Photocatalytic disinfection of surfaces with copper doped Ti02 nanotube coatings illuminated by ceiling mounted fluorescent light

High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. In this work we show that copper doped TiO2 nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights can retard the growth of Listeria Innocua by 80% in seven hours of exposure to the fluorescent lights at different places in a food processing plant or in the laboratory conditions with daily reinocuation and washing. The disinfection properties of the surfaces seem to depend mainly on the temperature difference of the surface and the dew point, where for the maximum effectiveness the difference should be about 3 degrees celsius. The TiO2 nanotubes have a potential to be employed for an economical and continuous disinfection of surfaces.


Introduction
Economic burden of $30-80 billion was estimated by the Center for Disease Control and Prevention (CDC) for the annual number of foodborne illnesses, affecting 48 million Americans [1,2]. Over 320.000 cases of food-borne zoonotic diseases were evidenced in humans each year, thus the measures in view of food safety have to be very strict especially on food and food premises hygiene [3]. Food can become contaminated at any point during production and distribution, as well as in consumers' own kitchens. Therefore, foodborne illness risk reduction and control interventions must be implemented at every step throughout the food preparation process [4]. Recent global developments are increasingly challenging international health security according to the World Health Organization (WHO). These developments include the growing industrialization and trade of food production and the emergence of new or antibiotic-resistant pathogens. Micro-organisms are known to survive on surfaces, for extended periods of time. Among the foodborne pathogens, Listeria monocytogenes has the highest a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 are needed [19]. Photo generated holes are highly oxidizing whereas photo generated electrons are reducing enough to produce superoxide from dioxygen [20]. After reacting with water, holes can produce hydroxyl radicals (·OH). Photo excited electrons can become trapped and loose some of their reducing power, but are still capable of reducing dioxygen to superoxide radical (·O 2 -), or to hydrogen peroxide H 2 O 2 . Hydroxyl radical, superoxide radical, hydrogen peroxide, and molecular oxygen could all play important roles in preventing proliferation of bacteria. Using TiO 2 surface coatings one should therefore be able to maintain clean surfaces with the use of UV light close to visible spectrum. Doping of TiO 2 with transition metals such as copper has already been sugested to enhance its photocatalytic properties [21,22]. For example, several research groups made antibacterial coatings based on TiO 2 containing copper and tested them at different light intensities [23][24][25][26][27][28][29]. Although the antibacterial effect of copper doped TiO 2 was greater compared to TiO 2 alone, much of the antibacterial effect is due to leached copper ions [25,26]. It turned out that higher amounts of copper than approximately 1% cannot be successfully incorporated into TiO 2 structure, but are deposited as clusters on the TiO 2 surface [30][31][32], from which copper can be released into the solution. Although this contributes to toxicity of the nanoparticles in absence of irradiation [33] it also leads to unacceptable development of copper resistant bacterial strains as well. Our goal was therefore to incorporate small amount of copper into TiO 2 structure, thus avoiding formation large copper aggregates, which might leach into the solution.
We have shown previously that Cu 2+ -doped TiO 2 nanotubes (Cu-TiO 2 NTs) coated polymer surfaces reduce number of seeded bacteria by 99.93% (i.e. Log 10 reduction = 3.2, when inoculated with 2.08 10 4 Listeria innocua) when illuminated with low power UVA diodes for 24 hours at 4˚C [34], where the intensity of UVA light needed to observe the antibacterial effect was only about 10 times more than it is usually present in common fluorescent lighting. In this manuscript we present antibacterial effect observed on polymer surfaces when illuminated with common fluorescent lights, which emit light with strong peak at 365 nm and were already present on a ceiling of a food processing plant. Coated surfaces innoculated with 10 7 bacteria exhibit similar antimicrobial effect as we observed previously on the TiO 2 nanotube coated petri dishes, reducing the number of Listeria innocua by 80% in seven hours of exposure to the fluorescent lights, compared to control surfaces without the nanotube coating.

