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Wettability modified aluminum surface for a potential antifungal surface
Materials Letters 161 (2015) 234–239
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Wettability modified aluminum surface for a potential antifungal surface Yeongae Kim, Woonbong Hwang n Department of Mechanical Engineering, POSTECH, Pohang 37673, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2015 Received in revised form 30 July 2015 Accepted 22 August 2015 Available online 24 August 2015
Circulation of contaminated air by air conditioning equipment promotes infection, notably bronchial diseases and the development of allergies. The evaporator of the air conditioning system has a key influence because its interior provides a favorable environment for growth of microbes. Most of the evaporator is manufactured from aluminum because of its mechanical properties, but it is poor at preventing contamination and growth of microorganisms. To overcome these problems, we use a non-wetting aluminum surface to retard microbial growth. This paper reports the extent of fungal growth on a superhydrophobic surface based on aluminum in comparison with superhydrophilic, weakly hydrophobic, and hydrophobic surfaces. Three common airborne fungi were used: Penicillium, Cladosporium, and Aspergillus. Separate experiments were performed direct and indirect contamination. In the direct contamination experiment, only the superhydrophobic surface was not contaminated. In the indirect contamination experiment, fungal contamination occurred on the superhydrophilic, weakly hydrophobic, and hydrophobic surfaces. There was a small amount of contamination on the superhydrophobic surface, but little spread. It is concluded that only the superhydrophobic surface is effective as an antifungal surface. & 2015 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Functional Contacts Interfaces Microstructure Surfaces
1. Introduction Modern life involves considerable exposure to Heating, Ventilating, and Air conditioning (HVAC) systems, because more people are spending more time indoors. HVAC systems may exacerbate diseases caused by airborne microbes, by circulating contaminated air, despite being intended to improve the indoor environment [1]. In particular, the evaporator, a key component of air conditioning equipment, has a major effect because it provides favorable conditions for the adhesion, growth, and spreading of contaminants [2]. Aluminum is used for the most of the evaporator because of its high heat conductivity and corrosion resistance and its light weight. It has no ability to prevent contamination and growth of microorganisms [3]. To control microbial infection two methods are used [4]. One is sterilization using disinfectant, and the other is use of antimicrobial surfaces. Disinfectant readily removes microorganisms. Most disinfectants are toxic chemicals, however, and are injurious not only to the environment but also the human body. It is therefore important to develop an antimicrobial surface n
Corresponding author. E-mail addresses: [email protected] (Y. Kim), [email protected] (W. Hwang). http://dx.doi.org/10.1016/j.matlet.2015.08.103 0167-577X/& 2015 Elsevier B.V. All rights reserved.
that obstructs adhesion and spreading of microbes. Various solutions have been proposed to this challenge, including the application of metallic nanoparticles [5–7] and antimicrobial polymers [8,9], and replacement of aluminum by antibiotic metals such as copper, silver, or titanium [3,10]. Unfortunately these methods have proved difficult to apply to evaporator because of the instability of the resulting chemical and physical structures, reduced heat exchange efficiency, and economic problems. It is difficult to apply nanoparticles on structures having complex shapes, and the antimicrobial properties of polymers are temporary. Copper and silver have higher thermal conductivity than aluminum but are expensive, while titanium reduce the thermal efficiency. To overcome these problems, we have fabricated an antimicrobial surface based on an anti-wetting aluminum surface. The anti-wetting characteristic is called superhydrophobicity. The superhydrophobic surface is composed of rough structures on both the nano and micro scales and a lower surface energy than the surface tension of water. The lower surface energy prevents the permeation of water into the surface by the formation of gas pockets between the rough structures. Because of this characteristic, water cannot attach to the surface. In the growth of biological life forms, water is a crucial factor; however, as these surfaces do not provide moisture, we expect that microbial cannot survive on them. Therefore, this research considers a superhydrophobic
Y. Kim, W. Hwang / Materials Letters 161 (2015) 234–239
surface as a potential antimicrobial surface [11]. In this research, the antifungal effect of a superhydrophobic surface based on aluminum was investigated in comparison with other surfaces. Superhydrophilic, hydrophobic, and superhydrophobic surfaces were fabricated, and a weakly hydrophobic surface acted as a control surface. Three species of fungus that comprise most airborne fungi were used: Penicillium implicatum, Cladosporium cladosporioides, and Aspergillus fumigatus [12]. In experiments involving direct inoculation of fungal spores, only the
