New insight into the disinfection mechanism of

Journal of Photochemistry & Photobiology, B: Biology 176 (2017) 17–24

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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

New insight into the disinfection mechanism of Fusarium monoliforme and Aspergillus niger by TiO2 photocatalyst under low intensity UVA light

MARK

Chonlada Pokhum, Duangamon Viboonratanasri, Chamorn Chawengkijwanich⁎ Hybrid Nanostructure and Nanocomposite Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand

A R T I C L E I N F O

A B S T R A C T

Keywords: Fusarium monoliforme Aspergillus niger TiO2 Disinfection mechanism

Titanium dioxide (TiO2) photocatalytic reaction has great potential for the disinfection of harmful pathogens. However, the disinfection mechanisms of TiO2 photocatalysis are not yet well-known for fungi and protozoa. In this work, the photocatalytic disinfection mechanism of Fusarium monoliforme and Aspergillus niger under low intensity UVA light (365 nm, < 10 W/m2) was studied at the ultrastructural level. Photocatalytic treatments showed that the photocatalytic oxidation of 10% TiO2 based paint was efficacious in the complete disinfection of F. monoliforme under low intensity UVA light. No growth of F. monoliforme was observed on agar plate in the subsequent dark. Transmission electron microscopy (TEM) of F. monoliforme exposed to TiO2 photocatalysis treatment showed a distinct damage to electron-dense outer cell wall, but not to an underlying electron-transparent layer cell wall. The TEM image revealed that the UVA-light only did not damage cell wall, cell membrane and cellular organelles. Unlike, A. niger was more sensitive to UVA-light. Serious destructions of cell membrane and cellular organelles were shown in A. niger exposed to UVA-light only and photocatalytic treatments. However, morphological change in A. niger cell wall was only observed in photocatalytic treatment. Changes to the outermost melanin like layer and cell wall of A. niger spore due to photocatalytic treatment were greatly apparent while the intracellular organelles of A. niger spore were not affected. Therefore, regrowth of A. niger on agar plate was expected from the germination of A. niger spore in the subsequent dark. These observations give a better understanding of the photocatalytic disinfection mechanism toward fungi.

1. Introduction So far, there has been much interest in using the disinfecting property of titanium dioxide (TiO2) photocatalyst because of its potential to use sunlight directly to drive the disinfection process [1–3]. The first study on the ability of TiO2 photocatalysis to destroy microbial pathogens in water has been documented in 1985 [4]. TiO2 has drawn much attention for its high photo-activity, low toxicity, good chemical and thermal stability, low cost, and insolubility in water [5–6]. Photocatalytic disinfection has been studied extensively in bacteria [7–10], but photocatalytic disinfection of fungi has not had much research. Ultraviolet (UV) radiation is typically classified into three major groups based on its wavelength: UVA (315–400 nm), UVB (280–315 nm) and UVC (100–280 nm). Direct UVC disinfection (254 nm) uses shortwave radiation to kill microbes, and this type of UV radiation is also harmful to human health [11]. But, TiO2 photocatalytic disinfection is driven by long-wave UVA radiation. The absorption of energy and the subsequent generation of electron–hole pairs (e−/h+) are the initiating steps of TiO2 photocatalysis [3,12–13]. These two ⁎

highly reactive existences are therefore concomitantly involved in all subsequent reactions in the system. Photogenerated electrons react with absorbed oxygen (O2) forming initially a superoxide radical (O2−•), while the photogenerated holes interact with surface water molecules or hydroxide ions to form the highly reactive hydroxyl radicals (•OH). This reactive oxygen species (ROS) acts as the primary oxidants in the photocatalysis and is considered to be the main oxidant to inactivate microorganisms, including viruses, bacteria, spores and protozoa [7,13–14]. The various targeted sites of microorganisms during photocatalytic inactivation can be grouped into extracellular and intracellular sites [13]. The cell membrane and cell wall are obvious targets in the extracellular site. The destruction of the cell membrane is an important process for inactivation [8,15–17]. The fundamental photocatalytic disinfection mechanisms are based on lipid peroxidation of membrane fatty acids. Unlike, the possibility of intracellular target sites for photocatalytic oxidation is rather limited because photocatalytic oxidation takes place through surface-bound radicals, which are not free to diffuse into the cell [13]. The attack of intracellular constituents, such as

