Trends Sci. 2026; 23(3): 11684

Harnessing Metal-Loaded Zeolites for Superior Antibacterial Activity

Sriatun^1,*, Tania¹, Siti Nursyamsiah¹, Adi Darmawan¹, Choiril Azmiyawati¹,

Damar Nurwahyu Bima¹ and Nor Aida Zubir²

¹Department of Chemistry, Faculty of Science and Mathematics, Diponegoro University, Semarang 50275, Indonesia

²Chemical Engineering Studies, College of Engineering, Universiti Teknologi MARA, Cawangan Pulau Pinang, Permatang Pauh 13500, Malaysia

(^*Corresponding author’s e-mail: [email protected])

Received: 25 August 2025, Revised: 2 October 2025, Accepted: 12 October 2025, Published: 15 December 2025

Abstract

Metal-loaded zeolites has gained significant interest for their antibacterial properties and potential applications in biomedical and environmental fields. In this study, silver- and copper-loaded zeolites were successfully synthesized using ion exchange and impregnation methods. Characterization techniques confirmed the successful incorporation of Ag⁺ and Cu²⁺ ions into the zeolite framework. X-ray diffraction (XRD) analysis demonstrated reduction in peak intensities at 2θ ≈ 26°, 34° and 42° for Ag-zeolite and 2θ = 19.59°, 32.22° and 37.97° for Cu-zeolite, indicating successful metal loading. Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDX) verified the presence of 3.98% Ag and 3.79% Cu in the modified zeolites. The antibacterial performance of Ag- and Cu-loaded zeolites was evaluated against Escherichia coli and Staphylococcus aureus using disk diffusion and well diffusion methods. Ag-zeolite (0.05 M, 9 h) exhibited inhibition zones of 12.23 mm and 11.35 mm for E. coli and S. aureus, respectively. Cu-zeolite showed even stronger antibacterial activity, with inhibition zones of 15.3 mm and 18.1 mm. These findings highlight the potential of Ag- and Cu-loaded zeolites as effective antimicrobial agents. This research demonstrates a simple and efficient approach for synthesizing antibacterial zeolites, offering promising applications in water treatment, wound healing and antibacterial coatings. This research can serve as a reference for further studies, including the evaluation of long-term stability and the exploration of practical applications for large-scale implementation.

Keywords: Zeolite, Antibacterial activity, Metal ion loading, Escherichia coli, Staphylococcus aureus

Introduction

Antibacterial materials have emerged as advanced materials, particularly in field of health, tropical diseases, nutrition and pharmaceuticals. The increasing prevalence of bacterial infections has necessitated the development of effective antibacterial agents. Traditionally, antibiotics have been widely used as antibacterial agents, aiding the immune system in halting bacterial infections. However, prolonged and improper use of antibiotics have led to the emergence of antibiotic-resistant bacteria, posing a serious global health concern [1]. This highlights the urgent need for the development of safer and environmentally friendly antibacterial materials.

Recent advancements have focused on the utilization of reliable and cost-effective antibacterial agents, particularly modified zeolite-based materials [2]. As an inorganic material, zeolite offers unique advantages as an antibacterial carrier due to its heat resistance, high chemical stability, prolonged durability, and safety for users [3]. The high cation-exchange capacity of zeolites, resulting from their large surface area and negatively charged framework, makes them suitable carriers for metal ions with antibacterial properties [4].

The antibacterial performance of zeolite can be significantly enhanced by modification with metal ions. Metals such as silver (Ag), copper (Cu) and zinc (Zn) exhibit potent antimicrobial properties [5]. Silver ions (Ag⁺), for instance, possess broad-spectrum antibacterial activity with low toxicity in humans. They disrupt bacterial proteins and cell membranes by interacting with disulfide (S–S) and sulfhydryl (–SH) groups, forming S–Ag bonds that lead to bacterial inactivation and cell death [6]. Copper ions (Cu²⁺) can disturb membrane integrity, while zinc ions (Zn²⁺) have been widely employed to inhibit cariogenic bacteria in dental applications [7].

