Size-dependent cytotoxicity of silver nanoparticles to Azotobacter vinelandii: Growth inhibition, cell injury, oxidative stress and internalization
Size-dependent cytotoxicity of silver nanoparticles to Azotobacter vinelandii: Growth inhibition, cell injury, oxidative stress and internalization
Li Zhang 0 1
Lingli Wu 0 1
Youbin SiID 0 1
Kunhui Shu 0 1
0 Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University , Hefei , China
1 Editor: Yogendra Kumar Mishra, Institute of Materials Science , GERMANY
The influence of nanomaterials on the ecological environment is becoming an increasingly hot research field, and many researchers are exploring the mechanisms of nanomaterial toxicity on microorganisms. Herein, we studied the effect of two different sizes of nanosilver (10 nm and 50 nm) on the soil nitrogen fixation by the model bacteria Azotobacter vinelandii. Smaller size AgNPs correlated with higher toxicity, which was evident from reduced cell numbers. Flow cytometry analysis further confirmed this finding, which was carried out with the same concentration of 10 mg/L for 12 h, the apoptotic rates were20.23% and 3.14% for 10 nm and 50 nm AgNPs, respectively. Structural damage to cells were obvious under scanning electron microscopy. Nitrogenase activity and gene expression assays revealed that AgNPs could inhibit the nitrogen fixation of A. vinelandii. The presence of AgNPs caused intracellular reactive oxygen species (ROS) production and electron spin resonance further demonstrated that AgNPs generated hydroxyl radicals, and that AgNPs could cause oxidative damage to bacteria. A combination of Ag content distribution assays and transmission electron microscopy indicated that AgNPs were internalized in A. vinelandii cells. Overall, this study suggested that the toxicity of AgNPs was size and concentration dependent, and the mechanism of antibacterial effects was determined to involve damage to cell membranes and production of reactive oxygen species leading to enzyme inactivation, gene down-regulation and death by apoptosis.
Data Availability Statement: All relevant data are
within the manuscript.
Funding: Funding for this study was provided by
the National Natural Science Foundation of China
[grant numbers 41430752, 41471405]. Youbin Si
was the funding recipient. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
Nanosilver (AgNPs) is currently the most widely used and fastest growing class of
nanomaterials. Due to its unique antibacterial properties, it is widely used in traditional industries and
emerging fields such as coatings, food, textiles, cosmetics, water purification, medical
]. Currently, there are more than 1,600 consumer goods containing nanomaterials,
4.2-fold higher than 380 goods containing AgNPs in 2010 (http://www.nanotechproject.org).
The large-scale commercial application of AgNPs has created great economic benefits, but
there are also risks of AgNPs entering the environment through various channels . The
biological toxicity and impacts of AgNPs on the environment have been a major focus of recent
Previous studies have shown that the microbial toxicity of AgNPs can be caused by
inhibition of cell growth, alteration of cell morphology and production of reactive oxygen species
(ROS). Beddow et al [
] investigated the effects of AgNPs on different bacteria, including three
nitrifying bacteria (Nitrosomonas europaea, Nitrosospira multiformis and Nitrosococcus ocean),
and Escherichia coli, Bacillus subtilis. They found that AgNPs caused significant inhibition to
nitrification potential rates (NPRs) in nitrifying bacteria, and adversely affect the growth of E.
coli and B. subtilis. Bryaskova et al [
] reported that AgNPs have a strong antimicrobial effect
against bacteria and fungi. AgNPs have extremely small diameter and large surface area, easily
enter into cells or bind to the bacterial surface [
]. Long et al [
] have detected the Ag elements
in E. coli cell membrane and cytoplasm as exposed to AgNPs, and found the cells were severely
damaged. Arnaout et al [
] determined that AgNPs cause damage to bacterial cell membranes,
leading to leakage of intracellular substances and induction of cell death. Numerous studies
have shown that the presence of AgNPs cause the generation of ROS, which can lead to
apoptosis, destroy cell membranes, interfere with intracellular antioxidant systems, and damage
intracellular components such as DNA [
]. Further researches have revealed that AgNPs
can enter cells by endocytosis or by other means after contact with bacteria [
], where it can
cause cell damage. In summary, after contact with the cells, the AgNPs are firstly adsorbed on
the cell surface and then enter into the cell. AgNPs pass through the inner membrane and
produce ROS, which leads to arrest of DNA replication, inactivates proteins, and destroys cell
membranes. Subsequently the cellular components leak from the damaged cell membranes,
and this eventually leads to cell death [
The toxicity of AgNPs is determined by many factors, including particle size, type of coating
material and morphological structure [
]. Numerous reports have demonstrated that the
cytotoxicity of AgNPs is size dependent. Gliga et al [
] proposed that as the size decreases, the
specific surface area of AgNPs increases, providing more contact sites for interaction with
bacteria. Compared to larger AgNPs, smaller size AgNPs are more likely to cross the cell
membrane and enter into the cell, increasing the toxicity to bacteria [
]. Studies have also
shown that smaller AgNPs has a larger specific surface area and more active sites, which can
produce more ROS and exert stronger antibacterial effects [
Azotobactervinelandii (A. vinelandii) is a gram-negative bacterium belonging to the
genus Azotobacter, which is easy to grow and fixes nitrogen under aerobic conditions [
Soil Azotobacter are functional bacteria composed of a variety of bacteria capable of fixing
N2 in the environment, these including many kinds of nitrogen-fixing forms such as
autotrophic nitrogen fixation, symbiotic nitrogen fixation and united nitrogen fixation. Because
this type of functional bacterium contains nitrogenase, it can assimilate N2 into its own
biomass and eventually release it into the soil as part of the soil nitrogen pool [
vinelandii can fix nitrogen in the atmosphere, which is also important for studying soil nitrogen
The aim of this study was to investigate (i) the role of AgNPs size on the growth inhibition
of A. vinelandii (ii) the mechanism of AgNPs toxicity on A. vinelandii, which was determined
by examining cell morphology and apoptosis, nitrogenase activity, gene expression, ROS and
▪OH production (iii) the possibility that the AgNPs are internalized in A. vinelandii cells.
