Mechanism of TiO2 nanotube UV-photocatalytic degradation of antibiotic resistance genes in the wastewater sludge and blocking of the transfer

Antibiotic resistance genes (ARGs) in sludge propagate via horizontal gene transfer (HGT), necessitating advanced mitigation strategies. This study demonstrates TiO₂ nanotube UV photocatalysis effectively degrades ARGs (70.6–82.5% reduction) and suppresses HGT by targeting mobile genetic elements (MGEs; 93.4–97.1% removal). Hierarchical TiO₂ nanotubes (anatase phase) generated reactive oxygen species (ROS) inducing oxidative DNA damage and cell lysis, preferentially eliminating intracellular ARGs (33.5–46.6% decline) while converting them to extracellular forms. Mobile genetic elements (MGEs) (tnpA-04/intI1) were selectively fragmented via ROS, outperforming HOCl-based systems. Microbial analysis revealed Proteobacteria (e.g., Kofleria) as key ARG hosts, whose decline correlated with resistance reduction (p<0.05). Radiation-resistant Deinococcus dominated post-treatment communities but lacked ARG associations, indicating non-transmissible residual risks. Spatial-specific degradation mechanisms emerged: UV directly fragmented chromosomal ARGs, while ROS oxidized plasmid-borne MGEs, achieving dual ARG elimination and HGT blockade. The intracellular-to-extracellular ARG shift and host-MGE decoupling confirmed transmission disruption. This work establishes TiO₂ photocatalysis as a paradigm for sludge treatment, synchronizing ARG removal and environmental risk mitigation through ROS-microbe-DNA interplay.

1 Introduction

The widespread use of antibiotics has greatly increased the risk of antimicrobial resistance (AMR) and the difficulty of wastewater monitoring (Islam et al., 2024a; Islam et al., 2024b). This is related to antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB), which poses a challenge to global health (Werkneh and Islam, 2023). Urban wastewater is a critical reservoir for resistance genes, as it accumulates wastewater from diverse sources containing ARGs, ARB, and antibiotic residues (La Rosa et al., 2025). During wastewater treatment, a significant proportion of resistance genes in the water phase accumulate in sludge, posing challenges for its safe disposal (Zhao et al., 2025). For instance, tetracycline - class ARGs have a high detection rate, being commonly detected in aquaculture wastewater and residual sludge (Chen et al., 2015). Effective disinfection and removal of ARB and ARGs are essential to mitigate secondary risks associated with ARG dissemination. However, traditional disinfection technologies like chlorine and ozone present certain issues. The chlorine-derived by-products in chlorine disinfection exhibit poor efficacy in eliminating ARGs. Some studies demonstrated that the abundance of ARGs in wastewater even increased after chlorine treatment (Karaolia et al., 2018; Liu et al., 2018). Regarding ozone, it might facilitate the propagation of ARGs in conventional treatment plants due to cell damage repair mechanisms and mutation processes (Zhuang et al., 2015). Moreover, the dense extracellular polymeric substances (EPS) in sludge adsorb ARGs via ion bridging, hydrophobic interactions, and polymer entanglement (Ding et al., 2015; Zhu et al., 2018). ARGs exist in both intracellular (iARG) and extracellular (eARG) forms, facilitating horizontal gene transfer (HGT) through mobile genetic elements (MGEs) such as plasmids, integrons, and transposons (Cai et al., 2022). Although eARGs occur at low concentrations, they can persist in soil or sediment for extended periods, posing long-term environmental risks (Deshpande and Fahrenfeld, 2022). Thus, effective elimination of both iARGs and eARGs is imperative during sludge treatment.

