Biotechnology Reports 40 (2023) e00816

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Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre

Research Article

Optimization of the biosynthesis of silver nanoparticles using bacterial

extracts and their antimicrobial potential
Iván Solís-Sandí a, Sara Cordero-Fuentes b, Reinaldo Pereira-Reyes c,
José Roberto Vega-Baudrit c, d, Diego Batista-Menezes c, Gabriela Montes de Oca-Vásquez c, e, *
a
School of Biology, Tecnológico de Costa Rica, Campus Central, 159-7050 Cartago, Costa Rica
b
School of Chemistry, Universidad Nacional, Campus Omar Dengo, 86-3000 Heredia, Costa Rica
c
National Nanotechnology Laboratory, National Center for High Technology, 10109 Pavas, San José, Costa Rica
d
Laboratory of Polymer Science and Technology, School of Chemistry, Universidad Nacional, Campus Omar Dengo, 86-3000 Heredia, Costa Rica
e
Center for Sustainable Development Studies, Universidad Técnica Nacional, 1902-4050, Alajuela, Costa Rica

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

Keywords: In the present study, silver nanoparticles (AgNPs) were biosynthesized using the supernatant and the intracel­
Biosynthesis lular extract of Cupriavidus necator, Bacillus megaterium, and Bacillus subtilis. The characterization of the AgNPs
Silver nanoparticles was carried out using UV–Vis spectroscopy, FTIR, DLS and TEM. Resazurin microtiter-plate assay was used to
Characterization
determine the antimicrobial action of AgNPs against Escherichia coli. UV–Visible spectra showed peaks between
Bacterial extracts
Antimicrobial effect
414 and 460 nm. TEM analysis revealed that the synthesized AgNPs showed mostly spherical shapes. DLS results
determined sizes from 20.8 to 118.4 nm. The highest antimicrobial activity was obtained with the AgNPs syn­
thesized with supernatant rather than those using the intracellular extract. Therefore, it was determined that the
bacterial species, temperature, pH, and type of extract (supernatant or intracellular) influence the biosynthesis.
This synthesis thus offers a simple, environmentally friendly, and low-cost method for the production of AgNPs,
which can be used as antibacterial agents.

1. Introduction cellular respiration, replication, and transcription; furthermore, it has

been demonstrated that AgNPs have antifungal and antioxidant prop­
Nanoscience and nanotechnology are research areas that involve the erties [5,8]. AgNPs have this antimicrobial property because they act
creation and modification of structures, devices, and systems with novel directly on the cell membrane of bacteria, which generates structural
properties and functions due to the atoms’ arrangement in the scale of changes such as the formation of membrane pores, increasing the
1–100 nm [1]. Within the different types of nanoparticles (NPs), the permeability of the membrane and, finally, bacterial death [9,10].
most employed are the metallics, which include Ag, Au, Pt, Pd, Cu, Ni, Furthermore, it has been reported that the small size of synthesized
Se, and Fe [2]. These metallics have been used widely because of their AgNPs increases their stability and biocompatibility, since their small­
optic, magnetic, catalytic, and antimicrobial properties, which allow ness facilitates internalization through cellular membranes, which is
them to be used in fields like medicine, agriculture, environmental related to efficient antimicrobial activity [11]. Additionally, multiple
studies, electronics, and more [3,4]. Silver nanoparticles (AgNPs) are studies indicate that the non-agglomeration of the nanoparticles plays a
currently one of the most used metallics, largely due to their antimi­ key role in their high efficiency in various nanoparticle applications, in
crobial properties against several infectious and pathogenic microor­ this case antimicrobial activity [12]. Currently, synthesized AgNPs are
ganisms, including multi-resistant bacteria [5]. It has been shown that used in food preservation, disinfection, water filtration systems, in­
AgNPs inhibit the bacterial growth on bacteria like Staphylococcus secticides, cosmetics, and antimicrobial agent in the medical field,
aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, among other applications [5,13].
and others [6,7]. This inhibition may occur because AgNPs can interact While AgNPs can be synthesized through physical or chemical pro­
with enzymes, proteins, and biological surfactants present on microbial cesses, green synthesis is most advantageous because it minimizes the
cells, affecting membrane permeability and certain processes like use of environmental hazardous substances, costs less than chemical

* Corresponding author at: Center for Sustainable Development Studies, Universidad Técnica Nacional, 1902-4050, Alajuela, Costa Rica.
E-mail address: [email protected] (G. Montes de Oca-Vásquez).

https://doi.org/10.1016/j.btre.2023.e00816
Received 8 June 2023; Received in revised form 14 October 2023; Accepted 20 October 2023
Available online 29 October 2023
2215-017X/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
I. Solís-Sandí et al. Biotechnology Reports 40 (2023) e00816