Preparation of bacterial inoculum
Antimicrobial properties were tested on non-pathogenic bacterium Listeria innocua, which is closely related to pathogenic species Listeria monocytogenes. Suspension of Listeria innocua strain, isolated during routine examination (RDK.), was supplied by the Institute of Microbiology and Parasitology, Veterinary faculty, University of Ljubljana. Strain was maintained frozen at -70˚C in sterile vials containing porous beads which serve as carriers to support microorganisms (Microbank, pro-lab Diagnostics) and kept at -70˚C. The inoculum was prepared in liquid medium and incubated aerobically for 24 h at 37˚C. After incubation the culture contain approximately 10 9 colony forming units (CFU) per milliliter. Working suspensions with appropriate concentrations were achieved by several 10-fold dilutions.

Preparation and properties of Cu 2+ -doped TiO 2 nanotubes
Cu 2+ -doped TiO 2 nanotubes (Cu-TiO 2 NTs) were prepared in several steps: (i) first sodium titanate nanotubes (NaTiNTs) were synthesized from anatase powder (325 mesh, ! 99.9%, Aldrich) and 10 M NaOH (aq) (Aldrich) at T = 135˚C for 3 days under hydrothermal conditions. Exact synthesis procedure is described previously [35], (ii) in the next step NaTiNTs were rinsed with 0.1 M HCl(aq) yielding protonated titanate nanotubes (HTiNTs), (iii) then 400 mg HTiNTs were dispersed in 100 mL of 0.5 mM solution of Cu 2+ (aq) (source of the Cu 2+ was CuSO 4 Á5H 2 O (Riedel de Haen)) using an ultrasonic bath (30 minutes) and stirred at room temperature for 3 hours. By centrifugation the solid material was separated from the solution, and (iv) finally isolated material was heated in air at 375˚C for 10 hours.
The powder X-ray diffraction (XRD) pattern was obtained on a Bruker AXS D4 Endeavor diffractometer using Cu Kα radiation (1.5406 Å; in the 2θ range from 10 to 65˚). Morphology of the particles in the sample was determined using transmission electron microscope (TEM, Jeol 2100). The specimen for the TEM investigation was prepared by dispersing the sample in MeOH with the help of an ultrasonic bath and depositing a droplet of the dispersion on a lacey carbon-coated copper grid.

Activity of TiO 2 nanotubes
The photocatalytic activity of synthesized titanate and TiO 2 nanomaterials was determined using electron paramagnetic resonance spectroscopy (EPR) with spin trapping, which was optimized for measurement of primary radicals generated in the vicinity of the nanomaterial surface. This was achieved by measuring primary hidroxyl radicals in the presence of 30% ethanol with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trap (DEPMPO). EPR spin trapping was applied to measure the generation of reactive oxygen species (ROS) production.

Deposition of Cu-TiO 2 NTs on PET surface and testing of the deposition stability
The deposition of Cu-TiO 2 NTs was made on different surfaces: 2.5 cm × 7.5 cm polyethylene terephthalate (PET) slides, polystyrene petri dishes (8 cm diameter), aluminum oxide slides with the same dimensions as PET slides. The surfaces were washed before deposition. They were soaked in 20% NaOH solution, rinsed with distilled water, and finally with ethanol vapor.
The suspension of the nanotubes with concentration of 1 mg/mL was processed with ultrasonic liquid processor (Sonicator 4000, Misonix) prior to the deposition on the surfaces. Sonication was performed using 419 Microtip™ probe, 15 min process time, 10 s pulse-ON time, 10 s pulse-OFF time and maximum amplitude (resulting in 52 W of power).
The surfaces were treated with compressed air 3 times for 3 s. 150 μL of nanoparticle suspension was applied on each surface, immediately after compressed air treatment, and smeared evenly. The same number of surfaces with nanoparticle deposition and control surfaces were prepared for each experiment. On control surfaces, only 150 μL of solution was applied. After the deposition, the surfaces were left in the oven at 50˚C for 2 h. Then they were rinsed with distilled water and put back in the oven at 50˚C for another 2 h.
The photocatalytic activity of the nanodeposit on the surfaces was tested using EPR spectroscopy. Three measurements were performed on the surfaces, with or without the nanodeposit.
On each surface, small pool, proportionate to the size of the sample, was made with silicon paste and was filled with 2 μL of 0,5 M DEPMPO and 18 μl of 30% ethanol and irradiated with 290 nm diode for 5 min. The diode was 1-2 mm above the surface of the sample. The solution with short-lived radicals being trapped in the form of stable DEPMPO spin adducts was then drawn into the quartz capillary of 1 mm diameter, which was put in the 5 mm wide quartz tube and transferred into EPR spectrometer. All EPR measurements were performed on an X-band EPR spectrometer Bruker ELEXYS, Type W3002180. All measurements were recorded at room temperature using 1 Gauss (10 −4 T) modulation amplitude, 100 kHz modulation frequency, 20 ms time constant, 15 x 20 seconds sweep time, 20 mW microwave power and 150 G sweep width with center field positioned at 3320 G.
The amount of deposited material was estimated from EPR signal decrease when rinsing the deposit of 150 μL of 1 mg/mL applied to a 2.5 × 7.5 = 18.8 cm 2 surface. With EPR signal being decreased to about 1/3, we estimated that the amount of deposited nanomaterial was about 2 μg/cm 2 .