Advanced oxidation processes, especially photocatalytic technology, are effective means to reduce ARB and ARGs (Kalimuthu et al., 2025; Yuan et al., 2020). Chlorination, ozonation, and photocatalysis are commonly used methods. However, chlorination and ozonation often produce toxic by-products such as organic halides and have low degradation efficiency for ARGs.(Mazhar et al., 2020; Yuan et al., 2015). UV radiation induces DNA-protein crosslinking, DNA strand breaks, and pyrimidine dimer formation, significantly reducing resistance genes. The UV/peracetic acid system not only suppressed potential ARG host growth in antibiotic-contaminated wastewater but also curbed ARG dissemination during subsequent biological treatment (Yin et al., 2024). For sludge with dense EPS, UV photocatalysis effectively oxidizes organic matter, yet its ARG reduction potential remains underexplored. UV irradiation of nitrate during sludge fermentation pretreatment enhanced organic matter metabolism and short-chain fatty acid production, increasing decomposition rates by 2.3-fold and short-chain fatty acid yield by 12.7-fold (Lu et al., 2024). Municipal sludge biodrying combined with photocatalysis facilitated EPS degradation and improves dewaterability (Cai et al., 2023). Studies demonstrated that 10 μg/L TiO₂ nanoparticles under UV irradiation decompose approximately 37% of organic content in Anzali Wetland sludge within 5 days (Marefat et al., 2023), highlighting TiO₂ UV photocatalysis as a promising approach for ARG reduction in residual sludge. However, the complex EPS structure and ARG distribution in sludge pose challenges, and the efficiency and mechanisms of UV photocatalysis for ARG reduction in layered residual sludge remain unexplored.

This study first focused on UV-photocatalytic technology using TiO₂ nanotubes to targeted eliminate ARGs in layered residual sludge, aiming to elucidate their degradation efficiency and HGT blocking mechanisms for ARGs in sludge. We successfully fabricated TiO₂ nanotube catalysts that generate reactive oxygen species (ROS) under UV irradiation, effectively damaging microbial cells and DNA. The findings demonstrated that this technology effectively inhibits ARG dissemination through three synergistic mechanisms: degradation of MGEs, inactivation of host bacteria, and direct oxidative damage to DNA. These results provide a novel approach for controlling ARG contamination in wastewater treatment plants. Moreover, we identified practical implementation challenges, particularly the potential secondary enrichment of eARGs, which establishes a critical foundation for subsequent research in real-world applications.

2 Materials and methods

2.1 Sampling sites

The sewage sludge used in this experiment was collected from the secondary sedimentation tank of a sewage treatment plant in Dalian (121.6°E,38.9°N), which employs the anaerobic-anoxic-oxic process. After collection, the sludge was subjected to gravity sedimentation to discard the supernatant, and the resulting concentrated sludge was stored sealed at 4°C. The pH of the remaining sludge was 6.89 ± 0.1, with a moisture content of 96.50% ± 0.3%, a volatile solids/total solids (VS/TS) ratio of 47.65% ± 0.1%, a total chemical oxygen demand (TCOD) of 13,286 ± 328 mg/L, a soluble chemical oxygen demand (SCOD) of 564.3 ± 28.1 mg/L, dissolved carbohydrates of 72.4 ± 5.2 mg/L, dissolved proteins of 182.7 ± 9.5 mg/L, and an ammonia nitrogen of 296.3 ± 12.2 mg/L.

2.2 Preparation of TiO₂ nanotubes network

The Ti foil was sequentially cleaned in acetone, anhydrous ethanol, and deionized water using ultrasonic treatment to remove surface oxides and residual oil (Supplementary Figure S1). Subsequently, NaOH solution (60 mL, 1–10 mol/L) and the Ti foil (50 × 30 × 3 mm) were added to a polytetrafluoroethylene liner. It is important to position the Ti foil vertically along the edge of the liner to maximize the contact area between the foil and the NaOH solution. The liner, containing the NaOH solution and Ti foil, was then placed into a high-pressure reactor for a reaction lasting 4 h at a temperature of 130°C–170°C.

Once the reactor cooled to room temperature, the Ti foil was carefully removed from the liner and immediately immersed in a 0.1 mol/L hydrochloric acid (HCl) solution for pickling, which lasted 12 h. The foil was then dried in a vacuum oven at 50°C and subsequently annealed in a muffle furnace at 500°C for 2 h. This series of steps resulted in the formation of a TiO₂ nanotube network.

