Applied Surface Science 438 (2018) 66–73

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Applied Surface Science

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Full Length Article

Characterization and antimicrobial activity of silver nanoparticles,

biosynthesized using Bacillus species
I. Ghiuţă a , D. Cristea a,∗ , C. Croitoru b , J. Kost c , R. Wenkert d , I. Vyrides e , A. Anayiotos e,f ,
D. Munteanu a
a
Transilvania University of Brasov, Material Science Department, 29 Eroilor Blvd., 500036 Brasov, Romania
b
Transilvania University of Brasov, Materials Engineering and Welding Department, 29 Eroilor Blvd., 500036 Brasov, Romania
c
Ben-Gurion University of the Negev, Department of Chemical Engineering, P.O.B 653, Beer-Sheva, 84105, Israel
d
Soroka University Medical Center, Beer-Sheva, Israel
e
Department of Environmental Science and Technology, Cyprus University of Technology, Limassol 3036, Cyprus
f
Department of Mechanical Engineering and Materials Science and Engineering, Cyprus University of Technology, Limassol 3036, Cyprus

a r t i c l e i n f o a b s t r a c t

Article history: In this work, the biosynthesis of silver nanoparticles, using AgNO3 as a precursor, by two Bacillus
Received 3 June 2017 species, namely Bacillus amyloliquefaciens and Bacillus subtillis, is reported. After the synthesis stages,
Received in revised form 3 September 2017 the absorbance of the brown nanoparticle colloidal solutions was assessed by UV–vis spectrophotome-
Accepted 19 September 2017
try, which showed the peak absorbance values at 418 nm and 414 nm, corresponding to surface plasmon
Available online 22 September 2017
resonance of silver nanoparticles. The EDX, SEM and DLS analyses confirmed the formation of spher-
ical silver nanoparticles with an average diameter smaller than 140 nm. XRD confirmed the presence
Keywords:
of face-centered cubic silver crystals, with the highest intensity peak at 2␪ = 38.12◦ , which corresponds
Biosynthesis
Silver nanoparticles to the (111) diffraction planes. The antibacterial activity after 24 h of incubation was observed against
Bacillus amyloliquefaciens gram negative bacteria: Escherichia coli, Pseudomonas aeruginosa, Salmonella, as well as gram positive:
Bacillus subtillis Staphylococcus aureus, Streptococcus pyogenes. Furthermore, the antifungal activity was assessed against
Candida albicans. The inhibition zone was clearly observed on the plates containing silver nanoparticles,
either standalone or in combination with antibiotics, thus showing their potentiating antibacterial effect.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction or magnetron sputtering. These top-down methods can often lead

to nanoparticle imperfections [3–5].
Nanotechnology has gained increased interest in the last years AgNPs can be synthesized by bottom-up methods as well,
because of a broad scope of applications and promising results, such as: pyrolysis, sol-gel method and chemical reduction
mostly due to the properties of nanomaterials, which are most often [6,7]. However, the physiochemical approaches used for metallic
different than those of bulk materials. Generally, a particle is con- nanoparticles synthesis involve the use of toxic, hazardous chem-
sidered to be nano-sized if its dimensions are close to 100 nm, or icals, which can be harmful to the environment because of the
smaller [1]. reducing agents used for metal salt solutions and the usage of non-
Ideally, the synthesis methods of nanoparticles should be cost polar solvents. Among these, one can consider: sodium dodecyl
effective, should not involve toxic hazards, and most importantly, sulfate, sodium borohydrate, hyperbranched polyester, hydrocar-
should offer superior properties to the synthesized nanomaterials. bons and so forth. These toxic chemicals absorbed in trace amounts
Several reports can be found concerning the synthesis of metallic on the surface of the nanoparticles limits their applications in
nanoparticles, such as: Au, Ag, Co, Cd, but the research interest has clinical fields [8]. Therefore, development of clean, biocompati-
grown also to some oxide metals: TiO2 , ZnO [2]. ble, non-toxic and eco-friendly methods for nanoparticles synthesis
Silver nanoparticles (AgNPs) can be obtained by different tech- deserves merit.
niques, such as laser ablation, Tollens process, anisotropic etching Consequently, the synthesis of silver nanoparticles by biolog-
ical means was reported, using bacteria from the Acinetobacter,
Escherichia, Lactobacillus, or Staphyloccocus genera, which have
shown that they have the ability to reduce silver nanoparticles from
∗ Corresponding author at: Transilvania University of Brasov, Material Science salts [9–11]. Moreover, Aspergellius, Penicillium and Fusarium gen-
Department, 29 Eroilor Blvd., 500036 Brasov, Romania. era of fungi contain species capable of synthesizing AgNPs [12,13].
E-mail address: [email protected] (D. Cristea).

