Visible-light-driven photocatalytic inactivation of MS2

Water Research 106 (2016) 249e258

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Visible-light-driven photocatalytic inactivation of MS2 by metal-free g-C3N4: Virucidal performance and mechanism Yi Li a, *, Chi Zhang a, Danmeng Shuai b, Saraschandra Naraginti a, Dawei Wang a, Wenlong Zhang a a

Key Laboratory of Integrated Regulation and Resource Development of Shallow Lakes, Ministry of Education, College of Environment, Hohai University, Xikang Road #1, Nanjing, 210098, PR China Department of Civil and Environmental Engineering, The George Washington University, 800 22nd St NW Suite 3530, Science and Engineering Hall, Washington, DC 20052, USA

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 July 2016 Received in revised form 1 October 2016 Accepted 3 October 2016 Available online 5 October 2016

The challenge to achieve effective water disinfection of pathogens, especially viruses, with minimized harmful disinfection byproducts calls for a cost-effective and environmentally benign technology. Here, polymeric graphitic carbon nitride (g-C3N4), as a metal-free robust photocatalyst, was explored for the first time for its ability to inactivate viruses under visible light irradiation. MS2 with an initial concentration of 1
108 PFU/mL was completely inactivated by g-C3N4 with a loading of 150 mg/L under visible light irradiation of 360 min. g-C3N4 was a robust photocatalyst, and no decrease in its virucidal performance was observed over five cycles of sequential MS2 photocatalytic inactivation. The reactive oxygen species (ROSs) were measured by a range of scavengers, and photo-generated electrons and its derived ROSs (O- 2) were found to be the leading contributor for viral inactivation. TEM images indicated that the viral particle shape was distorted and the capsid shell was ruptured after photocatalysis. Viral surface proteins, particularly replicase proteins and maturation proteins, were damaged by photocatalytic oxidation. The loss of proteins would result in the leakage and rapid destruction of interior components (four main types of RNA genes), finally leading to viral death without regrowth. Our work opens a new avenue for the exploration and applications of a low-cost, high-efficient, and robust metalfree photocatalyst for green/sustainable viral disinfection. © 2016 Published by Elsevier Ltd.

Keywords: g-C3N4 Viruses Visible light Photocatalysis Inactivation mechanism

1. Introduction Viruses that may pose significant health risks to human have been found to be widely present in drinking water sources (both surface water and groundwater) (Liga et al., 2011; Prevost et al., 2016). Many recent disease outbreaks are associated with viruses, including SARS, Avian Influenza, Ebola, and Zika Fever. Therefore, efficient removal of viruses is urgently needed to guarantee the safety of drinking water. Unfortunately, viruses are generally difficult to be fully inactivated due to their small size and high resistance to harsh environmental conditions. Hijnen et al. (2010) demonstrated that protozoan (oo)cysts were removed significantly and bacteria/the anaerobic spores were removed moderately, while no viruses were removed by granular activated carbon

* Corresponding author. E-mail address: [email protected] (Y. Li). http://dx.doi.org/10.1016/j.watres.2016.10.009 0043-1354/© 2016 Published by Elsevier Ltd.

adsorption filtration. Conventional water treatment uses chemical disinfectants or UV irradiation for viral inactivation. An increased dosage of disinfectants is needed for the inactivation of viruses that are persistent, and it will increase the likelihood of producing more disinfection byproducts (DBPs) (Zheng et al., 2015). UV irradiation also effectively inactivates viruses without DBP production, while the energy consumption and operational cost is higher to prevent viral regrowth (Koivunen and Heinonen-Tanski, 2005). Therefore, there is necessary for the development of a cost-effective, environmentally benign, sustainable disinfection strategy with minimized chemical and energy footprint for virus removal. Photocatalysts activate oxygen in the air or water to produce a series of oxidative species under an ambient condition, and then effectively destruct chemical contaminants as well as inactivate microbial pathogens in water. TiO2 is the most studied semiconductor for photocatalytic inactivation of viruses in aqueous solution (Lee and Ko, 2013; Li et al., 2014; Liga et al., 2013); whereas it only works under UV irradiation that accounts for ~4% of the solar

