Accepted Manuscript

Fast and efficient inactivation of antibiotic resistant Escherichia coli by iron

electrode-activated sodium peroxydisulfate in a galvanic cell

Lingling Zhang, Hongkun Ma, Xinmei Huang, Zhengxu Yan, Wei Ding, Zifu
Li, Daqiang Cang

PII: S1385-8947(18)31539-0
DOI: https://doi.org/10.1016/j.cej.2018.08.065
Reference: CEJ 19679

To appear in: Chemical Engineering Journal

Received Date: 10 April 2018

Revised Date: 9 August 2018
Accepted Date: 10 August 2018

Please cite this article as: L. Zhang, H. Ma, X. Huang, Z. Yan, W. Ding, Z. Li, D. Cang, Fast and efficient inactivation
of antibiotic resistant Escherichia coli by iron electrode-activated sodium peroxydisulfate in a galvanic cell,
Chemical Engineering Journal (2018), doi: https://doi.org/10.1016/j.cej.2018.08.065

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Fast and efficient inactivation of antibiotic resistant Escherichia coli

by iron electrode-activated sodium peroxydisulfate in a galvanic cell

Lingling Zhanga,b , Hongkun Maa,b, Xinmei Huangb, Zhengxu Yana,b, Wei Dinga,b , Zifu Lia,b,

Daqiang Cang c
a
Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, Beijing

100083, PR China
b
School of Energy and Environmental Engineering, University of Science and Technology

Beijing, Beijing 100083, PR China

c
School of Metallurgy and Ecological Engineering, University of Science and Technology

Beijing, Beijing 100083, PR China

Abstract

Antibiotic overuse has led to the emergence of antibiotic resistant bacteria. However,

ordinary disinfection methods are not environment friendly and are inefficient in preventing

the spread of antibiotic resistant bacteria. Therefore, this study investigated a novel and

efficient electrolytically enhanced sulfate radical-based advanced oxidation process to

disinfect antibiotic resistant Escherichia coli (AR E. coli) in aqueous solution. Sterilization of

AR E. coli using the coupled process of /peroxydisulfate (PDS) and electrolysis

treatment of galvanic cell (galvanic cell- /PDS, named GFP) was evaluated. Various

process parameters, including temperature, PDS dosage, electrolyte concentration, and

concentration, were investigated. The active radicals involved in the GFP process were

identified, and the changes in cell substances were determined by Fourier transform infrared

spectroscopy and flow cytometry. Intracellular protein leakage and acute effluent biotoxicity

were also analyzed to evaluate the performance of the proposed method. The GFP system

demonstrated a high disinfection efficiency (7.882-log inactivation within 20 min), indicating

that the novel system is a promising treatment for the removal of AR E. coli in water.


Corresponding author: Tel/Fax: +86- 010-82376239; E-mail: linglingzhangll@hotmail.com

