Rapid Aerobic Inactivation and Facile Removal of Escherichia coli with

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Rapid Aerobic Inactivation and Facile Removal of Escherichia coli with Amorphous Zero-valent Iron Microspheres: Indispensable Roles of Reactive Oxygen Species and Iron Corrosion Products Hongwei Sun, Jian Wang, Yao Jiang, Wenjuan Shen, Falong Jia, Shaohui Wang, Xiaomei Liao, and Lizhi Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06499 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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Environmental Science & Technology

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Rapid Aerobic Inactivation and Facile Removal of Escherichia

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coli

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Indispensable Roles of Reactive Oxygen Species and Iron

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Corrosion Products

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Hongwei Sun†, Jian Wang§, Yao Jiang§, Wenjuan Shen†, Falong Jia†,*, Shaohui Wang§, Xiaomei

6

Liao§,*, Lizhi Zhang†,*

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†

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Environmental & Applied Chemistry, College of Chemistry, Central China Normal University,

9

Wuhan 430079, P. R. China

with

Amorphous

Zero-valent

Iron

Microspheres:

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, Institute of

10

§

11

China Normal University, Wuhan 430079, P. R. China

Hubei Key Lab of Genetic Regulation and Integrative Biology, School of Life Sciences, Central

12 13

* To whom correspondence should be addressed.

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Fax: +86 027 67867535; E-mail: [email protected]. (F. J.)

15

Fax: +86 027 67861936; E-mail: [email protected]. (X. L.)

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Fax: +86 027 67867535; E-mail: [email protected]. (L. Z.)

17

1

ACS Paragon Plus Environment


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ABSTRACT: Zero valent iron (ZVI) is recently regarded as a promising alternative for water

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disinfection, but still suffers from low efficiency. Herein we demonstrate that amorphous zero valent

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iron microspheres (A-mZVI) exhibit both higher inactivation rate and physical removal efficiency

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for the disinfection of Escherichia coli (E. coli) than conventional crystalline nanoscale ZVI

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(C-nZVI) under aerobic condition. The enhanced E. coli inactivation performance of A-mZVI was

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mainly attributed to more reactive oxygen species (ROSs), especially free •OH, generated by the

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accelerated iron dissolution and molecular oxygen activation in bulk solution. In contrast, C-nZVI

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preferred to produce surface bound •OH, and its bactericidal ability was thus hampered by the

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limited physical contact between C-nZVI and E. coli. More importantly, hydrolysis of dissolved iron

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released from A-mZVI produced plenty of loose FeOOH to wrap E. coli, increasing the dysfunction

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of E. coli membrane. Meanwhile, this hydrolysis process lowered the stability of E. coli colloid and

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caused its rapid coagulation and sedimentation, favoring its physical removal. These findings clarify

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the indispensable roles of ROSs and iron corrosion products during the ZVI disinfection, and also

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provide a promising disinfection material for water treatment.

32 33

Keywords: Amorphous zero valent iron; Disinfection; E. coli; Reactive oxygen species; Physical

34

removal;

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INTRODUCTION

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Over the past decades, nanoscale zero-valent iron (nZVI) has been widely used to remove various

38

environmental pollutants, including heavy metals,1, 2 halogenated organics and nitroaromatics,3-5 etc,

39

owing to its high redox activity, facile synthesis and environmental benignancy.6-8 Recently, nZVI

40

was also reported to inactivate a broad spectrum of microbes such as virus, coliphage and bacteria, 2


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making it a promising antimicrobial agent for water disinfection.9-11 In comparison with chlorine,

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nZVI is attractive due to free of carcinogenic disinfection byproducts (DBPs) such as

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trihalomethanes (THMs), haloacetic acids (HAAs), bromate and chlorite, which are listed as primary

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contaminants in drinking water by the United States Environmental Protection Agency (USEPA),

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European Union and World Health Organization (WHO). Meanwhile, nZVI disinfection is capable

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of removing co-existing contaminants like heavy metals and organic pollutants simultaneously,

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which is advantageous over chlorination especially in the scenario of wastewater disinfection, where

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large amounts of pollutants are present. These pollutants might act as precursors of DBPs and must

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be removed in advance in the case of chlorination, which would increase the operation cost.12,

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More importantly, the concomitant physical removal of bacteria from water along with inactivation

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through magnetic separation,14 was strikingly advantageous for practical disinfection by nZVI,

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because this physical removal process could reduce the chemical oxygen demand (COD) of water by

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removing the debris of inactivated microbes, and eliminate the risk from regrowth of inactivated

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bacteria and potential spread of antibiotic resistance genes through horizontal gene transfer.15-18 In

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addition, nZVI is easier for handling during transport, storage and deployment than chlorine,

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therefore benefits its application in the disinfection of non-central water supply, and point-of-use

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water purification.19

13

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Despite its merits, the disinfection efficiency of nZVI was still inadequate for practical

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application, especially under aerobic conditions. Therefore, relatively high doses were required to

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achieve satisfactory disinfection effects, which might improve cost and cause iron sludge.20, 21 It was

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reported that the aerobic inactivation of microorganism by nZVI mainly involved the generation of

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reactive oxygen species (ROSs) via oxygenation of nZVI, and the direct physical disruption of cell

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membrane components by adhered nZVI particles.9,

22

Normally, the oxygenation of nZVI would 3



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produce surface oxide layers, which subsequently passivated nZVI and decelerated the further

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outward electron transfer from Fe0 core to the oxide surface for activation of oxygen and production

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of ROSs.7, 23 In addition, easy aggregation of nZVI particles, which was driven by electrostatic and

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magnetic attractions, resulted in poor dispersibility of nZVI and less adhesion onto cells.24 Thus,

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these two reasons accounted for the unsatisfactory disinfection reactivity of nZVI. Although doping

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nZVI with precious metals such as Pt and Pd could avoid the unsatisfactory corrosion of iron core

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and accelerate outward electron transfer through galvanic mechanism,25, 26 the high cost of precious

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metals disfavored their field application. Surface coating with polymeric stabilizers such as starch

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and cellulose was a common strategy to improve the dispersion and mobility of nZVI,27, 28 but was

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always faced with a trade-off between the stability and reactivity of nZVI, and the decreased

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bactericidal effect after polymer coating.29-32 Obviously, it is vital to develop new strategies to

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improve the disinfection performance of nZVI.