Corresponding author. E-mail address: [email protected] (C. Chawengkijwanich).

http://dx.doi.org/10.1016/j.jphotobiol.2017.09.014 Received 20 July 2017; Received in revised form 8 September 2017; Accepted 13 September 2017 Available online 15 September 2017 1011-1344/ © 2017 Elsevier B.V. All rights reserved.


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purchased from RCI Labscan Co., Ltd. Bangkok, Thailand. Isopropyl alcohol was purchased from Fisher Scientific Limited, UK. Toluene and ethyl acetate were purchased from Carlo Erba Reagent SPA, Rodano, Italy. Polypropylene (PP) film (25 μm in thickness) was used as the substrate for TiO2 deposition. Two 20-watt blacklight blue lamps (BBL, peak wavelength emission of 365 nm were applied as UVA light source.

enzymes, coenzymes and DNA, takes place through generation of other oxidants in the cytoplasm or direct surface-bound radicals as a result of large perforations in the cell membrane [7,13,18–20]. Microbes are omnipresent pollutants and present significant health effects. Compared to bacteria, fungi are more resistant to photocatalytic inactivation due to the complexity and strength of the cell envelopes and existing spores [21–22]. The Aspergillus niger is mainly selected for challenging test because it is ubiquitous in the air and its resistance spores to common means of disinfection [23]. For example, 99.9% of Escherichia coli are killed in about 1 h spores under photocatalytic treatment at 104 W/m2 of UVA light intensity while it takes 72 h to achieve about 90% killing of A. niger [23]. The inactivation of A. niger by TiO2/UVA light is directly dependent on the UVA light intensity [24]. The application of TiO2 and UVA light intensity at 10 W/m2 greatly reduced the surviving number of A. niger spores, but decreased slightly when the UVA intensity was reduced to 1 W/m2 [24]. Even though here was no visible A. niger growth during the TiO2/UVA process (UVA intensity of 1.78 W/m2), regrowth appeared once the UVA irradiation was stopped, indicating that the photocatalytic reaction was insufficient for total disinfection against A. niger but only temporarily suppressed its growth [25]. Fusarium is a large genus of filamentous fungi and has a worldwide distribution. Fusarium species are mycotoxigenic fungi, and the mycotoxins produced by these organisms are often associated with animal and human health. Several researchers have studied the detrimental effect of TiO2 photocatalytic against Fusarium using solar light [21,26–27]. Solar photocatalytic disinfection against F. solani under strong solar UV light (300–400 nm, 200 W/m2) using a TiO2 suspension could achieve at least a 4-Log reduction in cell viability [21]. Solar photocatalytic disinfection of various Fusarium strains under high solar UV irradiance (300–400 nm, 25–40 W/m2) was studied using TiO2 suspension [26–27]. Total photocatalytic disinfection of F. solani, F. Anthophilum, F. equisetti, F. verticillioides (=F. monoliforme), and F. Oxysporum have been reported. From above literatures, it suggests that the sensitivity of Fusarium and Aspergillus to TiO2 photocatalysis is different. The main purpose of this research is to study a detrimental effect of TiO2 photocatalytic disinfection against F. monoliforme and A. niger exposed onto TiO2 based paint with varying TiO2 concentrations (0, 1, 5 and 10% w/v) under low intensity UVA light (< 10 W/m2). The amount of F. monoliforme and A. niger was evaluated by the colony forming count method, and the cell morphology was analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