Antimicrobial activity of nanotube coated PET surface in a meat processing plant
Four measurement points were selected in a poultry slaughterhouse with regards to different air microclimate conditions (humidity, temperature, airflow) as well as intensity of UV irradiation. PET slides were innoculated with 10 7 bacteria in 10 μL droplet and placed either vertically or horizontally at different altitudes (0.5 or 2 m) and exposed for 7 hours. After exposure, the samples were washed in saline (NaCl 0.9%) and examined bacteriologically to determine the number of bacteria. Survival of bacterial culture of Listeria innocua has been measured for samples with and without germicidal Cu-TiO 2 NTs coating. Reduction ratio was expressed in percentage and logarithm (Log 10 ). % reduction was calculated using the following equation: Where N final control is the number of bacteria after the exposure on a control surface, and N final Cu-TiO 2 NTs is the number of bacteria after the exposure on a surface coated with Cu-TiO 2 NTs nanotubes. Log reduction was calculated using the following equation: LR = À log 10 (N final CuÀ TiO 2 NTs /N final control ) = log 10 (N final control ) À log 10 (N final CuÀ TiO 2 NTs ) ð2Þ

Antimicrobial activity in presence of repeated daily contamination and washing
Effect of long term illumination was studied only on PET surfaces, by placing uncoated (control) PET slides and PET slides covered by Cu-TiO 2 NTs on cooled (4˚C) aluminum plates in order to mimic cold and condensing conditions at the cooling walls commonly present in food processing plants. Bacterial suspension (10 μl) of living microorganism Listeria innocua in concentration of 1.5 to 5.0 x 10 9 CFU/mL was applied daily on each PET slide. The slides were then cooled to the dew point, which prevented the drying of microorganism containing droplets on the slides. Slides were washed with 100 mL of sterile saline solution (0.9 weigth % NaCl) at different time intervals and the number of surviving microorganisms was determined. The remaining PET slides were stored in the dark at 4˚C until the next day when the above described process was repeated. The whole experiment with daily washing and bacteria application lasted for 28 days.

