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Citation: Liu, Z.; Yu, X.; Zhou, Z.;
Zhou, J.; Shuai, X.; Lin, Z.; Chen, H.
3D ZnO/Activated Carbon Alginate
Beads for the Removal of
Antibiotic-Resistant Bacteria and
Antibiotic Resistance Genes. Polymers
2023,15, 2215. https://doi.org/
10.3390/polym15092215
Academic Editors: Qina Sun, Lichun
Xiao and Liazhou Song
Received: 23 April 2023
Revised: 6 May 2023
Accepted: 6 May 2023
Published: 7 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
polymers
Article
3D ZnO/Activated Carbon Alginate Beads for the Removal of
Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes
Zhe Liu 1, Xi Yu 1, Zhenchao Zhou 1, Jinyu Zhou 1, Xinyi Shuai 1, Zejun Lin 1and Hong Chen 1,2,3,*
1College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China;
22014059@zju.edu.cn (Z.L.); 22114054@zju.edu.cn (J.Z.)
2International Cooperation Base of Environmental Pollution and Ecological Health,
Science and Technology Agency of Zhejiang, Zhejiang University, Hangzhou 310058, China
3Key Laboratory of Environment Remediation and Ecological Health, Ministry of Education,
College of Environmental Resource Sciences, Zhejiang University, Hangzhou 310058, China
*Correspondence: chen_hong@zju.edu.cn; Tel./Fax: +86-571-8898-2028
Abstract:
The worldwide prevalence of antibiotic-resistant bacteria (ARB) and antibiotic resistance
genes (ARGs) have become one of the most urgent issues for public health. Thus, it is critical to
explore more sustainable methods with less toxicity for the long-term removal of both ARB and ARGs.
In this study, we fabricated a novel material by encapsulating zinc oxide (ZnO) nanoflowers and
activated carbon (AC) in an alginate biopolymer. When the dosage of ZnO was 1.0 g (
≈
2 g/L), the
composite beads exhibited higher removal efficiency and a slight release of Zn
2+
in water treatment.
Fixed bed column experiments demonstrated that ZnO/AC alginate beads had excellent removal
capacities. When the flow rate was 1 mL/min, and the initial concentration was 10
7
CFU/mL, the
removal efficiency of ARB was 5.69-log, and the absolute abundance of ARGs was decreased by
2.44–2.74-log. Moreover, the mechanism demonstrated that ZnO significantly caused cell lysis,
cytoplasmic leakage, and the increase of reactive oxygen species induced subsequent oxidative
stress state. These findings suggested that ZnO/AC alginate beads can be a promising material for
removing ARB and ARGs from wastewater with eco-friendly and sustainable properties.
Keywords:
antibiotic resistance; calcium alginate; zinc oxide/activated carbon; fixed bed; reactive
oxygen species
1. Introduction
Since their discovery in the 1940s, antibiotics have been widely used in aquaculture,
livestock breeding, and controlling human infectious diseases [
1
–
3
]. However, the abuse
and overuse of antibiotics have caused the progressive increase of antibiotic-resistant
bacteria (ARB) and antibiotic resistance genes (ARGs), which have led to high biological
risks worldwide [
4
–
6
]. Owing to their potential risks, ARB and ARGs are characterized as
emerging environmental contaminants [
7
] and recognized as one of the three severe threats
to public health [8].
Conventional disinfection, such as chlorination and ultraviolet, are mostly used in
wastewater treatment [
9
–
11
]. Nevertheless, previous studies have reported that these meth-
ods have limited efficiency in removing ARGs [
12
,
13
], forming disinfection byproducts [
14
],
and inducing ARB to enter viable but nonculturable states [
15
]. Therefore, it is essential
to develop efficient and eco-friendly disinfection methods. To date, metal oxides, includ-
ing TiO
2
and ZnO, have been frequently reported because of their bactericidal properties
without any toxic byproducts [
16
]. Compared to the other reported materials, ZnO has
more advantages, such as simple preparation, low cost, and high stability [
17
]. Recently,
the performance of ZnO was optimized by modifying its structure [
18
] or exploring green
synthesis methods [
19
]. However, in the case of water treatment, ZnO still exhibits issues
such as limitation of aggregation, difficulty in recycling, and high leaching of Zn
2+
into
Polymers 2023,15, 2215. https://doi.org/10.3390/polym15092215 https://www.mdpi.com/journal/polymers
Polymers 2023,15, 2215 2 of 14
water [
20
]. To address these issues, biopolymer matrices of alginate or chitosan, which
were extracted from nature with non-toxicity and biodegradability, were introduced to
immobilize and slow the leaching of metal ions [
21
–
23
], while activated carbon (AC) was
added to enhance mechanical strength and adsorption capacity [24].
Some researchers have investigated the potential of ZnO-alginate beads for disinfection
treatment in water [
25
,
26
]. In contrast, a few reports have studied the efficiency of the
composite in removing ARB and ARGs. In addition, few reports have been made on fixed
beds to remove ARB and ARGs, and fixed bed study is a fundamental step for engineered
applications. In this context, column experiments with the composite were designed and
used for efficient removal under the influence of bed depths, initial bacterial concentrations,
and flow rates. Considering technologies for low-cost and point-of-use treatment [
27
], these
filtration and disinfection systems are expected to remove ARB and ARGs from highly
contaminated wastewater independently without extra energy input.
In this paper, synthesized ZnO nanoflowers and AC were encapsulated in sodium
alginate to form composite beads. This study aimed to (1) characterize the alginate compos-
ites and compare the performance of ZnO/AC alginate beads against a model ARB E. coli
HB101, (2) examine the efficiency of removing ARB and ARGs of fixed bed with different
parameters, and (3) illustrate the possible mechanism of ARB removal.
2. Materials and Methods
2.1. Materials
ZnO nanoflowers were simply prepared through a one-step hydrothermal method
published by Xu [
28
]. Briefly, 2.5 mmol zinc acetate dihydrate and 2.5 mmol sodium citrate
were dissolved in deionized (DI) water with the addition of 4.0 M NaOH. Then, the above
mixture was transferred into a 100 mL Teflon-lined autoclave and heated at 120
◦
C for
8 h
. The industrial ZnO (99.99% purity) was purchased from Aladdin (Hangzhou Bangyi
Chemical Co., Ltd., Hangzhou, China). Sodium alginate (CP) was purchased from SCR Co.,
Ltd. (Shanghai, China), and activated carbon (AR, 200 mesh) was purchased from Xianding
Biotechnology Co., Ltd. (Shanghai, China). The model antibiotic-resistant bacteria strain
was E. coli HB101 which carried three plasmid-encoded ARGs (tetA, bla
TEM
, and aph(3
0
)-Id).
