Synthesis of Ag/TiO₂ photocatalysts via direct photodeposition of silver from industrial electroplating wastewater

Abstract

Silver-containing wastewater from cyanide-based electroplating processes represents both an environmental burden and an underutilized resource. Conventional silver recovery methods often generate intermediate products that require further refining before reuse. This study demonstrates a proof-of-concept waste-to-material strategy in which real silver-containing electroplating wastewater was used directly as the Ag source for photodeposition onto TiO₂, without adding high-purity silver precursors. Structural analyses confirmed the formation of Ag-modified TiO₂ with increase Ag nanoparticle sizes from 5.29 ± 3.49 to 13.19 ± 5.60 nm, corresponding to Ag contents of 0.82, 3.54, and 8.69 wt% for Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3, respectively. Ag incorporation also reduced the apparent bandgap from 3.14 eV for pristine TiO₂ to 2.96 eV for Ag/TiO₂-3. Under UV irradiation, all photocatalysts achieved a substantial decrease in Staphylococcus aureus viability (99.9%) at 1 mg/mL, while Ag/TiO₂-3 showed the strongest antibacterial activity at the lowest tested concentration of 0.1 mg/mL. Consequently, Ag/TiO₂-3 was identified as the optimal composite and chosen for further mechanistic studies. These studies indicated a non-lytic killing pathway driven by enhanced intracellular reactive oxygen species generation and membrane permeabilization. This work demonstrates the feasibility of converting silver-containing industrial wastewater into functional Ag/TiO₂ photocatalysts and support broader strategies for environmental remediation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-60689-2.

Introduction

The jewelry industry is economically significant but generates substantial wastewater containing heavy metals, particularly silver ions (Ag⁺), which are commonly associated with cyanide-based silver-plating processes¹. If these effluents are not properly treated, they may be released into the sewage system or contaminate surface and groundwater, thereby disrupting aquatic ecosystems, and posing risks to human health through bioaccumulation and toxicity^2,3.

Silver recovery from industrial wastewater has traditionally relied on physicochemical separation techniques such as precipitation, ion exchange, membrane filtration, and adsorption. While these methods are effective in reducing silver concentrations in effluents, the recovered silver is typically obtained in forms such as insoluble precipitates, metal-loaded resins, or concentrated brines^3–5. These recovered forms are not directly suitable for reuse in industrial applications and generally require additional refining processes, including chemical reduction, purification, and re-formulation into high-purity silver salts or metallic silver before reintroduction into electroplating or electronic manufacturing processes. Such refining steps are often energy-intensive, chemically demanding, and economically unfavorable, limiting the overall value of the recovered silver^6,7. Therefore, alternative strategies that directly transform waste-derived metals into functional materials have attracted increasing interest as a route to both pollution mitigation and resource valorization.

Recent studies have demonstrated the growing potential of innovative nanomaterials for photocatalytic wastewater treatment and antimicrobial applications. For example, green-synthesized Ag nanoparticles (NPs) have been investigated as antimicrobial agents against Pseudomonas aeruginosa and Staphylococcus aureus, highlighting the ability of biologically derived materials to produce functional silver-based nanostructures⁸. Similarly, biosynthesized TiO₂ NPs have been evaluated for photocatalytic dye degradation, cytotoxicity, and antimicrobial activity, demonstrating the importance of integrating material characterization, biological response, and photocatalytic performance when developing nanomaterials for environmental applications^9,10. Other emerging photocatalytic systems, including Nb₂O₅ NPs synthesized using plant-derived extracts and supported CuO-based nanocatalysts for methylene blue degradation, further demonstrate the broad movement toward greener synthesis routes, heterogeneous photocatalysis, and predictive evaluation of pollutant degradation pathways^11–13. However, most of these approaches still rely on controlled precursor systems, plant extracts, or model synthetic routes rather than the direct conversion of real industrial metal-containing wastewater into functional photocatalytic materials.

Among silver-based materials, silver-modified titanium dioxide (Ag/TiO₂) has attracted considerable attention due to its enhanced photocatalytic and antimicrobial properties. TiO₂ is widely recognized for its chemical stability, low toxicity, and strong oxidative capability under light irradiation, while the incorporation of silver can improve charge separation, extend light absorption, and introduce additional antimicrobial effects through silver species^14,15. As a result, Ag/TiO₂ composites have been explored for applications ranging from water purification, self-cleaning surfaces, antimicrobial coatings, and biomedical devices^16–19. Reported Ag/TiO₂-based antimicrobial materials include Ag-TiO₂ nanocomposite coatings, green-synthesized Ag-TiO₂ catalysts, Ag/Ag₂O/Ag₃PO₄/TiO₂ composite films, and TiO₂-Ag-AgCl photocatalysts for microbial inactivation and water disinfection¹⁸. Importantly, the antibacterial activity of Ag/TiO₂ is generally attributed to the generation of reactive oxygen species (ROS) under light irradiation, which induce oxidative stress and damage to essential cellular components in microorganisms^20,21.