https://doi.org/10.1016/j.apsusc.2017.09.163
0169-4332/© 2017 Elsevier B.V. All rights reserved.
 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73 67

Microbial synthesis of metal nanoparticles has been reported as 1 g/l yeast extract; 18 g/l agar-agar; 5 g/l sodium nitrate; 0.2 g/l glu-
taking place both intra- or extracellularly, depending on the local- cose (all supplied by Scharlau Chemicals), which was autoclaved at
ization of the reductive components of the cells. While intracellular 128 ◦ C in the volume of 100 ml distilled water. Culture media plates
synthesis of metallic nanoparticles occurs inside the cell, in the obtained in petri dishes of 55 mm diameter from 12 ml of media
cytoplasm, the extracellular synthesis take place when the cell wall after gelification, have been inoculated with 1 ␮l of each bacterium
reductive enzymes or soluble secreted enzymes are involved in the and further incubated at 33 ◦ C for 48 h.
reductive process of metal ions [14].
As a further matter, the AgNPs synthesis using plant extracts was 2.2. Biosynthesis of AgNps using B. amyloliquefaciens 1853 and
also reported. The most widely used are Alpinia calcarata, Dioscorea B. subtillis 10833
bulbifera, Heliotropium crispum, and Aloe vera extracts [2,15–17].
Silver nanoparticles have significant perspectives as antimi- To synthesize the silver nanoparticles, 1 ␮l of bacterial strains
crobial agents against pathogenic bacteria and fungi [18–23]. It were freshly inoculated in conical flasks containing 100 ml liquid
is well known that Escherichia coli is one of the most common media (0.6 g/l yeast extract; 1 g/l sodium nitrate; 3 g/l glucose) at
causes of bloodstream infection. K. B. Laupland [24] state that 33 ◦ C for 48 h, 150 rpm. After 48 h, the cultures were centrifuged at
the emergence of antibiotics resistance, especially ciprofloxacin, 4000 rpm for 30 min using an angle rotor centrifuge. 10 ml of super-
and their adverse effects on patient outcome was a major con- natant cell-free cultures were mixed with 90 ml precursor (1 mM
cern. Furthermore, Morten Helms [25] reported that there were AgNO3 aqueous solution). The precursor was firstly autoclaved at
strains of Salmonella resistant to important antibiotics like, ampi- 128 ◦ C for 30 min. The procedure is schematically presented in
cillin, tetracycline, quinolone, which led to a higher mortality rate. Fig. 1. The cell free supernatant without silver nitrate was kept as
The combination of silver nanoparticles with antibiotics can be ben- a control. The samples were incubated for 48 h at 33 ◦ C (to ensure
eficial, increasing the synergistic antibacterial efficiency [9,26–29]. maximum enzymatic activity of the extract) and 150 rpm. In addi-
Furthermore, a current interest is represented by the interaction tion, a control sample containing the precursor and fresh liquid
between metallic nanoparticles and viruses. Due to the interaction media, without any bacteria, was prepared in order to assess its
between silver nanoparticles and HIV, the inhibition of the virus behavior regarding nanoparticle synthesis. The control sample was
from binding to host cells has been shown [30]. kept in the same conditions as the samples containing supernatant.
The activity of silver nanoparticles in medicine is not limited For the purification of biosynthesized AgNPs, the samples were cen-
by its antimicrobial effect. Another important property of silver trifuged at 4000 rpm for 30 min. The collected pellets were washed
nanoparticles is their cytotoxicity. After some tests against the with 25 ml distilled water, and dried at 80 ◦ C until the liquid had
human cervical cancer cell lines, it has been reported that AgNPs evaporated. This last step was performed for 4 times, then the sam-
have a significant role in apoptosis, thus having potential benefits ples were dried in an oven at 200 ◦ C for 6 h.