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spectrum (Zhang et al., 2015). To date, TiO2 has been extensively tailored through molecular doping or surface engineering (such as TiON/PdO, Ag/TiO2, Mn/Co-TiO2) to improve its utilization of solar energy in viral inactivation (Li et al., 2008; Liga et al., 2011; Venieri et al., 2014). Besides, several other visible-light-driven photocatalysts such as Ag-AgI/Al2O3 (Hu et al., 2010) and Pt-WO3 (Takehara et al., 2010) were also investigated for viral inactivation. Nevertheless, the reactivity is still limited under visible light irradiation, the cost of material fabrication is high when noble metal loading is involved, and the incidental leaching of toxic metals in treated water cannot be ignored (Zhu et al., 2013). The ideal photocatalyst is expected to be visible-light-active, chemically stable, highly effective and economically feasible in engineering applications. Thus far, there are only a few metal-free, visible-light-active photocatalysts investigated for viral inactivation in water, including GO-aptamer nanosheets (Hu et al., 2012) and C60 derivatives (Cho et al., 2010; Lee et al., 2010; Moor et al., 2015). Unfortunately, the fabrication of these materials is complicated and high cost, and thus limits their mass production and engineering applications. Recently, graphitic carbon nitride (gC3N4) attracts great attention for environmental applications (Liu et al., 2015) since it was first reported as a photocatalyst for water splitting under visible light irradiation (Wang et al., 2009). The material can be directly synthesized from earth abundant, low cost nitrogen rich precursors, e.g., heating the melamine (Yan et al., 2009). It can absorb visible light up to 460 nm, be biocompatible with negligible toxicity, be resistant to photo-corrosion and air oxidation (up to 600  C), and remain chemically stable in most solvents (e.g. bases and diluted acids) (Cao et al., 2015; Lin et al., 2014). Previous studies have suggested that g-C3N4 is promising for the oxidation of organic contaminants (Li et al., 2016; Yan et al., 2009) and bacterial inactivation (Huang et al., 2014; Li et al., 2015), however, no systematic works have been conducted for viral inactivation on g-C3N4. Viruses are more resistant than bacteria to
mez et al., 2015), and conventional disinfection methods (Ortega-Go the results of bacterial disinfection cannot be translated to viral disinfection. Furthermore, the mechanism of the photocatalytic inactivation of viruses is largely unknown, impelling us to conduct the present study. In this work, we evaluated the photocatalytic inactivation of viruses on g-C3N4 under visible light irradiation for the first time. Bacteriophage MS2, a widely used surrogate for waterborne pathogenic viruses due to their similar size, structure and surface properties (Badireddy et al., 2012; Venieri et al., 2014), was selected as a model virus in the present work. The performance of g-C3N4 was compared with other visible-light-active photocatalysts, i.e., NTiO2, Bi2WO6 and Ag@AgCl, for viral inactivation. Scavengers were used to mechanistically understand the roles of ROSs in photocatalytic viral disinfection. In addition, the response of viruses to photocatalytic disinfection, including the virus morphology, total organic components, surface proteins and ribonucleic acids (RNA) of live and inactivated viruses, was systematically examined to provide insights of viral inactivation mechanisms.

diffuse reflectance spectra (UVeVis DRS) of the sample powders were performed on a Varian Cary 500 UVeVis spectrophotometer equipped with an integrating sphere. The morphologies of the samples were recorded on a Keyence VE-8800 scanning electron microscope with an accelerating voltage of 5 kV and a Tecnai G2 spirit Biotwin transmission electron microscope with an accelerating voltage of 120 kV.

2. Experimental

To investigate the roles of ROSs generated by g-C3N4 during photocatalytic inactivation of viruses, a sub-experiment was conducted in the presence of six scavengers. And their applied concentrations were determined empirically to ensure the maximum scavenging effect without viral inactivation (Fig. S2). Then Cr(VI) (0.05 mmol/L), Fe(II)-EDTA (0.1 mmol/L), TEMPOL (1 mmol/L), Lhistidine (0.5 mmol/L), isopropanol (0.5 mmol/L) and sodium oxalate (0.5 mmol/L) were used as scavengers for electrons (e), hydrogen peroxides (H2O2), superoxide radicals (O- 2), singlet oxygen (1O2), hydroxyl radicals (OH) and holes (hþ), respectively. The scavenger experiments were similar to the photocatalytic

2.1. g-C3N4 preparation and characterization The g-C3N4 was prepared by directly heating the low-cost melamine, according to the previous report of Yan et al. (2009). Briefly, melamine as the sole source material was put into a semiclosed system to be heated with two-step treatment (shown in Supporting Information). X-ray diffraction patterns for the photocatalysts were obtained on a Rigaku D/max 2500 V X-ray diffractometer with Cu-Ka1 radiation (l ¼ 1.54056 Å). Ultravioletevisible