Despite extensive studies on Ag/TiO₂ photocatalysts, most reported synthesis routes use reagent-grade Ag precursors under controlled laboratory conditions. This limits their relevance to industrial wastewater valorization, where real effluents contain residual cyanide species, organic additives, and competing ions that may affect Ag deposition, material structure, optical properties, and biological performance. Therefore, an unresolved challenge is whether silver present in real electroplating wastewater can be directly converted into Ag-modified TiO₂ photocatalysts while retaining the structural and functional characteristics required for photocatalytic antimicrobial activity.

The novelty of this study is the direct photodeposition of Ag from cyanide-based silver electroplating wastewater onto TiO₂ without adding high-purity silver salts. This approach differs from conventional silver recovery, which commonly generates intermediate products requiring further refining, as well as traditional conventional Ag/TiO₂ synthesis, which typically depends on purified Ag precursors. The present work therefore links industrial wastewater valorization with photocatalyst fabrication by evaluating Ag deposition, crystalline phase retention, optical-property modification, antibacterial activity against Staphylococcus aureus, and ROS-associated bacterial inactivation. This integrated analysis establishes a focused proof-of-concept path for transforming silver-containing electroplating wastewater into functional Ag/TiO₂ photocatalysts for antimicrobial environmental applications.

Results and discussion

Properties of all synthesized photocatalysts

In this study, TiO₂ was prepared and then used to recover Ag⁺ from silver-plating effluent, resulting in 3 photocatalysts designated as Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3. These correspond to initial silver ion concentrations in the effluent of 40, 100, and 280 mg/L used to decorate the TiO₂ NPs, respectively. The microstructure of synthesized samples was first examined using SEM analysis. As shown in Fig. 1(a), the pristine TiO₂ consisted of uniform, sphere-like particles with a smooth surface and consistent porosity. The Ag/TiO₂-3 sample exhibited a similar external microstructure compared to the original TiO₂ (Fig. 1(b)). The EDX spectrum of Ag/TiO₂-3 confirmed the presence of the expected elements, including Ti, O, and Ag (Fig. 1(c)), with a uniform distribution of these elements (Fig. 1(d)).

Fig. 1.

(a) SEM image of pristine TiO₂. (b) SEM image of Ag/TiO₂-3. (c) EDX spectrum of Ag/TiO₂-3, (d₁–d₃) FESEM–EDX elemental maps of Ti, O, and Ag, and (d₄) merged elemental mapping.

By TEM analysis, all Ag/TiO₂ samples exhibited lattice spacings of 0.350, 0.290, and 0.230 nm (Fig. 2(a), (c), (e)), corresponding to the (101) crystal plane of anatase TiO₂¹⁹, the (211) crystal plane of brookite TiO₂²⁰, and metallic Ag²¹, respectively. As expected, Ag NPs were well dispersed on the surface of TiO₂ (Fig. S1(a)–(c)).

Fig. 2.

(a, c, e) High-resolution TEM analysis and (b, d, f) corresponding particle size distribution of Ag NPs of (a) Ag/TiO₂-1, (b) Ag/TiO₂-2 and (c) Ag/TiO₂-3 photocatalysts.

The average particle sizes, determined from measurements of 100 particles, were 5.29 ± 3.49 nm, 7.67 ± 3.35 nm, and 13.19 ± 5.60 nm for Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3, respectively (Fig. 2(b), (d), (f)). The average size of Ag NPs increased with increasing Ag concentration in the effluent, which may reflect greater Ag availability during photodeposition and surface growth on TiO₂. Based on XRF analysis, the bulk Ag contents of the dried Ag/TiO₂ powders were 0.82, 3.54, and 8.69 wt% for Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3, respectively (Table 1). Because XRF provides bulk elemental composition, the Ag contents reported in Table 1 were used as the quantitative composition of the Ag/TiO₂ composites. SEM–EDX mapping further confirmed the spatial distribution of Ti, O, and Ag in Ag/TiO₂-3, while HRTEM confirmed Ag NPs on the TiO₂ surface. However, quantitative EDX was used as supporting evidence rather than the primary composition measurement because EDX is more sensitive to local sampling area and particle heterogeneity.

Table 1.

Physicochemical properties of TiO₂ and Ag/TiO₂ photocatalysts.

Catalyst types	Silver content (wt%)	Crystallite size of TiO2 (nm)		Anatase TiO2 content (%)	Bandgap values (eV)
		Anatase	Brookite
TiO2	-	11.12	8.74	83.90	3.14
Ag/TiO2-1	0.82 ± 0.16	11.22	9.20	83.93	3.05
Ag/TiO2-2	3.54 ± 0.44	11.18	8.92	84.06	3.00
Ag/TiO2-3	8.69 ± 0.86	11.15	10.16	84.31	2.96

The crystallite size of anatase TiO₂ was estimated from the (101) crystal plane (Fig. 3), using the Debye–Scherrer equation (Eq. (1))²² and the anatase content in all synthesized samples was quantified using Eqs. (2) and (3)²³. As summarized in Table 1, all synthesized Ag/ TiO₂ samples exhibited slightly larger crystallite sizes and higher anatase phase contents compared to the original TiO₂. This indicates that the photodeposition of silver species did not significantly alter the crystal structure or polymorphic composition of the pristine TiO₂.

