Simultaneous Enhancement of Photocatalytic Bactericidal Activity and Strength Properties of Acrylonitrile-Butadiene-Styrene Plastic Via a Facile Preparation with Silane/TiO2

This work aims to enhance the photocatalytic antibacterial performance of plastics according to the JIS Z 2801:2010 standard, and to determine their mechanical properties by studying: (i) the influence of calcination on titanium dioxide (TiO2); (ii) modification with different TiO2 concentrations, and; (iii) the effect of silane as a coupling agent. Acrylonitrile-butadiene-styrene plastics (ABS) and Escherichia coli (E. coli) were chosen as the model plastic and bacteria, respectively. The 500 °C calcined TiO2 successfully provided the best photoantibacterial activity, with an approximately 62% decrease of E. coli colony counts following 30 min of exposure. Heat treatment improved the crystallinity of anatase TiO2, resulting in low electron-hole recombination, while effectively adsorbing reactants on the surface. ABS with 500 °C-calcined TiO2 at the concentration of 1 wt % gave rise to the highest performance due to the improved distribution of TiO2. At this point, blending silane coupling agent could further improve the efficacy of photoantibacterial activity up to 75% due to greater interactions with the polymer matrix. Moreover, it could promote a 1.6-fold increase of yield strength via increased adherent bonding between TiO2 and the ABS matrix. Excellent photocatalytic and material stability can be achieved, with constant photocatalytic efficiency remaining for up to five reuse cycles without loss in the yield strength.


Introduction
Thermoplastic polymers are widely used for appliances such as sanitary ware, medical appliances, furniture and children's toys due to their favorable properties, such as excellent impact resistance, good machinability and excellent aesthetic qualities [1]. Durable plastics with a useful life of three years or more, such as Acrylonitrile-Butadiene-Styrene (ABS) plastics, could be potentially colonized by myriad microorganisms. Therefore, one of the biggest problems these plastics pose is microorganism Polymers 2020, 12, 917 3 of 12

Calcination of TiO 2 Powder
Anatase TiO 2 particles were preheated in an oven at 90 • C for 2 h, and then calcined at temperatures of 300, 500 and 800 • C for 2 h with a ramping rate of 5 • C min −1 .

Surface Modification of TiO 2 with Silane
A 0.5 g mass of calcined TiO 2 was dispersed in 50 cm 3 of 2.5% v/v ethanol. Then, 0.15 g of the silane-coupling agents (APTES) at standard conditions were added in the dispersion, and stirred for 45 min. The resulting slurry was centrifuged and dried in an oven for 24 h at 80 • C

Preparation of TiO 2 /ABS Compositions
Prior to blending polymers, ABS plastic was dried in an oven at 90 • C for 2 h to remove moisture. The TiO 2 or calcined TiO 2 particles (concentration of 0.5, 1 and 2 wt %) or calcined TiO 2 treated with silane, and ABS plastic were loaded into a twin-screw extruder, and then mixed well in an internal mixer at 250 • C with speed round mixing at 60 rpm for 6 min. The melting material was put in the mold (5 × 5 cm 2 ) by using a compression molding at a temperature of 250 • C and pressure of 125 kg cm −2 for 5 min, and rapidly cooled for 5 min. The resulting TiO 2 /ABS compositions were air-cooled at room temperature before being tested.

E. coli Bacteria Preparation and Photoantibacterial Activity
Photoantibacterial activity and efficacy assessments followed the standard JIS Z 2801 (The test for antibacterial activity and efficacy on surfaces of antibacterial products). A colony of E. coli bacteria was transferred into TSB solution, and then incubated at 32 ± 0.5 • C for 24 h. The bacterial suspensions were diluted into 2.5 × 10 8 cfu cm −3 , dropped on TiO 2 /ABS workpieces, and covered with a polyethylene (PE) film size 4 × 4 cm 2 . The sample was illuminated by UVC light (15 W) for 30 min. The bacterial population was determined by plated serial dilution, and plates were incubated at 32 ± 0.5 • C for 24 h. All photoantibacterial activity experiments were performed with three replicates (independent experiments), and the data were represented as the average mean ± SD (error bar).