2.2. Photocatalytic inactivation experiments The details of viral culture and analysis are provided in Supporting Information. The MS2 stock suspension was diluted into 100 mL of PBS in a 250 mL conical flask to obtain the initial virus concentration of 1
108 PFU/mL. After that, 5, 10, 15 or 20 mg of freshly prepared g-C3N4 photocatalysts was added to achieve the desired concentration for viral inactivation. The reaction suspension was kept at a room temperature (~25  C) and stirred at a constant speed with a magnetic stirrer throughout the experiment. The light source was a 300 W Xenon lamp (Philips) with a UV cutoff filter to provide visible light (l  400 nm) with an average illumination intensity of 150 mW/cm2, and its irradiation spectrum is shown in Fig. S1. Before irradiation, the system was left in the dark under continuous stirring for 30 min to establish an adsorption-desorption equilibrium. Viruses were sampled following 0, 60, 120, 180, 240, 300 and 360 min of illumination, and then they were immediately enumerated to avoid further inactivation. A virus suspension without photocatalysts was irradiated under the same conditions as a light control. And a reaction solution containing viruses and 15 mg of photocatalysts without visible light illumination was used as a dark control. The efficiency of photocatalysis was reported as Log (Ct/C0), where C0 and Ct were the concentration of survival viruses before and after inactivation, respectively. All materials used in the experiments contacting viruses were sterilized by autoclaving at 121  C for 30 min. In addition, to compare the photocatalytic performance for viral inactivation with g-C3N4, nitrogen-doped TiO2 (N-TiO2), Bi2WO6 and Ag@AgCl were also prepared using the method described by Cong et al. (2007), Tang et al. (2004) and Wang et al. (2008), respectively. 2.3. Photocatalytic stability experiments Inactivation of MS2 by g-C3N4 was conducted in five consecutive cycles to evaluate the stability of the photocatalyst. The experiments were similar to the photocatalytic activation experiments using 15 mg of g-C3N4 in 100 mL of MS2 solution with an initial concentration of 1
108 PFU/mL. At the end of each run, the g-C3N4 was collected by centrifugation (4000 rpm for 10 min), and then washed twice with distilled water. Finally, the used photocatalysts were dried in the vacuum drying oven at 60  C before another run. 2.4. ROS analysis

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inactivation experiments. Nitroblue tetrazolium (NBT) (0.1 mmol/L) was used to identify the generation of O- 2 produced from the g-C3N4 samples. The yield of O- 2 was quantitatively analyzed by monitoring the decrease in the concentration of NBT on a Hach DR-6000 UVeVis spectrophotometer. Terephthalic acid (TA) (3 mmol/L in a 10 mmol/ L NaOH solution), which can react with OH to produce a highly fluorescent product 2-hydroxyterephthalic acid (2-HTA), was used as a probe molecule to detect the level of OH on a Hitachi F-7000 fluorescence spectrophotometer. The concentration of H2O2 was determined by redox titration with potassium permanganate (KMnO4). Furfuryl alcohol (FFA) (0.1 mmol/L), a well-known 1O2 probe molecule, was used to measure the amount of 1O2 on an Agilent 1260 high-performance liquid chromatography system equipped with a reverse-phase C18 column.

2.5. Inactivation mechanism exploration 2.5.1. Virus morphology The bacteriophage MS2 is an icosahedral and single-stranded RNA phage, of which the diameter is ~26 nm and the isoelectric point is 3.9 (Thompson et al., 1998). The preparation process for transmission electron microscopy (TEM) samples was similar to the description in our previous report (Wang et al., 2015; Zhang et al., 2013). TEM images of viruses were obtained before and after treatment by g-C3N4 for given time intervals on a JEOL 2011 operated at the voltage of 200 kV.

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2.5.2. Total organic carbon and total protein degradation The total organic carbon (TOC) content of the viral suspension was measured using a Total Organic Carbon Analyzer (TOC-Vcsn, Shimadzu, Japan), and details are shown in Supporting Information. The total protein concentration in the viral suspension was determined using a Bradford assay kit (Bio-Rad). The level of viral protein oxidation was measured using an OxiSelect™ Protein Carbonyl ELISA Kit (Cell Biolabs) by monitoring the concentration of protein carbonyls, following the manufacturer's protocol. 2.5.3. Surface protein analysis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to assess the degree of damage to surface proteins (Wu et al., 2015) caused by g-C3N4 under visible light irradiation. The gel was prepared according to the manufacturer's instruction and 1
Tris-glycine was used for the sample loading buffer. 25 mL of solution containing either untreated or treated MS2 (6 h) or a protein standard with a kDa scale of 20e120 was separately mixed with the prepared gel and then subjected to SDS-PAGE analysis by the electrophoresis equipment. Electrophoresis was conducted at a voltage of 90 V for 60 min, followed by staining with Coomassie brilliant blue R. 2.5.4. RNA analysis Viral total RNA quantification was measured to explore the RNA photocatalysis and verify the breakage of surface proteins. The viral RNA was extracted by the QIAamp Viral Mini Kit according to the

Fig. 1. A typical (A) XRD pattern, (B) UVeVis DRS spectrum, (C) SEM image, (D) TEM image of g-C3N4.

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manufacturer's instructions. Viral RNA levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), and the corresponding primers are shown in Supporting Information. The qRT-PCR was conducted as follows: reverse transcription at 50  C for 30 min, and then denaturation at 95  C for 15 min, followed by 45 cycles of 10 s for denaturation at 94  C, further annealing at 55  C for 30 s, and final extension at 72  C for 20 s. In addition, RNA agarose gel electrophoresis (AGE) combined with reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm RNA destruction. The viral RNA extraction procedures and RT-PCR cycle conditions including the complementary DNA, the forward primers and the reverse primers were chosen according to a previous report (Wu et al., 2015). Four viral surface protein genes namely replicase protein genes, maturation (A) protein genes, lysis protein genes and coat protein genes were amplified and then monitored using 3% agarose gels at a constant voltage of 60 V for 100 min, followed by staining with GelRed.