of using these kinds of nanoparticles in anticancer drugs [26,31,32]. The mechanism of synthesis is the following: Ag+ ions are
The cytotoxicity assay of silver nanoparticles shows in a dose- reduced in Ag0 atoms in the presence of aqueous enzymatic extracts
dependent manner that the viability of some cancer cell lines has from cell free supernatant of Bacillus species, as depicted in Eq. (1)
been reduced. Consequently, results against fibrosarcoma, fibro- [6].
blast, glioblastoma, Dalton’s lymphoma and breast carcinoma cell
lines can be found in the specialized literature [32–36]. A. Syed et al. Ag+ + e− (enzymes) → Ag0 (1)
have demonstrated in their work that silver nanoparticles synthe-
sized by biological approaches, are non-toxic to normal and cancer 2.3. Characterization of AgNPs
cells up to 50 ␮g/ml concentration. The same kind of nanoparti-
cles, at same concentration produced by other methods have been The UV–vis absorbance spectra were obtained by using an
reported to be toxic to normal cells. Moreover, M. I. Sriram et al. Infinite 200 microplate plate reader from Tecan using 250 ␮l of
showed that biologically synthesized silver nanoparticles present aqueous solution, placed on Greiner 96 flat bottom transparent
cytotoxic potential only in tumor cells, without affecting normal polystyrene plates. The UV–vis spectra were measured in the range
cells [34,35]. Furthermore, the possibility of using AgNPs to suc- 300–600 nm with a wavelength step size of 2 nm at a temperature
cessfully inhibit the growth of cancer cells and their cytotoxicity of 25 ◦ C.
for potential therapeutic treatments, was reported elsewhere [36], A JSM 7400f scanning electron microscope (SEM) was used for
Silver nanoparticles can potentially have an extended area of surface morphology and chemical composition analyses by Energy
application. Coupled with the promising effects in the medical Dispersive Spectroscopy (EDS). The silver nanoparticles powder
sphere, AgNPs provide advantages for biosensors, plasmonic, elec- was mounted on the sample holder using double-sided adhesive
tronic and catalysts applications. Furthermore, the treatment for carbon tape. The acceleration voltage was fixed to 10 kV.
waste water, agriculture or poultry industry can benefit from the The crystalline nature of silver nanoparticles was analyzed by
use of silver nanoparticles [32,37]. XRD using a Philips PW 1050/70 X-ray powder diffractometer with
The above-mentioned reasons led to studying the biosynthesis graphite monochromator using CuK␣1 (␭ = 1,54 Å), at a voltage of
of silver nanoparticles by green methods, using microorganisms. In 40 kV, a current of 28 mA, in the scan range 20–60◦ , in Bragg-
this paper, the Bacillus subtillis 10833 and Bacillus amyloliquefaciens Brentano geometry.
1853 strains were used to obtain AgNPs from silver nitrate through The nanoparticle size distribution was investigated by Dynamic
bio-reduction. Light Scattering (DLS) in aqueous samples. A CGS-3 Compact
Goniometer and LSE-5003 correlator were used. The results were
obtained after 15 cycles at a 90◦ angle and 26 ◦ C.
2. Materials and methods
2.4. Antimicrobial activity of AgNPs
2.1. Bacteria and culture conditions
Five microbial strains and one fungus strain were selected for
The Bacillus amyloliquefaciens 1853 and Bacillus subtillis 10833 antimicrobial activity studies. The strains were grown on Mac-
used in this study were provided from Cyprus University of Tech- Conkey and Sabouraud agar for 24 h in order to obtain fresh
nology. The bacteria were cultivated in the following solid medium: cultures. After this, several colonies of each culture were sus-
68 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73

Fig. 1. The procedure of silver nanoparticles synthesis using cell free supernatant as reductant and silver nitrate as precursor.