2.2. Synthesis of ZnO/AC Alginate Beads
The different amounts of ZnO (0 g, 0.25 g, 0.5 g, and 1 g) and 0.2 g AC (2 g/L),
the typical level used in previous studies, were added to 100 mL of DI water. Then,
sodium alginate (2.5% w/v) was mixed with the above solution under ultrasonic radiation
and stirred until a homogeneous mixture was obtained. After that, uniform ZnO/AC-
alginate solution was injected into 0.3 M CaCl
2
solution dropwise to form crosslinked
hydrogel beads (~3 mm). Subsequently, the yielded beads were left in the solution for
2 h to harden and then washed thoroughly to remove residual surface reagents. After-
ward, these ZnO/AC alginate beads were stored in sealed and sterilized containers at
room temperature.
2.3. Characterization of ZnO/AC Alginate Beads
X-ray diffraction (XRD, Bruker D8 Advance, Karlsruhe, Germany) and Fourier-transform
infrared spectroscopy (FTIR, Nicolet iS50FT-IR, Madison, WI, USA) patterns were recorded
to investigate the crystal structure and the functional groups of the synthesized material.
The morphology and elemental components were observed using scanning electron mi-
croscopy (SEM, Hitachi SU8010, Tokyo, Japan) and energy dispersive spectroscopy (EDS).
The UV
−
vis absorption spectra were estimated using a spectrophotometer (DR 5000, Hach,
Loveland, CO, USA), and the fluorescence emission spectra of samples was recorded using
a Fluorescence spectrometer (FLS1000, Edinburgh, UK).
To examine the release of Zn
2+
ions, samples were prepared after mixing 10 g beads
with 100 mL DI water while containers shaking at 37
◦
C for 240 min and collected at time
Polymers 2023,15, 2215 3 of 14
intervals. The Zn content was precisely analyzed with ICP-MS (PerkinElmer, NexION
300X, Waltham, MA, USA).
2.4. The Removal Efficiency of ARB by ZnO/AC Alginate Beads
A single colony of E. coli HB101 was inoculated in LB Broth for 16 h overnight at 37
◦
C
to reach on log phase. Bacteria were then harvested by centrifuging and resuspended in
0.01 M phosphate buffer solution (PBS, pH = 7.2) to maintain OD
600
around 0.6. At this
moment, the concentration of bacteria was measured using the cell counting method at
10
8
CFU/mL. Subsequently, the bacteria were mixed with different ZnO content (0 g,
0.25 g
, 0.5 g, and 1 g) and AC content (0 g, 0.2 g) of beads. Then an aliquot of 100
µ
L
was withdrawn every 2 h. The treated bacterial suspension was serially diluted to 10-fold
increments using PBS and coated onto selective LB agar plates containing tetracycline,
kanamycin, and ampicillin. Removal difference was identified using the cell counting
method, and the above experiments were done in triplicate.
2.5. Application of ZnO/AC Alginate Beads in Fixed Bed
Fixed bed experiments were conducted in glass columns, provided with an inlet,
outlet, and glass valve, with an internal diameter of 22 mm and length of 300 mm. Before
treatment, the glass tube, valve, inlet pipe, and packing tools were sterilized using UV
irradiation of clean bench. The bed depth was designed for 10 cm, while the removal
efficiency was investigated under different initial concentrations (10
8
, 10
7
, 10
6
CFU/mL)
with a hydraulic retention time (HRT) of 0.32 h and at varying flow rates (1, 2, 3 mL/min)
with HRT of 0.63 h, 0.32 h, and 0.21 h, respectively. Each fixed bed unit was operated in
downflow mode, and bacteria suspension flow was continuously driven by gravity. The
number of colony-forming units (CFU) per mL of effluent was recorded using the cell
counting method.
Breakthrough experiments were described as a ratio of N
t
/N
0
where N
0
and N
t
refer
to the concentrations of influent and effluent at time t, respectively. The breakthrough
point is when bacteria at the outlet reaches 10% (N
t
/N
0≈
0.1) of the initial concentration.
Therefore, the scatter graph of (N
t
/N
0
) versus t was plotted to visualize the performance.
AEC interprets the treated capacities against contaminated influent before the materials
were exhausted, which was calculated using Equation (1):
AEC =
M
Vt(1)
where Mrepresents the mass of dried material in g and Vtis the influent volume in L.
2.6. Absolute Abundance of Antibiotic Resistance Genes
Samples were collected from the outlet of the fixed bed and then filtered through
0.22
µ
m aquo-filter membranes. DNA has been extracted using FastDNA SPIN Kit (MP
Biomedicals, Santa Ana, CA, USA) and measured on NanoDrop LITE (Thermo, Waltham,
MA, USA). The removal efficiency of ARGs was detected by quantitative real-time PCR
(qPCR) using the Bio-Rad iQ5 (Bio-Rad, Hercules, CA, USA). The information of primers
and standard curves of tetA, bla
TEM
, and aph(3
0
)-Id were shown in Table S1. The qPCR
system of 15
µ
L contains 1
µ
L of DNA template, 7.5
µ
L of SYBR Premix Ex Taq (TaKaRa,
Dalian, China), 5.5
µ
L distilled H
2
O and 0.5
µ
L of each primer. After the reaction program
that followed, the absolute abundance of gene copy number was calculated.
2.7. Mechanism of ARB Removal
2.7.1. Microbial Morphology Using SEM
Microstructure before and after the exposure for 120 min was observed using SEM to
verify the progressive cellular damage of ARB. The precipitation of bacteria was collected
and sealed with 2.5% glutaraldehyde at 4
◦
C overnight. Afterward, the pretreatment and
details of sample detection were conducted as a laboratory manual.
