Red Phosphorus: An Earth-Abundant Elemental Photocatalyst for "Green" Bacterial Inactivation under Visible Light. - PDF Download Free (2024)

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Red phosphorus: An earth-abundant elemental photocatalyst for “green” bacterial inactivation under visible light Dehua Xia, Zhurui Shen, Guocheng Huang, Wanjun Wang, Jimmy C Yu, and Po Keung Wong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00531 • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 23, 2015

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Red phosphorus: An earth-abundant elemental photocatalyst for “green”

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bacterial inactivation under visible light

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Dehua Xia,†,+ Zhurui Shen,‡,

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Wong†,*

‖,+

Guocheng Huang,† Wanjun Wang,‡ Jimmy C. Yu,‡,*, Po Keung

6 7

8

China

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School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR,

Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong

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Kong, China & Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen,

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China

12 13

Key Laboratory for Advanced Ceramics and Machining Technology of Ministry of Education,

School of Material Science and Engineering, Tianjin University, Tianjin 300072, China

14 15

+Equal contribution

16 17

*Corresponding authors:

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Jimmy C. Yu: Tel: +852-3943-6268, Fax: +852-2603-5057, E-mail: [emailprotected]

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Po Keung Wong: Tel: +852-39436383, Fax: +852-2603-5767, E-mail: [emailprotected]

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Abstract

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Earth-abundant red phosphorus was found to exhibit remarkable efficiency to inactivate

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Escherichia coli K-12 under the full spectrum of visible light and even sunlight. The reactive

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oxygen species (•OH, •O2-, H2O2), which were measured and identified to derive mainly from

25

photogenerated electrons in the conduction band using fluorescent probes and scavengers,

26

collectively contributed to the good performance of red phosphorus. Especially, the inactivated-

27

membrane function enzymes were found to be associated with great loss of respiratory and ATP

28

synthesis activity, the kinetics of which paralleled cell death and occurred much earlier than those

29

of cytoplasmic proteins and chromosomal DNA. This indicated that the cell membrane was a

30

vital first target for reactive oxygen species oxidation. The increased permeability of the cell

31

membrane consequently accelerated intracellular protein carboxylation and DNA degradation to

32

cause definite bacterial death. Microscopic analyses further confirmed the cell destruction

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process starting with the cell envelope and extending to the intracellular components. The red

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phosphorous still maintained good performance even after recycling through five reaction cycles.

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This work offers new insight into the exploration and use of an elemental photocatalyst for

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“green” environmental applications.

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INTRODUCTION

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The ever-increasing standards for high-quality drinking water have put much emphasis on

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the removal of pathogenic microorganisms1 due to recent occurrences of severe diseases such as

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pervasive SARS, Ebola virus, avian influenzas and pneumonia.2,3 To address this urgent issue,

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semiconductor-mediated photocatalysis has been extensively studied and proposed to be an

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effective, safe disinfection process. The generated reactive oxygen species (ROS) can serve as

44

powerful oxidants to inactivate various microorganisms, including bacteria, viruses, spores and

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protozoa.4-11 Consequently, much effort has been devoted to searching for and developing cost-

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effective materials for disinfection.4-11 First, multiple titanium-based or novel visible-light-driven

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photocatalysts were synthesized and exhibited disinfection performance, but the difficulty of

48

mass production of such materials strongly limited their application.5-9 Second, earth-abundant

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natural minerals such as natural sphalerite were successfully used as alternatives for bacterial

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inactivation under visible light (VL), but their limited photocatalytic activity still made their

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application controversial.10-11 Most of these efficient photocatalysts are solely metal-based and

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would leak toxic metallic ions due to their instability. For example, plasmonic Ag/AgBr/Bi2WO67

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will release toxic Ag0 or Ag+, and natural magnetic sphalerite10 will leak Zn2+ and Fe2+ due to

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photocorrosion. Therefore, the ideal photocatalyst should be VL active and stable to further

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decrease the economic and environmental costs of potential pollution by secondary metallic ions.

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Elemental phosphorus is versatile and can exist in three forms: white (P4), black and red

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allotropes.12 The most available type is red phosphorus, which has been developed as a functional

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material with fascinating properties in semiconductors,13 rechargeable batteries14,15 and even

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photocatalysts.16-20 For instance, Wang et al. discovered that high-stability red phosphorus could

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constantly generate H2 from water for over 90-h under VL.16,17 Later, Yuan et al. further

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introduced g-C3N4 onto red phosphor surfaces, which led to considerable improvement in the 3 ACS Paragon Plus Environment

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photocatalytic activity for H2 production and CO2 conversion into valuable hydrocarbon fuel

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(CH4).18 Dang et al. also fabricated Ni(OH)2-modified red phosphorous that showed a 1.12-times

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higher photocatalytic activity for H2 evolution than that of Pt-deposited red phosphorous under a

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wide range of VL.19 Very recently, Shen et al. modified amorphous phosphorus into a single

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crystalline fibrous type with higher efficiency and better cycling stability for the degradation of

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rhodamine B.20 The activity, stability and low cost of red phosphorus make it a potential metal-

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free VL-driven photocatalyst. However, it remains unknown whether red phosphorus can cause

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the photocatalytic inactivation of bacteria, which impelled us to conduct the present study.

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Importantly, this elusive amorphous structure of red phosphorus is stable under ambient

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conditions and is eco-friendly as its toxicity level was reported in a chemical journal to be zero.12

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Thus red phosphorus is favorable as a means of “green” water disinfection without potential

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environmental risk.

