International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3073–3078
DOI 10.1099/ijs.0.000381
Celeribacter naphthalenivorans sp. nov., a
naphthalene-degrading bacterium from tidal
flat sediment
Young Taek Oh,1 Catherine Avedoza,2 Sang-Suk Lee,2
Sang Eun Jeong,3 Baolei Jia3 and Che Ok Jeon3
Correspondence
Che Ok Jeon
1
Department of Microbiology and Immunology, Yonsei University, Seoul 120-752,
Republic of Korea
[email protected]
2
Department of Animal Science & Technology, Sunchon National University, Sunchon 540-742,
Republic of Korea
3
Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea
A Gram-stain-negative, aerobic and moderately halophilic bacterium, designated strain
EMB201T, was isolated from tidal flat sediment of the South Sea in Korea. Cells were motile
rods with a single polar flagellum and had catalase- and oxidase-positive activities. Growth of
strain EMB201T was observed at 15–37 8C (optimum, 30 8C), at pH 5.0–9.5 (optimum,
pH 7.0–7.5) and in the presence of 1–7 % (w/v) NaCl (optimum, 2–3 %). Strain EMB201T
contained ubiquinone-10 as the sole isoprenoid quinone and summed feature 8 (comprising
C18 : 1v7c/v6c), C18 : 0v7c 11-methyl and C10 : 0 3-OH as the major fatty acids.
Phosphatidylglycerol and an unidentified amino lipid were identified as the major polar lipids and
an unidentified phospholipid and three unidentified lipids were detected as minor components.
The G+C content of the genomic DNA was approximately 58.4 mol%. Phylogenetic analysis
based on 16S rRNA gene sequences showed that strain EMB201T formed a phylogenetic
lineage with members of the genus Celeribacter. Strain EMB201T was related most closely to
Celeribacter halophilus ZXM137T with a 16S rRNA gene sequence similarity of 98.3 %, and
the level of DNA–DNA relatedness between the two strains was 17.0¡2.0 %. The combined
chemotaxonomic and molecular properties suggest that strain EMB201T represents a novel
species of the genus Celeribacter, for which the name Celeribacter naphthalenivorans sp. nov.
is proposed. The type strain is EMB201T (5KACC 18393T5JCM 30679T).
The genus Celeribacter was first proposed by Ivanova et al.
(2010) as a member of the family Rhodobacteraceae within
the class Alphaproteobacteria. Recently, Huaishuia halophila
was reclassified as a member of the genus Celeribacter
(Lai et al., 2014), and at the time of writing, the genus
Celeribacter includes five recognized species, Celeribacter
neptunius (Ivanova et al., 2010), C. baekdonensis (Lee et al.,
2012), C. halophilus (Wang et al., 2012; Lai et al., 2014),
C. marinus (Baek et al., 2014) and C. indicus (Lai et al.,
2014). The genus Celeribacter accommodates Gramnegative, catalase- and oxidase-positive, moderately halophilic and rod-shaped bacteria containing C18 : 1v7c/v6c and
C18 : 0v7c, ubiquinone-10 and phosphatidylglycerol as the
major fatty acids, isoprenoid quinone and polar lipid,
The GenBank accession number for the 16S rRNA gene sequence of
strain EMB201T is KP272156.
One supplementary table and two supplementary figures are available
with the online Supplementary Material.
000381 G 2015 IUMS
respectively. All members of the genus Celeribacter have
been isolated from marine environments including seawater
and marine sediment. Tidal flats consist of broad areas of
coastal plain sediments alternately exposed to air and seawater, and diverse pollutant-degrading bacteria have been
isolated from these flats, which suggest that they may play
an important role in the bioremediation of sea environments. We have isolated diverse pollutant-degrading bacteria from tidal flats (Jin et al., 2012, 2013, 2015; Lo et al.,
2014). In this study, a novel naphthalene-degrading bacterium, strain EMB201T, belonging to the genus Celeribacter
was isolated from tidal flat sediment and its taxonomic properties were characterized using a polyphasic approach.
