Indones. J. Chem.

, 2024, 24 (5), 1445 - 1455 1445

Green Synthesis of Silver Nanoparticles with Bioreductant from Lime Juice Powder
(Citrus aurantifolia): Effect of Concentration and pH

Septian Dwi Mulyana, Retno Sari*, and Agus Syamsur Rijal

Department of Pharmaceutical Science, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60115, Indonesia

* Corresponding author: Abstract: Significant development in antibacterial agents derived from metal
nanoparticles is currently underway. One commonly employed type is silver nanoparticles
email: [email protected]
(AgNPs), which display potential antibacterial activity at lower concentrations compared
Received: April 2, 2024 to other metals. Due to their high surface-to-volume ratio and small size, AgNPs can
Accepted: May 8, 2024 readily penetrate bacterial cell walls. Green synthesis methods of AgNPs, such as utilizing
DOI: 10.22146/ijc.95283 plants as reducing agents, offer substantial advantages over other synthesis techniques.
Lime (Citrus aurantifolia), containing compounds like flavonoids and saponins, can serve
as a natural reducing agent, converting Ag+ ions to Ag0. This study aims to evaluate the
effects of AgNO3 concentration, lime juice powder concentration, and pH on the
formation and characteristics of AgNPs, as well as their activity against the bacteria
Propionibacterium acnes, Staphylococcus aureus, and Escherichia coli. Results indicate
that at an AgNO3 concentration of 1 mM, lime juice extract at 1% with a pH of 9 produces
optimal AgNP formation, with an absorbance of 4.631 and particle size of 68.4 nm.
AgNPs exhibit higher antibacterial activity than AgNO3. AgNPs synthesized with lime
juice powder can increase their activity and have the advantages of being safe and
environmentally friendly since they use plant material as a reducing agent.

Keywords: silver nanoparticles; green synthesis; lime juice powder; pH; antibacterial
activity

■ INTRODUCTION Silver nanoparticles (AgNPs) have garnered

considerable attention due to their unique
In recent years, nanotechnology has garnered
physicochemical properties compared to other
significant attention in the realm of future science and
materials, characterized by a high surface area-to-
technology. Nano-sized materials (nanomaterials) offer
volume ratio, surface energy, and chemical reactivity [4].
distinct advantages such as large surface area and particle
With sizes ranging from 1–100 nm, AgNPs have
sizes ranging from 1–100 nm. Metal nanoparticles have
demonstrated potential applications in cancer therapy,
emerged as a focus of research within this domain. Metals
wound treatment, disinfection, and antibacterial agents
possess inherent antibacterial properties due to their [5]. Conventionally, AgNPs are synthesized through
ability to release metal ions that are toxic to bacteria [1]. chemical or physical means, which involve hazardous
Surface modification of metals is undertaken to enhance substances and high temperatures or pressures,
antibacterial activity and improve biocompatibility. The rendering them environmentally unfriendly and
antimicrobial efficacy of metal nanoparticles stems from expensive. Consequently, there has been a shift towards
their nano-sized dimensions and extensive surface area, green synthesis methods, using plants, algae, bacteria,
facilitating specific interactions with bacterial structures and fungi for more economical, eco-friendly, and non-
[2]. Various metals, including silver (Ag), gold (Au), zinc toxic AgNPs production [6].
oxide (ZnO), copper (Cu), and iron (Fe), have been Lime (Citrus aurantifolia) is a medicinal plant rich
investigated for their antibacterial properties [3]. in bioactive compounds such as saponins, alkaloids,

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1446 Indones. J. Chem., 2024, 24 (5), 1445 - 1455

