key: cord-0805088-m9zwekng
authors: Zhang, Chi; Li, Yi; Wang, Chao; Zheng, Xinyi
title: Different Inactivation Behaviors and Mechanisms of Representative Pathogens (Escherichia Coli Bacteria, Human Adenoviruses and Bacillus Subtilis Spores) in g-C3N4-Based Metal-Free Visible-Light-Enabled Photocatalytic Disinfection
date: 2020-09-30
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.142588
sha: 3b7231f758761612c0ec5e2a229506585b784000
doc_id: 805088
cord_uid: m9zwekng

Continuous economic loss and even human death caused by various microbial pathogens in drinking water call for the development of water disinfection systems with the features of environmentally friendly nature, high inactivation efficacy without pathogen regrowth, facile disinfection operation and low energy consumption. Alternatively, g-C3N4-based visible-light-enabled photocatalytic disinfection can meet the above requirements and thus has attracted increasing interest in recent years. Here, we explored for the first time the antimicrobial ability and mechanisms of a wide spectrum of representative pathogens ranging from bacteria (Escherichia coli), to viruses (human adenoviruses) and spores (Bacillus subtilis spores) by g-C3N4/Vis system with the assistance of two common oxidants (H2O2 and PMS), especially in a comparative perspective. Pristine g-C3N4 could achieve a complete inactivation of bacteria (5-log) within 150 min, but displayed negligible antimicrobial activity against human viruses and spores (< 0.5-log). Fortunately, simple addition of oxidants into the system could greatly enhance the inactivation of bacteria (5-log with PMS within 120 min) and human viruses (2.6-log with H2O2 within 150 min). Roles of reactive oxygen species were found to be quite different in the disinfection processes, depending on both types of chemical oxidants and microbial pathogens. Additionally, disinfection efficiency could be facilely and effectively improved by statistical optimization of two important operating factors (i.e., catalyst loading and oxidant addition). Selection of added oxidants was determined by not only the target pathogen but also the water matrix. As a proof of concept, this work can provide some meaningful and useful information for advancing the field of green and sustainable water disinfection.

The war between humans and pathogens has never been stopped over the past thousands of years and seems to continue endlessly, due to the fact that pathogenic microorganisms are ubiquitous in environment and can cause various infectious diseases and even mass death, such as the current COVID-19 pandemic (Baum et al., 2020) . Waterborne infectious diseases (e.g., diarrhoea, cholera, dysentery, typhoid, and polio) are generally associated with bacteria, viruses, protozoa and other microbes transmitted via water, and remain a global public health issue as a major source of morbidity and mortality in the world today (Fenwick, 2006; Leclerc et al., 2002; Semenza, 2020; Wang et al., 2019) . According to a recent report from WHO (2019), diarrhoea alone is estimated to kill 829,000 people per year, including 485,000 deaths caused by contaminated drinking water with rotavirus and Escherichia coli. It is clear to see that a significant proportion of waterborne diseases can be prevented through a safe drinking water supply. Therefore, there is a pressing need to effectively control microbial contamination in drinking water for human health. byproducts, possible complete inactivation of microbial pathogens and potential utilization of renewable solar energy (Fernandez-Ibanez et al., 2020; Liu et al., 2016; Zhang et al., 2019a) . In particular, metal-free visible-light-enabled photocatalytic water disinfection has a high solar light-harvesting capability while at the same time avoids the risk of metal leaching into the treated water (Li et al., 2017a; Yadav et al., 2019) . Specifically, g-C 3 N 4 is rapidly emerging as an easily available and metal-free photocatalyst for various applications in both energy conversion and environmental purification (Liu et al., 2015; Ong et al., 2016) , since it was first found to have the ability to split water and generate hydrogen under visible light irradiation (Wang et al., 2009) . More recently, g-C 3 N 4 has been identified with antimicrobial activity against bacteria (e.g. Escherichia coli and Staphylococcus aureus) (Huang et al., 2014; Orooji et al., 2020) , viruses (e.g. MS2 bacteriophages and human adenoviruses) Zhang et al., 2019c) , and spores (e.g. Bacillus anthracis endospores) (Thurston et al., 2017) via the attack of photogenerated reactive oxidative species (ROS), attracting increasing interest as a low cost and environmentally friendly antimicrobial agent candidate for next-generation sustainable water disinfection.