These findings would provide an integrative understanding of nanoparticle-bacterial cell
interaction and be of great significance for evaluation of the ecological risk of AgNPs.
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Materials and methods
Materials and chemicals
AgNPs of two different sizes were used in this study: 10 nm nanosilver (nano-Ag 10, coated
with polyvinyl pyrrolidone (PVP)) was purchased from Nanjing Xianfeng Nanomaterial
Technology Co. (Nanjing, China); 50 nm nanosilver (nano-Ag 50, without coating agent) was
purchased from Nanjing Empero Nanomaterials Co. (Nanjing, China). The purity of the AgNPs
were about 99%. The suspensions of AgNPs (1000 mg/L for both sizes) was stored in the dark
at 4˚C until use.
Annexin V-FITC and PI dye (Annexin V-FITC/PI Apoptosis Dual Stain Kit) was
purchased from BD Co. USA. DCFH-DA dye (Reactive Oxygen Species Assay Kit) was purchased
from Biyotime Co. China. RNA extraction (UNIQ-10 Column Trizol Total RNA Extraction
Kit), reverse transcription (Promega reverse transcription kit) and qPCR reagents (TransStrat
Top Green qPCR SuperMix Kit) were purchased from Sangon Biotech Co. Shanghai, Promega
Co. Shanghai and TransGen Biotech. Co. Beijing, respectively.
Characterization of two sizes of AgNPs
Transmission electron microscopy (TEM, H-7650, Hitachi, Japan) were used to determine the
primary sizes of the AgNPs at an accelerating voltage of 80kV. Dispersed aqueous solutions of
AgNPs were dropped onto a carbon-coated copper grid and dried at room temperature to
make TEM samples [
], and at least 5 photographs were taken forobserving. According to the
TEM images, the Nano Measure software was used to analyze the particle size of AgNPs to
obtain the size distribution. For further characterization of stability of AgNPs, the zeta
potential and hydrodynamic size were determined; the AgNPs stock solutions were resuspended in
the test media to achieve the desired concentrations (2 mg/L and 10mg/L for nano-Ag 10 and
10mg/L and 100 mg/L for nano-Ag 50, respectively), Dynamic Light Scattering (DLS; Malvern
Instrument Zetasizer Nano-ZS90, USA) was used, and samples were incubated at 30˚C for 12
h under shaking (220 rpm). For the purpose of silver ion release, suspensions of AgNPs were
centrifuged using Amicon Ultra Centrifugal Filters (3 kDa; USA), and the filtrates were
measured by inductively coupled plasma mass spectrometry (ICP-MS, 7500CX, Agilent. USA).
A. vinelandii culture
The A. vinelandii was a kind gift from Nanjing Institute of Soil Science, Chinese Academy of
Sciences. The A. vinelandii was grown in 1 L of nitrogen-free medium containing 0.5 g yeast,
20.0 g mannitol, 0.8 g K2HPO4, 0.2 g KH2PO4, 0.2 g MgSO4 7H2O, 0.1 g CaSO4 H2O, 1 ml
trace elements, containing 22 g/L ZnSO4 7H2O, 5 g/L MnCl2 4H2O, 5 g/L, FeSO4 7H2O, 1.6
g/L CoCl2 6H2O, 1.6 g/L CuSO4 5H2O, 7.5 g/L, Na2MoO4 2H2O and 60 g/L EDTA-Na2. The
culture temperature was maintained at approximately 30˚C and the pH remained around 7.2
throughout the experiment. After the bacteria were cultured to logarithmic phase (108 cells/
mL), they were collected for the subsequent studies.