2.3. Preparation of the TiO2-based Paint First, TiO2 nanopowder (1, 5 and 10 g ca.) was added to 100 mL of a mixture of organic solvents and acrylic resin, and the suspension was homogenized for 30 min at 10,000 rpm. Next, the TiO2-based paint was coated on the polypropylene (PP) film substrate (15 × 25 cm size, thickness of 25 μm) using a simple bar-coating process by a wire bar control coater (model K control coater, R K Print Coat Instrument Ltd., Royston, UK). Each painted specimen (TiO2-PP) was dried in air at 60 °C for 1 h. The obtained TiO2-PP specimens were characterized using a scanning electron microscope (SEM, Hitachi S-3400 N, Hitachi, Japan) and energy dispersive spectrometry (EDS, Horiba 7021, Hitachi, Japan) operating at 15 kV. 2.4. Photocatalytic Disinfection Experiment Photocatalytic experiments were performed by the previous method [14] with some modification. The initial concentration of F. monoliforme and A. niger suspension was approximately 105 CFU/mL. First, 50 μL of the suspension was inoculated onto each surface of four group sample specimens (20 × 20 mm size): uncoated PP, 1% TiO2-PP, 5% TiO2-PP and 10% TiO2-PP. Each inoculated specimen was covered with a sterile lid and placed in a sterile Petri dish at 28°C in the dark or under UVA illumination for 24 h. Illumination was carried out using two blacklight bulbs (20 W, 365 nm, UVA light intensity ca. 8 W/m2) located 15 cm above the test specimens. After 24 h, the F. monoliforme and A. niger were collected from the test specimens by gently washing with 10 mL of autoclaved 0.85% saline solution. Serial dilutions were then cultured on PDA plates and kept in the dark for 3 days at 28 °C. Analyses were conducted in triplicate per treatment, and the control treatment was uncoated PP in the dark. The number of colony forming units per milliliter (CFU/mL) of all treatments was determined. The efficacy of TiO2 photocatalytic disinfection was represented as percentage elimination of fungi as compared to control treatment and calculated using Eq. (1).

%Elimination = (A − B) × 100 A

(1)

where: A - the average number of colony forming units after exposure to control treatment for 24 h, B - the average number of colony forming units after exposure to other treatments for 24 h.

2. Experimental 2.1. Fungal Cultures

2.5. SEM Analysis The fungi used were laboratory strains of F. moniliforme (BCC 5747) and A. niger (BCC 17391) obtained from the National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand. F. monoliforme and A. niger were cultured on Potato Dextrose Agar (PDA, Difco, Becton, Dickinson and Company, USA) at ambient temperature for 5–7 days. From these plates, each suspension of F. monoliforme and A. niger was prepared by washing the mycelium with sterile deionized water containing Tween 80 (0.1% v/v). The concentration of each fungus was then adjusted to ~105 CFU/mL (CFU = colony forming unit).

F. monoliforme and A. niger were collected from the surface of test specimens and fixed with 2.5% glutaraldehyde in 0.1 M phosphatebuffer (pH = 7.2) for 2 h, washed twice with phosphate buffer and once with distilled water. Next, the samples were dehydrated through a graded series of ethanol solutions (30%, 50%, 70%, and 90%, each solution once for 10 min, and finally three times for 10 min each in 100% ethanol). Samples were then dried using a critical point dryer (Quorum, model K850, United Kingdom) and directly photographed with SEM (model JSM-S410LV, JEOL) at an accelerating voltage of 15 kV and instrumental magnifications of 500–5000 ×.

2.2. Materials

2.6. TEM Analysis

The photocatalyst used in this study was a commercial nano-TiO2 powder (P25, average size 20–30 nm) purchased from Evonik, Germany, and TiO2 was used as purchased. Acrylic resin was obtained from Ciba Specialty Chemical, Bangkok, Thailand. All organic solvents used in this study were of analytical grade. Methyl ethyl ketone was

The TEM imaging of F. monoliforme and A. niger was carried out as described by the previously [28]. After exposure to photocatalytic TiO2/PP surface, the fungi were collected and fixed with 2.5% 18


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Fig. 1. SEM images of (a) pure PP, (b) 1%TiO2-PP, (c) 5% TiO2-PP, (d) 10% TiO2-PP and (e) EDS results.