A tidal flat sediment sample was obtained from the
Yeosu coastal area of South Korea (348 489 330 N
1278 439 24.680 E) and strain EMB201T with naphthalene
degradation capability was enriched and isolated according
to the procedure described previously (Jin et al., 2012).
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3073
Y. T. Oh and others
Strain EMB201T was routinely grown aerobically on Marine
Agar 2216 (MA; BD) at 25 8C for 3 days, except where indicated, and stored at 280 8C in marine broth 2216 (MB; BD)
supplemented with 15 % (v/v) glycerol for long-term preservation. C. neptunius KCTC 23106T, C. baekdonensis KCTC
23497T, C. halophilus DSM 26270T, C. indicus DSM 27257T
and C. marinus KACC 17482T were obtained from their
culture collection centres and used as reference strains for
the comparison of phenotypic properties and fatty acid
compositions.
Stappia alba CECT 5094T (AJ878875)
Pseudoruegeria aquimaris SW-255T (DQ675021)
Pseudoruegeria sabulilitoris GJMS-35T (KJ729032)
Tropicimonas isoalkanivorans B51T (AB302379)
Celeribacter indicus P73T (EU440950)
Celeribacter naphthalenivorans EMB201T (KP272156)
100
Celeribacter neptunius H14T (FJ535354)
Celeribacter baekdonensis L-6T (HM997022)
100
T
The 16S rRNA gene of strain EMB201 was cloned into the
pCR2.1 vector using a TOPO cloning kit (Invitrogen)
according to the manufacturer’s instructions and sequenced
using the M13 reverse and T7 primers of the TOPO cloning
kit. Sequence similarities of the resulting 16S rRNA gene
sequence (1387 nt) were evaluated using the Nucleotide
Similarity Search program in the EzTaxon-e server (Kim
et al., 2012). The 16S rRNA gene sequences of strain
EMB201T and closely related type strains were aligned
using the fast secondary-structure aware Infernal aligner
available from the Ribosomal Database Project (Nawrocki
& Eddy, 2007). Phylogenetic trees based on the neighbourjoining and maximum-parsimony algorithms were reconstructed using PHYLIP software (ver. 3.695, Felsenstein,
2002) and their topologies were evaluated by bootstrap analyses using a 1000 resampled dataset. Maximum-likelihood
analysis was carried out using RAxML-HPC BlackBox (version 8.1.11) of the CIPRES Science Gateway (www.phylo.org;
Stamatakis et al., 2005).
Comparative analysis based on 16S rRNA gene sequences
showed that strain EMB201T was related most closely to
C. halophilus ZXM137T, C. neptunius H14T, C. baekdonensis
L-6T, C. indicus P73T and C. marinus IMCC 12053T with
similarities of 98.3, 97.3, 97.2, 97.1 and 95.5 %, respectively. Phylogenetic analysis using the neighbour-joining
algorithm revealed that strain EMB201T was tightly affiliated
with C. halophilus ZXM137T with a 100 % bootstrap
value (Fig. 1). Phylogenetic trees reconstructed using the
maximum-likelihood and maximum-parsimony algorithms
also showed that strain EMB201T formed a phyletic lineage
with members of the genus Celeribacter (Fig. 1).
Recently, it has been suggested that 98.65–98.7 % similarity
among 16S rRNA gene sequences should be used as a
threshold for using DNA–DNA hybridization in bacterial
classification (Stackebrandt & Ebers, 2006; Kim et al.,
2014). Nevertheless, C. halophilus, sharing 98.3 % sequence
similarity with strain EMB201T, was selected for evaluation
of DNA–DNA relatedness. DNA–DNA hybridization
analysis was reciprocally performed in triplicate to evaluate
DNA–DNA relatedness between strain EMB201T and
C. halophilus DSM 26270T using the DIG High Prime
DNA Labelling kit (Roche Applied Science), as described
previously (Lee et al., 2011). Hybridization signals were
analysed using Adobe Photoshop CS6 (ver. 13.0). Hybridization signals produced by hybridization of the probe to
the homologous target DNA were taken to be 100 %,
3074
Celeribacter halophilus ZXM137T (FJ436725)
Celeribacter marinus IMCC 12053T (KF146343)
Maritimibacter alkaliphilus HTCC2654T (AAMT01000002)
Roseovarius crassostreae CV919-312T (AF114484)
89
Roseovarius aestuarii SMK-122T (EU156066)
Litoreibacter meonggei MA1-1T (JN021667)
Ruegeria atlantica IAM 14463T (D88526)
82
0.01
Nautella italica CCUG 55857T (AM904562)
Lutimaribacter saemankumensis SMK-117T (EU336981)
Roseisalinus antarcticus EL-88T (AJ605747)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene
sequences showing the phylogenetic relationships between strain
EMB201T and related taxa. Bootstrap values are shown at nodes
as percentages of 1000 replicates; only values .70 % are shown.
