Insights into the Photocatalytic Bacterial Inactivation by Flower-Like Bi 2 WO 6 under Solar or Visible Light, Through in Situ Monitoring and Determination of Reactive Oxygen Species (ROS)

: This study addresses the visible light-induced bacterial inactivation kinetics over a Bi 2 WO 6 synthesized catalyst. The systematic investigation was undertaken with Bi 2 WO 6 prepared by the complexation of Bi with acetic acid (carboxylate) leading to a ﬂower-like morphology. The characterization of the as-prepared Bi 2 WO 6 was carried out by X-ray di ﬀ raction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), speciﬁc surface area (SSA), and photoluminescence (PL). Under low intensity solar light ( < 48 mW / cm 2 ), complete bacterial inactivation was achieved within two hours in the presence of the ﬂower-like Bi 2 WO 6 , while under visible light, the synthesized catalyst performed better than commercial TiO 2 . The in situ interfacial charge transfer and local pH changes between Bi 2 WO 6 and bacteria were monitored during the bacterial inactivation. Furthermore, the reactive oxygen species (ROS) were identiﬁed during Escherichia coli inactivation mediated by appropriate scavengers. The ROS tests alongside the morphological characteristics allowed the proposition of the mechanism for bacterial inactivation. Finally, recycling of the catalyst conﬁrmed the stable nature of the catalyst presented in this study.


Introduction
Over the last few decades, environmental contamination has shifted from the exclusive focus of organic and inorganic pollutants [1], towards the inclusion of bacteria and other organisms [2][3][4]. Therefore, well-organized methods are urgently required to control the spread [5] or eradicate microorganism-related issues [6]. In recent times, beside the traditional bacterial inactivation methods such as UV disinfection and chlorination, a green, efficient, and cost-effective semiconductor photocatalysis has appeared to be a more promising technique [7,8]. TiO 2 has been extensively reported as an effective bactericidal semiconductor photocatalyst due to its high stability, strong redox potential, low cost, and non-toxic nature, but its band-gap of 3.2 eV allows light absorption up to 387 nm which makes up just over 4% of the total solar spectrum [9][10][11]. Water 2020, 12, x FOR PEER REVIEW 3 of 19 Scheme 1. Schematic illustration of the preparation of flower-like Bi2WO6 by hydrothermal method.

Physical Characterization of the Bi2WO6 Flakes
The crystallinity and phase identification of the as-prepared samples were determined by powder X-ray diffraction (XRD) using an X'Pert MPD PRO (Panalytical) analyzer, equipped with a ceramic tube (Cu anode, λ = 1.54060 Å), and with a continuous scanning rate in the range of 5° < 2θ < 80°. The results were studied with Rietvield refinement by the FullProf program. The morphology developments of the samples were characterized using scanning electron microscopy (SEM, FEI Quanta 200). Before SEM imaging, the samples were coated with a thin layer of gold. The specific surface area and porosity size were obtained using Brunauer-Emmett-Teller (BET) analysis, performed with a BELSORP-mini II analyzer, Japan. The photoluminescence (PL) measurement was carried out using a fluorescence spectrophotometer (Perkin Elmer LS55) equipped with a xenon lamp at an excitation wavelength of λ = 340 nm. The surface atomic percentage of the element in the assynthesized sample was analyzed using an AXIS NOVA photoelectron spectrometer with a monochromatic Al Ka X-ray (hν = 1486.6 eV) source (Kratos Analytical, Manchester, UK). The interfacial in situ voltage and pH variation during the bacterial inactivation was monitored in a pH/mV/Temp meter (Jenco 6230N) equipped with a microprocessor and a RS-232-C IBM interface for data recording.

Photocatalytic Antibacterial Activity on Bi2WO6 and Light Sources
The bacterial strain used was a wild type E. coli K12, supplied by the German Collection of Microorganisms and Cell Cultures, DSMZ (No. 498). The master plate and stock solution were prepared according to previous research reported by our laboratory [24,25]. The bacterial concentration of the samples was measured in Colony Forming Units (CFU/mL) and was determined by plating on a non-selective cultivation media, namely, Plate Count Agar (PCA). A total of 1 mL of Scheme 1. Schematic illustration of the preparation of flower-like Bi 2 WO 6 by hydrothermal method.