1
Keywords: galvanic cell- /PDS(GFP); antibiotic resistant Escherichia. coli; disinfection;

sulfate radical

1. Introduction

Antibiotic resistance has been considered as one of the most serious threats to human health
in the 21st century[1]. Antibiotic overuse has led to the emergence of antibiotic resistant
bacteria (ARB). Bacteria carrying multiple antibiotic resistance genes (ARGs) can spread all
over the world through various environmental media, such as rivers[2, 3], lakes[4],
groundwater[5], wastewater treatment plants[6-8], soils[9], and air[10]. Owing to horizontal
gene transfer and genome evolution, ARB have even been found in areas of no human
activity[4]. In addition, several ARB have been detected in healthy people who have not been
treated with antibiotics[11, 12], indicating that humans can absorb ARGs through other means,
such as eating and drinking[13, 14]. Furthermore, ARB thrive rapidly and can increase the
spread of infectious diseases and mortality intensively. Therefore, effective measures to curb
this phenomenon are urgently needed.
Currently, reusing water and wastewater after effective disinfection is essential to coping
with water shortages. However, UV radiation and chlorination as popular water disinfection
methods cannot control the spread of antibiotic resistance effectively[15, 16]. Furthermore,
Guo et al.[17] and Pang et al.[18] testified that low doses of UV radiation or chlorine may
increase the risk of ARB reproduction. Moreover, after chlorination, wastewater containing
organic matter and other precursors releases carcinogenic disinfection by-products[19].
Ozonation disinfection was also proved to be an unsatisfactory treatment by the results of Sousa
et al.[20] and Zhuang et al.[21]. Gradually, many types of advanced oxidation processes (AOPs)
(e.g., Fenton, photo-Fenton, TiO2/UV, UV/O3, UV/H2O2, and UV/H2O2/TiO2) have been
developed for inactivating ARB[22, 23]. Fenton oxidation and related technologies are
effective but economically infeasible methods due to their high costs and chemical
requirements [21, 24]. The H2O2/UV process is fast but unsuitable due to effluent
cytotoxicity[23]. Meanwhile, the TiO2/UV process is considerably affected by influent quality
and requires a long processing time. Obvious regrowth of ARB also occurs after the TiO2/UV
process[25].
Activated peroxydisulfate (PDS) process has attracted considerable attention in recent
years as a reliable alternative to Fenton oxidation because of its stability and ease of
transportation. This process can produce , which has a similar oxidation potential
2
(2.5–3.1 V) to but has a higher half-life (30–40 μs vs. 20 ns) and stronger selectivity than
[26, 27]. Thus, sulfate radical-based advanced oxidation processes (SR-AOPs) may have
the potential to the overcome limitations of a conventional AOP including short lifetime and
less selectivity of , long processing time etc.[28-31]. To date, SR-AOPs have been widely
used in soil and groundwater remediation by in situ chemical oxidation and eliminating
refractory organic pollutants (e.g., dyes, herbicides, pharmaceuticals and personal care
products, and landfill leachate) [32-37]. In analogy, SR-AOPs may hold promise to be more
effective than conventional water disinfection processes in inactivating E. coli due to their
powerful oxidation capabilities. Samyoung et al.[38] examined the inactivation of marine
phytoplankton by using persulfate activated with zerovalent iron. Peizhe et al.[39] found that
using UV combined with PDS to inactivate bacteriophage MS2 and E. coli enhances the
inactivation rate of MS2 by threefold and achieves one additional log of E. coli inactivation due
to formation. In addition, the work of Michael-Kordatou et al.[40] revealed that
UVC-activated PDS oxidation could cause total inactivation of E. coli in secondary-treated
wastewater within 90 min. The above outcomes suggest that SR-AOPs have advantages in
disinfection.
Meanwhile, the galvanic effect could promote reactions in situ efficiently and thus has
been applied to deal with various environmental pollutants[41]. Nie et al.[42] invented iron
oxides coated on Fe0 core–shell nanospheres and verified that its galvanic cell-like performance
is through the generation of hydroxyl radicals in addition with H2O2. And in the work of Nie et
al.[43], ferrous ions and ferric ions were produced through a similar galvanic effect from
Fe0/iron oxide. Moreover, Alcalá-Delgado et al.[44] found that galvanic Fenton has a
synergistic effect to remove pollutants more efficiently. Thus, the galvanic effect has been
proved useful for alleviating environmental problems.
On the basis of the above findings, the inactivation activity of the coupled Fe2+/PDS and
electrolysis treatment in a galvanic cell is noteworthy of further studies. This new approach
named as “galvanic cell- /PD (G P)” proc ss by combining galvanic cell with
ferrous-activated PDS was investigated for the first time. The performance of the GFP to
inactivate antibiotic resistant E. coli (AR E. coli) was investigated through the examination of
various operational parameters, including temperature, PDS dosage, electrolyte concentration,
and concentration, from which the appropriate experimental conditions were identified.
Additionally, the predominant active radicals involved in the GFP process were identified
through radical quenching study and electron spin resonance (ESR) spectroscopy. Then,
Fourier transform infrared (FTIR) spectroscopy, flow cytometry, and intracellular protein
3
leakage analysis were also carried out to detect the influence of this treatment on AR E. coli
cells. Ultimately, the acute toxicity of the effluent was evaluated by its light inhibition over
luminescent bacteria (Vibrio fischeri).

2. Materials and methods

2.1 Chemical materials and reagents

All chemical materials used in this study were analytical reagents. Sodium peroxydisulfate

(PDS, Na2S2O8, 98%), sodium sulfate (Na2SO4, 99%), and methanol (CH3OH, 99.5%) were

purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Tert-butanol was produced by

Tianjin Chemical Works (China). Luria–Bertani (LB) agar and broth medium (Beijing Land

Bridge Technology Co., Ltd., China) were used for growing and maintaining bacterial cultures.