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The cell membrane is a crucial structure for the survival of bacteria. Many cellular

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functions, such as semi-permeability, respiration, and oxidative phosphorylation reactions, rely

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on an intact membrane structure.21 Cell membrane permeability has frequently been used as an

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indicator of bactericidal effects in conventional photocatalytic disinfection studies by measuring

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the leakage of intracellular ions such as K+ and intracellular enzyme activity such as that of β-D-

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galactosidase.22,23 Bosshord et al. reported that increased bacterial cell membrane permeability

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was caused by the loss of membrane potential resulting from damage to membrane-associated

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proteins, such as the enzymes involved in the respiration chain and ATPase, during solar

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disinfection, which subsequently inhibited the substrate transport system to maintain membrane

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potential.24-26 However, identification of the details behind the decay of membrane function

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enzymes and the subsequent damage to bacterial energy metabolism has rarely been attempted in

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photocatalytic systems.

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We constructed a metal-free elemental material by purifying commercial red phosphorus

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through a hydrothermal method and applied it under VL to inactivate Escherichia coli. The E.

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coli cells were found to be rapidly inactivated under full VL wavelengths and even sunlight by

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red phosphorus. ROS production was quantitatively determined and its roles explored. A

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systematic approach was used to gain an in-depth understanding of the inactivation process by

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tracking the membrane-functionalized enzymes and biomolecule leakage in the face of red

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phosphorous. It is envisaged that the red phosphorus-based disinfection technique will be an

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economically viable solution for practical wastewater treatment.

94 95

EXPERIMENTAL SECTION

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Preparation and characterization of the materials. Commercial red phosphorus (2 g) was

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added to 15 mL H2O, hydrothermally treated at 200oC for 12 h in a 25-mL autoclave to clear the

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oxide layers, then dried in a vacuum oven. Scavenger stock solutions included 1 M isopropanol

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(Riedel-de Haën®, Germany) for •OH, 1 M K2Cr2O7 (Merck, Germany) for e-, 1 M sodium

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oxalate (Fuchen, China) for h+, 100 mM Fe(II)-EDTA for H2O2 (prepared with FeSO4 and

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Na2EDTA, Ajax Chemicals, Australia) and 100 mM TEMPOL (Fuchen, China) for •O2-.7,10,11 X-

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ray diffraction patterns for the catalysts were recorded on a Rigaku SmartLab X-ray

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diffractometer with Cu Kα1 irradiation (λ = 1.5406 Å, 40 kV, 40 mA). Ultraviolet (UV)-visible

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diffuse reflectance spectra of the powders were obtained on a Varian Cary 500 UV-vis

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spectrophotometer equipped with an integrating sphere diffuse-reflectance accessory. The

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morphologies of the catalysts were recorded by a field emission scanning electron microscope

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(FEI, Quanta 400 FEG).

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ROS analysis. pCBA is a well-known specific probe to detect •OH due to its high reactivity 5 ACS Paragon Plus Environment

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with •OH (k = 5.2 × 109 M−1s−1). FFA is extremely reactive toward 1O2 (k = 1.2 × 108 M-1s-1) and

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adequately specific for 1O2. The decay of pCBA and FFA in the collected filtrate was analyzed

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with a high-performance liquid chromatography system (Dionex U3000, USA) equipped with a

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Thermo Scientific Hypersil BDS C18 column.27 The production of •O2- was quantitatively

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analyzed by detecting the decrease in the concentration of nitro blue tetrazolium (NBT, k = 5.88

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×104 M-1s-1) at a wavelength of 259 nm with a UV-Vis spectrophotometer (LabTech).28 H2O2 was

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analyzed on a Hitachi F-4500 fluorescence spectrophotometer based on the reaction of H2O2 with

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courmarin to form a high fluorescent compound (7-hydroxylcoumarin, 456 nm).10

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Photocatalytic bacterial inactivation. A 300-W Xenon lamp with a UV cutoff filter (λ < 400

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nm) as the light source with light intensity fixed at 193 mW cm-2 was used. The experiments

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under sunlight were conducted on sunny days (October 2014, between 15:00-16:30) at The

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Chinese University of Hong Kong with timely monitoring of light intensity. The experiments

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were conducted in phosphate buffer solution (PBS, pH 7.0), and the detailed procedure is

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described in the supporting information.

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Enzyme activity assay. Bacterial membrane permeability was defined following the o-

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nitrophenyl-β-D-galactosidase (ONPG) assay kit procedure (Cayman Chemical Company,

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USA).9,23 Cellular respiration ability was monitored with 2,3,5-triphenyl tetrazolium (TTC; Cat.

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No. 118388, Bailingwei, China) as the final electron acceptor to form a red supernatant. The

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absorbance of the supernatant at 485 nm was recorded on a Cary 5E spectrophotometer.21 The

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ATP synthesis ability of the treated cells was monitored with the BacTiter-GloTM Microbial cell

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viability assay (G8230, Promega Corporation, USA).21

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Biomolecule oxidation assay. The residual protein concentration was measured with the

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Bradford assay (SK3041, Sangon Biotech, China). Carbonylated protein levels were measured

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using an OxiSelect Protein Carbonyl ELISA Kit (STA-310, Cell Biolabs, USA). Chromosomal

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DNA was extracted using an Ezup Column Bacteria Genomic DNA Purification Kit (SK8255,

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Sangon Biotech), then verified by DNA agarose gel electrophoresis (0.6% agarose gel at 100 V

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for 30 min in 1 × TAE buffer).23

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Microscopic observations. (i) Fluorescence staining: Aliquot samples were concentrated and

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stained with a fluorescent dye mixture prepared with the LIVE/DEAD BacLight Bacterial

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Viability Kit (L7012, Molecular Probes, Inc., USA) and observed with a light microscope

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(ECLIPSE 80i, Nikon, Japan).10,23 (ii) Scanning electron microscope observation: Aliquot

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samples were collected and transferred onto poly-lysine-coated coverslips, pre-fixed in 5%