235
superhydrophobic surface was not contaminated. Hyphae and spores grew steadily on the weakly hydrophobic and the hydrophobic surfaces. On the superhydrophilic surface, active growth of vegetative hyphae took place. In the indirect contamination experiment, fungal contamination occurred on the superhydrophilic surface from the beginning, and also later on the weakly hydrophobic and hydrophobic surfaces. There was only a small amount of contamination on the superhydrophobic surface, and there was little spread. It can be concluded that superhydrophobic aluminum
Fig. 1. Surface morphology, chemical analysis, and wetting properties of prepared surfaces. (a) FE-SEM and (b) EDS images of the samples. The weakly hydrophobic and hydrophobic surface have the same topography, with micro-scale roughness. The superhydrophilic and superhydrophobic surface also have the same structures, with microscale valleys formed by bunches of nanofibers. However, their wetting properties are different because the HDFS coating changes the surface energy. (c) and (d) show advancing contact angles of the surfaces with (c) deionized water and (d) spore suspension. The contact angle of the spore suspension is the same as the contact angle of deionized water, because the suspension was largely water. (Scale bar: 5 μm, Droplet volume: 20 μl).
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Y. Kim, W. Hwang / Materials Letters 161 (2015) 234–239
surface with modified wettability is a potential antifungal surface.
2. Experimental section 2.1. Surface preparations An untreated aluminum surface was used as a control surface. This is a weakly hydrophobic surface, with an advancing contact angle of about 80°. The hydrophobic surface was fabricated by coating the weakly hydrophobic surface with a hydrophobic polymer (heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane, HDFS). The resulting surface has the same topography as the weakly hydrophobic surface, because the HDFS is coated on it as a monomolecular layer. The wetting properties are different, however, as a result of the low surface energy of the HDFS. The advancing contact angle of the hydrophobic surface is about 110°. The superhydrophilic and superhydrophobic surfaces were fabricated by anodization and HDFS coating. Industrial aluminum (99.5%, 30 mm * 30 mm * 1 mm) was connected to an anode, and a potential of 65 V was applied in 0.3 M oxalic acid for 10 min. The resulting surface has superhydrophilic properties, with an advancing contact angle of almost 0°. The superhydrophobic surface was fabricated by coating the superhydrophilic surface with HDFS. The advancing contact angle of this surface exceeds 165° [13]. The surface characteristics are shown in Fig. 1. The contact angle was measured following the international protocol [14] by a drop shape analyzer (Smart Drop, FemtoFab). The surface structures and chemical composition were observed by field emission scanning electron microscopy and energy-dispersive X-ray spectroscopy (FE-SEM; EDS; JSM-7401 F, JEOL, NINT). The surface wettability was categorized by the international classification [15]. 2.2. Culture of fungus Three species of fungi that comprise most airborne fungi were used for the study. These are Penicillium (Korea collection for type cultures, KCTC no. 26750), Cladosporium (KCTC no. 16680), and Aspergillus (KCTC no. 6145) [12]. These fungi were grown on potato dextrose agar (PDA) for one week [Fig. S1]. The temperature was maintained at 25 °C during cultivation. 2.3. Making a spore suspension Fungal spores were scraped from the fully grown fungi after addition of a little sterile deionized water so as to make a spore suspension. The suspension was filtered to remove hyphae. The number of spores in the filtered suspension was counted using a hemocytometer, and then diluted to approximately 108 spores per 1 ml. 2.4. Inoculation of spore suspension 2.4.1. Direct contamination The first experiment matched the situation when a contaminated evaporator stopped running without supplement of moisture, and then with moisture. 20 μl of the Aspergillus spore suspension was inoculated onto the center of each surface. These surfaces were maintained at ambient temperature (17–19 °C) and humidity (20–23%RH) with no further supply of moisture and nutrients for two weeks. Following observation the specimens were placed onto PDA to provide moisture and nutrients, and were sealed with parafilm for a further two weeks. In the second experiment, a clump of dried spores obtained from 20 μl of the spore suspension was placed at the center of each surface. Moisture and nutrients were provided for the spores using PDA, and the specimens were sealed. The experiment was
performed at ambient temperature for four weeks. 2.4.2. Indirect contamination from outside Migration and growth of the fungus was checked on the surfaces, which were placed in a contaminated environment. PDA (20 ml) was put in a sterile petri dish, and each surface was placed on the agar. The spore suspension was inoculated on agar 1 cm away from the surface edge at four points. Each suspension comprised 10 μl of the spore suspension. This experiment lasted four weeks, using Penicillium [Fig. S4].