5,5-Dimethyl-1-pyrroline N-oxide (DMPO, C6H11NO, 97%) and Coomassie brilliant blue

reagent G-250 (C47H48N3NaO7S2) were procured from J&K Scientific Ltd. (China) and Beijing

Solarbio Science Technology Co., Ltd. (China), respectively.

2.2 Bacterial preparation

The ARB for disinfection were AR E. coli (CICC 10664) with multiple antibiotic resistance

purchased from the China Center of Industrial Culture Collection. The AR E. coli carries ARGs,

such as SulⅡ and ampC. The two ARGs matching with known genes were confirmed by

BLAST searches on the GenBank database (see Supplementary data for detail). After

two-generation incubation, the third-generation bacteria were used for disinfection. AR E. coli

preparation was operated as follows: one bacterial colony was selected from LB agar medium

and then grown aerobically at 37.5 °C overnight with shaking at 200 rpm in LB broth medium

to approximately mid-exponential phase. Then, the bacteria were condensed by centrifuging at

5000 ×g for 10 min and rinsed with deionized water twice. Finally, the AR E. coli was

resuspended in 50 mL of deionized water, and the concentration of this bacteria in subsequent

experiments was 107-108 colony forming units per milliliter (CFU/mL).

2.3 Disinfection experiment design

4
 The galvanic cell system was composed of a commercial iron (Fe) sheet (2 cm × 5 cm) and a

graphite blade (2 cm × 5 cm) arranged parallel to each other at a distance of 3.0 cm and

connected by a wire, in which Na2SO4 was used as the electrolyte. The GFP disinfection

experiments were conducted in a 200 mL beaker containing deionized water at an original pH

of 6.5-7. Other doses of chemicals and AR E. coli were added into the liquid depending on

experimental needs. The beaker was placed in a magnetic-stirring water bath to maintain

constant temperature. Meanwhile, the plate count method was used to count the number of AR

E. coli cells in water samples at different moments. The disinfection effect was revealed by

log(C/C0), where C represents the concentration of AR E. coli at a time during treatment and C0

represents the primary concentration of AR E. coli. All experiments were duplicated, and

average values were presented.

2.4 ESR spectroscopy

Active radicals generated in the process were assayed by ESR spectroscopy. A 100 μL

sample was collected from the reactor without AR E. coli addition after reacting for 5 min.

Three samples were extracted from each of the following systems: galvanic cell system, GFP

system, and GFP+1M tert-butano syst m. Th samp s w r imm diat y mix d with 5 μL of

0.2 M DMPO to form a DMPO–radical adduct, which was then measured on an ESR spectrum

(Bruker EMX plus, Germany) with a microwave bridge (receiver gain, 5020; modulation

amplitude, 2 G; microwave power, 6.35 mW; modulation frequency, 100 kHz; and center field,

3525 G).

2.5 Cell substance inspection

During disinfection, some changes were observed between the bacterial cells before and after

treatments. Chemical bond changes, such as stretching and bending of AR E. coli cell substance,

were indicated by peak changes on the FTIR spectrum (Perkin-Elmer Thermo/Nicolet Nexus

470 FT-IR, USA). Three samples before and after GFP and electrolysis-only treatment were

centrifuged at 10,000 ×g (4 °C,10 min), and the bacterial precipitates were placed at 30 °C for

one day until they became dry blocks. Subsequently, 2% finely ground sample composed of
5
dried bacterial sample of 1 mg and KBr of 100 mg was placed in a KBr disc and analyzed using

FTIR spectroscopy. The infrared spectra of AR E. coli cells from three samples were recorded

in the wavenumber region of 2500-400 cm−1 at 2 cm−1 spectral resolution.

Moreover, the change in cellular membrane permeability was tested by LIVE/DEAD

BacLight Bact ria Viabi ity Kit (Invitrog n™, U A) purchased from Thermo Fisher Scientific

Inc. A 997 µL aliquot of reaction liquid in a flow cytometry analysis tube was added with 1.5

µL of 3.34 mM SYTO9 nucleic acid stain and 1.5 µL of 30 mM propidium iodide (PI). The

sample was incubated for 15 min at room temperature protected from light and then analyzed

by flow cytometry (Beckman coulter CytoFLEX, USA). Dot plots represented live/dead cells.

Intracellular protein leakage was analyzed using a modified Coomassie brilliant blue assay

(Bradford method). Samples (1 mL) collected from the reactor at different moments were

centrifuged at 10,000 ×g (4 °C, 10 min) to obtain the supernatants. Ev ry 10 μL sup rnatant

was b nd d with 990 μL of deionized water, and then absorbance was recorded on a UV–vis

spectrophotometer (HACH DR6000, USA) to determine the amount of protein released from

the cells.