Previous studies have demonstrated the potential of modified zeolites as antibacterial agents. For instance, Yao et al. [8] found that Cu-Zeolite X exhibited superior antibacterial activity against Escherichia coli and Staphylococcus aureus, while Milenkovic et al. [9] reported that Ag-Zeolite A showed the highest inhibition against Escherichia coli when compared to Cu- and Zn-modified zeolites. Furthermore, Ishak et al. [10] revealed the efficacy of silver-modified kaolinite zeolite in inhibiting the growth of both Gram-positive and Gram-negative bacteria. Lastly, Mintcheva, et al. [11] in his study investigates how exchangeable ions in natural and modified zeolites influence silver (Ag) content, Ag nanoparticles (AgNPs) formation, and their antibacterial effectiveness. The modified zeolites served as carriers for Ag ions, which were later reduced to form AgNPs within the zeolite structure. The study found that the type of exchangeable ion significantly affected the distribution and stability of AgNPs, influencing their antibacterial activity.

However, despite these promising findings, several critical gaps remain in the current literature. Few studies have provided a comprehensive analysis of the long-term stability of metal-loaded zeolites or their antibacterial performance under diverse environmental conditions and against multiple bacterial strains. Additionally, the environmental impact of their use, including potential metal ion leaching and toxicity, has not been thoroughly explored. Comparative analyses of modified zeolites with other antibacterial agents in terms of efficacy, cost-effectiveness and scalability are also limited. Moreover, challenges related to scaling up the synthesis process for industrial applications, including reproducibility, consistency and economic feasibility, have rarely been addressed [12].

From all this previous study, its sugested that zeolite, especially when modified with metal ions such as Ag, Cu and Zn, is an effective antibacterial material. The exchangeable ions in zeolites play a crucial role in determining their silver content, Ag nanoparticle (AgNP) formation and overall antibacterial performance. Ag-modified zeolites exhibit the highest antibacterial efficacy, particularly against Escherichia coli and Staphylococcus aureus, due to their ability to release Ag⁺ ions and form stable AgNPs within the zeolite structure [13]. Additionally, different metal modifications, such as Cu and Zn, also contribute to antibacterial activity, although their effectiveness varies depending on the zeolite type and ion-exchange process [14]. These findings highlight the potential of modified zeolites as promising antibacterial agents for various applications, but also underscore the need for further studies addressing stability, environmental impact, and scalability.

Given these promising results, this study aims to synthesize zeolites with a low Si/Al ratio through various synthesis methods and subsequently modify them with Ag⁺ and Cu²⁺ ions. The antibacterial performance of the modified zeolites will be evaluated against Escherichia coli and Staphylococcus aureus. This research not only seeks to develop highly efficient, stable and sustainable antibacterial materials but also aims to provide insights that could inform long-term applications and industrial-scale implementation.

Experimentals

Materials

The materials used in this study include sodium aluminate (Sigma-Aldrich), sodium silicate (Merck; density 1.35 g/cm³), SiO₂/LUDOX HS-40 (Sigma-Aldrich; colloidal silica), sodium hydroxide (NaOH, 98% purity, Merck; anhydrous pellets), ethanol (70% purity, Merck), deionized water, silver nitrate (AgNO₃, 99.0 - 99.97% purity, Merck), copper(II) nitrate trihydrate (Cu(NO₃)₂·3H₂O, Merck), zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, Sigma-Aldrich), Whatman No.42 filter paper (Whatman), universal pH indicator strips (pH range 0–14, Merck), nutrient agar (Technical grade, Merck), nutrient broth (Technical grade, Merck), test microbes (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923) and chloramphenicol (Technical grade, Merck).

Synthesis of zeolite

A total of 17.45 g of NaOH was dissolved in deionized water to prepare 266.17 mL of 1.69 M NaOH solution. Subsequently, 16.4 g of sodium aluminate (NaAl₂O) was added to the NaOH solution, resulting in a 0.75 M NaAl₂O solution. Next, 277.103 mL of the 0.75 M NaAl₂O solution was mixed with 11.6 mL of LUDOX. The mixture was stirred using magnetic stirrer at moderate speed and room temperature until it became homogeneous and slightly viscous.