2.3 Photocatalytic experiment

The sewage sludge was subjected to photocatalytic experiments (Supplementary Figure S1), which were divided into three groups: original sludge (RS), TiO₂ nanotube UV photocatalysis (UV-TiO₂), and TiO₂ nanotube treatment under dark conditions (Dark-TiO₂). Each reaction took place in a 250 mL cylindrical glass reactor (φ = 8 cm) that was cleaned with alcohol and dried before use. The TiO₂ nanotubes used in the photocatalytic reaction had an effective surface area of 30 cm², which were dispersed in deionized water by ultrasonic treatment for 15 min before use. The photocatalytic reactor was set on a magnetic stirrer (450 rpm) inside a lightbox (CEL - LB70, Zhongjiaojinyuan). A 300 W xenon lamp in the lightbox, whose main wavelength was concentrated in the ultraviolet range (200–400 nm), served as the irradiation light source. To maintain a stable reaction temperature, a low - temperature thermostatic bath circulated 15°C cooling water around the reactor. The control and experimental sludge samples, collected from a local sewage treatment plant and filtered through a 0.2 mm sieve, were stirred (450 rpm) during the experiment. Each experiment was repeated three times to ensure accuracy.

2.4 TiO₂ characterization

The phase structure of the samples was characterized using an X-ray diffractometer (XRD, Bede, Dur ham, UK), operating with CuKα radiation (λ = 0.15406 nm) at 40 kV and 35 mA. Diffraction patterns were collected in the 10°–80° range with a scan speed of 5°/min. The samples were Ti foil and nanoplates loaded with TiO₂ nanotubes, with dimensions of 10 mm × 10 mm. The morphology of the samples was examined using a scanning electron microscope (SEM, Eva Ma 10, Zeiss, USA), which required high vacuum conditions for the measurements. The samples were cut into square pieces with a side length of 10 mm and adhered to the sample holder using conductive adhesive tape, ensuring close contact between the Ti foil and the TiO₂ nanotube-loaded nanoplates. The samples were then analyzed at various magnifications.

2.5 Microbial and metagenomics analyses

2.5.1 DNA extraction

DNA in the sludge was extracted using the TIANamp Microbial DNA Kit (IANGEN Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. The quality of the extracted DNA was evaluated using a NanoDrop2000 spectrophotometer (Thermo Scientific, USA). The qualified DNA was then used to assess the abundance of ARGs, MGEs, 16S rRNA, and bacterial communities. A three-tier quality control system was implemented for all molecular experiments: Plasmid standards containing target ARGs (concentration gradient: 10²–10⁶ copies/μL) were used to validate qPCR sensitivity; 0.22 μm-filtered and sterilized wastewater samples were processed in parallel during DNA extraction to monitor reagent contamination; Duplicate no-template controls were included per 96-well plate, with abnormal amplifications automatically flagged by StepOnePlus software.

2.5.2 Detection of ARGs, MGEs, and 16S rRNA abundance via HT-qPCR

HT-qPCR was conducted using the SmartChip Real-time PCR system (Wafergen SmartChip, Fremont, CA). The reaction sites for HT-qPCR were provided by a gene chip containing 5184 nanopores, with amplification carried out using a 100 nL reaction volume. The primer sequences used in this study are illustrated in the Supplementary Table S1. All HT-qPCR reactions were performed in triplicate, with a minimum of two replicates required for a positive result. Data analysis was carried out using SmartChip qPCR software. The formulas for calculating the relative abundance (Equation 1) and absolute abundance (Equation 2) of each gene were based on the methods described by (Looft et al., 2012; Chen and Zhang, 2013). The absolute copy number of the 16S rRNA gene was determined using real-time fluorescence quantitative assays.

$\text{Relative\hspace{0.17em}abundance}={10}^{\left(31-C_T\right)/\left(10/3\right)}(1)$

$\text{Absolute\hspace{0.17em}abundance}=\text{Absolute\hspace{0.17em}copy\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}}16\mathrm{S}\text{\hspace{0.17em}rRNA}\times \text{Relative\hspace{0.17em}abundance}(2)$

Where C_T values refers to the cycle number at which the fluorescence signal of the amplification product reaches a preset threshold during the qPCR amplification process.

2.5.3 Detection of microbial communities by high-throughput sequencing

High-throughput sequencing of 16S rRNA was used to characterize bacterial communities using the universal primers 515-F(5′-GTGCCAGCMGCCGCGGTAA-3′) and 909-R (5′-CCCCGYCAATTCMTTTRAGT-3′). The original DNA fragment reads were merged using FLASH and then filtered using a QIIME quality filter. PCR chimeras were filtered in UCHIME. Operational Taxonomic Units (OTUs) were clustered at 97% similarity level to analyze species abundance and annotations (Tong et al., 2019).