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In 2005, Lowry et al. investigated the influence of nZVI annealing treatment on its catalyzed

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reductive trichloroethylene (TCE) transformation with H2. They found that poorly ordered pristine

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nZVI could activate H2 to reduce TCE to ethane, but annealed nZVI of good crystallinity could not.

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Meanwhile, the oxidative dissolution of nZVI was also retarded after annealing treatment.33 Later,

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Nurmi and his coworkers revealed that nZVI, being prepared by borohydride reduction, was poorly

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ordered, but outperformed its crystalline counterpart prepared through thermal reduction of goethite

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with H2, on reducing pollutants like benzoquinone and CCl4.34 Moreover, Wang et al. demonstrated

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that the recrystallization of aged nZVI was responsible for its activity loss of bromate reduction.35

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On the basis of these results, we proposed that the disordered structure of nZVI might benefit the

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electron donating capacity of iron core, and for the first time prepared amorphous zero valent iron

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microspheres (A-mZVI) through Fe(II) reduction with borohydride in the presence of 4


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ethylenediamine (EDA). However, it is still unknown how the amorphous structure will affect the

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ROSs generation and disinfection performances of ZVI, which is crucial for us to clarify the

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pollutant removal ability of A-mZVI.

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In this study, we compared the inactivation and physical removal performances of A-mZVI and

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zero-valent iron nanoparticles (C-nZVI) toward a typical Gram-negative bacterium Escherichia coli

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(E. coli) under aerobic condition, aiming to unravel the aerobic disinfection mechanism of A-mZVI

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through investigating the production and evolution of ROSs in the A-mZVI/air and C-nZVI/air

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systems, and clarified the effects of dissolution processes and corrosion products of A-mZVI and

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C-nZVI on the inactivation and physical removal of E. coli.

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EXPERIMENTAL SECTION

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Synthesis and Characterization of ZVI. To prepare A-mZVI, 75 mL of EDA (0.9 mol/L) was

99

added into 300 mL of 0.075 mol/L FeCl2 aqueous solution, after thoroughly mixed for 3 min under

100

stirring, 75 mL of 1.6 mol/L NaBH4 aqueous solution was introduced. The reduction process was

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finished in 25 min until no obvious bubble emission was observed. The obtained black precipitate

102

was collected, washed with deionized water and absolute ethanol for 3 times, and dried under

103

infrared lamp. All these procedures were conducted under argon gas protection to avoid oxidation of

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iron. C-nZVI was prepared with similar procedures without adding EDA. The as-prepared ZVI

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samples were characterized with scanning electron microscopy (SEM, TESCAN MIRA 3, Czech)

106

and X-ray diffraction (XRD, D8 Advance, Cu Kα radiation, λ = 0.15418 nm, Bruker, Germany). The

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content of nitrogen in the synthesized A-mZVI and C-nZVI was measured by elementary analysis

108

(vario Micro cube, Elementar) to check the residue of EDA, and the release of EDA from A-mZVI

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during use was evaluated by measuring total nitrogen (TN) concentrations with a TOC/TNb 5



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Analyzer (vario TOC select, Elementar). Fe0 content was quantified by acid digestion and hydrogen

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gas production assay.33 Specific surface area (SSA) was determined by Brunauer-Emmett-Teller

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(BET) method with a N2 adsorption system (Micromeritics Tristar 3000). Details of characterization

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methods are provided in Supporting Information (SI).

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Disinfection Experiments. E. coli strain BW25113 was purchased from Coli Genetic Stock Center

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of Yale University, USA. To prepare the bacterial suspension, E. coli was inoculated into 50 mL of

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autoclaved nutrient broth and incubated overnight, then the bacteria were harvested and washed. The

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pellet was resuspended in 0.85% NaCl and the optical density at 670 nm (OD670 nm) was adjusted to

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0.06. ZVI at predetermined doses were added into 50 mL of the obtained bacterial suspension and

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incubated at 250 rpm and 37 ℃ in an orbital shaker. To determine the survival ratio and physical

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removal efficiency of E. coli, 5 mL of the slurry samples were collected at regular intervals and

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magnetically separated to obtain the supernatant, which was subsequently stained with

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LIVE/DEADTM BacLightTM Bacterial Viability Kit and analyzed with flow cytometry

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(LSRFortessaTM, BD, USA). This Kit contains two fluorescent nucleic acids dyes, namely SYTOTM

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9 and propidium iodide (PI). SYTOTM 9 is cell membrane permeable and can bind with bacterial

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DNA to emit green fluorescence (530 nm) when excited at 488 nm. PI can quench SYTOTM 9 due to

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its higher DNA binding affinity to generate red fluorescence (excitation wavelength of 488 nm and

127

emission wavelength of 630 nm). Nevertheless, PI is not permeable to intact membrane, and thus the

128

live cells with intact membrane can only be stained green by SYTOTM 9, and dead cells with

129

disturbed membrane can be stained red by PI. The numbers of fluorescent particles (E. coli cells) in

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200 μL samples were recorded by volumetric counting hardware. In the two-parameter dot plots of

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PI versus SYTOTM 9, four clusters could be distinguished by quadrants gate (Q1-4), which were

6


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ascribed to dead, injured, live cells and negative background signals (SI Figure S1), respectively.

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The survival ratio and removal efficiency of E. coli were calculated according to Equation 1-3:

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Survival ratio = N3/(N1+N2+N3)

(1)

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C = (N1+N2+N3) / 200 μL

(2)

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Removal efficiency (%) = (C0-Ct)/C0

(3)

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where N1-3 were the cell numbers in Q1-3, respectively, and (N1+N2+N3) referred to the total cell

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numbers. C0 and Ct were the bacterial concentrations in the supernatant at time 0 and t, respectively.

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E. coli inactivation efficiency was also monitored by the heterotrophic plate count (HPC) method at

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a predetermined initial E. coli concentration of 1×106 colony forming unit per milliliter (CFU/mL) to

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facilitate the comparison of A-mZVI with other ZVI in the literature. Details of the method were

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provided in SI.