synthesis, and is considered an easy protocol for reproduction; in addi­ 2. Materials and methods
tion to producing nanoparticles with a greater antibacterial effect and
through more energy efficient processes [14–16]. Also, it has been 2.1. Microorganisms
determined that the synthesis using microorganisms is faster than using
plant extracts [17]. Two Gram-positive bacteria (Bacillus subtilis DSM 10 and Bacillus
Biological synthesis generally involves the reduction of metallic ions megaterium DSM 32) and one Gram-negative (Cupriavidus necator DSM
by biological organisms like plants [16–19], yeast, and fungal or bac­ 545) bacteria used for the AgNPs green synthesis, were obtained from
terial extracts [20,21].Otherwise, the synthesis of AgNPs is a complex the DSMZ (German Collection of Microorganisms and Cell Cultures
process that requires the optimization of different parameters such as GmbH). All bacterial strains were stored in 15 % glycerol containing
pH, temperature, agitation, metal concentration, microorganisms, nutrient broth medium at − 70 ◦ C. Prior to their use in green AgNPs
inoculum size, among others [22] Bacteria are the most used because of synthesis, the cryopreserved bacteria were grown on lysogeny broth
their large-scale production, fast cell growth rate, and easy handling. agar (LBA) at 37 ◦ C for 24 h, and 140 rpm, on an 211DS incubator with
There are numerous examples of AgNPs synthesized from diverse mi­ orbital agitator (Labnet International Inc., Edison, NJ, USA).
croorganisms, including, Paenibacillus anseongense [21], Bacillus pumilus
[23], Terrabacter humi [24], Pseudomonas alloputida [25], Bacillus sub­ 2.2. Extracellular synthesis of silver nanoparticles
tilis, and Bacillus megaterium [26,27]. To our knowledge, there are no
studies that use C. necator for the green synthesis of AgNPs using su­ The microorganisms were cultivated on 75 mL of lysogeny broth
pernatants and intracellular extracts [28]. Cupriavidus necator is a medium (LB) and maintained at 37 ◦ C on an 211DS incubator with an
facultative chemolithoautotrophic bacterium, it has the ability to use orbital agitator (Labnet International Inc., Edison, NJ, USA) at 120 rpm
nitrate/nitrite as an alternative electron acceptor and has resistance for 24 h. After the incubation period, the cultures were centrifuged at
mechanisms towards heavy metals [29–31]. Due to these characteristics, 10,000 rpm for 10 min, and the supernatants were used for the next
this bacterium was selected as a possible agent for the AgNPs synthesis. experiments. To carry out the synthesis, 7.5 ml of the supernatant was
Furthermore, B. subtilis is a fast-growing Gram-positive bacterium that is added drop by drop with constant agitation (550 rpm) on 30 mL of 1 mM
easy to cultivate, that has genes encoding for a respiratory nitrate aqueous AgNO3 solution. Subsequently, the mixture was maintained on
reductase [32,33]. Besides, B. megaterium is considered a powerful cell constant agitation at 550 rpm for 27 h under darkness conditions. The
factory widely studied and used in many biotechnological applications temperature (30 ◦ C and 60 ◦ C) and pH (7, 8, 9, and 10) effects were
such as small molecule biosynthesis, recombinant protein expression, studied on the AgNPs’ formation. The pH was adjusted with NaOH and
biotransformations, among others [34]. Due to the widely used Bacillus HCl. All the treatments were conducted in triplicate, using as control the
spp. in similar research, and the novelty in the use of C. necator for the microorganisms’ supernatant without AgNO3 under the same conditions
green synthesis of AgNPs, we decided to compare the AgNPs synthesis described above. The AgNPs green synthesis was visualized by colori­
between these bacteria. metric changes in the solution, turning from yellow pale to brownish
The synthesis and stability of these NPs is due to the biomolecules tones. Synthesis was also confirmed using the UV–vis spectrophotometer
present in the microorganisms, such as enzymes, proteins, and bio­ at 1 h intervals for 27 h.
tensoactives [4]. Furthermore, it has been determined that bacterial
supernatant and intracellular extract can synthesize stable AgNPs [23, 2.3. Green synthesis of silver nanoparticles using intracellular extracts
35,36]. Extracellular synthesis is a low-cost technique that has been
reported to facilitate large-scale production and that requires simpler The bacterial growth described in Section 2.2 was centrifuged at
downstream processing, otherwise the synthesis through intracellular 5000 rpm, and then 2 g of biomass was resuspended on 10 mL Milli-Q®
extracts requires a cellular lysis process (e.g. sonication) to release the water and centrifuged for 10 min at 5000 rpm. This process was con­
molecules needed to carry out the synthesis [4]. To date, many studies ducted three times to wash the biomass. Subsequently, the obtained
focus on extracellular methods for AgNPs synthesis [23,37], while few suspension was subjected to cellular lysis by sonication for 12 min, with
studies have compared synthesis processes using bacterial supernatants a 15 s pulse on and a 45 s pulse off, at 45 % amplitude, using the Qsonica
and those using intracellular extracts [35,38]. The study and compari­ Q700 sonicator (Cole-Parmer, Vernon Hills, EE. UU.). The lysed cultures
son of AgNPs green synthesis methods, using several bacterial strains were centrifuged for 30 min at 14,000 rpm. For the synthesis, 10 mL of
and experimental conditions, are fundamental to demonstrate the ad­ the lysed culture were mixed with 10 mL of a 1 mM aqueous AgNO3
vantages and drawbacks of the approaches, that can support the selec­ solution. The mixture was maintained on constant agitation at 550 rpm
tion of adequate synthesis protocols in future studies. at 60 ◦ C for 24 h under darkness conditions. The treatment was con­
In this study, we optimized the green synthesis of AgNPs mediated by ducted in triplicate. The AgNPs green synthesis was visualized by
two strains of Bacillus (B. megaterium and B. subtilis) and Cupriavidus colorimetric changes in the solution. Synthesis was also confirmed using
necator using two approaches (extracellular synthesis and synthesis the UV–vis spectrophotometer after 24 h.
using intracellular extracts). In addition, for the extracellular synthesis
we also evaluated the effect of pH (7, 8, 9, and 10) and temperature (30 2.4. Quantification of proteins and DNA from the intracellular extract
and 60 ◦ C) on the synthesis of AgNPs. Ultraviolet spectroscopy (UV–Vis),
Fourier-transform infrared spectroscopy (FTIR), dynamic light scat­ The proteins and double-stranded DNA of the intracellular extracts
tering (DLS), and transmission electron microscopy (TEM) were used to (1:10 dilution) of C. necator, B. subtilis, and B. megaterium were quanti­
characterize the synthesized AgNPs. These AgNPs were also studied for fied by a fluorimetric assay using the Qubit 4.0 Fluorometer (Life
their antimicrobial activity against the non-pathogenic E. coli ATCC Technologies, Carlsbad, California, US). For each sample, 2 μl was
25,922, which is a well-known strain usually used as quality control in examined using the Qubit dsDNA BR assay and protein assay kits, ac­
many fields [39–41], to demonstrate their potential application in the cording to the manufacturer’s instructions.
medical field. This work determines the use of C. necator as an agent for
the AgNPs synthesis. 2.5. Characterization of silver nanoparticles