Physical Characterization of the Bi 2 WO 6 Flakes
The crystallinity and phase identification of the as-prepared samples were determined by powder X-ray diffraction (XRD) using an X'Pert MPD PRO (Panalytical) analyzer, equipped with a ceramic tube (Cu anode, λ = 1.54060 Å), and with a continuous scanning rate in the range of 5 • < 2θ < 80 • . The results were studied with Rietvield refinement by the FullProf program. The morphology developments of the samples were characterized using scanning electron microscopy (SEM, FEI Quanta 200). Before SEM imaging, the samples were coated with a thin layer of gold. The specific surface area and porosity size were obtained using Brunauer-Emmett-Teller (BET) analysis, performed with a BELSORP-mini II analyzer, Japan. The photoluminescence (PL) measurement was carried out using a fluorescence spectrophotometer (Perkin Elmer LS55) equipped with a xenon lamp at an excitation wavelength of λ = 340 nm. The surface atomic percentage of the element in the as-synthesized sample was analyzed using an AXIS NOVA photoelectron spectrometer with a mono-chromatic Al Ka X-ray (hν = 1486.6 eV) source (Kratos Analytical, Manchester, UK). The interfacial in situ voltage and pH variation during the bacterial inactivation was monitored in a pH/mV/Temp meter (Jenco 6230N) equipped with a microprocessor and a RS-232-C IBM interface for data recording.

Photocatalytic Antibacterial Activity on Bi 2 WO 6 and Light Sources
The bacterial strain used was a wild type E. coli K12, supplied by the German Collection of Microorganisms and Cell Cultures, DSMZ (No. 498). The master plate and stock solution were prepared according to previous research reported by our laboratory [24,25]. The bacterial concentration of the samples was measured in Colony Forming Units (CFU/mL) and was determined by plating on a non-selective cultivation media, namely, Plate Count Agar (PCA). A total of 1 mL of the sample was withdrawn after each interval and then serial dilutions were made in a sterile 0.8% NaCl/KCl solution. A 100 µL aliquot was pipetted onto a nutrient agar plate and processed using the standard plate count method. The plates were incubated at 37 • C followed by the bacterial evaluation. Experimental results were carried in triplicate runs applying statistical analysis for the calculation of mean and standard deviation (reported in the graphs). Samples were irradiated in the cavity of a SUNTEST solar simulator CPS (Atlas GmbH, Hanau, Germany) with an overall light irradiance of 48 mW/cm 2 (~0.8 × 10 16 photons/s, Supplementary Figure S1). A cut-off filter was used in the SUNTEST cavity to filter the light <310 nm. A second cut-off filter was also used during bacterial inactivation under visible light with a cut-off blocking the wavelength < 405 nm rendering (Supplementary Figure S2). Finally, after the two filters, the visible light irradiance reaching the sample was 38 mW/cm 2 .

Results
3.1. Synthesis and Characterization of Bi 2 WO 6 : X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), X-Ray Photoelectron Spectroscopy (XPS), and SSA Determination Figure 1 depicts the XRD patterns of the as-synthesized Bi 2 WO 6 via the hydrothermal method at different reaction times and temperatures. All of the XRD patterns illustrated that characteristic peaks were in good agreement with the orthorhombic phased Bi 2 WO 6 in the standard JCPDS card (39-0256) [26]. No other diffraction peaks arising from possible impurities were detected. With the holding time increasing to 24 h, the characteristic peaks became much sharper due to an increase in crystallinity. Understandably, the increment of the temperature with the constant reaction time for 24 h resulted in the same trend because of grain growth. Table 2 illustrates the crystallite size of the samples (using the Scherrer formula based on the half-width of their (113) peak) calculated by the Rietveld method using the FullProf program.
Water 2020, 12, x FOR PEER REVIEW  4 of 19 the sample was withdrawn after each interval and then serial dilutions were made in a sterile 0.8% NaCl/KCl solution. A 100 μL aliquot was pipetted onto a nutrient agar plate and processed using the standard plate count method. The plates were incubated at 37 °C followed by the bacterial evaluation. Experimental results were carried in triplicate runs applying statistical analysis for the calculation of mean and standard deviation (reported in the graphs). Samples were irradiated in the cavity of a SUNTEST solar simulator CPS (Atlas GmbH, Hanau, Germany) with an overall light irradiance of 48 mW/cm 2 (~0.8 × 10 16 photons/s, Supplementary Figure S1). A cut-off filter was used in the SUNTEST cavity to filter the light <310 nm. A second cut-off filter was also used during bacterial inactivation under visible light with a cut-off blocking the wavelength < 405 nm rendering (Supplementary Figure  S2). Finally, after the two filters, the visible light irradiance reaching the sample was 38 mW/cm 2 . Figure 1 depicts the XRD patterns of the as-synthesized Bi2WO6 via the hydrothermal method at different reaction times and temperatures. All of the XRD patterns illustrated that characteristic peaks were in good agreement with the orthorhombic phased Bi2WO6 in the standard JCPDS card (39-0256) [26]. No other diffraction peaks arising from possible impurities were detected. With the holding time increasing to 24 h, the characteristic peaks became much sharper due to an increase in crystallinity. Understandably, the increment of the temperature with the constant reaction time for 24 h resulted in the same trend because of grain growth. Table 2 illustrates the crystallite size of the samples (using the Scherrer formula based on the half-width of their (113) peak) calculated by the Rietveld method using the FullProf program.      The scanning electron microscopy (SEM) images of the Bi 2 WO 6 samples prepared under different experimental conditions are shown in Figure 2. Heating at 160 • C for 12 h and 18 h (Figure 2a,b) led to aggregated irregular small Bi 2 WO 6 nanoparticles and flower-like microspheres. However, when the heating time was prolonged to 24 h (see Figure 2c), organized hierarchical flower-like Bi 2 WO 6 microspheres composed of nanoplates were obtained and the aggregated nanoparticles totally disappeared (Figure 2e,f). The SEM images of as-prepared Bi 2 WO 6 nanoparticles are also shown in Figure 2f. The joint effect of nanoparticles assembly followed by the localized ripening mechanism as well as the hierarchical assembly of nanoplates have been also previously reported for the formation mechanism of flower-like microspheres [27,28]. Owing to the absence of discrete nanoplates according to the SEM images at different reaction times (Figure 2), the former mechanism seems to predominate. Scheme 2 illustrates the proposed formation mechanism of flower-like Bi 2 WO 6 microspheres. Nanoparticles initially aggregated, then the self-assembled nanoparticles preferentially grew along <010>. Longer reaction times and higher temperatures result in dissolution of some nanoplates leading concomitantly to re-deposition by Ostwald ripening [27,28].