3. Results and discussion 3.1. Direct contamination Fig. 1(c) and (d) shows contact angles of the surfaces with deionized water and the spore suspension. The contact angle of the spore suspension is the same as the contact angle of deionized water, because the suspension was largely water. In the absence of an additional moisture supply, the suspension dried so that only the spores remained [Fig. S2(a)]. Fig. 2 shows the interface between the inoculated spot and the surface. The boundary of the inoculated suspension remained distinct on every specimens, from the first day to the fourteenth day, without fungal growth. After that time moisture and nutrients were supplied to the specimens for two weeks using PDA. The shape of the suspension changed, and fungal growth was observed as the inoculated spores took in moisture [Fig. S2(b)]. The results are shown in Fig. 2(b). Growth was observed from the first day. After two more weeks, fungal growth was observed on all differing types of surface. On the superhydrophilic surface, only vegetative hyphae grew actively along the surface. In the presence of abundant nutrition the fungus spread vegetative hyphae rapidly. Vigorous mycelial growth on the superhydrophilic surface was due to the supply of moisture and nutrients across the surface. On the weakly hydrophobic and hydrophobic surfaces, vegetative and aerial hyphae and spores grew. At first, spores germinate to produce vegetative hyphae that obtain nutrients, and aerial hyphae are then produced. The aerial hyphae bear reproductive spores. This is the life cycle of fungi [16,17]. On the superhydrophobic surface, only aerial hyphae and spores grew at the spore suspension, with no spreading of the fungi. When nutrients are lacking, fungi form spores for reproduction. The inoculated spores did not receive water or nutrition from the superhydrophobic surface. The results confirmed that the fungi did not develop in the absence of nutrients. More significant is that the fungi are able to grow after supplementation with nutrients, even after growth had stopped. In the second experiment, the initial contact area was controlled to be the same, regardless of the surface wetting properties, using dried spores. The results of both experiments were nevertheless the same for the first four weeks. Fig. 3 shows the results for Aspergillus. Results for Penicillium and Cladosporium are shown in Fig. S3(a) and S3(b), respectively. Vegetative hyphae grew actively on the superhydrophilic surface. On the weakly hydrophobic and hydrophobic surfaces, vegetative and aerial hyphae and spores were raised. In contrast, little growth took place on the superhydrophobic surface. Fungal growth is therefore related to surface wetting properties, not to the contact area of surface and fungus, because the surfaces have differing ability to supply moisture as a result of their differing wettabilities. 3.2. Indirect contamination from outside As the fungi grown on the agar spread, the specimens were also contaminated by the fungi. In the second experiment performed
Y. Kim, W. Hwang / Materials Letters 161 (2015) 234–239
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Fig. 2. Fungal growth at the boundary between the surface and the fungus grown from the point of inoculation with spore suspension (a) without supply of moisture and nutrients during the first two weeks, and (b) with moisture and nutrients using potato dextrose agar. (Scale bar: 50 μm).