Bacteria growth assays
The A. vinelandii suspensions were inoculated into solid medium containing two different
sizes of AgNPs (nano-Ag 10 and nano-Ag 50) at doses of 1, 2, 5, 10 mg/L, and 1, 10, 50, and
100 mg/L, respectively (Concentration selection came from pre-experiment optimization,
which is not shown in this study). At the same time, no AgNPs were added into the control
group medium. The bacterial suspension was spread evenly with a sterile inoculating loop.
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After 30 minutes, the culture dishes were inverted in a 30˚C incubator and cultured for 48
hours. The cell numbers were recorded periodically.
SEM and TEM imaging
The A. vinelandii were inoculated into culture media containing different doses of AgNPs (2
and 10 mg/L for nano-Ag 10, 10 and 100 mg/L for nano-Ag 50) and incubated for 12 h; the
control consisted of A. vinelandii grown to log phase in culture media without AgNPs. The
cells were collected by centrifugation, and all samples were fixed using 2.5% glutaraldehyde,
followed by post-fixed in 1% OsO4 for 1 h and 3 washes with phosphate buffer saline (PBS, pH
7.2). Finally, samples were subject to gradient dehydration in 30%, 50%, 70%, 80%, 90%, 100%
ethanol solution (20 min each time) at room temperature. The processed samples were
sectioned, stained with specific dyes and observed under scanning electron microscope and
transmission electron microscope (SEM, S-4800, Hitachi, Japan; TEM, H-7650, Hitachi, Japan).
Flow cytometry (FCM) analysis
Suspensions of A. vinelandii were inoculated into media containing nano-Ag 10 (2 and 10 mg/
L), nano-Ag 50 (10 and 100 mg/L) and medium without AgNPs; cultures were incubated for
12 h. After cultivation, cells were double-labeled with dye and analyzed for apoptosis according
to the manufacturer’s protocol. Briefly, the cultured cells were collected by centrifugation and
washed with 4˚C PBS and centrifuged again. The supernatant was removed, and cells were
resuspended in 300 μL diluted Binding Buffer; 5 μL Annexin V-fluorescein isothiocyanate
(FITC) was added and samples were incubated in the dark for 15 min. Propidium iodide (PI)
dye was added 5 min before the test and 200 μL of Binding Buffer was added. Finally, the
apoptosis rate was measured by FACSCalibur flow cytometry (BD Co. USA).
Nitrogenase activity and gene express studies
Suspensions of A. vinelandii were inoculated into media containing nano-Ag10 (2 and 10 mg/
L), nano-Ag50 (10 and 100 mg/L) and into medium without AgNPs; cultures were incubated
for 12 h at 30˚C. The nitrogenase activity of A. vinelandii was determined using the acetylene
reduction method [
]. Different A. vinelandii treatments were introduced into a 15 ml test
tube at the same inoculum volume, a 1/10th volume of air was drawn, and an equal volume of
10% acetylene gas was immediately injected, Tubes were sealed with a rubber plug and
cultured for another hour. From the test tube, 0.5 mL of gas was injected into a gas
chromatograph (7890B, Agilent. USA) to determine the amount of acetylene reduction. The nitrogenase
activity of A. vinelandii was calculated based on the amount of C2H4 produced / 108 cells.
In this study, the reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
technique was used to analyze whether AgNPs were genotoxic to A.vinelandii. After the 12 h
as per previous culture technique, the cells were collected by centrifugation. Approximately
108 cells were placed in a 1.5 mL microcentrifuge tube without RNase. Lysozyme solution
(100 μL) was added, and samples were digest at room temperature for 10 min, followed by
centrifugation at 12,000×g for 2 min at 4˚C. The supernatant was carefully removed and placed
into a fresh RNase-free 1.5 mL microcentrifuge tube. Bacterial total RNA was extracted
according to the UNIQ-10 Column Trizol Total RNA Extraction Kit procedure. Afterwards,
total RNA samples were combined with the random primer in RNase free dH2O, heated shock
at 70˚C for 3min, and immediately placed on ice for 5min. The first strand cDNA was obtained
by adding dNTP Mixture, RNase Inhibitor, M-MLV buffer, followed by incubated in a 42˚C
water bath for 1 h. Primers designed for this study were nifH-F (5'-ATCACCGCCATCAACTT
CCT-3') and nifH-R (5'-GATTTCCTGGGCCTTGTTCTC-3'). Quantitative PCR was
4 / 18
performed using a LightCycler 96 PCR system (Roche) in 20 μL of reaction mixture composed
of cDNA (2 μL), SYBR Green Master Mix (10 μL), 0.5 μL of each primer, and RNase Free
dH2O under following condition: 10 min at 95˚C, followed by 40 cycles of 30 s at 95˚C, 35 s at
61˚C. The results of the experiment were calculated using the 2-ΔΔCt method. Nitrogenase
activity and gene expression is shown as fold change.