The filled circle indicates that the corresponding node was also
recovered in trees generated by the maximum-parsimony and
maximum-likelihood algorithms. Stappia alba CECT 5095T
(AJ878875) was used as an outgroup. Bar, 0.01 changes per
nucleotide position.
and the signal intensities from self-hybridizations of serial
dilutions were used as a standard for the calculation of
DNA–DNA relatedness. The DNA–DNA hybridization
analysis was confirmed by reciprocal interchange of the
probe and the target DNA. The level of DNA–DNA relatedness between strain EMB201T and C. halophilus DSM
26270T was 17.0¡2.0 %, well below the 70 % threshold
generally accepted for species delineation (Stackebrandt
et al., 2002).
The naphthalene biodegradation ability of strain EMB201T
and other Celeribacter reference strains was evaluated in
serum bottles containing naphthalene and seawater as
described previously (Jin et al., 2012). Growth of strain
EMB201T was tested at 30 8C for 3 days on R2A agar
(BD), laboratory-prepared Luria–Bertani agar, nutrient
agar (NA; BD) and trypticase soy agar (TSA; BD), which
were supplemented additionally with NaCl to a final concentration of approximately 2 % (w/v). Growth of strain
EMB201T was assessed in MB at different temperatures
(4, 10, 15, 20, 25, 30, 37, 40 and 45 8C) and pH values
(5.0–11.0 at 0.5 pH unit intervals). MB with a pH value
below 8.0 and pH 8.0–11.0 was prepared using the
Na2HPO4–NaH2PO4 and Tris/HCl buffers, respectively
(Gomori, 1955). After sterilization (121 8C, 15 min), the
pH values were adjusted again if necessary. Growth at
different NaCl concentrations (0–15 % at 1 % intervals)
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Celeribacter naphthalenivorans sp. nov.
was investigated using MB prepared in the laboratory
according to the BD formula. Gram staining was tested
using the bioMérieux Gram stain kit according to the manufacturer’s instructions. Anaerobic growth was assessed on
MA under anaerobic conditions (with 4–10 % CO2) using
the GasPak Plus system (BBL) at 30 8C for 20 days. Cell
morphology and the presence of flagella were investigated
using phase-contrast microscopy and transmission electron
microscopy (JEM-1010; JEOL) with cells grown on MA at
30 8C. Oxidase and catalase activities were evaluated by the
oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine
(Merck) and the production of oxygen bubbles in 3 %
(v/v) aqueous hydrogen peroxide solution, respectively
(Smibert & Krieg, 1994). Nitrate reduction was assessed
according to the method of Lányı́ (1987). The following
properties of strain EMB201T and reference strains were
evaluated under the same conditions at 30 8C in this
study. Hydrolysis of Tween 80, Tween 20, aesculin,
casein, starch and tyrosine was tested on MA according
to the methods described by Lányı́ (1987) and Smibert &
Krieg (1994). Enzyme activities, biochemical features and
utilization of carbon sources were tested using the API
ZYM and API 20NE kits (bioMérieux) and GN2 MicroPlate system (Biolog), respectively, according to the
instructions of the manufacturers except that inocula
were prepared by resuspending cells in artificial seawater
(per litre: 20 g NaCl, 2.9 g MgSO4, 4.53 g MgCl2.6H2O,
0.64 g KCl and 1.75 g CaCl2.2H2O).
