Materials Science & Engineering C 104 (2019) 109968

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Materials Science & Engineering C

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

Antibacterial activity of ZnO and CuO nanoparticles against gram positive T

and gram negative strains
⁎
Rania Dadia,b, Rabah Azouanib, , Mamadou Traorea, Christine Mielcarekb, Andrei Kanaeva
a
LSPM -CNRS, Laboratoire des Sciences des Procédés et des Matériaux, Université Paris 13, Sorbonne Paris Cité, 99 Avenue Jean-Baptiste Clément, 93430 Villetaneuse,
France
b
EBI- Ecole de Biologie Industrielle, 49 Avenue des Genottes – CS 90009, F95895 Cergy Cedex, France

A R T I C LE I N FO A B S T R A C T

Keywords: This is a report on the antibacterial activity of ZnO and CuO nanoparticles synthesized via sol-gel method. The
Nanoparticles studies were performed on Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and
CuO Pseudomonas aeruginosa bacteria using disc and well diffusion methods, bioluminescence and optical density
ZnO analysis. The results show a strong decline of bacterial strains after a short contact with nanoparticles. The
Antibacterial
modelling allowed clarifying the bacterial sensitivity of toxic agents at different stages of their population
Diffusion
Modelling
evolution kinetics. It was concluded that the bacterial suppression is most effective at the exponential growth
phase while it is of a lower effectiveness at the lag and stationary phases. The CuO and ZnO nanoparticles showed
comparable effectiveness at the exponential growth phase. In the same time, ZnO was almost inactive at the lag
phase and of lower effectiveness at the stationary phase, at which CuO conserved a significant activity.

1. Introduction antibacterial potential [15], it has been reported the use of TiO2 [16],
ZnO [17], CuO [18], Ag [19], Au [20] and Al2O3 [21]. Our choice was
The emergence of infectious diseases in general causes a serious oriented considering antibacterial efficacy / low concentration (ZnO,
threat to public health worldwide, especially with the emergence of CuO), non-specific strain and economic cost.
antibiotic-resistant strains of bacteria. Gram-positive and Gram-nega- The antimicrobial activity of metallic Zn and Cu nanoparticles has
tive bacterial strains are considered presenting a major public health been studied with pathogenic bacteria such as Escherichia coli and
problem. Over the years, antibiotics have been used to control infec- Staphylococcus aureus [22,23]. The metal oxide ZnO and CuO nano-
tions resulting from community and hospital settings [1–3]. Recent particles have demonstrated an enhanced antimicrobial activity
advancements in the field of nanobiotechnologies, in particular the [24–26]. Escherichia coli and Staphylococcus aureus appeared to be very
ability to prepare metal oxide nanomaterials of specific morphologies, sensitive to the size and concentration of ZnO and CuO nanoparticles in
are likely to lead to the development of new antibacterial agents. the growth medium [27,28].
Especially, nanoparticles, whose functional response largely influenced The inhibition of the bacterial population growth can be explained
by their size and related surface to volume ratio [4–10], have received by a specific interaction with nanoparticles. In general, bacterial cell
much attention. The use of inorganic materials is advantageous because size is in the micrometer range, while its outer cell membranes have
of their chemical stability, their safety and biocompatibility [11,12]. pores in the nanometer range. Since nanoparticles may be smaller in
Several studies reported the use of functional nanomaterials such as size than bacterial pores, they will have a unique ability to penetrate
bone implants and in wound healing acceleration. In particular, hybrid the cell membrane, which is the case with CuO NPs [29–34]. For ZnO
hydrogel with modified CuS nanoparticles released copper ions pro- NPs the three most reported assumed mechanisms are: the generation of
viding a high antibacterial effect, stimulating fibroblast proliferation reactive oxygen species (ROS) including hydrogen peroxide (H2O2),
and angiogenesis [13]. Moreover, a synergistical effect was observed hydroxyl radicals OH– and peroxide O2−2 [35,36]. These species are
between silver and zinc ions, they enhance antibacterial activities and internalized into the bacteria cell membrane and induce the destruction
stimulate the immune function [14]. of cellular components such as DNA and proteins [37–39]. Another
Literature review dealing with antibacterial activity of nanoparticles proposed possibility is the release of ions in medium containing NPs
shows a growing interest for metal oxides, which has a high and bacteria [40]. In literature various studies consider that ZnO