whereas less have been dedicated to the inactivation of other common pathogens such as viruses and spores (Zhang et al., 2019b) . To the best of our knowledge, there is no comprehensive comparative study on the antimicrobial performance of the g-C 3 N 4 /Vis process for a broad spectrum of pathogens, involving bacteria, human viruses, and spores. Also, a major issue in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection is its slow microbial inactivation kinetics due to the limited photocatalytic activity of pure g-C 3 N 4 and the strong environmental resistance of many pathogens (e.g., human adenoviruses and bacterial spores) (Thurston et al., 2017; Zhang et al., 2020; Zhang et al., 2019c) . As a result, extensive efforts have been made to modify g-C 3 N 4 materials for enhanced photocatalytic disinfection performance (Wang et al., 2013; Xia et al., 2020) . Compared to modification of g-C 3 N 4 materials with additional processing steps and/or associated reagents, direct addition of commercially available oxidants (e.g., hydrogen peroxide (H 2 O 2 ) and peroxymonosulfate (PMS)) during photocatalysis has been proved to be a simple and efficient approach to improve the degradation efficiency of chemical pollutants (Cui et al., 2012; Tao et al., 2015) , whose resistance to disinfection treatment is quite different from microbial pathogens (Marugan et al., 2010; Zhang et al., 2018a) .

However, to date, neither inactivation behaviors nor mechanisms of these common pathogens in the g-C 3 N 4 /Vis process with the assistance of oxidants are known, especially in a comparative view.

Taking in consideration the above knowledge gaps and issues in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection, the aim of this work is to J o u r n a l P r e -p r o o f Journal Pre-proof i) test the microbicidal capacity of the g-C 3 N 4 /Vis process on a broad spectrum of representative pathogens (i.e., Escherichia coli bacteria, human adenoviruses and Bacillus subtilis spores), ii) comparatively evaluate the enhanced disinfection performance of the g-C 3 N 4 /Vis process with individual addition of two different oxidants (i.e., • OH-based H 2 O 2 oxidation and SO 4

•--based PMS oxidation), iii) explore the corresponding microbial inactivation mechanisms, which might be varied depending on the types of chemical oxidants and microbial pathogens, and iv) statistically optimize the composite disinfection process and attempt at its practicality in drinking water matrices.

Escherichia coli bacteria (ATCC 15597) were cultured and propagated in liquid nutrient medium, which contained 10 g of tryptone, 8 g of NaCl, 1 g of glucose, 1 g of yeast extract, 294 mg of CaCl 2 , 10 mg of thiamine and 1000 mL of deionized water.

Briefly, bacterial cultures were incubated at 37 °C for 24 h, and then centrifuged at 3000 rpm for 10 min. The resulting pellet was washed with phosphate-buffered solution (PBS), and ultimately stored at 4 o C before use. Bacterial titer was determined by the spread plate method using solid nutrient medium (15 g of agar), and expressed as colony forming units (CFU)/mL. Human adenoviruses (type 2, ATCC VR-846) were cultured and propagated in human lung cell line (A549, ATCC CCL-185) and Ham's F-12 K medium, which was supplemented with extra 10% fetal bovine serum and 1% antibiotics. Briefly, lung cell J o u r n a l P r e -p r o o f Journal Pre-proof cultures were incubated at 37 °C in humidified air with 5% CO 2 until the cells reached a confluence of 70-80%. Pre-warmed viral cultures were then added into the cellular monolayer, and the mixture was shaken for 45 min to allow sufficient contact between viruses and cells. After 4-day incubation at 37 o C, the infected cells were collected and freeze-thawed three times to release viruses. The mixture was then centrifuged at 1000 g for 10 min and the supernatant was filtered through a 0.45 μm membrane for virus purification. The resulting filtrate was concentrated and washed with PBS, and ultimately stored at -80 °C before use. Virus titer was measured by the most probable number (MPN) method using 96-well plate, and expressed as MPN/mL.

