Nanowire-Modified Three-Dimensional Electrode Enabling Low

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Nanowire-Modified 3D Electrode Enabling LowVoltage Electroporation for Water Disinfection Zheng-Yang Huo, Xing Xie, Tong Yu, Yun Lu, Chao Feng, and Hong-Ying Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01050 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016

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Environmental Science & Technology

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Nanowire-Modified 3D Electrode Enabling Low-

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Voltage Electroporation for Water Disinfection

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Zheng-Yang Huoa, Xing Xie*b, Tong Yua, Yun Lua, Chao Fengc and Hong-Ying Hu*ad

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a

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Environmental Protection Key Laboratory of Microorganism Application and Risk

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Control (SMARC), School of Environment, Tsinghua University, Beijing 100084, PR

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China. E-mail: [email protected]; Tel: +86-10-6279-4005.

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b

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Blvd, MC 131-24, Pasadena, CA 91125, USA. E-mail: [email protected]; Tel: +1-

Environmental Simulation and Pollution Control State Key Joint Laboratory, State

Linde+Robinson Laboratories, California Institute of Technology, 1200 E. California

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626-395-8716.

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c

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[email protected].

13

d

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Laboratory, Tsinghua-Berkeley Shenzhen Institute, Shenzhen 518055, PR China.

Institute for Advanced Study, Tsinghua University, Beijing 100084, PR China. E-mail:

Shenzhen Environmental Science and New Energy Technology Engineering

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ACS Paragon Plus Environment


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TABLE OF CONTENTS

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ABSTRACT

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More than 10% people in the world are still suffering from inadequate access to clean

25

water. Traditional water disinfection methods (e.g., chlorination and ultraviolet radiation)

26

have concerns of carcinogenic disinfection byproducts (DBPs) formation, pathogen

27

reactivation, and/or excessive energy consumption. Recently, a nanowire-assisted

28

electroporation-disinfection method was introduced as an alternative. Here, we develop a

29

new copper-oxide nanowire (CuONW)-modified 3D copper foam electrode using a facile

30

thermal oxidation approach. An electroporation-disinfection cell (EDC) equipped with

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two such electrodes has achieved superior disinfection performance (>7-log removal and

32

no detectable bacteria in the effluent). The disinfection mechanism of electroporation

33

guarantees an exceedingly low operation voltage (1 V) and energy consumption (25 J l -1)

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with a short contact time (7 s). Low operation voltage avoids chlorine generation, thus

35

reduces the potential of DBPs formation. Due to irreversible electroporation damage on

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cell membranes, no bacteria re-growth/reactivation occurs during storage after EDC

37

treatment. Water disinfection using EDCs has great potential for practical applications.

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INTRODUCTION

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Disinfection has protected people from bacterial infection for more than 100 years.

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Chlorination is the most common low-cost disinfection technology, but it inevitably

41

produces carcinogenic disinfection byproducts (DBPs), e.g. Trihalomethanes (THM) and

42

N-nitrosodimethylamine (NDMA), during disinfection and storage processes afterwards.

43

[1-9]

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chlorine disinfection. In order to avoid the formation of chlorinated DBPs, ultraviolet

45

(UV) disinfection, a non-oxidizing disinfection technique, has been used as a potential

46

alternative. However, due to high energy consumption and the issue of bacteria re-

47

growth/reactivation after UV inactivation,

48

limited.

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Electroporation, a common and efficient process to introduce DNA into bacteria, can be a

50

promising substitute of chlorination for water disinfection. [12-16] During electroporation,

51

the permeability of cell membrane increases due to a strong electric field (several kV cm-

52

1

53

electric field is high enough, the damage to cell membrane is irreversible and inactivation

54

occurs.

55

[16, 22, 23]

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electroporation tends to avoid the formation of harmful DBPs. However, to generate the

57

strong electric field, voltages as high as 1-10 kV are normally required, [25] which results

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in high cost and safety issues. [27]

Safety concerns regarding water reuse are driving the search of alternatives to

[10, 11]

applications of UV disinfection are

), thus the DNA transport across cellular membrane is enhanced.

[13, 16, 21]

[12-15, 17-20]

When the

Electroporation-based inactivation has been demonstrated with bacteria

, protozoa

[19, 24, 25]

and viruses

[26]

. Due to its disinfection mechanism,

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Nanostructures, e.g. nanowires, nanoparticles, nanobelts and nanotubes, have unique

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biological [28-31] and electronic [32-35] properties (e.g., field emission, high surface area and

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high conductivity) and may have specific interaction with bacteria

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control, or inhibit microbial activities).[38, 39] When one-dimension (1D) nanostructures

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are placed on the surface of a flat electrode, a much stronger electric field can be built up

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near the tip.