⁎
Corresponding author.
E-mail address: [email protected] (R. Azouani).

https://doi.org/10.1016/j.msec.2019.109968
Received 17 March 2019; Received in revised form 17 June 2019; Accepted 9 July 2019
Available online 09 July 2019
0928-4931/ © 2019 Elsevier B.V. All rights reserved.
R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

nanotoxicity is a result of the dissolution of ZnO-NPs into Zn2+ 2.3. Antibacterial activity
[27,41,42]. However, insolubility of ZnO in water is a limiting factor of
the distribution of zinc ions into the medium [43]. Jiang et al. [44] and The tests were carried out using pure strains: Pseudomonas aerugi-
Sawai [36] report that because of the low concentrations of solubilized nosa (ATCC 9027), Staphylococcus aureus (ATCC 6538) and Escherichia
Zn species released from ZnO dissolution, the contribution of Zn2+ to coli (ATCC 8739). Strains were maintained at −80 °C on cryobeads
the nanotoxicity of ZnO-NPs is minor. Finally, the accumulation and (AES Laboratoire, France). The test consisted of the artificial con-
dissolution of NPs in the membrane leads to changes in the bacterial tamination of the preparation by means of inoculum of microorganisms.
membrane permeability as progressive release of lipopolysaccharides The suspension of bacteria was grown in nutrient broth medium at
for Gram negative bacteria and cell lysis [45–47]. 35 °C.
The photocatalytic degradation of the bacterial wall membrane in
contact with TiO2 NPs has been reported [48]. 2.3.1. Disk diffusion method
Compared with published reports on chemical properties [15], very This is the reference method to test bacterial susceptibility to anti-
limited information is available on the antimicrobial properties of metal biotics. A similar test with a soil loaded disk of nanoparticles is used in
oxide nanoparticles. Despite much effort, peculiarities of the inhibition this study. To this end, 3.8 g of Muller Hinton powder was dissolved in
mechanisms and comprehension of the bacteria interaction with these 100 ml of water and then sterilized by autoclave at 121 °C for 15 min.
toxic agents on different phases of the population evolution still re- The pH of the medium is generally maintained around the physiological
quires detailed studies. The challenge was to connect the static nor- pH of 7.4. After cooling, about 20 ml of medium are added aseptically
malized measurements of the bacteria inhibition with population ki- to sterilized Petri dishes. A sterile swab is used to inoculate the surface
netics and draw conclusions about detoxification effectiveness at of the media in Petri dishes with a bacterial suspension of 107 UFC/mL
different phases of growth in a nutrient medium. In the present com- making three rotations with an angle of 60° to ensure a homogeneous
munication, we studied antimicrobial activity of metal oxide nano- growth. Filter discs 6 mm in diameter, are impregnated with nano-
particles, ZnO and CuO, against two Gram-negative bacteria P.aerugi- particle solutions, then placed inside the Petri dishes inoculated under
nosa and E.coli and one Gram positive S.aureus. In order to evaluate aseptic conditions. Four samples of different concentrations of the
antimicrobial activity, complimentary experimental techniques as disc copper oxide and zinc oxide solutions are used 0.5 M, 0.75 M, 1 M and
and well diffusion, bioluminescence and optical density measurements 1.5 M. A disk soaked only with solvent is used as a control. The dishes
were used. We applied a model to correlate active species diffusion were incubated at 35 ± 1 °C for 24 h to provide optimal conditions for
(ions and nanoparticles) with the bacteria inhibition zone, which al- the growth of the bacteria. A gradient of concentration around each disc
lowed concluding the time period of the strongest bacteria sensitivity to is established in the agar. Disk diffusion is based on the determination
the toxic agents. of inhibition zone proportional to the bacterial sensitivity and to the