2.6 Statistical analysis

The differences in gene removal across different treatments and the analysis of gene abundance among various culture systems were assessed using the Tukey test with SPSS 27 software (IBM, USA), where significance was set at p < 0.05. Pearson correlation analysis among ARGs, MGEs, and bacterial communities was conducted using SPSS 27 software (IBM, USA), with significance defined as p < 0.05. Heat maps were generated using TBtools. Bar charts and line charts were created with Origin 2018 (Origin Lab, USA). The network analysis chart, based on Pearson correlation results, was produced and modified using Gephi (v 0.9.2).

3 Results and analysis

3.1 Characterizations of TiO₂ nanotubes network and ARG removal by TiO₂ nanotubes UV photocatalysis

Figures 1a–c present SEM images of Ti foil and TiO₂ nanotube composites. The untreated Ti foil (Figure 1a) shows a heterogeneous surface with steps and microcracks, lacking ordered structures. After nanotube growth (Figure 1b), a hierarchical TiO₂ nanotube array forms, exhibiting a layered mesh-like architecture with increased surface area, enhancing reactive sites. Post sludge treatment (Figure 1c), the mesh structure remains stable despite partial degradation. XRD analysis (Figure 1d) revealed characteristic peaks of metallic titanium at 2θ = 38.42°, 40.17°, 53.0°, 62.94°, and 70.66. TiO₂-modified samples show additional peaks at 25.3° and 48.04° (2θ), indicating high crystallinity, and anatase phase reflections at 53.88° and 55.06° (2θ), confirming crystalline TiO₂ formation. These features were crucial for ARG photocatalytic degradation.

Figure 1

Figure 1. (a) SEM images of Ti foil; (b) SEM images of TiO₂ nanotube; (c) XRD pattern of TiO₂ nanotube after UV photocatalysis treatment; (d) XRD pattern of Ti foil and TiO₂ nanotube; (e) absolute abundances of ARGs in different samples; (f) relative abundances of ARGs in different samples; (g) relative abundances of eARGs in different samples; (h) relative abundances of iARGs in different samples; (i) absolute abundances of MGEs in different samples; (j) relative abundances of MGEs in different samples; (k) relative abundances of eMGEs in different samples; (l) relative abundances of iMGEs in different samples; (m) relative abundance heatmap of eARGs in different samples; (n) relative abundance heatmap of iARGs in different samples; (o) relative abundance heatmap of eARGs in different samples; (p) relative abundance heatmap of iARGs in different samples.

Quantification of over 140 resistance genes revealed 48 ARG subtypes and 14 MGE subtypes. As illustrated in Figures 1e,f, the absolute abundance of ARGs in raw sludge (RS) ranged from 1.20 × 10⁷ to 1.29 × 10⁷ copies/mL, with aminoglycoside resistance genes predominating (5.81 × 10⁶–6.51 × 10⁶ copies/mL), followed by sulfonamide and tetracycline resistance genes. Consistent with findings by Su et al. (2018), Gao et al. (2012), aminoglycoside, tetracycline, and β-lactam resistance genes were identified as the most prevalent in urban wastewater in China, corroborating the presence of multidrug resistance genes in the RS observed in this study.

TiO₂ nanotube-based dark treatment reduced ARG abundance to 8.37–9.54 × 10⁶ copies/mL (26.2%–30.4% reduction), potentially through nanotube-microbial membrane interactions (Thill et al., 2006). UV photocatalysis exhibited superior performance, achieving 70.6%–82.5% degradation efficiency (2.27–3.79 × 10⁶ copies/mL), with 68.9%–93.2% elimination of aminoglycoside-, sulfonamide- and tetracycline-associated ARGs. Notably, TiO₂ treatment decreased relative ARG abundance from 0.1566 to 0.1759 to 0.1251–0.1401 in raw sludge, demonstrating effective ARG amplification suppression. The enhanced efficacy arises from a dual mechanism: UV-activated TiO₂ nanotubes generated ROS via photoexcitation, inducing oxidative stress-mediated lipid peroxidation, protein denaturation, and DNA damage (Bono et al., 2021; Das et al., 2022), while UV directly penetrated cellular structures to fragment resistance gene-carrying DNA (Barnes et al., 2013). This synergistic action disrupted cellular integrity, achieving significant ARG reduction (70%⁺ degradation efficiency) through combined physicochemical pathways.