Fig. 2. (A) Photocatalytic inactivation efficiency against MS2 (1
108 PFU/mL, 100 mL) in the presence of different doses of g-C3N4 under visible light irradiation. (B) Photographs of MS2 plaques formation before and after photocatalytic disinfection with gC3N4 (150 mg/L) under visible light irradiation.

3. Results and discussion 3.1. Virucidal performance 3.1.1. Photocatalytic inactivation Highly graphitic-like structures are observed for the prepared samples with the typical dominant (002) diffraction peak at 27.6 corresponding to an interlayer distance of 0.33 nm (Fig. 1A), which is well known for g-C3N4 (Yan et al., 2009). The small (100) diffraction peak at 13.1 corresponding to an interlayer distance of 0.68 nm is associated with the in-plane repeated units (Yan et al., 2009). The prepared g-C3N4 samples possess an absorption edge located in the visible light region (Fig. 1B). The band gap energy was calculated to be 2.75 eV based on UVeVis DRS data (inset of Fig. 1B), further manifesting that the prepared samples can absorb visible light (450 nm). The morphology and microstructure of the prepared g-C3N4 powders were revealed to be layer-like structures with some irregular strips and sheet structures (Fig. 1C and D). The photocatalytic disinfection efficiency of viruses with different doses of g-C3N4 photocatalysts under visible light irradiation (l  400 nm) is exhibited in Fig. 2A. The results of dark control revealed that the concentration of surviving viruses remained almost unchanged after 360 min, demonstrating no toxic effects of g-C3N4 on MS2 virus particles in the dark (i.e., g-C3N4 is biocompatible in the dark). Also, the results of light control experiment indicated that no MS2 were inactivated when the g-C3N4 photocatalyst was absent, which manifested that the viruses were not inactivated by direct photolysis under visible light irradiation. However, MS2 could be efficiently inactivated in the co-existence of g-C3N4 and visible light. Hence the virucidal effects were attributed to the photocatalysis of g-C3N4 semiconductor. Compared with light control experiment without photocatalysts, the viral inactivation rate obviously increased even with a small amount of g-C3N4 (50 mg/L). The dosage of g-C3N4 showed a significant influence on the viral inactivation efficiency. After the introduction of photocatalysts, the inactivation level of MS2 increased from ~4.5 log at a photocatalyst concentration of 50 mg/L to ~6 log at a photocatalyst concentration of 100 mg/L, and reached the maximum value of ~8 log at a photocatalyst concentration of 150 mg/L with 360 min visible light illumination. Further increasing the g-C3N4 concentration to 200 mg/L decreased MS2 inactivation to 7.5 log at the end of the reaction. This reduction of the photocatalytic performance is

Fig. 3. Comparison of the photocatalytic performance of samples of g-C3N4, N-TiO2, Bi2WO6 and Ag@AgCl with the dose of 150 mg/L for MS2 inactivation (1
108 PFU/mL, 100 mL) under 300 min visible light irradiation.

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reasonable due to that the addition of a large amount of photocatalysts could lead to a great decrease of light penetration. An optimum dosage of g-C3N4 is critical for photocatalytic inactivation

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of viruses. All subsequent experiments were performed with the gC3N4 loading of 150 mg/L. MS2 with an initial concentration of 1
108 PFU/mL could be completely inactivated within 360 min under visible light irradiation when the concentration of g-C3N4 was 150 mg/L, as visualized in Fig. 2B. A viral regrowth test was next conducted to provide further insights of the virucidal effects. The results indicated that no visible plaques were formed even after incubation in the dark for 72 h, which suggested that MS2 had been totally inactivated in photocatalysis, instead of simply suppressing their growth and reproduction ability. Oppositely, for UV disinfection, the regrowth of resistant pathogens was reported to be significant after irradiation (up to 360 min) with high UV doses (147 and 439 mW s/cm2) (Gilboa and Friedler, 2008). The g-C3N4-based photocatalysis will be a more proficient solution for viral disinfection. The performance of g-C3N4 was compared with other visiblelight-active photocatalysts i.e., N-TiO2, Bi2WO6, and Ag@AgCl, for viral inactivation under the same experimental conditions. The photocatalytic inactivation of MS2 with different visible-lightactive photocatalysts is presented in Fig. 3. Under visible light irradiation for 300 min, more than 7 log of viruses were inactivated in the presence of g-C3N4, whereas the level of viruses was only decreased by ~1 log and ~4 log for N-TiO2 and Bi2WO6, respectively. The limited performance of N-TiO2 and Bi2WO6 is likely due to the inherent rapid recombination of photo-generated charges (Kumar et al., 2013; Zhang et al., 2012). It should be also noted that Ag@AgCl displayed a higher efficiency of viral inactivation, i.e., inactivating 8 log of viruses within 300 min. The observed high reactivity of Ag@AgCl is because Ag nanoparticles act as an electron sink to facilitate charge separation, surface plasmon resonance of Ag nanoparticles enhances photon harvesting, and the dissolved Agþ from Ag@AgCl is biocidal (Wang et al., 2015; Xia et al., 2016; Zhang et al., 2015). Nonetheless, the application of Ag-based photocatalysts is rather expensive and unsustainable, considering that the photocatalysts will eventually be exhausted through Agþ dissolution, and Agþ and/or Ag nanoparticles may pose health risks in the treated water. 3.1.2. Photocatalytic stability The stability of g-C3N4 plays a significant role in practical applications for drinking water disinfection. The photocatalyst was tested over multiple cycles of MS2 photocatalytic inactivation, and the durability and robustness of the material was evaluated. As illustrated in Fig. S4, the g-C3N4 exhibited similar virucidal effects under visible light irradiation for five cycles. No obvious reduction of the viral inactivation efficiency was observed (all viruses were completely inactivated within 6 h), demonstrating that the g-C3N4 is stable in the photocatalytic disinfection process. These promising results may suggest that g-C3N4 is able to serve as an alternative for viral disinfection under visible light. Moreover, g-C3N4 is abundant and easily fabricated through one-step polymerization of low-cost commercial precursors (i.e. urea, thiourea, melamine, cyanamide and dicyandiamide) (Cao et al., 2015). To extend the practical use of g-C3N4, it is needed to consider the toxicity of g-C3N4 toward human. The study of Lin et al. (2014) reported that the g-C3N4 possessed low cytotoxicity and could be used as drug carriers for cancer therapy. In a word, g-C3N4 is a relative safe and environmentally benign photocatalyst when using.