2.6 Acute toxicity of effluent evaluation

The acute toxicity of effluents was analyzed by a Modulus single-tube multimode reader

(Turner BioSystems, USA) based on the light emission inhibition of the luminescent bacteria V.

fischeri (in accordance with DIN EN ISO11348-2-2009, Germany). The bacterial freeze-dried

powder was rehydrated to 1 mL of bacterial suspension. A 0.4 mL sample with culture

suspension was diluted to 2 mL for later use as a test specimen. Test specimen ( 00 μL) added

with 20 μL of luminescent bacterial suspension was the test sample, whereas 2% NaCl (400

μL) added with 0 μL of bacterial suspension was the control sample. In general, the test was

performed at 15 °C. The decrease in bacterial luminescence (H%) due to addition of toxic

chemicals can be determined as follows:

100 - ITt / (f I 0 ) 100, ( 1)

f I t / I 0. ( 2)

where IC0 and IT0 stand for the luminescence of the control and test samples at 0 min,

6
respectively; ICt and ITt are the luminescence values for the control and test samples measured

after 15 min treatment, respectively; and f is the correction factor based on the initial bacterial

luminescence intensity of the control sample and the one measured after the bacteria reacted

with 2% NaCl for 15 min.

Γ was us d to express H% as follows:

Γ / (1− H%). (3)

Changing Γ and concentration of K2Cr2O7 (reference standard of toxicity evaluation) C into

d nary ogarithm, th r ation b tw n Γ and was shown by using a linear fit as follows:

og Γ b og og. (4)

The acute toxicity of effluents could be conveyed by the corresponding concentration of

K2Cr2O7 ca cu at d from Γ va u .

3 Results and discussion

3.1 Comparison of disinfection effects under different processes

The following tests were carried out to evaluate the efficiency of GFP in AR E. coli

inactivation: (1) electrolytic process in galvanic cell consisting of iron as the cathode and

graphite as the anode, (2) only 2 mM PDS as the oxidant, (3) only 3 mM PDS as the oxidant, (4)

GFP with 2 mM PDS: iron electrode-activated 2 mM PDS, and (5) GFP with 3 mM PDS: iron

electrode-activated 3 mM PDS. The results are compared in Fig. 1. The electrolysis-only

process of the galvanic cell and only PDS oxidation could not sterilize AR E. coli effectively.

During galvanic cell electrolytic process or only 2 mM/3 mM PDS treatment, the log(C/C0) was

fluctuant. After 60 min treatment, the final log(C/C0) values were −0.044, 0.035, and −0.152.

When the galvanic cell was coupled with 2 mM PDS, an apparently good disinfection effect

occurred (log(C/C0) was −5.426 within 60 min). Moreover, a strong sterilizing effect was

achieved within 20 min after adding 3 mM PDS to the galvanic cell (log(C/C0) declined to

−7.882). This result revealed that the sole electrolytic process of the galvanic cell or

non-activated PDS had limited disinfection effect on AR E. coli. While the combination of

electrocatalysis and Fe(II) catalysis is a highly effective alternative to activate the formation of

7
sulfate radicals from PDS, which was coincided with the study of Wang et al.[45]. The

disinfection efficiency in the work of Wordofa et al. is much lower than ours. In their work, less

than 4 log inactivation of E. coli was achieved after 180 min of Fe2+/3mM PDS treatment[46].

In the GFP system, was continuously released to the solution from the surface of the

iron electrode through negative oxidation. When PDS was poured into the liquid, sulfate

radicals were generated via the catalysis of transition metal as follows:

- (negative electrode), (5)

, ( 6)

(positive electrode), (7)

. ( 8)
Energy of this process was provided by the galvanic cell, and extra energy supply was not
needed. Fe2+ regeneration from the positive electrode was also conducive to the production of
sulfate radicals. Additionally, was easily transformed into OH, which has an effective
capacity to inactivating bacteria. Undoubtedly, this coupled GFP system has promising
disinfection effect.

Fig. 1 Inactivation of AR E. coli under various reaction conditions. Experimental conditions

were 25 °C; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment.

