3.4.3. Discussion of the Antifungal Mechanisms

In this work, the hybrid Ag/TiO<sup>2</sup> films grown on the bamboo surface produced a synergistic antifungal mechanism under both light and dark conditions. According to data from the experiments conducted in the dark, the Ag/TiO<sup>2</sup> NPs showed more effectiveness at inhibiting *A. niger* growth than pure Ag NPs or TiO<sup>2</sup> NPs, even though they could not completely inhibit the growth of *A. niger*. The mechanism for the enhanced antimicrobial effect of Ag/TiO<sup>2</sup> hybrids in the absence of light is still not completely understood. Their enhanced antimicrobial qualities originated from the generation of reactive oxygen species, the release of toxic Ag ions, and cell membrane damage through their contact with the Ag NPs.

Hoek et al. [24] reported that hybrid Ag/TiO<sup>2</sup> NPs exhibited stronger bactericidal activity than pure Ag and TiO<sup>2</sup> in the absence of light. The observed synergistic effects under dark conditions were most likely caused by the variation in the dissolution and reprecipitation kinetics and equilibrium between pure Ag NPs and Ag/TiO<sup>2</sup> NPs. Kim et al. [40] hypothesized that the toxicity of Ag NPs is mainly caused by oxidative stress and is not related to the activity of Ag ions. Perkas et al. [41] proposed that the antibacterial activity of Ag/TiO<sup>2</sup> composites originates from the presence of reactive oxygen species (ROS) as well as Ag ions on the surface of TiO<sup>2</sup> in the dark. Chen et al. [25] reported that the antibacterial activity of Ag/TiO<sup>2</sup> nanocomposites under dark conditions appears to be superior to that of some pure Ag NPs. They suggested that the smaller Ag particle size should account for the higher antibacterial activity of their Ag/TiO2. Perkas et al. [41] and Esfandiari et al. [42] both reported a similar observation, noting that the bactericidal capacity depended on the size characteristics of the Ag/TiO<sup>2</sup> coating. Under similar testing conditions, our previous work showed that TiO<sup>2</sup> thin films modified by Ag NP (diameter of 2–10 nm) have better antifungal activity for bamboo than those modified by large Ag NPs (diameter of 50–100 nm) [35]. In addition, the antifungal performance of Ag/TiO<sup>2</sup> nanocomposites was greater than that of AB and TiO2/bamboo in the absence of light, indicating that the Ag/TiO<sup>2</sup> nanocomposite produced a synergistic antifungal effect that was unrelated to photoactivity.

**Figure 7.** Antifungal properties of (**a**,**a1**) original bamboo (left) and TB (right), (**b**,**b1**) AB-10 (left) **Figure 7.** Antifungal properties of (**a**,**a1**) original bamboo (left) and TB (right), (**b**,**b1**) AB-10 (left) and ATB-5 (right), (**c**,**c1**) AB-30, (**d**,**d1**) ATB-10, (**e**,**e1**) ATB-30, and (**f**,**f1**) TB (left) and ATB-200 (right) to inhibit *A. niger* growth under LED light. We can clearly see the mycelia in the red rectangle. Incubation period: (**a**–**f**) 5 days, (**a1**–**f1**) 28 days. TB: TiO2/bamboo, ATB-*x*: the Ag-NP-decorated TiO2/bamboo samples were denoted as ATB-*x*, with *x* representing the solution concentration (5, 10, 30, and 200 mM) of AgNO<sup>3</sup> as one of the raw materials, AB-*x*: the Ag-NP-decorated bamboo samples were denoted as AB-*x*, with *x* representing the solution concentration (10 and 30 mM) of AgNO<sup>3</sup> as one of the raw materials.

As mentioned above, under dark conditions, the ATB samples could not completely inhibit *A. niger* growth on their surfaces. It is widely considered that photocatalytic microorganism disinfection depends on the interaction between microorganisms and ROS generated from photocatalysts under light illumination, such as •OH and •O<sup>2</sup> <sup>−</sup>, which can kill microorganisms [43]. Therefore, we further evaluated the antifungal activity of as-prepared samples to inhibit the growth of *A. niger* under light radiation. From the practical application perspective, photocatalysts should not be constantly exposed to light. Therefore, we attempted to perform our experiment under visible-light irradiation for 6 h every day and then turn off the light source. Interestingly, some of the as-prepared samples could achieve complete antimicrobial activity. The ATB samples exhibited strong visible-light absorption after the addition of Ag NPs owing to the localized surface plasmon resonance. They could generate electron–hole pairs under visible-light irradiation and then migrated to the surface of the catalyst to initiate redox reactions. Most interestingly, the as-prepared ATB samples could store electrons after visible light was removed.

Figure 8 presents the ESR spectra of the ATB-10 sample. After 10-min visible-light irradiation, the strong characteristic peak DMPO-•O<sup>2</sup> <sup>−</sup> signals were observed, which demonstrates the formation of •O<sup>2</sup> <sup>−</sup> radicals by ATB-10 under light illumination (Figure 8a). When illumination was turned off, the four peaks associated with DMPO-•O<sup>2</sup> <sup>−</sup> adducts for ATB-10 could still be distinguished. The intensity of the DMPO-•O<sup>2</sup> <sup>−</sup> signals was slightly reduced after the sample was kept in the dark for 20 min. This result demonstrates that •O<sup>2</sup> <sup>−</sup> could be produced by ATB-10 during a dark discharge process. Similarly, we also verified the formation of •OH radicals in the dark. The ATB-10 sample exhibited slower decay kinetics of DMPO-•OH adducts after being kept in the dark for 20 min, as shown in Figure 8b. This result indicates that a considerable number of electrons in ATB-10 may remain when illumination is stopped, providing additional •OH to mitigate the decay of DMPO-•OH, which is consistent with previous work [44]. Based on the experimental data and analysis, a possible mechanism for the memory antifungal activity can be proposed as follows (Figure 8c):

− **Figure 8.** Time evolution of (**a**) DMPO-•O<sup>2</sup> <sup>−</sup> and (**b**) DMPO-•OH ESR spectra for ATB-10. (**c**) Possible mechanism of the catalytic memory reaction. ATB-10: Ag/TiO2/bamboo; the solution concentration of AgNO<sup>3</sup> used is 10 mM.

During the photocatalytic disinfection period, excess electrons can be trapped on Ag NPs because of the capacitive nature of Ag nanomaterials. Stored electrons will be released in the dark and subsequently discharged to appropriate electron acceptors, such as O<sup>2</sup> and H2O, to produce the corresponding active free radicals to inhibit the growth of fungi [44]. The combination of photocatalytic disinfection and catalytic memory reaction provides a new pathway for producing novel catalysts to achieve round-the-clock pollutant removal.

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