Bacillus subtilis spores (ATCC 6633) were prepared from the corresponding strain in liquid broth medium. The culture procedures of Bacillus subtilis were similar to the above described for Escherichia coli. The difference is that Bacillus subtilis was next inoculated and incubated on solid medium with 10-fold dilutions of nutrients to induce sporulation. Bacillus subtilis spores were collected by rinsing with PBS, and then centrifuged at 3000 rpm for 10 min. The resulting pellet was washed and resuspended in PBS, but needed to be heated at 80 o C for 20 min to kill any remaining vegetative cells before use. Spore titer was detected by the spread plate method using solid nutrient medium, and also expressed as CFU/mL. previous studies Zhang et al., 2018b) . All microbial stock solutions were diluted into 20 mL of deionized water in 50-mL beakers to prepare the individual microbial suspensions with an initial concentration of 10 5 CFU/mL or MPN/mL. Then a certain dose of the freshly prepared g-C 3 N 4 photocatalysts and the oxidants (H 2 O 2 or PMS) were added for microbial inactivation with continuous stirring at room temperature. The light source was a 300 W Xenon lamp with a cutoff filter to provide visible light irradiation (≥ 400 nm, 150 mW/cm 2 ). Before illumination, the system was first kept in the dark for 30 min to reach adsorption-desorption equilibrium between photocatalysts and microorganisms.

Microbial samples were taken at time intervals of 0, 30, 60, 90, 120 and 150 min during photocatalytic disinfection, and were immediately counted to avoid further inactivation. Microbial solutions without g-C 3 N 4 photocatalysts were irradiated under the same experimental conditions as light control, and microbial suspensions with g-C 3 N 4 photocatalysts but without visible light irradiation was used as dark control.

The microbial inactivation efficiency was calculated as log [C t /C 0 ], where C 0 and C t were the microbial titer before and after photocatalytic disinfection, respectively. , and L-histidine (0.5 mM) for 1 O 2 , respectively, in order to exert the maximum scavenging effect but without toxicity to the target microbial pathogens (Rodríguez-Chueca et al., 2020; Zhang et al., 2018a; Zhang et al., 2019c) . Moreover, electron spin resonance spectroscopy (ESR) was performed for further verification of • OH and SO 4

•generation on a Bruker EMXplus spectrometer using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agents.

Bacterial samples were fixed by 2.5% glutaraldehyde, washed by PBS three times, and gradually dehydrated by ethanol series from 30% to 100%, followed by freeze-drying and gold-coating, for morphology observation by a SU8010 scanning electron microscope (SEM, Hitachi, Japan). In addition to fixation and washing procedures, viral samples were required to be negatively stained with 2% uranyl acetate for morphology observation by a FEI Talos F200X transmission electron microscope (TEM, Thermo Fisher Scientific, USA).

The two important operating parameters in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection, namely catalyst loading (X 1 , g/L) and oxidant addition (X 2 , mM), were interactively analyzed and optimized using response surface methodology (RSM) as a powerful statistical tool. The photocatalytic inactivation of bacteria (Y 1 ) and viruses (Y 2 ) after 60 and 120 min of Table   S1 . The obtained experimental results were fitted to a quadratic polynomial model:

where Y is the predicted response, β 0 is the constant, βi, βii, and βij are the linear, squared, and interaction coefficients, respectively. And k is the number of variables in the model (here k = 2). All statistical assessments, including analysis of variance (ANOVA), were conducted using Design-Expert® software (version 8.0.6.1).

To systematically evaluate the antimicrobial potency and efficacy of g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection, Escherichia

Coli, human adenoviruses, and Bacillus Subtilis spores were chosen as microbial representatives of pathogenic bacteria, viruses, and protozoa, respectively (Sun et al., 2016) . In detail, Escherichia Coli is the most commonly used and accepted indicator bacteria in photocatalytic disinfection processes, due to the prevalent occurrence in environment and the easy cultivation in lab. Human adenoviruses have been employed here when considering the controversy over the suitability of bacteriophages as surrogates for human viruses in water treatment processes. And

Bacillus subtilis spores have been tested because of their high resistance to various J o u r n a l P r e -p r o o f Journal Pre-proof disinfection treatments, including heat, UV radiation, and even chlorination.

The quite different microbial inactivation behaviors ranging from bacteria to human viruses and spores in the g-C 3 N 4 /Vis system have been clearly observed in Fig.   1 . As seen in the light and dark control groups, neither visible-light photolysis nor g-C 3 N 4 materials in dark could induce any inactivation of these microorganisms. The results suggest that, on one hand, most microbial pathogens are stable and can even proliferate under visible light irradiation without photocatalysts, on the other hand, the g-C 3 N 4 material alone is biocompatible in the dark and exert low or no toxicity to microorganisms in water. When the light was turned on in the presence of g-C 3 N 4 , all