3.2 Effect of parameters on inactivation of AR E. coli

3.2.1 Effect of temperature, electrolyte concentration and PDS concentration

8
 Temperature played a key role in the GFP process because it remarkably affected the reaction

rates in the system. To investigate the effect of different temperatures on inactivating AR E. coli,

several temperatures including 20, 25, 30 and 35 °C were chosen. After the water temperature

kept constant in a thermostat water bath, 50mM Na2SO4 and 2mM PDS were put into the

system with iron/ graphite galvanic cell. The control group was placed under the four

temperatures without any additional substance. Fig. 2 (a) reveals that high temperature

indicates good disinfection effect. From 20 °C to 30 °C, the disinfection performance of GFP

was promoted obviously. Previous works suggested that activation of PDS by thermal below

50°C may only slightly contribute to the generation of sulfate radicals [47-50].

The influence of electrolyte concentration on inactivation of AR E. coli was tested by

varying the concentration of Na2SO4 (30, 40, 50, and 60 mM) with other parameters unchanged.

As shown in Fig. 2 (b), the degradation rate slightly increased as the concentration of Na2SO4

was increased from 30 mM to 60 mM. The current produced by the galvanic cell increased to

peak value (10-15 mA) at 15-20 min and diminished with the reaction time (Table A.1). The

inactivation results with different concentrations of Na2SO4 were similar, suggesting that

electrolyte concentration might not carry weight for disinfection of AR E. coli.

To elucidate the effect of PDS in GFP, the initial concentration of PDS was increased from 1

mM to 4 mM and the final log(C/C0) of AR E. coli decreased from −4.083 to −7.711 in Fig. 2

(c). It can be suggested that more free radicals generated from activated PDS led to superior

sterilization. The disinfection performance of the GFP system with 3 mM PDS was extremely

better than that of the system added with 2 mM PDS, and similar to that in the treatment with 4

mM PDS within 30 min. This result may be ascribed to the sufficient number of active radicals

in the reactor. Consequently, 3 mM was the advisable concentration of PDS used in this

research.

9
Fig. 2 (a) Inactivation of AR E. coli after GFP treatment under various temperatures.
Experimental conditions were [PDS]=2 mM; [Na2SO4]=50 mM as supporting electrolyte; No
pH adjustment. (b) AR E. coli inactivation in the GFP process at various electrolyte
concentrations. Experimental conditions were 25 °C; [PDS]=2 mM; No pH adjustment. (c)
Sterilization of AR E. coli in the GFP process at various PDS concentrations. Experimental
conditions were 25 °C; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment.

3.2.2 Effect of concentration

To evaluate the influence of Cl− on the GFP system, GFP system addition with different

concentration was applied to inactivating AR E. coli. Three concentrations, namely, 0.2, 1,

and 5 mM, were selected to examine the disinfection influence of while keeping other

parameters unchanged in accordance with the procedures provided by references[51, 52] and

Surface Water Environment Quality Level of China. GFP + various dosage without PDS

were carried out as control experiments whose results indicated that the system had no

inactivation effect on bacteria with the absence of PDS (Table A.2). The results of these

processes were compared with the conventional GFP treatment without in Fig. 3. Without
10
 interference, at each moment, GFP system’s p rformanc was superior to that of other

systems with . The difference may arise because binds to a part of the sulfate radical

to generate other substances, which may have weaker disinfection effect than the sulfate radical

itself, as explained by Eqs. (9)-(12).

, (9)

, (10)

, (11)

. (12)

In addition, the concentration did not significantly affect disinfection. Fig. 3 indicated

that high concentration means good disinfection effect after 20 min in these systems with

extra . However, no significant difference was shown after 30 min. The impact of on

different treatment were varies depending on the objectives of the treatment. For example,

negatively influences the degradation of some refractory organic pollutants, including

perchloroethylene and azo dye Orange G, by the Fe2+/PDS process[53]. Nevertheless, the

positive role of on the reaction performance of sulfamethoxazole degradation by

electrochemically activated sulfate may be due to the generation of additional radicals, such as

and [54]. The different effects of on the degradation performance may be

attributed to the different molecular structures of pollutants and the presence of Fe2+ in

SR-AOPs.

11
Fig. 3 Sterilization of AR E. coli in GFP treatment at various concentrations.
Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting
electrolyte; No pH adjustment.