The resulting mixture was transferred into a closed Teflon vessel and heated in a microwave at medium power for 1 h. Afterward, the mixture was aged at room temperature for 24 h. The solid product was filtered and washed with deionized water until neutral pH was achieved. The solid was then dried at 80 °C until a constant weight was obtained, yielding synthetic zeolite.

For the combined microwave-hydrothermal method, the same composition was used. The mixture was heated in a microwave at medium power for 5 min, followed by hydrothermal treatment at 150 °C for 24 h

Metal loading onto zeolite

Variation in Ag⁺ Ion loading concentration

A total of 1 g of synthetic zeolite was mixed with 10 mL of AgNO₃ solution at varying concentrations (0.03, 0.04, 0.05, 0.06, 0.07 M) and stirred for 5 h. The product was filtered, washed and dried at 80 °C for 3 h to obtain Ag⁺-loaded zeolite (M).

Variation in Ag⁺ Ion loading time

A total of 1 g of synthetic zeolite was mixed with 10 mL of 0.05 M AgNO₃ solution and stirred using a magnetic stirrer for different durations (3, 6, 9, 12, 24 h). The product was filtered, washed and dried at 80 °C for 3 h to produce Ag⁺-loaded zeolite (t).

Variation in Cu²⁺ Ion loading concentration

The metal loading method on synthetic zeolite was adapted from Septommy et al. [15]. A total of 1 g of synthetic zeolite was mixed with 10 mL of Cu(NO₃)₂·3H₂O solution at varying concentrations (0.03, 0.04, 0.05, 0.06, 0.07 M) and stirred for 9 h. The product was filtered, washed and dried at 80 °C for 3 h to obtain Cu²⁺-loaded zeolite (M).

Variation in Cu²⁺ Ion loading time

A total of 1 g of synthetic zeolite was mixed with 10 mL of 0.05 M Cu(NO₃)₂·3H₂O solution and stirred using a magnetic stirrer at 400 rpm for different durations (3, 6, 9, 12, 24 h). The product was filtered, washed and dried at 80 °C for 3 h to produce Cu²⁺-loaded zeolite (t).

Characterization

All synthesized products were characterized for their crystallinity and phase using X-ray diffraction (XRD), functional groups using Fourier-transform infrared spectroscopy (FTIR) and surface morphology as well as elemental composition using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX). The zeolite loaded with metal ions was further characterized using XRD and SEM-EDX.

Antibacterial activity test of metal ion-loaded zeolites

Preparation of mcfarland standard solution

A total of 0.05 mL of 1% BaCl₂ was mixed with 9.95 mL of H₂SO₄ and stored away from direct sunlight. The absorbance value was then measured at a wavelength of 600 nm.

Preparation of Nutrient Agar (NA) media

A total of 2.3 g of NA powder was dissolved in 100 mL of distilled water, stirred, and heated on a hot plate. The solution was autoclaved at 121°C and 1.5 atm for 15 min. Subsequently, 10 mL of the medium was poured into sterile petri dishes.

Preparation of Nutrient Broth (NB) media

A total of 0.8 g of NB powder was dissolved in 100 mL of distilled water, stirred, and heated on a hot plate. The solution was autoclaved at 121 °C and 1.5 atm for 15 min. Subsequently, 10 mL of the medium was poured into sterile petri dishes.

Rejuvenation of S. aureus and E. coli bacteria

S. aureus or E. coli colonies were taken from bacterial stock on slanted agar medium using an inoculation loop and inoculated into an Erlenmeyer flask aseptically near a Bunsen burner in a laminar air flow (LAF). The culture was incubated in a shaker incubator at 37 °C for 24 h to obtain S. aureus or E. coli colonies. 2.6 Preparation of S. aureus and E. coli Bacterial Suspensions.

S. aureus or E. coli bacteria were inoculated into NB media aseptically near a Bunsen burner in a laminar air flow (LAF). The culture was incubated in a shaker incubator at 37 °C and absorbance at 600 - 625 nm was measured every 2 - 3 h until the absorbance matched that of the McFarland 0.5 standard solution, corresponding to a bacterial count of 1.5×10⁸ CFU/mL.