Polymers 2023,15, 2215 4 of 14
2.7.2. Live/Dead Fluorescence Assay
Bacterial viability was tested using confocal laser scanning microscopy (LSM880, Dres-
den, Germany) using the LIVE/DEAD BacLight Bacterial Viability Kit (L7012, Thermo
Fisher, Waltham, MA, USA). As per the operation manual, a 3
µ
L mixture (1:1) of fluo-
rescent nucleic acid dye (SYTO 9/PI) was reacted with 1 mL bacterial suspension in the
dark for
15 min
. Then, samples were identified with excitation/emission wavelengths of
488/537 nm and 561/624 nm.
2.7.3. Lipid Peroxidation Analysis
Malondialdehyde (MDA), which is the biomarker of lipid peroxidation reaction, was
detected according to the protocol of the MDA test kit (Solarbio, Beijing, China) using
thiobarbituric acid reactive substances assay (TBARS). Samples were collected at 0.5 h, 2 h,
and 4 h, and the absorbance was recorded at 532 nm and 600 nm.
2.7.4. Oxidative Stress Measurements
The oxidative stress of bacteria was evaluated using intracellular reactive oxygen
species (ROS) and the activity of corresponding antioxidant enzymes. Intracellular ROS
was analyzed using the fluorescent probe of 2
0
,7
0
-dichlorofluorescein diacetate (DCFH-DA)
(EMD Millipore, Burlington, MA, USA), which can be oxidized to fluorescent dichlorofluo-
rescein [
29
]. Flow cytometry (FCM, BD FACSVerse, Piscataway, NJ, USA) was employed to
monitor the fluorescence intensity of 20,000 cells under wavelengths of 488 nm and
525 nm
.
The activities of superoxide dismutase (SOD) and catalase (CAT) were measured using
detection kits (Solarbio, Beijing, China) based on absorbance at 560 nm and 240 nm via a
multimode reader (VLBL00D1, Thermo Fisher, Waltham, MA, USA).
2.8. Statistical Analysis
All the results were analyzed and calculated as mean and deviation values (n
≥
3)
using Microsoft Excel 2016. Data visualization was completed using Origin 2022b (Origin
Lab Corporation, Northampton, MA, USA), and significant differences were conducted in
SPSS V24 (IBM, Armonk, NY, USA) at two levels (* p< 0.05; ** p< 0.01).
3. Results and Discussion
3.1. Characterization of ZnO/AC Alginate Beads
SEM and EDS analysis were used to detect the morphology, size of the composite,
and dispersion of particles. Based on the SEM images, the diameter of ZnO/AC coated
in alginate beads was around 2 mm with a porous mesh structure after freeze-drying
(Figure 1a). ZnO nanoflowers exhibited uniform growth on the surface and between the
porosity (Figure 1b), as shown in high magnification (Figure 1c). As shown in Figure S1, the
diameter of synthesized ZnO was about 3.8
µ
m, as assembled from nanosheets of
30–40 nm
in thickness. As shown in Figure S1c, the bactericidal activity of synthesized ZnO was
also superior to that of industrial ZnO. From EDS analysis (Figure 1d–h), the elements of
Zn, C, and O were represented as clear peaks, and other elements like Cl and Ca were the
main elements of the solution during crosslinking. The Zn, C, and O elements were also
observed in elemental mapping images, further demonstrating the uniform distribution of
ZnO and AC in the composite.
XRD patterns of self-assembled ZnO and the composite beads are shown in Figure 2a.
The weakened peaks of bead powder can be attributed to the introduction of calcium
alginate and AC, while other diffraction peaks corresponded to ZnO (JCPDS 36–1451). The
FTIR spectra of the composite, calcium alginate, AC, and ZnO were recorded in Figure 2b
which composite beads exhibited O–H stretching vibration and –COO– stretching vibration
absorption peaks. In addition, C–O–C and –C–O–Ca–O–C–O– at 1030 cm
−1
were attributed
to the stretching vibration of calcium alginate. The optical property of the composite was
determined using UV
−
vis absorption spectra and the corresponding fluorescence (FL)
intensity as in Figure 2c,d.
Polymers 2023,15, 2215 5 of 14
Polymers 2023, 15, x FOR PEER REVIEW 5 of 14
Figure 1. SEM images of (a) low magnification and (b,c) high magnification of the composite. (d)
EDS analysis, (e) HAADF image and elemental mapping of Zn (f), O (g), and C (h).
XRD paerns of self-assembled ZnO and the composite beads are shown in Figure
2a. The weakened peaks of bead powder can be aributed to the introduction of calcium
alginate and AC, while other diffraction peaks corresponded to ZnO (JCPDS 36–1451).
The FTIR spectra of the composite, calcium alginate, AC, and ZnO were recorded in Figure
2b which composite beads exhibited O—H stretching vibration and—COO— stretching
vibration absorption peaks. In addition, C—O—C and —C—O—Ca—O—C—O— at
1030 cm−1 were aributed to the stretching vibration of calcium alginate. The optical prop-
erty of the composite was determined using UV−vis absorption spectra and the corre-
sponding fluorescence (FL) intensity as in Figure 2c,d.
Figure 1.
SEM images of (
a
) low magnification and (
b
,
c
) high magnification of the composite.
(d) EDS analysis, (e) HAADF image and elemental mapping of Zn (f), O (g), and C (h).
Polymers 2023, 15, x FOR PEER REVIEW 5 of 14
Figure 1. SEM images of (a) low magnification and (b,c) high magnification of the composite. (d)
EDS analysis, (e) HAADF image and elemental mapping of Zn (f), O (g), and C (h).
XRD paerns of self-assembled ZnO and the composite beads are shown in Figure
2a. The weakened peaks of bead powder can be aributed to the introduction of calcium
alginate and AC, while other diffraction peaks corresponded to ZnO (JCPDS 36–1451).
The FTIR spectra of the composite, calcium alginate, AC, and ZnO were recorded in Figure
2b which composite beads exhibited O—H stretching vibration and—COO— stretching
vibration absorption peaks. In addition, C—O—C and —C—O—Ca—O—C—O— at
1030 cm−1 were aributed to the stretching vibration of calcium alginate. The optical prop-
erty of the composite was determined using UV−vis absorption spectra and the corre-
sponding fluorescence (FL) intensity as in Figure 2c,d.
Figure 2.
(
a
) XRD patterns and (
b
) FTIR spectra. (Composite = the composite beads; CA = calcium
alginate; AC = activated carbon; ZnO = synthesized ZnO). (
c
) UV
−
vis absorption spectra (
d
) The
fluorescence (FL) intensity.