pended in 3 ml NaCl and used for further tests. The solid medium strated that the synthesis of silver nanoparticles can be mediated
used for antimicrobial activity was composed of 1 g/l yeast extract; by the alpha-amylase enzyme, and both species of Bacillus used in
18 g/l agar-agar; 5 g/l sodium nitrate; 0.2 g/l glucose. The silver this paper are well known as producers of this kind of enzyme [38].
nanoparticles activity was assessed against Staphylococcus aureus, Until now it was reported that B. amyloliquefaciens (LSSE 62)
Escherichia coli, Pseudomonas aeruginosa, Salmonella, Streptococ- is able to synthesize AgNPs in the presence of solar irradiation
cus pyogenes and Candida albicans. The potential of nanoparticles [39]. Moreover, the biosynthesis of AgNPs using the supernatant
regarding the antimicrobial activity was determined using the disk of B. subtillis (ATCC 6633) has been reported, the estimated 5 days
diffusion method (as described in CLSI Clinical and Laboratory process duration being significantly longer than the one from the
Standards Institute: M02-A11 standard (Performance Standards for results presented herein [40].
Antimicrobial Disk Susceptibility Tests)), by impregnating 15 ␮l An indication of complete reaction (i.e. bio-transformation of
AgNP-containing solution on each 6 mm diameter disk made of the entire amount of Ag+ from the precursor solution into AgNPs)
glass microfiber filters from WhatmanTM . Furthermore, the syner- has been assessed by the Mohr titrimetric method [41], using 10 ml
gistic effect of silver nanoparticles in combination with different of standard aqueous solution of 1 mM KCl, 0.5 ml of 0.25 M K2 CrO4
antibiotics against the microorganisms was investigated, using indicator and the precursor AgNO3 solution, respectively the AgNPs
6 mm disks containing Ciprofloxacin 5 ␮g for bacteria and Flu- colloidal solution as titrant (Fig. 2). While in the case of the pre-
conazole 25 ␮g for fungi, impregnated with 15 ␮l AgNP-containing cursor AgNO3 solution (1 mM, with a factor of 0.980), a brick-red
solution. Disks containing Ciprofloxacin 5 ␮g and Fluconazole precipitate of Ag2 CrO4 was observed in the solution, in the case
25 ␮g were used as control. The disks were placed on the surface of AgNPs colloidal solutions synthesized with the two bacterial
of the culture containing petri dishes. After 24 h of incubation at supernatants, no precipitate occurred, which could be an indica-
35 ◦ C, the inhibition diameter zone was measured and compared tive of complete reduction of Ag+ to Ag0 . The sensibility of the Mohr
to the controls. A series of three antibacterial activity tests were method is [Ag+ ] = 1.35·10−5 M [42] so it could be assumed that the
performed, the inhibition zone diameter was measured with digital reduction reaction is practically complete (the bacterial enzymatic
calipers and the standard deviation was calculated. extract is in excess).
Considering the complete bio-reduction of Ag+ , it could be con-
sidered that the same concentration of AgNPs exist in both Bacillus
3. Results and discussion amyloliquefaciens and Bacillus subtillis colloidal solutions, namely
8.82·10−4 mol AgNPs/l or 0.09513 mg AgNPs/ml.
The starting point for the formation of silver nanoparticles Fig. 3 shows the UV–vis spectra of the solutions containing
was observed after 6 h, when the color of the aqueous precursor- AgNPs obtained with the supernatant of Bacillus species. The
bacterial enzymatic extracts samples started to change from pale surface plasmon resonance peaks were observed at 418 nm and
yellow to light brown due to the surface-plasmon resonance phe- 414 nm for silver reduced by Bacillus amyloliquefaciens and Bacillus
nomenon [2]. After 48 h of incubation the final color of samples subtillis, respectively. Those peaks correspond to the plasmon res-
was changed to deep brown. This was the first sign of extracellu- onance of silver nanoparticles, according to other reports, which
lar synthesis of silver nanoparticles, taking into account that only present a peak around 420 nm [10,14,39].
the supernatant of cultures was used. The reduction of silver nitrate Some differences regarding the maximum intensity absorbance
could be produced by constituents of the cell supernatant. Peptides peak position for silver nanoparticles containing solutions,
or proteins may be responsible for the reduction of Ag+ ions and the obtained using the same species of Bacillus, but different pro-
subsequent formation of silver nanoparticles. It has been demon- cess parameters, including solar irradiation and microwave are
 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73 69

Fig. 2. Photographic images of the precursor solution after titration and respectively of the AgNPs colloidal solutions. The lack of precipitate is indicative for the complete
reduction of Ag+ –Ag0 .