Fig. 3.

X-ray diffraction (XRD) patterns of TiO₂ and Ag/TiO₂ photocatalysts.

1

2

3

Where D is the crystallite size, k is the Scherrer constant (0.9), λ is the wavelength of the X-ray radiation (0.15406 nm), β is the intensity of full width at half maximum (FWHM) of the peak and θ is the diffraction angle. W_A and W_B are the weight content of anatase and brookite, respectively, I_A(101) and I_B(211) are the integrated areas of the (101) anatase and (211) brookite, respectively. I_R represents the integrated area of rutile TiO₂, which is equal to zero in this case. K_A and K_B are correction coefficients, which are equal to 0.886 and 2.721, respectively.

The light absorption ability of all synthesized samples was evaluated based on their reflectance spectra in the wavelength range of 200–800 nm. As shown in Fig. 4 (a), the TiO₂ sample exhibited low reflectance at wavelengths below 380 nm and very high reflectance above 400 nm, indicating its strong UV light absorption and limited visible light response. In contrast, all Ag/TiO₂ samples showed low reflectance across the measured UV-visible range, indicating altered optical absorption after Ag deposition. This optical response may be associated with the localized surface plasmon resonance (LSPR) effect of Ag NPs. The estimated optical bandgap values of the Ag/TiO₂ samples were lower than that of pristine TiO₂, suggesting that Ag modification influenced the optical properties of the TiO₂-based photocatalysts. The enhanced visible light response of the Ag/TiO₂ samples may be attributed to the localized surface plasmon resonance (LSPR) effect of the Ag NPs, which enables them to resonate with visible light and thereby increase light absorption²³.

Fig. 4.

(a) UV-Vis diffuse reflectance spectra and (b) corresponding Tauc plots derived from the Kubelka–Munk function for bandgap energy estimation.

The optical bandgap of the synthesized samples was estimated using the Kubelka–Munk function (Eqs. (4)–(5))²⁴, by plotting F(R)⋅hv]^1/γ against the photon energy to generate a Tauc plot (Fig. 4 (b)). Notably, because free Ag NPs were not synthesized as an independent material in this study, standalone optical and crystallographic data for pristine Ag NPs are not included. This approach enabled the determination of the bandgap values of photocatalysts. As summarized in Table 1, the bandgap values of all Ag/ TiO₂ samples were, as expected, smaller than that of the original TiO₂ sample. This confirms that the presence of Ag NPs positively influenced the visible light absorption capacity of the synthesized samples.

4

5

Where R is reflectance of photocatalyst, h is Planck’s constant, ν is frequency of incident light, B is constant, E_g is bandgap value, and γ is transition-related exponent: γ = ½ for direct and γ = 2 for indirect allowed transitions²⁴.

The pristine TiO₂ synthesized in this study exhibited an apparent bandgap of 3.14 eV, which is consistent with the typical bandgap range reported for anatase- or mixed-phase TiO₂ photocatalysts. As summarized in Table 1, Ag incorporation progressively reduced the apparent bandgap from 3.14 eV for pristine TiO₂ to 3.05, 3.00, and 2.96 eV for Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3, respectively. Similar bandgap narrowing has been reported in modified TiO₂ photocatalysts, where interfacial modification, defect formation, or coupling with light-absorbing nanostructures altered the electronic structure and improved visible-light absorption. For example, N-CQD incorporation into mixed-phase TiO₂ reduced the bandgap from 3.30 to 2.99 eV, while plasmonic metal-TiO₂ interfaces have been shown to improve charge transfer and photocatalytic response through surface plasmon resonance and interfacial electron-transfer effects^25,26. In the present Ag/TiO₂ system, the decrease in apparent bandgap is therefore attributed mainly to Ag-related optical modification, including plasmonic absorption and metal-semiconductor interfacial effects, rather than TiO₂ phase transformation, as the XRD results showed that the anatase–brookite framework was retained after Ag deposition. The structural and optical characterization results obtained in this study indicate that the wastewater-derived photodeposition process successfully produced Ag-modified TiO₂ photocatalysts while preserving the crystalline framework of the TiO₂ support. The XRD analysis showed that the anatase–brookite structure of TiO₂ remained unchanged after silver incorporation, while TEM observations confirmed the presence of metallic Ag NPs dispersed on the TiO₂ surface. This finding indicates that the enhanced optical absorption and antibacterial activity observed for the Ag/TiO₂ samples are unlikely to originate from a major TiO₂ phase transformation. Instead, these changes are more plausibly associated with surface Ag deposition, increased Ag loading, and metal–semiconductor interfacial effects, as supported by XRF, HRTEM, and UV-Vis DRS analyses. Such structural preservation is consistent with previous reports showing that noble metal deposition typically modifies the surface electronic properties of TiO₂ without significantly altering its crystal lattice²⁷. The presence of metallic Ag NPs is particularly important because Ag can act as an electron sink at the metal–semiconductor interface, facilitating the separation of photogenerated electron–hole pairs and thereby suppressing recombination losses. This effect has been widely reported to enhance photocatalytic activity in Ag/TiO₂ systems²⁸. In addition, the optical analysis in this study revealed enhanced light absorption and a reduction in bandgap energy after Ag incorporation, which is commonly attributed to the LSPR effect of Ag NPs. LSPR can improve light harvesting and promote interfacial charge transfer between Ag and TiO₂, thereby increasing the generation of ROS under irradiation^18,29,30. Importantly, the successful formation of Ag-decorated TiO₂ using silver derived directly from industrial wastewater suggests that the complex chemical composition of electroplating effluent did not prevent Ag deposition onto TiO₂ under the tested conditions. The structural and optical properties observed here indicate that the resulting materials retained key physicochemical characteristics associated with Ag-modified TiO₂ photocatalysts. Although previous studies have suggested that impurities or competing ions in wastewater may influence nanoparticle growth or dispersion^31–33, the structural and optical properties observed here indicate that the resulting materials retain the key physicochemical characteristics required for effective photocatalytic performance.