previously, only Penicillium was detected on the superhydrophobic surface. This means that, of the three fungi, the Penicillium adhered most strongly to the surface. Hence the following experiment used Penicillium [Fig. S3(a)]. A photo was taken of each specimen under a microscope, and the ratio of the contaminated area to the total area was calculated. Fig. 4 shows the normalized results, relative to the value for the weakly hydrophobic surface at the first week. At the first week, all surfaces were polluted except the superhydrophobic surface. On the superhydrophilic surface the contaminated area was about ten times larger than on the control surface. On the superhydrophilic surface, the fungi-polluted area grew rapidly during the first two weeks, after which the rate of increase slowed. There was active growth of vegetative hyphae along the superhydrophilic surface
until the second week. On the weakly hydrophobic and hydrophobic surfaces the contaminated area increased explosively after the third week. This is because the aerial hyphae bear reproductive spores which germinate later on the surfaces. On the superhydrophobic surface, the fungus-polluted area increased linearly with time, although the largest value was less than 16% of the amount on other surfaces. Even though the surface was contaminated, the fungi did not spread because the surface did not provide nutrients. In summary, fungal infection on the superhydrophilic surface is critical at the start, but the superhydrophilic, weakly hydrophobic, and hydrophobic surfaces are vulnerable to contamination by fungi over the long term. Only the superhydrophobic surface, therefore, can block growth of fungi, as a result of lack of moisture supply.
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Fig. 3. Fungal growth on various surfaces. The experiment used a dried spore suspension of Aspergillus. Hyphae and spores were observed on all surfaces except the superhydrophobic surface. On the superhydrophilic surface, vegetative hyphae had grown. On the weakly hydrophobic and hydrophobic surfaces, hyphae and spores were produced, including aerial hyphae. (Scale bar: 50 μm).
4. Conclusions Surfaces widely differing in wettability were fabricated, and their antifungal effects were investigated. To observe fungal growth and spreading, spores were inoculated on the surfaces. On the superhydrophobic surface, there was little contamination or spreading. The other surfaces, experienced contamination with growth of fungi. It can be concluded from the results of these experiments that only the superhydrophobic surface has an antifungal effect. Therefore, this surface should be suitable for us as an antifungal surface for an evaporator.
Acknowledgment This work was supported by a grant from the National Research Foundation of Korea (NRF) (No. 2013R1A2A1A01016911), which is funded by the Ministry of Science, ICT and Future Planning, Korea. Fig. 4. Change of normalized contaminated area during four weeks. The ratio of the contaminated area to the total area was calculated. The normalization criterion was the value of the weakly hydrophobic surface after the first week.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matlet.2015.08. 103.
Y. Kim, W. Hwang / Materials Letters 161 (2015) 234–239
References [1] Y. Li, G.M. Leung, J.W. Tang, X. Yang, C.Y. Chao, J.Z. Lin, et al., Role of ventilation in airborne transmission of infectious agents in the built environment-a multidisciplinary systematic review, Indoor Air 17 (2007) 2–18. [2] M.G. Schmidt, H.H. Attaway, S. Terzieva, A. Marshall, L.L. Steed, D. Salzberg, et al., Characterization and control of the microbial community affiliated with copper or aluminum heat exchangers of HVAC systems, Curr. Microbiol. 65 (2012) 141–149. [3] L. Weaver, H.T. Michels, C.W. Keevil, Potential for preventing spread of fungi in air-conditioning systems constructed using copper instead of aluminium, Lett. Appl. Microbiol. 50 (2010) 18–23. [4] F. Siedenbiedel, J.C. Tiller, Antimicrobial polymers in solution and on surfaces: overview and functional principles, Polymers 4 (2012) 46–71. [5] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnol. Adv. 27 (2009) 76–83. [6] S.R. Choudhury, M. Ghosh, A. Mandal, D. Chakravorty, M. Pal, S. Pradhan, et al., Surface-modified sulfur nanoparticles: an effective antifungal agent against Aspergillus niger and Fusarium oxysporum, Appl. Microbiol. Biotechnol. 90 (2011) 733–743. [7] T. Ahmad, I.A. Wani, I.H. Lone, A. Ganguly, N. Manzoor, A. Ahmad, et al., Antifungal activity of gold nanoparticles prepared by solvothermal method, Mater. Res. Bul. 48 (2013) 12–20.