3.2. The Removal Efficiency of ARB Using ZnO/AC Alginate Beads
The ARB removal efficiency using alginate beads with different ZnO contents of 0 g,
0.25 g, 0.5 g, and 1.0 g (
≈
0, 0.5, 1, 2 g/L) was shown in Figure 3a. In the control group, the
bacterial activity decreased slightly without any treatment and was ultimately stabilized
Polymers 2023,15, 2215 6 of 14
around 10
8
CFU/mL after 24 h. The beads without ZnO only adsorbed in the early stage
and gradually reached an adsorption equilibrium of 1.13-log removal within 6 h, consistent
with previous studies of AC hydrogel [
30
,
31
]. As illustrated in Figure 3a, the removal
efficiency increased with the increase in ZnO concentration, and the 2 g/L ZnO composite
exhibited the highest removal efficiency of 2.3-log with the longest duration of 12 h. Given
that the beads with ZnO adsorbed ARB and also caused the inactivation of ARB. The
removal efficiencies of 1 g/L and 2 g/L of ZnO were significantly different from 2 h to 24 h
(p< 0.05) and showed highly significant differences from 6 h to 24 h (p< 0.01). In addition,
the initial concentration of bacteria was much higher, and 2 g/L ZnO-alginate beads were
more durable, with a removal efficiency of 99.5% among those previous studies [
25
,
26
]. For
the same content of ZnO, ZnO/AC alginate beads in this study also displayed excellent
efficiency in removing ARB compared to the others [
23
]. As shown in Figure 3b, adding
activated carbon increased the removal efficiency of 0.35–0.49-log.
Polymers 2023, 15, x FOR PEER REVIEW 6 of 14
Figure 2. (a) XRD paerns and (b) FTIR spectra. (Composite = the composite beads; CA = calcium
alginate; AC = activated carbon; ZnO = synthesized ZnO). (c) UV−vis absorption spectra (d) The
fluorescence (FL) intensity.
3.2. The Removal Efficiency of ARB Using ZnO/AC Alginate Beads
The ARB removal efficiency using alginate beads with different ZnO contents of 0 g,
0.25 g, 0.5 g, and 1.0 g (≈0, 0.5, 1, 2 g/L) was shown in Figure 3a. In the control group, the
bacterial activity decreased slightly without any treatment and was ultimately stabilized
around 108 CFU/mL after 24 h. The beads without ZnO only adsorbed in the early stage
and gradually reached an adsorption equilibrium of 1.13-log removal within 6 h, con-
sistent with previous studies of AC hydrogel [30,31]. As illustrated in Figure 3a, the re-
moval efficiency increased with the increase in ZnO concentration, and the 2 g/L ZnO
composite exhibited the highest removal efficiency of 2.3-log with the longest duration of
12 h. Given that the beads with ZnO adsorbed ARB and also caused the inactivation of
ARB. The removal efficiencies of 1 g/L and 2 g/L of ZnO were significantly different from
2 h to 24 h (p < 0.05) and showed highly significant differences from 6 h to 24 h (p < 0.01).
In addition, the initial concentration of bacteria was much higher, and 2 g/L ZnO-alginate
beads were more durable, with a removal efficiency of 99.5% among those previous stud-
ies [25,26]. For the same content of ZnO, ZnO/AC alginate beads in this study also dis-
played excellent efficiency in removing ARB compared to the others [23]. As shown in
Figure 3b, adding activated carbon increased the removal efficiency of 0.35–0.49-log.
Figure 3. The curves of viable cells in solution after exposure to different proportions of (a) ZnO or
(b) AC in beads (The proportions of ZnO is 2 g/L). (c) The amount of Zn2+ released from the compo-
site beads over 4 h (control = 2 g/L ZnO particles; composite = relative concentration of 2 g/L ZnO in
composite beads). (d–f) Effects of pH, temperature, and the concentration of antibiotics on removal
efficiency.
The removal efficiency above seems to depend on the concentration of ZnO, whereas,
for practical use, the maximum allowable concentration of Zn2+ in water is 3–5 mg/L [32].
Hence, the release of zinc ions was determined by ICP-MS, and Figure 3b showed the
amount of Zn2+ was 0.44 mg/L over 4 h, which was far below the limit and safe for water
use. The low leaching of ZnO is mainly aributed to the sustained release property of
calcium alginate [33]. Overall, our results suggested that 2 g/L ZnO of alginate beads
Figure 3.
The curves of viable cells in solution after exposure to different proportions of (
a
) ZnO
or (
b
) AC in beads (The proportions of ZnO is 2 g/L). (
c
) The amount of Zn
2+
released from the
composite beads over 4 h (control = 2 g/L ZnO particles; composite = relative concentration of 2 g/L
ZnO in composite beads). (
d
–
f
) Effects of pH, temperature, and the concentration of antibiotics on
removal efficiency.
The removal efficiency above seems to depend on the concentration of ZnO, whereas,
for practical use, the maximum allowable concentration of Zn
2+
in water is 3–5 mg/L [
32
].
Hence, the release of zinc ions was determined by ICP-MS, and Figure 3b showed the
amount of Zn
2+
was 0.44 mg/L over 4 h, which was far below the limit and safe for water
use. The low leaching of ZnO is mainly attributed to the sustained release property of
calcium alginate [
33
]. Overall, our results suggested that 2 g/L ZnO of alginate beads
exhibited a remarkable removal efficiency and could be an ideally packed material in
subsequent fixed bed experiments.
Various environmental factors may play an essential role in the removal difference
in practical application. As shown in Figure 3d~f, different groups of pH, temperature,
and the concentration of antibiotics were considered to determine the stability of ZnO/AC
alginate beads. Based on the results, environmental conditions of acidity (pH = 4), high
temperature (40
◦
C), and high antibiotic concentration (8 mg/L) led to higher removal
efficiency of ARB. However, the total removal efficiency became similar (90.9–99.9%) when
Polymers 2023,15, 2215 7 of 14
the composite reached plateaus. Thus, the ZnO/AC alginate beads could maintain high
removal efficiency at different pH, temperature, and antibiotic concentrations.