Antibacterial test with S. aureus and E. coli

Well diffusion method

A total of 0.1 mL of S. aureus or E. coli bacterial suspension was spread evenly on NA agar medium using a spreader. Five wells with a diameter of 6 mm were created in the agar medium. Each well was filled with 30 mg of metal ion-loaded zeolite dissolved in 50 µL of distilled water. The plates were incubated at 37 °C for 24 h and the diameter of the inhibition zones was measured using a ruler. The average diameter of the inhibition zones was recorded. The same procedure was applied to positive control (Chloramphenicol), negative control (distilled water) and sample control (AgNO₃, Cu(NO₃)₂ and Zn(NO₃)₂) using S. aureus and E. coli.

Disk diffusion method

The antibacterial test using the disk diffusion method was performed with Whatman No. 42 filter paper as the disk material. The agar medium, which had been inoculated with 0.1 mL of S. aureus or E. coli bacterial suspension, was overlaid with filter paper disks soaked in the sample solution. The plates were incubated at 37 °C for 24 h and the diameter of the inhibition zones was measured using a caliper. The average diameter of the inhibition zones was recorded. The same procedure was applied to positive control (Chloramphenicol), negative control (distilled water) and sample control (AgNO₃, Cu(NO₃)₂ and Zn(NO₃)₂) using S. aureus and E. coli.

Results and discussion

Characteristics of synthesized zeolite

The X-ray diffraction (XRD) analysis of the synthesized zeolite with a low molar ratio (Si/Al = 1) revealed the formation of Zeolite X, as evidenced by specific diffraction peaks at 2θ values of 10.31°, 24.13°, 26.30°, 27.26°, 30.09°, 30.94°, 32.66°, 34.32° and 42.77° as shown in Figure 1(a). These peaks were compared with the reference patterns from the JCPDS database and previous studies [16,17], which confirmed that the synthesized zeolite was predominantly Zeolite X, with a mixture ratio of 55.61% Zeolite X and 44.39% Zeolite A. This composition was determined using the Search and Match software, which matched the observed diffraction peaks to those of Zeolite X and A standards.

Because of its high ion exchange capacity due to the abundance of balancing cations, low molar ratio of Si/Al ~ 1 is chosen. Low Si/Al ratio zeolites (1 - 5) are typically classified as faujasite (FAU) structures with large pores (~7.4 Å) [18]. This zeolite used Na⁺ as the balancing cation which originated from the excess NaOH during synthesis that facilitates the dissolution of silica and alumina and accelerates crystallization process. Silica and alumina tetrahedral units underwent polymerization to form a negatively charged silica-alumina framework stabilized by Na⁺ [19]. Homogenized mixtures of NaAl₂O and LUDOX gradually thickened and turned white at room temperature, indicating the formation of zeolite precursors. NaAl₂O served as the alumina source, while LUDOX provided silica. Microwave heating followed by 24 h of aging stabilized the formation of synthetic zeolite crystals.

Figure 1 (a) XRD diffractogram of the synthesized zeolite, confirming the presence of Zeolite A (♦) and Zeolite X (▲) based on characteristic peaks. (b) SEM image showing the morphology of the synthesized zeolite particles in magnification 5,000×. Particle size distribution of (c) Zeolite A and (d) Zeolite X. Particle size measurements were obtained from the SEM image using ImageJ software.

The SEM image (Figure 1(b)) showed the morphology of the synthesized zeolite particles, revealing the characteristic crystal structures of Zeolite A and Zeolite X. Zeolite A exhibited a cubic morphology, whereas Zeolite X showed spherical to polyhedral particles. The well-defined crystal shapes indicated successful zeolite crystallization without significant amorphous phases [20].

The particle size distributions of Zeolite A and Zeolite X were analysed using ImageJ software (Figures 1(c) - 1(d)). Zeolite A particles have an average size of 1.31 µm, with a relatively broad distribution ranging from 0.6 to 2.4 µm. In contrast, Zeolite X particles obtained slightly smaller size with an average of 1.01 µm and exhibited a narrower size distribution (0.4 - 2.0 µm). The differences in particle size and morphology can be attributed to the synthesis conditions, particularly nucleation rate, growth time and NaOH concentration, which influence the crystallization pathways of different zeolite phases [21].