Synthesis and Characterization of Bi2WO6: X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), X-Ray Photoelectron Spectroscopy (XPS), and SSA Determination
The relevant reactions leading to the Bi 2 WO 6 synthesis in aqueous solutions when working in acetic acid media can be suggested as follows: Water 2020, 12, x FOR PEER REVIEW 6 of 19 Scheme 2 illustrates the proposed formation mechanism of flower-like Bi2WO6 microspheres. Nanoparticles initially aggregated, then the self-assembled nanoparticles preferentially grew along <010>. Longer reaction times and higher temperatures result in dissolution of some nanoplates leading concomitantly to re-deposition by Ostwald ripening [27,28]. The relevant reactions leading to the Bi2WO6 synthesis in aqueous solutions when working in acetic acid media can be suggested as follows: 2 ?
The initial complex between Bi and acetic acid presents a stability constant of 10 2.6-2.7 [29], which is not in the range found for insoluble complexes/precipitates >10 [11][12] [30][31][32]. This coordination complex is suggested in Equation (4) (Bi = M). The complex formation which is the precursor of Bi2WO6 does not lead to precipitate formation and gradually decomposes releasing Bi 3+ which reacts with WO4 2− . Therefore, the nanoplate formation leads to aggregates which present inner pores/voids and provide the required contact area for the photocatalytic bacterial inactivation.
In addition to the crystal structure and morphology, the surface chemical composition of the assynthesized flower-like sample at 200 °C for 24 h was examined by XPS. As shown in the survey XPS spectrum in Figure 3, the Bi, O, W, and C elements were present in the pure Bi2WO6. The C element peak can be attributed to adventitious carbon from the sample preparation and/or the XPS instrument (4) Water 2020, 12, x FOR PEER REVIEW 6 of 19 Scheme 2 illustrates the proposed formation mechanism of flower-like Bi2WO6 microspheres. Nanoparticles initially aggregated, then the self-assembled nanoparticles preferentially grew along <010>. Longer reaction times and higher temperatures result in dissolution of some nanoplates leading concomitantly to re-deposition by Ostwald ripening [27,28]. Scheme 2. Schematic illustration of the growth process of the flower-like Bi2WO6 microspheres.
The relevant reactions leading to the Bi2WO6 synthesis in aqueous solutions when working in acetic acid media can be suggested as follows: 2 ?
The initial complex between Bi and acetic acid presents a stability constant of 10 2.6-2.7 [29], which is not in the range found for insoluble complexes/precipitates >10 [11][12] [30][31][32]. This coordination complex is suggested in Equation (4) (Bi = M). The complex formation which is the precursor of Bi2WO6 does not lead to precipitate formation and gradually decomposes releasing Bi 3+ which reacts with WO4 2− . Therefore, the nanoplate formation leads to aggregates which present inner pores/voids and provide the required contact area for the photocatalytic bacterial inactivation.
In addition to the crystal structure and morphology, the surface chemical composition of the assynthesized flower-like sample at 200 °C for 24 h was examined by XPS. As shown in the survey XPS spectrum in Figure 3, the Bi, O, W, and C elements were present in the pure Bi2WO6. The C element peak can be attributed to adventitious carbon from the sample preparation and/or the XPS instrument (5) Bi2WO6 microspheres composed of nanoplates were obtained and the aggregated nanoparticles totally disappeared (Figure 2e,f). The SEM images of as-prepared Bi2WO6 nanoparticles are also shown in Figure 2f. The joint effect of nanoparticles assembly followed by the localized ripening mechanism as well as the hierarchical assembly of nanoplates have been also previously reported for the formation mechanism of flower-like microspheres [27,28]. Owing to the absence of discrete nanoplates according to the SEM images at different reaction times (Figure 2), the former mechanism seems to predominate.  The initial complex between Bi and acetic acid presents a stability constant of 10 2.6-2.7 [29], which is not in the range found for insoluble complexes/precipitates >10 11-12 [30][31][32]. This coordination complex is suggested in Equation (4) (Bi = M). The complex formation which is the precursor of Bi 2 WO 6 does not lead to precipitate formation and gradually decomposes releasing Bi 3+ which reacts with WO 4 2− .