Sample Characterization
Crystal and structural characteristics of the products and crystallinity were investigated using powder X-ray diffraction (XRD) system with monochromatized Cu kα radiation (λ = 1.5406 Å). Full width at half maximum (FWHM) derived from XRD patterns at 2θ = 25 • indicated the degree of crystallinity. Sample morphology was investigated by a scanning electron microscope, and the surface area and pore size distribution were determined by N 2 adsorption.

Mechanical Tensile Strength
The workpieces were tested using a universal testing machine according to the guidelines set in ASTM D639 Type I (Standard Test Method for Tensile Properties of Plastics). All experiments were performed with three replicates, and the data were represented as the average mean ± SD (error bar).

Effect of Calcination Temperatures on TiO 2
The crystal structure of TiO 2 was investigated by XRD analysis as shown in Figure 1 and corresponding crystallite size was calculated by using the Debye-Scherrer formula. The XRD pattern demonstrated that TiO 2 structures were influenced by calcination temperatures. The initial TiO 2 results included peaks at 2θ = 25.4 • , 37.8 • , 48 • , 54.5 • and 62.8 • , corresponding to the anatase phase of TiO 2 with the FWHM value of 0.5 and particle size of 6.12 nm. No peaks caused by impurities were observed. TiO 2 calcined at 300 • C showed that the intensity of crystallinity of anatase increased, with a lower FWHM value of 0.41 and particle size of 7.08 nm. Further increases in temperature to 500 and 800 • C increased crystallinity of anatase, as shown by recorded FWHM values of 0.27 and 0.08, respectively. Therefore, crystalline structure was slightly developed by increasing the calcination temperatures. The particle sizes of 500 and 800 • C calcined TiO 2 increased slightly to 7.13 and 7.15 nm, respectively. 8°, corresponding to the anatase phase of TiO2 with the FWHM value of 0.5 and particle size of 6.12 nm. No peaks caused by impurities were observed. TiO2 calcined at 300 °C showed that the intensity of crystallinity of anatase increased, with a lower FWHM value of 0.41 and particle size of 7.08 nm. Further increases in temperature to 500 and 800 °C increased crystallinity of anatase, as shown by recorded FWHM values of 0.27 and 0.08, respectively. Therefore, crystalline structure was slightly developed by increasing the calcination temperatures. The particle sizes of 500 and 800 °C calcined TiO2 increased slightly to 7.13 and 7.15 nm., respectively. The initial surface area of TiO2 was 10 m 2 g −1 . At 300 °C, the specific surface area was reduced to 9 m 2 g -1 . A continuous decrease in surface area with the rise of calcination temperature was observed in TiO2 calcined at 500 and 800 °C , with respective surface areas of 7 and 4 m 2 g −1 . The influence of calcination temperature on morphology was investigated by SEM imaging as presented in Figure 2. Initially, the TiO2 particles had a diameter of approximately 200 nm as shown in Figure 2a. When mixing TiO2 particles with ABS plastic, the particles appeared to be embedded in the ABS plastic, resulting in the rough surface, as observed in Figure 2b. Calcining at 300 °C led to structural aggregation of TiO2 particles (Figure 2c), whereby the surface of ABS plastic contained assemblies of particles in some areas (Figure 2d). At 500 °C, it was also observed that a continuous aggregation in TiO2 particles occurred ( Figure 2e) and these were visibly assembled on the ABS surface ( Figure 2f). When the temperature was increased to 800 °C , the TiO2 particles became obviously aggregating as seen in Figure 2g. Calcination of TiO2 results in partial or total collapse of the structure, decreasing of surface area, and the appearance of particle agglomerations. This resulted in poorly dispersed distribution of TiO2 particles on ABS plastic as shown in Figure 2h. The higher degree of agglomeration was ordered as follows: 800 °C-TiO2/ABS > 500 °C-TiO2/ABS > 300 °C-TiO2/ABS > uncalcined-TiO2/ABS, and could be affirmed by orderly lowering yield strength, as shown in the next section. The initial surface area of TiO 2 was 10 m 2 g −1 . At 300 • C, the specific surface area was reduced to 9 m 2 g −1 . A continuous decrease in surface area with the rise of calcination temperature was observed in TiO 2 calcined at 500 and 800 • C, with respective surface areas of 7 and 4 m 2 g −1 . The influence of calcination temperature on morphology was investigated by SEM imaging as presented in Figure 2. Initially, the TiO 2 particles had a diameter of approximately 200 nm as shown in Figure 2a. When mixing TiO 2 particles with ABS plastic, the particles appeared to be embedded in the ABS plastic, resulting in the rough surface, as observed in Figure 2b. Calcining at 300 • C led to structural aggregation of TiO 2 particles (Figure 2c), whereby the surface of ABS plastic contained assemblies of particles in some areas (Figure 2d). At 500 • C, it was also observed that a continuous aggregation in TiO 2 particles occurred (Figure 2e) and these were visibly assembled on the ABS surface ( Figure 2f). When the temperature was increased to 800 • C, the TiO 2 particles became obviously aggregating as seen in Figure 2g. Calcination of TiO 2 results in partial or total collapse of the structure, decreasing of surface area, and the appearance of particle agglomerations. This resulted in poorly dispersed distribution of TiO 2 particles on ABS plastic as shown in Figure 2h. The higher degree of agglomeration was ordered as follows: 800 • C-TiO 2 /ABS > 500 • C-TiO 2 /ABS > 300 • C-TiO 2 /ABS > uncalcined-TiO 2 /ABS, and could be affirmed by orderly lowering yield strength, as shown in the next section.