specimen blocks, which were sectioned using an ultramicrotome at a thickness of 60–90 nm. The section was deposited on carbon-coated copper grids and contrasted with uranyl acetate and lead citrate. Samples were directly observed by TEM (model JEOL-2100, JEOL).

glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature and postfixed with 1% osmium tetroxide (OsO4). Then, the samples were dehydrated in a graded series of ethanol solutions (35%, 50%, 70%, 95% and 100%, three times for 15–20 min each) and embedded in Spur resin. Samples were dried at 70 °C for 8–10 h to form

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dose and UVA intensity, whereas in the presence of TiO2 the required UVA dose or intensity was very low. Nevertheless, it was not possible to effectively reduce the numbers of F. monoliforme by photocatalytic treatment with low TiO2 concentration (1%). Therefore, the photocatalytic activity at low TiO2 concentrations was insufficient for disinfection of fungi. There was drastic increase in sensitivity of F. monoliforme to photocatalysis when TiO2 content increased and total disinfection of F. monoliforme was achieved when exposed to 10% TiO2-PP under UVA illumination. The sensitivity of F. monoliforme to UVA light-only (no TiO2) was much less. The efficiency of photocatalytic disinfection was dependent on the TiO2 concentration used. Increased TiO2 contents resulted in increased bacterial killing [29]. This is probably due to greater photon absorption sites of TiO2 particles and greater production of reactive oxygen species (ROS) on the surface of the TiO2. Therefore, it is possible that surfacebound ROS on TiO2 are responsible for elimination of F. monoliforme. Fig. 2 shows the TEM images of F. monoliforme after TiO2 photocatalytic disinfection treatment. Generally, fungal cells are surrounded by a rigid cell wall to provide shape and protection to the cell. Fig. 2a shows that the normal untreated F. monoliforme was surrounded by an electron-dense outer layer and an underlying electron-transparent layer. This observation demonstrates that F. monoliforme has a single cell wall. Compared to other Fusarium species, F. oxysporum has a double cell wall with an internal and external electron-dense outer layer [36]. The electron-dense character of the outer layer is enriched with glycoproteins, whereas the electron-transparent layer is probably enriched in carbohydrates [30–31]. Chitin seems to be localized in the electron-transparent layer of the cell wall [30]. After treatment with UVA-light only, the electron-dense outer layer was still present (Fig. 2b), indicating that UVA light and TiO2 particles do not damage the electron-dense outer layer. But, TiO2-PP under UVA illumination greatly affected the electron-dense outer layer (Fig. 2c), indicating that photocatalytic disinfection is capable of damaging the F. monoliforme cell wall. The role of cell wall degradation in inhibiting Fusarium growth has been reported previously [32]. Our results suggest that the electron-dense outer layer was sensitive to TiO2 photocatalysis, and ROS could attack glycoproteins in the outer layer of the cell wall. The primary step in photocatalytic decomposition is initiated by OH radicals attack on the cell wall, leading to cell punctures [33]. This is in agreement with our finding in Fig. 3. The SEM images clearly show that the surface of fungal hyphae was deformed after 8-h of UVA irradiation on TiO2-PP (Fig. 3c). At UVA-light only treatment, F. monoliforme completely retained its tubular-shaped hyphae with the normal appearance and smooth cell walls. This observation is consistent with the TEM results (Fig. 2). However, it was observed that the electron-transparent layer was not affected by TiO2 photocatalytic treatment as shown in Fig. 2c. This observation is consistent with the previous report [30] that the electron-transparent layer of F. oxysporum was not affected by either treatment, corresponding with it being enriched in carbohydrate polymers. The electron-transparent layer probably represents the skeletal layer. Likely, surface-bound radicals (mainly OH radicals) generated in

Table 1 The results of TiO2 content and EDS analysis of selected polypropylene substrate with TiO2 based paint (TiO2-PP) at different TiO2 concentrations (n = 12). Sample

Pure PP 1% TiO2-PP 5% TiO2-PP 10% TiO2-PP

TiO2 content (mg/m2)

% Weight

– 8.0 + 0.06 20.0 + 0.10 76 + 0.52

C

O

Ti

91.5 90.7 90.8 89.0

8.5 9.1 7.7 8.0

– 0.2 1.5 3.0

C - carbon element, O - oxygen element, Ti - titanium element.