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glutaraldehyde solution, washed with PBS (0.1 M, pH 7) and post-fixed with osmium tetraoxide

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(2%, E. M. grade, Fort Washington, USA). After dehydration in a graded series of ethanol (50, 60,

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70, 80, 90, 95 and 100%), the critical-point dried specimens were coated with gold and observed

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with a scanning electron microscope (Joel-JSM-6301-F).23

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RESULTS AND DISCUSSION

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Photocatalytic inactivation. The commercial red phosphorus was purified simply with a

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hydrothermal method before use (Materials preparation and characterization section). The

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resulting product was amorphous red phosphorus with wide diffraction peaks, which presented as

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2-10-µm microparticles (Figure 1). Ascribed to the imperfections and disordering on its surface,

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amorphous red phosphorus possesses better VL adsorption ability with a narrower band gap (1.42

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eV), indicating its potential excellent VL-driven photocatalytic activity. The valence and negative

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bands of this catalyst were determined to be +0.8 and -0.62 eV, respectively (inset of Figure 1B).

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Figure 2 shows the inactivation kinetics of E. coli by 100 mg L-1 red phosphorus, in which a cell

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loss of 2×107 cfu mL-1 was achieved within 90 min of VL irradiation. Control experiments

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confirmed that E. coli was not inactivated by red phosphorus alone (dark control) or by light

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alone (light control). The efficiency was faster than that of the extensively studied metal-free

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C3N49 and even metal-based photocatalysts like B-Ni-co-doped TiO2,29 which require several

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hours of irradiation.

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The inactivation kinetics were analyzed by fitting the data with a “shoulder + log-linear +

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tail” model for bacterial inactivation. They were characterized by the presence of an initial lag

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phase followed by a first-order reduction in cell viability with respect to time (R2 = 0.99 when log

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inactivation was plotted versus time; inset of Figure 2A), as shown in Eq. 1 proposed by

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Geeraerd et al.26

165 ,

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(1)

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where N0, N(t) and Nres are the initial, survival and residual number of cells (log, in cfu),

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respectively; Kmax is the inactivation rate; and S is the shoulder length (min). When the UV filter

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was removed (UV typically comprises about 4% of the total light emitted from a Xenon lamp),

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the inactivation was only slightly enhanced. These results suggest that red phosphorus inactivates

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bacteria primarily due to VL irradiation.

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The LED lamp is considered to be the next generation lighting source due to its long life-

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span and energy-efficient propertyies.30 To support the finding that the reaction proceeds through

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light absorption within red phosphorus, the dependence of the inactivation efficiency on the

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wavelength of incident light (the spectrum is shown in Figure S1) was investigated. As shown in

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Figure 2B, the inactivation efficiency decreased from 6 log reduction to 3 log reduction of E. coli 8 ACS Paragon Plus Environment

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as the wavelength of the light source increased from blue to red, matching well with that of the

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absorption in the optical spectra. Even the light with the longest wavelength (610-650 nm) was

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able to activate red phosphorus, suggesting that the photocatalyst could effectively use the full

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spectrum of VL for E. coli destruction. Similarly, all of the inactivation data also fit well with the

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three-stage kinetics and the calculated decreases in inactivation rates from 0.8 to 0.4 min-1 with

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the increasing wavelength of the irradiated light. The photon energy of a blue LED lamp is higher

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than that of other colored lamps. The wavelength of the blue LED lamp overlaps with the steep

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absorption edge of the red phosphorus, resulting in the best bactericidal performance.30

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Although artificial light-based photocatalysis can obtain excellent efficiency, solar-driven

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photocatalysis is more economically attractive in terms of energy consumption and hence has

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more application significance.31 Therefore, the inactivation of E. coli K-12 was also performed

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under sunlight irradiation in this study (Figure 2A). Because the spectrum of sunlight contains

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small amounts of UV light (Figure S2) and the temperature of the mixture increased from 25 to

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28oC, a minor reduction of E. coli (0.5 log10 cfu mL-1) was observed in the control experiment.

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When the suspension included 100 mg L-1 red phosphorous, a higher inactivation rate of 0.72

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min-1 was achieved. Complete inactivation was obtained within 60 min, which was much faster

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than that observed under Xenon lamp irradiation. This increased inactivation efficiency could be

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attributed to the appearance of UV and higher solar irradiance under sunlight.31

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These favorable results may indicate that red phosphorus can work as an alternative for

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disinfection under solar light. Moreover, red phosphorus is easily available, and the unit price of

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commercial red phosphorus on the market is generally low. For example, some available red

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phosphorus products with a purity of 90% in the China market are currently sold at USD$3,268

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(RMB20,000) per ton. To extend the practical use of red phosphorus, the consideration of the

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toxicity of red phosphorus toward human is needed. The study of Young reported that red phosphorus 9 ACS Paragon Plus Environment

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in its pure form, would not cause a significant health hazard, as its toxic level is set to be zero (none

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or very low toxicity).12 However, red phosphorus may contain white phosphorus, exposure to these

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contaminated red phosphorus by human, may result in adverse effects on health, including irritation

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of the skin, eyes, lungs, and gastrointestinal tract.12 Mamyrbaev et al. have reported the effect of red

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phosphorus for rat’s liver, mainly caused by the white phosphorus contaminant.32 To prevent this, the

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possible white phosphorus impurity can be cleared by treating the red phosphorus using sodium

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hydroxide solution. In addition, the red phosphorus is flammable, and its burning will generate the

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concentrated smoke, which will be harmful for the respiratory tract.33,34 So when we use it, the red

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phosphorus should avoid to mix with the strong oxidant or on fire. Besides the possible downside

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above, the red phosphorus is a relative safe photocatalyst when using.