bactericide present in the disk after 24 h of incubation. The diameter
was measured from the area around the disc where no bacterial growth
2. Materials and methods has been observed, called the inhibition zone. It indicates the value of
the MIC (minimum inhibitory concentration), which characterizes the
2.1. Nanoparticles synthesis bacteriostatic effect of nanoparticle soils. This diameter can be com-
pared to the critical diameters of antibiotics.
The sol-gel method was used to prepare ZnO and CuO Nanoparticles
(NPs) using zinc acetate (Zn(CH3COO)2, 2H2O, Merck Millipore, 99%) 2.3.2. Well diffusion method
and copper acetate (Cu(CH3COO)2, H2O, Alfa aesa, 98%) as precursors This technique is similar to the disk diffusion method but instead of
and monoethanolamine (MEA, Merck Millipore, 95%) as stabilizing depositing filter disk, wells are made aseptically with a cutting shape.
agent. The precursors were dissolved in a mixture of propan-2-ol Then 20 μL of the solutions of NPs of ZnO and CuO with different
(Sigma Aldrich, 99.5%) and MEA inside a Mbraun Labstar glove box to concentrations is dispensed into each well. After diffusion (20 min ap-
maintain atmosphere level < 1 ppm O2 and moisture. The solutions proximately), the cultures are incubated at a temperature of 35 °C for
were kept under constant stirring using a magnetic stirrer at 60 °C for 24 h. The inhibitions zones are measured and the diameter of the well
2 h (ZnO) and 4 h (CuO). Then, the solutions were filtered by paper (6 mm) is included in the results.
filter after 24 h of aging time. We obtained a homogenous transparent
sol for ZnO NPs and a homogenous dark-blue solution for CuO NPs. A 2.3.3. Bioluminescence measurements
range of solutions with the zinc acetate and copper acetate precursors The ATP measurement was performed in 96-well plates. Samples
concentrations, respectively CCu or CZn, equal to 1.5 mol/L, 1 mol/L, were analyzed using the promicol ATP reagent Kit (Promicol). The
0.75 mol/L, 0.5 mol/L, 0.4 mol/L, 0.3 mol/L, 0.2 mol/L and 0.1 mol/L) presence of ATP was analyzed by adding manually 50 μL of nano-
were prepared. particles solution at 1.5 mol/L and 50 μL of bacterial suspension at 109
The obtained nanoparticles are not crystalline and belong to so- UFC/mL to each well. Then the luciferin/luciferase reagent was added
called oxo-alkoxy phase species consisting of metal oxide core and a automatically via a pumping system on the luminometer (FLUOROS-
shell of surface groups including alkoxyls and hydroxyls. TAR OMEGA) after 2 s delay the light emission was measured. Samples
were measured in four replicates and the luminescence was measured
in relative light units (RLUs). All the samples were handled under
2.2. Nanoparticles characterization aseptic conditions; also ATP-free laboratory equipment and ATP-free
pipette tips were applied in the experiments.
The characterization of the nanoparticles solutions was performed
by TEM JEOL 2011, with maximal tension at 200 KeV corresponding to 2.3.4. Bacterial growth monitoring
a wavelength of electrons λ = 0.0251Ǻ°. The microscope is equipped The bacterial growth over time is performed with the measurement
with a camera GATAN Dual Vision (1300 × 1000 pixels). The particle of optical density in a relatively narrow spectral window at 600 nm.
size distributions were measured by Dynamical Light Scattering (DLS) Specific growth rate μmax and generation time tG was determined in the
method using. exponential phase. The cryopreserved microorganisms E.coli,
Zeta sizer Nano S90 from Malvern Instruments Ltd.; these mea- P.aeruginosa and S.aureus were regenerated by incubating a cryobead in
surements were performed at 90° scattering angle at 633 nm laser light a TCS liquid medium tube at 37 °C for 24 h. Subculture of micro-
wavelength. organisms in TCS broth by incubation at 37 °C for 15 h was done. All

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R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

Fig. 1. Size distribution of ZnO nanoparticles.