Characteristics of metal-modified zeolites

Crystall properties of metal-modified zeolites

The modification of zeolites with metal ions, such as silver (Ag⁺) and copper (Cu²⁺), enhances their functional properties, including antimicrobial activity and catalytic performance. Understanding the structural stability and ion-exchange behavior of metal-loaded zeolites is crucial for optimizing their applications [22]. In this study, X-ray diffraction (XRD) analysis was conducted to investigate the structural effects of silver and copper ion exchange on the synthesized zeolite.

Figure 2 (a) XRD patterns of the synthesized zeolite and silver-modified zeolite (AgZ) at different silver ion concentrations (0.03 M, 0.05 M and 0.07 M) with a fixed loading time of 9 h. (b) XRD patterns of AgZ with 0.05 M silver ion loading at different durations (3, 9 and 24 h). (c) XRD patterns of the synthesized zeolite and copper-modified zeolite (CuZ) at different copper ion concentrations (0.03 M, 0.05 M and 0.07 M) for 9 h. (d) XRD patterns of CuZ with 0.05 M copper ion loading at different loading time (3, 9 and 24 h).

The effect of silver ion modification on the zeolite structure was analyzed by loading silver ions with different concentrations (0.03 M, 0.05 M and 0.07 M) for varying times (3, 9 and 24 h). The XRD analysis, shown in Figures 2(a) - 2(b), indicated that despite the introduction of silver ions, no significant changes in the diffraction peaks occurred. This suggests that the overall crystalline structure of zeolite was well-preserved [23]. However, a notable decrease in the intensity of the diffraction peaks was observed, which indicates the ion exchange between sodium (Na⁺) ions in the zeolite framework with silver (Ag⁺) ions [24]. The most pronounced decrease in intensity was seen at 2θ values of ~26°, 34° and 42°, confirming successful silver incorporation [25]. The reduced peak intensities were attributed to a slight distortion in the zeolite lattice due to the difference in ionic radii between Ag⁺ and Na⁺, as well as the potential formation of Ag nanoparticles within the zeolite pores at higher silver concentrations.

Similarly, the XRD patterns of copper-modified zeolite in Figures 2(c) - 2(d) exhibited a reduction in diffraction peak intensities, particularly at 19.59°, 32.22° and 37.97° that indicate the successful incorporation of copper ions. The average crystallite size of the synthesized zeolite was calculated to be 35.67 nm using the Full Width at Half Maximum (FWHM) parameter [26]. Upon exchanging with copper ions, the decrease in peak intensity suggests partial substitution of Na⁺ with Cu²⁺, leading to minor structural modifications. Peak intensity decreased progressively with increasing ion-exchange duration, particularly at 24 h, implying a time-dependent effect on metal loading [27]. However, no new diffraction peaks corresponding to copper oxides or metallic copper were detected, confirming that Cu²⁺ remained in the ion-exchanged state within the zeolite framework. The relatively low intensity of Cu-loaded zeolite peaks suggests that the amount of incorporated copper was limited, potentially due to competition between ion exchange and adsorption onto external sites of the zeolite particles.

Surface morphology of metal-modified zeolites

The surface morphology and elemental composition of the silver-modified and copper-modified zeolite samples were analyzed using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX).

Figure 3 (1) Ag⁺-loaded zeolite at concentrations of (a) 0.03 M, (b) 0.05 M and (c) 0.07 M after 9 h of ion loading and (2) Ag⁺-loaded zeolite a (a) 3 h, (b) 9 h and (c) 24 h on 0,05 M.