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magnitude stronger. As a result, even when the applied voltage is low, the electric field

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near the tip structure can still be strong enough to cause irreversible electroporation.

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Based on this phenomenon, the Cui group developed a conducting filter, where the two

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electrodes were prepared by modifying carbon nanotube (CNT)-coated polyurethane

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sponges with Ag nanowires.

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electrodes when water flew through the device. The CNT-coated sponge functioned as a

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macroscale porous conducting matrix, while the silver nanowires on the surface provided

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millions of nanoscale tips to achieve the strong electric field. This conducting filter

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achieved >6-log removal efficiency for bacteria disinfection. The same research group

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also developed other electrodes for electroporation based water disinfection: an Ag

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nanowires-modified CNT-coated cotton textile

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(CuONW)-modified copper mesh

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The voltages applied in these studies were significantly lower than that normally required

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for irreversible electroporation, but still higher than the typical voltages for water

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electrolysis (>2 V). [45-47] Therefore, one concern for this new disinfection process is that

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notable energy would be consumed for unnecessary water decomposition. Another

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concern is on the persistence of the inactivation caused by electroporation. It is not clear

[40, 41]

[36, 37]

(e.g., promote

If the tip is small enough, the electric field can be several orders of

[42]

A voltage of 10-20 V was applied across the two

[44]

[43]

and a copper-oxide nanowire

. Similar disinfection performance was reported.

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whether the inactivated bacteria can be reactivated during the water storage after

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treatment.

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Here in this study, we introduced a new CuONW-modified 3D copper foam electrode

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prepared by a facile thermal oxidation approach (Fig. 1a). An electroporation-disinfection

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cell (EDC) equipped with two CuONW-modified copper foam electrodes achieved

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superior disinfection performance with low applied voltages, energy consumption and

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DBPs formation potential. In addition, no bacteria re-growth/reactivation occurred during

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the storage process. This novel disinfection process is promising for practical water

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treatment.

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MATERIALS AND METHODS

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Electrode

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construction. Copper foams were cut into Φ = 1 cm × 0.5 cm as cylinder electrodes.

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Electrodes were etched with 1 M hydrochloric acid to remove the oxide layer and washed

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with de-ionized (DI) water for 3 times. Then electrodes were heated in air at 400 ºC for

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120 minutes. Prepared electrodes were then put into a plexiglass coaxial electrode holder

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(5 cm × 5 cm × 2.5 cm) with a plastic mesh (~100 µ0) in the middle in case of short

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circuit.

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Copper-oxide nanowire (CuONW) Characterization. Field-emission scanning

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electron microscopy (FE-SEM) images were taken on a FEI STRATA DB235 microscope

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at voltage of 5 kV. The crystal characterization of the CuONW was determined using a

fabrication

and

electroporation-disinfection

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cell

(EDC)

device


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single nanowire device by sonicating a CuONW-modified 3D copper foam electrode in

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ethanol to form a suspension containing individual nanowires. Nanowires were drop-cast

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onto a transmission electron microscopy (TEM) copper gird for analysis. TEM was

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performed on a JEOL JEM-200CX microscope with an accelerating voltage of 160 kV.

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Total chlorine and current measurement. Total chlorine concentrations in both control

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and treated samples were measured by a Hanna HI96724 total chlorine pocket

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colorimeter. All the voltages applied to EDC treatment were provided by a direct current

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power supply (DG1718E-5). The anode of the power supply was connected to one

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electrode and the cathode to the other. During EDC treatment, water samples were passed

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through electrodes and certain voltages were applied to those two electrodes. Currents in

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the circuit were measured by a digital multimeter (UNI-T UT39C).

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Bacteria inactivation with EDC. Escherichia coli (ATCC 15597), Enterococcus faecalis

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(ATCC 19433) and Bacillus subtilis (ATCC 6633) were cultured to log phase (12 h) and

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then diluted using normal saline solution (9.0 g l-1 sodium chloride) to ~107 CFU ml-1.

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Secondary effluents were collected from two wastewater treatment plants (WWTPs) with

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bacteria concentration of ~104 CFU ml-1. Each water sample (250 ml) flew through the

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EDC device at different hydraulic retention times (HRTs) varied from 1 to 15 s. At the

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same time, voltages varied from 0 to 5 V were applied to the device. Effluents were

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collected in sterilized centrifugal tubes. Bacterial concentrations of effluents and control

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samples were measured using standard spread plating techniques. Each sample was

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serially diluted, and then plated in duplicate and incubated at 37 °C for 24 h. Control

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samples was passed EDC at the same flow rate with the experiment samples without

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applying voltage. Treated and control samples were compared to determine the

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inactivation efficiency.