Therefore, the nanoplate formation leads to aggregates which present inner pores/voids and provide the required contact area for the photocatalytic bacterial inactivation. In addition to the crystal structure and morphology, the surface chemical composition of the as-synthesized flower-like sample at 200 • C for 24 h was examined by XPS. As shown in the survey XPS spectrum in Figure 3, the Bi, O, W, and C elements were present in the pure Bi 2 WO 6 . The C element peak can be attributed to adventitious carbon from the sample preparation and/or the XPS instrument itself [33]. The surface atomic concentration ratio of Bi:W:O estimating from XPS peak areas is around 2.0:0.8:5.4, which further confirms its composition of Bi 2 WO 6 . Furthermore, the peaks centering at 164.7 and 159.4 eV are attributed to the binding energies of Bi 4f 5/2 and Bi 4f 7/2 , respectively (inset  Figure 3), confirming Bi 3+ ions in the crystalline structure [34][35][36]. The W4f energy region can be designated to be the +6 oxidation state of tungsten in accordance with previous reports [33,36].
Water 2020, 12, x FOR PEER REVIEW 6 of 19 Scheme 2 illustrates the proposed formation mechanism of flower-like Bi2WO6 microspheres. Nanoparticles initially aggregated, then the self-assembled nanoparticles preferentially grew along <010>. Longer reaction times and higher temperatures result in dissolution of some nanoplates leading concomitantly to re-deposition by Ostwald ripening [27,28]. The relevant reactions leading to the Bi2WO6 synthesis in aqueous solutions when working in acetic acid media can be suggested as follows: 2 ?
The initial complex between Bi and acetic acid presents a stability constant of 10 2.6-2.7 [29], which is not in the range found for insoluble complexes/precipitates >10 [11][12] [30][31][32]. This coordination complex is suggested in Equation (4) (Bi = M). The complex formation which is the precursor of Bi2WO6 does not lead to precipitate formation and gradually decomposes releasing Bi 3+ which reacts with WO4 2− . Therefore, the nanoplate formation leads to aggregates which present inner pores/voids and provide the required contact area for the photocatalytic bacterial inactivation.
In addition to the crystal structure and morphology, the surface chemical composition of the assynthesized flower-like sample at 200 °C for 24 h was examined by XPS. As shown in the survey XPS spectrum in Figure 3, the Bi, O, W, and C elements were present in the pure Bi2WO6. The C element peak can be attributed to adventitious carbon from the sample preparation and/or the XPS instrument Scheme 2. Schematic illustration of the growth process of the flower-like Bi 2 WO 6 microspheres.  Figure 3), confirming Bi 3+ ions in the crystalline structure [34][35][36]. The W4f energy region can be designated to be the +6 oxidation state of tungsten in accordance with previous reports [33,36]. The N2 adsorption-desorption isotherms of the well-organized flower-like (BWO5) and nanoparticles (BWO6) Bi2WO6 are presented in Figure 4. According to IUPAC classification, it can be seen that the isotherm shape for both samples exhibited a typical type IV isotherm with a clear hysteresis loop H3, suggesting the presence of mesopores in the size range of 2-50 nm [37].The insets show the Barrett-Joyner-Halenda (BJH) pore-size distributions and present the evidence for the existence of mesopores (2-50 nm). Table 3 summarizes the BET specific surface areas (SSA) and the pore volumes of BWO5 and BWO6.  The N 2 adsorption-desorption isotherms of the well-organized flower-like (BWO5) and nanoparticles (BWO6) Bi 2 WO 6 are presented in Figure 4. According to IUPAC classification, it can be seen that the isotherm shape for both samples exhibited a typical type IV isotherm with a clear hysteresis loop H3, suggesting the presence of mesopores in the size range of 2-50 nm [37].The insets show the Barrett-Joyner-Halenda (BJH) pore-size distributions and present the evidence for the existence of mesopores (2-50 nm). Table 3 summarizes the BET specific surface areas (SSA) and the pore volumes of BWO5 and BWO6.

