16 (70.03) 66 (76)
320
300
280
260
240
220
200
0 5 10 15 20
Time, (h)
J
Fig. 5  Effect of Ag NP concentrations ( , 0lM; K, 1.4 lM; ,
2.8 lM;  , 4.2 lM; &, 9.3lM) on the growth of E. coli PHL628-
gfp, as measured by relative fluorescence units.
Fig. 6  Silver nanoparticles adsorbed to the enriched
nitrifying culture on a copper grid using ESEM. Arrows
cause the quenching of the excited-state of GFP molecules on
show aggregated AgNPs that attached to microbial cells or
the Ag NPs.
embedded in microbial extracellular polymeric substances.
Bar size: 100 lm.
3.4. Microscopic observation of microbial/nanoparticle
interaction
3.5. Cell membrane integrity inferred from LIVE/DEAD
The microbial nanoparticle interaction was visualized by assays
ESEM, a specialized technique capable of imaging hydrous
samples without the need of pretreatment for conductive The fluorescence intensities of the stained microbial cells at
coating (Redwood et al., 2005; Priester et al., 2007). Commer- 535 nm (green) and 642 nm (red) represent live and dead cells,
cially available Ag NPs (Nanostructured & Amorphous Mate- respectively. The green/red fluorescence ratio, obtained by
rials, Inc., advertised powder size of 10 nm) aggregated in dividing the green and red intensities, was applied to
water and the nitrifying bacterial suspension. It appeared that compare the difference among various treatments by Ag
the particles were embedded in microbial extracellular species. At 1 mg/L Ag, the ratio obtained from the microbial
polymeric substances (Fig. 6). The true size (from 200 nm to suspensions treated with Ag NPs showed no significant
a few mm) of Ag NPs in water suspension was significantly difference compared to controls (P40.05), indicating that
different from the claimed size of commercial nanopowders, there is no evidence of cell membrane leakage caused by Ag
consistent with the results reported by others (Adams et al., NPs. Similar results were observed in samples treated with
2006). Ag+ ions or AgCl colloids.
Higher-resolution electron micrographs were obtained
using BSE mode. After mixing a freshly prepared Ag NP 3.6. Inhibition comparison and mode of antimicrobial
suspension with the mixed and enriched nitrifying cultures, action
it appeared that Ag NPs were adsorbed to the micro-
bial surfaces, probably causing cell wall pitting (Fig. 7). Among the Ag species tested, the freshly prepared Ag NPs
Additional work is underway to take higher-resolution images presented the highest inhibition to autotrophic nitrifying
in order to better understand the microbial nanoparticle organisms. In contrast, silver ion appeared to be most toxic to
interactions. heterotrophic growth. Different experimental assays chosen
RFU, (×1,000)
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3072 WATER RESEARCH 42 (2008) 3066 3074
Nanoparticles have substantially different physiochemical
properties from those of bulk materials of the same composi-
tion, possibly resulting in different toxicity mechanisms to
biological systems (Nel et al., 2006). The mode of antimicro-
bial action by Ag NPs could be the inhibition of the microbial
processes on the cell surface and in the cell. Previous research
demonstrated that Ag NPs attach to the surface of cell
membrane, causing the change of membrane permeability,
dissipation of the ATP pool and proton motive force, and
finally cell death (Sondi and Salopek-Sondi, 2004; Morones
et al., 2005; Lok et al., 2006). The results from our bacterial
viability tests indicated that there is no evidence of the cell
membrane leakage caused by any Ag species at 1 mg/L Ag.
The size of the Ag NPs used in this study was 1476 nm. These
particles would be too large to diffuse into the cell, as only the
smaller particles mainly in the range of 1 10 nm could enter
the cell based on indirect microscopic evidences (Morones
et al., 2005). Although electrophoresis studies indicated no
direct effect of Ag NPs on intracellular DNA or protein
expression (Gogoi et al., 2006), our recent results demon-
Fig. 7  Silver nanoparticles adsorbed to the enriched
strated that inhibition by the Ag NPs might be attributed to
nitrifying culture using a high-speed BSE detector. Arrows
the accumulation of intracellular ROS (Choi and Hu, 2008).
show spherical or hexagon types of Ag NPs that attached to
Bulk silver toxicity is generally governed by the total
the microbial cells, probably causing cell wall pitting. Bar
concentration of labile dissolved intracellular Ag species
size: 500 nm.
(Lee et al., 2005). In the cell, silver ions may deactivate cellular
enzymes and DNA by reacting with electron-donating groups
such as thiol ( SH) groups and generate ROS (Matsumura
et al., 2003; Sambhy et al., 2006). Because of its cationic nature
for the enriched nitrifying bacteria and heterotrophic E. coli and its strong association with various ligands in natural
cells made it difficult to compare the toxicity of Ag NPs to the waters, the toxicity of Ag+ ions depends largely on the
two different bacterial species. The difference in toxicity may strength and amount of the ligands present (Ratte, 1999).
be attributed to different growth conditions and cell proper- The freshly prepared AgCl colloids can be viewed as one of the
ties. The nitrifying bacteria were completely mixed in the labile species with respect to their small size and low stability
respirometric bottles, aerated with pure oxygen and their constant (log K1ź3.3) in solution (Stumm and Morgan, 1996).