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Bacteria storage experiment. Normal saline solution (9.0 g l-1 sodium chloride) and DI

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water were sterilized at 121 ºC for 20 minutes. Secondary effluents were filtered by 0.22

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µm membrane to remove all the indigenous bacteria. E. coli samples (~ 107 CFU ml-1)

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were passed through the EDC operated at 1 V with a HRT of 3 s, 5 s or 7 s. Treated E.

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coli were harvested by centrifugation (HITACHI RX2 series, 14500 rpm corresponding

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to 17600 g for 15 minutes at 15 ºC) and then re-suspended in different substrates

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(distilled water, saline or filtered secondary effluent). Treated samples were then stored at

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25 ºC, which represents a typical temperature of nature aquatic environment. Then E. coli

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concentrations of samples were measured using standard spread plating techniques.

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Bacteria sample preparation for scanning electron microscopy (SEM). All bacteria

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samples for SEM were harvested by centrifuging at 14500 rpm (17600 g) for 15 minutes

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at 15 ºC (HITACHI RX2 series), and supernatants were removed. Then bacteria were

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fixed overnight in the fixative containing 0.1 M phosphate buffered solution (pH 7.3), 2%

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glutaraldehyde and 4% paraformaldehyde at 4 ºC, then washed with DI water. Samples

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were then dehydrated in increasing concentrations of ethanol solution (50, 70, 90 and

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100%), and dried in 100% tert-butyl alcohol. Samples were dispersed on a metal grid in

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preparation of SEM characterization.

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Live/Dead baclight staining experiment. All bacteria samples were harvested by

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centrifuging at 14500 rpm (17600 g) for 15 minutes at 15 ºC (HITACHI RX2 series).

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Samples were re-suspended in 0.1 M phosphate buffered solution (pH 7.3) to 100 µL.

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Live/Dead Baclight kit (Molecular Probes®) was used to implement the staining

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experiment. Equal volumes (2.5 µl) of SYTO 9 (0.6 mM) and PI (3 mM) dye solutions

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were added into samples including control. Samples were stored in dark for 30 minutes

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and examined using fluorescent microscopy.

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RESULTS AND DISCUSSION

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Fabrication and characterization of electroporation-disinfection cells (EDCs) with

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copper-oxide nanowire (CuONW)-modified copper foam electrodes.

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Copper foams were selected as the initial substrates to fabricate electrodes due to

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electrical conductivity, high porosity and surface area (more than 95% and ~104 m2 m-3,

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respectively)

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modified copper foams were synthesized through a simple one-step thermal oxidation

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approach: heating in air at 400 ºC for 2 h (Fig. 1b). During oxidization, CuONWs grew

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on the copper substrate and copper foam changed the color from bronze to black (Fig.

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1b). Because the physical surface of the copper foam was continuous in 3D, the CuONW-

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modification layer was also 3D continuous (Fig. 1c). The pore size of the copper foam

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was ~500 µm (Fig. 1c). CuONWs were rooted on the foam surface with lengths more

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than 10 µm (Fig. 1d) and diameters mostly less than 30 nm (Fig. 1e). High-resolution

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TEM image and electron diffraction pattern (Fig. 1f) confirmed the monoclinic structure

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of CuONW. Estimated from the density and pore size of the copper foam, the porosity

[48-50]

and relatively low cost (~$65 per m2 of 1 cm thick sheet). CuONW-

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and specific surface area were ~98% and ~104 m2 m-3, respectively. The CuONW-

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modified copper foam still maintained a high conductance of ~0.4 S cm-1 as a solid bulk

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material.

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An EDC was built by placing two pieces of the CuONW-modified copper foam

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electrodes (φ1 cm × 0.5 cm) in a plexiglass coaxial electrode holder (5 cm × 5 cm × 2.5

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cm) (Figs. 1a and S1). For electroporation-based disinfection, our new electrode revealed

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obvious advantages. The 3D macroscale porous structure allowed water to easily flow

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through with little hydraulic resistance. Moreover, the complex flowing pattern within the

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porous electrode increased the opportunity for microbes to approach the electrode surface

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where electric filed was significantly enhanced by the CuONWs and electroporation-

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disinfection occurred. The big pores with sizes of several hundred µm were unlikely to be

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clogged by microbes, which were normally less than 10 µm. Since CuONWs were

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directly grown out of the copper foam by in situ thermal oxidation, the physical contact

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between them was stable and electrical connection was guaranteed.