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The survival states of the cells in sediment samples were determined by a fluorescent

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microscope (EVOS FL Auto, Life Technologies Corporation) after staining with LIVE/DEADTM

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Kit. Images of green (SYTOTM 9) and red (PI) fluorescent channels were captured simultaneously

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for the same sample, and their merged images were used to depict the viable states of E. coli. The

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intensities of green and red fluorescence were also integrated with ImageJ,36 and their ratio

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(Green/Red) was used as an index of the E. coli viability in the sediments. Note that the Green/Red

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ratio of E. coli cells at 0 min could not be calculated without red fluorescence signals. The ratio

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could only be obtained after E. coli cells were inactivated by ZVI and the bacterial membrane

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became disturbed and permeable to PI. Details of the method were described in the SI.

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Analytical Methods. The mixtures after ZVI disinfection were left to settle for different durations,

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and the turbidity of the supernatant was measured with a portable turbidimeter (2100Q, HACH).

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Concentrations of dissolved Fe2+ and Fe3+ were determined by the 1,10-phenanthroline colorimetric 7



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method.37

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5,5-dimethyl-1-pyrroline N-oxide (DMPO) were employed to detect •OH and •O2- with a JES FA

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200 X-band spectrometer (JEOL, Japan). H2O2 concentration was tested with a modified

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p-hydroxyphenylacetic acid (POHPAA) fluorescent method.38 Intracellular ROSs concentration was

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measured fluorescently by utilizing 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) as a

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probe.39 Catalase and superoxide dismutase (SOD) activities were measured using Catalase Assay

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Kit (S0051, Beyotime Institute of Biotechnology, China) and Superoxide Dismutase Assay Kit

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(S0101, Beyotime Institute of Biotechnology), respectively. Membrane potential of E. coli was

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measured with Rhodamine 123 (Rh123) probing method,22 and adenosine triphosphate (ATP)

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production by E. coli was assayed by a BacTiter-GloTM Microbial Cell Viability Assay Kit (G8230,

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Promega Corporation, USA). The sample preparation protocols of E. coli for SEM and transmission

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electron microscopy (TEM, JEOL-2010, 200 kV) characterization were provided in SI. Magnetic

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hysteresis loops of the oxidized ZVI were measured with a vibrating sample magnetometer (VSM) at

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ambient temperature within the magnetic field of ± 10,000 Oe. Particle size distribution of ZVI-E.

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coli suspension was measured by dynamic light scattering (Zetasizer Nano, Malvern), as described

170

in SI.

Electron

spin

resonance

(ESR)

spectra

and

the

radical

spin

trapper

171 172

RESULTS AND DISCUSSION

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Synthesis and Characterization of ZVI. As shown in the SEM images, the as-synthesized C-nZVI

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contained chains or aggregates assembled by spheres of 50-200 nm (Figure 1a), consistent with our

175

previous study.6 Differently, A-mZVI was composed of spheres in size of 0.8-1.2 μm (Figure 1b and

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1c). XRD pattern suggested C-nZVI exhibited an obvious diffraction peak around 2θ value of 44.5°,

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corresponding to α-Fe0 (JCPDS No. 87-722), which was the typical XRD pattern of nZVI produced 8


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by NaBH4 reduction in solution.40,

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A-mZVI, indicating the formation of amorphous structure of ZVI in the presence of EDA (Figure

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1d). This amorphous nature of A-mZVI might benefit the production of ROSs and aerobic

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disinfection of E. coli. The contents of N, C, H in C-nZVI were 0%, 0.47% and 0.48%, which were

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0.90%, 1.16% and 0.55% in A-mZVI, respectively (SI Table S1). This comparison suggested the

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EDA content in the as-prepared A-mZVI was rather low, as the washing with deionized water and

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ethanol might remove most of EDA residue. Subsequently, the release of EDA from A-mZVI during

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disinfection was evaluated. A-mZVI were subjected to aerobic corrosion for 1 h and then filtered.

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The TN concentrations of filtrates were found to be 1.5, 1.8 and 3.2 mg/L for A-mZVI dosage of

187

200, 500 and 1000 mg/L, respectively (SI Figure S2). These values were much lower than the US

188

national criterions of nitrate nitrogen (N) and total ammonia nitrogen (TAN), which were 10 mg/L

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for domestic water supply and 17 mg/L for fresh water, respectively.42, 43 TN concentrations of 1.5,

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1.8 and 3.2 mg/L corresponded to EDA concentrations of 3.2, 3.9 and 6.9 mg/L, respectively,

191

assuming that all the nitrogen elements were originated from EDA. Since there is no guideline

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concentration of EDA in water available, the lowest No Observed Adverse Effect Level (NOAEL, 9

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mg/kg body weight per day) obtained from rat experiments was used to evaluate the toxicity and

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overall risk of EDA oral intake, as recommended by WHO.44 The estimated daily intake (EDI) of

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EDA through water consumption was calculated according to equation 4:45

196

41

However, no obvious diffraction peak was acquired for

EDI = Ci × CR / BW

(4)

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where Ci is the concentration of EDA in water, CR represents the consumption rate of drinking

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water (2 L/day), and BW stands for the average body weight (70 kg for adults). The EDIs were 0.09,

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0.1 and 0.2 mg/kg body weight per day at A-mZVI dose of 200, 500 and 1000 mg/L, respectively,

200

which were far less than the NOAEL of 9 mg/kg body weight per day recommended by WHO. 9



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Therefore, the toxicity and risk induced by the EDA residual in water were negligible.

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Inactivation and Physical Removal of E. coli by ZVI. The as-prepared ZVI samples were then

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used for the disinfection of E. coli under aerobic condition. The initial concentration of E. coli was

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determined to be approximately 107 cells/mL by using flow cytometry (Figure 2a). In the

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supernatant samples after C-nZVI disinfection, both the total and viable E. coli concentrations

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decreased steadily, which were 8.4×105 and 5×105 cells/mL after 60 min, respectively. Impressively,

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A-mZVI induced much sharper concentration decrease of total and live cells in the supernatant,

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which were respectively down to 5×105 and 2×105 cells/mL after only 5 min (Figure 2a). The

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survival ratios and removal efficiencies of E. coli were calculated according to Eqs 1-3. We found

210

that A-mZVI inactivated E. coli much more rapidly, and only 18% of the cells remained viable after

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60 min, much lower than that (53%) of C-nZVI treatment (Figure 2b). More importantly, A-mZVI

212

also showed remarkably better physical removal performance. The removal efficiency of E. coli in

213

the C-nZVI system increased gradually to 95% after 1 h, but it just took 5 min for A-mZVI to reach

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a comparable removal efficiency (Figure 2c).