Escherichia coli bacteria (5-log) were inactivated within 150 min (Fig. 1a) , declaring a good bactericidal effect induced by the g-C 3 N 4 /Vis system. Unfortunately, almost no human adenoviruses (Fig. 1b) and Bacillus subtilis spores (Fig. 1c) were inactivated during the same process. Compared to common bacteria with fragile cell membranes, human viruses have highly rigid protein shells and bacterial spores have extremely thick cell walls, which can protect them well from a wide range of harsh environments, including chemical disinfectants, UV irradiation, and photocatalytic treatment (Płonka and Pieczykolan, 2020; Sun et al., 2016; Zhang et al., 2019b) . This is to say that g-C 3 N 4 can be served as a powerful antimicrobial weapon against bacteria rather than human viruses, protozoa, and not to mention other resistant pathogens with complex biological compositions and structures.

J o u r n a l P r e -p r o o f

As well known, H 2 O 2 is one of the most commonly used oxidants for enhanced water treatment, owing to its low-cost and green nature. Alternatively, PMS has also received increasing attention as a stable solid oxidant with the additional advantage of convenient storage and transportation. (Gligorovski et al., 2015) . Both of them have been confirmed to effectively improve the photocatalytic degradation activity of g-C 3 N 4 towards many chemical pollutants (Cui et al., 2012; Tao et al., 2015) . However, their enhancing effects on the photocatalytic disinfection activity of g-C 3 N 4 towards various microbial pathogens are still needed to be systematically studied and comparatively analyzed.

The viability of representative pathogens used in this study was found to be unaffected in the presence of H 2 O 2 or PMS alone at a low concentration (0.5 mM) under visible light irradiation. Expectedly, addition of H 2 O 2 or PMS into the g-C 3 N 4 /Vis system can accelerate and promote microbial inactivation, although in different degrees (Fig. 2) . For bacterial indicators, a complete inactivation of Escherichia coli (5-log) was quickly achieved within 120 min with the addition of either H 2 O 2 or PMS, reducing the disinfection time by 20% (Fig. 2a) . For viral indicators, a partial inactivation of human adenoviruses could be observed within 150 min, namely ~2.6-log with H 2 O 2 addition and ~1.7-log with PMS addition, J o u r n a l P r e -p r o o f respectively (Fig. 2b) . The more enhancement in adenovirus inactivation induced by H 2 O 2 might be attributed to the higher reaction rate and efficiency between • OH radicals and biological proteins, leading to rapid and irreversible modification of viral protein coats (Gau et al., 2010; Rinas et al., 2016) . While for protozoan indicators, a very slight inactivation of Bacillus subtilis spores (less than 1-log) was obtained only with the addition of H 2 O 2 instead of PMS (Fig. 2c) . The enhancing effects of addition of H 2 O 2 and PMS to the g-C 3 N 4 /Vis disinfection system are quite different, depending on the types of added oxidants and target microorganisms. On one hand, the added oxidants could assist the g-C 3 N 4 /Vis system to produce some more specific ROS.

Although certain ROS such as • OH are able to react unselectively and instantaneously with the surrounding compounds, the redox capacity of ROS towards one compound is closely related to their types and concentrations (Nosaka and Nosaka, 2017) . On the other hand, the target microorganisms with diverse biological structures and metabolic activities could have different resistance and response to a specific ROS attack. In view of both disinfection efficiency and operational simplicity, PMS is recommended for bacterial inactivation while H 2 O 2 is more preferred for viral inactivation as an effective antimicrobial accelerator in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection. As for some certain pathogens with high resistance such as protozoa, another more powerful antimicrobial agent or approach is required to be further explored. 

HSO 5 -+ e -→ SO 4

•-

In addition, considering that 1 O 2 as a non-radical ROS is capable of efficiently reacting with a wide range of biomolecules such as nucleic acids, proteins, and lipids,

Journal Pre-proof L-histidine as the specific chemical quencher was added in the systems (Di Mascio et al., 2019) . However, in contrast, no inhibitory effect was observed on the microbial inactivation in all systems, suggesting a negligible role of 1 O 2 in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection. This is probably due to the poor yields and selectivity for 1 O 2 generation from pristine g-C 3 N 4 with an insufficient inter-system crossing process .