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[8] F.X. Hu, K.G. Neoh, L. Cen, E.T. Kang, Antibacterial and antifungal efficacy of surface functionalized polymeric beads in repeated applications, Biotechnol. Bioeng. 89 (2005) 474–484. [9] A. Zumbuehl, L. Ferreira, D. Kuhn, A. Astashkina, L. Long, Y. Yeo, et al., Antifungal hydrogels, Proc. Natl. Acad. Sci. USA 104 (2007) 12994–12998. [10] G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl. Environ. Microbiol. 77 (2011) 1541–1547. [11] A. Marmur, Super-hydrophobicity fundamentals: implications to biofouling prevention, Biofouling 22 (2006) 107–115. [12] K.Y. Kim, C.N. Kim, Airborne microbiological characteristics in public buildings of Korea, Build. Environ. 42 (2007) 2188–2196. [13] Y. Kim, S. Lee, H. Cho, B. Park, D. Kim, W. Hwang, Robust superhydrophilic/ hydrophobic surface based on self-aggregated Al2O3 nanowires by single-step anodization and self-assembly method, ACS Appl. Mater. Interfaces 4 (2012) 5074–5078. [14] J. Drelich, Guidelines to measurements of reproducible contact angles using a sessile-drop technique, Surface Innov. 1 (2013) 248–254. [15] J. Drelich, A. Marmur, Physics and applications of superhydrophobic and superhydrophilic surfaces and coatings, Surface Innov. 2 (2014) 211–227. [16] K. Kavanagh, Fungi: biology and applications, John Wiley & sons Ltd;, Chichester, 2005. [17] G.J. Tortora, B.R. Funke, C.L. Case, Microbiology: an introduction, 10th ed., Pearson, San Francisco, 2010.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Wettability modified aluminum surface for a potential antifungal surface Yeongae Kim, Woonbong Hwang n Department of Mechanical Engineering, POSTECH, Pohang 37673, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2015 Received in revised form 30 July 2015 Accepted 22 August 2015 Available online 24 August 2015
Circulation of contaminated air by air conditioning equipment promotes infection, notably bronchial diseases and the development of allergies. The evaporator of the air conditioning system has a key influence because its interior provides a favorable environment for growth of microbes. Most of the evaporator is manufactured from aluminum because of its mechanical properties, but it is poor at preventing contamination and growth of microorganisms. To overcome these problems, we use a non-wetting aluminum surface to retard microbial growth. This paper reports the extent of fungal growth on a superhydrophobic surface based on aluminum in comparison with superhydrophilic, weakly hydrophobic, and hydrophobic surfaces. Three common airborne fungi were used: Penicillium, Cladosporium, and Aspergillus. Separate experiments were performed direct and indirect contamination. In the direct contamination experiment, only the superhydrophobic surface was not contaminated. In the indirect contamination experiment, fungal contamination occurred on the superhydrophilic, weakly hydrophobic, and hydrophobic surfaces. There was a small amount of contamination on the superhydrophobic surface, but little spread. It is concluded that only the superhydrophobic surface is effective as an antifungal surface. & 2015 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Functional Contacts Interfaces Microstructure Surfaces
1. Introduction Modern life involves considerable exposure to Heating, Ventilating, and Air conditioning (HVAC) systems, because more people are spending more time indoors. HVAC systems may exacerbate diseases caused by airborne microbes, by circulating contaminated air, despite being intended to improve the indoor environment [1]. In particular, the evaporator, a key component of air conditioning equipment, has a major effect because it provides favorable conditions for the adhesion, growth, and spreading of contaminants [2]. Aluminum is used for the most of the evaporator because of its high heat conductivity and corrosion resistance and its light weight. It has no ability to prevent contamination and growth of microorganisms [3]. To control microbial infection two methods are used [4]. One is sterilization using disinfectant, and the other is use of antimicrobial surfaces. Disinfectant readily removes microorganisms. Most disinfectants are toxic chemicals, however, and are injurious not only to the environment but also the human body. It is therefore important to develop an antimicrobial surface n
Corresponding author. E-mail addresses: [email protected] (Y. Kim), [email protected] (W. Hwang). http://dx.doi.org/10.1016/j.matlet.2015.08.103 0167-577X/& 2015 Elsevier B.V. All rights reserved.