Effect of TiO2 with and without Calcination on Photoantibacterial Activity and Yield Strength of ABS
The influence of TiO2 and calcined TiO2 mixed with ABS on photoantibacterial Escherichia coli (E. coli) as assessed by the JIS Z 2801: 2010 standard test are illustrated in Figure 3. Photoantibacterial effectiveness for E. coli was presented in the form of bacterial survival. The result obtained for pure ABS under UV illumination for 30 min showed little antibacterial activity, while the E. coli survival of 60 ± 2.8% was observed in ABS with TiO2, highlighting the dominant impact of TiO2 in influencing photocatalytic activity. Figure 4 illustrates the mechanism of photocatalytic antibacterial action proposed, in which the OH radicals and reactive oxygen species (ROS) generated by TiO2 would damage the cell membrane, resulting in the leakage of bacterial cytoplasm, leading to cell death [20][21][22]. Moreover, the calcined TiO2 mixed with ABS provided a greater reduction of E. coli compared to uncalcined TiO2/ABS. Calcining TiO2 at 300 °C could decrease E. coli on ABS by 45 ± 2.1% (remaining bacterial survival 55%), further decreasing to 60 ± 2.6% at 500°C (remaining bacterial survival 40%), despite a decrease in surface area. The photocatalytic improvement could be plausibly explained by