Fig. 4. (A) Photocatalytic inactivation efficiency against MS2 (1
108 PFU/mL, 100 mL) by g-C3N4 (150 mg/L) with different scavengers under visible light irradiation. (B) UVeVis absorption spectra of NBT degradation to monitor O- 2 and (C) Fluorescence spectra of 2-HTA concentration to monitor OH generated by g-C3N4 (150 mg/L) under visible light irradiation.

3.2. Viral inactivation mechanism 3.2.1. Roles of various ROSs It has been found that the photocatalytic viral inactivation is potentially caused by several main ROSs generated from

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Fig. 5. TEM images of MS2 photocatalytically (A and B) untreated or (C and D) treated with g-C3N4 for 6 h under visible light irradiation.

photocatalysts, such as OH from TiON/PdO (Li et al., 2008), Ag/TiO2 (Liga et al., 2011) and SiO2-TiO2 (Liga et al., 2013), hþ and O- 2 from Ag-AgI/Al2O3 (Hu et al., 2010), OH, O- 2 and 1O2 from GO-aptamer (Hu et al., 2012) and 1O2 from C60 derivatives (Badireddy et al., 2012; Cho et al., 2010; Lee et al., 2010; Moor and Kim, 2014; Moor et al., 2015). In order to understand the mechanism of gC3N4-induced viral disinfection, a series of scavengers were utilized individually during the photocatalytic inactivation of viruses for comparison. Cr(VI), Fe(II)-EDTA, TEMPOL, L-histidine, isopropanol and sodium oxalate were used to completely quench the reactions of MS2 with e, H2O2, O- 2, 1O2, OH and hþ, respectively. The preliminary experiments indicated that the presence of scavenger at the selected concentration did not inactivate viruses (Fig. S2). As shown in Fig. 4A, with the addition of TEMPOL or isopropanol, the photocatalytic inactivation efficiency for MS2 was greatly inhibited compared with no scavenger addition. This results suggested that O- 2 and OH were the main ROSs which played a vital role in the photocatalytic viral inactivation process. The production of O- 2 and OH was further quantified through the transformation of the probe molecules NBT and TA, respectively. An apparent decrease in UVeVis absorbance for NBT and increase in fluorescence for the highly fluorescent product 2-HTA of TA degradation were observed (Fig. 4B and C), and the corresponding concentrations of O- 2 and OH were calculated over the experimental time scale (Fig. S5), confirming that both O- 2 and OH radicals were involved in the g-C3N4-induced photocatalytic viral disinfection under visible light irradiation. The involvement of H2O2 was also identified by the decrease in the viral inactivation efficiency after adding the scavenger Fe(II)-EDTA (Fig. 4A). The concentration of H2O2 generated from g-C3N4 was calculated to be accumulated at around 0.7 mmol/L after 360 min visible light illumination (Fig. S6). And no H2O2 was measured with the addition of Fe(II)-EDTA, suggesting that H2O2 was fully quenched. The dominant virucidal effects of O- 2 rather than OH in Fig. 4A are expected, considering the fact that the e produced from the conduction band of g-C3N4 (1.35 eV vs. NHE) can directly reduce O2 into O- 2 (0.33 eV vs. NHE) whereas the hþ produced from the

