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OPEN Silver nanoparticles biosynthesis

using mixture of Lactobacillus sp.
and Bacillus sp. growth and their
antibacterial activity
Morad G. S. S. Al‑asbahi 1*, Bashir A. Al‑Ofiry 1, Fuad A. A. Saad 2, Adnan Alnehia 3 &
Murad Q. A. Al‑Gunaid 4
The biosynthesis of nanoparticles offers numerous advantages, including ease of production, cost-
effectiveness, and environmental friendliness. In our research, we focused on the bioformation of
silver nanoparticles (AgNPs) using a combination of Lactobacillus sp. and Bacillus sp. growth. These
AgNPs were then evaluated for their biological activities against multidrug-resistant bacteria. Our
study involved the isolation of Bacillus sp. from soil samples and Lactobacillus sp. from raw milk in
Dhamar Governorate, Yemen. The synthesized AgNPs were characterized using various techniques
such as UV–visible spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy
(FTIR), and transmission electron microscopy (TEM). The antibacterial properties of the AgNPs were
assessed using the modified Kirby Bauer disk diffusion method against multidrug-resistant strains
of Staphylococcus aureus and Pseudomonas aeruginosa. Our results demonstrated that the use of a
bacterial mixture for biosynthesis led to faster and more effective production of AgNPs compared
to using a single bacterium. The UV–visible spectra showed characteristic peaks indicative of silver
nanoparticles, while XRD analysis confirmed the crystalline nature of the synthesized particles. FTIR
results suggested the presence of capping proteins that contribute to the synthesis and stability of
AgNPs. Furthermore, TEM images revealed the size and morphology of the AgNPs, which exhibited
spherical shapes with sizes ranging from 4.65 to 22.8 nm. Notably, the antibacterial activity of
the AgNPs was found to be more pronounced against Staphylococcus aureus than Pseudomonas
aeruginosa, indicating the potential of these nanoparticles as effective antimicrobial agents. Overall,
our study highlights the promising antibacterial properties of AgNPs synthesized by a mixture of
Lactobacillus sp. and Bacillus sp. growth. Further research is warranted to explore the potential of
utilizing different bacterial combinations for enhanced nanoparticle synthesis.

The topic of nanoparticles in particular has attracted attention in a variety of areas in recent years. The word
“nano” refers to a dimension in the order of a billion (­ 109) metres and is derived from the Greek term “nanos”,
which means dwarf. The surface-to-volume ratio is the most important property of nanoparticles, enabling them
to interact more quickly with other p ­ articles1.
Due to the high biological, physical and chemical properties of silver nanoparticles, which are widely
researched and used in medicine, cultivation and environmental remediation, the synthesis method for silver
nanoparticles has recently undergone significant ­development2–5. These properties of silver nanoparticles are
mainly due to the crystallinity, composition, size, shape and structure of silver nanoparticles compared to their
large ­form6. Silver nanoparticles (AgNPs) have high antibacterial activity against susceptible and multi-drug
resistant (MDR) bacterial ­strains7, so they have long been used as broad-spectrum antimicrobials against many
pathogenic and non-pathogenic microbes in various industrial areas such as textiles and food p­ ackaging8. The use
of antibiotics is becoming increasingly ineffective due to the development of multi-resistant strains of ­bacteria9,
which means that innovative techniques are needed to cure bacterial infections. Silver nanoparticles are sub-
stances used in these t­ echniques10.

1
Department of Biology, Faculty of Sciences, Sana’a University, 12081, Sana’a, Yemen. 2Department of Biology,
Faculty of Applied Sciences, Thamar University, 87246, Dhamar, Yemen. 3Department of Physics, Faculty of
Applied Sciences, Thamar University, 87246, Dhamar, Yemen. 4Department of Chemistry, Faculty of Education,
Thamar University, 87246, Dhamar, Yemen. *email: [email protected]

