The antifungal efficacy of nano-metals supported TiO2 and ozone on the resistant Aspergillus niger spore

Journal of Hazardous Materials 261 (2013) 155–162

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The antifungal efficacy of nano-metals supported TiO2 and ozone on the resistant Aspergillus niger spore Kuo-Pin Yu ∗ , Yi-Ting Huang, Shang-Chun Yang Institute of Environmental and Occupational Health Sciences, National Yang-Ming University, No. 155, Li-Nong Street, Section 2, Taipei 11221, Taiwan, ROC

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Durable nano-metals prepared by simple and low-cost incipient wetness impregnation. • The prepared nano-Ag can inactivate mold spores better than several previous cases. • Ozone has a synergetic effect on the nano-metals antifungal effectiveness.

a r t i c l e

i n f o

Article history: Received 14 March 2013 Received in revised form 26 April 2013 Accepted 13 July 2013 Available online 22 July 2013 Keywords: Aspergillus niger spore Nano-metals Synergetic effect Antifungal effectiveness Ozone

a b s t r a c t Recently, antimicrobial efficacy of nano-metals has been extensively investigated. However, most of the related studies focused on the bactericidal effectiveness. Molds, especially their spores, are more resistant than bacteria, and can build a high concentration in houses due to dampness. Therefore, a comprehensive evaluation of the antifungal effectiveness of nano-metals is necessary. In this study, the nano-metals (Ag, Cu and Ni) supported catalysts were successfully prepared by the incipient wetness impregnation method, while the titanium dioxide (Degussa (Evonik) P25) nanoparticle was served as the support. The antifungal experiments of Aspergillus niger spores were conducted on two surfaces (quartz and putty) in the darkness with and without ozone exposure, respectively. The critical Ag concentration to inhibit the germination and growth of A. niger spores of 5 wt% nano Ag catalyst was 65 mg/mL, lower than several cases in previous studies. The inactivation rate constants (k) of A. niger spores on nano-metals supported catalysts in the presence of ozone (k = 0.475–0.966 h−1 ) were much higher than those in the absence of ozone (k = 0.001–0.268 h−1 ). However, on the surface of TiO2 particles, no antifungal effect was observed until 6-h exposure to ozone. Consequently, ozone has a synergetic effect on nano-metals antifungal efficacy. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Due to global climatic changes, more extreme rainfall events have occurred worldwide [1–4]. These abnormal precipitations have resulted in serious water damages and dampness in buildings which thus have caused molds growth. For example, several weeks after the flood caused by Typhoon Morakot in southern Taiwan, the indoor fungal concentration in remediated houses reached as high

∗ Corresponding author. Tel.: +886 2 28267933; fax: +886 2 28278254. E-mail address: [email protected] (K.-P. Yu). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.07.029

as 2 × 105 spores/m3 [5]. Exposure to home dampness and molds is a high risk factor for irritation, allergy, infection and respiratory diseases [6–8], and modern people spend most of their time (>80%) indoors [9]. Therefore, around 5% of individuals are estimated to have various allergic respiratory symptoms resulted from molds over their lifetime. These allergic symptoms include rhinitis and asthma; sinusitis may arise secondarily from obstruction [10]. In order to remediate microbial contaminations, many researchers had conducted numerous investigations on active biocidal materials such as TiO2 photocatalyst and nano-metals [11–29]. Most of these investigations focused on the antimicrobial efficiency on bacteria, particularly Escherichia coli, which is