The techniques of ultraviolet visible spectroscopy (UV–Vis), Fourier

transformed infrared spectroscopy (FTIR), transmission electron mi­
croscopy (TEM), dynamic light scattering (DLS) and zeta potential were
used for the characterization of the AgNPs.

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I. Solís-Sandí et al. Biotechnology Reports 40 (2023) e00816

2.5.1. UV–Vis characterization plotted on the OriginPro, version 2019b, Northampton, MA, USA.
To determine the formation of the AgNPs through time, the
measuring of the surface plasmon resonance (SPR) band using UV–vis 3. Results and discussion
spectroscopy (Shimadzu, UV-1800) was conducted from 200 to 800 nm,
operated at a resolution of 1 nm. 3.1. Green synthesis and characterization of silver nanoparticles

2.5.2. FTIR characterization 3.1.1. Visual observation and UV–Vis spectroscopy

The FTIR spectroscopy analysis (Nicolet 6700, Thermo Scientific) The green synthesis of AgNPs by reduction of silver nitrate using
was conducted to determine the main functional groups of the super­ supernatants and intracellular extracts of Cupriavidus necator, Bacillus
natants, intracellular extracts, and biosynthesized AgNPs. The analysis megaterium, and Bacillus subtilis was monitored based on the reaction
was done in duplicate. The results were analyzed on the OMNIC 8.1 mixture’s change in color. The appearance of a yellowish-brown color is
(OMNIC Series 8.1.10, Thermo Fischer Scientific) software. characteristic of the formation of AgNPs [23]. A clear difference in the
color of the reaction was observed between the synthesized nano­
2.5.3. DLS and zeta potential particles at 30 and 60 ◦ C, with the reaction mixture appearing darker at
The particle size measurement, polydispersity and zeta potential of 60 ◦ C (Fig. 1). As shown in Fig. 1a (60 ◦ C with B. subtilis supernatant),
the synthesized AgNPs were determined using a nanoparticle analyzer the color of the reaction slightly intensifies as the pH increases. With the
(NanoPartica SZ-100V2, Horiba, Japan). The analyses were carried out B. megaterium supernatant at 60 ◦ C (Fig. 1b), there was a noticeable
at a 90◦ dispersion angle at a temperature of 25 ◦ C. For the analysis, the difference between the color of the reaction at pH 10 and the reaction at
AgNPs suspension was diluted 1:3 using Milli-Q water. Histograms of the other pH values: at pH 10, a dark brown color was observed. Regarding
size distributions of AgNPs were plotted on the OriginPro, version the C. necator supernatant at 60 ◦ C (Fig. 1c), there was a darker color at
2019b, Northampton, MA, USA. pH 10 compared to the other treatments. The darker color of the reaction
mixtures could be associated with a major AgNPs concentration [42].
2.5.4. TEM characterization Otherwise, at 30 ◦ C and with C. necator supernatant (Fig. 1c), an intense
The size and morphology of the synthesized nanoparticles were coloration of the AgNPs solution was observed at pH 7 and pH 10.
measured by TEM (JEOL, model JEM 2011), coupled with energy Moreover, the AgNPs synthesized using the intracellular extract
dispersive X-ray fluorescence (EDS) using the Inca X-sight Si (Oxford showed a yellow pale color in the reaction after 27 h (Fig. 2). It should be
Instruments, model 7582-M). Window: ATW-2, resolution at 5.9 keV. noted that a more intense coloration occurred on the treatment using the
The analyzes were carried out employing an accelerating voltage of 120 C. necator intracellular extract (Fig. 2c). The lower color intensity of the
kV. For sample preparation before observing by TEM, 5 µl of AgNPs reaction for the AgNPs synthesized using the intracellular extract
suspensions were drop-casting over carbon-coated copper grids, and compared to those synthesized using the supernatant at 60 ◦ C agrees
dried for 16 h in a desiccator with silica. with the findings of [43], who observed a slight color change in the
reaction mixture in the intracellular synthesis of AgNPs compared to the
2.6. Determination of minimum inhibitory concentration brown color observed using the supernatant at pH 10 at 60 ◦ C.
The UV–Vis spectra of the biosynthesized AgNPs are shown in Fig. 3.
The antibacterial activity of the biosynthesized AgNPs, against E. coli
ATCC 25,922 was determined using a resazurin microtiter-plate assay
(minimum inhibitory concentration, MIC) in a 96-well round bottom
microtiter plate. A positive control with the commercial antibiotic
kanamycin was done. A previous bacterial suspension of E. coli was
prepared, and then centrifuged at 3500 rpm for 10 min. The resulting
pellets were resuspended in a saline solution of 0.85 % m/v NaCl. Then,
into the 96-well microtiter plates from column 12 to column 3, 5 × 104
UFC/well of E. coli was added, and the final volume was made up to 100
µL in each well, where the column 12 had the highest and column 3 the
lowest concentration of AgNPs. Next, in each well 10 µL of a resazurin
solution, with a concentration of 0.01 % (m/v), was added. Columns 1
and 2 served as the positive control (without AgNPs and kanamycin),
and rows A and H served as the negative control (without bacterial
suspension). For a period of 24 h at 37 ◦ C, the plates were incubated.
After that, an estimation, based on the change in color from blue to pink,
was used to determine the presence of viable microorganisms in each
well. The MIC value was taken at the lowest concentration of AgNPs that
inhibited bacteria growth (the color remained blue), and all experiments
were performed by triplicate, and then were reported the averages of the
MIC values.