E. Coli Inactivation Kinetics: Effect of the Bacterial Concentration, Amount of Catalyst, Light Dose, and
Applied Light Wavelength Figure 5 shows the complete bacterial inactivation mediated by the BWO5 being faster under low-intensity simulated solar light, compared to the other samples. The E. coli inactivation was 95% after 2 h. The effectiveness of a disinfection process resides in the time necessary to inactivate a determined percentage of bacteria. In the Chick-Watson model [38,39], the simplest inactivation model, the inactivation rate shown in Figure 5 is seen to be dependent on the residual bacteria after each specific time during the inactivation process and this allows comparing the effect of the different Bi2WO6 samples. Neither irradiation in the absence of Bi2WO6 (photolysis) nor runs in the presence of this catalyst in the dark lead to bacterial inactivation of up to 4 h. The latter provides the proof that Bi2WO6 is not toxic to E. coli and a photocatalytic process is required for their inactivation. As the treatment time increased, the photocatalytic process became more effective, owing to the formation of hierarchical flower-like Bi2WO6 microspheres and loss of aggregates and the higher crystallite size. Nevertheless, the nanoparticles (BWO6), which presented lower specific surface area than BWO5, led to lower inactivation rates. The pseudo first-order rates of the Bi2WO6 samples during flower-like development (BWO1 and BWO5) compared with Bi2WO6 nanoparticles (BWO6) are given in the supplementary material, Figure S3. The pseudo first-order rate constants ( ) of the BWO1, BWO5, and BWO6 were estimated to be 0.0331 min −1 , 0.0488 min −1 , and 0.0195 min −1 , respectively. As can be   Figure 5 shows the complete bacterial inactivation mediated by the BWO5 being faster under low-intensity simulated solar light, compared to the other samples. The E. coli inactivation was 95% after 2 h. The effectiveness of a disinfection process resides in the time necessary to inactivate a determined percentage of bacteria. In the Chick-Watson model [38,39], the simplest inactivation model, the inactivation rate shown in Figure 5 is seen to be dependent on the residual bacteria after each specific time during the inactivation process and this allows comparing the effect of the different Bi 2 WO 6 samples. Neither irradiation in the absence of Bi 2 WO 6 (photolysis) nor runs in the presence of this catalyst in the dark lead to bacterial inactivation of up to 4 h. The latter provides the proof that Bi 2 WO 6 is not toxic to E. coli and a photocatalytic process is required for their inactivation. As the treatment time increased, the photocatalytic process became more effective, owing to the formation of hierarchical flower-like Bi 2 WO 6 microspheres and loss of aggregates and the higher crystallite size. Nevertheless, the nanoparticles (BWO6), which presented lower specific surface area than BWO5, led to lower inactivation rates. The pseudo first-order rates of the Bi 2 WO 6 samples during flower-like development (BWO1 and BWO5) compared with Bi 2 WO 6 nanoparticles (BWO6) are given in the supplementary material, Figure S3. The pseudo first-order rate constants (k app ) of the BWO1, BWO5, and BWO6 were estimated to be 0.0331 min −1 , 0.0488 min −1 , and 0.0195 min −1 , respectively. As can be seen, the photocatalytic inactivation of bacteria mediated by as-developed flower-like Bi 2 WO 6 (BWO5) is around 2.5 times faster compared with nanoparticles. The photoluminescence spectrum of the prepared catalysts was used as a practical method to verify the separation efficiency of photo-generated electron-hole pairs in the semiconductors. Generally, a lower photoluminescence (PL) intensity represents a lower recombination rate of photogenerated charge carriers. The photoluminescence (PL) spectra of the Bi2WO6 samples during the flower-like development (BWO1 and BWO5) in comparison with Bi2WO6 nanoparticles (BWO6) is shown in Figure 6. The wide absorption-band was observed between 350 nm and 600 nm which is due to the Bi2WO6 electron-hole recombination giving rise to the free and bound-exciton luminescence [40]. The PL spectra of the as-synthesized samples through flower-like development (BWO1 and BWO5) exhibited significantly decreased PL intensity related to that of the Bi2WO6 nanoparticles. It could be ascribed that the recombination of photo-generated charge carriers is greatly inhibited in the hierarchically flower-like composed of nanosheets. Hence, the efficient separation of photo-generated electron-hole pairs and rapid transfer of electrons to the surface of crystal would be obtained. Moreover, the lower PL-intensity bands shown in BWO5 reflected a higher crystallinity in comparison with BWO1, allowing a lower amount of crystal defects, leading to a higher electron-hole separation and an increased photocatalytic activity [41], a fact that corroborates with the faster inactivation of bacteria ( Figure S3). The photoluminescence spectrum of the prepared catalysts was used as a practical method to verify the separation efficiency of photo-generated electron-hole pairs in the semiconductors. Generally, a lower photoluminescence (PL) intensity represents a lower recombination rate of photo-generated charge carriers. The photoluminescence (PL) spectra of the Bi 2 WO 6 samples during the flower-like development (BWO1 and BWO5) in comparison with Bi 2 WO 6 nanoparticles (BWO6) is shown in Figure 6. The wide absorption-band was observed between 350 nm and 600 nm which is due to the Bi 2 WO 6 electron-hole recombination giving rise to the free and bound-exciton luminescence [40]. The PL spectra of