Strain EMB201T and the other Celeribacter reference strains
except for C. baekdonensis L-6T exhibited naphthalene degradation ability in seawater. Strain EMB201T grew well on MA
and TSA with 2 % NaCl, but did not grow on NA or R2A agar
containing 2 % NaCl. Anaerobic growth was not observed
after 20 days of incubation at 30 8C. Cells were Gramstain-negative motile rods with a single polar flagellum
(0.8–1.2 mm in width and 1.2–3.4 mm in length) (Fig. S1,
available in the online Supplementary Material). Physiological and biochemical characteristics of strain EMB201T are
additionally described in the species description and compared with those of the closely related type strains of the
genus Celeribacter in Tables 1 and S1. Many properties of
strain EMB201T, such as oxidase and catalase activity, moderately halophilic nature and no hydrolysis activity of
casein and starch, were in common with those of other
related species of the genus Celeribacter, but many others,
such as nitrate reduction and motility, differentiated strain
EMB201T (Table 1).
Isoprenoid quinones of strain EMB201T were analysed
using a model LC-20A HPLC system (Shimadzu) equipped
with a diode array detector (SPD-M20A; Shimadzu) and
a reversed-phase column (25064.6 mm, Kromasil; Akzo
Nobel) as described by Komagata & Suzuki (1987).
Methanol-2-propanol (2 : 1, v/v) was used as an eluent at
a flow rate of 1 ml min21. For the analysis of cellular
fatty acids, strain EMB201T and three reference strains
were cultivated in MB at 30 8C and microbial cells were
harvested at the same growth phase (exponential phase,
http://ijs.sgmjournals.org
optical density of 0.8 at 600 nm). The cellular fatty acids
were saponified, methylated and extracted using the standard MIDI protocol. The fatty acid methyl esters were analysed by GC (Hewlett Packard 6890) and identified by
using the TSBA6 database of the Microbial Identification
System (Sherlock ver. 6.0B; Sasser, 1990). The DNA
G+C content of strain EMB201T was determined by
using a high-performance liquid chromatograph fitted
with a reversed-phase column (GROM-SIL 100 ODS-2FE;
GROM) according to the method of Tamaoka & Komagata
(1984). The polar lipids of strain EMB201T were analysed
by TLC using cells harvested during the exponential
growth phase as described by Minnikin et al. (1977). The
following reagents were used to detect different polar
lipids: 10 % ethanolic molybdatophosphoric acid (for total
polar lipids), ninhydrin (for amino lipids) and Dittmer–
Lester reagent (for phospholipids).
The only respiratory lipoquinone detected in strain
EMB201T was ubiquinone-10 (Q-10), which is in good
agreement with the only respiratory quinone detected in
all members of the genus Celeribacter. The major cellular
fatty acids (.5 %) of strain EMB201T were summed feature 8 (comprising C18 : 1v6c/v7c, 72.5 %), C18 : 0 v7c
11-methyl (8.2 %) and C10 : 0 2-OH (6.1 %). The overall
fatty acid profile of strain EMB201T was similar to those
of species of the genus Celeribacter although there were
some differences in the respective proportions of some
components (Table 2). C18 : 1v7c 11-methyl was detected
as a major component in all Celeribacter species except
C. indicus P73T. Phosphatidylglycerol (PG) and an unidentified amino lipid (AL) were identified as the major polar
lipids in strain EMB201T (Fig. S2). In addition, an unidentified phospholipid (PL) and three unidentified lipids were
also detected as minor polar lipids. The overall profile of
polar lipids allowed the differentiation of strain EMB201T
from other species of the genus Celeribacter (Table 1). The
DNA G+C content of strain EMB201T was 58.4 mol%,
slightly lower than the range (59.1–66.0 mol%) reported
for the genus Celeribacter (Lai et al., 2014). In conclusion,
the phenotypic and molecular features of strain EMB201T
support its description as representing a novel species of
the genus Celeribacter, for which the name Celeribacter
naphthalenivorans sp. nov. is proposed.
Description of Celeribacter naphthalenivorans
sp. nov.