tannins, phenolates, flavonoids, and terpenoids, exhibiting As one of the crucial factors, pH influences the size
antioxidant, anti-inflammatory, antitumor, and and morphology of nanoparticles prepared through green
antimicrobial activities [7]. The functional groups present synthesis. This is due to the electrical charge of biomass
in plant biomolecules can act as reducing agents in the and capping agents undergoing significant alteration
conversion of Ag+ ions to Ag0 [8]. Lime has antimicrobial under varying pH conditions, leading to alterations in
potential and is effective against both Gram-positive and their capability to bind and reduce metal ions [15-16].
Gram-negative bacteria, besides serving as an effective Several reports suggest that acidic environments yield
reducing and capping agent for AgNPs stability [9]. larger-sized AgNPs, whereas smaller-sized AgNPs are
AgNPs size and shape have a role in their formed in alkaline environments [17-18]. AgNPs
effectiveness as antibacterial agents. Smaller-sized AgNPs synthesized using Galega officinalis extract in an alkaline
(< 100 nm) demonstrate superior antibacterial activity pH environment result in smaller-sized AgNPs (10–
compared to larger nanoparticle sizes. Manipulating the 20 nm) [19]. This study investigated the effect of AgNO3
AgNP synthesis parameters such as AgNO3 concentration, lime juice powder concentration and pH
concentration, extract quantity, pH, and incubation time on AgNP formation. The AgNPs were evaluated for their
allows control over nanoparticle characteristics [10-11]. particle size, polydispersity index, and antibacterial
AgNO3 concentration significantly influences activity compared to AgNO3.
nanoparticle size and morphology. Higher AgNO3 ■ EXPERIMENTAL SECTION
concentrations yield higher peaks of surface plasmon
Materials
resonance (SPR) [11], indicating the rate of Ag+ to AgNP
conversion depends on AgNO3 concentration. Moreover, Materials in this study were silver nitrate (AgNO3,
AgNO3 concentration directly impacts synthesis yield, Merck KgaA, Germany), sodium hydroxide (NaOH,
with higher concentrations yielding greater yields [12-13]. Merck KgaA, Germany), and lime (C. aurantifolia)
AgNP synthesized using lime juice (C. aurantifolia) in taken from Ujung Pangkah, Gresik, East Java, Indonesia
liquid form with different incubation times showed the and has been verified by expert botanist researchers
formation of AgNP at different wavelengths [9]. from Materia Medika Indonesia, Batu, East Java, with
The concentration reductant significantly impacts document number 074/372/102.20-A/2022. Other
the biosynthesis process and the properties of AgNPs, materials, i.e., nutrient broth (Merck KgaA, Germany),
including their size and structure. A study by [14] nutrient agar (Merck KgaA, Germany), clindamycin
observed that elevating the concentration of Caulerpa hydrochloride (Xi'an Julong Bio-Tech Co., Ltd),
serrulata extract from 5 to 20% during AgNP synthesis Propionibacterium acnes (ATCC 11827) from BBLK
amplifies the intensity of SPR and results in a shift Surabaya, Staphylococcus aureus (ATCC 6538), and
towards a shorter wavelength (435 nm). This shift arises Escherichia coli (ATCC 8739) were obtained from the
from the reduction in the average size of AgNPs. Assessment Services Unit at Universitas Airlangga,
However, upon further escalation of the concentration to Indonesia.
25%, a decline in SPR intensity occurs owing to the Instrumentation
aggregation of nanoparticles. This investigation Analytical balance (Ohaus PA-2102 C, USA),
demonstrates that aggregation occurs beyond a certain Fourier transform infrared (FTIR) spectrometer Bruker
concentration threshold while augmenting the Eco-ATR (ALPHA II), magnetic stirrer (Thermo
concentration can boost AgNPs production. Such Scientific), UV-vis spectrophotometer (Hitachi
aggregation can detrimentally affect functional UH5300), Delsa Nano C particle analyzer (Beckman
properties. Consequently, for methods involving extracts, Coulter), scanning electron microscope (SEM, Hitachi
it is imperative to standardize the optimal concentration FlexSEM), micropipette, pH meter (SI Analytics Lab
alongside other parameters [13]. 865), drying oven binder ED 53 (Binder GmbH), freeze

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 Indones. J. Chem., 2024, 24 (5), 1445 - 1455 1447