3.3. Application of ZnO/AC Alginate Beads in Fixed Bed
3.3.1. Removal Efficiency of ARB under Different Parameters
The application of ARB removal was investigated using columns packed with the
composite, while experiment groups were designed using operational parameters, such
as flow rate and initial concentration. As shown in Figure 4b, the minimum flow rate
exhibited the best efficiency with only 10
2
CFU/mL residue in the effluent. In addition, the
bacteria concentration at the outlet increased by about 1-log progressively with increased
rates. Our results also showed significant differences (p< 0.01) among different flow rates,
probably because of the shorter contact and reaction time between the cell membrane
and beads surface under faster flow rates [
34
]. Meanwhile, fixed bed systems of different
initial concentrations (10
6
, 10
7
, 10
8
CFU/mL) were studied with the same bed depth
and a flow rate of 2 mL/min. As shown in Figure 4b, all three groups showed removal
efficiency of about 4.5-log, and the lower concentration resulted in fewer colonies of ARB
in the effluent. In addition, bacterial concentrations of 10
6
and 10
7
CFU/mL were both
significantly different (p< 0.05) from the concentration of 10
8
CFU/mL. Zhao reported that
the ceramic disk filter could remove 99.99% [
35
] of E. coli, while Huang reported that a
filter coated with nano ZnO removed 99.89% of E. coli [
36
]. Our findings showed that a
fixed bed with a flow rate of 1 mL/min and initial concentrations of 10
6
and 10
7
CFU/mL
exhibited higher bacterial removal, with a removal efficiency of 4.19-log~5.69-log. The
higher removal efficiency compared with the static experiments might be caused by a
comprehensive effect of inactivation, adsorption, and interception in the column.
Polymers 2023, 15, x FOR PEER REVIEW 7 of 14
exhibited a remarkable removal efficiency and could be an ideally packed material in sub-
sequent fixed bed experiments.
Various environmental factors may play an essential role in the removal difference in
practical application. As shown in Figure 3d~f, different groups of pH, temperature, and
the concentration of antibiotics were considered to determine the stability of ZnO/AC al-
ginate beads. Based on the results, environmental conditions of acidity (pH = 4), high tem-
perature (40 °C), and high antibiotic concentration (8 mg/L) led to higher removal effi-
ciency of ARB. However, the total removal efficiency became similar (90.9–99.9%) when
the composite reached plateaus. Thus, the ZnO/AC alginate beads could maintain high
removal efficiency at different pH, temperature, and antibiotic concentrations.
3.3. Application of ZnO/AC Alginate Beads in Fixed Bed
3.3.1. Removal Efficiency of ARB under Different Parameters
The application of ARB removal was investigated using columns packed with the
composite, while experiment groups were designed using operational parameters, such
as flow rate and initial concentration. As shown in Figure 4b, the minimum flow rate ex-
hibited the best efficiency with only 102 CFU/mL residue in the effluent. In addition, the
bacteria concentration at the outlet increased by about 1-log progressively with increased
rates. Our results also showed significant differences (p < 0.01) among different flow rates,
probably because of the shorter contact and reaction time between the cell membrane and
beads surface under faster flow rates [34]. Meanwhile, fixed bed systems of different initial
concentrations (106, 107, 108 CFU/mL) were studied with the same bed depth and a flow
rate of 2 mL/min. As shown in Figure 4b, all three groups showed removal efficiency of
about 4.5-log, and the lower concentration resulted in fewer colonies of ARB in the efflu-
ent. In addition, bacterial concentrations of 106 and 107 CFU/mL were both significantly
different (p < 0.05) from the concentration of 108 CFU/mL. Zhao reported that the ceramic
disk filter could remove 99.99% [35] of E. coli, while Huang reported that a filter coated
with nano ZnO removed 99.89% of E. coli [36]. Our findings showed that a fixed bed with
a flow rate of 1 mL/min and initial concentrations of 106 and 107 CFU/mL exhibited higher
bacterial removal, with a removal efficiency of 4.19-log~5.69-log. The higher removal effi-
ciency compared with the static experiments might be caused by a comprehensive effect
of inactivation, adsorption, and interception in the column.
Figure 4. (a) Schematic representation of fixed bed column. (b) The removal efficiency of ARB with
different flow rates (1, 2, 3 mL/min) (ARB samples of 50 mL were filtered with an initial bacterial
concentration of 108 CFU/mL) and initial concentrations (106, 107, 108 CFU/mL), (Flow rate was 2
mL/min; * for p < 0.05; ** for p < 0.01).
3.3.2. Removal Efficiency of ARGs under Different Parameters
The absolute abundance of ARGs was analyzed through qPCR. As shown in Figure
5a, three ARGs of E. coli HB101 were reduced to a certain extent, among which the absolute
abundance of ARGs with flow rates (1, 2, 3 mL/min) decreased by 2.06–2.61-log. For
Figure 4.
(
a
) Schematic representation of fixed bed column. (
b
) The removal efficiency of ARB with
different flow rates (1, 2, 3 mL/min) (ARB samples of 50 mL were filtered with an initial bacterial
concentration of 10
8
CFU/mL) and initial concentrations (10
6
, 10
7
, 10
8
CFU/mL), (Flow rate was
2 mL/min; * for p< 0.05; ** for p< 0.01).
3.3.2. Removal Efficiency of ARGs under Different Parameters
The absolute abundance of ARGs was analyzed through qPCR. As shown in Figure 5a,
three ARGs of E. coli HB101 were reduced to a certain extent, among which the absolute
abundance of ARGs with flow rates (1, 2, 3 mL/min) decreased by 2.06–2.61-log. For
different rates, our studies found that the removal efficiency of three genes decreased
steadily. Regarding the inlet concentration, the ARGs removal efficiency of 10
7
CFU/mL
(2.71-log, 2.44-log, and 2.74-log showed significant differences with the concentrations of
10
6
CFU/mL and 10
8
CFU/mL. These differences might be caused by the proportion of
bacteria captured on the composite surface based on the abundance of bacteria monitored
using the 16S rRNA gene [
37
]. According to previous studies, the ARGs removal efficiency
of chlorination and UV treatments [
12
,
13
] was often within 80%. This fixed bed seemed
Polymers 2023,15, 2215 8 of 14
highly effective, with more than 95% ARGs removal efficiency under different parameters.