3. Results and Discussion 3.1. Structural Characterization of TiO2-PP The structural characterizations of TiO2-PP specimens prepared using different TiO2 concentrations are provided in Fig. 1. In each TiO2PP specimen, the TiO2 particles were present in the form of agglomerates on the PP surface. The number size distribution of TiO2 agglomerates ranged from 200 nm to > 5000 nm with the peak of the distribution between 1000 and 5000 nm. The average size of TiO2 agglomerates was ~3560 nm in diameter. EDS analysis of TiO2-PP indicated the peaks of titanium (Ti), oxygen (O) and a large amount of carbon (C) element, where the C signal comes from the PP structure. The presence of Ti peak revealed the PP surface consisted of TiO2 particles. Comparing to 10%TiO2-PP, 1% TiO2-PP showed C, O, and a small amount of Ti elements. The weight percentage of Ti element increased with TiO2 coating concentration, in agreement with the TiO2 content (mg/m2) loaded on PP surface. Table 1 shows that TiO2 contents on PP ranged from 8 to 76 mg/m2. No cracks were formed on all TiO2-PP specimens, suggesting that the paint formulation and bar-coating process are practical for deposition of TiO2 on PP. Advantages of the bar-coating technique are that it is simple and cost-effective for production on an industrial scale.

3.2. Photocatalytic Disinfection of F. monoliforme The colony forming count method was used to investigate the photocatalytic disinfection of F. monoliforme. Table 2 shows the numbers of F. monoliforme after exposure to each treatment for 24-h. Fungi elimination values are the mean values of nine repetitions. The presence of UVA light could considerably promote the strong toxicity of TiO2-PP to F. monoliforme, whereas TiO2-PP had a little toxicity in the dark. Approximately 5 log CFU/mL of F. monoliforme was still detected after 24-h in all treatments, except for 10% TiO2-PP with UVA illumination where no surviving fungi were detected. As shown in Table 2, low intensity UVA light (8 W/m2) could promote highly efficient photocatalytic disinfection against F. monoliforme. Our results are in agreement with the previous work [26]: the authors reported that the solar light-only (no TiO2) disinfection of Fusarium required a high UVA

Table 2 Number of colony forming units (CFU) of F. monoliforme after exposure to each treatment for 24-h (n = 9). Sample

Without UVA illumination Numbers (CFU/mL)

Control With 1% TiO2 With 5% TiO2 With 10% TiO2

7.83 7.70 7.01 5.85

± ± ± ±

0.46 ×105 0.36 ×105 0.68 ×105 0.83 ×105

With UVA illumination % Elimination+

Numbers (CFU/mL)

% Elimination+

– 1.6 10.4 25.3

5.87 ± 1.28× 105 5.16 ± 0.62× 105 1.67 ± 0.48× 105 0

25.9 34.0 78.7 100

+ % Elimination = (A–B) × 100/A where A is number of CFU/mL without TiO2 and without UVA (control) after exposure for 24 h, B is number of CFU treatment = number of CFU in other treatments after exposure for 24 h.

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Fig. 2. TEM images of F. monoliforme cell wall after 24 h exposure onto (a) pure PP in the dark, (b) pure PP under low intensity UVA light, and (c) 10%TiO2-PP under low intensity UVA light. (a–b) The cell is surrounded by an external electron-dense outer layer cell (A) wall and an underlying electron-transparent layer (B). (c) TiO2 photocatalytic treatment affected the electron-dense outer layer (A) but not the electron-transparent layer (B).

