Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Isolation and characterization of atrazine-degrading strain

Shewanella sp. YJY4 from cornfield soil
J.Y. Ye, J.B. Zhang, J.G. Gao, H.T. Li, D. Liang and R.M. Liu
College of Life Science, Northeast Agricultural University, Harbin, China

Significance and Impact of Study: A new isolated strain, YJY4 showed high atrazine-degrading ability,
being able to degrade 100 mg l
1 atrazine completely in 36 h. Strain YJY4 was identified as Shewanella
sp. and contained atrazine-degrading atzA, atzB and atzC genes. This study examined the degradation
mechanism and metabolic ability of this strain and for the bioremediation of contaminated environ-
ments, provides more strain selection and determines the strain of atrazine bioremediation potential.

Keywords Abstract
atrazine, biodegradation, identify, metabolic
ability, Shewanella sp.. Atrazine has been used worldwide for over 50 years as a chemical herbicide. A
strain of bacteria, YJY4 which utilizes atrazine as its sole nitrogen source for
Correspondence growth was isolated from agricultural black soil in northeastern China. 16S
Rongmei Liu, College of Life Science, North- rDNA sequencing identified YJY4 as a Shewanella sp. PCR analysis and
east Agricultural University, Harbin 150030,
sequencing confirmed that YJY4 contained atrazine-degrading atzA, atzB and
China.
E-mail: lrm1975@sina.com
atzC genes. These genes revealed high similarity with those in Pseudomonas sp.
ADP and Arthrobacter sp. TC1. The strain YJY4 was observed to degrade
2015/2270: received 4 November 2015, atrazine (100 mg l
1) to cyanuric acid completely after 36 h. To the best of
revised 10 May 2016 and accepted 10 May our knowledge, YJY4 was the first reported Shewanella sp. to grow in pure
2016 culture with atrazine serving as a sole source of nitrogen. Therefore, YJY4 may
help with atrazine biodegradation and may become an abundant resource of
doi:10.1111/lam.12584
atrazine degradation strains.

To reverse atrazine pollution, many physical and chem-

Introduction
Atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine)
is a triazine herbicide principally used for controllingcer-
tain deep-rooted perennial herb that can kill annual
broadleaf and grass weeds, perennial weeds also has a
certain extent, primarily in corn (Zea mays L.) field and
sorghum (Chinese sorghum) field (Solomon et al. 2013).
Due to its long residual period and long-term heavy
use, atrazine has caused serious pollution in soil, water
and other natural environments and it has been detected
in high amounts in environmental waters (Ralebits et al.
2002; Tsui and Roy 2008). Currently, atrazine has been
identified as an endocrine disrupting chemical (EDC;
Safe 2005; Guerit et al. 2008). Studies have demonstrated
that long-term exposure to low doses of atrazine inter-
fere with the endocrine system of an organism (Bianchi
et al. 2006).
ical removal methods have been proposed. However,
these methods make toxic byproducts to spread easily
and cause water pollution. A biodegradable, cost-effective
and highly efficient method that causes no secondary pol-
lution would be an ideal solution to the problem of atra-
zine contamination.
Many bacteria that can partially degrade and even fully
mineralize atrazine have been isolated. These strains
include Pseudomonas sp. ADP (Siripattanakul et al. 2009),
Arthrobacter aurescens TC1 (Sajjaphan et al. 2004), Pseu-
daminobacter sp. C147 (Topp et al. 2000), Arthrobacter sp.
AD1 (Cai et al. 2003), Rhodococcus sp. MB-P1 (Fazlurrah-
man et al. 2009), Bacillus subtilis HB-6 (Wang et al. 2014)
and Pseudomonas aeruginosa (Fernandes et al. 2014).
Although these strains are from more than 10 genera
of Gram-positive and Gram-negative bacteria, their degra-
dation genes are highly conserved with nucleotide