measurements were done in triplicate from a suspension of 105 CFU/ 3.2. Antibacterial activity
mLevery 30 min and every 15 min when there is a strong increase of the
population. 3.2.1. Inhibition zone
The microbial sensitivity of metal oxide nanoparticles varies ac-
cording to the microbial species and the concentrations of the metal
3. Results and discussion oxides. Tested concentrations of 0.5 M, 0.75 M, 1 M and 1.5 M produce
an inhibition zone. The antimicrobial activity of CuO and ZnO nano-
3.1. Nanoparticles morphology particles were examined with various microorganisms using disk dif-
fusion (Fig. 3) and well diffusion (Fig. 4) methods. The inhibition zone
The DLS measurements of the ZnO particles size distribution are reflects the bacteria sensibility to toxic agents so that the disinfectant-
shown in Fig. 1. The particles have the mean size 2R = 3 nm with a sensitive strains show large inhibition radii and the resistant strains
relatively narrow polydispersity ΔR = 0.6 nm at the full width of half show a smaller or zero inhibition radii.
maximum. The same measurements in CuO colloids failed because of As our results show, both CuO and ZnO colloids behave as toxic
their dark-blue coloration prohibiting laser light propagation. Conse- agents developing inhibition zones. On the other hand, ZnO nano-
quently, reference TEM measurements were carried out to check the particles inhibit bacteria only at sufficiently high concentrations
CuO particles sizes and validate DLS results obtained in the ZnO col- ~0.5 mol/L while toxic behaviour of the CuO nanoparticles begins at
loids. any smaller concentration. The results obviously show that all tested
The ZnO and CuO colloids were deposited on grids by thermo- microorganisms possess the highest sensitivity to the most concentrated
sphere. A statistical analysis of the analyzed samples made it possible to colloids. It was observed that the inhibition zone in well diffusion
obtain the size distribution of ZnO nanoparticles. The TEM images measurements (Fig. 4) are larger compared with the disk diffusion
evidenced small ZnO nanoparticles of a mean diameter 3 nm (Fig. 2a), method (Fig. 3), which can be explained by the fact that in well dif-
which is in agreement with DLS measurements. The TEM analysis of fusion, we double the volume of NPs solutions and increase the diffu-
CuO nanoparticles showed the same mean size of 3 nm with a small sion of NPs through the medium. This confirms that higher con-
polydispersity comparable to the ZnO nanoparticles (Fig. 2b). centration and volume lead to better antibacterial activity [7,43,49].
The size of inhibition zones presented in Figs. 3 and 4 were aver-
aged over three independent series for each strain and the evolution of
the averaged inhibition diameter on colloids concentration for different

Fig. 2. TEM images a) ZnO and b) CuO nanoparticles.

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R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

Fig. 3. Disk diffusion measurements of ZnO/E.coli (a), ZnO/S.aureus (b), ZnO/P.aeruginosa (c), CuO/E.coli (d), CuO/S.aureus (e) and CuO/P.aeruginosa (f). The
numbers indicate concentration of the colloids in mol/l. The Petri dishes diameter is 9 cm.

bacteria strains are shown in Fig. 5. The inhibition of Gram-positive S. aureus shows signs of saturation
The detailed analysis of the obtained results showed that the bac- while the inhibition zone of Gram-negative E. coli and P. aeruginosa
teria inhibition begins in contact with CuO nanoparticles of any con- permanently expands, attaining larger size than in case of ZnO nano-
centration, at least above 0.1 mol/L, while ZnO nanoparticles suppress particles. In general, CuO nanoparticles were more effective against
the bacteria growth at concentrations above ~0.3 mol/L for E.coli, bacteria strains than ZnO nanoparticles.
P.aeruginosa and S.aureus. The inhibition diameter in contact with ZnO
nanoparticles increases with the concentration increase from 0.3 to
3.2.2. Bacteria population
0.5 mol/L and saturates at higher concentrations up to 1.6 mol/L for all
The quantification of the intracellular ATP concentrations was
considered strains. In contrast, in contact with CuO nanoparticles, the
carried out with bioluminescence method at 550 nm. The nanoparticle
behaviour of Gram-negative and Gram-positive strains was different.
concentrations similar to those in disk diffusion method were used. The

Fig. 4. Well diffusion measurements of CuO/E.coli (a), CuO/P.aeruginosa (b), CuO/S.aureus (c), ZnO/E.coli (d), ZnO/S.aureus (e) and ZnO/P.aeruginosa (f). The
Petri dishes diameter is 9 cm.