dryer (Buchi, L-LyovaporTM L-200), and glasswares were holder, which was then inserted into a sputter coater for
used in this study. gold-palladium coating lasting approximately 120 s.
Examination was carried out using an Inspect S50 type FP
Procedure
2017/12 (Hitachi FlexSEM) at various magnifications,
Sample preparation and freeze-drying of lime juice with measurements taken on multiple particles.
powder
Fresh limes were obtained from the Gresik area in AgNP antibacterial activity testing
East Java, Indonesia. The collected fruits were washed and The antibacterial activity of AgNPs was conducted
halved to extract the juice manually. After obtaining the using standard test organisms: Gram-positive bacteria
lime juice, it was filtered to obtain pure lime juice. (P. acnes, S. aureus) and Gram-negative bacteria (E.
Subsequently, the lime juice was frozen to produce lime coli). Bacterial suspensions were prepared by adding
juice powder using a freeze dryer. The freeze-drying 0.9% NaCl solution and the turbidity of the bacterial
process entailed freezing the lime juice at −40 °C, followed suspension was determined using a UV-vis
by vacuum drying for 3 weeks. spectrophotometer at 580 nm until a %transmittance of
25–26 ± 1% was achieved.
Synthesis of AgNPs with different concentrations of
To a 19.8 mL of sterile nutrient broth (NB)
AgNO3 and lime juice powder under various pH
solution, 20 μL bacterial suspensions were added to
conditions (8, 9, 10)
attain a total volume of 20 mL. AgNP solutions were
The lime juice powder solution was prepared with
then added in volumes of 5, 10, 15, and 20 mL to NB
concentrations of 1.00, 1.50, and 2.00%, while the AgNO3
solution and bacterial suspension. The mixture was then
solution was prepared with concentrations of 0.50, 0.75,
placed on an orbital shaker and incubated for 30 min.
and 1.00 mM. A total of 25 mL of lime juice powder
Further, 1 mL of the solution was extracted and added to
solution was added to 25 mL of AgNO3 solution and
9 mL of fresh NB solution, which was then incubated at
stirred at 250 rpm for 1 h. The mixture of AgNO3 solution
37 °C for 24 h. Similar testing was conducted using an
and lime juice powder was then adjusted to the desired pH
AgNO3 solution. Clindamycin at a concentration of
(pH 8–10) using 1 M NaOH and stirred at 250 rpm at
50 ppm served as a positive control. The %transmittance
room temperature for 1 h. Subsequently, the resulting
of all treatments was assessed using a UV-vis
mixture of AgNP solution was placed in an oven at 60 °C
spectrophotometer at 580 nm [20-21].
for 2 h, and color changes were observed. The influence
of concentration and pH values was examined to compare ■ RESULTS AND DISCUSSION
the characteristics obtained. Sample Preparation Lime Juice Powder
Characterization of AgNPs Lime juice powder from the freeze-drying process
The synthesized AgNPs were characterized by has a yellow color with a lime odor. Based on previous
assessing their absorbance using UV-vis spectroscopy research that has carried out phytochemical screening
across the wavelength range of 300–550 nm. Particle size using the thin-layer chromatography method, it is
and polydispersity Index (PDI) were determined using known that lime juice powder contains components
Delsa Nano C- Beckman Coulter. FTIR analysis was such as flavonoids, saponins, terpenoids, steroids, and
conducted to identify functional groups and ascertain the phenols [22].
presence of any chemical interactions. The sample was
appropriately positioned atop the attenuated total Synthesis of AgNP with Different Concentrations
of AgNO3 and Lime Juice Powder at Various pH
reflection (ATR) crystal. Subsequently, measurements
were conducted within the wavenumber range of 400– The green synthesis of AgNP was conducted with
4000 cm−1. Particle morphology was examined using variations in AgNO3 concentrations, namely 0.50, 0.75,
SEM. The sample was mounted on a carbon-coated and 1.00 mM, and lime juice powder solution

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1448 Indones. J. Chem., 2024, 24 (5), 1445 - 1455

concentrations of 1.0, 1.5, and 2.0% as a reductant, along UV-vis spectrum analysis confirms the formation
with the influence of different pH values (8, 9, and 10), to of AgNPs with a peak wavelength of 400 nm. The
determine the optimum conditions for AgNP formation, analysis results using a UV-vis spectrophotometer
evaluated based on absorbance and particle size. The display the peak of the SPR. Typically, the SPR peak is
formation of AgNPs was characterized by a color change influenced by the type of metal, particle size, shape, and
in the solution from clear to yellowish-brown (Fig. 1). The morphology [27]. The absorbance measurement of
color change in the solution occurred due to the reduction AgNPs was conducted in the wavelength range of 300–
of Ag⁺ ions to Ag0, resulting in the formation of AgNP 550 nm. This is illustrated in Table 1, which presents the
[23]. Several factors play a role in forming AgNP, SPR peak and absorbance values for various
including the concentration of AgNO3, concentration of concentrations of AgNO3 and lime juice powder,
the extract, reaction time, and pH, to accelerate the notably strong at 400 nm. Values of λmax below 400 nm
reaction rate. Additionally, secondary metabolites present indicate that the reduction process has not been
in plants can function as natural bioreductants, thus completed, suggesting that the formed nanoparticles are
contributing to nanoparticle formation by inducing the Ag⁺. The intensity of the SPR peak under basic pH
excitation of SPR in the particles [24-26]. Fig. 2 shows the conditions (8, 9, 10), reduced by lime juice powder, shifts
mechanism of AgNP synthesis using biocompound from towards shorter wavelengths and becomes narrower
lime. with increasing pH. This indicates a decrease in particle