valence band of g-C3N4 (1.40 eV vs. NHE) cannot directly oxidize H2O/OH (2.37 eV vs. NHE) into OH (Dong et al., 2014; Wang et al., 2014). Alternatively, the detected OH should be derived from the transformation of O- 2 by the e in the reductive site of g-C3N4. Therefore, feeding pure O2 could apparently enhance the viral inactivation (Fig. 4A) possibly by promoting the formation of O- 2 and OH. To affirm this pathway, a partition system having a semipermeable membrane with a molecular weight cutoff of 8000 Da was employed to prevent direct contact between the viral particles and g-C3N4 photocatalysts (Fig. S7). It can exclude the action of ROSs on the surface of g-C3N4 (such as hþ, e, O- 2 and surface OH) and allow only diffusing ROSs (such as free OH and H2O2) to pass across the membrane (Chen et al., 2011; Jin et al., 2013). In fact, the diffusion distance of OH is limited to several nm owing to its very short average lifetime in water (Gao et al., 2016; Wang et al., 2012), thus it is theoretically impossible for OH to pass through the semipermeable membrane with a thickness of several tens of mm. Only ~1.8 log reduction of viruses was achieved inside the partition system after 360 min of visible light illumination, declaring that the direct contact between viruses and photocatalysts is very crucial in the present system. It was found that the addition of Fe(II)-EDTA significantly inhibited the photocatalytic viral inactivation efficiency, demonstrating that the inactivation process is actually induced by the diffusing H2O2 in the partition system. In addition, Cr(VI) was added to fully quench the e production and their derived ROSs (Wang et al., 2012; Xia et al., 2015). Viral inactivation was completely prohibited, certifying that the virucidal ROSs were indeed produced in a reductive way from the conduction band of gC3N4. As a result, the photogenerated e and its derived ROSs, O- 2, are considered to be the leading effective reactive species for viral inactivation in the g-C3N4 photocatalytic system. Viral inactivation efficiency was slightly inhibited in the presence of sodium oxalate while greatly inhibited in the presence of Cr(VI) (Fig. 4A), showing a relatively minor function for hþ compared with e in the non-partition system. Noted that the degree of inhibition caused by Cr(VI) (e scavenger) was lower than

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that caused by TEMPOL (O- 2 scavenger). This is more likely due to that the e-hþ separation efficiency was enhanced by capturing e, facilitating the remaining hþ to inactivate viruses. Specially, there was almost no change in inactivation efficiency with the addition of 1 L-histidine, indicating the virucidal effects of O2 can be negligible in the photocatalytic system. Moreover, it was monitored that almost no degradation of FFA (a probe molecule for 1O2) was found during reaction time of 360 min (Fig. S8), illustrating that no 1O2 were formed in this photocatalytic system. It turned out that electron-transfer-generated species (O- 2) were more responsible for g-C3N4-induced photocatalytic viral disinfection than energytransfer-generated species (1O2), consistent with the results from Ag-AgI/Al2O3 and GO-aptamer photocatalysts (Hu et al., 2010, 2012). It has been known that O- 2 is an essential antibacterial agent which is potentially cytotoxic and causes damage to DNA (Hayyan et al., 2016). From our work, it is clear that O- 2 is also a crucial antiviral agent, and its longer lifetime and higher concentration were conducive to the efficient virucidal effects of g-C3N4. In recent years, many remarkable applications of O- 2 have been

Fig. 6. (A) Determination of protein concentration (-) in 100 mL concentrated viral particle lysates from 5 mL viral suspension (1
108 PFU/mL) and the protein carbonyl content ( ) during photocatalytic inactivation. (B) SDS-PAGE image of photocatalytically untreated and treated MS2 viral surface proteins. Lanes: 1 and 3, protein maker; 2, untreated MS2 virus control; 3, g-C3N4-treated MS2 viral proteins for 6 h visible light irradiation.

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reported, including the degradation of hazardous substances in both aqueous and non-aqueous phases and the remediation of polluted groundwater and soil (Hayyan et al., 2016). Here, an appealing potential application of O- 2 for viral inactivation in water will be of great interest and expected, which still needs more effort and direct evidence to identify. 3.2.2. Viral decomposition and biomolecule breakage To better confirm the disruption of viral particle integrity, the morphology and microstructure of MS2 before and after photocatalytic inactivation by g-C3N4 were examined by TEM. Fig. 5A shows that the bulk untreated MS2 displayed smooth sphere shapes. This is further verified in a representative TEM image (Fig. 5C), which exhibits that the MS2, possibly in contact with the layered photocatalyst, retained a well-preserved integrity of the particle before irradiation. After 6 h of visible light irradiation with g-C3N4, the structures of the treated MS2 were seriously damaged as there are no intact viral particles observed in Fig. 5B. Obviously, the viral particle shape is distorted and the capsid shell becomes ruptured, seen in a representative TEM image (Fig. 5D). Based on these observations, the capsid shell is supposed to suffer extreme destruction. Unlike bacteria, viruses do not possess their own lipids, cytoplasmic substances and genes encoding for proteins needed for translation, energy metabolism or membrane biosynthesis (Diehl and Schaal, 2013). Hence the destruction of viral particles was measured by the breakage of the building blocks of viruses, namely

Fig. 7. (A) RNA quantification of MS2 (1
108 PFU/mL) before and after visible light photocatalysis by g-C3N4. (B) AGE images of viral RNA genes coding for (a) replicase proteins, (b) maturation proteins, (c) lysis proteins and (d) coat proteins gene of MS2 (Lines 1) before and (Lines 2) after photocatalytic inactivation with g-C3N4 under visible light irradiation.