Reactive oxygen species (ROS) detection and ESR analysis
The presence of AgNPs stimulates production of reactive oxygen species (ROS), which leads to
oxidative stress in bacteria. DCFH2-DA (2,7-dichlorofluorescein diacetate) can detect ROS, and
is able to freely pass through cell membranes to react with intracellular ROS and produce the
fluorescent compound dichlorofluorescein (DCF). The level of ROS is determined by detection
of DCF fluorescence [
]. In this research, the A. vinelandii suspensions were separately
inoculated into the above AgNPs treatment medium and cells were collected at 0, 2, 4, 6, 12 h,
respectively and washed 3 times with PBS buffer. DCFH2-DA and nanosilver-free fresh medium were
added at a volume ratio of 1:2000 and incubated at 30˚C for 30 min. After the incubation, the
samples were collected by centrifugation, washed again with PBS and resuspended, and the
ROS production in A. vinelandii cells was measured by Flow Cytometry (BD Co., USA).
All electron spin resonance (ESR) were carried out using a JOEL ESR spectrometer (JES-FA
200, Japan) at room temperature. The acetate buffer solution system pH at 3.6 contained 0.5
mM H2O2, 50 mM DMPO, different doses of AgNPs and the control contained no AgNPs. A
spin trap, DMPO was used to determine that AgNPs can facilitate the generation of hydroxyl
radicals (•OH) in the presence of H2O2. The machine settings are as follows: Field modulation
of 1G, microwave power of 20mM and scan range of 100G. The final concentration of each
treatment was specified in the figure section.
Intracellular and membrane Ag contents
The cells were incubated in test media containing nano-Ag 10 (2 and 10 mg/L), nano-Ag 50
(10 and 100 mg/L) for 12 h. Equivalent amounts of bacteria from each sample were lysed via
intermittent sonication by an Ultrasonic Cell Crusher (XO-900D, Nanjing) for 1 min, followed
by centrifugation at 30,000×g for 30 min. After centrifugation, the supernatant was collected
and digested with 2 ml of the mixture (65% HNO3: 30% H2O2, 1:1) as a cell membrane
fraction of A. vinelandii, and the precipitate containing the cytoplasmic portion was treated in the
same manner. ICP-MS (7500CX, Agilent. USA) was then used to measure the Ag content [
SPSS19.0 software was used for the differential analysis of the experimental data with the
Student’s t-test. There was a significant difference when the P < 0.05.
Results and discussion
Characterization of two different sizes of AgNPs
The characterization results of the sizes and properties of supplied nanoparticles are presented
in Fig 1. As shown in the TEM image (Fig 1a and 1c), the shapes of AgNPs in this study was
relatively uniform, and both were approximately spherical. Under the same magnification, the
size of nano-Ag 10 was obviously smaller than nano-Ag 50, and had a better dispersibility. In
addition, nano-Ag 50 agglomerated more than nano-Ag10 due to the absence of stabilizer in
the solution [
]. The size distribution of two different AgNPs from TEM images was
determined and average sizes were about 10.48±0.94 and 48.08±3.44 nm for nano-Ag 10 and
nano5 / 18
Fig 1. The characterization of different sizes of silver nanoparticles by TEM. TEM images of (a) nano-Ag 10 and (c)
nano-Ag 50. Frequency of size distribution for (b) nano-Ag 10 and (d) nano-Ag 50; scale bar: 100nm.
Ag 50, respectively, these values were similar to those specified by the manufacturer. Since the
characterization between two used concentrations of the two sizes of AgNPs was not
significantly different, the highest concentrations data were representative. The Z-average
hydrodynamic diameter was 162.9±3.5 nm with polydispersity index (PDI) 0.4±0.02 for nano-Ag 10
and 750.7±65.8 nm with PDI 0.91±0.11 for nano-Ag 50 as monitored by DLS. The surface
charges of the both AgNPs were highly negative as indicated by zeta potential values:
-22.5±0.11 mV for nano-Ag 10 and -18.1±0.15 mV for nano-Ag 50, respectively (Table 1).
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As for dissolution, the nano-Ag 10 at the highest working concentration of 10 mg/L was found
to contain 0.12% Ag ions, and the nano-Ag 50 at a dose of 100 mg/L contained less than 0.02%
release (shown in Table 1), indicating that smaller AgNPs release more Ag in biological
medium. The amount of Ag+ from both AgNPs were less than 0.5%, which may be due to the
agglomeration reduce the surface-to-volume ratio of AgNPs [
]. Because of this, extremely
low Ag+ release in our research would not be considered in toxicity mechanism of AgNPs. In
sum, these results indicated that nano-Ag 10 in tested medium showed a moderate stability
and agglomeration while the nano-Ag 50 was less stable.