4
R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

4
ZnO

Inhibition zone (2R), cm

2

4
CuO

2
E.coli
S.aureus
P.aeruginosa
0
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

Concentration, mol/l
Fig. 5. Dependence of inhibition zone diameter on concentration of ZnO and CuO nanoparticles.

Fig. 6. ATP measurement of bacteria in contact with ZnO and CuO nanoparticles (1.5 mol/l).

measurements were realized in 96-well plates after depositing in each confirms the conclusions of disk diffusion method. This difference
well of 50 μL bacterial suspension of 109 CFU/mL and 50 μL of NPs might be related to a weaker aggregation of the CuO nanoparticles
solution. The instantaneous ATP signal in ZnO and CuO colloids was (Fig. 2) that result in a larger contact area. The ZnO and CuO nano-
almost zero for all three bacterial strains. The positive control (bacterial particles manifested significant activity against the Gram positive and
suspensions without nanoparticles) was measured for each strain as Gram negative microorganisms.
reference. Fig. 6 shows a high value of ATP in positive control with all
strains in stationary phase. In contrast, a sharp decrease of the ATP
signal was observed with all samples in contact with ZnO and CuO 3.2.3. Modelling of NPS/ions antibacterial effect
nanoparticles, which evidenced their toxicity. Most known antibacterial nanomaterials interact electrostatically
To evaluate the effect of NPs concentration on bacterial viability, we with the bacterial membrane causing disruption of the membrane
performed quadruplicate measurements with different NPs concentra- [33,38,43]. Often, reactive oxygen species (ROS) are produced due to
tion between 0.5 and 1.5 mol/L, which results are shown in Fig. 7. nanomaterial-membrane interactions, which can cause damage to sec-
Fig. 7 shows that an increase of the nanoparticles concentration ondary membranes, hinder the function of proteins, destroy DNA
leads to a decrease of the bacterial viability. In the same time, at any [37–39]. Other antibacterial nanoparticles like ZnO are photoactivated
concentration CuO nanoparticles show the stronger activity against becoming a strong oxidant that destroys various bacterial strains and
E.coli, S.aureus and P.aeruginosa compared to that of ZnO, which organic compounds through the photocatalytic mechanism. Recent
studies have shown that active oxygen species destroy the outer

5
R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

Fig. 7. ATP measurement of ZnO and CuO colloids (1 = 1.5 mol/l, 2 = 1 mol/l, 3 = 0.75 mol/l, 4 = 0.5 mol/l) against E. coli, S. aureus, P. aeruginosa.