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Role of various reactive species. The above results suggested that the E. coli inactivation was

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caused by the photocatalytic production of ROS by red phosphorus. The reactive species were

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generated by separation of electrons and holes, whose critical roles were determined by the

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addition of scavenger chemicals (Figure 3A). Scavenger is a useful method to determine

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bactericidal contribution for each reactive species (charged or oxidative), including sodium

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oxalate for h+, isopropanol for •OH, Cr(VI) for e−, TEMPOL for •O2-, and Fe(II) for

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H2O2.7,9,11,23,29 Before conducting the experiment, the applied concentration of each scavenger

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was optimized to ensure their maximum scavenging effect but did not cause any inactivation to

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the bacterial cell.7,9,11,23,29

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Interestingly, after adding Cr(VI) to capture e- in the suspension, the inactivation process

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was elevated a little mainly due to enhancement of the h+-e- separation efficiency, thus kinetically

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favoring the remaining h+ to inactivate cells in the system. Note that photogenerated h+ in the

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valence band was significantly involved in the reaction, as shown by the obvious inhibition of

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bacterial inactivation after the addition of oxalate. When argon gas was bubbled through the 10 ACS Paragon Plus Environment

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mixture to remove the residual oxygen, the observed inactivation of inhibition (Kmax = 0.13 min-1)

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may have been caused by insufficient e- trapping by the limited O2 and thus easier recombination

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of the e--h+ pair (Figure 3A).

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Among the ROS, 1O2 had a negligible role in E. coli inactivation. First, when excess 1O2

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scavenger L-histidine (1 mM) was added, there was no significant change in the inactivation

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kinetics (Figure 3A). Second, no measurable degradation of FFA, a probe for 1O2, was observed

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over the experimental time scale (Figure 3B). The results indicated that the weak contribution of

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1

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possesses a 0.65-eV oxidation potential and an approximately 3-µs lifetime in water. Its bubbles

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can effectively inactivate E. coli through an oxygen gradient inside and outside of the bubble,

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allowing 1O2 to solvate and diffuse through the aqueous solution until it reacts with the target

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organism.35

O2 was mainly attributable its non-formation. Actually, generating through energy transfer, 1O2

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•O2- and •OH were generated through e- transfer and are well known to have great

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oxidative potential for inactivating E. coli in nonselective reactions.7,10 With the addition of (1

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mM) TEMPOL (•O2- scavenger) or (0.5 mM) isopropanol (•OH scavenger), the inactivation of E.

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coli was virtually prohibited compared with no scavenger addition, confirming that both the

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determined •OH and •O2- were primary ROS accounting for the bacterial inactivation process

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(Figure 3A). Upon VL excitation with NBT sodium salt (0.15 mM, absorbance peak at 260 nm),

244

a probe specifically reacting with •O2- to form a precipitate, a decrease in absorbance at 260 nm

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after photocatalytic degradation was observed (Figure S3).27 The calculated concentration of

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steady-state •O2- was approximately 1.05 × 10-9 M (Figure 3C). The results obtained with

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terephthalic acid (0.4 mM) as the •OH probe showed increasing fluorescence intensities emitted

248

at 440 nm, confirming the involvement of •OH radicals in the photocatalytic pathway (Figure

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S3).26 The steady-state •OH concentration measured using pCBA was approximately 3.27 x 10-14

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M (Figure 3D). Only limited amounts of ROS were determined in the presence of scavengers,

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suggesting that the amounts of the scavengers added were adequate and efficient to quench the

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corresponding ROS.

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H2O2 is stable, with a half-life of several days in pure water, and can diffuse across the cell

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membrane into the cytoplasm to induce long-range bactericidal effects.23 The involvement of

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H2O2 was affirmed by the significant decrease in the inactivation efficiency after adding Fe(II)-

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EDTA (Figure 3A), which can be thermodynamically generated by either two-electron reduction

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of surface-adsorbed O2 (0.68 V vs SHE) or two-electron oxidation of surface-adsorbed H2O (1.78

258

V vs SHE) in the conduction band (CB).36 The absorbance (at 465 nm) of the fluorescent

259

compound (a product of coumarin reacting with H2O2) against VL irradiation time demonstrated

260

that H2O2 accumulated to 7 mM (Figure 3E).10 Similarly, with the addition of Fe(II)-EDTA, no

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obvious H2O2 was detected, suggesting that all of the H2O2 was quenched.

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The leading bactericidal effect of •O2- rather than •OH in Figure 3A was expected

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because •O2- production is thermodynamically favored by directly reducing O2 into •O2- (E0

264

(O2/•O2- = -0.33 eV vs NHE) by the CB (-0.61 eV vs NHE) of red phosphorous. •OH generation

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is thermodynamically forbidden as its valence band (+0.8 eV vs NHE) cannot directly oxidize

266

H2O/OH- (E0(OH/•OH = 2.38 eV vs NHE)).9,16,17 Therefore, the determined •OH and H2O2 were

267

likely generated from the reductive site, as electrons in the CB can reduce the absorbed oxygen to

268

generate •O2- and subsequently undergo facile disproportionation to produce •OH and H2O2.23,37

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To confirm this pathway, a partition system (a semipermeable membrane with a molecular weight

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cutoff of 12000 Da to separate bacterial cells and the photocatalyst) was used to block direct

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contact between the bacterial cell and thus exclude the function of h+. Only some of the •OH and

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H2O2 diffused across the membrane as a bactericide.23 Inactivation of 5 log10 cfu mL-1 was still

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achieved, indicating that the e- of the catalyst could induce enough ROS to function. Cr(VI) was

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added to the suspension to completely eliminate the photogenerated electrons and their derived

275

reactive species. Under this condition, inactivation was totally inhibited, in contrast with the

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partition system without scavengers (Figure 3A). No obvious signals were displayed in the inner

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part of the membrane with the addition of probes (•OH and H2O2), indicating that the diffusible

278

ROS were fully eliminated. These results are direct evidence that •OH and H2O2 were generated

279

in a reductive way in the CB of red phosphorous. Therefore, the photogenerated e- and its derived

280

ROS, •O2-, are suggested to be the dominant effective species and induce the decomposition of

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microorganisms in the present system. Since the contribution for each ROSs revealed by

282

scavenger study is semi-quantitative, the proposed mechanism for the photocatalyst is

283

conservatively reasonable, which still needs more direct evidences to identify.