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Performance of EDC for water disinfection.

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The performance of our EDC was evaluated by treating the water samples that contained

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~107 colony forming units (CFU) ml-1 (Cin - concentration in influent) E. coli. To obtain

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specifics hydraulic retention times (HRTs), water samples flew through the EDC with

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aimed flow rates that were controlled by a peristaltic pump. Considering that the volume

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of electrodes (φ1 cm × 1 cm) is 0.785 cm3, flow rates were kept in the range of 6.72 to

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47.1 ml min-1, corresponding to hydraulic retention times (HRTs) of 7 to 1 s. At a

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specific HRT, a voltage varied from 0 to 10 V was applied across the two copper foam

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electrodes. The E. coli concentrations in the effluent (Ceff) were carefully analyzed and

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log removal efficiencies were calculated (E = -log (Ceff/Cin)). As the results shown in Fig.

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2a, removal efficiencies generally increased with HRT and applied voltage. When the

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HRT was 1 s, the EDC equipped with CuONW-modified copper foam electrodes

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achieved >7-log removal efficiency at 5 V and no E. coli detection in the effluent.

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Increasing the HRT to 5 and 7 s effectively lowered the voltages requirement for

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achieving the same performance to 2 and 1 V, respectively. Scanning electron

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microscopy (SEM) images confirm the occurrence of membrane damage. Compared to

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untreated E. coli whose cell membranes were complete and smooth (Fig. 2b), treated E.

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coli (1 V, 7 s) had obvious electroporation holes on the surface (Figs. 2c and S2)

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indicating lethal membrane damage. The results of staining test with Live/Dead Baclight

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kit (Figs. 2d and e) also suggested that E. coli cells lost the membrane integrities during

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treatment, because Syto 9 entered all the cells despite of membrane integrity and PI only

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entered the cells with damaged membrane (see supporting information for additional

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discussion). [51] When EDC equipped with non-treated copper foams (no-CuONWs on the

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surface), the removal efficiency was marginal if the applied voltages were less than 5 V.

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To investigate the energy consumption and potential health risk from formation of DBPs,

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currents in the circuit and total chlorine concentrations in the effluent were measured.

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The sample was normal saline, which contained 9.0 g l-1 sodium chloride. The HRT was

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fixed at 7 s corresponding to the fastest flow rate for complete inactivation after 1 V

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treatment and applied voltages varied from 0 to 5 V. As the results shown in Fig. 2f,

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when applied voltages were not high than 1 V, currents were minimum ( secondary effluent > DI water. The E. coli concentration at

246

specific storage time over 24 h were analyzed. As the results shown in Figs. 3a-c, E. coli

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was inactivated immediately and completely with 1 V, 7 s treatment. During the storage

248

process in normal saline (Fig. 3a), sterile secondary effluent (Fig. 3b) and DI water (Fig.

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3c) afterwards, no E. coli was detected, indicating no re-growth/reactivation.

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When the water sample was treated with 1 V, 3 s, E. coli was almost not inactivated

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directly after treatment and the concentrations of re-suspended E. coli were similar with

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the initial cell concentrations (~107 CFU ml-1). However, cell inactivation gradually

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occurred during storage. When treated E. coli were cultured in normal saline, sterile

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secondary effluent and DI water for 24 h, their concentrations decreased to 104, 102 CFU

255

ml-1 and no live bacteria detection, respectively (Fig. 3d). The extension of membrane

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damage caused by electroporation would be enlarged by great difference of salinities

257

between cytoplasm and substrate (Figs. 3d and e). Hence, bacteria inactivation during

258

storage were varied in different storage substrates: DI water > secondary effluent >

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normal saline. Such inactivation phenomenon was more obvious after 1 V, 5 s treatment

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(Fig. 3e). For untreated samples, E. coli inactivation during storage was insignificant

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(Fig. 3f). These results suggested that although not inactivating E. coli cells immediately,

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the short time (3 s and 5 s) EDC treatment had caused cell damage, which was

263

irreversible and eventually resulted in cell inactivation when E. coli were stored in the

264

tested substrates.

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Characterization of E. coli in EDC-treated water during storage.

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Results of staining experiment (Figs. 4a and b) suggested that treated (1 V, 3 s) E. coli

267

cells lost membrane integrities during storage. As shown in Fig. 4a, compared with

268

untreated sample containing few PI stained (membrane-compromised) bacteria, the

269

sample right after 1 V, 3 s treatment (0 h) contained considerable PI stained E. coli. More

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bacteria were stained by PI after 4 h, indicating more E. coli had compromised

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membranes. The number of PI stained bacteria decreased after 12 h, because the total

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number of integral bacteria decreased, indicated by the Syto 9 staining results (Fig. 4b).