3.3 Possible disinfection mechanism analysis of GFP

To investigate the possible disinfection mechanism in GFP, the dominating radical species

were confirmed during this system treatment. Two radical scavengers, methanol and

tert-butanol, were employed to evaluate the contribution of and OH. On the basis of

references, the reaction rate constants of methanol with and OH are 1×107 M−1 s−1[55]

and 9.7×108 M−1 s−1[56], respectively. Moreover, tert-butanol is ordinarily utilized as an OH

scavenger because its rate constant with OH (3.8-7.6×108 M−1 s−1) is considerably greater than

that with (4.0-9.1×105 M−1 s−1)[57]. Thus, tert-butanol can quench OH more easily than

. As shown in Fig. 4, the initial log(C/C0) was near −8 in the absence of radical scavengers.

The disinfection was suppressed when radical scavengers were added into the reaction system.

For example, in the presence of 1 M methanol and 2 M methanol, the last log(C/C0) increased to

−3.649 and −3.460, respectively. The addition of 1 or 2 M methanol notably inhibited the

disinfection performance of the GFP system with log(C/C0) increased from −7.882 to −3.6485

and −3.4595 in 20 min, respectively. Meanwhile, log(C/C0) at the same moment was unaffected

by the addition of 1 M tert-butanol. The great differences in log(C/C0) rise by the two radical

12
scavengers suggested the presence of both hydroxyl and sulfate radicals in the system as

discussed previously. Furthermore, the results suggest that was the dominant radical to

sterilization.

Fig. 4 Effect of different radical scavengers on sterilization during GFP treatment.

Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting
electrolyte; No pH adjustment.
Furthermore, the reactive radicals were measured by ESR assay. Fig. 5 reveals that the

signals for the combination of GFP and 1 M tert-butanol were much higher than the signals for

the independent galvanic cell or GFP. No radical signals were observed in the electrolysis-only

treatment without PDS. Nevertheless, a DMPO/ OH adduct showing its characteristic 1:2:2:1

spectrum with hyperfine coupling constants of aN=14.9 G and aH=14.9 G was clearly identified

in the two other GFP systems. The signal of the DMPO/ adduct (see black arrows) was

also clearer in the GFP + 1 M tert-butanol system than in the GFP without tert-butanol system,

which has six peaks with hyperfine coupling constants of aN=12.79, aH=9.02, aH=1.4, and

aH=0.24 G. The DMPO/ adduct in water was unstable and can easily convert into the

DMPO/ OH adduct. Thus, the spectra were recorded during the transformation from

DMPO/ to DMPO/ OH[58]. ESR analysis results prove that the and OH radicals

were generated in the GFP system.

13
Fig. 5 ESR signals for different processes including electrolysis-alone treatment of galvanic
cell, GFP treatment, and GFP+1 M tert-butanol treatment. Experimental conditions were
25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment. The
samples were taken at 5 min.
In addition, the pH values of the liquid in the GFP system were tested at different times (Fig.

A.1). All pH values decreased intensively within 30 min treatment regardless of the added PDS

concentration. Moreover, increased PDS of the GFP system indicated low final pH after 30 min

reaction. The effect of low pH (pH 3) on disinfection was studied because the final pH of the

above processes was not lower than 3. The results are demonstrated in Table A.3. Low pH may

not contribute to the inactivation of AR E. coli. Therefore, and OH arising from GFP

treatment play a primary role in AR E. coli sterilization.

3.4 Cell substance damage after treatment with GFP

The FTIR spectra of AR E. coli cells before and after treatment with electrolysis and galvanic

cell–Fe(II)/PDS were recorded (Fig. 6), and the main peaks were linked to the corresponding

substances or groups (Table A.4) on the basis of previous studies[59, 60]. Five obvious changes

could be concluded from the comparison among the three FTIR spectra: (1) peak changes at

1651.6/cm and 1544.2/cm indicated that this treatment would influence amide I and amide II

bands of proteins and peptides; (2) stretching and bending of methyl and methylene in lipids,

14
proteins, and carbohydrates were indicated by peak changes at 1470–1250/cm; (3) peak change

at 1241.2/cm revealed (P═O) asymmetric stretching of phospholipids, whereas peak change at

1084.4/cm showed (P═O) symmetric stretching of phospholipids, DNA, and RNA; (4) peaks at

1200–900/cm illustrated the vibrations of C–OH, C–O–C, and C–C from carbohydrates present

in the cell wall; and (5) nucleotide-related peak changes at 900–800/cm. The FTIR analysis

results indicated that iron electrode-activated PDS might not only damage the cell wall and cell

membrane but also affect the intracellular substances, such as proteins and nucleic acid.