AgNPs had a large specific surface area and high surface potential, thus, they tended to
agglomeration, which was affected by various environmental factors such as the coating agent,
the type of ions in the solution, the ionic strength, pH [
]. Studies have shown that PVP as a
coating material is non-toxic [
], which enhance the dispersability of AgNPs. Several reports
have demonstrated that the reduction in toxicity may be attributed to a decrease in release of
silver ions and a reduction in surface area when AgNPs form agglomerates [
]. Thus, the
characterization of AgNPs may provide a possible reason for differences in antibacterial
actions of the two sizes.
Effect of AgNPs on the growth of A. vinelandii
Cultures of A. vinelandii were treated for 48 hours with 2, 5, 10 mg/L of nano-Ag 10 and 10,
50, 100 mg/L of nano-Ag 50, respectively (Fig 2). As shown in the Fig 2b, nano-Ag 50 below 50
mg/L did not produce significant toxicity to cells relative to the control, whereas a treatment of
nano-Ag 10 at 5 mg/L resulted in marked inhibition; this suggested that the bactericidal effect
of AgNPs may be related to dose and particle size.
Numerous studies have indicated that NPs exhibit strong biotoxicity. Yu et al [
the toxicity of four widely used NPs (TiO2, SiO2, Ag and CdTe/CdS) to Chlamydomonas
reinhardtii and found different cytotoxicity effects. Reyes et al [
] revealed that growth rate of
A. vinelandii biofilms was significantly reduced as exposed to CuNPs. Similarly, Gambino et al
] explored the toxicity of AgNPs (10 nm) on soil bacteria A. vinelandii and Bacillus subtilis
(B. subtilis). The authors found that the 0.1 mg/L of AgNPs significantly inhibited growth of
Fig 2. Effect of two different sizes of AgNPs on the growth of A. vinelandii. (a) nano-Ag 10 and (b) nano-Ag 50. The bacteria were cultured
for 48 h in media with and without AgNPs.
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A.vinelandii; however, the growth rate of B. subtilis showed a significant decline only when the
concentration of AgNPs reached 100 mg/L. It seems that increased AgNPs concentration serve
as an effective form of antibacterial action [
]. Our results also provide conclusive evidence
for the toxicity of AgNPs is dose-dependent. In addition to concentration, size is also an
important parameter for evaluating the biotoxicity of nanomaterials [
investigations demonstrated that smaller sizes of AgNPs had higher toxicities [
]. Because smaller
AgNPs have higher surface areas and particle numbers per unit mass, the contact area with
organisms is expanded and more silver ions are released. Moreover, smaller AgNPs have been
shown to penetrate the cell membrane more easily [
]. The present study indicates that
the tested AgNPs exert antibacterial activities in both size and concentration dependent
Effect of AgNPs on cell morphology of A. vinelandii
The changes in cell morphology were observed observed with 10 mg/L of nano-Ag10 and 100
mg/L of nano-Ag 50 (Fig 3), which were also the concentrations where an effect on cell growth
was noticed. Cells exposed to AgNPs differed significantly from the control. The SEM results
showed that there were small holes on the surface of the AgNPs-treated cells, indicating that
the bacterial cell surface was damaged after 12 h interaction with AgNPs. Additional TEM
observations confirmed this finding, as the figures indicated that the cell membrane edges of
A. vinelandii were rough and fuzzy and there were substances leak inside the cells. These
might due to the attachment of AgNPs to the cell membrane surface, disrupting the cell
membrane and cell wall. Part of AgNPs may even reach the cytoplasm and intereact with the
intracellular components, caused leakage of the contents.
The main components of the cell membrane are proteins and lipids, and AgNPs can
interact with proteins to form complexes with substances containing oxygen, phosphorus, sulfur or
nitrogen atoms [
]. Studies have shown that the reaction of AgNPs with sulfur-containing
Fig 3. The effect of AgNPs on the cell morphology of A. vinelandii observed by SEM (upper) and TEM (lower). (a) SEM and (b)
TEM images of a control group in which no AgNPs are added; (c) SEM and (d) TEM images of nano-Ag 10 at a concentration of 10
mg/L; (e) SEM and (f) TEM images of nano-Ag 50 at a concentration of 100 mg/L. All treatment groups were exposed for 12 h.