Letters in Applied Microbiology 63, 45--52 © 2016 The Society for Applied Microbiology 45
Bioremediation of atrazine
sequence similarity between 97 and 100%. According to
the composition of the degrading genes, atrazine-degrad-
ing bacteria can be classified into two types. One type
contains the degradation genes atzA, atzB, atzC, atzD,
atzE and atzF, which can completely degrade atrazine.
Another type contains the degradation genes trzN/atzA,
atzB and atzC, which can transform atrazine into non-
toxic cyanuric acid. Atrazine-degrading genes atzA and
trzN, atzD and trzD have the same functionality (Meyer
et al. 2009). The genes trzN/atzA, atzB and atzC encode
for three hydrolytic enzymes that catalyse the degradation
of atrazine to cyanuric acid. The genes trzD/atzD, atzE
and atzF encode the necessary enzymes for ring cleavage
that catalyse the degradation of cyanuric acid to CO2 and
NH3. To date, two of the most extensively studied atra-
zine degradation strains are Pseudomonas sp. ADP, which
contains the gene atzABCDEF located on plasmid pADP-
1, and A. aurescens TC1, which contains degrading genes
trzN-atzBC located on plasmid pTC-1.
In recent years, Shewanella sp. are widely distributed in
various environments (soil, rivers, oceans and sediments;
Hau and Gralnick 2007). They are capable of degrading
including, nitroaromatics, azo dyes and polyhalogenated
compounds (Wang et al. 2015). However, there has never
been a report of Shewanella sp. capable of degrading tri-
azine compounds.
In this study, a high-efficiency atrazine degrader, She-
wanella sp. YJY4, was isolated from a cornfield where
atrazine had been used for a long time, and the strain
was characterized. Strain YJY4 was the first reported She-
wanella sp. to grow in pure culture with triazine herbicide
atrazine serving as a sole source of nitrogen. The purpose
of this study was to explain the degradation mechanism
and metabolic ability of this strain and for the bioremedi-
ation of contaminated environments provide more strain
selection and indicate the Shewanella sp. YJY4 of organic
pollution bioremediation potential.
Results and discussion
Enrichment and isolation of strain YJY4
At the end of the enrichment cycles, the bacterial consor-
tium was plated on LB medium to isolate the organisms.
Five distinct members were isolated. The degradation
experiments showed that these strains exhibited a poten-
tial ability to degrade atrazine in the AMSM medium
with the interval of 123–100% in 7 days. Strain YJY4 had
the highest degradability, which was at 100%. Physiologi-
cal and morphological characteristics indicate YJY4 was a
Gram-negative bacterium, and can take advantage of atra-
zine as its sole nitrogen source. Strain YJY4 formed a
46 Letters in Applied Microbiology 63, 45--52 © 2016 The Society for Applied Microbiology
Y. Jinyu et al.
smooth colony, orange in colour, on LB agar plates after
48 h of incubation. It was selected for further studies.
Identification and 16S rDNA analysis of strain YJY4
The 16S rDNA sequences of strain YJY4 was size of
1515 bp (GenBank accession number KP231202). The
BLAST calculation results showed that strain YJY4’s 16S
rDNA gene sequence has 99% similarity with the She-
wanella sp. RB5-M4 (GenBank accession number
JN019028). Subsequently, similar 16S rDNA phylogenetic
tree was constructed with neighbour-joining methods and
the results showed that the strain YJY4 had a close
relationship with many Shewanella sp. (Fig. 1). On the
basis of its physiological characteristics and the
constructed phylogenetic tree, strain YJY4 was identified
as Shewanella sp.
Members of the phylogeny Shewanella sp. are metaboli-
cally versatile bacteria, and many Shewanella sp. strains
have been reported to degrade pollutants such as ben-
zoapyrene, naphthylaminesulfonic azo dye amaranth, hex-
ahydro- 1, 3, 5- trinitro- 1, 3, 5- triazine, as well as
others (Zhao et al. 2005; Hong et al. 2007; Chang et al.
2014). Several reports have also found that Shewanella sp.
strains can degrade acetaldehyde, phenol, malachite green
and methyl violet B (Chen et al. 2010; Liu et al. 2015).
However, strain YJY4 was the first reported Shewanella
sp. found to grow in pure culture with atrazine as a sole
nitrogen source (de Souza et al. 1998a; Getenga et al.
2009; Wang et al. 2014). All of these indicate that the
Shewanella sp. has great ecological diversity and possesses
potential capacity for bioremediation of organic
pollution.
Detection of atrazine-degrading genes in strain YJY4
Atrazine-degrading enzyme gene trzN, atzA, atzB, atzC,
atzD, atzE, atzF and trzD, using the primers listed in
Table 1. After amplification, we obtained 05 kb atzA,
05 kb atzB and 06 kb atzC, and there were no other
products for other atrazine-degrading genes such as atzD,
atzE, atzF, trzD and trzN (Fig. 2). We also attempted to
amplify cyanuric acid genes that were unsuccessful, and
the results were alike as those presented in Fig. 2. The
nucleotide sequences of atzA from Shewanella sp. YJY4
showed 98% sequence similarity with the Pseudomonas
sp. ADP (Genbank accession number U66917) and the
nucleotide sequences of atzB and atzC from Shewanella
sp. YJY4 showed 99% sequence similarity with the
Arthrobacter sp. TC1 (Genbank accession number
CP000475). This indicates that Shewanella sp. harbours
atzA, atzB and atzC genes.
Y. Jinyu et al. Bioremediation of atrazine