Celeribacter naphthalenivorans (naph.tha.le.ni.vo9rans. N.L.
neut. n. naphthalenum naphthalene; L. part. adj. vorans
devouring; N.L. part. adj. naphthalenivorans degrading
naphthalene).
Cells are Gram-stain-negative, aerobic, motile rods with a
single flagellum (0.8–1.2 mm wide and 1.2–3.4 mm long).
Colonies on MA are white, circular, convex and irregular.
Growth occurs at 15–37 uC (optimum, 30 uC), at
pH 5.0–9.5 (optimum, pH 7.0–7.5) and with 1–7 % (w/v)
NaCl (optimum, 2–3 %). Oxidase- and catalase-positive.
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Y. T. Oh and others
Table 1. Phenotypic characteristics of strain EMB201T and related type strains of species of the genus Celeribacter
Strains: 1, EMB201T (data from this study); 2, C. halophilus DSM 26270T (Wang et al., 2012); 3, C. neptunius KCTC 23106T (Ivanova et al., 2010); 4,
C. baekdonensis KCTC 23497T (Lee et al., 2012); 5, C. indicus DSM 27257T (Lai et al., 2014); 6, C. marinus KACC 17482T (Baek et al., 2014). All
strains are positive for the following characteristics: catalase, oxidase and leucine arylamidase. All strains are negative for Gram-staining, hydrolysis
of casein, starch, trypsin, a-chymotrypsin, a-mannosidase and a-fucosidase, indole production, arginine dihydrolase, and assimilation of D -maltose,
L -arabinose, D -mannose, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. +, Positive;
2, negative; W , weakly positive.
Characteristic
Nitrate reduction
Motility
Growth temperature (8C)
NaCl range (%) for growth
Naphthalene degradation*
Hydrolysis* of:
Tween 80
Tween 20
Aesculin
Enzyme activity (API ZYM)* of:
Alkaline phosphatase, esterase (C4), a-glucosidase
Esterase lipase (C8)
Lipase (C14)
Valine arylamidase
Cystine arylamidase, a-galactosidase
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
b-Galactosidase
b-Glucuronidase
b-Glucosidase
N-Acetyl-b-glucosaminidase
Assimilation (API 20NE)* of:
Adipic acid
D -Glucose, malic acid
D -Mannitol
Polar lipids:
Phosphatidylcholine
Phosphatidylglycerol
Diphosphatidylglycerol
Unknown glycolipid
Unknown aminolipid
Unknown phospholipid
Unknown lipid
DNA G+C content (mol%)
1
2
3
4
5
6
2
+
10237
1.027.0
+
2
2
4245
0.5211.0
+
+
+
5235
1.028.0
+
2
2
4237
0213.0
2
2
2
10241
0.5212.0
+
2
2
4–37
0.5–5.0
+
+
+
+
+
2
+
2
2
2
2
+
+
2
2
2
2
+
+
+
+
W
W
2
+
+
W
+
+
2
2
W
W
W
W
W
2
W
W
W
W
W
2
2
2
2
2
2
+
W
+
+
W
W
W
W
W
2
+
W
+
+
2
2
W
+
2
+
+
2
2
2
+
+
W
W
W
W
W
2
2
2
+
2
2
2
2
2
2
+
2
2
2
2
2
2
2
+
+
+
2
2
2
+
2
2
+
+
+
58.4
2
+
2
2
+
+
+
60.1
+
+
2
+
2
2
+
2
+
60.9
2
+
2
+
+
+
+
66.0
+
+
+
2
+
+
+
61.0
W
W
2
+
+
+
59.1
*These analyses were conducted in this study.