that obstructs adhesion and spreading of microbes. Various solutions have been proposed to this challenge, including the application of metallic nanoparticles [5–7] and antimicrobial polymers [8,9], and replacement of aluminum by antibiotic metals such as copper, silver, or titanium [3,10]. Unfortunately these methods have proved difficult to apply to evaporator because of the instability of the resulting chemical and physical structures, reduced heat exchange efficiency, and economic problems. It is difficult to apply nanoparticles on structures having complex shapes, and the antimicrobial properties of polymers are temporary. Copper and silver have higher thermal conductivity than aluminum but are expensive, while titanium reduce the thermal efficiency. To overcome these problems, we have fabricated an antimicrobial surface based on an anti-wetting aluminum surface. The anti-wetting characteristic is called superhydrophobicity. The superhydrophobic surface is composed of rough structures on both the nano and micro scales and a lower surface energy than the surface tension of water. The lower surface energy prevents the permeation of water into the surface by the formation of gas pockets between the rough structures. Because of this characteristic, water cannot attach to the surface. In the growth of biological life forms, water is a crucial factor; however, as these surfaces do not provide moisture, we expect that microbial cannot survive on them. Therefore, this research considers a superhydrophobic
Y. Kim, W. Hwang / Materials Letters 161 (2015) 234–239
surface as a potential antimicrobial surface [11]. In this research, the antifungal effect of a superhydrophobic surface based on aluminum was investigated in comparison with other surfaces. Superhydrophilic, hydrophobic, and superhydrophobic surfaces were fabricated, and a weakly hydrophobic surface acted as a control surface. Three species of fungus that comprise most airborne fungi were used: Penicillium implicatum, Cladosporium cladosporioides, and Aspergillus fumigatus [12]. In experiments involving direct inoculation of fungal spores, only the
235
superhydrophobic surface was not contaminated. Hyphae and spores grew steadily on the weakly hydrophobic and the hydrophobic surfaces. On the superhydrophilic surface, active growth of vegetative hyphae took place. In the indirect contamination experiment, fungal contamination occurred on the superhydrophilic surface from the beginning, and also later on the weakly hydrophobic and hydrophobic surfaces. There was only a small amount of contamination on the superhydrophobic surface, and there was little spread. It can be concluded that superhydrophobic aluminum
Fig. 1. Surface morphology, chemical analysis, and wetting properties of prepared surfaces. (a) FE-SEM and (b) EDS images of the samples. The weakly hydrophobic and hydrophobic surface have the same topography, with micro-scale roughness. The superhydrophilic and superhydrophobic surface also have the same structures, with microscale valleys formed by bunches of nanofibers. However, their wetting properties are different because the HDFS coating changes the surface energy. (c) and (d) show advancing contact angles of the surfaces with (c) deionized water and (d) spore suspension. The contact angle of the spore suspension is the same as the contact angle of deionized water, because the suspension was largely water. (Scale bar: 5 μm, Droplet volume: 20 μl).
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surface with modified wettability is a potential antifungal surface.