Structure and photochemical activity of copper doped TiO 2 nanotubes (Cu-TiO 2 NTs)
Cu 2+ -doped TiO 2 nanotubes used in this study were prepared by conversion of H 2 Ti 3 O 7 nanotubes doped with Cu 2+ by calcination at 375˚C. H 2 Ti 3 O 7 nanotubes upon calcination above 350˚C transform to anatase nanotubes [36]. As it can be observed from transmission electron microscopy (TEM) image ( Fig 1A) during this transformation the nanotube morphology is preserved while nanotubes become shorter [37]. Porous structure of Cu 2+ -doped TiO 2 nanotubes is clearly seen from both insets to Fig 1A. The outer diameter of nanotubes varies from 8 to 10 nm while in length they reach up to 300 nm. The phase identification of the sample was performed using X-ray powder diffraction (XRD). All diffraction peaks (Fig 1B) Fig 1A) nanoparticles caught in the inner side or outer sided of the tubes were observed but the number of this particles is low due to the low copper doping. When copper content is higher, that is about 11 wt. %, CuO nanoparticles are clearly observed as it was shown in our previous study [31]. More detailed characterization, using advanced high resolutiom transmission electron microscopy techniques, of the copper doped TiO 2 nanotubes is reported in Koklic et al. [34] (article submitted).
We have previously shown that the Cu-TiO 2 NTs deposited on polystyrene petri dishes reduce up to 99.93% (3.2 log 10 reduction, initial number of bacteria 2.08Á10 4 ) Listeria innocua in 24 hours in a refrigerator at 100% humidity, illuminated with UVA light emitting diodes [34]. However, Usage of additional illumination results in additional costs associated with application of such disinfection methods. On the other hand, ceiling mounted fluorescent lights, which are already in use in many food processing plants, contain a small portion of emitted light in UVA range. Fig 1C shows the spectrum of emitted light by a ceiling mounted fluorescent lamp. Three peaks in the spectrum are clearly visible, with one spectral peak at 365 nm. It is this peak which is absorbed by the Cu-TiO 2 NTs, as it is evident from the absorption of light as a function wavelength (Fig 1C, closed squares). The absorption of light versus wavelength of the light is consistent with a bandgap of the TiO 2 , a property of a semiconductor such as TiO 2 . Matsunaga et al. showed already in 1985 that Escherichia coli cells were completely sterilized when TiO 2 was irradiated with high intensity UV light. [38] Since then the antibacterial effect of photoexcited TiO 2 was shown against a wide range of microorganisms. [27] Photocatalytic mechanism and related photochemistry of TiO 2 is well researched [24,[39][40][41][41][42][43][44][45], antibacterial action seems to depend mainly on ÁOH radicals, which are produced on the surface of TiO 2 when illuminated with light consisting of wavelengths below TiO 2 's bandgap. Due to this semiconductor property of the Cu-TiO 2 NTs nanotubes the production of hydroxyl (ÁOH) radicals on the surface of nanotubes increases with decreasing wavelength. We measured the amount of produced radicals as a function of different wavelengths of light, by using a DEPMPO spin trap (Fig 1C, closed squares), which is commonly used for efficient trapping of the hydroxyl radicals [46]. Since the spectrum of the emitted light from a ceiling mounted common fluorescent light (Fig 1C, black line, the peak at 365 nm) overlaps with the spectrum of the light needed for efficient photoexcitation of the nanotubes (the closed squares), we expected that the nanotube coated surfaces could be excited by fluorescent lights, which are already present on ceilings at food processing plants. Especially due to Photocatalytic surface disinfection by ceiling mounted fluorescent lights intense peak at 365 nm, which is present in the emitted spectrum of the fluorescent light bulb and represents about 1% of total light emitted by the lamp. The scale in Fig 1C is set to emphasize the fluorescent light emission peak at 365 nm, which is overlapping with the ÁOH radical production efficiency curve (Fig 1C closed squares) and seems to be responsible for the antimicrobial activity of the nanotubes.

Deposition stability of copper doped TiO 2 nanotubes on different surfaces
Next we tested the stability of the nanotubes deposited on different surfaces, which are commonly used in food processing industry. The dispersion of Cu-TiO 2 NTs was added to the clean surface (see Materials and methods) and left to dry. No special chemical modification of either nanotubes or surface was necessary. Unattached nanoparticles were washed away under a stream of water and the production of hydroxyl radicals was measured as described in Materials and Methods section. Since the amount of the produced radicals is proportional to the amount of Cu-TiO 2 NTs still remaining on the surface after extensive washing, we used the measurement of the the quantity of radicals produced by illuinated surfaces as a measure for the stability of the deposition. That is, if the amount of produced radicals remains constant throughout the washing cycles, then the Cu-TiO 2 NTs nanoparticles should remain attached to the surface. We tested different materials: polyethylene terephtalate (Fig 2, row A), polystirene (Fig 2, row B), and aluminum oxide (Fig 2, row C). All surfaces were repeatedly soaked at different pH conditions neutral (pH7, Fig 2, first column, blue color), basic (pH10, Fig 2, second column, violet color), and acidic (pH4, Fig 2, third column, red color) and extensively washed under a stream of water after each soaking. The amount of material versus washing step was fit with a linear curve using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). The area, which contains a linear fit, which describes the data with 90% certainty is shown on each graph. In all of the graphs, except for aluminum oxide washed at pH10, linear fit is contained around 100% deposited material (horizontal dotted lines), thus indicating that Cu-TiO 2 NTs nanoparticles deposited to various surfaces should withstand daily washings with different detergents commonly used in food processing industry. However the material will not provide long term disinfection of aluminum oxide surfaces, when washed with basic detergent (Fig 2, frame C2). The Fig 2, frame C2 shows that deposited nanotubes can be washed off the aluminum oxide surface when washed in alkaline conditions. Recently Berg et al. [47] showed that both TiO 2 and aluminum oxide nanoparticle surfaces have high negative surface charge at pH 10, which might result in a repulsive force between the two surfaces.