Fig 1. AgNO3 solution and lime juice powder (a) before reaction and (b) after reaction

OH
OH Ag+
OH
O-

HO O
2Ag+ + HO O

OH
O-
OH O Ag+
OH O
Flavonoid (quercetin)

2e-

OH
O

HO O

AgNPs +
O
OH O
Fig 2. Illustration of the mechanism for the formation of AgNPs by green synthesis

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 Indones. J. Chem., 2024, 24 (5), 1445 - 1455 1449

Table 1. Absorbance value and particle size of AgNP synthesized with various AgNO3 concentrations, lime juice
powder concentrations and pH
Variable Lime Juice Powder
Observation pH 8 pH 9 pH 10
AgNO3
1.0% 1.5% 2.0% 1.0% 1.5% 2.0% 1.0% 1.5% 2.0%
Particle size (nm) 308.00 146.60 189.30 260.70 239.40 141.70 83.90 130.80 128.30
0.50 mM
Absorbance 2.46 2.41 2.53 2.70 2.70 3.03 2.42 2.76 2.83
Particle size (nm) 77.30 100.40 174.60 61.90 107.90 172.70 82.50 110.00 152.10
0.75 mM
Absorbance 2.49 3.21 2.44 3.67 3.23 2.55 1.88 3.32 2.95
Particle size (nm) 75.90 117.10 175.00 68.40 76.20 106.50 83.60 77.00 88.10
1.00 mM
Absorbance 3.06 3.40 2.47 4.63 3.76 3.77 4.07 3.87 3.87

Fig 3. Histogram (a) particle size and (b) absorbance value of AgNP synthesized with various AgNO3 concentrations,
lime juice powder concentrations and pH

4.5
pH 8 absorbance peak of AgNPs falls within the range of
pH 9 1.875–4.631. A low absorbance value indicates a small
4.0 pH 10
quantity of formed nanoparticles. However, the higher
the absorbance value obtained, the greater the quantity
3.5
of AgNP formed [29-35].
Absorbance

Increasing the concentration of lime juice powder

3.0
as a reducing agent can expedite the formation rate of
AgNPs. This acceleration is attributed to the heightened
2.5
availability of biomolecules such as alkaloids, glycosides,
2.0
flavonoids, saponins, and tannins, which serve as
reductants, capping agents, and stabilizing agents,
1.5
shielding the nanoparticles from aggregation [33,35].
300 350 400 450 500 Moreover, pH emerges as a pivotal factor influencing
Wavelength (nm)
Fig 4. Spectra of AgNPs synthesized with 1 mM AgNO3 nanoparticle synthesis in this green synthesis method
and 1% lime juice powder at different pH owing to its capability to modify the electric charge of
biomolecules. This alteration may impact the capping
size and polydispersity of the nanoparticles [28]. Based on ability, subsequently affecting nanoparticle stabilization
Table 1, Fig. 3 and Fig. 4, it can be observed that the and growth. Under low pH conditions, the SPR peak