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relatively vulnerable. The high resistance of fungi, especially the fungal spores, to the common means of disinfection, such as heat, irradiation, and chemical methods had been reported [26]. Fungi have also been proven to be less sensitive to photocatalytic disinfection than bacteria owing to the complexity and thickness of their cell envelopes [23,30,31]. Chen et al. [30,31] demonstrated that UV light photocatalytic disinfection method could efficiently inhibit the germination of Aspergillus niger spores. However in their experiments, the spores were still viable and would recover and re-grow after the illumination stopped, indicating that the inactivation effect was primarily induced by UV irradiation but not TiO2 photocatalyst. Our previous study showed a similar experimental result [32]. However, there were not many concentrated studies focusing on the antifungal effectiveness of nano-metals, and studies regarding the inactivation of resistant mold spores were few [25]. Ozone has been used as a disinfectant for air purification, water treatment and food processing owing to its strong oxidizing power [33–35]. Ozone can cause cell membrane damage of food-related microorganisms and destroy the lipopolysaccharide layer of Gram-negative bacteria. It can also damage the enzymes and genetic materials in the cells [36]. Ozone has also been applied for antimicrobial treatment in dental therapy and oral cavity and can effectively reduce the total number of microorganisms and the infection of Streptococcus mutans in oral cavity [37,38]. Kowalski et al. [39] demonstrated that more than 99.99% of E. coli and Staphylococcus aureus cells would die when being exposed to ozone between 300 and 1500 ppm for 10 to 480 s. Fan et al. [40] proved that when Pseudomonas fluorescens was exposed to ozone of 100 ppb and negative air ion of 106 mL–1 for 6 h, the cell viability would reduce to 76%. While many attempts have been made to investigate the antifungal activity of TiO2 under UV irradiation or using the ozone as an oxidizer with antifungal ability, studies on the effects of simultaneous use of nano-metals catalyst and ozone are rather few. The combination of nano-metals catalyst and ozone may be beneficial to the inactivation of fungi because nano-metals catalyst can catalyze the decomposition of ozone and then generate many reactive oxygen species (ROS) with antifungal ability [41,42]. In this study, a commercial TiO2 nanoparticle was served as the support to prepare the nano-metals (Ag, Cu and Ni) catalysts by using the simple and low-cost incipient wetness impregnation method. We assessed the antifungal effectiveness of these nanometals supported TiO2 catalysts on A. niger spores. A. niger was selected because it is ubiquitous in the air in indoor environments. Its negative health effects include infections such as allergic bronchi pulmonary aspergillosis, allergic symptom caused by the spores and toxic symptoms resulted from the mycotoxic (aflatoxin). The antifungal experiments were carried out in two conditions: (1) in the darkness (Dark); (2) exposed to ozone (O3 ); and on two surfaces: (1) quartz; (2) putty (a kind of building material). The experimental results provided an evaluation of the antifungal effectiveness of the nano-metals supported TiO2 catalysts and the effect of ozone on the antifungal efficacy.

2. Methodology 2.1. Preparation and characterizations of nano-metals supported TiO2 catalysts In the study, the incipient wetness impregnation method was utilized to prepare oxidized nano-metals supported TiO2 catalysts with silver nitrate (AgNO3 purity: 99.99%, J.T. Baker, USA), copper (II) nitrate, 2.5-hydrate (Cu(NO3 )2 ·2.5H2 O, purity: 99.9%, J.T. Baker, USA), and nickel (II) nitrate hexahydrate (Ni(NO3 )2 ·2.5H2 O, purity:

99.9%, Lot No.: 10156297, Alfa Aesar, UK) as the precursors of nanoAg, Cu and Ni, respectively. Degussa (Evonik) P25 TiO2 nanoparticle was used as the catalyst support. This TiO2 powder has a major particle size of 30 nm, and a mixed phase of 30% rutile and 70% anatase. In the beginning of the preparation, the metal precursor containing appropriate amount of metal was dissolved in distilled deionized water with the same volume as the pore volume of the TiO2 support (0.4 mL of water/per gram of P25 TiO2 ). Then the metal-containing solution was added to the TiO2 support. The resultant mixture was stirred for 2 h. After that, the catalysts were dried at 120 ◦ C overnight and then were grinded into catalyst powders with an agate mortar. The prepared catalysts were denominated as 0.5 wt%Ag/P25, 2 wt%Ag/P25, 5 wt%Ag/P25, 2 wt%Cu/P25, 5 wt%Cu/P25 and 5 wt%Ni/P25, respectively, according to the species and the amount of loaded metals. The prepared catalysts were characterized by a JEM-2000EXII transmission electron microscope and a JSM-7600F scanning electron microscope to determine the size distribution of the nano-metals particles. 2.2. Culturing of A. niger spores The strain of A. niger fungus from Bioresource Collection and Research Center in Taiwan (BCRC 30310) was shipped in the freeze-dried form and needed to be activated before use. The activation procedures followed the description in the BCRC product instruction sheets. After being activated, the A. niger culture was transferred to the malt extract agar (MEA, DifcoTM ) plate and then incubated at 25 ◦ C for more than one week for the production of spores. These spores were harvested with autoclaved 0.05% Tween 80 solution by a shaker with the shaking rate of around 80 rpm for ten minutes prior to being used for the subsequent experiments. The concentration of the A. niger spores in the suspension was around 107 CFU/mL (CFU = colony forming unit). 2.3. Inactivation of mold spores by nano-metals supported TiO2 catalysts on dry surface The inactivation of A. niger spores by the nano-metals supported TiO2 catalysts was conducted under two conditions: (1) in the darkness without ozone (Dark); (2) exposed to 5 ppm of ozone (O3 ). Before the inactivation experiments of A. niger spores, 10 ␮L of the aqueous suspension containing 5% catalyst and 0.005% dioctyl sulfossuccinate (purity: 99.9%, Sigma) was dropped on each 0.5 cm × 0.5 cm quartz chip. Then, these quartz chips were baked on a hot plate until a smooth catalyst layer was formed. After that, these chips were sterilized by an autoclave, and then, 10 ␮L of suspension of A. niger spores (around 105 spores) was inoculated on each sterilized chip. These chips were moved to a 2-l stainless steel test chamber that had been disinfected with 75% alcohol beforehand. The supply air of this test chamber was provided by a zero air system as shown in Fig. 1. The relative humidity inside the chamber was controlled at 50% and the air change rate at 5 h−1 . After the suspension of A. niger spores on the quartz chips were dry (It took about 1 h), the experiments started (Dark). In the cases of O3 , it took about 5 min for the ozone concentration to achieve the required level (5 ppm). The spores on the chips were harvested with autoclaved 0.05% Tween 80 solution at the time of 0, 1, 2, 3, 4 and 6 h in the case of O3 and were harvested at the time of 0, 2, 4, 6 and 8 h in the case of Dark. These suspensions were series diluted, transferred to the MEA plate, and then incubated for 48 h at 25 ◦ C. Finally, we enumerated the CFU on the agar plates to quantitate viable A. niger spores on the quartz chips and catalysts. We also tested the antifungal effectiveness of these catalysts on putty (a kind of building material that contains mainly calcium carbonate and some resins and preservatives). Putty was first coated