the as-synthesized samples through flower-like development (BWO1 and BWO5) exhibited significantly decreased PL intensity related to that of the Bi 2 WO 6 nanoparticles. It could be ascribed that the recombination of photo-generated charge carriers is greatly inhibited in the hierarchically flower-like composed of nanosheets. Hence, the efficient separation of photo-generated electron-hole pairs and rapid transfer of electrons to the surface of crystal would be obtained. Moreover, the lower PL-intensity bands shown in BWO5 reflected a higher crystallinity in comparison with BWO1, allowing a lower amount of crystal defects, leading to a higher electron-hole separation and an increased photocatalytic activity [41], a fact that corroborates with the faster inactivation of bacteria ( Figure S3). Following, the effects of initial catalyst or bacterial concentration were studied, and the results are summarized in Figure 7. The effect of the Bi2WO6 concentration on E. coli inactivation is shown in Figure 7a. Although increasing Bi2WO6 concentration of up to 0.2 mg/mL resulted in higher inactivation rates, increasing the catalyst concentration to 0.4 mg/mL resulted in a slower bacterial inactivation kinetics, most possibly due to a loss in surface area by catalyst agglomeration (particleparticle interactions), as well as a decrease in the penetration of the photon flux by the solution opacity, thereby decreasing the photocatalytic inactivation rate [42]. The effect of the initial concentration on the E. coli kinetics mediated by Bi2WO6 catalysts is presented in Figure 7b, showing a delay in the time necessary for bacterial inactivation at higher bacterial concentrations. Although this effect can be ascribed to the exhaustion of surface active sites due to opacity in solution [43], we note here that in absolute numbers, the higher the amount of bacteria in solution, the higher the number of available bacteria (for inactivation). Hence, by calculating the amount of cells inactivated in 4 h per mg of catalyst and per minute, we get 2075, 208, and 21 cells min −1 mg −1 for 10 8 , 10 7 , and 10 6 , respectively. As a result, we report that this catalyst can effectively disinfect higher amounts of microorganisms, albeit in a higher residence time. Following, the effects of initial catalyst or bacterial concentration were studied, and the results are summarized in Figure 7. The effect of the Bi 2 WO 6 concentration on E. coli inactivation is shown in Figure 7a. Although increasing Bi 2 WO 6 concentration of up to 0.2 mg/mL resulted in higher inactivation rates, increasing the catalyst concentration to 0.4 mg/mL resulted in a slower bacterial inactivation kinetics, most possibly due to a loss in surface area by catalyst agglomeration (particle-particle interactions), as well as a decrease in the penetration of the photon flux by the solution opacity, thereby decreasing the photocatalytic inactivation rate [42]. The effect of the initial concentration on the E. coli kinetics mediated by Bi 2 WO 6 catalysts is presented in Figure 7b, showing a delay in the time necessary for bacterial inactivation at higher bacterial concentrations. Although this effect can be ascribed to the exhaustion of surface active sites due to opacity in solution [43], we note here that in absolute numbers, the higher the amount of bacteria in solution, the higher the number of available bacteria (for inactivation). Hence, by calculating the amount of cells inactivated in 4 h per mg of catalyst and per minute, we get 2075, 208, and 21 cells min −1 mg −1 for 10 8 , 10 7 , and 10 6 , respectively. As a result, we report that this catalyst can effectively disinfect higher amounts of microorganisms, albeit in a higher residence time. Next up in the operational parameters investigation, we assessed the possibility of photonic limitation or saturation of the system. As such, Figure 8 a,b shows the effects of the light intensity and composition (UVA-vis or Vis only) on the bacterial degradation kinetics. A higher light dose accelerated the bacterial inactivation because of a higher amount of charges generated in the semiconductor during bacterial disinfection under band-gap irradiation (Figure 8a), since the direct inactivation by light was previously excluded. Figure 8 b illustrates that under visible light, a solution containing 0.2 g/L of Bi2WO6 was still efficiently inactivating bacteria and was more effective compared to commercial TiO2 P25 Degussa (used as reference). These results come from the optical absorption of up to ~450 nm in the visible region by Bi2WO6, which is significantly wider than that of Next up in the operational parameters investigation, we assessed the possibility of photonic limitation or saturation of the system. As such, Figure 8a,b shows the effects of the light intensity and composition (UVA-vis or Vis only) on the bacterial degradation kinetics. A higher light dose accelerated the bacterial inactivation because of a higher amount of charges generated in the semiconductor during bacterial disinfection under band-gap irradiation (Figure 8a), since the direct inactivation by light was previously excluded. Figure 8 b illustrates that under visible light, a solution containing 0.2 g/L of Bi 2 WO 6 was still efficiently inactivating bacteria and was more effective compared to commercial TiO 2 P25 Degussa (used as reference). These results come from the optical absorption of up to~450 nm in the visible region by Bi 2 WO 6 , which is significantly wider than that of TiO 2 P25 Degussa with an absorption of up to 387 nm for the 20 nm particles, making up the bulk of this mixed TiO 2 P25 Degussa rutile-anatase [44].