Shewanella sp. WW001 (AB111109)

79
59 Shewanella sp. THREE-12 (KF650764)

Shewanella sp. BC01 (JX119023)

81
Shewanella sp. 184 (AF387349)

55 Shewanella sp. PIM1 (GQ359960)

56
66 Shewanella sp. LHB20 (GQ850519)
80
Shewanella sp. Hac319 (DQ307732)

Shewanella sp. MK03 (AY690713)

Shewanella sp. PIC1 (GQ359955)

36
Shewanella sp. YJY4 (KP231202)
86
71 Shewanella sp. RB5-M4 (JN019028)

90 Shewanella sp. CN-32 (NR 074817)

Shewanella sp. SIC1 (AB039738)

Shewanella sp. ALL-2 (KC211018)

93 Shewanella sp. MS1 (KM522840)

0·001

Figure 1 Phylogenetic tree of the 16S rDNA sequences of Shewanella sp. YJY4. The calculations were performed according to a neighbour-join-
ing analysis (bootstrap number = 1000), and the bar indicates 0001 substitution per nucleotide position.

The atzA, atzB, atzC, atzD, atzE and atzF genes were
found in atrazine-degrading strain ADP (de Souza et al.
1998b; Garcia-Gonzalez et al. 2005). The first three genes
encode the enzymes required for the conversion of atra-
zine to cyanuric acid. The next three genes encode the
enzymes required for removal of cyanuric acid to NH3
and CO2. The biochemical mechanism is the same,
despite different hydrolysing enzymes (AtzA vs. TrzN;
Meyer et al. 2009). Therefore, the genetic potential for
atrazine degradation seems to be close to that of strain
TC1. This also implies that the strain YJY4 cannot take
advantage of cyanuric acid as its sole nitrogen source for
growth.
Atrazine and cyanuric acid utilization of Shewanella sp.
YJY4
To determine the growth ability of Shewanella sp. YJY4
and measure whether it can utilize atrazine or cyanuric
acid as its sole nitrogen source, we measured the growth
of strain YJY4 in medium where its sole nitrogen source
was ammonium nitrate, atrazine or cyanuric acid
(Fig. 3a). Strain YJY4 was able to utilize ammonium
nitrate and atrazine as its sole nitrogen source in the 24
and 28 h, respectively, required to reach a stationary
phase. The medium with ammonium nitrate as its sole
nitrogen source had a lag phase of nearly 4 h, while med-
ium with atrazine as its sole nitrogen source had a lag
phase nearly 8 h. This indicates that strain YJY4 with
atrazine as its sole nitrogen source in the growth requires
certain adaptability. In addition, with the medium with
cyanuric acid as the sole nitrogen source, strain YJY4 did
not substantially grow. This indicated that strain YJY4
cannot utilize nitrogen from cyanuric acid for growth.
Atrazine biodegradation analysis
We also investigated the ability of Shewanella sp. YJY4 to
degrade atrazine (Fig. 3b). When the strain utilized atra-
zine as its sole nitrogen source, over time, the concentra-
tion of atrazine decreased and at 12–16 h had been
degraded by half. After 36 h, atrazine could not be
detected by HPLC. This indicates that 100% of atrazine
(100 mg) was degraded after 36 h incubation with the
strain YJY4. In contrast, we also detected the concentra-
tion of cyanuric acid by HPLC, where concentration was

Letters in Applied Microbiology 63, 45--52 © 2016 The Society for Applied Microbiology 47
Bioremediation of atrazine Y. Jinyu et al.