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Since the physical removal effect of ZVI would precipitate most of E. coli cells into the

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sediment, it is essential to understand the fate of E. coli in this phase. Nonetheless, the survival status

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of bacteria physically removed by ZVI has never been studied in previous reports.14, 46 Herein, we

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utilized fluorescent microscope to check the viability of E. coli in the sediment samples, and found

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that these cells were inactivated gradually, following similar trends with those in the supernatant (SI

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Figure S3). As an index of E. coli viability, the fluorescent intensity ratio of Green/Red decreased

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steadily from 4.82 at 5 min to 0.67 at 60 min in the C-nZVI/E. coli system, whereas the ratio was

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1.24 after 5 min and further reduced to 0.46 at 40 min in the A-mZVI/E. coli system (Figure 2d).

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This comparison confirmed that A-mZVI could also more efficiently inactivate E. coli in the 10


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sediments.

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The influence of ZVI dose on the inactivation and removal of E. coli was also investigated. As

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expected, the inactivation of E. coli showed an obvious dose-dependent manner in the range of

227

20-400 mg/L, but A-mZVI exhibited apparent advantage over C-nZVI at identical dosage. For

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example, virtually complete inactivation of E. coli was achieved at 400 mg/L of A-mZVI, whilst

229

16% of E. coli still survived at the same dose of C-nZVI (Figure 3a). Similar trends were observed in

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the sediment samples via fluorescent microscopic images (SI Figure S4). The minimum doses of

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C-nZVI and A-mZVI to physically remove 90% of E. coli from water were 50 and 20 mg/L,

232

respectively. This difference further confirmed the superiority of A-mZVI for physical removal of E.

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coli (Figure 3b). Moreover, we conducted additional control experiments with crystalline

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micro-scale zero valent iron (C-mZVI), which was prepared by annealing the A-mZVI at 500 °C for

235

4 h in Ar. SEM images indicated that the size of C-mZVI was in the range of 1-2 μm (SI Figure

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S5a), similar with that of A-mZVI. As expected, the crystallinity of C-mZVI greatly improved after

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annealing treatment, as revealed by the sharp diffraction peak at 2θ = 45° (JCPDS No. 87-722) in the

238

XRD pattern (SI Figure S5b). We thus compared the E. coli inactivation performance of A-mZVI,

239

C-nZVI and C-mZVI, and found that the disinfection capacity decreased following the order of

240

A-mZVI > C-nZVI > C-mZVI (SI Figure S5c). As both C-nZVI and C-mZVI were crystallized, the

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lower disinfection activity of C-mZVI with larger particle size was consistent with previous reports

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about the size effect on bactericidal capacity of ZVI.47 Although A-mZVI and C-mZVI had similar

243

particle size, C-mZVI of crystalline nature possessed a dramatically reduced antibacterial activity.

244

Therefore, we concluded that the improved disinfection performance of A-mZVI was arisen from its

245

amorphous structure, rather than the size effect. Moreover, the Fe0 content of A-mZVI (67%) was

246

only slightly higher than that (60%) of C-nZVI (SI Table S1), which further confirmed that the 11



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improved E. coli inactivation of A-mZVI was not related to the Fe0 content, but to the amorphous

248

structure.

249

The E. coli inactivation efficiency of A-mZVI was also compared with previous reports. Since

250

most of the literatures determined the inactivation efficiencies of E. coli by using the HPC method

251

with initial E. coli concentration of 1×106 CFU/mL, we conducted similar experiments by using

252

A-mZVI for better comparison. The inactivation efficiency (log (N0/N)) was 2.4 ± 0.2 after treated

253

with 100 mg/L of A-mZVI for 60 min under aerobic condition. After settling for 0.5 h, the E. coli

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concentrations in the supernatant decreased by 3.8 ± 0.04 log (SI Table S2). This inactivation

255

efficiency of A-mZVI was higher than those of many ZVI reported in the literature, including nZVI,

256

electrosprayed nZVI, commercial Nanofer 25 (Nano Iron s.r.o., Czech Republic), commercial

257

reactive nanoscale iron particles (RNIP, Toda Kogyo Corp.), and microscale iron powder.14, 21, 29, 47, 48

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The cost of the A-mZVI synthesized was estimated to be ca. 0.6 USD/g (SI Table S3), which was

259

relatively high because the synthesis of A-mZVI was conducted in laboratory scale and the reagents

260

used were supplied in small packages and of high purity (AR). Nevertheless, the price of A-mZVI

261

could be significantly decreased in industrial production by scaling-up the fabrication and by

262

adopting much cheaper reagents of technical grade to meet the need of practical disinfection.

263

Subsequently, the turbidity of the ZVI treated water was also monitored, which were 158.0 and

264

88.6 nephelometric turbidity unit (NTU) for A-mZVI and C-nZVI right after the batch experiments.

265

The turbidity of the supernatant in the A-mZVI system decreased sharply to 19.2 NTU after only 0.5

266

h of settling, much faster than that (45.4 NTU) in the C-nZVI system. The turbidity further

267

decreased with prolonged settling duration (SI Figure S6a). Interestingly, a linear relationship was

268

revealed between Ln (turbidityt/turbidity0) and settling time, where turbidityt and turbidity0 were the

269

turbidity at settling time t and 0 h, respectively (SI Figure S6b). Therefore, the turbidity of effluent 12


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after A-mZVI disinfection can be controlled by tuning the settling duration according to the purpose

271

of application. In the case of wastewater disinfection, the wastewater discharge standard of China

272

(GB 8978-1996) regulates the total suspended solids (TSS) from 20 to 100 mg/L in different

273

scenarios, which corresponds to turbidity values of ca.13-100 NTU according to a general rule of

274

thumb. This suggests that 1 mg/L of TSS approximately equals to 1.0-1.5 NTU of turbidity.49 Then

275

less than 1.3 h of settling after A-mZVI disinfection was required to meet the wastewater discharge

276

standard (13 NTU, SI Figure S6b). In the context of drinking water disinfection, the criteria of

277

turbidity for central water supply and non-central water supply were 1 and 3 NTU, which could be

278

raised to 3 and 5 NTU when the quality of water source or the technical conditions of water

279

purification were limited, according to the drinking water standard of China (GB5749-2006). Then

280

the necessary settling durations were 6.8, 9.7 and 16 h to meet the turbidity standards of 5, 3 and 1

281

NTU, respectively (SI Figure S6b). Therefore, A-mZVI is potentially used for water disinfection

282

with post-disinfection settling process. Alternatively, settling process might be omitted if

283

disinfection be carried out in a flow-through column packed with ZVI.