niger photocatalytic inactivation was reached when 15–17 W/m2 of UVA light intensity was used [37]. In our study, the photocatalytic disinfection of A. niger was found to be much less potential under low intensity UVA light. TEM images of the internal morphology of TiO2 photocatalysistreated A. niger spore and hyphae are shown in Fig. 4. A. niger hyphae showed a distinct morphological difference among treatments, but the morphological difference in A. niger spores was much less. The regular internal structure of A. niger can be observed in the cells of the control treatment (Fig. 4a, b). The A. niger spore was 2.5 μm in diameter with a smooth surface, and its cell wall has an outermost melanin-like layer as found in other Aspergillus species [38]. Melanized layer is an effective absorber of UV radiation and results in protecting the spore from UV radiation damage. The melanin-like layer could be affected by 24 h exposure to UVA radiation layer as shown in Fig. 4c. This result is in agreement with SEM observations that the A. niger spore was deformed after UVA irradiation (data not shown). Such deformation of A. niger spore was however not observed by 6 h UVA irradiation. Fig. 4e presents that in the presence of TiO2 photocatalysis the melanized layer and cell wall could be destroyed more effective than UVA light-only. The appearance of A. niger spore exposed to TiO2 photocatalysis was distinctly aberrant with a thinner cell wall compared to normal and UVA-treat A. niger spores. However, the intracellular organelles of A. niger spore were not affected by TiO2 photocatalysis and UVA radiation (Fig. 4a, b, e), even though the outermost melanin-like layer and cell wall were damaged. This might explain the fact that damaged A. niger spore can recover their germination, resulting in the cell cultivability of A. niger as observed in the subsequent dark on agar plate (Table 3). Normal A. niger hyphae is shown in Fig. 4b. A. niger was surrounded by an electron-transparent layer cell wall, whereas an electron-dense outer layer cell wall was not found in A. niger. This observation was similar to the previous work [39]. Plasma membrane was intact with

the extracellular environment can travel only very short distances to encounter fatty acids [13]. The outer electron-dense layer and the electron-transparent layer of F. monoliforme together span a distance of approximately 150 nm (Fig. 2a). The cytoplasm is the intracellular target site of photocatalytic disinfection [13]. Usually, the cytoplasm is protected by the cell membrane. Therefore, the attack of intracellular constituents may take place through further generation of other oxidants in the cytoplasm. Alternatively, the lipid peroxidation of cell membrane can form large perforation that leads to the entry of OH radicals into the cells [19]. In the present study, the cytoplasm of F. monoliforme was found to have some obvious structural changes after photocatalytic treatment (Fig. 2c). Highly electron-translucent vacuole-like structures in the cytoplasm are apparent. Fungal vacuoles are involved in the diverse range of cellular function and play a fundamental role in growth and control of cellular morphology [34]. Alteration of vacuole morphology has been linked with impairment of hyphae [34–35]. This is in agreement with our result in Fig. 3c. Mycelium had aberrant and distorted hyphae, and lost their smoothness. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear [36]. 3.3. Photocatalytic Disinfection of A. niger The results of A. niger inactivation after exposure to each treatment for 24-h are shown in Table 3. There are no major differences between with and without TiO2 photocatalytic disinfection under low intensity UVA light against A. niger regardless of TiO2 content. Approximately 4 log CFU/mL of A. niger was still detected after 24-h in all treatments. As shown in Table 3, low intensity UVA light (ca. 8 W/m2) could not promote high photocatalytic activity against A. niger. Our results are in agreement with the previous work [24] that the inactivation of A. niger is directly dependent on the UVA light intensity. Previously, 50% of A.

Fig. 3. SEM images of F. monoliforme hyphae after 8 h exposure onto (a) pure PP in the dark, (b) pure PP under low intensity UVA light and (c) 10%TiO2-PP under low intensity UVA light.