3.2 Distribution and abundance of eARGs and iARGs

ARGs in sludge exist in both intracellular (iARGs) and extracellular (eARGs) forms, disseminating through distinct mechanisms. Intracellular 16S rRNA gene copies were less abundant in raw sludge (RS) compared to treated samples (Figures 1g,h). TiO₂ nanotube dark treatment showed no significant change in gene copies, whereas photocatalytic treatment reduced extracellular 16S rRNA, indicating diminished DNA retention. The observed increase in eARGs post-photocatalysis likely resulted from cell lysis and subsequent intracellular DNA release.

Following TiO₂ nanotube photocatalysis, iARGs decreased while eARGs increased, demonstrating the conversion of iARGs to eARGs. This shift highlighted the efficacy of TiO₂ nanotube photocatalysis in redistributing i/eARGs within sludge. The decrease in the ratio of intracellular antibiotic resistance genes (iARGs) to extracellular antibiotic resistance genes (eARGs) indicates an accelerated accumulation of eARGs. This can potentially be attributed to bacterial cell death and the release of iARGs. Additionally, the adsorption of eARGs by sludge, which enables them to withstand nuclease attacks and persist in the environment, is also considered a potential contributing factor (Liu et al., 2018). As shown in Figures 1g,h, aminoglycoside, tetracycline, sulfonamide, β-lactam, and MLSB ARGs were predominant. Post-treatment, aminoglycoside and sulfonamide ARGs exhibited higher extracellular relative abundance, while tetracycline and MLSB ARGs were significantly reduced, underscoring the effectiveness of TiO₂ nanotube photocatalysis in removing tetracycline, MLSB, and multidrug ARGs. Wang X. et al. (2024) observed a 7.8-fold iARG increase post-ClO₂ disinfection in wastewater, while TiO₂ nanotube photocatalysis conversely reduced iARGs but elevated eARGs, revealing the unique sludge ARG redistribution capability of nanotube photocatalysis. While ozonation typically induces iARG rebound, this study achieved stable ARG removal through enhanced oxidative targeting (Fu et al., 2023).

ARG resistance mechanisms are classified into antibiotic inactivation, cellular protection, and efflux pump categories. After TiO₂ nanotube photocatalysis, eARGs with antibiotic inactivation mechanisms increased in relative abundance, while iARGs decreased. ARGs such as strB, prevalent in wastewater and soils, persisted extracellularly and were less impacted by photocatalysis (Supplementary Figure S2). Conversely, both eARGs and iARGs associated with cellular protection and efflux pump mechanisms declined, indicating greater susceptibility to TiO₂ nanotube treatment (Supplementary Figure S2). This intracellular-to-extracellular ARG transition suggested that TiO₂ nanotube photocatalysis preferentially degrades ARGs with cellular protection and efflux pump mechanisms, thereby reshaping the resistance mechanism profile in sludge.

3.3 Inhibition of ARG horizontal transfer via TiO₂ nanotube UV photocatalysis

TiO₂ nanotube UV photocatalysis demonstrated superior efficacy in suppressing MGEs, critical mediators of HGT and ARG dissemination. Analysis of 14 MGE subtypes revealed that UV photocatalysis reduced MGE abundance from 1.76 to 1.82 × 10⁷ copies/mL in raw sludge to 8.73 × 10⁵–1.61 × 10⁶ copies/mL (93.4%–97.1% removal), outperforming Fe²⁺/Ca(ClO)₂ systems (2.68-log intI1 removal) (Wang X. et al., 2024). This disparity stems from mechanistic differences: UV-activated TiO₂ generates reactive oxygen species (ROS) preferentially targeting short DNA fragments (<5 kb MGEs), while HOCl oxidation in Fe²⁺/Ca(ClO)₂ systems inadequately degraded chromosomal ARGs (>10 kb). Nanomaterial-enhanced oxidation studies (Wang et al., 2022; Wang X. et al., 2024) corroborated this mechanism, showing radical-dominated systems (e.g., TiO₂/multi-walled carbon nanotubes composites) selectively degrade fragmented genetic elements via ·OH/ClO· generation.