membranes of bacteria and cause the bacterial death [36,48]. With Because of a complex variation of the concentration, which can attain a
respect to ZnO and CuO nanoparticles, their antibacterial activity may maximum and decrease afterwards, we also indicated in Table 1 the
be due to the release of Cu2+ and Zn2+ ions that interact with the time τ, at which the maximum concentration was attained after the
negatively charged cell [50]. The biochemical process would be af- beginning of process (t = 0). We notice that the diffusion of species was
fected and could damage the membranes. In addition, the small size of limited by the process duration of t0 = 86,400 s (24 h). Our compara-
the NPs would help to adhere to the bacterial wall, which would de- tive measurements of the solutions containing CuO and ZnO nano-
stroy the cell. The vital bacterial enzyme would be damaged by nano- particles showed that it is significantly lower than that of the respective
particles that can enter the cell membrane [51,52]. Physiology, meta- reference samples CuSO4 and ZnCl2. Assuming that the nanoparticle
bolism and degree of contact can contribute to differences in the contribution can be neglected, we estimated the concentration of Zn2+
bacterial activity of NPs to different bacteria. In the following, we de- and Cu2+ ions in the solutions to be about 19% of the initial precursor
scribe a model that permits to clarify toxic effect of the nanoparticles at concentration, either CZn or CCu. Although more precise measurements
different phases of the bacterial growth. of the ions concentration are required, this value was taken into ac-
The proposed model is based on the diffusion of biocide species, count in the first time to model the diffusion of ions, resumed in
which can be ions and/or nanoparticles. For each colloid (Zno or CuO), Table 1.
the inhibition zone expansion curves for different strains E.coli, We assumed that the maximal concentration of the diffused species
P.aeruginosa and S.aureus in Fig. 6 are quite similar and, for simplicity, (ions and/or nanoparticles) provokes the inhibition of the bacterial
we replace them by an average curve, which contains main features of growth and, therefore, defines the size of the inhibition zone. For this
the inhibition mechanism. The obtained two general curves of ZnO and reason, we can call it critical concentration Ccrit; the smaller con-
CuO nanoparticles were used for the model analysis. The spatial pro- centrations maintained at the inhibition radius R before and after τ do
pagation of the active species was calculated according to the Fick's law not contribute to the zone extension. The dependence of the critical
[53] with the diffusion coefficients of ions DCu ≈ 1.2·10−5 cm2/s [54] concentrations of Cu2+ and Zn2+ ions on time τ of achievement are
and DZn ≈ 1.1·10−5 cm2/s [5]. The diffusion coefficients of the CuO plotted in Fig. 8. Concerning participation of nanoparticles, their direct
and ZnO nanoparticles of size 2R = 3 nm at room temperature interaction with the bacteria seems unlikely. Indeed, because of the
DNP = 1.63·10−6 cm2/s was estimated from the Stokes-Einstein equa- smaller diffusion coefficient the characteristic path length of the na-
tion, supported by autocorrelation curves (ACF) measurements of the noparticles during the test duration is δ = (DNPt0)1/2 ≈ 0.4 cm, which
similar size nanoparticles using DLS method [6]. The maximal con- cannot account for the larger inhibition radii r > δ observed in the
centration of active species Cmax attained at the inhibition radius δ experiment. Our model calculations showed that the maximum local
during the Brownian random walk propagation is given in Table 1. concentration of 3-nm particles at the inhibition radii above 0.3 cm is

Table 1
Maximal concentration of active species Cmax attained at the inhibition radius δ at time t during the random walk propagation in aqueous solutions at room
temperature.
C0 Cu2+ Zn2+ CuO nanoparticles (2R = 3 nm)
mol/l
r Cmax T r Cmax τ r Cmaxb τ
(cm) (mol/L) (s) (cm) (mol/L) (s) (cm) (mol/L) (s)

0.1 0.37 0.0247 1650 – – – 0.37 0.0247 12,380

0.0102a
0.2 0.56 0.0211 5625 – – – 0.56 0.0211 41,630
0.0148a
0.3 0.81 0.0153 12,750 0.36 0.0821 1430 0.81 0.0153 t0
0.0149a
0.4 1.01 0.0131 20,250 0.93 0.0152 18,820 1.01 0.0111 t0
0.5 1.31 0.0097 34,500 1.33 0.0093 39,680 1.31 0.00435 t0
0.75 1.42 0.0124 41,250 1.43 0.0122 45,820 1.42 0.00393 t0
1 1.77 0.0105 64,500 1.54 0.0140 53,180 1.77 0.000780 t0
1.5 1.99 0.0126 83,250 1.72 0.0169 66,270 1.99 0.000296 t0

a
Concentration attained at t = 4.5 h.
b
Equivalent precursor concentration.