For the Ag-zeolite samples, modifications were performed with varying silver nitrate concentrations (0.03 M, 0.05 M, 0.07 M) and different loading times (3, 9 and 24 h) (Figure 3). The SEM images revealed uniform morphology, with crystal shapes corresponding to Zeolite X and Zeolite A, specifically octahedral and cubic forms, which is consistent with previous studies [28]. Upon modification with silver ions, the SEM micrographs showed that Ag⁺ ions were well-dispersed on the zeolite surface and the crystals formed aggregates with varying sizes [29,30]. However, the presence of larger aggregates suggested some agglomeration due to the ion exchange process. The EDX analysis revealed in Table 1 that the synthesized zeolite contained Si, Al, Na, O and Ag. The molar ratio of Si/Al in the unmodified zeolite was found to be relatively low (1.11 to 1.16). When modified with silver ions, a decrease in sodium content was observed, indicating that Na⁺ ions were exchanged with Ag⁺ ions during the modification process. The sample modified with 0.07 M AgNO₃ showed a higher Ag⁺ content but also a relatively high Na⁺ content, indicating incomplete ion exchange, where silver ions may still be bound to the zeolite surface.

Table 1 Elemental analysis results using EDX on synthesized zeolite and synthesized Ag-zeolite.

For the Cu-zeolite samples in Figure 4, similar modifications were carried out, with varying copper ion concentrations (0.03 M, 0.05 M, 0.07 M) and different loading times (3, 9 and 24 h). SEM analysis of the Cu-zeolite samples revealed no significant changes in morphology compared to the unmodified zeolite, with small octahedral-shaped particles forming aggregates. The EDX results on synthesized zeolite in Table 2 confirmed that copper ions had successfully exchanged with Na⁺ ions in the zeolite structure, as evidenced by the decrease in sodium content and the incorporation of copper. The Si/Al molar ratio for the Cu-zeolite samples ranged from 0.88 to 0.95, indicating the ion exchange was successful [31]. However, the sample modified with 0.07 M CuSO₄ showed the highest copper content, suggesting that a higher concentration of copper led to more copper ions being loaded onto the zeolite. Despite this, the sodium content remained somewhat high, indicating that the ion exchange was not fully completed and some sodium ions were still strongly bound within the zeolite structure.

Figure 4 Morphology of Cu-zeolite samples: (a) Cu-Z 0.03 M, (b) Cu-Z 0.05 M, (c) Cu-Z 0.07 M, (d) Cu-Z 3 H, (e) Cu-Z 9 H and (f) Cu-Z 24 H.

In both cases, the ion exchange processes were influenced by the concentration of metal ions and the duration of loading. While silver and copper ions were successfully incorporated into the zeolite, some incomplete ion exchange and surface adsorption of metal ions were observed, which could impact their further applications [32].

Table 2 Elemental analysis results using EDX on synthesized zeolite and synthesized Cu-zeolite.

Antibacterial activity of metal-modified zeoliyes

The antibacterial activity of metal-loaded zeolites was evaluated using both the disc diffusion and well diffusion methods against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Silver- and copper-loaded zeolites were synthesized by ion exchange with varying concentrations of AgNO₃ and Cu(NO₃)₂ and the inhibition zones were measured to assess antibacterial efficacy. The results of this antibacterial activity is shown in Figure 5.

The results demonstrate that increasing silver nitrate (AgNO₃) concentration and loading time enhanced antibacterial activity. In the disc diffusion method, the largest inhibition zones were observed at a 0.05 M AgNO₃ concentration, with values of 12.23 mm for E. coli and 11.35 mm for S. Aureus as shown in Figures 5(a) - 5(b). While, in the well diffusion method, the inhibition zones reach up to 13.32 mm for E. coli and 13.24 mm for S. aureus. On the other hand, copper-loaded zeolite exhibited stronger antibacterial properties, with the best performance observed at a 0.05 M Cu(NO₃)₂ concentration, similar to the silver-loaded sample, where inhibition zones reached 15.3 mm (E. coli) and 18.1 mm (S. aureus) in the disc diffusion method. In the well diffusion method, the inhibition zones were even larger, at 19.3 mm and 22.6 mm, respectively. When comparing the two methods, the well diffusion assay produced larger inhibition zones than the disc diffusion method, suggesting that metal ions diffuse more effectively when directly incorporated into the agar medium. In contrast, the disc diffusion method may have limited ion mobility due to the fixed amount of metal-loaded zeolite in the disc.