From Fig. 3 it could be seen that at roughly the same AgNPs

concentration, different values for the absorbance at maximum
absorption wavelength could be registered, the NPs colloidal solu-
tion synthetized with B. amyloliquefaciens having a 35% higher
absorbance value than in the case of B. subtilis. This could be
explained by the multiple scattering theory, which relates the
experimental absorbance values (A) to particle radius (R), density
of particles per unit volume (N), according to Eq. (2). [44]:
R2 Qext d0 N
A= (2)
2.303
In Eq. (2) Qext is the extinction coefficient (assumed constant for
a type of NP) and d0 is the optical path length of the spectropho-
tometer (kept constant for all measurements).
The geometric mean radius of the NPs (Rmean ) has been deter-
mined from DLS (Fig. 7a and b) with Eq. (3):
n
i=1
(IDLS,i · RDLS,i )
Rmean = (3)

n

Fig. 3. UV–vis absorption spectra of the obtained colloidal AgNPs solutions showing IDLS,i
similar surface plasmon resonance peaks.
i=1

where IDLS.i represents the relative dynamic light scattering (DLS)

intensities and R,DLS,i the corresponding NP radius ascribed to each
Ag b.a.
b.s.
DLS maximum.
The density of nanoparticles per unit volume (N) has been cal-
culated by the method proposed by Turkevich [45] as follows:
mAg,synt
N= (4)
C  · VNP
where mAg,synt represents the mass of silver ions from the
precursor solution (9.513 mg), ␳Ag represents the density of sil-
ver (10.49 g/cm3 ) and VNP , the volume of a single nanoparticle
(assumed spherical).
3
4Rmean
VNP = (5)
O Na Ag 3
The Rmean = 31.11 nm and N = 7.19 × 1011 for the NPs synthesized
with B. amiloliquesfaciens, while for B. subtilis Rmean = 58.54 nm and
N = 1.07 × 1011 . As the Rmean 2 × N product value is 72% higher for
B. amiloliquesfaciens, higher values for absorbance are expected for
this colloidal AgNPs solution, in accordance with the experimental
Fig. 4. Energy Dispersive X-ray spectra for the AgNPs synthesized using B. amyloliq- UV–vis spectra.
uefaciens (B. a.), and B. subtillis (B. s.). The quantitative elemental analysis shows that the silver con-
tent is predominant. The other elements shown on the EDS
spectrum (Table 1 and Fig. 4) appear mostly due to the impurifi-
reported elsewhere: by using B. amyloliquefaciens the absorbance cation of the samples from two sources: firstly, due to the carbon
has decreased from 423 nm to 418 nm [39,40] while the absorbance tape used for mounting the nanoparticles on the sample holder, sec-
of AgNPs colloids using B. subtillis has been presented to have higher ondly due to possible remnants from the media used for the growth
values from 410 nm to 414 nm [39,40]. Those aspects show the first of the bacteria. This last situation can be avoided either by reducing
sign of different results regarding the geometry (shape, size) of the the amount of glucose and sodium nitrate in the liquid media, or
nanoparticles. Generally, a longer wavelength for the absorbance by performing a calcination process, prior to sample mounting. The
peak position would indicate the formation of bigger particles [43]. AgNPs were found to have spherical shape with dimension values
70 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73

Table 1
The elemental composition of the AgNPs from EDS analyses: Sample 1 = AgNPs syn- B. amyloliquefaciens
thesized by B. amyloliquefaciens; Sample 2 = AgNPs synthesized by B. subtillis. B. subtillis Ag (111)
Sample Weight%

C O Na Ag

Sample 1 12.38 ± 0.30 9.42 ± 0.78 0.76 ± 0.13 77.44 ± 1.93

Sample 2 5.21 ± 0.79 – 3.77 ± 0.51 91.01 ± 5.71

Ag (200)
NaNO3

Fig. 6. X-Ray Diffraction patterns of biosynthesized silver nanoparticles.