Nitrogen adsorption-desorption analysis was not included in the present study. Nevertheless, SEM, TEM, XRD, XRF, and UV-Vis DRS collectively confirmed Ag deposition, preservation of the TiO₂ crystalline framework, increased Ag loading, and optical-property modification after photodeposition. Because surface area and porosity can influence photocatalytic and antibacterial performance, future work should evaluate BET surface area, pore volume, and pore-size distribution to further clarify the relationship between textural properties and photocatalytic activity.

Bactericidal activity

The structural and optical characterization studies presented in the previous section indicate that the synthesized Ag/TiO composites possess key features associated with enhanced photocatalytic activity. Specifically, the presence of metallic Ag NPs improves light absorption properties, which are known to promote interfacial charge separation and facilitate the generation of ROS under irradiation. While direct electrochemical measurements such as EIS or photocurrent response and ROS scavenger experiments were not performed in this work, the relationship between optical modification and antibacterial activity is interpreted based on UV-Vis DRS, ROS fluorescence, and established mechanisms in the previous Ag/TiO₂ studies^21,34–37.

To assess antibacterial efficacy, S. aureus cells were incubated with TiO₂, Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3 at concentrations of 0.1, 0.25, 0.5, and 1 mg/mL for 1 h under UV irradiation. Cells treated in the absence of photocatalysts, either without or with UV exposure, served as untreated controls and UV control, respectively. Following treatment, bacterial suspensions were plated on agar, and colony formation was quantified after 24 h of incubation (Fig. S2). Results were expressed as log CFU/mL (Fig. 5). The untreated S. aureus control exhibited 4.36 ± 0.03 log CFU/mL, whereas UV irradiation alone reduced the bacterial count to 3.90 ± 0.13 log CFU/mL, indicating that UV exposure by itself caused a modest reduction in bacterial viability. At 1 mg/mL, all photocatalysts achieved > 3-log reductions relative to the UV treated control (UV Control = 3.89 ± 0.13; TiO₂ = 1.17 ± 0.13; Ag/TiO₂-1 = 0.97 ± 0.17; Ag/TiO₂-2 = 0.87 ± 0.16; Ag/TiO₂-3 =  0.86 ± 0.06 log CFU/mL). At the lowest concentration, Ag/TiO₂-3 produced the strongest reduction in bacterial counts, whereas TiO₂, Ag/TiO₂-1, and Ag/TiO₂-2 showed limited changes relative to the UV control (UV control = 3.89 ± 0.13; TiO₂ = 3.99 ± 0.14; Ag/TiO₂-1 = 3.89 ± 0.15; Ag/TiO₂-2 = 3.43 ± 0.03; Ag/TiO₂-3 = 2.10 ± 0.42 log CFU/mL). The stronger antibacterial effect of Ag/TiO₂-3 is likely associated with its higher Ag loading and enhanced optical absorption, which can contribute to improved photocatalytic ROS generation under UV irradiation. This observation is consistent with previous reports showing that Ag-modified TiO₂ can improve the antibacterial performance of TiO₂ through combined Ag-associated and TiO₂-mediated photocatalytic effects^11,38,39. These results indicate that Ag/TiO₂-3 was the most active photocatalyst and was selected for further evaluation.

Fig. 5.

S. aureus cells were treated with TiO₂, Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3 at 0.1–1 mg/mL. Controls included cells without UV (black bar) and cells exposed to UV alone (grey bar). Bacterial viability was determined by colony counting after 24 h and expressed as log CFU/mL (mean ± SD, n = 3). Statistical significance was assessed by twotailed Student’s ttest vs. UV control (*p < 0.05, **p < 0.01, **p < 0.001; n.s., not significant).