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Fig. 1. Experimental system for testing the inactivation of Aspergillus niger spores.

on the quartz chips. After the putty was dry, 10 ␮L of aqueous suspension containing 5% catalyst and 0.005% dioctyl sulfossuccinate was dropped on the chips. After the quartz chips were dry and sterilized, the A. niger spores were inoculated on the chips to test the antifungal effectiveness with the same procedure described above. We used the initial survival fraction (SFi) to evaluate the fraction of viable A. niger spores harvested from the quartz chips at the beginning of experiment: SFi =

N(0) Ninoculated

(1)

where N(0) is the concentration of viable A. niger spores harvested from the quartz chips at the beginning of the experiment; Ninoculated is the concentration of viable A. niger spores inoculated on the quartz chips. The antifungal effectiveness of the nano-metals supported TiO2 catalysts on the inactivation of A. niger spores was evaluated by the survival ratio (SR): SR =

N(t) N(0)

(2)

where N(t) is the concentration of viable A. niger spores harvested at time t; N(0) is the concentration of viable A. niger spores harvested from the quartz chips at the beginning of the experiment. 2.4. Disk diffusion assay [43] The suspension of A. niger spores was applied equally on the malt extract agar plates. Then we hollow wells (0.6 cm in diameter) which served as reservoirs for loading catalyst suspensions onto these agar plates. The plates were then incubated at 25 ◦ C for 48 h

in darkness. The inhibition zones around the wells were determined based on five replicated experiments. 3. Results and discussion 3.1. Characterizations of nano-metals supported TiO2 catalysts Fig. 2 shows the SEM images of nano-Ag supported TiO2 catalysts, and the bright spots in the COMBO mode images are the Ag particles. Fig. S1 (supplementary data) demonstrates the TEM images of nano-metals supported TiO2 catalysts and the size distribution of the nano-metals particles. The sizes of the nano-metals particles range from 3 to 33 nm with an average size of 14.51–17.34 nm (Table S1). Fig. S2 shows the SEM images of nano-Cu and nano-Ni supported TiO2 catalysts. Fig. S3 demonstrates the size comparison of the P25 (∼30 nm). These microscopic images demonstrate that the TiO2 nanoparticles served as a good support for the preparation of nano-metals catalysts by the simple and low-cost incipient wetness impregnation method. The characterizations of these nano-metals particles remained unchanged for more than 2 years, which indicates that the TiO2 nanoparticles can avoid coagulation of the nano-metals particles. 3.2. Inactivation of the mold spores in darkness As shown in Fig. 3(a), after staying on the surface of bare quartz chip, P25 TiO2 and 5 wt%Ni/P25 (loading level was 2 mg/cm2 ) in the darkness for 8 h, A. niger spores were still viable with survival ratios (SRs) higher than 0.91. According to previous studies, the relatively vulnerable E. coli and yeast were totally inactivated under the same condition (SR decreased to zero within 1 h) [44]. Therefore,