A Peak Diameter Size [nm]

1.0 1 1.012
2 73.86

0.8
Sample (a)

0.6

0.4

0.2

0.0
1 10 100 1000 10000

B Peak Diameter size [nm]

1.0 1 1.836
2 10.866
3 135.78
Sample (b)
0.8

Fig. 5. Scanning Electron Microscopy images of the synthesized silver nanoparti- 0.6
cles: (a) AgNPs synthesized by culture supernatant of B. amyloliquefaciens 1853; b)
AgNPs synthesized by culture supernatant of B.subtillis 10833.
0.4

in good agreement with the results from DLS, according to the SEM
micrographs (Fig. 5). 0.2
X-ray diffraction was performed in order to obtain information
about the structure of the crystalline material. On the spectrum
0.0
ranging between 2␪ = 10◦ ÷ 60◦ (Fig. 6), 2 peaks for each sample can
be observed, at 2␪ = 38.09◦ , and 2␪ = 44.25◦ , which coincide with
the (111) and (200) diffraction planes for face-centered cubic silver
(00-004-0783). The third peak present in both diffractograms, at Fig. 7. DLS results for particles size distribution of AgNPs, synthesized by green
2␪ = 29.28◦ corresponds to sodium nitrate (00-079-2056). The rea- chemistry methods using Bacillus amyloliquefaciens (a) and Bacillus subtillis (b).

sons for the presence of this compound as well as ways to eliminate

it were mentioned previously. After deconvolution of the 2 diffrac- mean crystallite size of 10.93 nm, while for the (200) orientation
tion patterns with a Pearson7 function, the exact peak position, the mean crystallite size is 6.55 nm. The samples obtained with B.
intensity and FWHM have been obtained. Using the Debye-Scherrer subtillis 10833 exhibit slightly larger crystallite sizes of 23.11 nm
equation, the crystallite size was calculated, for all the diffraction and 9.53 nm, for the (111) and (200) peaks, respectively.
peaks corresponding to silver. The silver nanoparticles synthesized The DLS graph of colloid silver nanoparticles obtained using B.
using B. amyloliquefaciens 1853, for the (111) orientation have a amyloliquefaciens (Fig. 7a) shows that the colloid includes 2 pop-
 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73 71

diameter, for the disks containing AgNPs, the control antibiotic

AgNPs - B. a. disks, and AgNPs in conjunction with antibiotics. Although there
AgNPs - B. s.
Antibiotic is a clear difference between the activity of the AgNPs infused disks
50
Antibiotic + AgNPs - B. a. and the control samples in the case of the 5 bacteria strains, the
Antibiotic + AgNPs - B. s. antimicrobial activity in the case of C. albicans, is noticeably higher
compared to the control antibiotic. The Fluconazole disks exhibited
40
little to no activity on the C. albicans strain, only in conjunction with
AgNPs the inhibition zone was measurable. The beneficial effect
30 of AgNPs concerning the antibiotic activity was calculated as the
percentage fold increase in antibacterial effect, shown in Fig. 9.
While using different species of microorganisms for nanoparti-
20 cles synthesis the results can vary in regard to their size and shape.
This observation is relevant even when using the same species,
but different parameters of the process, like pH of the precursors,
10 incubation time, precursor concentration. As Mathew et al. have
shown, the antimicrobial effect of biosynthesized silver nanopar-
ticles was found to be size and shape dependent [43]. Moreover,
0
s l i s s K. Gudikandula and S. C. Maringant have demonstrated that sil-
u co sa ne n el
la
re E. no e ica on
ver nanoparticles synthesized by biological methods using fungi
au gi og al
b
S. ru py lm (Pycnoporus sp.) showed higher antibacterial activity, compared to
ae S. C. Sa silver nanoparticles synthesized by chemical routes. The tests were
carried out against both gram-positive and gram-negative bacteria
Fig. 8. Inhibition diameter variation for the tested strains, for the AgNPs disks, the
[49].
antibiotic disks and the synergic effect of the AgNPs + antibiotic combination.
The effect of silver particles against bacteria is due to their
interaction with thiol group compounds, which are found in the
B. a. respiratory enzymes of bacterial cells. Silver binds to the bacterial
B. s. cell wall and cell membrane and inhibits the respiration process.
60 In case of E. coli, silver inhibits the uptake of phosphate, while
releasing succinate, proline, phosphate, mannitol, and glutamine
50 from E. coli cells. Due to their much larger surface area, AgNPs
should show increased antimicrobial activity. It was reported that
AgNPs get attached to the cell membrane of the bacterium and also
40
penetrate inside the bacteria. The bacterial membrane contains
sulfur-containing proteins and the silver nanoparticles interact
30 with these proteins in the cell as well as with the phosphorus
containing compounds like DNA. AgNPs preferably attack the res-
20
piratory chain [43].