Both silver- and copper-loaded zeolites exhibited optimal antimicrobial performance at a precursor concentration of 0.05 M. Although antimicrobial activity generally increases with higher metal loading, a decline in efficacy was observed when the concentration exceeded this threshold. This reduction can be attributed to pore blockage by excess metal ions at elevated concentrations, which decreases the material’s effective surface area. This interpretation is consistent with the study of Fanta, et al. [33], who reported that the adsorption capacity of copper-doped zeolite increased with loading in the range of 0.63 - 6.3 g, but declined at higher amounts, as evidenced by reduced iodine values that indicate fewer accessible active sites. Our EDX analysis further supports this explanation: At precursor concentrations above 0.05 M, the spectra revealed substantially higher metal contents within the zeolite structure, confirming that additional ions were incorporated beyond the optimal level. Such excessive loading likely results in partial pore clogging and a reduction in exchangeable sites, thereby limiting ion release and decreasing the overall antimicrobial activity. Since surface area and ion availability are both critical for bacterial contact and inhibition, the combination of restricted diffusion and diminished active sites explains the observed plateau or decline in antibacterial performance despite higher metal incorporation.

Figure 5 (a) Inhibition zone diameters for E. coli at varying metal-loaded concentrations. (b) Inhibition zone diameters for S. aureus at varying metal-loaded concentrations. (c) Inhibition zone diameters for E. coli at different metal-loading times. (d) Inhibition zone diameters for S. aureus at different metal-loading times. The antibacterial activity was assessed using disc diffusion and well diffusion methods. Chloramphenicol was used as the positive control, while distilled water served as the negative control.

The time-dependent study of silver- and copper-loaded zeolites revealed that the optimal loading time for antibacterial activity was 9 h. At this condition, silver-loaded zeolite exhibited inhibition zones of 12.26 mm against E. coli and 11.33 mm against S. aureus in the disc diffusion method (Figures 5(b) - 5(c)). A comparable trend was observed in the well diffusion assay, with inhibition zones of 13.15 mm and 12.23 mm, respectively. In contrast, copper-loaded zeolite achieved inhibition zones of 14.6 mm (E. coli) and 18.1 mm (S. aureus) in the disc diffusion method, while the well diffusion method yielded even larger zones, measuring 20.5 mm and 22.6 mm, respectively. Longer loading times beyond 9 h showed diminishing returns. This phenomenon may be attributed to the saturation of the zeolite structure, which reduces the release rate of Ag⁺ and Cu²⁺ ions. Prolonged loading increases the amount of metal ions within the pores, thereby limiting their subsequent release into solution or agar. The fraction of leachable metal further decreases, due to stronger ion–framework interactions or the full occupation of exchangeable sites.

Silver and Copper ions exhibit antibacterial activity through two primary mechanisms: (1) disruption of bacterial membranes via electrostatic interactions, leading to leakage of intracellular contents and (2) generation of reactive oxygen species (ROS), which cause oxidative damage to DNA and protein [34]. Compared to silver, copper displayed stronger antibacterial effects, particularly against S. aureus. This effect may be attributed to the smaller ionic radius of Cu²⁺ (73 pm) compared to Ag⁺ (116 pm). The reduced size facilitates more efficient penetration through outer membrane porin channels, thereby enhancing bactericidal activity relative to Ag⁺ [35]. In addition, the electrochemical properties of Ag⁺ contribute to its lower stability in biological environments. With a standard reduction potential of +0.80 V (Ag⁺/Ag), Ag⁺ is more readily reduced to metallic silver under physiological conditions [36]. This reduction decreases the concentration of bioavailable ions capable of interacting with bacterial cells, ultimately limiting its antimicrobial performance. By contrast, Cu²⁺ possesses a lower reduction potential, allowing it to remain in its ionic form for longer periods and sustain ion release, thereby maintaining stronger antimicrobial efficacy.