2. Experimental section 2.1. Surface preparations An untreated aluminum surface was used as a control surface. This is a weakly hydrophobic surface, with an advancing contact angle of about 80°. The hydrophobic surface was fabricated by coating the weakly hydrophobic surface with a hydrophobic polymer (heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane, HDFS). The resulting surface has the same topography as the weakly hydrophobic surface, because the HDFS is coated on it as a monomolecular layer. The wetting properties are different, however, as a result of the low surface energy of the HDFS. The advancing contact angle of the hydrophobic surface is about 110°. The superhydrophilic and superhydrophobic surfaces were fabricated by anodization and HDFS coating. Industrial aluminum (99.5%, 30 mm * 30 mm * 1 mm) was connected to an anode, and a potential of 65 V was applied in 0.3 M oxalic acid for 10 min. The resulting surface has superhydrophilic properties, with an advancing contact angle of almost 0°. The superhydrophobic surface was fabricated by coating the superhydrophilic surface with HDFS. The advancing contact angle of this surface exceeds 165° [13]. The surface characteristics are shown in Fig. 1. The contact angle was measured following the international protocol [14] by a drop shape analyzer (Smart Drop, FemtoFab). The surface structures and chemical composition were observed by field emission scanning electron microscopy and energy-dispersive X-ray spectroscopy (FE-SEM; EDS; JSM-7401 F, JEOL, NINT). The surface wettability was categorized by the international classification [15]. 2.2. Culture of fungus Three species of fungi that comprise most airborne fungi were used for the study. These are Penicillium (Korea collection for type cultures, KCTC no. 26750), Cladosporium (KCTC no. 16680), and Aspergillus (KCTC no. 6145) [12]. These fungi were grown on potato dextrose agar (PDA) for one week [Fig. S1]. The temperature was maintained at 25 °C during cultivation. 2.3. Making a spore suspension Fungal spores were scraped from the fully grown fungi after addition of a little sterile deionized water so as to make a spore suspension. The suspension was filtered to remove hyphae. The number of spores in the filtered suspension was counted using a hemocytometer, and then diluted to approximately 108 spores per 1 ml. 2.4. Inoculation of spore suspension 2.4.1. Direct contamination The first experiment matched the situation when a contaminated evaporator stopped running without supplement of moisture, and then with moisture. 20 μl of the Aspergillus spore suspension was inoculated onto the center of each surface. These surfaces were maintained at ambient temperature (17–19 °C) and humidity (20–23%RH) with no further supply of moisture and nutrients for two weeks. Following observation the specimens were placed onto PDA to provide moisture and nutrients, and were sealed with parafilm for a further two weeks. In the second experiment, a clump of dried spores obtained from 20 μl of the spore suspension was placed at the center of each surface. Moisture and nutrients were provided for the spores using PDA, and the specimens were sealed. The experiment was
performed at ambient temperature for four weeks. 2.4.2. Indirect contamination from outside Migration and growth of the fungus was checked on the surfaces, which were placed in a contaminated environment. PDA (20 ml) was put in a sterile petri dish, and each surface was placed on the agar. The spore suspension was inoculated on agar 1 cm away from the surface edge at four points. Each suspension comprised 10 μl of the spore suspension. This experiment lasted four weeks, using Penicillium [Fig. S4].
3. Results and discussion 3.1. Direct contamination Fig. 1(c) and (d) shows contact angles of the surfaces with deionized water and the spore suspension. The contact angle of the spore suspension is the same as the contact angle of deionized water, because the suspension was largely water. In the absence of an additional moisture supply, the suspension dried so that only the spores remained [Fig. S2(a)]. Fig. 2 shows the interface between the inoculated spot and the surface. The boundary of the inoculated suspension remained distinct on every specimens, from the first day to the fourteenth day, without fungal growth. After that time moisture and nutrients were supplied to the specimens for two weeks using PDA. The shape of the suspension changed, and fungal growth was observed as the inoculated spores took in moisture [Fig. S2(b)]. The results are shown in Fig. 2(b). Growth was observed from the first day. After two more weeks, fungal growth was observed on all differing types of surface. On the superhydrophilic surface, only vegetative hyphae grew actively along the surface. In the presence of abundant nutrition the fungus spread vegetative hyphae rapidly. Vigorous mycelial growth on the superhydrophilic surface was due to the supply of moisture and nutrients across the surface. On the weakly hydrophobic and hydrophobic surfaces, vegetative and aerial hyphae and spores grew. At first, spores germinate to produce vegetative hyphae that obtain nutrients, and aerial hyphae are