Effect of TiO 2 with and without Calcination on Photoantibacterial Activity and Yield Strength of ABS
The influence of TiO 2 and calcined TiO 2 mixed with ABS on photoantibacterial Escherichia coli (E. coli) as assessed by the JIS Z 2801: 2010 standard test are illustrated in Figure 3. Photoantibacterial effectiveness for E. coli was presented in the form of bacterial survival. The result obtained for pure ABS under UV illumination for 30 min showed little antibacterial activity, while the E. coli survival of 60 ± 2.8% was observed in ABS with TiO 2 , highlighting the dominant impact of TiO 2 in influencing photocatalytic activity. Figure 4 illustrates the mechanism of photocatalytic antibacterial action proposed, in which the OH radicals and reactive oxygen species (ROS) generated by TiO 2 would damage the cell membrane, resulting in the leakage of bacterial cytoplasm, leading to cell death [20][21][22]. Moreover, the calcined TiO 2 mixed with ABS provided a greater reduction of E. coli compared to uncalcined TiO 2 /ABS. Calcining TiO 2 at 300 • C could decrease E. coli on ABS by 45 ± 2.1% (remaining bacterial survival 55%), further decreasing to 60 ± 2.6% at 500 • C (remaining bacterial survival 40%), despite a decrease in surface area. The photocatalytic improvement could be plausibly explained by a higher degree of crystallinity as mentioned earlier. That is, the more-active crystal phase was improved and surface defects were reduced as documented by several studies [23][24][25][26]. However, when increasing calcination temperature up to 800 • C, the photoantibacterial performance decreased. This may derive from sintering and agglomeration effects during calcination at high temperature, as shown in SEM imagery ( Figure 2).
Polymers 2020, 12, x FOR PEER REVIEW 6 of 12 a higher degree of crystallinity as mentioned earlier. That is, the more-active crystal phase was improved and surface defects were reduced as documented by several studies [23][24][25][26]. However, when increasing calcination temperature up to 800°C, the photoantibacterial performance decreased. This may derive from sintering and agglomeration effects during calcination at high temperature, as shown in SEM imagery ( Figure 2).    Polymers 2020, 12, x FOR PEER REVIEW 6 of 12 a higher degree of crystallinity as mentioned earlier. That is, the more-active crystal phase was improved and surface defects were reduced as documented by several studies [23][24][25][26]. However, when increasing calcination temperature up to 800°C, the photoantibacterial performance decreased. This may derive from sintering and agglomeration effects during calcination at high temperature, as shown in SEM imagery (Figure 2).     The yield strengths of 300 • C TiO 2 /ABS, 500 • C TiO 2 /ABS and 800 • C TiO 2 /ABS were 17.1 ± 0.3, 17.0 ± 0.3 and 16.9 ± 0.2 MPa, respectively. The decrease of yield strength may be attributed to increasing agglomeration and reduced surface area of TiO 2 (e.g., a lower interfacial area for bonding to the ABS polymer) with increasing calcination temperature, as seen from SEM imagery (Figure 2), which contributed to formation of fewer crosslinks among the polymer chains.
Polymers 2020, 12, x FOR PEER REVIEW 7 of 12 strengths of 300 °C TiO2/ABS, 500 °C TiO2/ABS and 800 °C TiO2/ABS were 17.1 ± 0.3, 17.0 ± 0.3 and 16.9 ± 0.2 MPa, respectively. The decrease of yield strength may be attributed to increasing agglomeration and reduced surface area of TiO2 (e.g., a lower interfacial area for bonding to the ABS polymer) with increasing calcination temperature, as seen from SEM imagery (Figure 2), which contributed to formation of fewer crosslinks among the polymer chains.

Effect of Concentration of Calcined TiO2 on Photoantibacterial Activity and Yield Strength of ABS
The previous section demonstrates that optimum performance on photocatalytic performance occurred at 500 °C for calcined TiO2/ABS. In this section, the influence of 500 °C -calcined TiO2 concentration in ABS on photocatalytic performance was considered. From the 40% bacterial survival by 1 wt % calcined TiO2/ABS, changing of TiO2 concentration had been considered, as shown in Figure 6. The reduced concentration of TiO2 resulted in the increase of bacterial survival by 50 ± 2.2 %. This indicated that the smaller amount of TiO2 was not enough to produce OH radicals and reactive oxygen species (ROS) to inactivate the bacteria. However, by increasing the concentration to 2 wt %, the bacterial survival increased to 70 ± 2.5%. This showed that presence of large amounts of TiO2 did not always lead to the high photocatalytic activity, but may in fact suppress the activity due to their aggregation. Figure 7 shows that yield strength decreased with increasing of TiO2 loading. A 0.5 w t% TiO2/ABS had the highest yield strength of 18.0 ± 0.3 MPa. This was to be expected given the higher TiO2 inducing the effective matrix reduction. In other words, higher TiO2 loading (1 and 2 wt %) increase "particle-to-particle" interactions rather than "particle-to polymer" interactions, thus lowering yield strength [27].

Effect of Concentration of Calcined TiO 2 on Photoantibacterial Activity and Yield Strength of ABS
The previous section demonstrates that optimum performance on photocatalytic performance occurred at 500 • C for calcined TiO 2 /ABS. In this section, the influence of 500 • C-calcined TiO 2 concentration in ABS on photocatalytic performance was considered. From the 40% bacterial survival by 1 wt % calcined TiO 2 /ABS, changing of TiO 2 concentration had been considered, as shown in Figure 6. The reduced concentration of TiO 2 resulted in the increase of bacterial survival by 50 ± 2.2%. This indicated that the smaller amount of TiO 2 was not enough to produce OH radicals and reactive oxygen species (ROS) to inactivate the bacteria. However, by increasing the concentration to 2 wt %, the bacterial survival increased to 70 ± 2.5%. This showed that presence of large amounts of TiO 2 did not always lead to the high photocatalytic activity, but may in fact suppress the activity due to their aggregation. Figure 7 shows that yield strength decreased with increasing of TiO 2 loading. A 0.5 w t% TiO 2 /ABS had the highest yield strength of 18.0 ± 0.3 MPa. This was to be expected given the higher TiO 2 inducing the effective matrix reduction. In other words, higher TiO 2 loading (1 and 2 wt %) increase "particle-to-particle" interactions rather than "particle-to polymer" interactions, thus lowering yield strength [27].