284

Mechanism of Inactivation. To unravel the mechanism of promoted E. coli inactivation

285

performance of A-mZVI, we first investigated the contribution of various ROSs by scavenging

286

experiments, since ROSs played vital roles in the aerobic inactivation of bacteria by ZVI.9,

287

Intriguingly, the inactivation performance of the two ZVI samples changed in significantly different

288

manners after ROSs quenching. In the C-nZVI/E. coli system, the scavenging effects descended

289

following the order of SOD > catalase > tert-butyl alcohol (TBA), which were used to quench •O2-,

290

H2O2 and •OH, respectively. This trend suggested that •O2- and H2O2 were responsible for the

291

inactivation of E. coli, whereas •OH was less important, which was consistent with previous

292

reports.9, 47 As for the A-mZVI/E. coli system, the addition of SOD, catalase and TBA resulted in

50

13



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293

similar changes of survival ratio, highlighting the indispensable role of •OH for the E. coli

294

disinfection (Figure 3c and SI 　Figure S7), and also explaining the better bactericidal performance

295

of A-mZVI, because •OH is a much stronger oxidant than •O2- and H2O2.

296 297

Subsequently, we attempted to clarify how the amorphous structure altered ROSs production of ZVI. Generally, the evolution of ROSs by ZVI oxygenation could be described by Eqs 5-11:8, 51, 52

298

Fe0 → ≡Fe2+ + 2e-

(5)

299

≡Fe2+ → Fe2+

(6)

300

O2 + 2e- + 2H+ → H2O2

(7)

301

O2 + e- → •O2-

(8)

302

•O2- + e- + 2H+ → H2O2

(9)

303

≡Fe2+ + H2O2 → ≡Fe3+ + •OHsurface + OH-

(10)

304

Fe2+ + H2O2 → Fe3+ + •OH + OH-

(11)

305

where ≡ indicated surface bound species. As stated above, •OH more contributed to the disinfection

306

in the A-mZVI/E. coli system than in the C-nZVI/E. coli system, whereas •O2- and H2O2 served

307

mainly as the precursors of •OH in the A-mZVI/E. coli system. To clarify the ROSs production

308

processes, we first investigated the dissolution behavior of the two ZVI samples. The dissolution rate

309

of A-mZVI was higher, indicating that A-mZVI possessed stronger electron donating capacity than

310

C-nZVI (Figure 4a). Obviously, this stronger electron donating capacity should facilitate the ROSs

311

production via reduction of O2 by A-mZVI (Eqs 6, 7), which was confirmed by more •O2- generated

312

in the A-mZVI system (SI Figure S8 and Figure 4b). Moreover, more accessible electrons from iron

313

core and dissolved Fe2+ in the A-mZVI system would further accelerate the transformation of •O2-

314

into H2O2 and finally •OH (Eqs 9-11), and thus shorten the lifetime of •O2- and H2O2. Consequently,

315

•O2- and H2O2 would act as intermediates of •OH rather than direct bactericidal agents in the 14


Page 15 of 35


316

A-mZVI system. To validate this hypothesis, we measured the concentrations of H2O2 and •OH.

317

Although the steady-state H2O2 were not detected due to its rapid in-situ decomposition, the

318

accumulated H2O2 concentrations produced by A-mZVI were higher than C-nZVI (Figure 4c),

319

confirming the enhanced transformation of •O2- into H2O2 by A-mZVI. A-mZVI also showed much

320

faster H2O2 depletion performance (Figure 4d), and the H2O2 decomposition rate constant of

321

A-mZVI was 0.708 min-1, much higher than that (0.109 min-1) of C-nZVI (SI Figure S9). Finally, the

322

•OH levels, as reflected by the ESR intensities of DMPO-OH adduct, were significantly elevated in

323

the A-mZVI system (Figure 4f). These results proved the accelerated transformation of H2O2 into

324

•OH by A-mZVI. The shortened lifetime of •O2- and H2O2 was further supported by the lowered

325

intracellular ROSs levels induced by A-mZVI (Figure 4e), because the limited lifespan rendered it

326

less likely for •O2- and H2O2 to penetrate the cytomembrane into cytoplasm before their

327

transformation, whereas their derivative •OH was also too active and short-lived to enter cells.53

328

As for the C-nZVI system, most of Fe2+ generated by oxidation of Fe0 would be

329

surface-associated rather than dissolved,6, 51 resulting in the decomposition of H2O2 dominantly at

330

the surface of C-nZVI to produce surface-bound •OH (•OHsurface, Eq. 10).54 In such a case, the

331

interaction between •OHsurface and E. coli would largely depend on the physical contact between

332

C-nZVI and cells. However, the poor dispersion of C-nZVI in the disinfection system disfavored the

333

direct contact between C-nZVI and E. coli and thus impeded the attack of •OHsurface to the cells.

334

Whereas in the case of A-mZVI, more Fe2+ was dissolved, resulting in more decomposition of H2O2

335

in the bulk solution to produce free •OH (Eq. 11), which could kill E. coli without the direct contact

336

between A-mZVI and cells. To verify the proposed mechanism, the fractions of •OHsurface among the

337

overall •OH (sum of •OHsurface and free •OH) were further investigated by using DMPO-trapped EPR

338

technique. F- was used to desorb the •OHsurface by forming strong hydrogen bond,55 thus the EPR 15



Page 16 of 35

339

intensities without F- (I0) represented free •OH levels, and the intensities with F- addition (IF) was

340

attributed to overall •OH levels, assuming that F- could disassociate all the •OHsurface. We defined the

341

•OHsurface fractions as (IF-I0)/IF, and found the •OHsurface fractions (78.7-92.5%) of C-nZVI system

342

was much higher than those (36.1-57.9%) of A-mZVI system (Figure 4f). These results confirmed

343

that C-nZVI was dominated by •OHsurface whereas A-mZVI favored free •OH.