Hydrolyses aesculin, Tween 20 and Tween 80, but not starch,
casein or tyrosine. Nitrate is not reduced. Alkaline phosphatase, esterase (C4), acid phosphatase, leucine arylamidase, aglucosidase, b-glucosidase and N-acetyl-b-glucosaminidase
activities are positive. Activities of naphthol-AS-BI-phosphohydrolase, b-galactosidase and esterase (C8) are weakly
positive, but urease lipase (C14), valine arylamidase, cystine
arlyamidase, trypsin, a-chymotrypsin, a-galactosidase,
b-glucuronidase, a-mannosidase and a-fucosidase are negative. Negative for indole production, arginine dihydrolase,
assimilation of D -glucose, L -arabinose, D -mannose,
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D -mannitol,
N-acetylglucosamine, D -mannitol, malic acid,
capric acid, adipic acid, trisodium citrate, phenyl acetic
acid and potassium gluconate. The following substrates are
oxidized: D -fructose, D -psicose, sucrose, turanose, pyruvic
acid methyl ester, succinic acid monomethyl ester, acetic
acid, cis-aconitic acid, a-ketoglutaric acid, citric acid,
a-hydroxybutyric acid, b-hydroxybutyric acid, DL -lactic
acid, succinic acid, bromosuccinic acid, succinamic acid
and L -glutamic acid. Weakly positive for dextrin, D -arabitol,
cellobiose, maltose, trehalose, a-ketobutyric acid, propionic
acid, hydroxy-L -proline, inosine, uridine and thymidine.
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Celeribacter naphthalenivorans sp. nov.
Table 2. Cellular fatty acid compositions of strain EMB201T
and related type strains of species of the genus Celeribacter
References
Strains: 1, EMB201T; 2, C. halophilus DSM 26270T; 3, C. neptunius
KCTC 23106T; 4, C. baekdonensis KCTC 23497T; 5, C. indicus DSM
27257T; 6, C. marinus KACC 17482T. All data were from this study.
Data are expressed as percentages of the total fatty acids. Major components (.5.0 %) are highlighted in bold; TR , trace amount
(,1.0 %); 2, not detected.
sp. nov., isolated from coastal seawater. Int J Syst Evol Microbiol 64,
1323–1327.
Fatty acid
1
2
3
4
5
6
Saturated
C12 : 0
2
2
2
2
TR
TR
C16 : 0
2.6
2.0
2.9
3.2 7.7 3.4
C17 : 0
2
TR
2
2
2
2
3.1
2.3
2.6
1.6
1.6
2.2
C18 : 0
Hydroxy
2
2
2
2
TR
2
C10 : 0 2-OH
6.1 4.6 8.7 4.5 6.2 2.8
C10 : 0 3-OH
C16 : 0 3-OH
TR
TR
2
1.1 TR
2
1.4 TR
1.6
1.6 TR
2
C18 : 0 3-OH
Unsaturated
TR
TR
2
TR
2
2
C14 : 1v5c
8.2 8.8 8.0 18.0 2
9.8
C18 : 1v7c 11-methyl
2
2
2
2
2
TR
C18 : 1v9c
C19 : 0v8c cyclo
2
2
2
2 14.8 2
3.6
4.4
2.8 2
3.7 TR
C19 : 0 10-methyl
C20 : 1v7c
2
TR
2
2
2
TR
C20 : 4v6c (6, 9, 12, 15) 1.4 2
5.1 4.9 2
2
Summed features*
TR
TR
2
2
2
TR
3
4
2
2
2
2
1.2 2
8
72.5 75.7 68.5 64.6 61.8 79.4
*Summed features represent groups of two or three fatty acids that
could not be separated by GLC with the MIDI system. Summed features 3, 4 and 8 consist of C16 : 1v6 and/or C16 : 1v7c, anteiso-C17 : 1 B
and/or iso-C17 : 1 I, and C18 : 1v7c/v6c, respectively.
Contains phosphatidylglycerol, an unknown amino lipid, an
unknown phospholipid and three unknown lipids as polar
lipids. The major cellular fatty acids are summed feature
8 (comprising C18 : 1v7c/v6c), C18 : 1v7c 11-methyl and
C10 : 0 3-OH. The only isoprenoid quinone is Q-10.
The type strain is EMB201T (5KACC 18393T5JCM
30679T), isolated from tidal flat sediment in the Yeosu
coastal area of the South Sea in South Korea. The DNA
G+C content of the type strain is 58.4 mol%.
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Acknowledgements
This study was supported by grants from the National Institute of
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Environment (MOE) and the ‘Cooperative Research Program for
Agriculture Science & Technology Development (Project No.
PJ010906)’ of Rural Development Administration, Republic of Korea.
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