Thus, the filter system showed high efficiency in removing both ARB and ARGs, which can
be considered a favorable alternative to purifying wastewater.
Polymers 2023, 15, x FOR PEER REVIEW 8 of 14
different rates, our studies found that the removal efficiency of three genes decreased
steadily. Regarding the inlet concentration, the ARGs removal efficiency of 107 CFU/mL
(2.71-log, 2.44-log, and 2.74-log showed significant differences with the concentrations of
106 CFU/mL and 108 CFU/mL. These differences might be caused by the proportion of
bacteria captured on the composite surface based on the abundance of bacteria monitored
using the 16S rRNA gene [37]. According to previous studies, the ARGs removal efficiency
of chlorination and UV treatments [12,13] was often within 80%. This fixed bed seemed
highly effective, with more than 95% ARGs removal efficiency under different parameters.
Thus, the filter system showed high efficiency in removing both ARB and ARGs, which
can be considered a favorable alternative to purifying wastewater.
Figure 5. The absolute abundance of ARGs (tetA, blaTEM, and aph(3′)-Id) and 16S rRNA at the outlet
with different (a) flow rates (1, 2, 3 mL/min) (CK = control group; ARB samples of 50 mL were fil-
tered with an initial bacterial concentration of 108 CFU/mL) and (b) initial concentrations (106, 107,
108 CFU/mL) (CK1,2,3 = control groups at different concentration and flow rate was 2 mL/min).
3.3.3. The Performance Analysis of Fixed Bed
The performance of the fixed bed was expressed through the breakthrough curve,
which reveals the time-dependent variation of bacterial concentration at the outlet [38,39].
Three kinds of bed curves (10 cm, 20 cm, and 30 cm) with a concentration of 106 CFU/mL
and flow rate of 1 mL/min were described in Figure S2. The breakthrough curves moved
from left to right with the increase in bed depths, showing that penetration and saturation
time increased, which led to more removal of ARB and ARGs. Moreover, the slope of the
breakthrough curves became lower, suggesting that the saturation point reached slowly
at higher bed depths. As shown in Figure S2, the breakthrough point and the saturation
point were highly dependent on the bed depths. The results showed that the breakthrough
point increased from 75 min to 150 min, and the saturation point increased from 400 min
to 1000 min when the depths increased from 10 cm to 30 cm. The AEC of material was
indicated by 4.25, 4.07, and 3.74, respectively. Compared to the previous study [40], this
performance was superior to a fixed bed with zirconium-caged activated biochar alginate
beads in which the saturation point was 680 min for 30 cm bed height. And beyond that,
the durability of this fixed bed was examined through the removal efficiency of E. coli
HB101 and ARGs (tetA, blaTEM, aph(3′)-Id) in five cycles (Figure 6a,b). The ARB removal
efficiency reached 6-log at first, and the removal rates decreased gradually by 1-log before
the fourth cycle. In the subsequent two cycles, the removal efficiency gradually stabilized
to around 2-log. As depicted in Figure 6b, the efficiency decreased with the increase in the
cycle and then with more than 90% removal of ARGs at the last operation cycle. Based on
the durability analysis, the composite beads in fixed beds were reusable for removing ARB
and ARGs.
Figure 5.
The absolute abundance of ARGs (tetA, bla
TEM
, and aph(3
0
)-Id) and 16S rRNA at the outlet
with different (
a
) flow rates (1, 2, 3 mL/min) (CK = control group; ARB samples of 50 mL were
filtered with an initial bacterial concentration of 10
8
CFU/mL) and (
b
) initial concentrations (10
6
, 10
7
,
108CFU/mL) (CK1,2,3 = control groups at different concentration and flow rate was 2 mL/min).
3.3.3. The Performance Analysis of Fixed Bed
The performance of the fixed bed was expressed through the breakthrough curve,
which reveals the time-dependent variation of bacterial concentration at the outlet [
38
,
39
].
Three kinds of bed curves (10 cm, 20 cm, and 30 cm) with a concentration of 10
6
CFU/mL
and flow rate of 1 mL/min were described in Figure S2. The breakthrough curves moved
from left to right with the increase in bed depths, showing that penetration and saturation
time increased, which led to more removal of ARB and ARGs. Moreover, the slope of the
breakthrough curves became lower, suggesting that the saturation point reached slowly
at higher bed depths. As shown in Figure S2, the breakthrough point and the saturation
point were highly dependent on the bed depths. The results showed that the breakthrough
point increased from 75 min to 150 min, and the saturation point increased from 400 min
to
1000 min
when the depths increased from 10 cm to 30 cm. The AEC of material was
indicated by 4.25, 4.07, and 3.74, respectively. Compared to the previous study [
40
], this
performance was superior to a fixed bed with zirconium-caged activated biochar alginate
beads in which the saturation point was 680 min for 30 cm bed height. And beyond that,
the durability of this fixed bed was examined through the removal efficiency of E. coli
HB101 and ARGs (tetA, bla
TEM
,aph(3
0
)-Id) in five cycles (Figure 6a,b). The ARB removal
efficiency reached 6-log at first, and the removal rates decreased gradually by 1-log before
the fourth cycle. In the subsequent two cycles, the removal efficiency gradually stabilized
to around 2-log. As depicted in Figure 6b, the efficiency decreased with the increase in the
cycle and then with more than 90% removal of ARGs at the last operation cycle. Based on
the durability analysis, the composite beads in fixed beds were reusable for removing ARB
and ARGs.
3.4. Mechanism of ARB Removal
3.4.1. Morphology Observation Using SEM
As indicated using SEM (Figure S3), the control group showed normal morphology
of E. coli HB101, a rod shape with an intact and smooth membrane. In contrast, after
treatment for 30 min, the membrane became wrinkled, and ZnO particles adhered to the
surface. After being exposed for 120 min, obvious deformation of cell walls was observed
(
Figure S3c
). The morphology was basically changed, as well as cell lysis and cytoplasmic
Polymers 2023,15, 2215 9 of 14
leakage in high magnification. These results imply that ZnO assembled by nanosheets can
cause physical damage when attached to the surface, and the release of Zn
2+
will form
E. coli HB101/ZnO aggregation, which accelerates cell death [41].
Polymers 2023, 15, x FOR PEER REVIEW 9 of 14
Figure 6. (a) The number of colonies of E. coli HB101 after filtration in five cycles; (b) The proportion
of removal efficiency (%) of ARGs after filtration in five cycles. (ARB Samples = 50 mL, initial bacte-
rial concentration = 108 CFU/mL, and flow rate = 1 mL/min).