then produced. The aerial hyphae bear reproductive spores. This is the life cycle of fungi [16,17]. On the superhydrophobic surface, only aerial hyphae and spores grew at the spore suspension, with no spreading of the fungi. When nutrients are lacking, fungi form spores for reproduction. The inoculated spores did not receive water or nutrition from the superhydrophobic surface. The results confirmed that the fungi did not develop in the absence of nutrients. More significant is that the fungi are able to grow after supplementation with nutrients, even after growth had stopped. In the second experiment, the initial contact area was controlled to be the same, regardless of the surface wetting properties, using dried spores. The results of both experiments were nevertheless the same for the first four weeks. Fig. 3 shows the results for Aspergillus. Results for Penicillium and Cladosporium are shown in Fig. S3(a) and S3(b), respectively. Vegetative hyphae grew actively on the superhydrophilic surface. On the weakly hydrophobic and hydrophobic surfaces, vegetative and aerial hyphae and spores were raised. In contrast, little growth took place on the superhydrophobic surface. Fungal growth is therefore related to surface wetting properties, not to the contact area of surface and fungus, because the surfaces have differing ability to supply moisture as a result of their differing wettabilities. 3.2. Indirect contamination from outside As the fungi grown on the agar spread, the specimens were also contaminated by the fungi. In the second experiment performed
Y. Kim, W. Hwang / Materials Letters 161 (2015) 234–239
237
Fig. 2. Fungal growth at the boundary between the surface and the fungus grown from the point of inoculation with spore suspension (a) without supply of moisture and nutrients during the first two weeks, and (b) with moisture and nutrients using potato dextrose agar. (Scale bar: 50 μm).
previously, only Penicillium was detected on the superhydrophobic surface. This means that, of the three fungi, the Penicillium adhered most strongly to the surface. Hence the following experiment used Penicillium [Fig. S3(a)]. A photo was taken of each specimen under a microscope, and the ratio of the contaminated area to the total area was calculated. Fig. 4 shows the normalized results, relative to the value for the weakly hydrophobic surface at the first week. At the first week, all surfaces were polluted except the superhydrophobic surface. On the superhydrophilic surface the contaminated area was about ten times larger than on the control surface. On the superhydrophilic surface, the fungi-polluted area grew rapidly during the first two weeks, after which the rate of increase slowed. There was active growth of vegetative hyphae along the superhydrophilic surface
until the second week. On the weakly hydrophobic and hydrophobic surfaces the contaminated area increased explosively after the third week. This is because the aerial hyphae bear reproductive spores which germinate later on the surfaces. On the superhydrophobic surface, the fungus-polluted area increased linearly with time, although the largest value was less than 16% of the amount on other surfaces. Even though the surface was contaminated, the fungi did not spread because the surface did not provide nutrients. In summary, fungal infection on the superhydrophilic surface is critical at the start, but the superhydrophilic, weakly hydrophobic, and hydrophobic surfaces are vulnerable to contamination by fungi over the long term. Only the superhydrophobic surface, therefore, can block growth of fungi, as a result of lack of moisture supply.
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Fig. 3. Fungal growth on various surfaces. The experiment used a dried spore suspension of Aspergillus. Hyphae and spores were observed on all surfaces except the superhydrophobic surface. On the superhydrophilic surface, vegetative hyphae had grown. On the weakly hydrophobic and hydrophobic surfaces, hyphae and spores were produced, including aerial hyphae. (Scale bar: 50 μm).
4. Conclusions Surfaces widely differing in wettability were fabricated, and their antifungal effects were investigated. To observe fungal growth and spreading, spores were inoculated on the surfaces. On the superhydrophobic surface, there was little contamination or spreading. The other surfaces, experienced contamination with growth of fungi. It can be concluded from the results of these experiments that only the superhydrophobic surface has an antifungal effect. Therefore, this surface should be suitable for us as an antifungal surface for an evaporator.
Acknowledgment This work was supported by a grant from the National Research Foundation of Korea (NRF) (No. 2013R1A2A1A01016911), which is funded by the Ministry of Science, ICT and Future Planning, Korea. Fig. 4. Change of normalized contaminated area during four weeks. The ratio of the contaminated area to the total area was calculated. The normalization criterion was the value of the weakly hydrophobic surface after the first week.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matlet.2015.08. 103.
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