Antimicrobial activity of TiO 2 nanotube coated surfaces placed in a food processing plant
We exposed polyethylene terephthalate (PET) surfaces with or without Cu-TiO 2 NTs antibacterial coating at different locations in the food processing plant to test whether the intensity of ceiling mounted fluorescent lights in a food processing plant is sufficient to provide measurable antibacterial activity of surfaces coated with the nanotubes. We applied 10 μL of bacterial suspension of Listeria innocua on the PET surfaces (10 7 bacteria), as shown schematically in Fig 3A, and placed the PET surfaces at different places in the food processing plant with respect to performed tasks (evisceration, meat cut up, cold room, Depo-meat storage, and butchering) for 7 hours. The plant used common ceiling mounted fluorescent lights with a light emitting spectrum shown in Fig 3B. The air microclimate conditions (UV light Intensity, humidity, and temperature) were followed and the number of remaining bacteria was determined (S1 and S2 Tables). Microclimatic air conditions measured at different places in the food processing plant were different with respect to temperature, humidity, ambient light intensity emitted from ceiling mounted fluorescent lights, and airflow (S1 Fig). The reduction of the number of bacteria compared to a control surface was achieved at two places: 1) Evisceration (90%±61%), and 2) Cold room (73%±44%) (Fig 3D, grey bars). As seen in S1 Table log 10 (CFU) = 0 and cannot be shown on the logarithmic scale of the Fig 3C. Since the error in calculating %R is very large due to errors in determining colony forming units (Fig 3C) we can only speculate that the disinfection properties of the surfaces might depend on the temperature difference of the surface and the dew point (Fig 3D, grey bars), where for maximum effectiveness of the photocatalytic effect the difference should be about 3˚C (Fig 3D, black dashed  line). This is not supprising, since fogs of all types start forming when the air temperature and dewpoint of the air become nearly identical. This occurs through cooling of the air near the cool surface to a little beyond its dewpoint and the precipitation of water droplet from the air seldom forms when the dewpoint spread is about 3˚C. Other microclimatic parameters (S1 Fig) didn't follow the relative reduction of L. innocua. This results shows that photocatalytic   (Fig 3E) when we averaged reductions of bacteria across all the places in the food processing plant. The average number of bacteria on the surfaces with the nanotube coating was reduced more (Log 10 = 2.92) than on the control surfaces (Log 10 = 3.90), which can be expressed as relative percent reduction of % R = 80%. This is similar to the antibacterial activity of copper doped TiO 2 coatings on Methicillin Resistant Staphylococcus aureus (MRSA) (Log 10 = 4.2 under visible light irradiation and Log 10 = 1.8 in darkness) owing to the generation of reactive oxygen species which should be a major factor which substantially reduces bacterial growth on glass surfaces [48]. However the demonstrated antibacterial activity was also high even in the dark, probably due to presence of copper particles alone. Similarly Xiaojin et al. [49] developed an innovative coating system based on polymers containing fine particles Cu and TiO 2 nanoparticles. Coatings were applied to various substrates, including plastics, glass, and steel. During investigation antimicrobial properties and microhardness of coatings were studied. Coatings showed an effective antibacterial activity (reduction of E. coli by 10 6 in 2 hours), where Cu and TiO 2 particles contributed to the strengthening of the resistance, persistence and microhardness of surfaces to which they were applied.
For further quantification of the results we calculated the ratios of bacteria from the coated surfaces versus the control surfaces for all the measurements (Fig 3F, white bars). In such presentation of the results the antibacterial effect is reflected in the ratio to of less than 1. The distributions of survival ratios (Fig 3F, white bars) were clearly not normal. Since biological mechanisms often induce lognormal distributions [50], for example when exponential growth is combined with further symmetrical variation such as initial concentration of bacteria [51][52][53][54], we fit our data with a log normal distribution (Fig 3F, dashed line). The lognormal fits of the histograms fit best the survival ratios also when compared to other distributions.