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There are various methods used to synthesis silver nanoparticles, including biological, chemical and physical
methods. However, the chemicals used in many of these methods are toxic, flammable and not easily removed,
posing a risk to individuals and the ­environment11,12. Biosynthesis seems to be a promising area to synthesize
nanoparticles in an environmentally friendly, simple, safe, cost-effective and time-saving ­way13–15. Fungi, bacteria,
algae and plants are increasingly being used to biosynthesize n ­ anoparticles16. Interestingly, when silver nanopar-
ticles are synthesized from silver metal using biological methods, it has been observed that their antimicrobial
activity increases while their toxicity d ­ ecreases17. Some of the bacteria that have been used on a large scale for
the bioformation silver nanoparticles are Escherichia coli, Lactobacillus casei, Aeromonas sp. Pseudomonas pro-
teolytica, Bacillus cereus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus indicus, and Bacillus cecembensis18.
Bacteria synthesize nanoparticles in different ways, depending on the type of bacteria. Some bacteria synthesize
nanoparticles via extracellular synthesis and other bacteria via intracellular s­ ynthesis1.
The reduction of metal ions to NPs by plant extractors is swifter than the use of ­microorganisms19, but the
synthesis of nanoparticles by plant extractors may require heating to 85 °C, which can be expensive when large
amounts of nanoparticles are ­synthesized1. Not getting raw materials and harvesting the plants at the wrong time
may be also a challenge to biosynthesis of the nanoparticles by plant ­extractors20. To improve the efficiency of
microorganism in biosynthesis, pharmaceuticals production, biodegradation and food industry, a mixture of
microorganisms is used, which is called coculture, co-culture, binary culture, heteroculture, heterotypic culture,
mixed culture, dual culture, bi-culture, tri-cultivation, co-localization or co-cultivation. The System of mixed
cultivation will be fundamental importance for the advancement of synthetic biology. The study of natural inter-
action between cells will spotlight new ways of reengineering even in organisms that are difficult to ­cultivate21.
The synthetic/artificial co-culture systems surpass the limitations of monocultures or consortia in nature with
the added advantages in exploring allelopathic interactions in food industries involving fermentation and natural
product/drug ­discovery22. The use of a mixture of different microorganism cultures is gaining great interest in
bioleaching, decontamination and bioelectrical production, where the use of a single microorganism has so far
not yielded good ­results23. The results of Grujić et al.24 show that the mixed biofilm (Escherichia coli and Rhodoto-
rula mucilaginosa) has the highest efficiency in the removal of ­Zn2+ (99.26%), ­Pb2+ (99.52%) and ­Cu2+ (99.88%).
The metal removal efficiency was within the limits of 94.99–99.88% for the mixed biofilm and 81.56–97.85% for
the single biofilm. The experiments of Yang et al.25 have shown that the degradation of antibiotics in wastewater
is better than the use of a single antibiotic and that the use of mixed aerobic and anaerobic conditions practerial
species. Nwokoro and D ­ ibua26 also found that the use of a mixture of Pseudomonas stutzeri and Bacillus subtilis
was more effective in removing cyanide from soil than the use of a single bacterium.
Most studies on the biosynthesis of silver nanoparticles by bacteria using a bacterial genus. D ­ akhil27 used a
mixture of Lactobacillus species and found that silver nanoparticles were biosynthesized by a mixture of Lacto-
bacillus acidophillus and Lactobacillus plantarum and had a typical shape (spherical) and a size of 30–100 nm,
he also found that the distribution of silver nanoparticles formed by this mixture was more consistent than the
nanoparticles formed by a mixture of Lactobacillus acidophillus and Lactobacillus bifidus.
The use of more than one bacterial genus in the synthesis of silver nanoparticles can improve and accelerate
the synthesis process. Therefore, in the present study, the synthesis of AgNPs by mixing the growth of Lacto-
bacillus sp. and Bacillus sp. was investigated and the biological activities of AgNPs against multidrug-resistant
bacteria were evaluated.

Materials and methods

Materials
AgNO3, nutrient agar, nutrient broth, MRS agar, Mueller–Hinton agar, UIM medium, methyl red (MR) medium,
egg yolk agar and Simmon citrate medium from Himedia Laboratories. Carbohydrate discs, H ­ 2O2, Gramme
stain and Ziehl-Nelseen stain. Sterile physiological solution (NS; 0.9 w/v% sodium chloride; pH 4.5–7.0). Tubes,
absorbent cotton, swabs and loops. Deionized water (DW) was used to prepare the culture media and wash the
silver nanoparticles.