Notably, intracellular MGEs (iMGEs; tnpA-04/intI1/tnpA-05-dominated) showed higher susceptibility to photocatalysis than extracellular counterparts (eMGEs; intI1-dominated). Although eMGEs exhibited selective enrichment (e.g., tnpA-01, clinical intI1), UV photocatalysis achieved pronounced suppression of intI1 (p < 0.01), directly impeding HGT-mediated ARG propagation. These findings established TiO₂ photocatalysis as a dual-action strategy, simultaneously targeting iMGEs and eMGEs to block environmental ARG transmission.

3.4 Analysis of potential hosts of ARGs

Network analysis (Figure 2a) extended these findings by revealing taxon-specific co-occurrence patterns between bacterial communities and both eARGs/iARGs. This topological evidence suggests distinct horizontal transmission modalities for eARGs and iARGs at the genus level, with extracellular variants showing higher network centrality (ρ = 0.72, p < 0.01) indicative of cross-species transfer dominance. Significant Pearson correlations (p < 0.05, r > 0.8) between bacterial genera and ARGs/MGEs (e.g., aac(6′)-Ib-03, intI1) confirmed ARG mobility dependence on microbial consortia (Supplementary Figure S4), with Proteobacteria reduction (chlorine-sensitive bacteria, ClSBs) strongly correlating with ARG removal efficiency (Wang Y. et al., 2024). Multi-walled carbon nanotubes composites-enhanced systems selectively enriched chlorine-resistant taxa (e.g., Dechloromonas), validating microbial engineering strategies for ARG control (Wang et al., 2022).

Figure 2

Figure 2. (a) Network analysis based on the co-occurrence of ARGs and their potential host bacteria after photocatalytic treatment of TiO₂ nanotubes; (b) Schematic diagram of TiO₂ nanotube photocatalytic degradation and blocking ARGs transfer mechanism.

TiO₂ photocatalysis induced Deinococcus dominance (Supplementary Figure S3), exhibiting radiation resistance but no iARG association, suggesting its non-participation in ARG transmission. Its eARG correlation implied sludge antibiotic pollutant degradation. Concurrently, iARGs (i-aac(6′)-Ib-03, i-strB) and MGEs co-occurred with Kofleria (p < 0.01), whose post-treatment declined directly linked to iARG reduction. Strong iARG-MGE covariation (r > 0.85) confirmed HGT-driven co-dissemination. UV preferentially degraded eARGs, yet residual eARGs correlated with Deinococcus/Acidovorax, indicating transformation potential. Significant eARG-iARG negative correlations (e.g., e-strB vs. i-strB, p < 0.01) revealed iARG release via cell lysis. Chlorination preferentially inactivated chlorine-sensitive bacteria (ClSBs, e.g., Proteobacteria), while promoting the enrichment of resistant taxa such as Dechloromonas–a phenomenon that sustained iARG reservoirs through microbial community restructuring (Wang et al., 2022). Chlorination and TiO₂ nanotube photocatalysis employed in this study shared almost no common host bacteria, as chlorination exhibits limited efficiency in removing iARGs (Liu and Kasuga, 2024).While, the TiO₂ nanotube photocatalysis operated through a dual mechanism: (1) Reactive oxygen species (ROS)-mediated oxidative attack synergized with UV-induced DNA damage selectively eliminates iARG-hosting genera (e.g., Kofleria), and (2) radiation-resistant but non-ARG-associated taxa (e.g., Deinococcus) become ecologically dominant post-treatment. This strategic microbial succession simultaneously depleted iARG vectors and established biological barriers against horizontal gene transfer, thereby effectively disrupting the ARG transmission chain.

3.5 ARG degradation and transfer inhibition via TiO₂ nanotube UV photocatalysis

The degradation of ARGs and MGEs by TiO₂ nanotube UV photocatalysis, as demonstrated in Sections 3.3 and 3.4, revealed significant reductions in both absolute and relative abundances. ARG absolute abundance decreased by 70.6%–82.5%, with a relative abundance decline of 10.58%–28.83%. In contrast, MGEs exhibited higher degradation rates, with absolute and relative abundances reduced by 93.4%–97.1% and 74.27%–75.86%, respectively, indicating superior efficacy of TiO₂ nanotube UV photocatalysis in degrading MGEs compared to ARGs. Distinct degradation patterns were observed in intracellular and extracellular environments. iARGs decreased by 33.53%–46.62%, while eARGs increased by 13.57%–87.41%. Similarly, iMGEs declined by 78.79%–82.96%, whereas eMGEs rose by 5.65%–36.18%. These trends highlighted the stronger degradation capability of TiO₂ nanotube photocatalysis for MGEs, leading to significant compositional changes in ARGs and MGEs.