Photocatalysts are known to exert antimicrobial effects primarily through ROS generation, which can induce oxidative stress in microbial cells^40–42. To elucidate the underlying mechanism of Ag/TiO₂-3-mediated bacterial inactivation, intracellular ROS levels were evaluated using the DCFH-DA fluorescent probe in S. aureus cells after different treatment conditions (Fig. 6). Untreated control cells exhibited low fluorescence intensity, indicating basal ROS levels under normal physiological conditions. UV irradiation alone resulted in a modest increase in fluorescence intensity, which is consistent with previous reports describing UV-induced oxidative stress in bacterial cells^43–45. Treatment with 0.1 mg/ml of Ag/TiO₂-3 in the absence of UV irradiation resulted in a slight increase in intracellular fluorescence relative to the untreated control, implying that Ag/TiO₂-3 exerts limited oxidative stress even without photoactivation, potentially due to the intrinsic antimicrobial properties of silver species^46–48. In contrast, the combination of Ag/TiO₂-3 and UV irradiation exhibited a pronounced increase in fluorescence intensity. The ROS level observed under this condition approached that the H₂O₂ treated positive control, indicating substantial oxidative stress. This enhancement indicates a cooperative effect between Ag/TiO₂-3 and UV irradiation, which significantly promotes intracellular ROS accumulation and that ROS-associated oxidative stress is a major contributor to the observed bactericidal activity.

Fig. 6.

Intracellular ROS levels were quantified using the DCFH-DA fluorescent probe. Cells treated with H₂O₂ as a positive control. Fluorescence intensity is expressed as arbitrary units (a.u.). Data represent mean ± S.D. from at least three independent experiments. Statistical significance was evaluated using a two-tailed Student’s t-test compared to the control (***P < 0.001); n.s., not significant.

In Ag/TiO₂ photocatalytic system, ROS can be generated at the photocatalyst surface following light-induced electron-hole separation, and these reactive species may interact with the bacterial envelope, disturb membrane-associated processes, and promote oxidative damage to intracellular components⁴⁹. These extracellular ROS are powerful oxidizing agents that are extremely reactive, immediately attacking the bacterial cell membranes¹¹. This process initiates lipid peroxidation, a chain reaction in which ROS abstract electrons from membrane phospholipids, progressively compromising membrane integrity and increasing membrane permeability. This creates a chain reaction that damages the integrity of the membrane, leading to increased permeability⁵⁰. Disruption of bacterial cell membrane represents a critical turning point in the bactericidal process. Once the membrane’s protective barrier is compromised, the intracellular environment becomes increasingly vulnerable to oxidative stress. Enhanced membrane permeability facilitates the penetration of extracellular ROS and oxygen molecules into the cytoplasm and, under severe damage conditions, may contribute to leakage of intracellular components. These effects disrupt osmotic balance and impair metabolic functions, further destabilizing cellular homeostasis⁵¹.

To assess structural integrity and oxidative modifications induced by the photocatalyst on key biomolecular targets, genomic DNA and total proteins were isolated from treated cells. Because the photocatalyst is water-insoluble, cell normalization via turbidity measurement was not feasible. Therefore, equal volumes of the treated bacterial suspension were processed for extraction and subsequently analyzed for relative integrity.

The control samples not exposed to UV irradiation exhibited intact genomic DNA and protein patterns. UV-exposed samples, both with and without the photocatalyst, retained similar intact profiles but displayed reduced recovered amounts (Fig. 7 (a, b)). However, the absence of extensive DNA smearing and the retention of major protein bands suggest that the treatment did not primarily induce random genomic DNA fragmentation or broad degradation of cellular proteins. Rather, the reduced recovery of biomolecules is consistent with reduced viable cell numbers and oxidative stress-associated cellular injury after photocatalytic treatment. Therefore, the Ag/TiO₂-3/UV system induces substantial bacterial injury through ROS-associated oxidative stress, resulting in reduced viable cell recovery without clear evidence of extensive DNA or protein degradation^52–54.

Fig. 7.

Effect of photocatalytic treatment on genomic DNA and total protein integrity of S. aureus. (a) genomic DNAs were run on 1% Agarose gel electrophoresis (b) Proteins were run on 12% SDS–PAGE. Lane L: DNA ladder (λ DNA/HindIII), and lane M: protein marker (All Blue Regular Range). Both uncropped images are shown in Fig. S3.

Ag/TiO₂ photocatalysts have been reported to inhibit bacterial growth through photoactivation-induced oxidative stress, which leads to cell envelope permeabilization and ultimately cell death^55,56. Because ROS-associated antimicrobial activity may involve changes in membrane permeability, fluorescence microscopy was performed to evaluate membrane-associated effects in S. aureus (Fig. 8 (a)). Treatment with Ag/TiO₂-3 alone produced a phenotype comparable to the untreated control, indicating that Ag/TiO₂-3 by itself, without UV irradiation did not noticeably alter cell morphology or membrane integrity. Similarly, UV irradiation alone did not produce discernible changes in fluorescence staining relative to the control. In contrast, the combination of Ag/TiO₂-3 and UV irradiation resulted in markedly increased intracellular DAPI fluorescence. Quantitative analysis showed that Ag/TiO₂-3 and UV irradiation increased DAPI uptake by 3.15 ± 0.69-fold relative to the untreated control, whereas Ag/TiO₂-3 alone (0.92 ± 0.07) and UV alone (1.00 ± 0.05) did not cause a significant change (p > 0.05) (Fig. 8 (b)). These results suggest that the combination of Ag/TiO₂-3 photocatalyst and UV irradiation enhances cell envelope permeability, leading to increased DAPI penetration, similar to the effects observed for antibiotics that compromise bacterial membrane permeability^57–59.