2.7. Statistical analysis

The statistical analyses of the results of the MIC assay were carried
out using a one-way ANOVA, followed by post hoc analyses using
Tukey’s significant difference test. Differences at P < 0.05 were regarded
as statistically significant. All results are reported as means ± standard
deviation. These statistical analyses were performed using IBM SPSS
(version 20.0; SPSS Inc, Chicago, IL, USA). Prior to performing the one- Fig. 1. Silver nanoparticles synthesized using the bacterial supernatants. (a)
way ANOVA, the normality and the homogeneity of variances were AgNPs synthesized using B. subtilis, (b) AgNPs synthesized using B. megaterium,
tested by the Ryan-Joiner and Levene tests, respectively. The data were and (c) AgNPs synthesized using C. necator at 30 ◦ C and 60 ◦ C.

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I. Solís-Sandí et al. Biotechnology Reports 40 (2023) e00816

wavelength of 407 nm at pH 10. The highest absorbance was obtained at

pH 7; however, similar to the findings with the other bacterial super­
natants, a wider band was observed at this pH, which could be related to
the higher polydispersity of the AgNPs [44]. Regarding the AgNPs
synthesized at 30 ◦ C, the typical absorption band indicative of AgNPs
formation was not detected for any of the evaluated bacteria, except for
the synthesis using the C. necator supernatant at pH 10, which showed
an absorbance of 0.5 and wavelength at 453 nm. The lack of absorbance
at pH 7 and 30 ◦ C agrees with the results of El-Bendary et al. (2021), who
found that absorbance did not occur in the reduction of silver ions under
the same pH and temperature conditions [45]. Therefore, according to
the results of UV–Vis, the best AgNPs synthesis was obtained at pH 10
and 60 ◦ C. Previous investigations have determined that the green
synthesis on basic mediums produce AgNPs that are more stable and
Fig. 2. Silver nanoparticles synthesized using the intracellular extract at 30 ◦ C
monodisperse and that show high growth rate and major absorbance
for 27 h. (a) AgNPs synthesized using B. subtilis, (b) AgNPs synthesized using
[46], which agrees with the present findings. Also, it has been reported
B. megaterium, and (c) AgNPs synthesized using C. necator.
that temperatures above 60 ◦ C disfavor the reduction process of silver
ions because high temperature degrades the biomolecules responsible
The absorbance was found to constantly increase in intensity as a
for the synthesis process and generates NP aggregates [45]. On the other
function of time, in both the synthesis using the supernatant and the
hand, regarding AgNPs synthesis using intracellular extracts, the present
synthesis using the intracellular extract. Regarding the synthesis using
study found that the highest absorbance occurred in the synthesis using
the C. necator supernatant (Fig. 3a), the highest absorbance was found at
C. necator, followed by B. megaterium and B. subtilis, respectively
pH 10 and 60 ◦ C, followed by the treatment at pH 7 and 60 ◦ C. On the
(Fig. 3d). Similarly, some authors have reported the use of intracellular
other hand, the synthesis using the B. megaterium supernatant (Fig. 3b)
extracts and supernatants from different bacteria to synthesize AgNPs
showed a similar wavelength, about 425 nm, between the AgNPs syn­
[23,25].
thesized at most pH levels at 60 ◦ C; however, the highest absorbance was
obtained at pH 10. The synthesis using the B. subtilis supernatant showed
3.1.2. FTIR spectroscopy
a wavelength close to 414 nm with treatments at pH 7, 8, and 9, and a
The FTIR characterization determined the presence of different

Fig. 3. UV–visible absorption spectra of AgNPs synthesized using the supernatant at 60 ◦ C and 30 ◦ C at different pH values for 27 h, and the intracellular extract at
30 ◦ C for 27 h. (a) AgNPs synthesized using C. necator supernatant, (b) AgNPs synthesized using B. megaterium supernatant, (c) AgNPs synthesized using B. subtilis
supernatant, and (d) AgNPs synthesized using the intracellular extracts.