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Fig. 2. SEM images of nano-Ag supported TiO2 catalysts (a) 0.5 wt%Ag/P25, SEI (secondary Electron Image) (b) 0.5 wt%Ag/P25, COMBO mode (SEI and Back Scatted Electron Image) (c) 2w t%Ag/P25, SEI (d) 2 wt%Ag/P25, COMBO mode (e) 5 wt%Ag/P25, SEI (f) 5 wt%Ag/P25, COMBO mode.

A. niger spores are more resistant to dryness and environmental stress than bacteria and yeast. Nevertheless, increasing the loading level of catalysts would enhance the antifungal effectiveness. As shown in Fig. 3(a), when the loading levels of P25 and 5 wt% Ni/P25 increased to 4 mg/cm2 , the SRs(8 h) of A. niger spores would decrease to 0.844 ± 0.181 and 0.807 ± 0.194, respectively. The similar result was also found in the cases of 0.2 wt% Ag/P25 and 2 wt% Cu/P25 catalysts.

The antifungal kinetics fitted the Chick-Watson model (firstorder decay model) [45]: SR = exp(−kt)

(3)

in which k is the inactivation rate constant; t is the elapsed time. The fitting result of Eq. (3) is listed in Table 1. Accordingly, nanoCu supported TiO2 catalysts were better than nano-Ni supported ones on the inactivation of A. niger spores, but worse than nano-Ag

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Fig. 3. The time profiles of the survival ratio of Aspergillus niger spore on the surface of catalysts and quartz chip (a) in the darkness (Dark); (b) exposed to 5-ppm ozone (O3 ).

supported ones. However, the nano-Cu could be a low-cost alternative for the inactivation of A. niger spores, since copper is much more inexpensive than silver. The initial survival fraction (SFi) shown in Table 1 reflects the effectiveness of catalysts on the inactivation of A. niger spores in darkness when the catalysts were wet. Both the SFi and SRs (8 h) in the cases of 0.5 wt%Ag/P25 and 2 wt%Cu/P25 were larger than those in the cases of P25 TiO2 and 5 wt%Ni/P25, indicating that the antifungal effectiveness of 0.5 wt%Ag/P25 and 2 wt%Cu/P25 was higher. In this study, the inactivation of A. niger spores by 5 wt%Ag/P25 (4 mg/cm2 ), 2 wt%Ag/P25 (2 mg/cm2 ) and 5 wt%Cu/P25 (4 mg/cm2 ) catalysts were also investigated. As. niger spores were inactivated completely by 5 wt%Ag/P25 at the beginning of the experiment (SFi = 0), and were entirely inactivated by 2 wt%Ag/P25 (SFi = 0.015 ± 0.003; SR(2 h) = 0) and 5 wt%Cu/P25 (SFi = 0.018 ± 0.004; SR(2 h) = 0) after 2 h. This result demonstrated the considerable antifungal effectiveness of nano-Ag and -Cu on the inactivation of A. niger spores in darkness. We also used the disk diffusion assay to evaluate the antifungal efficiency of 5 wt%Ag/P25 and 2 wt%Ag/P25 catalysts. As shown in Fig. 4(a), the inhibition zone surrounding the reservoir of 5 wt%Ag/P25 catalyst increased with the Ag concentration. The radii of the inhibition zone (ri ) was fitted to the solution of the one-dimensional, radial diffusion equation: (ri − rr )2 = 4Dt ln(C/Cc )

(4)

where rr is the radius of the catalyst reservoir (4.5 mm); D is the diffusion constant of nano-Ag/Ag+ ; t represents time; C is the Ag concentration; Cc is the critical Ag concentration expected to inhibit any germination and growth of A. niger spores. Rearranging Eq. (4), we had: ln C = (ri − rr )2 /4Dt + ln Cc

(5)