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Table 3 Number of colony forming units (CFU) of A. niger after exposure to each treatment for 24-h (n = 9). Sample

Control With 1% TiO2 With 10% TiO2

Without UVA illumination

With UVA illumination

Numbers (CFU/mL)

% Elimination+

Numbers (CFU/mL)

% Elimination+

6.47 ± 1.50 ×104 4.83 ± 1.79 ×104 4.36 ± 1.09 ×104

– 25.3 32.6

4.58 ± 1.98× 104 4.50 ± 2.03× 104 4.19 ± 1.20× 104

29.2 30.5 35.2

+ % Elimination = (A–B) × 100/A where A is number of CFU/mL without TiO2 and without UVA (control) after exposure for 24 h, B is number of CFU treatment = number of CFU in other treatments after exposure for 24 h.

death [40]. Our result showed that A. niger hyphae is very sensitive to UVA light even at low intensity. Compare to F. monoliforme, lack of electron-dense outer layer cell wall might be not able to protect A. niger cell from UVA penetration into the cell. A. niger treated with TiO2

the cell wall and the cytoplasm. Healthy cellular organelles were seen within the cytoplasm. A. niger exposed to UVA light-only showed severe damage to the plasma membrane and cellular organelles, but not to cell wall (Fig. 4d). The loss of the integrity of the cell finally leads to the cell

Fig. 4. TEM images of A. niger spores (left) and hyphae (right) after 24 h exposure onto (a–b) pure PP in the dark, (c–d) pure PP under UVA radiation, and (e–f) 10% TiO2-PP under UVA radiation. A - Melanin like layer; B - cell wall of A. niger conidia; C - electron transparent layer cell wall of A. niger hyphae; D cell membrane.

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Fig. 5. SEM images of A. niger (a) before and after 6-h treatment on 10% TiO2-PP (b) under UVA light and (c) in the dark.

Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, for supporting fungi strains and a laminar flow cabinet. Furthermore, we are special grateful to Professor Dr. Wiwut Tanthapanichakun for his advice.

photocatalysis showed severe morphological alteration (Fig. 4f). The damaged cells seemed to be approaching each other by sharing the cell walls to attempt for nourishment. This phenomena was similar to the previous work that A. niger was treated by essential oil [39]. The fungal cell wall performs an essential role during the interaction with its environment. This evidence suggested that the cell wall remodeling of A. niger was in response to cell wall threatening by ROS radicals from TiO2 photocatalysis treatment. Hence, the death of A. niger might be probably resulted from disruption of cell membrane and cellular organelles. This is in agreement with our result in Fig. 5. A. niger hyphae on TiO2PP surface under UVA light was much more decreased in amount than that on TiO2-PP surface in the dark. The present work showed that although A. niger hyphae was severely damaged, A. niger spores survived and continued to germinate (Table 3). Our finding was in agreement with the previous work that regrowth of A. niger exposed onto TiO2 coated film on moist wood did appear when UVA illumination was stopped [25]. The photocatalytic inactivation mechanism of A. niger spores is further considerably studied.

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4. Conclusion This study was focused on the TiO2 photocatalytic disinfection mechanism against F. monoliforme and A. niger. The results indicated that it was effective to total disinfect F. monoliforme using TiO2 based paint under low intensity UVA light (< 10 W/m2). The photocatalytic oxidation led to considerably destroy the electron-dense outer layer cell wall and promote vacuolization in cytoplasm of F. monoliforme while UVA light-only did not. In contrast, A. niger was more susceptive to UVA radiation than F. monoliforme. Melanin like layer of A. niger spore, cell membrane and cellular organelles of A. niger hyphae were distinctly sensitive to UVA radiation. The photocatalytic oxidation increased morphological alteration of the A. niger hyphae and spore as compared to UVA-light only. However, the spore killing might not be achieved by TiO2 photocatalytic disinfection under low intensity UVA light, resulting in the growth of A. niger on agar plate in subsequent dark. Experimental results demonstrated the difference between F. monoliforme and A. niger in response to TiO2 photocatalytic disinfection. These factors become important and help to improve a potential benefit of photocatalytic application against fungi. Conflicts of Interest The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by a grant from Thailand Research Fund (TRF) (Grant number: TRG5080010) and National Nanotechnology Center (NANOTEC), Thailand (Project no. P1202012), which are greatly appreciated. We would like to thank the National 23


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