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functional groups associated with the green synthesis process and sta­ Table 1
bilization of the AgNPs. The FTIR spectra showed peaks on the Dynamic light scattering (DLS) and zeta potential of AgNPs.
3500–3200 cm− 1, 1600–1500 cm− 1, and 1300–1200 cm-1 regions Treatment Hydrodynamic Z- Polydispersity
(Fig. 4a). The peaks in the 3500–3200 cm− 1 region could be associated diameter (nm) potential
with the -OH of the hydroxy functional groups [6], and the -NH of the (mV)
amine functional groups [47]. Regarding the identified peaks in the Intracellular AgNPs- 70.5 ± 2.1 − 21.4 ± 0.329 ±
1600–1500 cm− 1 region, those could be related with the presence of extract C. necator 1.2 0.025
C=O links from amides or esters [6]. On the other hand, the peaks be­ AgNPs- 118.4 ± 1.9 − 34.1 ± 0.170 ±
B. subtilis 1.9 0.020
tween 1300 and 1200 cm− 1 and 1036 cm− 1 are related to aromatic and AgNPs- 91.2 ± 2.4 − 33.9 ± 0.322 ±
aliphatic amines [48]. It has been determined that the peak at 1078 B. megaterium 1.8 0.012
cm− 1 could be a C–O stretch and the peak at 1038 cm− 1 a C–N stretch Supernatant AgNPs- 85.9 ± 5.9 − 19.0 ± 0.605 ±
and aliphatic amines vibrations [49]. The presence of these functional C. necator 0.5 0.050
AgNPs- 20.8 ± 0.6 − 21.9 ± 0.907
groups in the synthesized AgNPs suggests the presence of proteins, en­ ±
B. subtilis 0.7 0.144
zymes, peptides, polysaccharides, and organics acids, which are AgNPs- 103.4 ± 7.6 − 18.1 ± 0.617 ±
involved in the reduction and stabilization of metallic ions in metallic B. megaterium 2.0 0.086
nanoparticles [47,50]. Likewise, with the AgNPs synthesized using the
*Values are presented as means ± standard deviation (SD).
intracellular extract (Fig. 4b), similar absorbance bands were observed
as those obtained for AgNPs synthesized using the supernatants. Peaks
compared to the AgNPs synthesized with the supernatant. Considering
were observed in the 3350–3290 cm− 1, 1650–1600 cm− 1, 1450–1350
this information, it is possible to associate the different values of the zeta
cm− 1, and 1090–1050 cm− 1 regions. It should be noted that the
potential to the greater availability of stabilizing factors that can be
stretching vibration of C–O increased to higher wavenumbers, shifting
found in the intracellular extract, which can be corroborated by means
from 1078 to 1087 cm− 1 in the synthesis using the intracellular extract,
of the protein and DNA quantification results in Section 3.2. According
suggesting the presence of higher protein content [51,52].
to these results, it is important to take into account the stability over
time of nanoparticles for the desired application, for example, nano­
3.1.3. DLS and zeta potential
particle formulations that need to be applied constantly, such as disin­
As shown in Table 1, the AgNPs synthesized using the intracellular
fectant sprays, may not require high stability over time compared to
extracts of B. subtilis and B. megaterium were found to be more stable,
nanomedicines. Nanoparticles can also be modulated to generate better
with zeta potential values of − 34.1 mV and − 33.9 mV, respectively,
stability, using precision electrostatic/charge or precision polymer
compared with C. necator, which yielded a zeta potential value of − 21.4
methods, as mentioned in the work by Sperling and Parak [56]. In
mV. Fig. 5 shows the histograms of AgNPs synthesized. According to
addition, other factors such as antimicrobial activity should be taken
Dhiman et al. (2021), zeta potential values above − 30 mV indicate high
into account in order to determine the potential use of AgNPs.
NPs stability [53]. On the other hand, in the AgNPs synthesized using
On the other hand, several authors have suggested that there are
the supernatant, all the zeta potential values were between − 18.1 and
different proteins that can be attached to nanoparticles through amine
− 21.9 mV, which indicates a lower stability compared with the AgNPs
or cysteine groups, which can contribute to their stabilization [57]. In
synthesized using the intracellular extracts. These differences could be
addition, in the green synthesis mediated by bacteria, it has been
due to the different biomolecules present in the intracellular and su­
determined that proteinous molecules with –OH, –NH2, –SH2, –CHO, or
pernatant extracts, such as carbohydrates, proteins and lipids [54]. In
–COOH functional groups are responsible for the reduction of the silver
the case of the intracellular extract, the presence of DNA, c-type cyto­
nitrate and stabilization of AgNPs [58–61]. The secretion of extracel­
chromes, peptides, cellular enzymes such as nitrate reductase, and
lular enzymes, specifically nitrate reductase, has been suggested to be
reducing cofactors could favor the synthesis and stabilization of AgNPs
responsible for the reduction of Ag ions to AgNPs, which has been
[55], preventing their aggregation and providing greater stability
confirmed in studies that use extracellular extracts of B. subtilis and

Fig. 4. FTIR spectra of bacterial supernatants, intracellular extracts and synthesized AgNPs. (a) FTIR of bacterial supernatants and AgNPs synthesized using the
bacterial supernatants, at pH 10 and 60 ◦ C, and (b) FTIR spectra of intracellular extracts and AgNPs synthesized using the intracellular extracts.