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1450 Indones. J. Chem., 2024, 24 (5), 1445 - 1455

tends to be diminished due to absorption by larger-sized reductant [9], this study can yield considerably smaller
nanoparticle forms [27]. Adjusting the pH during AgNP particle sizes under basic conditions. Consequently, the
synthesis can be leveraged to govern specific findings suggest that escalating pH values can culminate
characteristics such as nanoparticle morphology and size. in smaller particle sizes.
Notably, at higher pH levels, there is heightened From the synthesis outcomes, it is discerned that a
competition between protons and metal ions to establish concentration of 1 mM AgNO3 and 1% lime juice
bonds with negatively charged regions, leading to powder at pH 9 yields optimal results in AgNP synthesis.
enhanced synthesis efficacy at alkaline pH [31]. It is This is evidenced by a high absorbance value (4.631) and
apparent from Table 1 that the synthesis of AgNP with an small particle size (68.400 nm) with a PDI of 0.251.
AgNO3 concentration of 1 mM and a lime juice powder
Spectroscopy ATR-FTIR Analysis
concentration of 1% manifests an absorbance of 4.63 at
pH 9 as seen in Fig 3 and 4. Spectroscopy ATR-FTIR analysis was undertaken
Evaluation using the dynamic light scattering (DLS) to ascertain the functional groups of biomolecules
method to obtain Z-average values (d.nm) and PDI engaged in reduction. The ATR-FTIR spectra of AgNPs
constitutes a significant parameter in nanoparticle are depicted in Fig. 5. The peak observed at the
characterization [32]. The PDI value can gauge the degree wavenumber of 3317 cm−1 indicates the presence of
of particle size distribution non-uniformity, stemming hydroxyl (–OH) groups and hydrogen bonding within
from the ratio of the average weight to the average the silver nanoparticles. Additionally, the peak at
molecular weight generated [34,36]. Furthermore, a PDI 2123 cm−1 suggests the involvement of stretching
value approaching 0 denotes homogeneous particle size, vibrations of symmetric and asymmetric C–H bonds
while a PDI value < 0.3 signifies heterogeneous particle from aliphatic –CH and –CH2 groups in forming silver
size [37]. Based on DLS measurements, the size of AgNP nanoparticles. Carbonyl groups are also discerned in the
ranging from 61.9 to 308.0 nm on average was determined absorption band at the wavenumber of 1636 cm−1.
(Table 1). For AgNPs to traverse cell membranes and Compared with the ATR-FTIR spectra of lime juice
impede bacterial growth, the particle size should be powder, a minor shift from the wavenumber of 3389–
smaller than 100 nm [38]. Various factors influence the 3317 cm−1 is noted. This shift implies the potential role
formation of AgNP size, encompassing temperature, pH, of amino groups, alkaloids, or terpenoids present in the
AgNO3 concentration, and reductant concentration, in
achieving the desired size. The influence of AgNO3
concentration can impact the size and morphology of
synthesized nanoparticles. For example, in a study [11] AgNO3
Transmittance (a.u.)

employing AgNO3 concentrations of 1–5 mM, it was

noted that the higher the concentration, the larger the
resulting size obtained. With increasing pH, the charge on Lime Juice Powder

biomolecules shifts from positive to negative, leading to

robust repulsion between negatively charged AgNO3 ions AgNP
and biomolecules. The presence of larger nanoparticles at
higher pH can be attributed to uncontrolled aggregation
towards acidic pH owing to augmented AgNO3 ion
interactions. Conversely, heightened repulsion between
4000 3500 3000 2500 2000 1500 1000 500
ions with increasing pH may have hindered further Wavenumber (cm-1)
nucleation, thus yielding smaller nanoparticles [11]. In Fig 5. ATR spectra of AgNP formed from 1 mM AgNO3
comparison to AgNPs synthesized utilizing lime juice as a and 1% lime juice powder at pH 9

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 Indones. J. Chem., 2024, 24 (5), 1445 - 1455 1451