344

The intracellular ROSs did not play important role in E. coli inactivation in the A-mZVI-E. coli

345

system, because most of ROSs were generated and transformed in the extracellular bulk solution to

346

•OH, the predominant bactericidal ROSs. In the scavenging experiments, the scavengers

347

impermeable to the membrane were supposed to scavenge the extracellular ROSs. Therefore, the

348

significant inhibition of disinfection by TBA suggested the indispensable role of extracellular •OH

349

(Figure 3c). Although A-mZVI could inactivate E. coli much faster than C-nZVI, the intracellular

350

ROSs levels in the A-mZVI-E. coli system was much lower than those in the C-nZVI-E. coli system

351

(Figure 4e). In contrast, intracellular ROSs might play relatively more important role in the

352

C-nZVI-E. coli system because of its much higher intracellular ROSs concentrations. The

353

scavenging experiments also indicated the vital roles of extracellular •O2- and H2O2 for E. coli

354

inactivation, but neither •O2- nor H2O2 were efficient for the direct oxidative damage of E. coli cells.

355

Comparatively, •OH is a much stronger oxidant, but the contribution of extracellular •OH was

356

negligible in the C-nZVI-E. coli system because most of •OH were produced in the form of surface

357

associated species and had limited physical contact with E. coli cells. Therefore, it is highly possible

358

that •O2- and H2O2 entered E. coli cells and thus became intracellular •OH to inactivate E. coli, as

359

revealed by the high intracellular ROSs levels induced by C-nZVI.56, 57 Nevertheless, it is still a great

360

challenge to elucidate the role of intracellular ROSs because of the limited techniques to quantify or

361

quench intracellular ROSs. 16


Page 17 of 35


362

The responses of E. coli to the oxidative stress were then checked to further reveal the

363

promotion of ROSs mediated disinfection by using A-mZVI. Increasing concentrations of H2O2 and

364

•O2- would respectively trigger the biosynthesis of SOD and catalase, which in turn quench

365

excessive •O2- and H2O2 to keep their concentrations at acceptable levels.56,

366

elevation of SOD and catalase activities are supposed to indicate more ROSs production. As

367

expected, the activities of both SOD and catalase increased more quickly when E. coli were treated

368

with A-mZVI, in agreement with its greater ROSs generating capacity. Moreover, the

369

overwhelmingly yielded ROSs by A-mZVI, especially the •OH which cannot be quenched

370

effectively by catalase or SOD, would then cause the oxidative damage of the antioxidative

371

enzymes,39, 56 resulting in the sharp decrease of enzymatic activities after only 10 min. In contrast,

372

the activities of both enzymes kept increasing until 40 min and decreased more slowly in the

373

C-nZVI/E. coli system (Figure 5a and 5b). These results further confirmed the indispensable role of

374

ROSs on the enhanced E. coli inactivation of A-mZVI.

58

Therefore, the

375

Besides the oxidative inactivation induced by ROSs, the physical disruption of cell membrane

376

by adhered ZVI particles might also contribute to the disinfection of E. coli,9, 22, 59, 60 since quenching

377

ROSs could not inhibit the inactivation of E. coli completely (Figure 3c). Therefore, we checked the

378

physical contact between ZVI and E. coli by using SEM and TEM. In the C-nZVI/E. coli system,

379

quasi-spherical nanoparticles were found to adhere to the outer surface of E. coli loosely after 1h,

380

similar with previous reports.46,

381

numerous interconnected flakes to wrap the E. coli cells more tightly (Figure 6). The different

382

shapes and adsorption of reacted A-mZVI suggested the corrosion process of ZVI was greatly

383

changed by the amorphous structure. It is well documented that the corrosion of C-nZVI took place

384

mainly at its outer surface, featured by slow leaching of Fe2+ and the onsite growth of oxide shells.61

47

Interestingly, A-mZVI formed a thick envelope assembled by

17



Page 18 of 35

385

As a result, the adsorption of pristine or oxidized C-nZVI on the bacterial surface was hampered

386

owing to the poor dispersibility of C-nZVI. Differently, the fast dissolution of A-mZVI resulted in

387

more Fe2+ release, which was then hydrolyzed and oxidized to iron (hydro)oxide in the bulk

388

solution, thus the corrosion products of A-mZVI had easier access for the adsorption of E. coli. This

389

process was supported by the clear spatial separation of the spherical A-mZVI (denoted as

390

A-mZVI-2) from the laminar corrosion products (denoted as A-mZVI-1) in the SEM images (SI

391

Figure S10). More importantly, the iron (hydro)oxides species after corrosion were also altered by

392

the amorphous structure of A-mZVI. The XRD analysis showed that C-nZVI was converted to a

393

mixture of magnetite (Fe3O4, JCPDS No. 85-1436), akaganéite (β-FeOOH, JCPDS No. 75-1594) and

394

lepidocrocite (γ-FeOOH, JCPDS No. 70-0714), with magnetite as the dominant species, whereas

395

A-mZVI was mainly oxidized to lepidocrocite (Figure 6g), whose structure was more loosely than

396

that of magnetite and thus favored the sorption of E. coli cells. Meanwhile, it was reported that the

397

hydrolysis and oxidation of dissolved Fe2+ were able to produce polymeric Fe(OH)2 and Fe(OH)3

398

flocs as the precursors of lepidocrocite,62 and these flocs were also voluminous and favored the wrap

399

of E. coli cells.63 According to energy dispersive spectroscopy (EDS) analysis, the corrosion

400

products of A-mZVI (A-mZVI-1) possessed significantly higher carbon atomic percentage (36.41%)

401

than those (27.61% and 26.80%) of the remained spherical A-mZVI (A-mZVI-2) and used C-nZVI,

402

further confirming the higher E. coli adsorption capacity of the loose corrosion products of A-mZVI

403

(SI Figure S11).