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Fig. 8. Schematic illustration of proposed mechanism of viral inactivation by g-C3N4 under visible light irradiation.

proteins and nucleic acids. The protein concentration of 5 mL gC3N4 treated viruses (1
108 PFU/mL) reduced from 9.8 mg/mL to 0 mg/mL after 360 min visible light irradiation, confirming the damage of proteins from viral particles during photocatalytic inactivation process (Fig. 6A). The decrease of viral protein levels occurred at the initial of photocatalytic disinfection. In contrast, bacterial protein levels have been reported to be maintained unchanged at the initial of photocatalytic inactivation (Xia et al., 2015). This may be due to the fact that viruses do not own the defense mechanisms, like the antioxidant enzymes (catalase and superoxide dismutase), against the attack by ROSs. As a result, the defense system of viruses was easily overwhelmed by ROSs at the initial stage. Considering that ROSs may oxidize the protein side chains, particularly arginine, lysine and threonine (Sun et al., 2014; Xia et al., 2015), the protein carbonyl content was detected for evaluating the oxidative damage to viral proteins in the photocatalytic treatment system. Coincidentally, the protein carbonyl content began to rise from 0 to 240 min and remarkably rise after 240 min (Fig. 6A), implying that the viral proteins were highly oxidized and then broken in that period. Damage to viral proteins will lead to the generation of protein fragments, protein carbonyls and other unknown substances in the ROSs mediated photocatalytic disinfection reaction. The products are speculated to aggregate at the end of inactivation process, but this needs further validation from additional studies. In order to provide more details of the viral protein breakage, the SDS-PAGE was carried out with extracted surface proteins from untreated and treated MS2 respectively. Fig. 6B presents the electrophoresis image of the standard protein marker with molecular weights ranging from 20 to 120 kDa. The control image (untreated MS2) demonstrated molecular weights of MS2 surface proteins were between 35 and 85 kDa. However, after photocatalytic disinfection, the protein band intensities were significantly decreased or even disappeared compared to that for the control, declaring the remarkable breakage of surface proteins by g-C3N4 photocatalysis. The maturation (A) proteins (44 kDa, seen in Fig. 6B) were considered to be responsible for RNA packing and host identification (Hu et al., 2012). Besides, the replicase proteins (60 kDa, seen in Fig. 6B) were involved in replicase production that functions in viral RNA synthesis (Wu et al., 2015). Unfortunately, some bands below 20 kDa (i.e. coat and lysis proteins) did not show up in the image because of their small sizes. Nonetheless, any damage to maturation proteins and replicase proteins from the external attack will cause the loss of virus infectivity and the

leakage of enveloped RNA from viruses. It is well-known that the oxidative damage of proteins may not be totally lethal to the viruses, because they possess a capability to self-repair and regrow under the suitable conditions, bringing the potential risks related with drinking water. Comparatively, the severe loss or damage of another vial building block, RNA, will result in definite viral death. Hence, the effect of g-C3N4 photocatalysis on viral RNA was investigated so as to ascertain the cause of viral death from a genomic point of view, namely RNA leakage or damage. RNA of MS2 treated with g-C3N4 photocatalysts was quantified by qRTPCR (Fig. 7A), and the viral RNA was ensured to be destroyed with a ~4.5 log reduction. Also, breakage to the viral RNA genes coding for four main viral surface proteins (replicase proteins, maturation proteins, lysis proteins and capsid proteins) was determined by AGE and intuitively observed in Fig. 7B. The RNA band intensities of these three types of viral surface proteins, maturation proteins, lysis proteins and capsid proteins, were obviously diminished and those of replicase proteins vanished almost completely. This is, even if all viral surface proteins are intact after the g-C3N4 photocatalysis, breakage solely to the RNA genes can prevent viral reproduction within the infected host cells. The above results collectively reveal the photocatalytic inactivation mechanism of MS2 by g-C3N4 under visible light irradiation. As illustrated in Fig. 8, viral inactivation by photocatalysis has been described as a nonselective reaction resulted from photo-generated e and its derived ROSs (mainly O- 2 and OH). Loss and oxidative damage of viral surface proteins results in the leakage and rapid destruction of interior substances namely RNA, finally leading to viral death with no regrowth. The g-C3N4-based photocatalysts with enhanced antiviral properties, such as the g-C3N4/TiO2 hybrid material which has been reported with an excellent performance for both pollutant degradation (Li et al., 2016) and bacteria inactivation (Li et al., 2015), need to be developed and validated in further research. 4. Conclusion In the present study, a metal-free visible-light-driven photocatalyst, g-C3N4, exhibited long term, effective and stable virucidal effects in a dose dependent manner on MS2. The results of scavenger experiments suggested that the photo-generated e and its derived ROSs (O- 2) dominated viral inactivation process. Furthermore, viruses were primarily decomposed due to that viral particle shapes were distorted and surface proteins (especially