Fig. 8.

Fluorescence microscopy analysis of membrane integrity and permeability in S.aureus. (a) Experimental cells were stained with FM4-64 (red), DAPI (blue), and SYTOX Green (green). Scale bar = 1 μm. (b) Quantification of relative DAPI fluorescence intensity represent as mean ± SEM calculated from individual nucleoids across single cells (three biological replicates). Statistical significance was evaluated using a two-tailed Student’s t-test (P < 0.05); n.s., not significant.

Importantly, none of the tested conditions produced detectable SYTOX Green fluorescence. Because SYTOX Green is commonly used as an indicator of severe membrane disruption and loss of membrane barrier function, the absence of SYTOX Green staining suggests that the Ag/TiO₂-3 plus UV treatment did not induce rapid gross membrane rupture under the tested condition^60,61. This interpretation is further supported by the undetectable of DNA and protein leakage into the supernatant, as measured by NanoDrop spectrophotometry. The DNA and protein concentrations remained comparable to those of the control groups, indicating that substantial macromolecular leakage did not occur despite the observed reduction in bacterial viability. Taken together, the plate-count, intracellular ROS determination, biomolecular integrity, and imaging results indicate that Ag/TiO₂-3 under UV irradiation inactivates S. aureus primarily through ROS-associated oxidative stress accompanied by increased membrane permeability. However, the lack of SYTOX Green staining and the absence of detectable DNA/protein leakage suggest that the bactericidal mechanism is not dominated by immediate lytic membrane destruction. Instead, the data support a non-lytic or weakly permeabilizing oxidative killing pathway in which photocatalytically generated ROS disturb cellular homeostasis and reduce bacterial viability. Based on these combined results, a proposed photocatalytic antibacterial mechanism of wastewater-derived Ag/TiO₂-3 under UV irradiation is summarized in Fig. 9.

Fig. 9.

Proposed photocatalytic antibacterial mechanism of wastewater-derived Ag/TiO₂-3 under UV irradiation. Graphic components were sourced from Freepik (www.freepik.com) and combined using Adobe Illustrator to generate the final figure.

Although the present study experimentally focused on antibacterial activity, the physicochemical characteristics of the wastewater-derived Ag/TiO2 photocatalysts suggest potential relevance to broader wastewater-treatment applications. Recent studies on advanced water-treatment systems have shown that ROS-mediated catalytic processes can effectively degrade emerging pollutants and improve water quality, including electrocatalytic PMS activation for antibiotic degradation, VUV-based advanced oxidation pretreatments for natural organic matter control, and TiO₂-integrated solar interfacial evaporation systems for simultaneous water production and organic pollutant decomposition^62–64. In addition, current advances in nature-inspired electrocatalysts, electrocatalytic H₂O₂ production, and durable functional adsorbents highlight the importance of catalyst design, operational stability, and sustainable process engineering for environmental and energy-related applications^65–67. Therefore, the optical modification observed in the wastewater-derived Ag/TiO2 photocatalysts may support future applications in organic pollutant degradation; however, pollutant degradation kinetics, mineralization efficiency, catalyst recovery, and reusability were not evaluated in the present study. The broader wastewater-treatment potential of the material should therefore be regarded as a future application.

The potential toxicity of Ag/TiO₂ photocatalysts should also be considered before environmental deployment. Although TiO₂ is widely used because of its chemical stability and relatively low toxicity, Ag-containing nanomaterials may pose dose-, size-, surface-chemistry-, and release-dependent risks to non-target organisms. Recent studies on environmental nanomaterials have emphasized the need to evaluate cytotoxicity, phytotoxicity, and ecotoxicity alongside photocatalytic performance. Therefore, although the present study demonstrates antibacterial activity and wastewater-derived photocatalyst formation, future studies should assess Ag release, mammalian-cell cytotoxicity, aquatic ecotoxicity, and phytotoxicity before practical use in open environmental systems^68,69.

Conclusion

This study demonstrates a proof-of-concept strategy for converting silver-containing electroplating wastewater into Ag-modified TiO₂ photocatalysts through direct photodeposition. The use of real cyanide-based industrial effluent as the Ag source distinguishes this approach from conventional Ag/TiO₂ synthesis routes that rely on high-purity silver precursors. Structural and optical characterization confirmed that Ag NPs were successfully deposited onto TiO₂ while the anatase–brookite crystalline framework was retained. Increasing Ag loading from Ag/TiO₂-1 to Ag/TiO₂-3 was associated with larger Ag NPs, higher Ag content, and a reduced apparent bandgap, indicating that wastewater-derived Ag deposition effectively modified the physicochemical properties of TiO₂.