Table 1 PCR primers, annealing temperatures and expected product sizes in the amplification of atrazine-degrading genes

Gene Primer Sequence (50 ?30 ) Annealing (°C) Size (kb)

atzA atzA-1 CCA TGT GAA CCA GAT CCT 56 526

atzA-2 TGA AGC GTC CAC ATT ACC
atzB atzB-1 TCA CCG GGG ATG TCG CGG GC 54 500
atzB-2 CTC TCC CGC ATG GCA TCG GG
atzC atzC-1 GCT CAC ATG CAG GTA CTC CA 50 600
atzC-2 GTA CCA TAT CAC CGT TTG CCA
atzD atzD-1 GGA GAC ATC ATA TGT ATC ACA TCG ACG TTT TC 55 1100
atzD-2 CCA ATA AGC TTA GCG CGG GCA ATG ACT GCA
atzE atzE-1 TAC GCG GTA AAG AAT CTG TT 52 1000
atzE-2 GGA GAC CGG CTG AGT GAG A
atzF atzF-1 CGA TCG GAA AAA CGA ACC TC 53 900
atzF-2 CGA TCG CCC CAT CTT CGA AC
trzD trzD-1 CCT CGC GTT CAA GGT CTA CT 58 750
trzD-2 TCG AAG CGA TAA CTG CAT TG
trzN trzN-1 ATG ATC CTG ATC CGC GGA CTG A 50 1370
trzN-2 CTA CAA GTT CTT GGG AAT GAG TG

M 1 2 3 4 5 6 7 8 The pathway for atrazine degradation is well established

5000 bp
3000 bp
2000 bp
1000 bp
750 bp
500 bp
250 bp
100 bp
Figure 2 Agarose gel electrophoresis (1%) of atrazine-degrading
gene PCR product generated from strain YJY4. Lane M corresponds
to DM 2000 Plus DNA molecular weight marker. Lane 1 to lane 8 pre-
sents the PCR amplification products for trzN, atzA, atzB, atzC, trzD,
atzD, atzE and atzF.
increased over time. The reduction of the amount of atra-
zine was inversely proportional to the increase in the
amount of cyanuric acid. Due to these results, we specu-
late that strain YJY4 may only be able to degrading atra-
zine to cyanuric acid and cannot use cyanuric acid as its
nitrogen source for growth. Therefore, the final product
of strain YJY4 degrading atrazine might be the cyanuric
acid.
Given this result, the proposed pathway for atrazine
degradation by Shewanella sp. YJY4 was shown in Fig. 4.
(Wackett et al. 2002; Zhang et al. 2012). Several atrazine-
degrading strains were isolated from agricultural soils in
France, USA, Canada, China and India. But Shewanella
sp. YJY4 was the first reported strain that utilizes atrazine
as its sole nitrogen source for growth. Because it can uti-
lize many kinds of organic pollutants, it may possess great
potential as bioremediation for organic pollution.
Materials and methods
Soils
The bacteria described in this study were isolated from
the surface soil layer of a cornfield (Yichun City, Hei-
longjiang Province, China) where the use of atrazine was
for long-term (0–15 cm). Soil samples were sieved (50-
mm mesh) to remove stones and plant debris and then
stored at 4°C in plastic bag until use.
Chemicals and media
Atrazine standard sample (≥99% purity) was purchased
from Harbin Limin Technology Ltd (Harbin, China).
Cyanuric acid standard sample (≥98% purity) was
purchased from Sinopharm Chemical Reagent Co., Ltd.
(Beijing, China). Other chemicals used in this paper were
analytical grade reagents, and those used for HPLC analy-
ses were of HPLC grade reagents.
The liquid enrichment media used was mineral salts
medium (MSM). MSM contained 10 g l
1 of KH2PO4,
60 g l
1 of Na2HPO412H2O, 02 g l
1 of MgSO47H2O,
05 g l
1 of NaCl, 3 g l
1 of glucose and 1 ml l
1 of
trace element concentrate solution (pH 70). Trace

48 Letters in Applied Microbiology 63, 45--52 © 2016 The Society for Applied Microbiology
Y. Jinyu et al. Bioremediation of atrazine