Besides, the differences between spectra before and after disinfection might also be attributed

to the destruction of intracellular organic matter outflowing from the cell in the system.

Fig. 6 FTIR spectra of AR E. coli cells before and after treatment with electrolysis and GFP

for 30 min. Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as

supporting electrolyte; No pH adjustment.

3.5 Flow cytometry study of cell membrane integrity in AR E. coli

LIVE/DEAD BacLight Bacterial Viability Kit was composed of SYTO9 and PI. Stains in

living and dead Gram-negative bacteria can be shown by nucleic acid dyestuff SYTO9,

15
whereas PI only enters permeabilized cytoplasmic membranes. Thus, only after the change of

cell membrane integrity was PI able to enter the cells and quench SYTO9 fluorescence. In Figs.

7(a)–7(d), those cells dyed by PI were regarded as dead. Cell structures destroyed by active

radicals could not be shown by PI as dead bacteria because those cell membranes were not only

permeabilized but also were wrecked into cell debris. The shapes in the following three figures

were different from the first one possibly because several cell debris appeared during

disinfection treatment. Furthermore, abundant black dots stained by SYTO9 appeared in the

lower right section of the figures. Thus, the nucleotide of the bacteria was possibly changed by

this treatment, which agrees with the disinfection influence of GFP treatment on the bacteria as

indicated in the FTIR spectra. In addition, Fig. 7(b) – (d) show several dead cells stained by PI

from 10 min to 30 min. At 30 min, only 17.31% bacteria were detected as dead cells on the base

of permeabilized membranes, but most dead bacteria possibly lost their cell structure. Thus,

they were unable to grow in LB agar and impossible to be counted.

(a) (b)

16
 (c) (d)

Fig. 7 Changes in membrane permeability during GFP treatment. Experimental conditions

were 25 °C; [PDS]=2 mM; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment.

3.6 Intracellular protein leakage test

Protein leakage from AR E. coli cells was determined after contacting with GFP for different

times (Fig. 8). During GFP with 3 mM PDS treatment, intracellular protein leakage increased

intensively to 2238.71 μg mL−1 in the first 10 min because of the cell membrane permeability

disorder resulting from the destruction of radical oxidation. Then, with further destruction by

active radicals, the protein leakage values decreased because of the injury of protein, which

could not be dyed by Coomassie brilliant blue reagent. Gau et al.[61] also indicated that sulfate

radical is a great reagent for fast oxidation of proteins because of its potent, nonspecific, and

tunable characteristics. Moreover, sulfate radical has been applied in sludge dewatering,

reflecting its strong oxidizing property to damage proteins and shorten

It can be easily found that the values in the galvanic cell–Fe(Ⅱ)/3 mM PDS system

were higher than those in the one with 2 mM PDS within 30 min. The protein leakage results

indicated clearly that GFP treatment under normal temperature could cause cell death with

thorough cracking of the cell wall and membrane. This result was in consistent with FTIR and

flow cytometry analysis results.

17
Fig. 8 Intracellular protein leakage measurement during GFP treatment. Experimental

conditions were 25 °C; [PDS]=3 mM/2 mM; [Na2SO4]=50 mM as supporting electrolyte; No

pH adjustment.

3.7 Acute toxicity change in the GFP treatment

Considering that the degradation intermediates or low pH value might be toxic, the effluent

toxicity was investigated on the luminescent bacteria V. fischeri. The toxicity was reflected by

the concentration of standard material K2Cr2O7 based on the experiment method. Fig. 9 reveals

that the toxicity values at different moments in the system without were lower than those

in the system with before 20 min. The formation of some intermediates and products led

to high acute toxicity due to the generation of and in the presence of [54].

Besides, univariate analysis of variance was conducted to evaluate the impacts of time and Cl¯

concentration on toxicity. Both time and Cl¯ concentration had obvious impact on acute

toxicity of effluents (Ptime= 01, PCl¯= 0.022). According to pairwise comparison of different Cl¯

1
P<0.01 represents extreme significant influence.
2
P <0.05 represents significant influence.

18
concentration, toxicity might be more influenced by 5 mM than 2 mM (P5mM= 0.007,

P2mM= 0.053). However, might not make a large contribution to the acute toxicity after 30

min treatment, which is likely to be induced by decreasing pH and not by the toxicity of

intermediates generated in the reactor[63]. The change in pH is shown in Fig. A.2. Moreover,

the initial acute toxicity could not be detected by this method because the toxicity of the initial

test sample was weaker than that of the control sample as evidenced by their light emission

inhibition of the luminescent bacteria.