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Process of bacterial cell damage

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Destruction of bacterial metabolism. The bacterial cell membrane contains essential protein

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components such as the permease system and respiratory chain, which transport substrates and

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generate energy with functionalized electron chains, playing a vital role in bacterial

288

metabolism.22,23 Therefore, it is reasonable to identify the inactivation patterns of membrane-

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bounded proteins during photocatalysis because ROS oxidation may lead to conformational

290

changes in membrane architecture, and subsequent membrane dysfunction is the original cause

291

underlying cell death.22,23

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Respiration is fundamental for cellular energy production through oxidative

293

phosphorylation of substrate with O2 as the final electron acceptor. TTC in its oxidized form is

294

reducible to 1,3,5-triphenyl formazan (insoluble red precipitate) by the cytochrome systems of

295

bacteria during respiration. It is frequently used as an artificial acceptor to assess metabolic 13 ACS Paragon Plus Environment

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activities in various microorganisms.38,39 Glucose was used as the electron donor in this assay.

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Comparatively, light or catalyst alone did not have any significant effect on the reduction of TTC

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activities. When E. coli cells were treated for various periods of time, the kinetics (Figure 4A)

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revealed an initial lag phase for 15 min, then drastic loss of respiratory activity thereafter. Failure

300

to reduce TTC with the following treatment implies that the damaged cell membrane could no

301

longer generate or maintain a sufficiently negative redox potential. The spatial organization of the

302

electron mediators had been disrupted, resulting in short-circuiting of the electron transport

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pathway from substrate to oxygen. Interestingly, the rate of loss of respiratory activity (30 min,

304

56.6%; 40 min, 9.3%), was much faster than that of cell viability (30 min, 71.4%; 40 min, 29.7%)

305

in Figure S4 at a cell density of 108 cfu mL-1. This comparison may suggest that some injured

306

cells in the initial phase can still self-repair their respiratory ability and then supply enough

307

energy for regrowth after culture in nutrients, whereas longer destruction causes irreversible

308

death.

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A damaged respiration chain directly inhibits substrate transportation, subsequently

310

causing a rapid decrease in proton motive force-dependent and -independent transport across the

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cell membrane, which is the driving force for ATP synthesis.11 As the material for storing both

312

vital and direct energy, the synthesis of ATP is directly responsible for cellular metabolic activity.

313

Therefore, ATPase (the ATP synthesis enzyme) activity was monitored to identified damaged

314

metabolism, which is in proportion to the elevated fluorescence intensity after luciferin oxidizes

315

into fluorescent oxyluciferin.25,40 The accumulated ATP content in the cells decreased

316

instantaneously after treatment (the amount of ATP at an incubation time of 5 min is shown in

317

Figure 4B): untreated cells contained almost 30 × 10-7 pmol ATP cell-1, which then decreased to

318

14.1 × 10-7 pmol ATP cell-1 after 30 min and to almost 0 pmol ATP cell-1 at 60 min. The energy

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consumption rate exceeded the production rate in the cells undergoing a prolonged reaction. With

320

the further addition of 0.1 mL nutrient broth to culture the treated cells, the amount of ATP

321

gradually increased, suggesting that the damaged cells could effectively make use of the added

322

nutrients for ATP generation. Particularly in cells undergoing photocatalytic treatment between 0

323

and 10 min, there was a slight decrease in ATP in the initial incubation period. Bosshard et al.

324

suggested that cells may temporarily use large amounts of energy for vigorous metabolism, such

325

as membrane self-repair or transmembrane potential maintenance.24,25 However, after a long

326

period of treatment, the lysed cells lost almost all ability to synthesize ATP even after 60 min

327

incubation in broth. The calculated ATP generation rates for treated cells after various periods is

328

shown in Table S1. After the initial lag phase, it is noteworthy that the rate of ATP generation

329

rapidly decreased to ~0.11 pmol min-1 cell-1 in 40 min, only 20.4% of the initial value. This is

330

consistent with the 23.7% of cells that survived the inactivation kinetics at a cell density of 108

331

cfu mL-1 (Figure S4), but is less that of the 9.3% of cells with loss of respiration ability. This may

332

be evidence that facultative E. coli can still synthesize ATP through other methods, such as

333

substrate fermentation-substrate level phosphorylation, when the functionalized electron chains

334

for aerobic respiration have been disturbed.40 However, given that all metabolized pathways are

335

damaged, cells will ultimately die because of the lack of energy available for regrowth, repair and

336

proliferation.