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Fig. 5. Histogram of AgNPs synthesized using the supernatant at 60 ◦ C pH 10, 60 ◦ C, for 27 h (a–c), and the synthesized using the intracellular extract at 30 ◦ C for 27
h (d-f). (a and d) AgNPs synthesized using C. necator, (b and e) AgNPs synthesized using B. megaterium, (c and f) AgNPs synthesized using B. subtilis.

Cupriavidus sp. [48,62]. Besides, other molecules such as oxidases, and nucleus of the microorganisms [70–72], however it is important to
reducing cofactors, cyclic ring of monosaccharides, biosurfactants and take into account that the antimicrobial activity can also be influenced
exopolysaccharides are produced by bacteria [60,63–68]. by another factors, such as concentration, shape and charge of the
Regarding the hydrodynamic diameter of the synthesized AgNPs nanoparticles [73,74].
using the intracellular extract, the AgNPs with a larger size were bio­
synthesized by B. subtilis, followed by B. megaterium and C. necator, with 3.1.4. Morphological characterization of AgNPs
values of 118.4, 91.2, and 70.5 nm, respectively. On the other hand, TEM images (Figs. 6 and 7) revealed different shapes and sizes be­
according to the polydispersity data, the AgNPs synthesized using the tween the AgNPs synthesized with the different microorganisms and the
intracellular extract from B. subtilis presented higher polydispersity. type of extract (supernatant or intracellular extract), spherical being the
Concerning the AgNPs produced using the supernatant, the AgNPs most predominant shape. Similarly, Ameen et al. (2020), obtained
formed by B. subtilis had an average size of 20.8 nm, which was signif­ AgNPs using Cupriavidus sp. with spherical morphology and sizes be­
icantly smaller than those formed by C. necator and B. megaterium, which tween 10 and 50 nm, while Stancu, 2021 performed a similar experi­
had average sizes of 85.9 and 103.4 nm, respectively (Table 1). Ac­ ment using B. megaterium and obtained spherical AgNPs with a size
cording to our results of zeta potential and particle size, using the su­ between 1 and 85 nm [48,75]. It has been demonstrated that different
pernatant of B. subtilis at pH 10, Zhao et al. (2019), obtained similar data microorganisms secrete different biomolecules in different proportions
for these parameters using B. subtilis [26]. However, different results [76], such as carbohydrates, proteins, lipids, and polysaccharides [54,
were obtained with respect to polydispersity values. This could be 57], which have been reported as molecules responsible for the green
associated with differences in the experimental conditions, such as synthesis of AgNPs. In addition, associated with the EDS analyzes pre­
temperature, agitation, light conditions, and pH, which influence the sent in Fig. 6 (g, h and i) and Fig. 7 (g, h and i), silver was observed by a
size, potential and polydispersity of AgNPs [45,53]. Ag peak in the EDS spectrum of all samples. Furthermore, the presence
The variations in the particle size obtained between the different of elements such as copper (Cu), aluminum (Al), carbon (C) and oxygen
bacteria and type of extract, could be associated to the distinct bio­ (O) was also detected, however, it is important to emphasize that the
molecules present in each extract, as explained previously. In addition, it other elements observed in the analyzes come from the chemical
has been reported that differences in the hydrodynamic diameter could structure of the carbon tape and the copper grid used in the assembly of
be associated with some chemical characteristics of the nanoparticles, the samples.
such as the rapid agglomeration of groups with low stabilization and the
anisotropy of this type of AgNPs. Furthermore, part of the AgNPs could
be very unstable due to their energy content and their reactivity, which 3.2. Quantification of proteins and DNA from the intracellular extract
explains the presence of different population sizes [69]. According to
our results, it is important to highlight that the size of AgNPs has an The protein and DNA concentration of the intracellular extract was
impact on the antimicrobial activity, which has been reported by several determined by a fluorimetric assay (Table 2). The intracellular extract of
studies, that found that smaller sizes of AgNPs seems to exhibit higher B. subtilis had the highest concentration of protein and DNA, followed by
antimicrobial activity, since them can reach more easily the cytoplasm C. necator and B. megaterium. Various studies have determined that these
biomolecules are responsible for the reduction and stabilization of

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Fig. 6. Transmission electron micrograph and energy dispersive X-ray fluorescence of AgNPs synthesized using bacterial supernatant. (a, b and g) AgNPs from
C. necator at pH 10, 60 ◦ C, (c, d and h) AgNPs from B. megaterium at pH 10, 60 ◦ C, and (e, f and i) AgNPs from B. subtilis at pH 10, 60 ◦ C.

Fig. 7. Transmission electron micrograph and energy dispersive X-ray fluorescence of AgNPs synthesized using the intracellular extract (a, b and g) AgNPs from
C. necator at pH 10, 60 ◦ C, (c, d and h) AgNPs from B. megaterium at pH 10, 60 ◦ C, and (e, f and i) AgNPs from B. subtilis at pH 10, 60 ◦ C.