8 / 18
membrane proteins may lead to inactivation of membrane-bound enzymes and proteins [
]. AgNPs can also interfere with the respiratory chain and reduce energy production when
attacking the membrane [
]. Several investigations have reported that AgNPs could attack
unsaturated fatty acids on cell membranes and change the membrane fluidity which can
hinder membrane function by destroying membrane permeability and integrity [
researchers have indicated that cells treated with AgNPs are deformed. Michels et al .
studied the inhibition of two different nanomaterials (silver and magnetite) on ammonia-oxidizing
bacteria and found that the nanomaterials would continue to adsorb on the surface of the
bacterial cell membrane until the surface morphology was completely destroyed. This finding was
supported by Giao et al [
]. AgNPs caused severe aberration to the cell morphology of
ammonia-oxidizing bacteria and could cause bacterial death by damaging the cell membrane. Li
et al’s study found that the AgNPs treated E. coli cells were deformed, with pits and voids on
the surface, and some cells also showed a large leakage [
In our previous studies, many small pits were found in the cell wall of bacteria which may
have been caused by penetration of AgNPs . After passing through the cell wall, AgNPs
can accumulate on the surface of the cell membrane, disrupt the cell membrane structureand
alter its permeability. AgNPs can also enter into the cell, causing the DNA to become
concentrated and stressed, and DNA accumulates in combination with the cytoplasm of the damaged
bacteria; this eventually results in leakage of cellular components, leading to bacterial death
]. In summary, the direct destruction of cell wall and cell membrane by AgNPs may be
an important antibacterial mechanism, which needs further study.
Effect of AgNPs on cell apoptosis of A. vinelandii
Annexin V is a Ca+-dependent protein that binds phosphatidylserine (PS) with high affinity
outside the cell membrane to label cells during early-stage apoptosis. PI is a nucleic acid dye
that can pass through the membrane of cells in late-stage apoptosis and necrosis to stain the
]. After exposured to AgNPs for 12 h, the apoptosis ratios were observed. The
apoptosis rate was slightly elevated for A.vinelandii treated with nano-Ag10 at 2 mg/L (Fig 4a),
relative to the control group (3.35% and 2.58%, respectively). The apoptosis rate was 20.23%
when when A. vinelandii were exposed to 10 mg/L of nano-Ag 10 (Fig 4b). However, the
apoptosis rate of cell treated with the same concentration of nano-Ag 50 treatment was 3.14% (Fig
4c). Only the concentration was increased to 100 m/L did the apoptosis rate reach 21.06% (Fig
4d). This result is consistent with those of the bacterial growth assays, which indicated that the
toxicity of AgNPs to A. vinelandii significantly increased with decreasing nominal particle size.
Apoptosis is a genetically regulated and orderly death caused by certain physiological or
pathological stimulation of cells [
]. A wide variety of compounds, from antibacterial
organics to nanoparticles, have been shown to cause bacterial apoptosis [
], and our previous
research also explored the apoptosis of Nitrosomonas europaea under AgNPs exposure .
Apoptosis may be present in most bacteria but activated differentially depending on the
]. ROS are considered to be a major mechanism of apoptosis in nanoparticles
treatments, and DNA damage is a common apoptotic mark [
]. Piao et al [
] reported that
the destruction of mitochondrial transmembrane potential is one of the earliest events in the
process of apoptosis. Zapo´r et al [
] confirmed the finding that AgNPs can cause an increase
in ROS production to induce mitochondria-mediated apoptosis. Furthermore, AgNPs will
cause autophagy function disorder by generating ROS; excessive autophagy can also lead to
]. The apoptotic features in bacteria and eukaryotic cells have been found in high
], but further research is needed to characterize how ROS induce apoptosis in
9 / 18
Fig 4. Effect of AgNPs on cell apoptosis of A. vinelandii. (a) nano-Ag 10 at a concentration of 2 mg/L; (b) nano-Ag 10 at a
concentration of 10 mg/L; (c) nano- Ag50 at a concentration of 10 mg/L; (d) nano-Ag 50 at a concentration of 100 mg/L; (e) no AgNPs
added. All treatments were exposed for 12 h.
Effect of AgNPs on nitrogenase activity and nifH expression of A.
The assay result showed that the nitrogenase activity and expression of the nitrogenase gene
nifH were significantly inhibited (Fig 5) after exposure of A.vinelandii to two sizes and
different doses of AgNPs for 12 h; the effect of nano-Ag 10 treatment was more obvious. These
results were well corresponded with the observations of a reduced effect in apoptosis rates
from nano-Ag 50 exposure, nano-Ag 10 has a stronger destructive effect on cells. Our research
suggested that AgNPs not only induced cytotoxicity, but also caused genotoxicity and
impaired nitrogen fixation in A.vinelandii cells.