337

Bacterial decomposition and biomolecule leakage. . The damaged bacterial energy metabolism,

338

which was insufficient to maintain membrane potential, was accompanied by a quick change in

339

the permeability of the cell membrane.23 ONPG hydrolysis is an effective method to observe a

340

change in cell permeability. The yellow-colored product o-nitrophenol (420 nm) only forms when

341

ONPG penetrates the cell and reacts with the intracellular E. coli enzyme β-D-galactosidase

15 ACS Paragon Plus Environment

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342

(Figure 5A).23 During the treatment, ONPG hydrolysis in permeabilized cells decreased quickly

343

from 39.1 to 17.9 nmol min-1 mL-1 within the first 30 min and then was totally lost, indicating

344

that the β-D-galactosidase was degraded, which coincided well with the inactivation kinetics of 8

345

log10 cfu mL-1 (Figure S4). In contrast, no measurable ONPG hydrolysis was observed in the non-

346

permeabilized cells because the traverse of ONPG across the cell membrane barrier is limited in

347

intact cells depending on the activity of permease.24,25 Therefore, the observed cell permeability

348

increased slightly in the first 15 min and then sharply within the next 15-30 min because the

349

counteraction between the increase in permeability and the decrease in permease activity in the

350

transport system of the lysed cells was interrupted. These results also confirm that the presence of

351

the initial shoulder was attributable to the time required for the oxidant to disrupt the cell

352

envelope components. In contrast, the diffusion of leaking K+ during the treatment also

353

confirmed this.10,23 The sharply increasing amount of K+ leaking from the cytoplasm during the

354

initial 30 min indicated the aggravated damage to and elevated permeability of the cell

355

membranes and coincided well with the exponential disinfection period (Figure S5). The great

356

diffusion of K+ may be attributable to the inactivation of the Na+-K+ pump because the cellular

357

metabolism collapsed and could no longer generate enough ATP.24,25

358

The processed bacterial membrane integrity was also visually observed through

359

microscopy in Figure 6.10,23 After staining with a dye mixture, except for the orange fluorescence

360

emitted by red phosphorous, the red fluorescent cells increased in number to replace the green

361

fluorescent cells after prolonged treatment, indicating cell membrane rupture (Figure 6A). As

362

shown in Figure 6B, untreated E. coli cells displayed plump rod shapes with an intact cell

363

envelope. After 30 min of treatment, the cell surface started to wrinkle and become rougher. By

364

90 min, hollows and holes occupied almost the entire cell surface, indicating the extreme

365

destruction of the cell envelope. 16 ACS Paragon Plus Environment

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366

The damaged membrane motivated us to investigate the leakage of cytoplasmic substances,

367

the building blocks of bacteria, particularly the protein side chains lysine, arginine and threonine,

368

which are vital for cell construction and biochemical function.23 The protein content of 10 mL of

369

treated cells (108 cfu mL-1) was maintained at around 170 µg mL-1 in the initial 60 min but then

370

decreased to 0 µg mL-1 after 180 min of treatment (Figure 5B), confirming the leakage of protein

371

during the disinfection process. The protein carbonyl level was further monitored to assess the

372

oxidative damage of the leaked proteins. Conversely to the pattern of protein leakage, the

373

carbonyl protein level started to rise until 60 min of reaction, implying that the oxidative defense

374

enzymes (SOD, CAT) were highly broken by that time, following the great destruction of

375

proteins.10,11 Coincidentally, a remarkable decrease in both SOD and CAT activities was observed

376

after 30 min and continued thereafter (Figure S6), further confirming that the defense capacity

377

was overwhelmed by ROS at the initial stage and then decomposed rapidly. Damage to the

378

antioxidative defense system will result in the fragmentation of proteins, release of ions and

379

generation of protein carbonyl derivatives, among others.23 Therefore, the elevated protein

380

oxidation level and fragments suggested a tendency to aggregate at the end of the process.

381

However, cells still can self-repair and regrow even when their proteins are damaged, and

382

the occurrence of a viable but non-culturable (VBNC) state in bacteria may dramatically

383

underestimate the health risks associated with drinking water.41,42 Only severe damage or loss of

384

chromosomal DNA is lethal to the cells. The leakage and decomposition of genomic DNA could

385

be observed in Figure 5C because the fluorescent intensity of the DNA bands decreased within

386

the first 2 h of the process, continued to fade thereafter, and then totally disappeared after 6 h. In

387

contrast, no leakage of genomic DNA was observed in the control.

388

The above evidence collectively suggests the photocatalytic inactivation mechanism of E.

389

coli by red phosphorous under VL. E. coli inactivation by ROS has been delineated as a sequence 17 ACS Paragon Plus Environment

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Page 18 of 32

390

of nonselective reactions with the cell envelope as the first major target. As shown in Figure 7,

391

ROS first oxidize cell membrane-associated proteins such as enzymes of the respiratory chain

392

and ATPase, and thus the bacterial energy metabolism becomes insufficient to maintain the cell

393

membrane potential. Loss of the cell membrane potential results in an increase in cell membrane

394

permeability, followed by the leakage and rapid decay of cytoplasmic contents such as proteins

395

and DNA, ultimately leading to cell death with no regrowth.21

396

Stability of red phosphorus. To evaluate the stability of red phosphorous, five cycling reactions

397

of bacterial inactivation were allowed to proceed. The catalyst was recycled through a 2-µm filter

398

paper and dried in a vacuum oven before reuse. The results showed a virtually constant pattern of

399

cell loss over the duration of the reactions under VL irradiation, indicating the great potential of

400

red phosphorus to be used in practical water disinfection. Only slight deterioration was observed

401

in the fifth cycle (Figure S7). After five cycles, the quantity of red phosphorus had decreased, but

402

its chemical nature remained unchanged. The loss of activity was mainly ascribed to mass loss in

403

the recycling process. Unknown organic intermediates generated in the photocatalytic bacterial

404

inactivation could also have contaminated the red phosphorus and decreased its activity.10 To

405

further confirm its photostability, the possible eluted phosphate ions in ultrapure water were

406

measured with ion chromatography. The amount of residual HPO43-/PO43- in the first to fifth

407

cycles stayed at a low level and remained almost constant at 80 µg L-1, indicating that the catalyst

408

was maintained well during the reaction. The determined phosphate ions may also have included