3.4. Mechanism of ARB Removal
3.4.1. Morphology Observation using SEM
As indicated using SEM (Figure S3), the control group showed normal morphology
of E. coli HB101, a rod shape with an intact and smooth membrane. In contrast, after treat-
ment for 30 min, the membrane became wrinkled, and ZnO particles adhered to the sur-
face. After being exposed for 120 min, obvious deformation of cell walls was observed
(Figure S3c). The morphology was basically changed, as well as cell lysis and cytoplasmic
leakage in high magnification. These results imply that ZnO assembled by nanosheets can
cause physical damage when aached to the surface, and the release of Zn2+ will form E.
coli HB101/ZnO aggregation, which accelerates cell death [41].
3.4.2. Investigation of Cell Damage by CLSM
Two fluorescent nucleic acid dyes of different penetrability were performed to inves-
tigate the bactericidal mechanism. In this method, SYTO 9 stains viable cells and cyto-
plasm in green fluorescence, while PI stains nucleic acids of dead bacteria in red fluores-
cence. As shown in Figure 7a, the untreated E. coli HB101 showed green fluorescent rod-
shaped and evenly dispersed spots with few red spots. This could be because of its natural
death in the absence of nutrients. In contrast, after 30 min, bacteria appeared in agglom-
eration, which contributed to the depolarization of the cell membrane [42]. Moreover, the
easier penetration of exogenous ROS into cells facilitated oxidative stress response and
cell damage [43]. As illustrated in Figure 7b,c, the increased intensity of red fluorescence
indicated a considerable number of bacterial death and serious damage to cell integrity
after exposure for 120 min.
Figure 6.
(
a
) The number of colonies of E. coli HB101 after filtration in five cycles; (
b
) The proportion
of removal efficiency (%) of ARGs after filtration in five cycles. (ARB Samples = 50 mL, initial bacterial
concentration = 108CFU/mL, and flow rate = 1 mL/min).
3.4.2. Investigation of Cell Damage by CLSM
Two fluorescent nucleic acid dyes of different penetrability were performed to investi-
gate the bactericidal mechanism. In this method, SYTO 9 stains viable cells and cytoplasm
in green fluorescence, while PI stains nucleic acids of dead bacteria in red fluorescence.
As shown in Figure 7a, the untreated E. coli HB101 showed green fluorescent rod-shaped
and evenly dispersed spots with few red spots. This could be because of its natural death
in the absence of nutrients. In contrast, after 30 min, bacteria appeared in agglomeration,
which contributed to the depolarization of the cell membrane [
42
]. Moreover, the easier
penetration of exogenous ROS into cells facilitated oxidative stress response and cell dam-
age [
43
]. As illustrated in Figure 7b,c, the increased intensity of red fluorescence indicated a
considerable number of bacterial death and serious damage to cell integrity after exposure
for 120 min.
3.4.3. Lipid Peroxidation and Oxidative Stress Response
Lipid peroxidation reaction is an important indicator when generated ROS attacks cell
membranes [
44
] and can be quantified by the concentration of the major product MDA [
45
].
From Figure 8a, the degree of lipid peroxidation of ARB was time-dependent and increased
after exposure to composite beads over a long period. In addition, a significant (
p< 0.05
)
increase in the degree of lipid peroxidation of ARB was observed after treatment for
120 min
.
The highest concentration was observed at 4 h for 0.0033 nmol/10
4
cells, and this correlated
trend also occurred in intracellular ROS production (Figure 8b).
FCM was employed to measure the fluorescence intensity of DCFH-DA, which was
positively related to the intracellular ROS content. As time passed (Figure 8b), the fluores-
cence intensity increased significantly compared to the control group, with no significant
difference at time intervals. ROS generally maintains a dynamic equilibrium and regulates
intracellular redox homeostasis in the respiratory process [
46
]. However, our results indi-
cated that the intracellular ROS of ARB was out of balance and that the excessive ROS might
induce cells into an oxidative stress state. Intracellular ROS was mainly produced due
to cytotoxic response when ZnO particles or Zn
2+
entered the cells [
47
]. Previous studies
have also proven that ZnO solution produced a significant quantity of ROS even in dark
conditions [
48
]. Moreover, the enzyme levels of SOD and CAT depicted the antioxidant
activity against ROS. As shown in Figure 8c, both antioxidant enzyme activities were
Polymers 2023,15, 2215 10 of 14
significantly higher than those of the control before 4 h. However, these activities decreased
at 6 h, indicating that SOD and CAT were in high expression at the beginning to main-
tain ROS balance until the antioxidant defense system collapsed under the accumulation
of ROS.
Polymers 2023, 15, x FOR PEER REVIEW 10 of 14
Figure 7. Confocal laser scanning microscopy (CLSM) images of E. coli HB101 after treatment for
different times. (a) 0 min; (b) 30 min; (c) 120 min. (The green fluorescence represents viable cells;
The red fluorescence represents dead cells.)
3.4.3. Lipid Peroxidation and Oxidative Stress Response
Lipid peroxidation reaction is an important indicator when generated ROS aacks
cell membranes [44] and can be quantified by the concentration of the major product MDA
[45]. From Figure 8a, the degree of lipid peroxidation of ARB was time-dependent and
increased after exposure to composite beads over a long period. In addition, a significant
(p < 0.05) increase in the degree of lipid peroxidation of ARB was observed after treatment
for 120 min. The highest concentration was observed at 4 h for 0.0033 nmol/104 cells, and
this correlated trend also occurred in intracellular ROS production (Figure 8b).
FCM was employed to measure the fluorescence intensity of DCFH-DA, which was
positively related to the intracellular ROS content. As time passed (Figure 8b), the fluores-
cence intensity increased significantly compared to the control group, with no significant
difference at time intervals. ROS generally maintains a dynamic equilibrium and regulates
intracellular redox homeostasis in the respiratory process [46]. However, our results indi-
cated that the intracellular ROS of ARB was out of balance and that the excessive ROS
might induce cells into an oxidative stress state. Intracellular ROS was mainly produced
due to cytotoxic response when ZnO particles or Zn2+ entered the cells [47]. Previous stud-
ies have also proven that ZnO solution produced a significant quantity of ROS even in
dark conditions [48]. Moreover, the enzyme levels of SOD and CAT depicted the antioxi-
dant activity against ROS. As shown in Figure 8c, both antioxidant enzyme activities were
Figure 7.