Isolation and identification of bacteria

The soil samples were collected in the vicinity of the car repair shops in Dhamar City, Yemen. The collected soil
samples were transported to the laboratory in sterile polyethylene bags. Ten grams of soil were soaked in 90 ml
of sterile physiological solution (0.9% w/v) and to kill non-spore forming bacteria, the soil solution was treated
in a water bath at 60 °C for one hour to isolate Bacillus sp. fill loop from each of the soil solutions, was cultured by
swabbing on nutrient agar medium and incubated at 35 °C for 1 ­day28. The raw milk samples from the cows were
collected in rural Dhamar Governorate and stored in a sterilized glass bottle. The milk samples were stored at 4
°C and then transported to the laboratory. After the formation of whey in the milk, Lactobacillus sp. was isolated
by plating the whey milk on MRS agar whose PH was modulated to 5.4 by acetic acid to facilitate the isolation
of the genus Lactobacillus sp.29. The isolated bacteria were identified on the basis of their colonies, their form
on the culture media, Gram staining, biochemical and physiological characteristics according to the procedures
described in Bergey’s Manual of Systematic ­Bacteriology30,31.

Bacteria growth in nutrient broth

Lactobacillus sp. was cultivated in a flask with 100 ml nutrient broth and a flask with 50 ml nutrient broth. The
same was repeated with Bacillus sp. All flasks were incubated at 30 °C on a shaker and shaken at 120 rpm for 24 h.

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Bio‑synthesized silver nanoparticles

After the bacteria had passed into the logarithmic phase, silver nitrate was added to the flask containing 100 ml
of Lactobacillus sp., to the flask containing 100 ml of Bacillus sp. and to the flask containing 100 ml of mixed
growth of Lactobacillus sp. and Bacillus sp., the concentration of silver nitrate in each flask being 2 mM. The
flasks were incubated at 30 °C on a shaker at 140 rpm for 24 h to obtain the reaction, noting a color change in the
flasks every half hour. This work was carried out twice. The color change of the solution determines the forma-
tion of AgNPs. To obtain silver nanoparticles from the growth medium solution, the solution was centrifuged
at 10,000 rpm for 20 min. To prevent any component of the growth medium from influencing the properties of
the nanoparticles, they were washed three times with distilled water. The silver nanoparticles were dried at 70
°C for 15 h and processed into fine powder for their c­ haracterization32,33.

Characterization
The silver nanoparticles were analyzed using different analytical techniques, i.e. UV–visible, X-ray diffraction
(XRD), Fourier transform infrared (FTIR) and transmission electron microscopy (TEM). Five ml (5 mL) of the
supernatant of the reaction solution was analyzed using a UV–Vis spectrophotometer (Hitachi, Tokyo, Japan)
at room temperature to determine the optical properties of the silver nanoparticles. The powder samples of
silver nanoparticles were subjected to X-ray diffraction (XRD) analysis. The XRD style was registered by an
XD–2 X-ray diffractometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) with CuKα radia-
tion of λ = 1.5418Å, over 2-theta 15–75° and scanning rate of 0.02 m­ in−1. Fourier-Transform Infrared was used
to identify the interaction between biomolecules and AgNPs. FTIR pattern was registered on FTIR in the extent
of 400–4000 c­ m−1 at a precision of 4 c­ m−1. AgNPs shapes were evaluated by TEM. Diluted sliver nanoparticles
were placed onto carbon-coated copper TEM meshs.

Antibacterial activity
The antibacterial activities of AgNPs were investigated using the modified Kirby-Bauer disk diffusion ­method34.
The antibacterial test was carried out against multi-resistant Staphylococcus aureus and Pseudomonas aeruginosa.
After activation of the bacteria by cultivation on nutrient agar for 24 h and separate turbidity of the bacteria
of each species in sterile normal saline solution, the turbidity corresponded to the turbidity of barium sulfate
(turbidity standard according to McFarland 0.5). Muller–Hinton agar plates were inoculated with a sterile swab
with bacterial turbidity. Sterilized filter leaf disks with a diameter of 5 mm impregnated with biosynthetic silver
nanoparticles of different concentrations (10, 20 and 40 μg) were placed on the Muller–Hinton agar plates. The
inhibition area was measured in mm to determine the inhibition zone.