All kinds of nitrogen-fixing organisms are able to fix the molecules of ammonia in the air at
room temperature and atmospheric pressure and transform them into combined nitrogen
which can be used by organisms; these include symbiotic rhizobia, autogenous, combined
nitrogen-fixing bacteria, cyanobacteria, and photosynthetic nitrogen-fixing bacteria. Nitrogen
fixation is accomplished by a catalytic protein-nitrogenase enzyme [
]. It has been found that
all nitrogen-fixing microorganisms contain the ferritin-encoding nifH gene, and the
phylogenetic tree of the nifH gene is highly consistent with that of 16s RNA [
]. The nifH gene is one
of the key areas for scientists to study biological nitrogen fixation. Molecular biology analysis
based on the nifH gene provided a more sensitive and accurate analytical method for the
nitrogen fixation than traditional culture.
10 / 18
Fig 5. Nitrogenase activity (a) and relative expression of nifH (b) of A. vinelandii exposed to AgNPs for 12 h.
An important feature of nitrogenase is their high sensitivity to oxygen. The result of gene
assay showed that AgNPs inhibited the expression of nifH gene, thereby decreasing the activity
of nitrogenase. Moreover, exposure of bacteria to AgNPs increased ROS level in bacteria, also
affected the activity of nitrogenase. Abd-Alla et al found there was a significant reduction in
nitrogenase activity of faba bean Rhizobium leguminosarum exposed to AgNPs, and they
suggested that presence of AgNPs decreasd the viability of the rhizobial cell [
examined the physiological response of Azolla filiculoides to the presence two sizes of ZnO
NPs (26.7±1 nm and 238±30.7 nm), and found the nitrogenase activity of Azolla filiculoides
decreased under treatment with all the ZnO NPs concentrations regardless of particle size
Reactive oxygen species (ROS) generation and free radicals induced by
The reactive oxygen species (ROS) are a type of oxygen-containing highly reactive molecules,
including hydroxyl radicals ( OH)s,uperoxide anions ( O2-), singlet oxygen (1O2), and
hydrogen peroxide (H2O2) [
]. The generation of ROS is one of the mechanisms by which
nanomaterials exert their toxicity. Electron spin resonance spectroscopy is the standard method for
quantification of ROS [
]. As shown in Fig 6. Both nano-Ag 10 and nano-Ag 50 (Fig 6a)
significantly increased ROS production in A. vinelandii cells in dose-dependent. It is observed
that the amount of ROS produced is higher for nano-Ag 10 than nano-Ag 50. Obviously, the
ROS production at 4 h appeared to be greater compared to 6 h which is higher than control.
And the reduction of ROS probably due to the ROS generation triggered the antioxidant
system in the bacteria cells which decreased ROS levels subsequently [
]. Results for ESR spectra
obtained for solutions containing DMPO spin trap without or with AgNPs are shown in Fig
6b. The ESR spectra of the two sizes of AgNPs are consistent with changes in ROS levels. The
observation rationalized that nano-Ag 10 has higher antimicrobial activity than nano-Ag 50.
Several previous studies have demonstrated that mechanism by which AgNPs induce
hydroxyl radicals in the presence of hydrogen peroxide. The current model involves AgNPs
acting as a reagent and generates hydroxyl radicals through a Fenton-like reaction (Ag +
H2O2 + H+ = Ag+ + •OH + H2O) [
]. Thus the level of AgNPs would affect the production
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Fig 6. Changes in intracellular ROS levels of A.vinelandii (a) and generation of hydroxyl radicals by two sizes of AgNPs (b). Electron spin
resonance (ESR) spectra were obtained from samples containing 50 mM DMPO + 0.5 mM H2O2 + 10 mM pH 3.6 HAc-NaAc in the presence of
control (1), 2 mg/L (2), 10 mg/L (3) of nano-Ag 10, and 10 mg/L (4), 100 mg/L (5) of nano-Ag 50, respectively.