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R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

16 10
2+
Cu
corr for t=4.5 hours
12 2+ 1
Zn
a
Ccrit, mmol/l

8
0.1

< Ccrit > = 2.3 mmol/l 0.01

4

OD
0
0 10 20
b
Time, hours 0.1
2+ 2+
Fig. 8. Maximal concentration of Cu and Zn ions at the inhibition radii
versus time of achievement.
0.01
attained at the end of the test and decreases exponentially with the
following increase of the inhibition radius. Such behaviour cannot be
rationalised in framework of known inhibition mechanisms. 1
By inspecting this temporal behaviour of the bacteria inhibition,
different domains can be clearly distinguished: c
0 ≤ t < 6 (hours): Ccrit is rather high and decreases with the time of 0.1
attaining.
6 ≤ t < 15 (hours): Ccrit is almost constant and minimal.
15 ≤ t (hours): Ccrit slightly increases with the time of attaining.
Although in CuO colloids with low C0∈[0.1,0.3] the maximal con-
0.01
centration at the inhibition radii were relatively high, we used to re- 0 5 10 15
calculate the respective concentrations attained at the end of the lag Time, hours
phase (4.5 h). A comparison showed that these values are quite similar
to the minimal Ccrit observed at the phase of exponential growth (see Fig. 9. Growth kinetics of E.coli (a), S.aureus (b) and P.aeruginosa (c).
points in Fig. 8). The same procedure applied to ZnO colloids was not
successful and the recalculated concentrations attained at the end of the (OD) measurement. Since OD is proportional to the concentration of
lag phase were significantly higher than Ccrit. This may indicate that, in bacteria, their exponential growth phase of the process kinetics could
contrast to ZnO, bacteria do not develop resistance to toxic agents de- be easily distinguished by plotting the experimental data in a semi-
livered by CuO during lag phase. logarithmic frame log(OD) versus time. The results are shown in Fig. 9.
These domains correspond to different stages of the bacteria popu- The phase of exponential growth can be easily identified in the ex-
lation evolution. In fact, as Fig. 9 shows lag phase occupies about 5 h perimental points. The linear fit with log(OD) = k + b·t permitted to
followed by the phase of exponential growth and terminating by the obtain the exponential factor k an process onset time τ, which are listed
stationary phase after 10–15 h from the beginning of process (t = 0). in Table 2.
Accordingly, several conclusions can be drawn concerning the species The bacterial growth rates differ for the three strains and do not
activity. depend on the bacteria type, Gram positive or Gram negative. Indeed
E.coli has the fastest bacterial multiplication rate, while S.aureus and
1) Bacteria decrease is ineffective during lag phase. P.aeruginosa have rather similar lower growth rates. An important result
2) Bacteria decrease is most effective during exponential growth phase concerns the turnover point from lag to exponential growth phase, which
and requires minimal concentration Ccrit = 2.3 ± 0.2 mmol/L of is τ = 5 ± 1 h for the considered strains. This value is in agreement
either Cu2+ or Zn2+ ions. with the critical concentration analysis in Fig. 9. These results of the OD
3) Cu2+ ions conserve the high effectiveness of the bacterial inhibition measurements confirm the hypothesis of different sensitivities of bac-
at stationary phase. teria to toxic action of nanoparticles during principal phases of growth.
4) Bacteria decrease by Zn2+ ions during stationary phase is of low The strongest effect is produced at the exponential growth phase
effectiveness requiring higher ions concentrations. during the active bacteria reproducibility, which required an increased
income from a nutrient medium containing toxic species. In contrast,
We notice that CuO and ZnO nanoparticles are an excellent source
of ions for bacteria decrease, since in a complementary test with ZnO
crystal of size 1 × 1 × 0.1 cm3 no antibacterial activity has been ob- Table 2
Fit parameters of the exponential growth phase.
served.
Bacteria Slope (k) τ, hours

E.coli 0.47 ± 0.02 4 ± 0.5

3.2.4. Bacterial growth
S.aureus 0.36 ± 0.02 5.5 ± 0.5
In order to confirm the model conclusions, the monitoring of bac- P.aeruginosa 0.33 ± 0.01 5 ± 0.5
terial growth in Mueller Hinton broth was performed by optical density

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R. Dadi, et al. Materials Science & Engineering C 104 (2019) 109968

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