Hemolytic assay
In order to evaluate the biological safety of silver nanoparticles biosynthesized by bacteria, these particles were
added to red blood cells in different concentrations, according to Guowei et al.35 with a slight modification of his
method. Half a millilitre of fresh blood was taken from a young person of group ­O+ in a tube containing an anti-
coagulant. Then the red blood cells (RBCs) were separated by centrifugation at 5000 rpm for 10 min, the plasma
was discarded and the blood cells were washed three times with normal saline. The erythrocyte suspension was
then prepared in physiological solution (pH 4.5–7.0) to obtain a 2% cell suspension. Silver nanoparticles were
prepared in physiological solution to final concentrations of 3.9–500 μg/ml. 0.5 ml of the erythrocyte suspen-
sion was added to 9 tubes, then 0.5 ml of different concentrations of silver nanoparticles were added separately
to 7 tubes, while 0.5 ml of the physiological solution was added to tube No. 8 as a negative control and 0.5 ml of
distilled water as a positive control. After incubation for 1 h at 37 °C and gentle shaking, the tubes were placed
in the centrifuge for 5 min at 5000 rpm and then the hemolysis was measured with the spectrometer at 540 nm.
The hemolytic activity was evaluated by the following equation:
Percentage of hemolysis = (AS − AN / AP − AN ) × 100
where ­AS is the absorbance of the test sample, A
­ N is the absorbance of the negative control and A
­ P is the absorb-
ance of the positive control.

Results and discussion

Isolated bacteria features
Morphological characterization revealed that both the cultures of Lactobacillus sp. and Bacillus sp. contained
Gram-positive bacilli. Biochemical characterization revealed that both the Lactobacillus sp. and Bacillus sp.
(Table 1).

Silver nanoparticles biosynthesis

The ability to biosynthesis of silver nanoparticles by the culture of Lactobacillus sp. and Bacillus sp. separately
was showed, and the ability to biosynthesis of silver nanoparticles by mixture of Lactobacillus sp. and Bacillus
sp. growth also was showed. The silver nanoparticles formation in the culture liquid is observed by the appear-
ance of brown ­color36. The brown color appeared in the aqueous solution of AgNPs due to agitation by surface
­plasmons37. In the flask containing a mixture of Lactobacillus sp. and Bacillus sp., a dark orange color appeared
within less than 1 h (Fig. 1), while in the flasks containing only Lactobacillus sp. and Bacillus sp., the appearance
of a dark orange color was observed only after 16 h. The use of a mixture of bacteria for biosynthesis is faster
and more effective than the use of a single bacterium. This could be due to the fact that the metabolic products
of one bacterium can be utilized by other bacteria and that the interaction between the bacteria increases the

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Test Lactobacillus sp. Bacillus sp.

Shape Rod Rod
Gram stain + +
Motility − +
Catalase test − +
Sporing − +
Anaerobic growth −
Voges proskauer − +
Citrate Utilization +
Egg yolk reaction −
Fermentation
Arabinose + +
Lactose +
Mannose +
Mannitol − +
Sorbitol +
Fructose −
Galactose −
Xylose +

Table 1.  Biochemical characterization of Lactobacillus sp. and Bacillus sp. +  = positive; − = negative.

Figure 1.  Color change to a dusky orange in the middle flask that contains a mixture of Lactobacillus sp. and
Bacillus sp. growth which indicates the formation of AgNPs.

­ iosynthesis23,38. In a study on the biosynthesis of AgNPs by Bacillus cereus, the color changed
effectiveness of b
to deep orange after the addition of A ­ gNO3 to the Bacillus cereus supernatant after 24 h
­ 39. In another study, the
Ag ions ­(Ag+) were minimized to AgNPs within 18 h after the addition of ­AgNO3 to the cell-free supernatant
of Bacillus subtilis40.

Characterization of AgNPs
UV–VIS analysis
The bioformation of silver nanoparticles was characterized by UV–visible spectroscopy in the range of 200–500
­nm41. In our study, we observed a broad peak in the range between 410 and 430 nm in silver nanoparticles
biosynthesized by bacteria due to their surface plasmon resonance absorption band (Fig. 2). Our results are
close to the findings of previous studies, which suggested that a common AgNps surface plasmon resonance
­ m42. Our study showed that the peak of
(SPR) absorption band exists at a wavelength in the range of 400–480 n
silver nanoparticles formed by mixed growth of Lactobacillus sp. and Bacillus sp. was sharper and more intense
(absorbance at 3.2) (Fig. 2A). Increasing the number of nanoparticles synthesized in aqueous solution leads to
­ eak27.
an increase in the intensity of the SPR p

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LB 2.0
3.5 B L
B
3.0
1.5
2.5
Absorbance (a.u.)