Fig. 9 Change in acute toxicity during GFP treatment at different concentrations.

Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting

electrolyte; No pH adjustment.

4. Conclusion

In this study, the iron electrode of a galvanic cell was used to activate PDS, and

generated from iron electrode formed by the transformation of in the positive

electrode. The role of the GFP system on the removal of AR E. coli was distinguished, with

7.882 log units and 5.426 log units of bacterial reductions under the GPF treatment added

with 3 and 2 mM PDS at 25 °C within 30 min, respectively. Although had high

19
oxidizing power and could be converted from , the great disinfection effect was

attributed to in the GFP system. Destructions of cell membrane and nucleotide by GFP

were revealed by the FTIR spectra and figures in flow cytometry. This study indicates that

the GFP system can eliminate almost all AR E. coli in water at the laboratory scale in a short

time. Further research is required to survey its effectiveness and overcome its shortcomings at

the field scale to apply this method for real water disinfection.

Acknowledgements

This work was supported by the “th undam nta R s arch unds for th ntra

Univ rsiti s” [grant numbers FRF-IC-17-007] in China.

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24
Figures

Fast and efficient inactivation of antibiotic resistant Escherichia coli

by iron electrode-activated sodium peroxydisulfate in a galvanic cell


Lingling Zhanga,b , Hongkun Maa,b, Xinmei Huangb, Zhengxu Yana,b, Wei Dinga,b , Zifu Lia,b,

Daqiang Cang c
a
Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, Beijing

100083, PR China
b
School of Energy and Environmental Engineering, University of Science and Technology

Beijing, Beijing 100083, PR China

c
School of Metallurgy and Ecological Engineering, University of Science and Technology

Beijing, Beijing 100083, PR China


Corresponding author: Tel/Fax: +86- 010-82376239; E-mail: linglingzhangll@hotmail.com

25
Fig. 1 Inactivation of E. coli under various reaction conditions. Experimental conditions were
25 °C; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment.

Fig. 2 (a) Inactivation of E. coli after GFP treatment under various temperatures.
Experimental conditions were [PDS]=2 mM; [Na2SO4]=50 mM as supporting electrolyte; No
pH adjustment.

Fig. 2 (b) AR E. coli inactivation in the GFP process at various electrolyte concentrations.
26
Experimental conditions were 25 °C; [PDS]=2 mM; No pH adjustment.

Fig. 2 (c) Sterilization of E. coli in the GFP process at various PDS concentrations.
Experimental conditions were 25 °C; [Na2SO4]=50 mM as supporting electrolyte; No pH
adjustment.

27
Fig. 3 Sterilization of E. coli in GFP treatment at various concentrations. Experimental
conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting electrolyte; No pH
adjustment.

Fig. 4 Effect of different radical scavengers on sterilization during GFP treatment.

Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting
electrolyte; No pH adjustment.

Fig. 5 ESR signals for different processes including electrolysis-alone treatment of galvanic
cell, GFP treatment, and GFP+1 M tert-butanol treatment. Experimental conditions were
28
25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment. The
samples were taken at 5 min.

Fig. 6 FTIR spectra of E. coli cells before and after treatment with electrolysis and GFP for 30
min. Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting
electrolyte; No pH adjustment.

29
 (a) (b)

(c) (d)
Fig. 7 Changes in membrane permeability during GFP treatment. Experimental conditions
were 25 °C; [PDS]=2 mM; [Na2SO4]=50 mM as supporting electrolyte; No pH adjustment.

30
Fig. 8 Intracellular protein leakage measurement during GFP treatment. Experimental
conditions were 25 °C; [PDS]=3 mM/2 mM; [Na2SO4]=50 mM as supporting electrolyte; No
pH adjustment.

Fig. 9 Change in acute toxicity during GFP treatment at different concentrations.

Experimental conditions were 25 °C; [PDS]=3 mM; [Na2SO4]=50 mM as supporting
electrolyte; No pH adjustment.
31
Highlights

 A novel system used from galvanic cell to activate sodium peroxydisulfate.

 7.882 log inactivation of antibiotic resistant E. coli within 20 min in the system.
 The dominant radicals were identified as sulfate radicals and hydroxyl radicals.
 Destructions of bacteria even their nucleotide were discovered by flow cytometry.

32