AgNPs [77,78]. Similarly, some studies have identified that certain

Table 2
bacterial enzymes (silver-binding proteins, metalloproteins, reductases)
Concentration of proteins and DNA from the intracellular extract of the
can perform the intracellular synthesis of AgNPs [43]. One of the most
microorganisms.
studied enzymes is the nitrate reductase, which has been reported to
Microorganism Protein (mg/ml) DNA (ng/ml) synthesize AgNPs [79]. This enzyme gains NADH electrons, which pass
B. subtilis 0.127 10.6 to NAD+ oxidized form and reduce the silver ions to AgNPs [3].
B. megaterium 0.024 0.868 Furthermore, some biomolecules such as soybean and milk proteins
C. necator 0.0906 2.24
have been used for the synthesis of AgNPs [78]. However, some studies
have determined that the reduction of silver using soy protein occurs
thanks to the tyrosine residue, while in milk protein the reduction occurs
due to interactions between casein and whey protein. In addition, it has

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been reported that DNA can be used as a capping molecule to control the - 12.5 μg/mL using AgNPs biosynthesized by bacteria [89,90].
morphology of metal nanomaterials such as gold and silver, among Regarding the AgNPs synthesized by using plant extracts, some research
others [69]. Similarly, Chumpol & Siri, 2018 reported that bacterial has found MIC values between 7.8 and 20.0 μg/mL [91–93]. Besides,
genomic DNA obtained from a previous lysis and purification can syn­ other biological extracts from fungus have been used for the synthesis of
thesize AgNPs [77]. AgNPs, Mohanta et al. (2022) synthesized AgNPs from the extract of
Talaromyces funiculosus and determined a MIC value of 43.94 μg/ml
against E. coli [94].
3.3. Antimicrobial activity of silver nanoparticles On the other hand, some studies have compared the MIC value of the
synthesized AgNPs with commercial antibiotics. For example, Vazquez
According to the best results of green synthesis, the antibacterial (2017) have found major antimicrobial activity of commercial antibi­
activity was tested for the AgNPs synthesized using the bacterial su­ otics (ampicillin, chloramphenicol, kanamycin, biapenem, aztreonam,
pernatant at pH 10 and 60 ◦ C, as well as for those synthesized using the amphotericin B, fluconazole and caspofungin) compared to commercial
intracellular extract. The AgNPs displayed antimicrobial activity toward AgNPs, with MIC values of 0.05–2 μg/ml, and 10 μg/ml to inhibit the
the tested strain E. coli (Table 3). According to the results of the MIC growth of E. coli, respectively [95]. Similarly, Wypij et al. (2022), found
(Table 3), the highest antimicrobial activity against E. coli was obtained MIC values of AgNPs of 64 μg/ml and between 0.5 and 16 μg/ml for
using the AgNPs synthesized using the supernatant of C. necator. Among antibiotics [96]. The results of these research are in line with our find­
the treatments using intracellular extracts, the highest antimicrobial ings, were the MIC values of the synthesized AgNPs were higher than the
activity was obtained with the AgNPs synthesized with B. megaterium. value of kanamycin (0.55 μg/ml), however it is important to highlight
These results prove the capacity of synthesized AgNPs to inhibit the that this value was similar to the MIC value obtained with the synthe­
growth of E. coli, confirming that these AgNPs have great potential as sized AgNPs from the supernatant of C. necator (0.69 μg/ml). Our results
antimicrobial agents, a finding supported by other studies [36,80,81]. It revealed that the synthesized AgNPs exhibited potent activity against
has been reported that nanoparticles’ antibacterial activity is deter­ E. coli and can be used as antibacterial agents.
mined by their size, concentration, shape, charge, and stabilizing agent
and depends on the microbial strain. In similar investigations, it has 4. Conclusions
been determined that the size of AgNPs influences the inhibition of
microbial growth. Research has evidenced that small AgNPs have a This study demonstrated that the optimal conditions to synthesize
greater surface area/volume ratio, which increases the interaction of AgNPs were pH 10 and 60 ◦ C using the bacterial supernatant of
NPs with the membrane surface of bacteria, which facilitates penetra­ C. necator. In addition, a series of known techniques were used to
tion through the membrane and causes its death [18,82,83]. characterize the AgNPs, including UV–Vis spectroscopy, indicating the
In our research, it was clear that the AgNPs synthesized using the highest absorbance in C. necator, followed by B. megaterium and
supernatant had lower MIC values compared to those synthesized using B. subtilis. Regarding the intracellular extracts, C. necator presented the
the intracellular extract. This difference in the antibacterial activity highest absorbance, followed by B. subtilis and B. megaterium. The FTIR
could be due to the different biomolecules that are present in the analysis indicated the possible presence of functional groups of proteins,
different extracts, such as carbohydrates, proteins, lipids, poly­ enzymes, peptides, and polysaccharides, among others, which is rele­
saccharides, enzymes, and DNA, which could produce AgNPs with vant because they are responsible for reducing and stabilizing the silver
different stabilizing molecules [54,84]. In the same way, it has been ions, thus carrying out the AgNPs’ formation. Furthermore, the AgNPs
demonstrated that different microorganisms secrete different bio­ synthesized with the supernatant showed greater antimicrobial activity
molecules in distinct proportions, which could affect the stabilization of against E. coli compared to those synthesized with the intracellular
AgNPs [76]. AgNPs with different stabilizing molecules influence anti­ extract, whereas C. necator presented a lower MIC value when compared
bacterial activity, becoming more or less antimicrobial by interacting with B. megaterium and B. subtilis. Therefore, this green synthesis method
differently with certain molecules of the microbial membrane, such as appears to be an eco-friendly way to obtain stable AgNPs with antimi­
proteins [85–87]. crobial properties. Besides, the extracellular synthesis is preferred over
On the other hand, the MIC values obtained in our research are lower the intracellular synthesis because it is easier to recover the molecules
than those found by other authors who have used plant extracts for the responsible for the reduction and stabilization. In addition, according to
synthesis of AgNPs and have evaluated the antimicrobial activity against several investigations, the advantage of the biological method over
E. coli. For example, some research found MIC values between 7.8 and chemical synthesis of nanoparticles is that with the green synthesis of
20.0 μg/mL with AgNPs synthesized from plant extracts [76–78]. AgNPs a greater antimicrobial activity is obtained. On the other hand, to
Otherwise, Rolim et al. [87], determined a MIC value of 15 ug/mL using our knowledge, this investigation is the first to use C. necator, both its
AgNPs synthesized from green tea extract. While, Das et al. (2019), extracellular supernatant and intracellular extracts, for AgNPs synthesis.
obtained a MIC value of 60 µg/ml using AgNPs synthesized from Morus Likewise, it was possible to demonstrate that its use would be promising
alba leaf extract [88]. On the other hand, other investigations that have due to its greater antimicrobial activity compared to AgNPs synthesized
carried out AgNPs synthesis by microorganisms have found similar re­ with Bacillus. Finally, this investigation underscores the need for more
sults to those obtained in our investigation. Akter and Huq (2020), ob­ analysis based on AgNPs green synthesis from intracellular extracts in
tained a MIC value against E. coli of 6.25 ug/mL using AgNPs order to elucidate the synthesis mechanisms with biomolecules such as
synthesized from the extracellular extract of Sphingobium sp. MAH-11T DNA and intracellular proteins, an area which, to date, remains
[81], and other investigations have obtained MIC values between 3.12 understudied.