404

The enhanced adsorption of A-mZVI corrosion products should cause more severe membrane

405

dysfunction of E. coli cells by physical disruption. Normally, the membrane potential and adenosine

406

triphosphate (ATP) synthesis capacity of cells were used as indicators of membrane dysfunction.

407

Membrane potential is crucial for the selectivity of plasma membrane,64 and the closely adhered ZVI 18


Page 19 of 35


408

might block the ion channels and prevent the efflux of charged ions to construct the transmembrane

409

potential.65 After E. coli was treated with C-nZVI, the membrane potential maintained constant

410

during the initial 10 min, and dropped thereafter, but A-mZVI induced an instant membrane

411

potential decline as soon as E. coli was in contact with A-mZVI (Figure 5c). Moreover, the adsorbed

412

particles might isolate E. coli cells from their environment and prevent their consumption of

413

extracellular nutrients to synthesize ATP.59, 66 We collected the E. coli cells after different duration

414

of ZVI disinfection and measured their ATP synthesis rate (SI Figure S12), and found 5 min of

415

A-mZVI treatment drastically declined the rate from 4.75 to 0.87 nM/min, much faster than the case

416

of C-nZVI (Figure 5d). We thus concluded that the enhanced adhesion of corrosion products on E.

417

coli outer surface, originated from the amorphous structure of A-mZVI, also contributed to its better

418

disinfection performance.

419

It was known that the physical bacteria removal of nZVI relied on the adsorption of magnetic

420

iron oxide over bacterial surface and magnetic separation.14 However, the major corrosion product of

421

A-mZVI was lepidocrocite, whose saturation magnetization value was two orders of magnitude

422

lower than that (80-100 Am2/kg) of magnetite.62 As expected, the corrosion products of A-mZVI

423

were virtually non-magnetic (Figure 6h), and the sedimentation of the A-mZVI/E. coli mixture after

424

1 h of aerobic reaction was not affected by a magnet, different from the case of C-nZVI/E. coli

425

system (Figure 6i). These phenomena suggested that A-mZVI altered the E. coli physical removal

426

process thoroughly. Intrinsically, the suspension of E. coli is a kind of active colloid, and the rotation

427

of helical flagella endows E. coli with swimming motility, which is essential to maintain the cells

428

suspended against gravitational sedimentation.67 Therefore, the loss of motility would result in fast

429

precipitation and easy physical removal of E. coli. Although the inactivation of cells might decrease

430

motility, this reason could be ruled out because live cells were removed simultaneously with dead 19



Page 20 of 35

431

cells by ZVI and ROSs scavengers had little impact on the removal efficiencies (Figure 3d). As

432

stated above, A-mZVI could form thick envelope to wrap the E. coli cells tightly. This might inhibit

433

the rotation of helical flagella, thus decrease the mobility of E. coli cells and the stability of the E.

434

coli colloid, and subsequently cause the aggregation and gravitational sedimentation of bacterial

435

cells. To support this hypothesis, the particle size distribution of the ZVI/E. coli mixture was

436

measured. C-nZVI treatment induced a slow increase of particle size from ca. 750 nm (bare E. coli

437

cells) at 0 min to ca. 1800 nm (nZVI bearing E. coli cells and their aggregates) at 60 min, indicating

438

the slow adsorption of C-nZVI onto E. coli. In contrast, A-mZVI caused a drastic increase of particle

439

size from ca. 750 nm to ca. 5300 nm within 5 min, confirming the effective generation of

440

voluminous corrosion products and their fast coating of cells. After 5 min, the particle size decreased

441

steadily, corresponding to the precipitation of large E. coli-iron coagulate (SI Figure S13). The

442

possible physical removal processes of two systems are illustrated in Scheme 1.

443 444

Environmental Implications. ZVI is a promising antimicrobial agent because of its low cost, high

445

activity and low toxicity. However, the antimicrobial activity of ZVI is often hampered greatly by

446

the presence of oxygen due to the growth of passivation layer and the subsequent decreased outward

447

electron transfer from iron core, which limit its use. In this study, we have revealed that

448

amorphorization is an effective strategy to improve the bactericidal ability of ZVI under aerobic

449

conditions by enhancing the electron donating capacity of iron core to produce more ROSs,

450

especially free •OH in bulk solution. More importantly, the production of abundant loose corrosion

451

products by oxygenation of A-mZVI facilitates the simultaneous inactivation and physical removal

452

of E. coli, very promising for water self-cleanup during disinfection. Since the bacteria can be

453

immobilized by the iron hydroxides and easily separated from the treated water, the release of 20


Page 21 of 35


454

antibiotic resistance genes (ARGs) from these bacteria can be suppressed. With further

455

detoxification processes of the bacteria-containing iron sludge, the spread of ARGs in water systems

456

might be eliminated. Therefore, A-mZVI might reduce our dependence on chlorine and antibiotics to

457

tackle problems caused by carcinogenic disinfection byproducts, resistant bacteria and ARGs, etc.

458 459

AUTHOR INFORMATION

460

Corresponding Author

461

*Phone/Fax: +86-27-6786 7535; e-mail: [email protected]; [email protected];

462

[email protected].

463

Notes

464

The authors declare no competing financial interest.

465 466

Acknowledgments: This research was financially supported by Natural Science Funds for

467

Distinguished Young Scholars (Grant 21425728), National Natural Science Foundation of China

468

(Grant 41601543 and 21777050), China Postdoctoral Science Foundation (Grant 2018T110782 and

469

2017M620327), Science Funds for Outstanding Postdocs of Hubei Province, China (Grant Z13), the

470

program of China Scholarship Council (Grant 201706775080), the 111 Project (Grant B17019), and

471

the CAS Interdisciplinary Innovation Team of the Chinese Academy of Sciences.