Effect of Silane on Photoantibacterial Activity and Yield Strength of ABS
The influence of silane on TiO2/ABS was observed in the SEM images shown in Figure 8, in which the 500 °C -calcined TiO2/ABS without silane possessed rough surfaces and smaller particles on the ABS surface, as illustrated in Figure 8a. After mixing silane, the morphology of blended polymer in Figure 8b shows the presence of a smooth surface and better dispersion, rather than TiO2/ABS. The difference was attributed to a greater interactions with the polymer matrix, which silane coupling agent showed the compatible behaviour of ABS/TiO2 by creating more adherent

Effect of Silane on Photoantibacterial Activity and Yield Strength of ABS
The influence of silane on TiO2/ABS was observed in the SEM images shown in Figure 8, in which the 500 °C -calcined TiO2/ABS without silane possessed rough surfaces and smaller particles on the ABS surface, as illustrated in Figure 8a. After mixing silane, the morphology of blended polymer in Figure 8b shows the presence of a smooth surface and better dispersion, rather than TiO2/ABS. The difference was attributed to a greater interactions with the polymer matrix, which silane coupling agent showed the compatible behaviour of ABS/TiO2 by creating more adherent

Effect of Silane on Photoantibacterial Activity and Yield Strength of ABS
The influence of silane on TiO 2 /ABS was observed in the SEM images shown in Figure 8, in which the 500 • C-calcined TiO 2 /ABS without silane possessed rough surfaces and smaller particles on the ABS surface, as illustrated in Figure 8a. After mixing silane, the morphology of blended polymer in Figure 8b shows the presence of a smooth surface and better dispersion, rather than TiO 2 /ABS. The difference was attributed to a greater interactions with the polymer matrix, which silane coupling agent showed the compatible behaviour of ABS/TiO 2 by creating more adherent bonding between TiO 2 and ABS matrix, as similarly explained for the modified SiO 2 with silane [28][29][30].
bonding between TiO2 and ABS matrix, as similarly explained for the modified SiO2 with silane [28][29][30]. To investigate the effect of silane on photocatalytic activity, the ratio of TiO2 to silane during catalyst preparation was varied over the weight ratio of 1:0.2-1:0.5. The optimum photocatalytic activity occurred for samples at a ratio of TiO2 to silane of 1:0.3 as presented in Figure 9. Figure 9 reveals that 500 °C-calcined TiO2/ABS with silane (called "silane-TiO2/ABS" for brevity) displays a better photobacterial activity compared with 500 °C-calcined TiO2/ABS without silane. The silane-TiO2/ABS could reduce 75% of E. coli (remaining bacteria survival 25 ± 2.6 %). The higher silane-TiO2/ABS photobacterial activity was attributed to the comparatively greater distribution of TiO2 on ABS which promoted good UV absorption; however this result did not agree with the findings of Pazokifard et al. [31], who reported that in a case of degradation of rhodamine, TiO2 P25 nanoparticles showed a better activity than silane-treated particles due to the reduced surface area of TiO2 affecting poor photon absorption. The differences between the photocatalytic activity findings for bacteria and rhodamine likely originate from the operating conditions when blending the composite and the particle itself. The inset of Figure 9 shows that the yield strength of silane-TiO2/ABS was 1.6 times higher than TiO2/ABS without silane. The yield strength improvement was ascribed to better dispersion and adhesion of TiO2 in the ABS matrix, which arose from the silane coupling agent, enhancing interfacial bonding between the TiO2 and the matrix. We could highlight one key finding here, that the incorporation of silane results in improvement of TiO2 particls dispersion within the ABS polymeric matrix (shown as higher photocatalytic activity) and increasing possible interactions between TiO2 particles and the matrix (shown as higher yield strength).