Antimicrobial activity in presence of repeated daily contamination and washing
Next we repeatedly inoculated and washed PET surfaces with Listeria innocua daily in laboratory conditions, in order to mimic daily contamination in food processing industry or surfaces in a household, as shown schematically in Fig 4A. After application, we left the bacteria on the surface for 7 hours while being exposed to low intensity light from fluorescent lamps on the ceiling (t = 7 h, j = 2.5 W/m 2 , A = 8 J (total light), A <380nm = 80 mJ). As it can be seen from the measured light intensity spectrum of the fluorescent lamp (Fig 4B), the intensity of light with wavelengths below 380 nm (80 mJ) is only 1 percent of the total light intensity, the corresponding energy of the illumination of the nanotubes, which can induce the photocatalytic process of hydroxyl radical production, is therefore around 80 mJ in our experimental setup. Methods section; Grey bars represent the microclimatic parameter (difference between surface temperature and dew point temperature-dew point spread) which correlates best with the %R. E) Boxplot presents log10 of number of bacteria on either control PET surface without antibacterial nano coating (PET) or the number of bacteria on a PET surface coated with copper doped TiO 2 nanotubes (PET+Cu-TiO 2 NTs). Median value of each distribution is shown with horizontal line within each box, while the dashed line marks the mean. The boundary of the box closest to zero indicates 25th percentile, and the boundary farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. The outliers are shown as dots. Note the LOG scale. Since normality test (Shapiro-Wilk) failed (P < 0.050), Mann-Whitney rank sum test was performed on the control group (N = 16, median = 4) and on the PET+Cu-TiO2NTs group (N = 32, median = 3.1). The difference in the median values between the two groups is greater than would be expected by chance (P = 0.003) Ã . F) Histogram of the distribution (probability density function-PDF) of survival ratios, calculated as a ratio between the number of Listeria innocua from the surface with antibacterial coating (PET+Cu-TiO 2 NTs) and the number of Listeria innocua from a control surface without the coating (PET). For inefficient antibacterial coating survival ratio of 1 is expected. Dotted line is lognormal fit of the distribution with a maximum of the PDF below 0.1 for the reduction of the number of bacteria on the surface. https://doi.org/10.1371/journal.pone.0197308.g003 Photocatalytic surface disinfection by ceiling mounted fluorescent lights Although the antimicrobial effect was not as pronounced as in the food processing plant, average number of CFU/mL in eluate from the control surface was 146 ± 38, which is significantly higher than the number of CFU/mL in the eluate from the TiO 2 nanotubes coated surface, which decreased to 43 ± 13 (Fig 4C, eluate) (t = 3.018, with 38 degrees of freedom, twotailed P-value = 0.00453, power of performed two-tailed test with alpha = 0.050 : 0.837). In the 28 days lasting experiment the average number of CFU remaining on the control surface was 74 ± 31 CFU/mL, whereas the average number remaining on the nanotube coated surface was 45 ± 22 CFU/mL, which was not statistically different (Fig 4C, surface). The relative reduction of the number of Listeria Innocua in the eluate was 70% ± 39 in seven hours of exposure to the fluorescent lights, compared to a control surface (Fig 4D, eluate), whereas the reduction of bacteria remainig on a surface was negligible (Fig 4D, surface, see error bar extending to zero). All the data, from which averages were calculated, are shown in in the supplement (S1 Table) and in the Fig 4E in a form of a box plot, from which can be easily seen that the distribution of measurements is not normal (the mean and median are not overlaping). For more detailed quantification of the results we therefore calculated also the ratios of bacteria from the coated surfaces versus the control surfaces for all measurements (Fig 4F). The effect of the antibacterial coating on the survival of Listeria innocua is indicated by the ratio of less than 1. The distributions of survival ratios were also not normal as for the data in Fig 3F. We again fit the histograms of the survival ratios in the eluate and on the surface. The best fits of the data to the lognormal distribution indicate that the maximum of the probability density function is around 0.1, thus confirming that antibacterial coating is inhibiting the growth of Listeria innocua on TiO 2 nanotube coated surfaces.
To test whether a small addition of copper is solely responsible for the antibacterial properties of the nanotube coated surface, the above experiments were also performed in the dark (see the Supplement, S2 Fig). In the experiment in the dark the number of colony forming units of Listeria innocua on nanotube coated as well as on control surface was the same, indicating that the reduction of bacteria that we observed originates from photocatalytic process of the TiO 2 nanotubes.