Absorbance (a.u.)
2.0
1.0
1.5

1.0 0.5

0.5

0.0 0.0

300 350 400 450 500 550 600 650 700 750 800 300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm) Wavelength (nm)

Figure 2.  UV–Vis absorption spectra of AgNPs synthesized using (A) mixture Lactobacillus sp. and Bacillus sp.
growth (B) the growth each bacteria individually.

Surface plasmon resonance and UV–Vis spectra analysis according to time. After the appearance of a dusky
orange color in the flask containing a mixture of Lactobacillus sp. and Bacillus sp. 5 ml of each flask was sampled
and measured by UV–visible spectroscopy and measured every 8 h Fig. 3.
When measured by UV at less than an hour of incubation, we found that silver nanoparticles were not
formed by each bacterium separately, while we found the formation of nanoparticles when using a mixture of
Lactobacillus sp. and Bacillus sp. growth. The maximum time to complete the synthesis of silver nanoparticles
by a mixture of Lactobacillus sp. and Bacillus sp. growth was 16 h. The maximum time for complete synthesis by
Bacillus sp. alone is 40 h and more than 40 h by Lactobacillus sp. alone. The passage of reaction time increases
the intensity of absorption until the reaction is c­ ompleted43. Lv et al.44 found that the depletion of silicon from
electrolytic manganese residues with a mixture of Paenibacillus mucilaginosus and Bacillus circulans was better
and faster than using the individual bacteria.
In their study, El-Saadony et al.45 found that the maximum time for the synthesis of silver nanoparticles by
Bacillus subtilis ssp spizizenii MT5 was 40 h. In another study, Tariq et al.46 found that the reaction in the synthesis
of silver nanoparticles by Bacillus subtilis was completed after 72 h. Matei et al.47 reported in their study that the
synthesis of AgNPs by Lactobacillus plantarum was completed after 24 h, while Chaudhari et al.48 found that the
reaction was completed after 3 days when 2 isolates of Lactobacillus species were used.

XRD of AgNPS
The crystalline properties and purity of the silver nanoparticles synthesized by bacteria were determined by
XRD. The X-ray diffraction pattern of silver nanoparticles biosynthesized by the growth of Lactobacillus sp.
and Bacillus sp. Nine peaks were detected on XRD at 2θ values of 27.87°, 32.32°, 38.23°, 44.44°, 46.22°, 54.8°,
57.34°, 64.63° and 77.39°, which correlate with the reflectance of (111), (200), (220), (222) and (311), respectively
(JCPDS-ICDD files No 04-0783), as shown in Fig. 4. Our results were close to the results of previous studies on
the characterization of AgNPs by ­XRD17,49. The purity of silver nanoparticles was achieved in the absence of other
external peaks, suggesting that the use of two types of bacteria is an effective method for obtaining high-purity
silver nanoparticles. However, the undefined peaks at 2θ° of 9.538 and 10.45 in the L and B profiles, respectively,

LB
3.5 B
L
3.0
Absorbance at 410 nm

2.5

2.0

1.5

1.0

0.5

0.0

0 4 8 12 16 20 24 28 32 36 40 44 48 52
Less than 1h Time (h)

Figure 3.  Rate synthesis silver nanoparticles according to time.

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L
(111)
B
(200) LB

(200) (220)
(220)
(311)
(311) (222)

Intensity (a.u.)
(111)

10 20 30 40 50 60 70 80
2 Theta

Figure 4.  XRD spectra of AgNPs synthesized using mixture of Lactobacillus sp. and Bacillus sp. growth (LB)
and Lactobacillus sp. and Bacillus sp. growth separately (L and B).

could be due to some organic matter in the growth ­medium50. Regarding the silver nanoparticles biosynthesized
in our study, we found that the particles biosynthesized by a mixture of Lactobacillus sp. and Bacillus sp. bacteria
were purer than the particles synthesized by each bacterium individually, with peaks at 2θ° with values of 38.23°,
­ anoparticles17.
44.44°, 64.63° and 77.39° indicating the high purity of silver n