Reusability and Robustness
The stability and reusability in terms of photocatalytic activity and the robustness of the composite material are crucial for practical applications. Therefore, the antibacterial experiments were repeated without any treatment on the specimen between the cycle runs. Constant photocatalytic efficiency was observed after five reuse cycles without loss in the yield strength of material, as shown in Figure 10. This suggests that blending calcined TiO2 into ABS prevented the loss of TiO2 photocatalyst particles from the surface, which is usually observed when coating TiO2 on the polymer surface. As expected, Ti content in the ABS plastic after the fifth run was identical to the as-prepared material (as determined by inductively coupled plasma atomic emission spectroscopy (ICP-OES). This robust ABS material could minimize detachment and release of TiO2 leading to lessening of the possibly negative environmental fates, transport, transformation and toxicity. To investigate the effect of silane on photocatalytic activity, the ratio of TiO 2 to silane during catalyst preparation was varied over the weight ratio of 1:0.2-1:0.5. The optimum photocatalytic activity occurred for samples at a ratio of TiO 2 to silane of 1:0.3 as presented in Figure 9. Figure 9 reveals that 500 • C-calcined TiO 2 /ABS with silane (called "silane-TiO 2 /ABS" for brevity) displays a better photobacterial activity compared with 500 • C-calcined TiO 2 /ABS without silane. The silane-TiO 2 /ABS could reduce 75% of E. coli (remaining bacteria survival 25 ± 2.6%). The higher silane-TiO 2 /ABS photobacterial activity was attributed to the comparatively greater distribution of TiO 2 on ABS which promoted good UV absorption; however this result did not agree with the findings of Pazokifard et al. [31], who reported that in a case of degradation of rhodamine, TiO 2 P25 nanoparticles showed a better activity than silane-treated particles due to the reduced surface area of TiO 2 affecting poor photon absorption. The differences between the photocatalytic activity findings for bacteria and rhodamine likely originate from the operating conditions when blending the composite and the particle itself. The inset of Figure 9 shows that the yield strength of silane-TiO 2 /ABS was 1.6 times higher than TiO 2 /ABS without silane. The yield strength improvement was ascribed to better dispersion and adhesion of TiO 2 in the ABS matrix, which arose from the silane coupling agent, enhancing interfacial bonding between the TiO 2 and the matrix. We could highlight one key finding here, that the incorporation of silane results in improvement of TiO 2 particls dispersion within the ABS polymeric matrix (shown as higher photocatalytic activity) and increasing possible interactions between TiO 2 particles and the matrix (shown as higher yield strength).

Reusability and Robustness
The stability and reusability in terms of photocatalytic activity and the robustness of the composite material are crucial for practical applications. Therefore, the antibacterial experiments were repeated without any treatment on the specimen between the cycle runs. Constant photocatalytic efficiency was observed after five reuse cycles without loss in the yield strength of material, as shown in Figure 10. This suggests that blending calcined TiO 2 into ABS prevented the loss of TiO 2 photocatalyst particles from the surface, which is usually observed when coating TiO 2 on the polymer surface. As expected, Ti content in the ABS plastic after the fifth run was identical to the as-prepared material (as determined by inductively coupled plasma atomic emission spectroscopy (ICP-OES). This robust ABS material could minimize detachment and release of TiO 2 leading to lessening of the possibly negative environmental fates, transport, transformation and toxicity. Polymers 2020, 12, x FOR PEER REVIEW 10 of 12

Conclusions
Titanium dioxide (TiO 2 ) was found to influence antibacterial performance and yield strength enhancement when blended with acrylonitrile-butadiene-styrene plastics (ABS). The optimum photoantibacterial activity occurred for ABS in the 500 • C-calcined TiO 2 at a concentration of 1 wt %. At this temperature and concentration, the high degree of crystallinity and optimal amount of TiO 2 , were sufficient to produce OH radicals and reactive oxygen species (ROS), resulting in damage to bacterial cell membranes. The photoantibacterial performance for 500 • C calcined TiO 2 at 1 wt % in ABS was more efficient than plain ABS over 62%. With optimal conditions, silane addition could further improve TiO 2 dispersion on ABS. This resulted in a decrease of bacterial survival by 75%. Moreover, the benefit of TiO 2 -embedded ABS plastic could improve yield strength than pure ABS. The yield strength of TiO 2 /ABS with silane was 67.7% higher than of pure ABS. The efficiency of TiO 2 /ABS with silane photocatalyst showed an excellent photocatalytic antibacterial stability after five reuses, without loss in the yield strength.