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The proportion of PI stained bacteria continuously increased to ~97% after 24 h (Fig. 4c),

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which confirmed no bacterial re-growth/reactivation. SEM images (Figs. 4d-k) further

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showed the morphologies of E. coli stored in normal saline for 0-24 h after 1 V, 3 s

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treatment. Similar with untreated E. coli (Fig. 4d), E. coli cells right after treatment still

277

had complete and smooth membranes (Fig. 4e). However, significant damages could be

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observed on the surface after 4 h storage (Fig. 4f), and after 12 h storage, the treated E.

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coli strains could hardly maintain their cell configuration due to severely compromised

280

membranes (Figs. 4g and h). After 24 h, cells were disintegrated completely: cell

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membranes twisted together and their structures could hardly be observed (Figs. 4i-k).

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These evidences demonstrated that no bacteria re-growth/reactivation but inactivation

283

occurred during storage after EDC treatment. These outstanding features enabled EDC

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treatment become a promising substitution for the common UV irradiation disinfection

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by avoiding bacteria re-growth/reactivation after treatment.

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With sufficient EDC treatment (e.g., 1 V, 7 s), bacteria were inactivated immediately and

287

became uncultivable (Fig. 2). The mechanism has been reveled previously. Nanowire-

288

enhanced electric field has strong dipole-dipole interaction with the lipid bilayer of cell

289

membrane, which lead to thinning of membrane and finally large electroporation pores.

290

[12, 18, 20]

291

inactivation occurs (Fig. 2c).

292

or high voltage) EDC treatment, low dosage treatment (1 V, 5 s in our case) would form

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primary small electroporation pores on cell membrane with radii of at most several

Consequently, cell inclusion flows out exceedingly and immediate bacteria [12, 53]

Compared to high dosage (either long treatment time

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hundred nanometers (Fig. 5).[12] These primary electroporation pores were too small for

295

PI intercalation (Fig. 4c) and cell inclusion outflow, thus treated bacteria were detectable

296

when cultured in nutrient rich substrate right after treatment (e.g., nutrient agar) (Fig. 3e).

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However, stabilized by strong electric field, these primary small electroporation pores

298

were irreversible due to adhesion of hydrophilic groups of phospholipid from cell

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membrane and became water pathways (Step 1, Fig. 5).

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water inflow and cell inclusion outflow were prompted by difference of salinities

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between cytoplasm and substrate, which enlarged the electroporation “pores” during

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storage (Fig. 4c and Figs. 4f-h). [17] As a result, secondary damage of membrane formed,

303

cell structure broke down (Figs. 4i-k) and the cell died subsequently (Step 2, Fig. 5). It

304

was worth noting that the extension of membrane damage would be enlarged by great

305

difference of salinities between cytoplasm and substrate (Figs. 3d and e). Hence, bacteria

306

inactivation during storage were varied in different storage substrates: DI water >

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secondary effluent > normal saline. Considering that nature water bodies (rivers and

308

lakes) had similar salinity as secondary effluent, EDC effluent would be safe and reliable

309

when was discharged into environment.

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Since UV disinfection destroys the bacteria DNA structure instead of the membrane

311

integrity, the UV treated bacteria that become uncultivable right after treatment can

312

maintain the integrate membranes for over 24 h.

313

recover and become cultivable again in suitable environment.

314

treated bacteria will lose membrane integrities and cannot recover during storage due to

315

irreversible electroporation damages. Thus, no bacteria re-growth/reactivation occurs

[10]

[17]

Through the water pathway,

These uncultivable bacteria can

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[11]

Conversely, the EDC


316

during storage if bacteria have been inactivated and become uncultivable after EDC

317

treatment, which makes EDC a more reliable disinfection technology compared to UV.

318

In summary, we have developed a new CuONW-modified 3D copper foam electrode by a

319

facile thermal oxidation approach. An EDC equipped with two such electrodes has

320

achieved superior disinfection performance (> 7 log removal and no detectable bacteria in

321

the effluent) with an applied voltage as low as 1 V and energy consumption of 25 J l-1,

322

and with low potential of DBP formation. No bacteria re-grow during storage after EDC

323

treatment due to irreversible electroporation damage on cell membrane. EDCs can be

324

easily powered by batteries or solar cells. The energy consumption is

Nanowire-Modified Three-Dimensional Electrode Enabling Low

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