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replicase proteins and maturation proteins) were oxidatively damaged. Importantly, four major types of RNA genes were severely destroyed, resulting in definite viral death. The g-C3N4 photocatalyst is hence identified as a good candidate for viral disinfection. Acknowledgement The study was financially supported by the National Natural Science Foundation of China (No. 51322901 and 51479066), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (51421006), the Fundamental Research Funds for the Central Universities (2016B10614), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2016.10.009. References Badireddy, A.R., Budarz, J.F., Chellam, S., Wiesner, M.R., 2012. Bacteriophage inactivation by UV-A illuminated fullerenes: role of nanoparticle-virus association and biological targets. Environ. Sci. Technol. 46 (11), 5963e5970. Cao, S., Low, J., Yu, J., Jaroniec, M., 2015. Polymeric photocatalysts based on graphitic carbon nitride. Adv. Mater. 27 (13), 2150e2176. Chen, Y., Lu, A., Li, Y., Zhang, L., Yip, H.Y., Zhao, H., An, T., Wong, P.K., 2011. Naturally occurring sphalerite as a novel cost-effective photocatalyst for bacterial disinfection under visible light. Environ. Sci. Technol. 45 (13), 5689e5695. Cho, M., Lee, J., Mackeyev, Y., Wilson, L.J., Alvarez, P.J.J., Hughes, J.B., Kim, J.H., 2010. Visible light sensitized inactivation of MS-2 bacteriophage by a cationic aminefunctionalized C60 derivative. Environ. Sci. Technol. 44, 6685e6691. Cong, Y., Zhang, J., Chen, F., Anpo, M., 2007. Synthesis and characterization of nitrogen-doped TiO2 nanophotocatalyst with high visible light activity. J. Phys. Chem. C 111, 6976e6982. Diehl, N., Schaal, H., 2013. Make yourself at home: viral hijacking of the PI3K/Akt signaling pathway. Viruses 5 (12), 3192e3212. Dong, F., Wang, Z., Li, Y., Ho, W.K., Lee, S.C., 2014. Immobilization of polymeric gC3N4 on structured ceramic foam for efficient visible light photocatalytic air purification with real indoor illumination. Environ. Sci. Technol. 48 (17), 10345e10353. Gao, M., Ng, T.W., An, T., Li, G., Yip, H.Y., Zhao, H., Wong, P.K., 2016. The role of catalase and H2O2 in photocatalytic inactivation of Escherichia coli: genetic and biochemical approaches. Catal. Today 266, 205e211. Gilboa, Y., Friedler, E., 2008. UV disinfection of RBC-treated light greywater effluent: kinetics, survival and regrowth of selected microorganisms. Water Res. 42 (4e5), 1043e1050. Hayyan, M., Hashim, M.A., AlNashef, I.M., 2016. Superoxide ion: generation and chemical implications. Chem. Rev. 116 (5), 3029e3085. Hijnen, W.A., Suylen, G.M., Bahlman, J.A., Brouwer-Hanzens, A., Medema, G.J., 2010. GAC adsorption filters as barriers for viruses, bacteria and protozoan (oo)cysts in water treatment. Water Res. 44 (4), 1224e1234. Hu, X., Hu, C., Peng, T., Zhou, X., Qu, J., 2010. Plasmon-induced inactivation of enteric pathogenic microorganisms with Ag-AgI/Al2O3 under visible-light irradiation. Environ. Sci. Technol. 44, 7058e7062. Hu, X., Mu, L., Wen, J., Zhou, Q., 2012. Covalently synthesized graphene oxideaptamer nanosheets for efficient visible-light photocatalysis of nucleic acids and proteins of viruses. Carbon 50 (8), 2772e2781. Huang, J., Ho, W., Wang, X., 2014. Metal-free disinfection effects induced by graphitic carbon nitride polymers under visible light illumination. Chem. Commun. 50 (33), 4338e4340. Jin, Y., Dai, Z., Liu, F., Kim, H., Tong, M., Hou, Y., 2013. Bactericidal mechanisms of Ag2O/TNBs under both dark and light conditions. Water Res. 47 (5), 1837e1847. Koivunen, J., Heinonen-Tanski, H., 2005. Inactivation of enteric microorganisms with chemical disinfectants, UV irradiation and combined chemical/UV treatments. Water Res. 39 (8), 1519e1526. Kumar, N., Maitra, U., Hegde, V.I., Waghmare, U.V., Sundaresan, A., Rao, C.N., 2013. Synthesis, characterization, photocatalysis, and varied properties of TiO2 cosubstituted with nitrogen and fluorine. Inorg. Chem. 52 (18), 10512e10519. Lee, J., Mackeyev, Y., Cho, M., Wilson, L.J., Kim, J.H., Alvarez, P.J.J., 2010. C60 aminofullerene immobilized on silica as a visible-light-activated photocatalyst. Environ. Sci. Technol. 44, 9488e9495. Lee, J.E., Ko, G., 2013. Norovirus and MS2 inactivation kinetics of UV-A and UV-B with and without TiO2. Water Res. 47 (15), 5607e5613.

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