Table 3 CRediT authorship contribution statement

Minimum inhibitory concentration of the synthesized AgNPs against E. coli.
Supernatant Intracelular extract Iván Solís-Sandí: Investigation, Writing – original draft, Visualiza­
Microorganism MIC (μg/ml) MIC (μg/ml) tion. Sara Cordero-Fuentes: Investigation, Writing – original draft,
B. megaterium 2.750 ± 0.487a 5.087 ± 0.615a Visualization. Reinaldo Pereira-Reyes: Visualization, Data curation,
B. subtilis 1.880 ± 0.340b 5.500 ± 0.001a Writing – review & editing. José Roberto Vega-Baudrit: Resources,
C. necator 0.687 ± 0.151c 8.800 ± 1.230b Writing – review & editing. Diego Batista-Menezes: Investigation,
*Values are presented as means ± standard deviation (SD). Values with different Supervision, Writing – original draft, Visualization. Gabriela Montes de
letters in the same column indicate significant differences (P < 0.05). Oca-Vásquez: Investigation, Supervision, Data curation, Writing –

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original draft, Visualization. Nanotechnol. Environ. Eng. 8 (2022) 377–397, https://doi.org/10.1007/s41204-

022-00276-8.
[16] R.A. Alvarez-Bayona, M. Cortez-Valadez, F. Martínez-Suárez, J.J. Cruz-Rivera,
Declaration of Competing Interest M. Flores-Acosta, Green synthesis approximation of Au/Li nanoparticles with
Opuntia ficus-indica extract, Physica E: Low-Dimens. Syst. Nanostruct. 108 (2018)
169–173, https://doi.org/10.1016/j.physe.2018.12.008.
The authors declare that they have no known competing financial [17] J.G. Bocarando-Chacon, M. Cortez-Valadez, D. Vargas-Vazquez, F. Rodríguez,
interests or personal relationships that could have appeared to influence M. Flores-Acosta, P.G. Mani-Gonzalez, E. Leon-Sarabia, A. Navarro-Badilla,
the work reported in this paper. R. Ramírez-Bon, Raman bands in Ag nanoparticles obtained in extract of Opuntia
ficus-indica plant, Physica E: Low-Dimens. Syst. Nanostruct. 59 (2014) 15–18,
https://doi.org/10.1016/j.physe.2013.12.006.
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López, M. Flores-Acosta, Silver nanoparticle-decorated silver nanowires: a
nanocomposite via green synthesis, Appl. Phys. A 126 (2020) 15, https://doi.org/
Data will be made available on request.
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jechem.2021.10.013.
[20] Huq MdA, Ashrafudoulla Md, M.M. Rahman, S.R. Balusamy, S. Akter, Green
The authors would like to express their thanks for the financial synthesis and potential antibacterial applications of bioactive silver nanoparticles:
support provided by Laboratorio Nacional de Nanotecnología (LAN­ a review, Polymers (Basel) 14 (2022) 742, https://doi.org/10.3390/
polym14040742.
OTEC), Centro Nacional de Alta Tecnología (CeNAT-CONARE). They [21] Huq MdA, Paenibacillus Anseongense Sp. Nov. a silver nanoparticle producing
also thank Frank Solano-Campos, Escuela de Ciencias Biológicas, Uni­ bacterium isolated from rhizospheric soil, Curr. Microbiol. 77 (2020) 2023–2030,
versidad Nacional, Costa Rica, for carrying out the DNA and protein https://doi.org/10.1007/s00284-020-02086-0.
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quantification. Optimization for biogenic microbial synthesis of silver nanoparticles through
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