FTIR analysis
The dried silver nanoparticles were analyzed by FTIR to determine the presence of a protein around the silver
nanoparticles that could be responsible for the synthesis and stability of the silver nanoparticles. Figure 5 shows
the FTIR spectra of the nanoparticles synthesized using a mixture of Lactobacillus sp. and Bacillus sp. (LB), Lac-
tobacillus sp. (L) and Bacillus sp. (B). The FTIR spectrum of AgNps shows that the peaks at 3448.1, 3433.05 and
3432.67 ­cm−1 are due to the existence of the –NH or –OH group and some free ­amides51,52, but these peaks were
weak, indicating the reduction of A ­ g+ by –OH ­groups53 and the amine –NH chelated by ­Ag+ ­ions54.
The observed peaks show from 2935 to 2920 c­ m−1 and 2851.13 ­cm−1 due to the tremolization of the sym-
metric and asymmetric stretching of the C-H functional group, which represent the status of the aliphatic and
aromatic ­styles55,56.
The absorption around1735.62 and 1708.81 ­cm−1 is due to the C = O stretching vibration absorption of
­amides57, and the peaks at 1640.15, 1634.95, and 1632.67 c­ m−1 show the presence of alkenyl C=C in the proteins
(amide I)56. These peaks appear weak in Fig. 5, indicating the oxidation of C=O and C=C ­bonds54. The absorbance

1640.15
1453.64
LB
2851.13
2934.54 1735.62 628.07
3448.1
1530.28 1075.76
Transmittance (%)

1360

1453.24
B

3433.05 2920.30 1708.81

1632.67 1087.01

1357.87
L

3432.67 2924.56 1634.95

1075.34
1518.53
1356.95

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

Figure 5.  FTIR spectra of AgNPs synthesized using mixture of Lactobacillus sp. and Bacillus sp. growth (LB)
and Lactobacillus sp. and Bacillus sp. growth separately (L and B).

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around 1530.28 and 1518.53 ­cm−1 as a result of NH stretching in amide ­II53, and the peaks at 1453.64 and 1453.24
­cm−1 were attributed to C-H binding of methylene in p ­ roteins53,55. The strong peaks at 1360, 1357.87 and 1356.95
­cm are due to aromatic amine or CN stretches. The observed peak at 1075.76 ­cm−1 probably indicates the
−1

tensile tremolo of the C–O s­ tretch58. FTIR results have demonstrated the presence of a capping protein in silver
nanoparticles from growth ­bacteria59. The carboxyl group C=O, the hydroxyl group –OH and the free amine
–NH of the bacterial protein may be responsible for the synthesis and stability of the silver n ­ anoparticles60–62.
Figure 5 shows many convenient groups on the outside of silver nanoparticles synthesized using a mixture of
Lactobacillus sp. and Bacillus sp. compared to silver nanoparticles synthesized by the individual bacteria. In the
mixed growth of two types of bacteria, the total amount of protein produced is higher than the protein produced
when each bacterium grows s­ eparately63.

TEM
The TEM image of the silver nanoparticles synthesized by bacteria is shown in Fig. 6. The TEM micrograph
shows the size of the silver nanoparticles ranging from 4.65 to 11.3 nm for the nanoparticles synthesized by a
mixture of Lactobacillus sp. and Bacillus sp., 7.97–14.3 nm for the nanoparticles synthesized by Lactobacillus sp.
and 11–22.8 nm of the nanoparticles synthesized by Bacillus sp. Electron micrographs showed that the silver
nanoparticles synthesized by a mixture of Lactobacillus sp. and Bacillus sp. were well distributed, indicating that
these nanoparticles are more stable than those synthesized by each bacterium individually. The discrete distribu-
tion of AgNPs seen in the TEM could be due to clogging by ­proteins36.

Inhibition zones of AgNPs on two multidrug resistant bacteria

The biosynthesized AgNPs were tested for their antibacterial activity against multidrug-resistant bacteria (Staphy-
lococcus aureus (Gram positive) and Pseudomonas aeruginosa (Gram negative)) isolated from clinical samples.

Figure 6.  TEM images of AgNPs synthesized by (a) mixture Lactobacillus sp. and Bacillus sp. growth; (b)
Lactobacillus sp. (c) Bacillus sp.