409

leakage from the phospholipid bilayer of the constituent cell membrane after oxidation. No

410

obvious bactericidal effect occurred after 8 h of stirring (under VL) in the presence of an equal

411

dose of eluted PO43-. These results further suggest that the total decrease in cell viability should

412

be attributed to the attack of the photo-generated ROS and that the phosphate ions had no toxic

413

effect on the bacterial cells. 18 ACS Paragon Plus Environment

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414 415

Acknowledgement. This work was supported by grants from the General Research Fund (GRF

416

476811) of the Research Grants Council of the Hong Kong SAR Government, Hong Kong SAR,

417

China, the Theme-Based Research Scheme (T23-407/13-N) of the University Grant Committee,

418

Hong Kong SRA Government, Hong Kong SAR, China, and the Shenzhen Basic Research

419

Scheme (JCYJ20120619151417947), Shenzhen, China. P.K. Wong is also supported by the

420

CAS/SAFEA International Partnership Program for Creative Research Teams of Chinese

421

Academy of Sciences, China.

422 423

Supporting Information Available. Additional detail information including setup of ATP

424

generation rate, light spectrum of LED lamp, Real-time environmental conditions, determination

425

of •OH and •O2-, cell viability of 8 Log10 cfu/mL, K+ leakage, SOD and CAT activity and cycle

426

runs experiments. This material is available free of charge via the Internet at http://pubs.acs.org/.

427 428

References

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549 550 551

Figure 1. A typical (A) XRD pattern, (B) UV-vis DRS spectrum, (C) SEM image, (D) HRTEM

552

image of red phosphorus.

553 554 555 556 557 558

25 ACS Paragon Plus Environment

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Page 26 of 32

Cell density (Log 10cfu/mL)

8

6

Xenon lamp Xenon lamp + UV Filter Sunlight Dark control Light control (Sunlight) Light control (Xenon lamp)

A 4

k = 0.33 min-1 R2 = 1

2

k = 0.72 min-1 R2 = 0.99 0 0

20

40

60

80

100

120

Time (min)

8

Cell density (Log10cfu/mL)

7 6

B

5 4 3

-1

2 1 0 0

559

2

White k = 0.06 min , R = 0.99 -1 2 Blue k = 0.08 min , R = 0.98 -1 2 Green k = 0.08 min , R = 0.99 -1 2 Yellow k = 0.05 min , R = 0.99 -1 2 Red k = 0.04 min , R = 0.99

60

120 Time (min)

180

240

560

Figure 2. (A) Photocatalytic inactivation kinetics under Xenon lamp and sunlight irradiation in

561

the presence of red phosphorous; (B) Photocatalytic inactivation efficiencies under different

562

singlet colored LED lamp irradiation (red LED 610–650 nm, yellow LED 570–620 nm, green

563

LED 470–570 nm, and blue LED 440–490 nm).

26 ACS Paragon Plus Environment

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Environmental Science & Technology

8

Cell density (Log10cfu mL-1)

A 6

Fe-EDTA TEMPOL Isopropanol Oxalate Cr(VI) L-histine Argon Partition+Cr(VI) Partition No scavenger

4

2

20

40

60

80

100

120

Time (h)

1.0

0.7 Red phosphorous Red phosphorous + L-histine

0.8

Red phosphorous Red phosphorous + TEMPOL

0.6

-

1-[NBT]/[NBT]0

1-[FFA]/[FFA]0

0.6

B

0.4

0.2

-9

[•O2 ]=1.04875×10 M

0.5 0.4 0.3

C

0.2 0.1 0.0

0.0 -0.1 0

20

40

60 80 Time (min)

100

120

0.8 Red phosphorous Red phosphorous + Isopropanol

0.7

40

60 J1

80

100

120

[•OH]=3.267×10

Red phosphorous Red phosphorous + Fe(II)EDTA

6

0.6

5

-14 M

0.5

4 H2O2 (µ µM)

1-[pCBA]/[pCBA]0

20

7

0.4

D

0.3

3

E

2

0.2 1 0.1 0 0.0 0

564

20

40

60

80

100

-1

120

20

Time (min)

40

60 Time (min)

80

100

120

565

Figure 3. (A) Photocatalytic inactivation efficiencies, (B) Level of 1O2, (C) Level of •O2-, (D)

566

Level of •OH, (E) Level of H2O2, were measured with red phosphorus in the presence of various

567

scavengers (L-histidine, 0.5 mM; Fe-EDTA, 0.1 mM; Cr(VI), 0.05 mM; TEMPOL, 1mM;

568

Sodium oxalate, 0.5 mM; Isopropanol, 0.5 mM) under a Xenon lamp irradiation.

27 ACS Paragon Plus Environment

Environmental Science & Technology

60

100

Relative respiration rate (%)

Page 28 of 32

0 min

50

ATP (10-7pmol per cell

80 Dark Control

60

Light Control Treatment

40

A

20 0

15

30

45 60 75 Time (min)

90 105 120

40

40 min

10 min

50 min

20 min

60 min

30 min

30

B

20 10 0

10

20

30

40

50

60

Incubation time after treatment (min)

569 570

Figure 4. (A) Bacterial cell respiration activity assay during photocatalytic inactivation; (B) The

571

ATP generation potential after photocatalytic inactivation for 0, 10, 20, 30, 40, 50 and 60 min, the

572

inactivated bacterial cells were mixed with 10% NB and incubated at 37 oC. E. coli K-12

573

concentration is 1.2×108 cfu mL-1.