The study highlights distinct antibacterial profiles for silver- and copper-loaded zeolites. While both metals showed strong antibacterial effects, copper demonstrated superior inhibition zones, particularly against S. aureus. This difference can be attributed to structural features of Gram-positive cell envelopes. Unlike Gram-negative bacteria, which possess a thin peptidoglycan layer (~8 nm), Gram-positive bacteria have a much thicker peptidoglycan matrix (20 - 80 nm) enriched with anionic wall teichoic acids (WTAs) [37]. These glycopolymers, composed of polyribitol or polyglycerol phosphates, carry a high density of negatively charged phosphate groups that readily bind divalent cations [38]. Such binding sites act as reservoirs for metal ions and play a key role in charge neutralization and cell wall stability. Consequently, Cu²⁺ ions exhibit strong electrostatic affinity toward these anionic sites, particularly phosphate and carboxylate groups, leading to enhanced accumulation and interaction with the Gram-positive cell wall. This preferential binding likely contributes to the greater antibacterial efficacy of copper against S. aureus.

Although Ag⁺ exhibited lower overall antibacterial activity compared to Cu²⁺, it showed stronger inhibition against E. coli (Gram-negative) than S. aureus (Gram-positive). This selectivity can be explained by differences in cell wall structure. Gram-negative bacteria possess a relatively thin peptidoglycan layer, which allows Ag⁺ ions, despite their larger ionic radius, to diffuse more readily through the cell envelope. In contrast, Gram-positive bacteria have a much thicker peptidoglycan matrix, which acts as a stronger physical barrier and limits Ag⁺ penetration. As a result, silver-loaded zeolite demonstrates greater antibacterial efficacy against E. coli than S. aureus.

Both materials show potential for use in antibacterial coatings, water treatment, and medical applications. Silver-loaded zeolite may be preferable in scenarios requiring controlled ion release, whereas copper-loaded zeolite is more effective for applications requiring strong antibacterial action, particularly against Gram-positive bacteria. The findings provide valuable insights for optimizing metal-modified zeolites for specific antibacterial applications based on bacterial strain and required ion release kinetics.

Conclusions

In this study, silver- and copper-loaded zeolite materials were successfully synthesized using ion exchange and impregnation methods, as described in the methodology. Characterization results confirmed the successful incorporation of metal ions into the zeolite framework. XRD analysis revealed a pronounced decrease in peak intensity at 2θ = 26°, 34°, and 42° for Ag-zeolite, confirming silver incorporation. Similarly, Cu-zeolite exhibited a reduction in diffraction peak intensities at 2θ = 19.59°, 32.22° and 37.97°, indicating the successful integration of copper ions. SEM images demonstrated uniform particle distribution with minimal aggregation, while EDX analysis verified the elemental composition, indicating 3.98% Ag and 3.79% Cu in the modified zeolites. Antibacterial testing revealed that both Ag-zeolite and Cu-zeolite exhibited significant inhibition against E. coli and S. aureus. In the disk diffusion method, Ag-zeolite (0.05 M, 9 h) produced inhibition zones of 12.23 mm for E. coli and 11.35 mm for S. aureus, while Cu-zeolite (0.05 M, 9 h) achieved inhibition zones of 15.3 mm and 18.1 mm, respectively. The well diffusion method showed even larger inhibition zones. These results demonstrate the potential of metal-loaded zeolites as effective antibacterial materials for biomedical and environmental applications. Future work will focus on optimizing synthesis conditions and assessing long-term stability in real-world applications.

Acknowledgment

The authors gratefully acknowledge the financial support provided by the Dean of the Faculty of Science and Mathematics, Diponegoro University for funding this research under contract No. 21/UN7.F8/PP/II/2023.

Credit Authorship Contribution

Sriatun: Conceptualization; Funding acquisition; Project administration; Resources; Supervision; Validation; Writing – review & editing. Tania: Data curation; Formal analysis; Methodology; Writing – original draft. Siti Nursyamsiah: Data curation; Methodology; Investigation; Writing – original draft. Adi Darmawan: Supervision; Validation; Conceptualization; Writing – review & editing. Choiril Azmiyawati: Supervision; Validation; Visualization; Writing – original draft. Damar Nurwahyu Bima: Supervision; Validation; Writing – review & editing. Nor Aida Zubir: Resources; Validation; Writing – review & editing.

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