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The antibacterial activity was determined using the disc diffusion method at three different concentrations of
10, 20 and 40 μg. The diameter of the inhibition zone (mm) around each disc with silver nanoparticles is shown
in Table 2. The silver nanoparticles synthesized with a mixture of Lactobacillus sp. and Bacillus sp. and each
bacterium individually showed antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa
(Table 2 and Fig. 7). Our results were very close to the results of previous s­ tudies43,64.
In this study, we found that silver nanoparticles synthesized by a mixture of Lactobacillus sp. and Bacillus sp.
exhibited greater antibacterial activity than silver nanoparticles synthesized by each bacterium individually. This
greater efficacy could be due to the small size of the silver nanoparticles synthesized by the bacterial mixture
(4.65–11.3 nm). The increase in the effectiveness of silver nanoparticles in killing bacteria depends on the size of
the NPs, as smaller NPs have a larger outer surface area, so their effectiveness in killing bacteria is greater than
larger ­NPs42,63,65,66, and this effectiveness also depends on the reducing agents and ­stabilizers67.

Inhibition zone (mm)

(mean ± SD of 3 replicates) Control negative
Ag ­NO3
Bacteria 10 µg/ml 20 µg/ml 40 µg/ml 40 µg/ml
Staphylococcus aureus 12 ± 0.4 15 ± 0.1 20 ± 0.2 –
AgNps from LB
Pseudomonas aeruginosa 10 12 ± 0.1 16 ± 0.22 –
Staphylococcus aureus 10 ± 0.43 12 ± 0.24 16 ± 0.34 –
AgNPS from L
Pseudomonas aeruginosa 8 ± 0.76 11 ± 0.5 13 ± 0.3 –
Staphylococcus aureus 9 ± 0.43 11 ± 0.12 14 ± 0.22 –
AgNPs from B
Pseudomonas aeruginosa - 10 ± 0.23 12 ± 0.25 –

Table 2.  Antibacterial activity of AgNPs using the disc diffusion method. LB Lactobacillus sp. and Bacillus sp.,
L Lactobacillus sp., B Bacillus sp.

AgNPs from LB
AgNPs from L
P. aeruginosa AgNPs from B
20
Zone inhibition (mm)

16
15
13
12 12
11
10 10
10
8

0
0
10 µg/ml 20 µg/ml 40 µg/ml
AgNPs concentration AgNPs from LB
AgNPs from L
S. aureus AgNPs from B
20
20
Zone inhibition (mm)

16
15
15 14
12 12
11
10
10 9

0
10 µg/ml 20 µg/ml 40 µg/ml
AgNPs concentration

a b

Figure 7.  (a) Effectiveness tests against bacteria for AgNPs synthesized by a mixture Lactobacillus sp.
and Bacillus sp. (LB), Lactobacillus sp. (L) and Bacillus sp. (B): (b) Diagram showing silver nanoparticles
synthesized by a mixture of Lactobacillus sp. and Bacillus sp. growth had greater antibacterial activity than silver
nanoparticles synthesized by each bacteria separately.

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In our study, the antibacterial activity of AgNPs synthesized by bacteria was higher against Staphylococcus
aureus than against Pseudomonas aeruginosa. The reason that Pseudomonas aeruginosa was less susceptible to
silver nanoparticles may be that these Gram-negative bacteria have an outer membrane that is difficult to pen-
etrate and accumulate silver nanoparticles in the cell by forming efflux pumps with outer membrane proteins.
These efflux pumps excrete harmful substances, including biocides and ­antibiotics68. The mechanism of resist-
ance through efflux pumps may be naturally present in bacteria or may have arisen through mutation or gene
­transfer69.
The increased efficacy of AgNPs against Staphylococcus aureus could also be due to the fact that these AgNPs
were synthesized by Gram-positive bacteria, as Singh and M ­ ijakovic8 found in their study that silver nano-
particles produced by a bacterial species are more effective against bacteria closely related to that species. The
effectiveness of silver nanoparticles against microbes is due to several mechanisms, adhesion to the cell wall and
plasma membrane of the microbial cell, resulting in increased cell ­permeability57 or destruction of the plasma
membrane and leakage of cell ­components70,71. Formation of reactive oxygen species (ROS) and free radicals
and modification of microbial signal transduction pathways, which damage DNA and the cell wall and increase
the permeability of the plasma ­membrane6,72. Prevention of DNA replication through the release of silver ­ions73
which are bound to the phosphorus in the D ­ NA74.