(a) (b)
100
0·7
90
0·6
80
0·5 70
60
0·4
50
0·3 40
0·2 30
20
0·1
10
0 0
0 4 8 12 16 20 24 28 32 36 0 4 8 12 16 20 24 28 32 36

Figure 3 (a) Growth of strain Shewanella sp. YJY4 in MSM containing atrazine, cyanuric acid and ammonium nitrate (100 mg l
1), respectively,
as the sole nitrogen source. Error bars represent the mean (SD) of three independent samples. (b) Metabolism of atrazine by Shewanella sp.
YJY4 in MSM containing atrazine (100 mg l
1) as the sole nitrogen source. Error bars represent the mean (SD) of three independent samples.
(a) ( ) Ammonium nitrate, ( ) Atrazine, ( ) Cyanuric acid. (b) ( ) Atrazine, ( ) Cyanuric acid.

Cl OH OH OH

N N atzA N N atzB N N atzC N N

H7C3HN N NHC2H5 H7C3HN N NHC2H5 H7C3HN N OH HO N OH

Atrazine Hydroxyatrazine N-isopropylammelide Cyanuric acid

Figure 4 Proposed pathways of atrazine degradation by Shewanella sp. YJY4.

element concentrate solution contained 04 g l
1 of
Na2B4O710H2O, 05 g l
1 of Na2MoO42H2O, 08 g l
1
of CuSO45H2O, 2 g l
1 of FeSO47H2O, 2 g l
1 of
MnSO4H2O, 10 g l
1 of ZnSO47H2O and 5 g l
1 of
EDTA. As required, atrazine (atrazine mineral salts med-
ium, AMSM) or cyanuric acid (cyanuric acid mineral salts
medium, CMSM) was added as the sole nitrogen source.
Lysogeny broth (LB) medium contained 10 g l
1 tryp-
tone, 5 g l
1 yeast extract and 10 g l
1 NaCl. Preparation
of solid medium included the addition of 16 g l
1 of
agar. The pH of media was regulated to 72 before steril-
ization. Sterilization conditions were 115°C for 30 min.
Enrichment and isolation
Five grams of the soil specimen was added to a 250-ml
Erlenmeyer flask with 100 ml AMSM (50 mg l
1 of atra-
zine). The specimen was incubated aerobically and shaken
at 150 rev min
1 at 30°C. Ten millilitres of enrichment
culture was then inoculated into fresh AMSM for 7 days,
where the atrazine concentration was increased to
100 mg l
1. This process was repeated eight times with the
exception of the final enrichment incubation, where the
atrazine concentration was increased to 500 mg l
1. The
eventually culture was diluted and coated on AMSM agar
plates (100 mg l
1 of atrazine). Mature consortiums were
repeatedly cross-vaccinated on AMSM plates to separate
the pure culture. Among the selected isolates, a bacterium
showed high atrazine-degrading activity. This strain was
named YJY4 and was selected for further experiments.
Identification of atrazine-degrading strain YJY4
Identification of strain YJY4 was based on 16S rDNA gene
sequence analysis. Extraction of total genomic strain YJY4
was the use of GenElute bacterial genome DNA extraction
kit (Sigma-Aldrich, Shanghai, China). PCR analysis for
bacterial was using 16S universal primers. Universal pri-
mers were used—27F (50 -AGAGTTTGATCCTGGCTCAG-
30 ) as the forward primer and 1492R (50 -TACGGC-
TACCTTGTTACGACT T-30 ) as the reverse primer (Frank
et al. 2008). The PCR conditions were as follows: at 94°C
preheated 5 min, 30 cycles of three steps at 94°C denatu-
ration 1 min, at 54°C annealed 1 min, and at 72°C exten-
sion 2 min, respectively, and at 72°C final extension
10 min. The products of PCR were divided on a 1% agar-
ose gel electrophoresis. The resulting DNA fragments were
purified through AxyPrepTM DNA gel extraction kit
(Axygen, Hangzhou, China), and then cloned into a
pMD18-T vector (TaKaRa, Dalian, China). The vector