For bacterial indicators, the direct oxidative damage from photogenerated h + cannot be ignored, based on a moderate inhibition of Escherichia coli inactivation with the addition of oxalate in these two systems of g-C 3 N 4 /Vis/H 2 O 2 (Fig. 3a) and g-C 3 N 4 /Vis/PMS (Fig. 3b) 

While for viral indicators, the role of photogenerated h + seemed to be dispensable in these two systems of g-C 3 N 4 /Vis/H 2 O 2 (Fig. 3c) and g-C 3 N 4 /Vis/PMS (Fig. 3d) ) can contribute to viral inactivation in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection.

Notably, a unique feature of microbial pathogens, different from chemical compounds, is that they have the ability to self-repair damage under favorable conditions. The microbial regrowth tests of 48-h dark incubation have been conducted and found that neither Escherichia coli bacteria nor human adenoviruses could be visualized in culture (Fig. S1) (Fig. 4) . Obviously, the biological structures of Escherichia coli bacteria and human adenoviruses were severely destroyed, especially for bacteria, without the possibility of regeneration after the photocatalytic treatment of g-C 3 N 4 /Vis/PMS and g-C 3 N 4 /Vis/H 2 O 2 , respectively.

Can there be a convenient way to achieve satisfactory disinfection?

The statistical tool RSM has been successfully used for optimizing photocatalytic processes, but mainly involving the degradation of non-living chemical pollutants in which reaction kinetics and mechanisms are quite different from living microbial pathogens. Here, two easy-to-operate but important disinfection parameters (i.e., catalyst loading X 1 and oxidant addition X 2 ) have been chosen for achieving satisfactory inactivation towards microbial pathogens (Y) in the g-C 3 N 4 -based photocatalytic disinfection system (Tables S2 and S4 ). Then, two empirical second order polynomial models were developed for bacterial inactivation in the g-C 3 N 4 /Vis/PMS system (Eq. 8) and viral inactivation in the g-C 3 N 4 /Vis/H 2 O 2 system (Eq. 9), respectively. 

Significance and adequacy of the established models were statistically checked J o u r n a l P r e -p r o o f by ANOVA (Tables S3 and S5 ). Both of the models exhibited a high F-value (35.54 and 28.18) along with a low P-value (< 0.0001 and 0.0002), implying that they are statistically significant. The lack of fit F-value (4.07 and 3.20) was not significant relative to the pure error, which is good for a model. Again, the predicted R 2 of 0.7823 and 0.7407 was in reasonable agreement with the adjusted R 2 of 0.9350 and 0.9189, respectively. Meanwhile, the adequate precision (20.105 and 15.659) was desirable here (> 4), indicating an adequate signal to noise ratio. All the above results of statistical analysis have strongly suggested that these two constructed models can be successfully used to navigate the design space. This conclusion was further supported by the linear plots in Figs. 5 and 6. On one hand, the predicted values matched well with the actual responses (Figs. 5a and 6a) . On the other hand, neither response transformation was required nor apparent problem with normality would occur (Figs.

5b and 6b), all confirming the high accuracy of the established models.

According to the analysis data in Table S3 , the terms of X 1 , X 2 , X 1 X 2 , and X 1 2 with P-values < 0.0500 were significant, representing a great contribution to bacterial inactivation response. And the order of contribution was: X 1 > X 2 > X 1 X 2 > X 1 2 , describing a greater contribution of catalyst loading than oxidant addition towards bacterial inactivation in the g-C 3 N 4 /Vis/PMS system. While according to the analysis data in Table S5 , the order of significant contribution was: X 2 > X 1 > X 1 2 > X 2 2 > X 1 X 2 , demonstrating a larger proportion of oxidant addition than catalyst loading towards viral inactivation in the g-C 3 N 4 /Vis/H 2 O 2 system. This opposite trend can be related to a higher sensitivity of Escherichia coli bacteria in comparison of human J o u r n a l P r e -p r o o f Journal Pre-proof adenoviruses to g-C 3 N 4 photocatalysts under visible light irradiation.

Besides, the interaction effect between catalyst loading and oxidant addition (X 1 X 2 ) cannot be neglected, which was further elucidated by 2D contour (Figs. 5c and   6c ) and 3D response surface analysis (Figs. 5d and 6d) . From an individual viewpoint, the increase of catalyst loading and oxidant addition can enhance the microbial inactivation owing to the increase of surface reaction active sites and ROS for photocatalytic disinfection. However, the further increase of either catalyst loading or oxidant addition can slightly deteriorate microbial inactivation because of the shading effect of excess catalyst loading to limit light penetration and the scavenging effect of excess oxidant addition to produce less reactive ROS (Eqs. 10 and 11). From an interactive viewpoint, the increase of catalyst loading along with oxidant addition provides sufficient active sites for generating photogenerated eto capture and activate oxidants (Eqs. 6 and 7), which can reduce and even eliminate their scavenging effects.