The synthesized photocatalysts exhibited antibacterial activity against S. aureus under UV irradiation. Specifically, the Ag/TiO₂-3 delivered the most potent inhibitory effect at minimal concentrations. Mechanistic analysis indicated that Ag/TiO₂-3 under UV irradiation promoted intracellular ROS accumulation and increased cell envelope permeability. However, the absence of detectable SYTOX Green staining and DNA/protein leakage suggests that bacterial inactivation was not dominated by rapid lytic membrane destruction. Instead, the results support a ROS-associated oxidative killing pathway accompanied by moderate membrane permeabilization.

Overall, this work shows that silver-containing industrial wastewater can be directly transformed into a functional antimicrobial photocatalyst, providing a waste-to-material strategy relevant to circular resource utilization and environmental disinfection. Further studies should evaluate catalyst reusability, Ag release, ecotoxicity, cytotoxicity, and pollutant degradation performance in real wastewater matrices before practical environmental deployment.

Materials and methods

Materials

Titanium isopropoxide (C₁₂H₂₈O₄Ti, Sigma-Aldrich) was used as the titanium precursor for TiO₂ synthesis. Isopropanol (i-C₃H₈O, QReC), hydrochloric acid (HCl, QReC), ethanol (C₂H₆O, QReC), and distilled water were used during hydrothermal synthesis, washing, and photodeposition. Cyanide-based silver electroplating wastewater obtained from the jewelry industry was directly utilized as the Ag source for photodeposition. The wastewater exhibited a highly alkaline pH (∼11) and contained 400 mg/L of Ag, along with trace amounts of Cu, Si, Fe, Zn, and Ni at concentrations of 2.0, 1.5, 0.3, 0.12, and 0.03 mg/L, respectively. To prepare Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3, the wastewater was diluted with deionized water to achieve Ag concentrations of 40, 100, and 280 mg/L, respectively. Barium sulfate (BaSO₄) was used as the reference material for UV-Vis diffuse reflectance measurements. 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from sigma-Aldrich. Hydrogen peroxide was purchased from Merck. All chemicals were used as received unless otherwise stated.

Synthesis of TiO₂ and Ag/TiO₂

Before synthesizing the Ag/TiO₂ photocatalysts, the TiO₂ NPs were first prepared using a sequential hydrothermal and calcination method, adopted from Zaw et al. Briefly, approximately 8.9 mL of titanium isopropoxide (C₁₂H₂₈O₄Ti, Sigma-Aldrich) and 11.5 mL of isopropanol (i-C₃H₈O, QReC) were mixed and stirred at 400 rpm for 10 min. Subsequently, 50 mL of distilled water was added dropwise while maintaining the same stirring rate for 4 h. Then, 1 mL of 3 M hydrochloric acid (HCl, QReC) was carefully added, followed by continued stirring for an additional hour. The resulting solution was transferred to a Teflon-lined cylinder, sealed in a stainless-steel autoclave, and heated at 200 °C for 2 h in an oven (Memmert, UF55). After natural cooling, the solid product was separated by centrifugation at 9,000 rpm for 15 min, washed three times with ethanol (C₂H₆O, QReC) and twice with distilled water, and then dried at 100 °C for 12 h. The final TiO₂ NPs were obtained by calcination in air at 500 °C for 2 h.

To synthesize Ag/TiO₂, the photodeposition was conducted using a cyanide-based silver-plating effluent obtained from the jewelry industry as the silver source. In each experiment, approximately 0.45 g of TiO₂ NPs was dispersed in 300 mL of wastewater containing the required silver ion concentration, and ethanol was added at 3.0% (v/v) as a hole scavenger. The solution was stirred continuously at 400 rpm for 30 min in the dark to ensure uniform dispersion of the solid catalyst and effective adsorption of active species on the surface of the TiO₂ NPs. The system was then irradiated using a high-pressure mercury lamp (400 W; RUV 533 BC) at a constant light intensity of 5.8 mW/cm² for 1 h. The resulting Ag/TiO₂ photocatalyst was recovered by centrifugation, followed by drying at 80 °C for 4 h. The synthesized photocatalysts were designated as Ag/TiO₂-1, Ag/TiO₂-2, and Ag/TiO₂-3, corresponding to silver ion concentrations in the cyanide-based silver-plating effluent of 40, 100, and 280 mg/L, respectively.

Nanoparticle characterization

Field emission scanning electron microscopy (FESEM, JSM-7610FPlus) coupled with energy-dispersive X-ray spectroscopy (EDX, ULTIM MAX 65, JEOL) was conducted to observe the microstructure and elemental composition of the synthesized samples. Transmission electron microscopy (TEM, JEM-3100 F, JEOL) was used to examine the lattice fringes of the Ag/TiO₂ samples at an accelerating voltage of 200 kV. An X-ray fluorescence (XRF) instrument (Horiba XGT-9000 Pro) was employed to estimate the bulk Ag content of the dried powder samples. X-ray diffraction (XRD, D2 Phaser, Bruker) analysis was performed to investigate the crystal structure using Cu-Kα radiation. UV–Vis spectrophotometry (UV-2600, Shimadzu) was carried out to evaluate the light absorption capacity, using BaSO₄ as the reference. Ag particle size distributions were obtained from TEM images using ImageJ. For each Ag/TiO₂ sample, at least 100 clearly distinguishable Ag NPs were measured. The particle size is reported as the average diameter ± standard deviation. Gaussian fitting was not applied because the measured distributions were not assumed to follow a normal distribution.