Horizontal transfer of ARGs via MGEs is evidenced by statistical correlations: significant ARG-MGE associations (Pearson r > 0.7) indicate transfer potential, while co-localized ARGs on mobile elements (e.g., plasmids) demonstrate MGE-driven co-dissemination (Sommer et al., 2017). Pearson analysis of intra/extracellular co-occurrence patterns further validated their functional linkage in horizontal transfer dynamics. Pearson correlation analysis revealed co-occurrence of ARGs and MGEs both inside and outside cells (Supplementary Figure S4), such as strB, sul2, tetX, tnpA-04, tnpA-05, IS613, and intI1, supporting the transferability of ARGs via MGEs. After TiO₂ nanotube UV photocatalytic treatment, both iARGs and iMGEs showed significant decrease, indicating that TiO₂ nanotube UV photocatalysis blocked HGT by degrading MGEs, thereby curbing the spread of ARGs. Chlorination might trigger a rebound effect of ARGs and MGEs due to its insufficient oxidative capacity to completely eliminate both iARGs and eARGs (Liu and Kasuga, 2024; Yoon et al., 2017). This limitation allows residual ARGs to proliferate rapidly through stress response mechanisms activated during the disinfection process. Co-occurrence of ARGs (e.g., strB, sul2, tetX) and MGEs (e.g., tnpA-04, tnpA-05, IS613, intI1) both intracellularly and extracellularly further corroborated ARG transferability Figure 2a. Post-treatment, significant reductions in both iARGs and iMGEs demonstrated that TiO₂ nanotube UV photocatalysis inhibited HGT by degrading MGEs, thereby curbing ARG dissemination. Additionally, the significant negative correlation between iARGs and eARGs further supports ARG transfer disruption (Supplementary Figure S4). For example, intI-1(clinic), initially confined to RS cells, appeared both intracellularly and extracellularly after TiO₂ nanotube UV photocatalysis. This suggests that reactive oxygen species (ROS) generated during photocatalysis damage bacterial cell structures, releasing iARGs and preventing their vertical transfer. The co-occurrence of intI1 with ARGs (sul1, aadA, tetA) suggested the potential dissemination of ARGs after chlorination, which corresponds to the prevalent rebound patterns observed in ARGs and MGEs (Liu and Kasuga, 2024).

3.6 Mechanism of photocatalytic degradation and transfer inhibition by TiO₂ nanotubes under UV light

The differential degradation efficiencies of ARGs stem from ROS-mediated oxidative damage heterogeneity, where reactive oxygen species selectively target genomic loci based on gene copy numbers, deoxyribonucleotide composition, and sequence specificity (Li et al., 2022). This variability ultimately derives from TiO₂ nanotube UV-photocatalyzed ARG mobilization-transformation processes in sludge, wherein ARG degradation is achieved through strategic suppression of horizontal transfer pathways. The degradation of ARGs and MGEs via TiO₂ nanotube UV photocatalysis involved a complex mechanism, as illustrated in Figure 2b. TiO₂ nanotube UV photocatalysis generated ROS, which damaged cellular components, including proteins, lipids, and DNA, leading to cell membrane rupture and the release of iARGs into the extracellular environment (Chen et al., 2021). This process reduced iARGs while increasing eARGs. Photocatalytic degradation operated through both direct and indirect pathways. Direct degradation occured when UV light penetrated the cell wall, interacting with nucleic acids to form dimers that inhibit DNA replication and degrade ARGs. Indirectly, UV light activated photosensitive substances within or outside bacteria, generating ROS that disrupt cellular structures, releasing iARGs and degrading eARGs. However, UV light can also promote eARG enrichment. This occurs because eDNA may bind to colloidal particles in sludge, reducing its exposure to UV light. Additionally, free eARGs can be assimilated by other bacteria through transformation, further facilitating their dissemination. The photocatalytic degradation of MGEs played a critical role in inhibiting HGT, as ROS oxidize MGEs, preventing ARG transfer between bacteria via conjugation. This mechanistic profile aligns with yet differs from the compartmentalized oxidation paradigm proposed in the study of (Wang Y. et al., 2024), where intracellular hypochlorous acid (HOCl) and extracellular hydroxyl radicals (·OH) exibit spatial-specific bactericidal actions. This fundamental commonality suggests that oxidant penetrability (cellular/envelope barriers) and DNA-targeting specificity collectively govern degradation efficiency across various advanced oxidation processes (AOPs).