The plot lnC vs. (r i − rr )2 has a slope of 1/4Dt and a y-intersection of lnCc as shown in the inset of Fig. 4(b). According to the plotting, the critical Ag concentrations of 5 wt%Ag/P25 and 2 wt%Ag/P25 catalysts were 65 and 135 ␮g/mL, which was close to the minimum inhibitory concentration (MIC) [43]. MIC is defined as “the lowest concentration not associated with visible growth on malt extract agar after 72 h of incubation at 30 ◦ C” [25]. Comparing our results with previous studies, we found that the antifungal effectiveness of 5 wt%Ag/P25 catalyst may be better than the novel silver nanocomposite material (MIC = 2000 ␮g/mL, 20%wt of Ag), silver zeolite (MIC = 125 ␮g/mL, 38 wt% of Ag) reported by Egger et al. [25], and fluconazole (a triazole antifungal drug, MIC =128 ␮g/mL) [46], but worse than silver nitrate (MIC = 15.6 ␮g/mL, 63.5 wt% of Ag)[25]. In addition, the antifungal efficiency of 2 wt%Ag/P25 catalyst might be comparable to the silver zeolite. The disk diffusion assay was also conducted with nano-Cu catalyst, however, no inhibition zone was observed. This might result from that the Cu ion formed complexes with the alkalinity of the malt extract agar which limited the diffusion of the Cu ion/nano-Cu. We also tested the antifungal effectiveness of the 0.5 wt%Ag/P25, 2 wt%Cu/P25, and 5 wt%Ni/P25 catalysts on putty. As shown in Fig. 5(a), nano-Ag and -Cu catalysts did inactivate the A. niger spores when being applied on putty, but the inactivation rate constants were lower than those applied on the quartz chips. This might result from the fact that the Ag and Cu ions can form complexes with the alkalinity of the putty which reduced the diffusivity and antifungal effectiveness of the nano-Ag and Cu. The inactivation rate constant (k) of A. niger spores on putty applied on bare chips were higher than those without putty applied indicating the moderate antifungal ability of the calcium carbonate and preservatives in the putty. Although the mechanisms of the inactivation of A. niger spores by nano-Ag and nano-Cu have not been fully understood yet, they may involve in the interactions with sulphydryl groups in proteins.

Table 1 The inactivation rate constant (k) and initial survival fraction (SFi) of Aspergillus niger spores on catalysts under various conditions.

*

Catalyst Loading level = 2 mg/cm2

Condition SFi

Bare chip P25 TiO2 0.5 wt%Ag/P25 2 wt%Cu/P25 5 wt%Ni/P25

0.104 0.117 0.102 0.099 0.114

Putty applied on bare chip.

± ± ± ± ±

0.02 0.01 0.017 0.019 0.009

Dark k

O3 k

Dark (on putty) k

O3 (on putty) k

0.005 h−1 (R2 = 0.35) 0.005 h−1 (R2 = 0.45) 0.268 h−1 (R2 = 0.9) 0.093 h−1 (R2 = 0.87) 0.01 h−1 (R2 = 0.44)

0.304 h−1 (R2 = 0.98) 0.072 h−1 (R2 = 0.46) 0.475 h−1 (R2 = 0.97) 0.966 h−1 (R2 = 0.99) 0.77 h−1 (R2 = 0.97)

0.044 h−1 (R2 = 0.89)* 0.009 h−1 (R2 = 0.25) 0.162 h−1 (R2 = 0.93) 0.076 h−1 (R2 = 0.91) 0.051 h−1 (R2 = 0.9)

0.150 h−1 (R2 = 0.75)* 0.085 h−1 (R2 = 0.13) 0.182 h−1 (R2 = 0.96) 0.156 h−1 (R2 = 0.9) 0.214 h−1 (R2 = 0.87)

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Fig. 4. (a) The zones of inhibition around the reservoir of 5 wt%Ag/P25 catalyst. (b) Radii of the zone of inhibition is a function of Ag concentration. The inset demonstrates the semilog plot of the experimental data. Critical inhibitory concentration 65 ␮g/mL is estimated from the y-intercept.

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considerably improve the efficiency of ozonation via the production of hydroxyl radical (OH·) described as the following Eqs. [41,42]:

Fig. 5. The antifungal kinetics of the 0.5 wt%Ag/P25, 2 wt%Cu/P25 and 5 wt%Ni/P25 catalysts against Aspergillus niger spore when being applied on the putty (a) in the darkness (Dark); (b) exposed to 5-ppm ozone (O3 ).

Silver ions can interact with electron donors and other cellular components such as nucleic acids. Silver ions may also cause chloride or anion restriction within the cells due to the silver salt formation [20,24,25]. Oxidative DNA damage mediated by copper (II) Fenton reaction may also lead to the inactivation of fungal spores [47].