Antibacterial activity studies

Staphylococcus aureus (MSSA) ATCC 29,213 was used in this study. The bacterial stock was streaked onto agar plate and incubated at 37 °C overnight. A single colony was then picked and grown in LB broth overnight in an incubator shaker at 37 °C. The overnight cultures were diluted 100-fold and further grown in the incubator shaker for around 2 h. Cell density was determined by measuring absorbance at 600 nm (OD₆₀₀). The cultures were then adjusted to a final concentration of 2 × 10⁴ CFU/ml in 10 ml of bacterial cultures. The photocatalytic activity was evaluated under UV light irradiation using a 400 W UVA lamp. The cell suspension flask was placed in the reactor, with the temperature maintained below 30 °C throughout the experiment, at a distance of approximately 30 cm from the UV light source for irradiation. A magnetic stirring bar was placed at the bottom of the flask to ensure adequate mixing of the contents throughout the irradiation period. Fifty microliters of treated bacterial suspension were streaked onto LB agar plates and incubated overnight at 37 °C to determine bactericidal activity under the tested photocatalytic conditions. The controls were the cells exposed to UVA light in the absence of a photocatalyst. The remaining treated cells were divided into 4 parts for DNA, protein, ROS determination and imaging analysis.

DNA and protein extraction

After treatment, the cell suspensions were centrifuged at 14,000 rpm at 4 °C for 10 min. The cell pellets were further processed for genomic DNA and protein extraction, while the culture media were collected to assess the presence of leaked DNA and proteins resulting from compromised cell membranes, using a NanoDrop spectrophotometer.

The genomic DNA was extracted from the cell pellet using PrimeWay Genomic II DNA Extraction Kit following the manufacturer instruction. The genomic DNA concentrations were measured at A260/A280 by NanoDrop spectrophotometer. 2 µl of each DNA sample was run on 1% agarose gel electrophoresis.

For protein, the cells were lysed in lysis buffer containing 50 mM Tris, pH 8.0, 200 mM NaCl, 1 mg/mL lysozyme and 0.1% SDS. The cells were incubated on ice for 30 min with periodically vortexing every 5 min and then sonication. The lysates were centrifuged at 14,000 rpm at 4 °C for 30 min. The supernatants were collected and prepared as SDS sample and run on 10% SDS-PAGE.

Intracellular reactive oxygen species (ROS) determination

Intracellular ROS was detected using fluorescent probe-based assay. The S. aureus cells were prepared as mentioned above. After treatment, the cells were centrifuged at 12,000 rpm for 5 min. The pellets were resuspended in 1 mL of PBS and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to a final concentration of 10 µM. The suspension was incubated in the dark at 37 °C for 1 h. After incubation, the cells were washed with PBS for 3 times. Finally, the cells were resuspended in PBS for 200 µl and put onto a black 96-well plate. The fluorescence intensity was measured at Ex485/Em535 by multi-mode microplate reader. Cells treated with 30 mM H₂O₂ for 30 min was used as positive control.

Fluorescent microscopy

The S. aureus cells were prepared as mentioned above. After treatment, bacterial cells were stained with 2 µg/mL FM4-64, 0.5 µM SYTOX Green and 2 µg/mL DAPI. Cells were harvested by centrifugation at 3,300 × g for 1 min and resuspended in one-tenth of the original culture volume. 5 µL of the concentrated suspension was spotted onto an agarose pad (1.2% agarose in 20% LB broth) mounted on a concave glass slide. All experimental conditions and imaging parameters were kept constant across experiments. Raw fluorescence microscopy images were preprocessed using ImageJ software⁷⁰, and cellular features such as DAPI and SYTOX Green intensities were quantified with CellProfiler 4.0⁷¹. For each condition, experiments were conducted in three biological replicates. Within each replicate, fluorescence intensity was quantified from 50 to 350 nucleoids across three images (n = 3). To account for batch-to-batch variation, all intensity values were normalized to the mean intensity of the corresponding untreated control.

Statistical analysis

CFU values were converted to log₁₀ CFU/mL before analysis and are presented as mean ± standard deviation. ROS fluorescence data are presented as mean ± standard deviation from at least three independent experiments. For microscopy analysis, the normalized measurements for each replicate were then averaged, yielding three final values per condition (N = 3). Statistical significance between conditions was subsequently determined using a two-tailed Student’s t-test.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

ST: Methodology, Data analysis, and Writing-review & editing & original draftAO: InvestigationPN: Resource and and Writing-review & editing & original draftTS: InvestigationNS: InvestigationAK: Writing-review & editing & original draftCS: Methodology, Investigation, Data analysis, Supervision and Writing-review & editing & original draftMH: Funding acquisition, Supervision and Writing-review & editing & original draft.

Funding

The research project is supported by the Mahidol University (MU’s Strategic Research Fund: 2023).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.