28 ACS Paragon Plus Environment

Permeability (%)

100 200

25

Protein concentration

Permeabilized Not Permeabilized

20

30

A

20

80 60 40

10 20 0

15 30 45 60 75 90 105 120

150

15 100

B

50

5 0

0 0

30

60

90

120

Time (min)

C

29

10

150

180

Carbonyl protein

40 Permeability (%)

ONPG hydrolysis rate (nmol min-1 mL-1)

50

Environmental Science & Technology

Page 30 of 32

581 582 583

Figure 6. Fluorescence microscopic images of E. coli K-12 (2×107 cfu/mL, 50 mL) and SEM

584

images after photocatalytically treated with red phosphorus at (A) 0, (B) 60, (C) 90 min.

585

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586 587

Figure 7. Illustration summarizing the proposed bactericidal mechanism of red phosphorous

588

under visible light: Red phosphorous quickly generate ROSs; ROSs subsequently inhibits

589

bacterial surface metabolism and oxidize intracellular components.

590 591

31 ACS Paragon Plus Environment

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592

TOC (Graphical Abstract)

593

594 595 596

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FAQs

What is red phosphorus used for? ›

Red-P is also used in electroluminescent coatings, in flame retardants for polymers, in the production of safety matches, pyrotechnics, incendiary shells, smoke bombs, and tracer bullets; as well as in the manufacture of fertilizers, pesticides, and semiconductors.

How do you prepare red phosphorus? ›

Red phosphorus is prepared by heating white phosphorus to 4820F in an inert atmosphere (like in presence of nitrogen) and in presence of iodine (I2) as catalyst.

What is the most active photocatalyst? ›

The most widely used photocatalyst is the polycrystalline titanium dioxide, TiO2, due to its strong catalytic activity, long lifetime of electron hole–pairs, high (photo)chemical stability in aqueous media and in a large range of pH (0–14), low cost (due to the abundance of Ti in the earth's crust), and harmlessness.

What's the difference between white phosphorus and red phosphorus? ›

The two main forms of phosphorus are white phosphorus and red phosphorus. White phosphorus is a poisonous waxy solid and contact with skin can cause severe burns. It glows in the dark and is spontaneously flammable when exposed to air. Red phosphorus is an amorphous non-toxic solid.

Why is red phosphorus illegal? ›

It is unlawful for any person knowingly or intentionally to possess or distribute red phosphorus, white phosphorus, or hypophosphorous acid, knowing, or having reasonable cause to believe, these substances will be used to illegally manufacture methamphetamine.

Is red phosphorus harmful to humans? ›

Ingestion: Red phosphorous is consider non-toxic in its pure form. However, nausea, vomiting, abdominal pain or garlic odor on breath will indicate poisoning by the yellow allotrope. DO NOT INDUCE VOMITING.

What is the natural source of red phosphorus? ›

Red phosphorus are found in which material? It is not found in free state but in nature is available in certain minerals called phosphates. It is 11th most abundant element in earth's crust. It is also found in human body.

What happens if you put red phosphorus in boiling water? ›

Red phosphorus can react with hydrogen to produce phosphine - but not with hot water. White phosphorus can react with sodium hydroxide (a chemical he would have had) but you can see he throws in a red powder, rather than a white substance.

How do you activate red phosphorus? ›

Red phosphorus ignites in the air at temperatures above 240°C whereas white phosphorus ignites air at low temperatures such as 50°C. This is produced via heat-treating white phosphorus where this is produced via heating phosphorus rock in an electric or fuel-fired furnace, i.e., in the presence of carbon and silica.

What is the drawback of photocatalyst? ›

But some of their drawbacks such as, hard to recover the powder photocatalyst, leakage of metals which further contaminate the water and difficulties in handling, are limiting their practical applications [19] [20] [21][22].

What is photocatalysis in simple terms? ›

Photocatalysis is a chemical reaction that takes place in the presence of a photocatalyst and suitable light. In other words, it is a reaction that uses light to generate a pair of excited electrons and positive holes to induce redox reactions as the first step with both positive and negative Gibbs-energy change.

Are photocatalysts reusable? ›

The photocatalytic MoS2 films are easily recoverable and reusable. The films exhibit high structural and chemical stability even after 5-cycles of the degradation studies.

What is the advantage of red phosphorus? ›

Red phosphorus has many advantages such as good flame retardancy and low in dosage, but it is flammable, easy to absorb moisture in the air and release harmful gas when it burns. Therefore, the application of red phosphorus is restricted [48,49].

What is the function of red phosphorus? ›

Red Phosphorus is an excellent flame retardant, particularly in thermoplastics (e.g. polyamide) It is utilised in the manufacture of drugs. It may be used as an elemental photocatalyst to produce Hydrogen ( ) from water ( H 2 O ).

What happens when red phosphorus is heated? ›

When red phosphorus is heated a series of phases of black phosphorus is formed which does not react with air upto 673 K. Q.

What is red phosphorus used for in the military? ›

Military Applications

In Army field use, red phosphorus smoke is deployed explosively from grenades and mortar shells. Suggested Citation:"4 - Red Phosphorous Smoke." National Research Council.

What does red phosphorus do in Breaking Bad? ›

In the 2008 pilot of the crime drama television series Breaking Bad, Walter White poisons two rival gangsters by adding red phosphorus to boiling water to produce phosphine gas. However, this reaction in reality would require white phosphorus instead, and for the water to contain sodium hydroxide.

What does phosphorus do for the body? ›

Phosphorus is needed for the growth, maintenance, and repair of all tissues and cells, and for the production of the genetic building blocks, DNA and RNA. Phosphorus is also needed to help balance and use other vitamins and minerals, including vitamin D, iodine, magnesium, and zinc.

What happens when you burn red phosphorus? ›

Red phosphorus is a flammable solid but does not ignite spontaneously on exposure to air. At high temperatures (~300 °C), red phosphorus is converted to the white form. White phosphorus reacts with a number of substances to form explosive mixtures.

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