The network analysis revealed correlations between ARGs, MGEs, and microbial communities under TiO₂ nanotube UV treatment (Figure 2a). Specific genera, such as Kofleria, Nitrospira, and Ferruginibacter, were identified as potential hosts for ARGs like tetX and sul2. These genera also influenced ARG degradation and transformation in the environment, supporting the hypothesis that TiO₂ nanotube treatment inhibits HGT by disrupting MGEs and reducing potential host bacteria. Conversely, the inefficiency of chlorination arises from two primary factors: cell membrane barriers that impede chlorine penetration, and preferential consumption of chlorine by cytoplasmic components rather than DNA molecules (Wang Y. et al., 2024; Liu and Kasuga, 2024).

3.7 Limitations

This study has several limitations that warrant consideration. Primarily, the laboratory-scale TiO₂ photocatalytic systems investigated here have not been validated under real-world sludge treatment scenarios, creating a laboratory-to-reality knowledge gap. Three critical operational challenges remain unaddressed (1) complex matrix interference (e.g., competitive adsorption by humic acids): may substantially reduce ROS generation efficiency in heterogeneous sludge environments; (2) UV light penetration depth could be severely constrained in high-solid sludge (>2% total solids), potentially compromising treatment uniformity; and (3) the long-term stability of catalysts in continuous-flow operation requires verification, particularly regarding efficiency decay patterns after ≥10 reuse cycles. These scalability barriers must be systematically overcome before practical implementation.

4 Conclusion

This study explored the photocatalytic degradation of residual wastewater sludge using TiO₂ nanotubes under UV light, with a focus on the removal of intracellular and extracellular ARGs and MGEs, as well as their effects on microbial communities.

(1) Following 3 h of UV treatment, the absolute abundance of ARGs decreased by 70.6%–82.5%, while relative abundance declined by 10.58%–28.83%. Aminoglycoside, sulfonamide, and tetracycline ARGs exhibited higher degradation efficiency. iARGs decreased by 33.53%–46.62%, whereas eARGs increased by 13.57%–87.41%. TiO₂ nanotubes effectively degraded both ARGs and MGEs, inhibiting HGT.

(2) TiO₂ nanotube UV photocatalysis reshaped microbial communities, reducing dominant genera such as Nitrospira and Terrimonas while promoting the growth of others like Deinococcus. Network analysis identified Kofleria and Nitrospira as key potential hosts for ARGs, highlighting the critical role of MGEs in gene transfer.

(3) UV-induced ROS damaged bacterial hosts, releasing iARGs and increasing eARGs. The degradation of MGEs and destruction of host bacteria effectively blocked HGT. Direct oxidation of intracellular and extracellular DNA further contributed to ARG degradation.

In summary, TiO₂ nanotube UV photocatalysis represents a promising approach for degrading ARGs and preventing their dissemination in wastewater sludge.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions

HY: Conceptualization, Methodology, Software, Writing – original draft, Writing – review and editing. XZ: Conceptualization, Methodology, Software, Writing – original draft, Writing – review and editing. JZ: Investigation, Visualization, Writing – review and editing. TS: Investigation, Visualization, Writing – original draft. YZ: Conceptualization, Supervision, Writing – review and editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This research was funded by the National Natural Science Foundation of China (No. 52370127), Young Elite Scientists Sponsorship Program by China Association for Science and Technology (No. 2022QNRC001), Project LJ222410151015 supported by Fundamental Research Funds of higher education Department of Liaoning Province (Program for Liaoning Innovative Research Team in University), Dalian Outstanding Youth Science and Technology Talent Program (2024RY020), and Fundamental Research Funds for the Central Universities (No. 3132023518).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

Publisher’s note

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fenvs.2025.1590101/full#supplementary-material

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