plant in the formation of AgNPs, functioning as reducing evaluate the impact of the formation and concentration
and stabilizing agents [9]. of AgNPs on activity against Gram-positive (P. acnes, S.
aureus) and Gram-negative (E. coli) bacteria. AgNP
SEM Analysis
synthesized with 1 mM AgNO3 and 1 lime juice powder
The results of the observations utilizing SEM with at pH 9 has the smallest particle size and highest
magnifications of 10,000× and 20,000× for AgNPs are absorbance, which is used in this evaluation.
depicted in Fig. 6. The morphology of AgNP at pH 9, as Determination of AgNP antibacterial activity involved
observed with SEM, reveals small spherical shapes that comparing %transmittance values of samples with the
tend to aggregate, with particle sizes ranging between 50– positive control. An antibiotic (clindamycin) was used as
100 nm (Fig. 6). Nanoparticles are prone to aggregation a positive control, while the negative control remained
due to their high surface area, particularly when stored in untreated. The results of the antibacterial activity test of
liquid form. Various factors during synthesis are known AgNP are presented in Table 2 and Fig. 7.
to influence the physicochemical properties of AgNPs, Based on the results of the AgNP antibacterial
leading to diverse actions and applications. The study activity test in Table 2 and Fig. 7, it can be seen that the
conducted by Traiwatcharanon et al. [11] indicates that %transmittance of AgNP is higher than AgNO3 for P.
pH is a critical reaction parameter directly affecting size acnes, S. aureus, and E. coli. Antibacterial activity is
distribution, aggregation, particle morphology, and concentration-dependent. The bactericidal properties of
antimicrobial activity. As evidenced in the nanoparticle AgNPs depend on their physicochemical properties,
characterization in this study, pH variations result in such as size, shape, particle surface charge, and
AgNPs with distinct sizes and shapes [32]. characteristics of the bacterial species. Smaller particles
are known to have more excellent antibacterial activity
AgNP Antibacterial Activity Test because they can easily reach the bacterial core, and their
The antibacterial activity of AgNPs was assessed to larger surface area leads to stronger interactions and

Fig 6. The SEM image of AgNPs with magnifications of (a) 10.000× and (b) 20.000×

Table 2. Antibacterial activity of AgNP, AgNO3 against P. acnes, S. aureus, and E. coli
Transmittance (%)
Bacteria Control (+) Control (−) AgNO3 concentration (mM) AgNP concentration (%)
0.125 0.250 0.375 0.500 0.125 0.250 0.375 0.500
P. acne 95.3 31.4 41.7 46.7 50.7 53.6 50.2 69.8 77.5 79.3
S. aureus 93.5 30.5 49.4 54.1 57.0 59.8 40.8 67.0 78.7 80.0
E. coli 89.6 28.4 35.5 37.3 40.1 43.6 38.6 44.8 59.1 74.0

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1452 Indones. J. Chem., 2024, 24 (5), 1445 - 1455

Fig 7. The result of the antibacterial activity test of AgNP against (a) P. acnes, (b) S. aureus, and (c) E. coli

bactericidal effects. Different forms may exhibit different 040/E5/PG.02.00.PL/2024 and 1680/B/UN3.LPPM/PT.
antibacterial activities [39-40]. This effect may be related 01.03/2024.
to differences in effective surface area and number of
■ CONFLICT OF INTEREST
active sites. So, the antibacterial activity of AgNPs
synthesized with lime juice powder is higher than AgNO3 The authors declared no conflict of interest.
against P. acnes, S. aureus, and E. coli. ■ AUTHOR CONTRIBUTIONS
■ CONCLUSION Conceptualization, Septian Dwi Mulyana, Retno
The concentration of AgNO3 and lime juice extract Sari, Agus Syamsur Rijal; Methodology, Septian Dwi
as the reductant determines the synthesis outcomes of Mulyana, Retno Sari; Software, Septian Dwi Mulyana;
AgNPs, including absorbance values and particle size. Validation, Retno Sari, Agus Syamsur Rijal; Formal
Increasing the pH enhances the absorbance value and Analysis, Septian Dwi Mulyana; Investigation, Septian
diminishes the particle size of AgNPs, with pH 9 being the Dwi Mulyana; Resources, Retno Sari, Agus Syamsur
optimal condition. AgNPs synthesized using 1 mM Rijal; Data Curation, Septian Dwi Mulyana; Writing -
AgNO3 and 1% lime juice powder at pH 9 exhibit a size of Original Draft, Septian Dwi Mulyana, Retno Sari; Writing
68.4 nm. The antibacterial activity of AgNPs against P. - Review & Editing, Retno Sari, Agus Syamsur Rijal;
acnes, S. aureus, and E. coli bacteria demonstrates higher Visualization, Septian Dwi Mulyana, Retno Sari, Agus
growth inhibition compared to AgNO3 solution and Syamsur Rijal; Supervision, Retno Sari; Project
escalates with increasing AgNP concentration. Administration, Retno Sari; Funding Acquisition, Retno
Sari.
■ ACKNOWLEDGMENTS
This research was financially supported by the ■ REFERENCES
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