Hemolytic of AgNPs
The hemolytic assay was performed with different concentrations of AgNPs (3.9–500 μg/ml) to evaluate the
potential toxicity of the nanoparticles on human RBCs and used normal saline as negative control and distilled
water as positive control. Figure 8 illustrates the averaged data obtained from the two experiments. In our study,
the hemolysis of RBCs by AgNPs at concentration 500 µg/ml, 250 µg/ml, 125 µg/ml. 62.5 µg/ml and 31.25 µg/
ml were 8.8, 8.3, 7.77, 7.16 and 6.7% respectively, while slightly hemolysis at concentration 15.62 and 7.8 were
3.97 and 2.28% (Table 3). The non-hemolysis effect at 3.9 µg/ml, where the hemolysis was 1.8%. Substances
causing hemolysis > 5% were classified as toxic, while those causing hemolysis 2–5% were less toxic and while
that cause hemolysis < 2% were non-toxic50,75. Our results showed that the silver nanoparticles had no toxicity
compared to the silver nanoparticles in the study by Rajora et al.59 where they found that hemolysis was 3.58%

Figure 8.  Hemolysis of RBCs caused by AgNPs at different concentrations (3.9–500 μg/mL), physiological
solution (negative control), and distilled water (positive control).

No Transactional Hemolysis
1 500 µg/ml 8.8%
2 250 µg/ml 8.3%
3 125 µg/ml 7.77%
4 62.5 µg/ml 7.16%
5 31.25 µg/ml 6.7%
6 15.6 µg/ml 3.97%
7 7.8 µg/ml 2.28%
8 3.9 µg/ml 1.8%
9 CN (NS) 0
10 CP (DW) 100%

Table 3.  Hemolytic efficacy of silver nanoparticles on RBCs. CN control negative, CP control positive, NS
normal slain, DW distilled water.

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at a concentration of 0.5 for silver nanoparticles, 4.7% at 1 µg and 8.2% at 2 µg. The rate of hemolysis increases
with increasing concentration and small size of the silver n­ anoparticles76.

Conclusions
In our study, we observed that the combination of Lactobacillus sp. and Bacillus sp. in the biosynthesis of silver
nanoparticles (AgNPs) resulted in a faster and more effective process than the use of a single bacterium. Interest-
ingly, the AgNPs produced by the mixed growth of Lactobacillus sp. and Bacillus sp. were found to be purer than
those synthesized by each individual bacterium. Furthermore, these AgNPs exhibited enhanced antibacterial
properties, particularly against Staphylococcus aureus, compared to Pseudomonas aeruginosa. It is noteworthy that
the silver nanoparticles generated in our study showed no signs of toxicity, unlike those reported in other studies.
Additionally, the silver nanoparticles biosynthesized by bacteria in our research were deemed safe and non-toxic.

Data availability
The data used to support the findings of this study are included within the article.

Received: 22 January 2024; Accepted: 16 April 2024

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Acknowledgements
The authors would like to thank Al-Hikma University Dhamar, represented by Dr. Salah Misfer, for providing
their facilities and laboratories for this research. The authors would also like to thank Sabaa Bagah, Amani Al-
Aghbari and Mayada Al-Asbahi for facilitating the work in the university’s laboratory.

Author contributions
M.G.S.S.A. and B.A.A.: Conceptualization; M.G.S.S.A. and A.A: Methodology; B.A.A., F.A.A. and M.Q.A.A.:
Validation; M.G.S.S.A. and A.A.: Formal Analysis; M.G.S.S.A. and A.A.: Investigation; M.G.S.S.A. and A.A.:
Resources; M.G.S.S.A. and A.A.: Data curation; M.G.S.S.A., B.A.A. and F.A.A.: Writing-Original Draft Prepara-
tion; M.G.S.S.A., B.A.A. and F.A.A.: Writing-Review & Editing; M.G.S.S.A. and B.A.A.: Visualization; B.A.A. and
F.A.A.: Supervision. All authors have read and agree to the published version of the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
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