Conclusion
To implement advantages of germicidal disinfection with the use of ultraviolet light as well as antibacterial properties of copper-containing surfaces, we used recently characterized Cu 2+doped TiO 2 nanotubes and achieved a stable deposition on several materials, including on the line). C) Number of Listeria innocua shown on a logarithmic scale. On each surface 10 7 bacteria were placed on PET slides. After 7 hours of exposure to ceiling mounted fluorescent lights remaining bacteria were transferred from the surfaces and colony forming units (CFU) were counted. Number of CFU on control surfaces is shown with white bars (PET control); Number of CFU on the nanoparticle coated surfaces is shown with black bars (PET + Cu-TiO 2 NTs); D) Relative reduction (%R) of bacteria as a consequnce of disinfecting action of nanoparticle coated surface, illuminated with ceiling mounted fluorescent lights (grey bars), calculated according to the Eq 1 in Materials and Methods section. E) Number of Listeria innocua in eluate from either control surface without nano coating (PET) or from a surface coated with copper doped TiO 2 nanotubes (PET+Cu-TiO 2 NTs) presented in a boxplot, where the line in a box marks the median number of bacteria, while the dashed line marks the mean. The boundary of the box closest to zero indicates 25 th percentile, and the boundary farthest from zero indicates the 75 th percentile. Whiskers (error bars) above and below the box indicate the 90 th and 10 th percentiles, respectively. The outliers are shown as dots. Note the LOG scale. Reduction in number of bacteria in eluate for 0.6 orders of magnitude was statistically significant Ã (t = 3.018, with 38 degrees of freedom, two-tailed P-value = 0.00453, power of performed two-tailed test with alpha = 0.050 : 0.837). The reduction of bacteria on a surface was smaller than in eluate (t = 1.338, with 20 degrees of freedom, two-tailed P-value = 0.196, power of performed two-tailed test with alpha = 0.050 : 0.247). F) Histograms of the distribution (probability density function-PDF) of survival ratios in eluate (red) or on a surface (blue). Survival ratio was calculated as a ratio between the number of Listeria innocua from the surface with antibacterial coating (PET+Cu-TiO 2 NTs) and the number of Listeria innocua from a control surface without the coating (PET). For inefficient antibacterial coating survival ratio of 1 is expected. Dotted line is lognormal fit of the distributions with a maximum of the PDF at 0.02 and 0.13 for the reduction of the number of bacteria in eluate and on the surface, respectively. https://doi.org/10.1371/journal.pone.0197308.g004 Photocatalytic surface disinfection by ceiling mounted fluorescent lights surface of polyethylene terephthalate (PET), as the one of the synthetic polymers commonly used in food processing industry. More importantly, we showed that such coating has disinfecting effect, with the number of remaining microorganisms significantly decreased on the surface coated with Cu 2+ -doped TiO 2 nanotubes as well as in eluate from the coated surface, when illuminated with common ceiling mounted fluorescent lights. The disinfection properties of the nanotube coated surfaces depend on the intensity of the light, which should include wavelengths at about 370 nm, as well as on the temperature difference of the surface and the dew point, where for maximum effectiveness of the photocatalytic effect the difference should be about 3˚C.
Our results show that one dimensional nanomaterials, such as TiO 2 nanotubes, can be employed for disinfection of polymer surfaces in the food industry, using cost effective illumination with existing fluorescent lights or additional low power light emitting diodes. Future use of such surfaces with antibacterial nano-coating and resulting sterilizing effect holds promise for such materials to be used in different environments or in better control of critical control points (HACCP) in food production as well as an improved biosecurity during the food manufacturing process.