3.3. Inactivation of the mold spores exposed to ozone Ozone is a strong oxidizer, and the survival ratio of microbes may decrease with the increase of the exposure duration to ozone. As shown in Fig. 3 (b), the survival ratio of A. niger spores on bare chips decreased to 0.19 ± 0.12 after being exposed to ozone for 8 h, and the deactivation rate constant was 0.304 h−1 . Comparing the results of Fig. 3(a) and Fig. 3(b), we found that the inactivation rate of A. niger spores by nano-metals supported TiO2 catalysts was also significantly enhanced by ozone. When the ozone concentration increased from 0 to 5 ppm, the inactivation rate constants of A. niger spores by 0.5 wt%Ag/P25, 2 wt%Cu/P25 and 5 wt%Ni/P25 would increase from 0.268 to 0.475 h−1 , from 0.093 to 0.966 h−1 and from 0.01 to 0.77 h−1 , respectively, as demonstrated in Table 1. The increment of the inactivation rate was relevant to the amount of nano-metals loading. Thus, the enhancement effect of ozone on the inactivation rate of A. niger might be resulted from that the ozone-decomposition catalysts (nano-metal oxide) could

O3 + ∗ → ∗ · O− + O2

(6)

∗ · O− + H2 O → 2OH·

(7)

where * is the active site of nano-metals oxide catalyst. The generated hydroxyl radical could attack the A. niger spores, which enhanced the inactivation rate of these spores. However, the inactivation rate of A. niger spores by TiO2 was not enhanced by ozone as significantly as in the cases of nano-metals supported TiO2 catalysts. As shown in Fig. 3(a) and (b), there was no significant changes of the SRs in the case of P25 when the ozone concentration increased from 0 to 5 ppm, except the SRs(8 h) deceased from 0.923 ± 0.081 to 0.095 ± 0.045 (P25). Therefore, the ozone decomposition and hydroxyl radical production by P25 was not as efficient as that by nano-metals supported TiO2 catalysts. Furthermore, the TiO2 particles may cover the A. niger spores and protect them from the damages caused by ozone. The shoulder-shape inactivation curve in the cases of P25 might result from the clump of A. niger spores or multiple attacks of critical sites by ozone [45]. We also tested the synergetic effect of ozone and nano-metals on the antifungal efficacy on A. niger spores when the 0.5 wt%Ag/P25, 2 wt%Cu/P25, and 5 wt%Ni/P25 catalysts was applied on putty. As shown in Fig. 5(b) and Table 1, the inactivate rate constant of A. niger spores of O3 condition was higher than that of the Dark condition, indicating that the antifungal effectiveness of nano-metals catalysts were obviously enhanced by ozone. The synergetic effect of ozone and nano-metals on the antifungal efficacy was also observed in the cases of O3 condition, in which the inactivate rate constant of the nano-metals catalysts was much higher than that of P25. However the inactivation rate constants were lower than those without being applied on the putty. This might be caused by the fact that the formation of metal complexes with the alkalinity of the putty can reduce the activity of nano-metals catalysts. The shoulder-shape inactivation curve in the case of “Putty” might be caused by the clump of A. niger spores or manifold attacks of critical sites by ozone [45]. Putty applied might reduce the exposure to ozone and resulted in the decrease of inactivation rate constant of A. niger spores. 3.4. Limitations 1. Practicality of treatment – although there was some antifungal effect, there were still survivors. These survivors are likely to grow and may still cause issues and generate more resistant offspring. Therefore, for practical applications, prolong treatments are necessary to completely inactivate the survival fungus. 2. Incipient wetness impregnation is a simple method and cannot synthesize nano-metals catalyst with the precise particle sizes. The sizes of the nano-metals particles were normally distributed from 3 to 33 nm. Therefore, only the overall antifungal effect of nano-metals particles was observed in our experiments. The effect of particles size, which is relevant to the penetration of the particles into the fungal cells, was not considered. 4. Conclusions 1. The nano-Ag and -Cu loaded TiO2 catalysts can reduce the survival ratio of A. niger spores in the darkness. The loading level of Ag or Cu is important and the antifungal property of nano-Ag or -Cu supported TiO2 in the darkness is obtained in higher loading level. The antifungal effectiveness of 5 wt%Ag/P25 catalyst is better than several cases reported in previous studies. 2. In the presence of ozone, the nano-metals oxide can catalyze the decomposition of ozone and then produce hydroxyl radicals

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