Letters in Applied Microbiology 63, 45--52 © 2016 The Society for Applied Microbiology 49
Bioremediation of atrazine
was transformed into Escherichia coli strain DH5a.
Sequenced gene fragments was completed by the Suzhou
Genewiz Biotechnology Co., Ltd. (Suzhou, China).
The sequences were analysed using the GenBank
nucleotide database. Sequence alignments and phyloge-
netic analysis were performed using the CLUSTAL W and
MEGA ver. 5.0 (Philadelphia, PA). A phylogenetic bootstrap
analysis using 1000 replicates for each sequence was used
with a 50% minimum for branch reliability.
PCR detection of atrazine-degrading genes
Extraction of total genomic strain YJY4 was the use of
GenElute (Shanghai, China) bacterial genome DNA
extraction kit and it was used as a template to amplify
atzA, atzB, atzC, atzD, atzE, atzF, trzD and trzN genes,
respectively. The primers have been previously described
(Mulbry et al. 2002; Devers et al. 2004; Zhang et al. 2011).
The primer sequences, annealing temperature, products
size and references are shown in the Table 1. The PCR
conditions were as follows: at 94°C preheated 5 min, 30
cycles of three steps at 94°C denaturation 1 min, at 54°C
annealed 1 min, at 72°C extension 45, 45, 45, 60, 60, 60,
60 and 90 s to atzA, atzB, atzC, atzD, atzE, atzF, trzD and
trzN genes, respectively, and at 72°C final extension
10 min (Table 1). Sequencing and sequence analysis were
showed as described in Materials and methods 24.
Growth capacity of strain YJY4
Three 100 ml cultures of liquid medium containing
100 mg l
1 atrazine (AMSM), 100 mg l
1 cyanuric acid
(CMSM), or 100 mg l
1 ammonium nitrate (NH4NO3)
(MSM) were grown. AMSM and CMSM were used to
determine whether strain YJY4 could utilize atrazine and
cyanuric acid as its sole nitrogen source. In contrast, MSM
was used to understand whether atrazine and cyanuric acid
inhibited the growth of the strain YJY4. Bacteria were cul-
tured for 24 h and later centrifuged at 10 752 g. The result-
ing cell pellet was resuspended in sterile water and adjusted
to OD600 = 01. The three cultures were inoculated at 1%
concentration. Bacterial growth was determined using a
spectrophotometer (U-2910; HITACHI, Tokyo, Japan) at
600 nm (OD 600) absorbance. Each treatment was incu-
bated aerobically and shaken at 150 rev min
1 at 30°C. To
establish the growth curve, samples were taken every 4 h,
and the OD600 was measured over a period of 36 h. Each
experiment was performed in triplicate.
Mineralization experiments by strain YJY4
Bacteria were cultured for 24 h and later by centrifuga-
tion at 8000 rpm for 10 min to collected strain. The
50
Y. Jinyu et al.
resulting precipitate was resuspended in sterile water to
OD600 = 01. AMSM medium containing 100 mg l
1
atrazine was inoculated using 1% concentration of the
resuspension. Aliquots were taken every 4 h, and atrazine/
cyanuric acid content was measured over a period of
36 h. All experiments were performed in triplicate.
Atrazine concentration and cyanuric acid yield was
quantified by HPLC (Waters 600; Waters) equipped with a
dual k absorbance detector (Waters 2487; Waters, Milford,
MA) and a reversed phase C18 column (Agilent Eclipse
XDB-C 18, 46 mm 9 150 mm; Agilent, Beijing, China).
For the detection of atrazine (Kadian et al. 2008), the
flow rate was 10 ml min
1 (acetonitrile : water =
80 : 20, v : v), the column temperature was 25°C, and
the UV detector set at a wavelength of 221 nm. The
retention time of atrazine is 26 min. For detection of the
yield of cyanuric acid (Vaclavik et al. 2010), the flow rate
was 10 ml min
1 (50 mM K2HPO4/methanol = 95 : 5,
v : v), the column temperature was 35°C, and the UV
detector set at a wavelength of 213 nm. The retention
time of atrazine is 15 min.
Acknowledgements
This study was supported by the Heilongjiang Provincial
National Science Foundation (12521021), Dr Start-up
Fund in Northeast Agricultural University of China (No.
2010RCB54).
Conflict of Interest
We declare that we have no financial and personal rela-
tionships with other people or organizations that can
inappropriately influence our work, there is no profes-
sional or other personal interest of any nature or kind in
any product, service and/or company that could be con-
strued as influencing the position presented in, or the
review of the manuscript entitled.
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