of hydroxyl radicals and ROS. This is also consistent with the dose-dependent inhibition on
A. vinelandii growth after AgNPs exposure. Recent studies suggest that ROS generation and
oxidative stress are the likely mechanism of AgNPs toxicity . There are antioxidant systems
in the cells, which mainly include antioxidant enzymes and antioxidants such as SOD and
GSH . Studies have shown that the presence of ROS will activate SOD or GSH to maintain
a dynamic balance between oxidation and antioxidant levels [
]. Oxidative stress occurs
when ROS generation exceeds the capacity of the antioxidant defense mechanism. ROS and
oxidative stress harm cell membrane, leading to lipid peroxidation [
]. In addition, DNA
damage, apoptotic cell death, reduction in ATP generation are also induced by ROS [
and eventually lead to cell death. Thus, it is important to study antibacterial effects of AgNPs
and the underlying mechanisms of ROS-mediated damage in AgNPs treated cells should be
AgNPs distribution in bacteria
The intracellular and cell-wall-bound Ag contents of A. vinelandii were determined after the
cells were treated with two different sizes of AgNPs for 12h (Fig 7). Compared with the
treatment group, the control group cells had almost no AgNPs present, which further
demonstrated the internalization of AgNPs in A. vinelandii. In addition, the intracellular Ag content
in the nano-Ag 10 (Fig 7a) treatment group was significantly higher than that on the cell
membrane, whereas the nano-Ag 50 (Fig 7b) treatment group had the opposite result, with most of
the Ag on the cell membrane. This phenomenon was attributed to that smaller AgNPs entered
into cell more easily [
Similarly, Long et al [
] observed that the amounts of cytoplasmic Ag were slightly higher
than membrane Ag, which proved that regardless of the form (particles or ions) of AgNPs, it
can enter into the cells. Thus, they concluded that the AgNPs exert antibacterial activities
though the combination of internal and external methods; Ag could bind to the cell membrane
and destroy the respiratory chain protein, or it could enter into the cells to cause bacterial
damage. Uptake of AgNPs has been investigated in different cells and found to occur through
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Fig 7. Intracellular and membrane Ag contents of control cells and cells treated with two size of AgNPs for 12h. (a) nano-Ag 10 and (b)
the following steps: initially, they bind on the cell membrane, with the internalization
occurring secondly [
]. Many analyses using flow cytometry and electron microscopy showed that
AgNPs could enter into cells by penetration or phagocytosis [
]. Studies suggested that
the internalization of Ag might exert toxicity on cell membranes and cytoplasm through a
Trojan horse type mechanism, which explains the release of Ag ions from AgNPs [
17, 48, 67
Combining Ag distribution results with previous electron microscopy images showed that
smaller Ag nanoparticles with higher surface areas could damage cell membranes by direct
interaction with cell membranes to allow Ag atoms easy entry into the cells. As earlier
researches reported, the internalization of NPs was size- and concentration-dependent [
]. Additionally, the internalized AgNPs can induce cell death by causing macromolecular
damage such as DNA fragmentation and protein denaturation [
]. Further, AgNPs can
enter into the cells through diffusion, endocytosis or the action of carrier proteins, and react
with intracellular components, leading to the disintegration of cells and cell contents [
Several studies have reported that after exposure of bacteria to NPs, cell wall damage and NPs
were also found in passaged cells; these findings suggested that nanoparticles could be
internalized and delivered to newly formed cells as a component of the cytoplasm [
Passible mechanism for toxicity
The antibacterial activities of AgNPs were observed by a cell growth inhibition assay. Damages
to cell morphology upon exposure to AgNPs are shown in Fig 3. Apoptosis of cells were
determined by FCM. The impact of AgNPs on bacterial function was detected by enzyme activity
and RT-qPCR assays. The intracellular ROS production was measured using the fluorescent
dye H2DCFDA. Based on the results, we speculate that the antibacterial mechanisms of AgNPs
(Fig 8) involve adherence to the surface of bacteria and destruction of the cell membrane
structure. Smaller AgNPs more easily enter into the cell and react with sulfur-containing
substances, such as DNA and proteins. These intracellular contents finally flow out along the pits
in the damaged cell membrane; AgNPs can bind to sulfhydryl group (-SH) of enzymatic
proteins in bacteria, inactivate the enzyme and inhibiting gene expression related to the enzyme;
this ultimately interferes with the particular function of the bacteria. The presence of AgNPs
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Fig 8. Possible toxicity mechanism for AgNPs in A.vinelandii.
also leads to the generation of ROS, which further damages cell structure and leads to bacterial
apoptotic death [
8, 13, 65
]. We believe that the toxicity mechanism of AgNPs may be much
more complicated than hypothesized. It may be that one or more mechanisms work together
and even lead to more serious cell damage. Thus, detailed bactericidal activities still require
more researches to verify.
The possible mechanisms of AgNPs effects were explored and AgNPs were found to have
significant inhibition on A. vinelandii. In addition, the toxicity of AgNPs was directly dose- and
size- dependent. AgNPs inhibited the growth of bacteria and accelerated the cell apoptosis
rate; it also affected the nitrogenase activity and expression of nifH. ROS detection and ESR
analysis revealed that the presence of AgNPs could generate ROS and hydroxyl radicals.
Electron microscopy combined with the distribution assay for AgNPs showed that AgNPs tend to
attach to cell membrane, and damage the cells. At the same time, AgNPs could interact with
A.vinelandii and could be internalizated in cells.
This work was supported by the National Natural Science Foundation of China [grant
numbers 41430752, 41471405].
Investigation: Li Zhang, Lingli Wu, Kunhui Shu.
Methodology: Li Zhang.
Resources: Youbin Si.
Supervision: Youbin Si.
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Writing – original draft: Li Zhang.
Writing – review & editing: Li Zhang, Youbin Si.
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16 / 18
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