activities were monitored by oxygen uptake rate measure- Depending on their size and bioavailability, the inhibition
ments. Conversely, the E. coli cells were mixed intermittently caused by AgCl colloids can be as significant as that of Ag+
in microwells, aerated with ambient air and their activities ions.
were inferred from fluorescence measured over a prolonged
period of time ( 1 d). Because they have a faster growth rate 3.7. Ag NP dissolution
than nitrifying bacteria, E. coli cells may have stronger
oxidizing/reducing power and interact with Ag species to The time-dependent increases of Ag+ concentrations and
form cell-particle aggregates (Sondi and Salopek-Sondi, 2004; associated color changes of the Ag NP suspension demon-
Kahraman et al., 2007), as visible from Figs. 6 and 7, or strated the complexity of various processes such as oxidation,
produce extracellular or intracellular Ag NPs (Efrima and crystallization, dissolution and aggregation involved in mi-
Bronk, 1998), causing more complex growth problems.
crobial nanoparticle interactions. Previous research showed
Nitrifying bacteria have remarkably complex internal mem- that Ag NPs were susceptible to oxidation by oxygen, and the
brane systems where ammonia monooxygenase (AMO, re- partially oxidized particles appeared more toxic than the
sponsible for ammonia oxidation to produce hydroxylamine, freshly prepared nanoparticles (Lok et al., 2007). Others found,
NH2OH) is located, whereas hydroxylamine oxidoreductase however, that the concentration of Ag+ decreased by 80% after
(HMO) is located in the periplasm (Madigan et al., 2000). 24 h, possibly due to Ag0 cluster formation (Morones et al.,
Therefore, we speculate that Ag NPs may have a direct impact 2005). When the Ag NPs were added into a liquid medium, the
on nitrifying cell membranes where key ammonia oxidation antimicrobial effectiveness appeared to decrease when com-
enzymes are located. pared to that on the agar plates, presumably because of
The use of PVA during the synthesis of Ag NPs to control the microbial-induced coagulation of nanoparticles (Sondi and
nanoparticles size could affect the antibacterial activity by Salopek-Sondi, 2004). Experiments involving synthetic zinc
the coverage of PVA on AgNPs to prevent the direct contact sulfide nanoparticles and representative amino acids also
with bacteria. Due to the significant dilution (1:25 or higher) of indicated a driving role of microbially derived extracellular
the Ag NP suspension and solvent competition in cell proteins in rapid nanoparticle aggregation (Moreau et al.,
cultures, the effect of PVA on nanosilver toxicity, however, 2007). Further research is required to investigate nanoparticle
would be minimal. properties such as size, shape, dissolution/aggregation,
ARTICLE IN PRESS
WATER RESEARCH 42 (2008) 3066 3074 3073
surface coating, and solubility that may affect the specific (3) There was no evidence of change in cell membrane
physicochemical and transport properties, which could exert integrity at 1 mg/L Ag for all of the Ag species tested
a significantly different impact on microbial growth (Nel et al., based on the results from the bacterial LIVE/DEAD assays.
2006).
3.8. Environmental application and implication
Acknowledgments
The numerous engineered nanomaterials with different sizes,
This research work was supported by the University of
shapes, compositions, and coatings require high-throughput
Missouri Research Board and the National Science Foundation
benchmarked protocols to screen for potential hazards in the
under Grant no. 0650943. Any opinions, findings, and conclu-
environment (Maynard et al., 2006). The developed extant
sions or recommendations expressed in this material are
respirometric assay and the automatic microtiter assay
those of the author(s) and do not necessarily reflect the views
employed in this research are suitable for toxicity assessment
of the National Science Foundation.
of nanomanterials to microorganisms. The bacteria selected
for each assay, however, are generally not exchangeable
R E F E R E N C E S
between the two assays. Because of the intrinsic slow
growth rate (about an order of magnitude lower than that
of heterotrophs) of autotrophic nitrifying bacteria and their
Adams, L.K., Lyon, D.Y., Alvarez, P.J.J., 2006. Comparative eco-
high oxygen uptake (4.3 mg O2/mg of NH+-N oxidized to toxicity of nanoscale TiO2, SiO2, and ZnO water suspensions.
4
Water Res. 40, 3527 3532.
nitriate) (Grady et al., 1999), the enriched nitrifying
Blum, D.J.W., Speece, R.E., 1991. A database of chemical toxicity to
cultures are particularly useful in respirometric assays, but
environmental bacteria and its use in interspecies compar-
failed to show significant growth in the cell-enumeration-
isons and correlations. J. Water Pollut. Control Fed. 63,
based microtiter assay in this study. In contrast, E. coli
198 207.
cells were easily determined with the automatic microtiter
Chandran, K., Smets, B.F., 2000. Single-step nitrification models
assay because of their fast growth rate, but failed to pro-
erroneously describe batch ammonia oxidation profiles when
duce meaningful oxygen profiles from the extant respioro- nitrite oxidation becomes rate limiting. Biotechnol. Bioeng. 68,
396 406.
metric assay because of the low biomass concentrations
Choi, O.K., Hu, Z.Q., 2008. Size and ROS dependent nanosilver
from overnight batch cultivation and their low oxygen
toxicity to nitrifying bacteria, Environ. Sci. Technol., submitted
uptake constants ( 0.5 mg O2/mg COD removed) (Grady
for publication.
et al., 1999).
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The results of nanosilver toxicity to environmentally
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independent microbial growth assays extant respirometric
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