Water 2020, 12, x FOR PEER REVIEW 12 of 19 TiO2 P25 Degussa with an absorption of up to 387 nm for the 20 nm particles, making up the bulk of this mixed TiO2 P25 Degussa rutile-anatase [44].

Mechanistic Interpretation: ROS-Species Involvement, Interfacial Charge Transfer, and Catalyst Reuse During Bacterial Inactivation
The reactive oxygen species (ROS) such as ⦁ OH, O2 ⦁− , and vb (h + ) play a pivotal role in the photodegradation of organic pollutants and bacterial inactivation [22,[45][46][47]. To determine the main ROS followed by the photodegradation mechanism, appropriate radical-scavengers such as isopropanol ( ⦁ OH scavenger), sodium oxalate (a vbh + hole scavenger), and superoxide dismutase (O2 ⦁− scavenger) were used in the present study. Figure 9 depicts the results of scavenging experiments mediated by the optimized flower-like Bi2WO6 (BWO5). The photocatalytic bacterial inactivation could be remarkably suppressed by the addition of isopropanol and sodium oxalate. It is very likely that ⦁ OH

Mechanistic Interpretation: ROS-Species Involvement, Interfacial Charge Transfer, and Catalyst Reuse During Bacterial Inactivation
The reactive oxygen species (ROS) such as · OH, O 2 ·− , and vb (h + ) play a pivotal role in the photo-degradation of organic pollutants and bacterial inactivation [22,[45][46][47]. To determine the main ROS followed by the photodegradation mechanism, appropriate radical-scavengers such as isopropanol ( · OH scavenger), sodium oxalate (a vbh + hole scavenger), and superoxide dismutase (O 2 ·− scavenger) were used in the present study. Figure 9 depicts the results of scavenging experiments mediated by the optimized flower-like Bi 2 WO 6 (BWO5). The photocatalytic bacterial inactivation could be remarkably suppressed by the addition of isopropanol and sodium oxalate. It is very likely that · OH and h + intervene jointly in the bacterial inactivation. Meanwhile, the addition of SOD (O 2 ·− scavenger) inhibits the bacterial inactivation to a smaller degree compared to vb(h + ) and the · OH-radical as shown in Figure 9, traces (a) and (b).
Water 2020, 12, x FOR PEER REVIEW 13 of 19 and h + intervene jointly in the bacterial inactivation. Meanwhile, the addition of SOD (O2 ⦁− scavenger) inhibits the bacterial inactivation to a smaller degree compared to vb(h + ) and the ⦁ OH-radical as shown in Figure 9, traces (a) and (b). The possible reaction mechanism for the inactivation of E. coli mediated by Bi2WO6 can be proposed as the following, which is shown in Scheme 3. Under visible-light irradiation, the photoexcitation of Bi2WO6 implies the transfer of an electron from the valence band (Equation (6)). + ℎ → ( ) + ℎ ( ).  The possible reaction mechanism for the inactivation of E. coli mediated by Bi 2 WO 6 can be proposed as the following, which is shown in Scheme 3. Under visible-light irradiation, the photo-excitation of Bi 2 WO 6 implies the transfer of an electron from the valence band (Equation (6)).
As mentioned before, the valence band of Bi 2 WO 6 is a hybrid band made up by the O2p and Bi6s orbitals. Under light irradiation, the O2p and Bi6s hybrid orbitals increase the charge transfer in the W5d orbitals of Bi 2 WO 6 . This moves the valence band (VB) potential to a more positive potential energy narrowing the band-gap and inducing a higher photocatalytic activity [48].
Based on the references, CB and VB potentials of Bi 2 WO 6 are 3.08 and 0.36 eV, respectively [49,50]. The redox potential for the dissolved oxygen/superoxide couple (E 0 (O 2 / O 2 ·− )), O 2 /HO 2 · , and OH -/ · OH are −0.33 eV, −0.046 eV, and 1.98 eV vs NHE [49], respectively. Comparing the band edge energy level of Bi 2 WO 6 with the redox potentials of ROS, it is obvious that the excited holes in the valence band of Bi 2 WO 6 were sufficiently more positive than that of OH -/ · OH, suggesting that the photogenerated holes on the surface of Bi 2 WO 6 could react with OH -/H 2 O to form "non-selective" · OH radicals (Equation (7)). However, the conduction band edge potential of Bi 2 WO 6 , which is more positive than the standard redox potential of O 2 / O 2 ·− and O 2 /HO 2 · , cannot directly reduce O 2 to O 2 ·− or HO 2 · .
As shown in Figure 9, the bacterial inactivation is reduced in the presence of SOD-scavengers, which confirms the presence of the HO 2 · radicals. Considering the redox potential of O 2 /H 2 O 2 = +0.682 eV vs NHE [51], H 2 O 2 seems to be generated initially (Equation (8)) which is followed by the formation of different species according to the relations 9-10 in the photocatalytic reaction. It is worth noting that the powerful hole can directly attack bacteria cells in the photocatalytic oxidation process, which was also confirmed by the hole scavenger [45,52].