472 473

ASSOCIATED CONTENT

474

Supporting Information: characterization of ZVI; E. coli inactivation efficiency monitored by HPC

475

method; fluorescence images intensity processing procedures by using ImageJ; measurement of

476

dissolved iron; H2O2 determination; intracellular ROSs level detection; CAT and SOD activity 21



Page 22 of 35

477

analysis; membrane potential measurement; ATP production rate assay; bacterial sample preparation

478

protocols for SEM and TEM; dot plots of bacterial samples in flow cytometer; TN concentrations;

479

fluorescent images of E. coli in sediment; SEM, XRD and E. coli inactivation performance of

480

C-mZVI; turbidity profile of treated water; ESR spectra of DMPO trapped •O2-; kinetic fitting of

481

H2O2 decomposition; SEM images and EDS of used ZVI; kinetics of ATP generation; particle size

482

distribution of E. coli-ZVI mixture; Comparison of the elementary and Fe0 content between the

483

C-nZVI and A-mZVI; Comparison of the E. coli inactivation performance by employing ZVI in

484

different studies.

485 486

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487

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dechlorination of TCE and PCBs. Environ. Sci. Technol. 1997, 31, (7), 2154-2156.

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1,2-diaminoethane (ethylenediamine), Geneva, 1999

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health risk assessment of SVOC pollution in small streams in Pearl River Delta, China. Environ. Sci.

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(46) Chen, Q.; Li, J.; Wu, Y.; Shen, F. X.; Yao, M. S., Biological responses of Gram-positive and

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Gram-negative bacteria to nZVI (Fe0), Fe2+ and Fe3+. RSC Adv. 2013, 3, (33), 13835-13842.

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(47) Lee, C.; Kim, J. Y.; Lee, W. I.; Nelson, K. L.; Yoon, J.; Sedlak, D. L., Bactericidal effect of

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zero-valent iron nanoparticles on Escherichia coli. Environ. Sci. Technol. 2008, 42, (13), 4927-4933.

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implications of in situ remediation by nanoscale zero valent iron (nZVI): Behavior, transport and

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impacts on microbial communities. Sci. Total Environ. 2016, 565, 889-901.

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(51) He, D.; Ma, J. X.; Collins, R. N.; Waite, T. D., Effect of structural transformation of

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approach to in-depth understanding of photoelectrocatalytic bacterial inactivation mechanisms by

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(61) Liu, A. R.; Liu, J.; Zhang, W. X., Transformation and composition evolution of nanoscale zero

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valent iron (nZVI) synthesized by borohydride reduction in static water. Chemosphere 2015, 119,

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(64) Shin, E. H.; Li, Y.; Kumar, U.; Sureka, H. V.; Zhang, X.; Payne, C. K., Membrane potential

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potential. RSC Adv. 2015, 5, (18), 13660-13666.

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(66) Xiu, Z. M.; Jin, Z. H.; Li, T. L.; Mahendra, S.; Lowry, G. V.; Alvarez, P. J. J., Effects of

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nano-scale zero-valent iron particles on a mixed culture dechlorinating trichloroethylene.

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Bioresource Technol. 2010, 101, (4), 1141-1146.

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(67) Schwarz-Linek, J.; Arlt, J.; Jepson, A.; Dawson, A.; Vissers, T.; Miroli, D.; Pilizota, T.;

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657 658

Figure 1. The typical SEM images of (a) C-nZVI and (b, c) A-mZVI. (d) XRD of A-mZVI and

659

C-nZVI.

660 661

Figure 2. (a) The temporary change of E. coli concentrations in the supernatants during ZVI

662

inactivation processes. (b) The ratio of survived E. coli among the total cells in the supernatants 30


Page 31 of 35


663

treated by C-nZVI and A-mZVI, respectively. (c) The removal efficiencies of total E. coli cells from

664

the supernatants by C-nZVI and A-mZVI as a function of time. (d) The temporary trend of the

665

bacterial viability in the sediment samples as reflected by the intensity ratios of green and red

666

fluorescence. The ZVI dose was 100 mg/L, and the initial concentration of E. coli was OD670nm =

667

0.06 in 0.85% NaCl.

668 669

Figure 3. (a) The survival ratio and (b) removal efficiency of E. coli in the supernatant as a function

670

of ZVI dose. The impacts of ROSs scavengers on (c) inactivation of E. coli and (d) removal

671

efficiency of E. coli in the supernatant. The duration of disinfection was 60 min, and the initial

672

concentration of E. coli was OD670nm = 0.06 in 0.85% NaCl. For scavenging experiments, the ZVI

673

dose was 200 mg/L and the concentrations of SOD, catalase and TBA were 100 mg/L, 100 mg/L and

674

1% (v/v), respectively.

31



Page 32 of 35

675 676

Figure 4. (a) The temporary change of dissolved iron concentrations from C-nZVI and A-mZVI

677

during the disinfection process. (b) Time profiles of ESR intensities of DMPO-•O2- adduct. (c)

678

Accumulation of H2O2 as a function of time. (d) Decomposition profile of H2O2 by C-nZVI and

679

A-mZVI. (e) Evolution of intracellular ROSs level within E. coli treated by C-nZVI and A-mZVI. (f)

680

Time profiles of ESR intensities generated by DMPO trapped •OH (symbols + lines), and the

681

proportions of surface bound •OH among overall •OH production (bars). The ZVI dose was 100

682

mg/L and the concentration of F- was 2 × 10-3 mol/L.

32


Page 33 of 35


683 684

Figure 5. The evolution of enzymatic activities of (a) catalase and (b) superoxide dismutase (SOD)

685

as a function of ZVI inactivation time. (c) The time profile of bacterial membrane potential

686

represented by the fluorescent intensity of Rh123 stained cells. (d) The impact of ZVI on ATP

687

synthesis capacity of E. coli. The ZVI dose was 100 mg/L.

33



Page 34 of 35

688 689

Figure 6. The SEM (a, b, c) and TEM (d, e, f) images of the E. coli cells before (a, d) and after 1 h

690

of disinfection treating by C-nZVI (b, e) and A-mZVI (c, f). (g) The XRD patterns and (h) the

691

magnetic hysteresis loops of the corrosion products generated by C-nZVI and A-mZVI after 1 h of

692

aerobic disinfection. (i) The sedimentation curves of ZVI-E. coli mixture after 1 h of aerobic

693

disinfection, w/ and w/o a magnet.

694 695

Scheme 1. The possible physical removal processes of E. coli from supernatant by C-nZVI and

696

A-mZVI. 34


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TOC Art Figure

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35


Rapid Aerobic Inactivation and Facile Removal of Escherichia coli with

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