The analysis highlights the regulation effectiveness of these two operating parameters, namely catalyst loading and oxidant addition, towards microbial pathogen inactivation in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection. 

The optimal solutions were obtained by a numerical optimization method to achieve the best inactivation performance for representative pathogens in g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection. The two developed models predicted the maximum bacterial inactivation of 4.49-log at 60 min J o u r n a l P r e -p r o o f in the g-C 3 N 4 /Vis/PMS system with a solution of 0.20 g/L and 1.00 mM for g-C 3 N 4 loading and PMS addition, and maximum viral inactivation of 3.91-log at 120 min in the g-C 3 N 4 /Vis/H 2 O 2 system with a solution of 0.19 g/L and 1.00 mM for g-C 3 N 4 loading and H 2 O 2 addition, respectively. Moreover, the verification and stability experiments have been carried out under the optimized conditions (Fig. 7) . The actual bacterial inactivation at 60 min (Fig. 7a) and viral inactivation at 120 min (Fig. 7c) were tested to be ~4.4-log and ~3.8-log, respectively. The actual measurements are very close to the predicted values, identifying the validity of these prediction models.

The disinfection ability of g-C 3 N 4 /Vis/PMS was stable against bacteria ( Fig. 7b) , while the disinfection ability of g-C 3 N 4 /Vis/H 2 O 2 was slightly but acceptably decreased against human viruses after five cycles (Fig. 7d) . After optimization by RSM, the disinfection time for a complete inactivation of Escherichia coli bacteria was reduced by 25%, and the disinfection efficiency for inactivation of human adenoviruses was enhanced by 46%, stating a convenient way to achieve satisfactory disinfection.

Flexible selection of the established system in various waters.

( Fig. 8a) . However, bacterial inactivation was notably inhibited in source water (Fig.   8b ), which might be attributed to the presence of a higher level of dissolved organic matters for occupying active sites and consuming ROS from photocatalysts. An interesting observation was that the g-C 3 N 4 /Vis/PMS system prominently outperformed the g-C 3 N 4 /Vis/H 2 O 2 system for bacterial inactivation (~5 log versus ~4.5 log) in the relatively complex water matrix (Fig. 8b ). This disinfection superiority of g-C 3 N 4 /Vis/PMS is possibly ascribed to a highly selective reactivity of SO 4 •with the electron-rich moieties on the surface of bacterial cell membranes (Wordofa et al., 2017) . Additionally, more than 3-log of human adenoviruses was inactivated in tap water (Fig. 8c) , whereas only 2-log inactivation was achieved in source water by g-C 3 N 4 /Vis/H 2 O 2 (Fig. 8d) , not mention to by g-C 3 N 4 /Vis/PMS. This requires that the established disinfection system is able to bind to human viruses both effectively and selectively in the complex real water matrices.

Given the difficult recovery of powdered g-C 3 N 4 materials from water, the integration of g-C 3 N 4 photocatalysis and membrane separation into multifunctional membranes is considered as promising alternatives for practical water disinfection 

In this study, g-C 3 N 4 -based metal-free visible-light-enabled photocatalytic disinfection with addition of common oxidants (H 2 O 2 versus PMS) was comparatively examined against a broad spectrum of representative pathogens, including

Escherichia coli Bacteria, Human Adenoviruses and Bacillus subtilis spores. The major conclusions can be drawn as follows:

 Although the pristine g-C 3 N 4 material exhibits a good antibacterial activity towards the bacterial indictors under visible light irradiation, it cannot be a universal antimicrobial weapon due to a very limited disinfection activity against the viral and protozoan indicators.

 Simple addition of common low-cost oxidants can significantly enhance the disinfection efficiency and efficacy of the g-C 3 N 4 /Vis system against bacteria and human viruses, while inactivation of some highly resistant pathogens (e.g., bacterial spores and protozoa) is still a challenge. 

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Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing -Original Draft, Writing -Review & Editing, Visualization

Supervision, Project administration, Funding acquisition

Writing -Review & Editing

Xinyi Zheng: Writing -Review & Editing

This study was supported by the National Key R&D Program of China