Confocal laser scanning microscopy (CLSM) images of E. coli HB101 after treatment for
different times. (
a
) 0 min; (
b
) 30 min; (
c
) 120 min. (The green fluorescence represents viable cells; The
red fluorescence represents dead cells.)
3.4.4. Contribution of Inactivation and Adsorption
The inactivation capacity of the ZnO/AC alginate beads was greater than its adsorp-
tion (Figure 8d). The results showed that each AC bead could adsorb 3.95
×
10
4
CFU/mL,
while the number of colonies decreased significantly on ZnO/AC beads (7
×
10
3
CFU/mL).
It indicated that the contribution of inactivation and adsorption upon each composite bead
was around 17.7–31.6% and 68.4–82.3%, respectively. To visually characterize the morphol-
ogy of ARB with and without ZnO, SEM images of bacteria on two kinds of material surface
were acquired. The cell membrane was intact when exposed to AC beads. In contrast,
bacterial fragments were observed in ZnO/AC beads which suggested that ZnO caused
the inactivation of bacteria.
Polymers 2023,15, 2215 11 of 14
Polymers 2023, 15, x FOR PEER REVIEW 11 of 14
significantly higher than those of the control before 4 h. However, these activities de-
creased at 6 h, indicating that SOD and CAT were in high expression at the beginning to
maintain ROS balance until the antioxidant defense system collapsed under the accumu-
lation of ROS.
Figure 8. (a) Lipid peroxidation. Columns marked by the same leer are not significantly different
(p < 0.05); (b) ROS; Different color bars represent different exposure times. (c) CAT-SOD activity; (d)
Contribution of inactivation and adsorption.
3.4.4. Contribution of Inactivation and Adsorption
The inactivation capacity of the ZnO/AC alginate beads was greater than its adsorp-
tion (Figure 8d). The results showed that each AC bead could adsorb 3.95 × 104 CFU/mL,
while the number of colonies decreased significantly on ZnO/AC beads (7 × 103 CFU/mL).
It indicated that the contribution of inactivation and adsorption upon each composite bead
was around 17.7–31.6% and 68.4–82.3%, respectively. To visually characterize the mor-
phology of ARB with and without ZnO, SEM images of bacteria on two kinds of material
surface were acquired. The cell membrane was intact when exposed to AC beads. In con-
trast, bacterial fragments were observed in ZnO/AC beads which suggested that ZnO
caused the inactivation of bacteria.
4. Conclusions
In summary, our research provided efficient and stable ZnO/AC alginate beads with
less environmental impact. A designed fixed bed system of the composite can significantly
remove ARB and ARGs from highly contaminated wastewater. It was demonstrated that
the system exhibited the highest removal efficiency of ARB (5.69-log) and ARGs (99.5%)
when the flow rate was 1 mL/min, and the initial bacterial concentration was 107 CFU/mL.
In addition, after exposure to the composite beads, the direct contact or release of ZnO,
lipid peroxidation, and oxidative stress caused by generated ROS would collectively lead
to membrane disruption and cell death.
Figure 8.
(
a
) Lipid peroxidation. Columns marked by the same letter are not significantly different
(p< 0.05); (
b
) ROS; Different color bars represent different exposure times. (
c
) CAT-SOD activity;
(d) Contribution of inactivation and adsorption.
4. Conclusions
In summary, our research provided efficient and stable ZnO/AC alginate beads with
less environmental impact. A designed fixed bed system of the composite can significantly
remove ARB and ARGs from highly contaminated wastewater. It was demonstrated that
the system exhibited the highest removal efficiency of ARB (5.69-log) and ARGs (99.5%)
when the flow rate was 1 mL/min, and the initial bacterial concentration was 10
7
CFU/mL.
In addition, after exposure to the composite beads, the direct contact or release of ZnO,
lipid peroxidation, and oxidative stress caused by generated ROS would collectively lead
to membrane disruption and cell death.
Considering the DBPs production during conventional disinfection, this study has put
forward a novel biopolymer and its application system that can be deemed as a potential
pattern in water purification, especially for point-of-use in areas with poor sanitary condi-
tions. Moreover, antimicrobial resistance, which threatens public health, has always been
ignored in wastewater treatment. Therefore, the composite beads with high effectiveness
of adsorption and disinfection combined have overcome the limitations of removing ARB
and ARGs.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/polym15092215/s1, Table S1: The standard curves of Qpcr;
Figure S1:
(a) SEM image and (b) enlarged view of the as-prepared hierarchical ZnO nanoflowers.
(c) The antibacterial property of synthesized ZnO (ZnO* = ZnO nanoflowers); Figure S2: Break-
through curves of the composite beads during ARB removal at different bed depth (Initial bacterial
concentration = 106 CFU/mL; flow rate = 1 mL/min); Figure S3: SEM images of E.coli HB101 after
treatment for different time. (a) 0 min, (b) 30 min and (c) 120 min.
Polymers 2023,15, 2215 12 of 14
Author Contributions:
Conceptualization, H.C. and Z.L. (Zhe Liu); methodology, Z.L. (Zhe Liu);
validation, H.C.; formal analysis, H.C., Z.L. (Zhe Liu), Z.Z. and X.S.; investigation, Z.L. (Zhe Liu),
X.Y. and Z.L. (Zejun Lin); resources, Z.L. (Zhe Liu) and X.Y.; writing—original draft preparation, Z.L.
(Zhe Liu); writing—review and editing, H.C., Z.L. (Zhe Liu) and Z.Z.; visualization, Z.L. (Zhe Liu),
X.Y. and J.Z.; supervision, H.C.; project administration, H.C.; funding acquisition, H.C. All authors
have read and agreed to the published version of the manuscript.
Funding:
This research was funded by “Pioneer” and “Leading Goose” R&D Program of Zhejiang
grant number 2023C03136 and National Natural Science Foundation of China grant numbers 52270201.
And The APC was funded by “Pioneer” and “Leading Goose” R&D Program of Zhejiang.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not Applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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