10 4. Conclusions

0 B. amyloliquefaciens 1853 and B. subtillis 10833 present the ability

us li a es ns a
el l
to synthesize extracellularly silver nanoparticles by bioreduction
e co os en ca
a ur E. u gin og lbi on from 1 mM AgNO3 precursor solution. During UV–vis analysis,
S. er py a lm
a S. C. Sa peaks for both colloids at 418 nm, 414 nm, respectively, were
observed. SEM analysis showed the spherical shape of nanoparti-
Fig. 9. Percentage fold increase in antibacterial effect of antibiotics with AgNPs cles with size less than 100 nm. X-ray diffraction analysis confirmed
against test strains, calculated with equation ((b-a)/a) × 100 (%), where b repre- that the highest peak of silver crystal corresponds to the (111)
sents the inhibition zone diameter for the AgNPs + antibiotic combination, and a
diffraction plane. DLS size distribution confirms the SEM results in
represents the inhibition zone diameter for the antibiotic disk [48].
regard to nanoparticle size. The AgNPs synthesized using B. amy-
loliquefaciens 1853 and B. subtillis 10833 have presented significant
ulations of nanoparticles. The mean size of the first population is antibacterial and antifungal activity.
0.506 nm/radius and for the second one is around 36.93 nm/radius, The advantage of the proposed synthesis method consists in its
supporting the results obtained from SEM analyses. The mean size economic efficiency (the wide ability of the B. amyloliquefaciens
of each population is 0.918, 5.433 and 67.89 nm/radius. Besides and B. subtilis strains, the one-step synthesis approach, consisting
supporting the SEM observations, the DLS results show that even in reduction of silver ions by the bacteria-free enzymatic extract).
smaller particles can be synthesized by this method. Potential drawbacks of our method may include the high synthesis
To the best of our knowledge, the antimicrobial activity of silver duration (48 h), reproducibility issues (due to different behavior in
nanoparticles synthesized by Bacillus subtillis has been investigated bacterial strain enzymatic activities, the presence of trace impu-
against a relatively limited number of different bacteria strains (K. rities which may inhibit bacteria activity, the careful maintaining
pneumoniae, P. aeruginosa, V. cholera, S. pneumonia) and fungi (C. of identical synthesis conditions). Through the proposed method,
keratinophilum) [46,47]. number concentrations of silver nanoparticles in the range of
The results of antibacterial and antifungal activities of both solu- 1–7·1011 have been obtained at mass concentrations of 0.095 mg/L,
tions containing silver nanoparticles against five bacterial strains similar to other commercial silver nanoparticle products [x5].
and one fungus strain, using the disk diffusion method, can be Further research in this domain will be focused on perfecting the
observed in Figs. 8–10. Fig. 8 shows the variation in inhibition zone synthesis duration by using other bacterial strains and in assess-
72 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73

Fig. 10. Photographs of the inhibition zone of S. aureus (1), E. coli (2), P. aeruginosa (3), S. pyogenes (4), C. albicans (5) and Salmonella (6) cultures: a – antibiotic, b – antibiotic + B.s.
AgNPs, c – antibiotic + B.a. AgNPs.
 I. Ghiuţă et al. / Applied Surface Science 438 (2018) 66–73 73

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