The possible reaction mechanism for the inactivation of E. coli mediated by Bi2WO6 can be proposed as the following, which is shown in Scheme 3. Under visible-light irradiation, the photoexcitation of Bi2WO6 implies the transfer of an electron from the valence band (Equation (6)). + ℎ → ( ) + ℎ ( ).  Figure 10 shows the variation of the interfacial potential and the local pH shift under simulated solar light. At pH~6, the bacterial inactivation preferentially proceeds via the O 2 ·− species over HO 2 −· as shown in Equation (11) and Figure 9, trace (c).
The initial pH at time zero in Figure 10 was observed to decrease slightly from 6.0 to 5.9 within four hours of irradiation. The initial pH of 6.0 in this figure is seen to decrease drastically to 5.4 after 8000 s due to the concomitant production of long-lived intermediates carboxylic acids, owing to the degradation of the bacterial membrane. The interface potential is shown to drastically drop within 8000 s (2.2 h) when the bacterial reduction is reduced by 99.90%, which is equivalent to 3 logs as shown in Figure 5. The interface potential recovers to its initial value as shown in Figure 10 after the inactivation of bacteria [52]. The recovery to the initial pH-level occurs when the intermediate acids are mineralized to CO 2 by the photo-Kolbe reaction according to Equation (12) [53,54]. Finally, we provide the evidence for synthesizing a stable Bi2WO6 flower-like photocatalyst by a repetitive inactivation of a E. coli test, which results are shown in Figure 11. In order to evaluate the bacterial inactivation after each cycle, the pseudo first-order rate constants ( ) were calculated and are reported in Table 4. The recycled sample used in Figure 11 was thoroughly washed after each cycle. Practically, no loss of bacterial inactivation was observed. These results show the stable repetitive bacterial inactivation mediated by flower-like Bi2WO6 up to five cycles and confirm the potential for the practical application of this photocatalyst in E. coli inactivation.   Figure 11. Finally, we provide the evidence for synthesizing a stable Bi 2 WO 6 flower-like photocatalyst by a repetitive inactivation of a E. coli test, which results are shown in Figure 11. In order to evaluate the bacterial inactivation after each cycle, the pseudo first-order rate constants (kapp) were calculated and are reported in Table 4. The recycled sample used in Figure 11 was thoroughly washed after each cycle. Practically, no loss of bacterial inactivation was observed. These results show the stable repetitive bacterial inactivation mediated by flower-like Bi 2 WO 6 up to five cycles and confirm the potential for the practical application of this photocatalyst in E. coli inactivation. Finally, we provide the evidence for synthesizing a stable Bi2WO6 flower-like photocatalyst by a repetitive inactivation of a E. coli test, which results are shown in Figure 11. In order to evaluate the bacterial inactivation after each cycle, the pseudo first-order rate constants ( ) were calculated and are reported in Table 4. The recycled sample used in Figure 11 was thoroughly washed after each cycle. Practically, no loss of bacterial inactivation was observed. These results show the stable repetitive bacterial inactivation mediated by flower-like Bi2WO6 up to five cycles and confirm the potential for the practical application of this photocatalyst in E. coli inactivation.   Figure 11.

Conclusions
In the present study, Bi 2 WO 6 flower-like samples were prepared at 200 • C attaining a high crystallinity and led a low amount of crystal by hydrothermal growth in acetic acid media. By SEM, XRD, XPS, and PL analysis, the properties of the flower-like Bi 2 WO 6 samples and nanoparticles were investigated. These catalysts resulted in effective bacterial inactivation even under visible light and were faster than TiO 2 . In addition to higher SSA of flower-like Bi 2 WO 6 , its lower PL intensity leads to lower recombination of photo-generated electron-hole pairs as a consequence of more efficient photocatalytic activity. The photocatalytic inactivation of bacteria mediated by as-developed flower-like Bi 2 WO 6 (BWO5) is around 2.5 times faster when compared with nanoparticles. The samples under light lead to effective Bi 2 WO 6 charge separation and the generation of ROS inducing bacterial inactivation. The intermediate ROS species produced by Bi 2 WO 6 were identified by the use of the appropriate scavengers, and the · OH-radical was identified to be the dominant inactivation mechanism. Finally, the stable performance of the synthesized catalyst during recycling indicates its robustness and may suggest practical application potential.
Funding: Minoo Karbasi obtained exchange scholarship from the Ministry of Science, Research and Technology of Iran and the Isfahan University of Technology, and its contribution is hereby acknowledged. Stefanos Giannakis would like to acknowledge the Spanish Ministry of Science, Innovation and Universities (MICIU) for the Ramón y Cajal Fellowship (RYC2018-024033-I).

Conflicts of Interest:
The authors declare no conflict of interest.