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Biodegradation of naphthalene,
BTEX, and aliphatic hydrocarbons
by Paraburkholderia
aromaticivorans BN5 isolated from
petroleum-contaminated soil
Yunho Lee, Yunhee Lee & Che Ok Jeon
To isolate bacteria responsible for the biodegradation of naphthalene, BTEX (benzene, toluene,
ethylbenzene, and o-, m-, and p-xylene), and aliphatic hydrocarbons in petroleum-contaminated
soil, three enrichment cultures were established using soil extract as the medium supplemented with
naphthalene, BTEX, or n-hexadecane. Community analyses showed that Paraburkholderia species
were predominant in naphthalene and BTEX, but relatively minor in n-hexadecane. Paraburkholderia
aromaticivorans BN5 was able to degrade naphthalene and all BTEX compounds, but not n-
hexadecane. The genome of strain BN5 harbors genes encoding 29 monooxygenases including two
alkane 1-monooxygenases and 54 dioxygenases, indicating that strain BN5 has versatile metabolic
capabilities, for diverse organic compounds: the ability of strain BN5 to degrade short chain aliphatic
hydrocarbons was veried experimentally. The biodegradation pathways of naphthalene and BTEX
compounds were bioinformatically predicted and veried experimentally through the analysis of
their metabolic intermediates. Some genomic features including the encoding of the biodegradation
genes on a plasmid and the low sequence homologies of biodegradation-related genes suggest that
biodegradation potentials of strain BN5 may have been acquired via horizontal gene transfers and/
or gene duplication, resulting in enhanced ecological tness by enabling strain BN5 to degrade all
compounds including naphthalene, BTEX, and short aliphatic hydrocarbons in contaminated soil.
Over the last few decades, diverse organic pollutants including petroleum fuels, pesticides, solvents, pharma-
ceuticals, and other organic chemicals have been produced industrially and released intentionally or uninten-
tionally during their transports or storages1,2. ese organic pollutants are generally highly persistent, have low
degradability, and can become trapped for long periods of time in the soil minerals of contaminated sites, and
subsequently bioaccumulated, due to their hydrophobic and stable chemical properties3–5. Because they have
potentially adverse eects on human health and multiple environments (aquatic, terrestrial, and atmospheric),
the contamination of these organic pollutants is a cause of great environmental concerns, which necessities the
need to restore contaminated sites1,6–10.
Bioremediation, a technique that employs microorganisms to remove organic pollutants, has been proven
to be sustainable, eco-friendly, and cost-eective among remediation technologies for contaminated sites11–14.
Many diverse microorganisms that have good pollutant-degrading ability have been previously isolated through
enrichment processes using synthetic basal media, which has led to extensive genomic and metabolic studies on
pollutant biodegradation15–20. However, bioremediation of contaminated sites by these microorganisms oen
results in failure due to unfavorable several biotic and abiotic factors including low degradability, low viabil-
ity, low pollutant availability, depletion of nutrients, and unfavorable pH, oxygen, temperature and moisture at
contaminated sites21–23. erefore, it has been suggested that the use of microorganisms mainly responsible for
pollutant degradation at contaminated sites is requisite for successful bioremediation of contaminated sites15,24–30.
Therefore, the identification and characterization of key players responsible for the degradation of organic
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea. Correspondence and requests
for materials should be addressed to C.O.J. (email: cojeon@cau.ac.kr)
Received: 23 April 2018
Accepted: 16 November 2018
Published: xx xx xxxx
OPEN
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pollutants at contaminated sites are of necessity, and enrichment cultures mimicking contaminated environ-
ments are good approaches to identify and isolate microorganisms having an ability to actively degrade organic
pollutants at contaminated sites27,31–35.
To date, biodegradation studies have been mostly focused on the isolation and characterization of micro-
organisms having an ability to degrade single organic compounds although environments are generally con-
taminated with mixed organic compounds33,36,37. erefore, a microorganism with a wide range of degradation
abilities on mixed organic pollutants or a consortium of microorganisms with a degradation ability on each
component of mixed organic pollutants is required for the successful bioremediation of contaminated sites. Soil
of gas station is oen contaminated with mixed organic compounds including BTEX (benzene, toluene, ethyl
benzene, and o-, m-, p-xylene), polycyclic aromatic hydrocarbons (PAH), and aliphatic hydrocarbons by leak-
ing gasoline and diesel fuels. ese compounds have been recognized as environmental priority pollutants that
should be removed from polluted environments because they are toxic, genotoxic, mutagenic or carcinogenic to
human6,8. erefore, to remediate gas station soil contaminated with gasoline and diesel fuels, microorganisms
able to degrade BTEX, PAH, and aliphatic hydrocarbons may be necessary. However, bacterial members harbor-
ing degradation potentials for dierent types of compounds such as PAH, BTEX, and aliphatic hydrocarbons
have been very rarely reported. It was reported that Rhodococcus strains showing broad degradation capabilities
toward various compounds, including naphthalene, BTEX, aliphatic hydrocarbons were successfully isolated, but
their genomic and biochemical features for their biodegradation were not investigated38. In this study, to identify
and isolate bacteria mainly responsible for the biodegradation of BTEX, naphthalene, and aliphatic hydrocarbons
from soil contaminated with gasoline and diesel fuels, we established three enrichment cultures using soil extract,
supplemented with naphthalene-, BTEX-, or hexadecane, as an enrichment medium to mimic environmen-
tal conditions and isolated a bacterial strain, Paraburkholderia aromaticivorans BN5, which had naphthalene-,
BTEX-, and short chain aliphatic hydrocarbon-biodegrading ability. In addition, in this study we investigated the
genomic and biochemical features of strain BN5 for the biodegradation of naphthalene-, BTEX-, and short chain
aliphatic hydrocarbons.
Results
Enrichment cultures and microbial community analysis. e use of microorganisms that have good
pollutant-degrading ability in the contaminated site of interest, not just in the laboratory or in other sites, is a
prerequisite for successful bioremediation. In this study, to isolate bacteria mainly responsible for the biodegra-
dation of naphthalene, BTEX, and aliphatic hydrocarbons in a contaminated site, soil extract was prepared using
soil from a gas station that had been contaminated with gasoline and diesel fuels for a long period of time. e
contaminated soil extract, rather than a dened minimal medium, which has been commonly used previously for
the enrichment of pollutant-degrading bacteria, was used as an enrichment medium.
e enrichment cultures supplemented with naphthalene, BTEX mixture, and n-hexadecane as the sole car-
bon source were sub-cultured three times, and the bacterial communities of the contaminated soil used for the
enrichment and the nal enrichment cultures were analyzed by PacBio RS II sequencing. e bacterial commu-
nity analysis showed that members of the genera Paraburkholderia and Rhodococcus were present at low rela-
tive abundances (approximately 0.9 and 0.3%, respectively) in the contaminated soil (Fig.S1). However, aer
the enrichments, the genus Paraburkholderia became predominant in both enrichment cultures supplemented
with naphthalene and BTEX, accounting for 98.4% and 79.3% of their total bacterial communities, respectively
(Fig.1). On the other hand, the enrichment culture supplemented with n-hexadecane was dominated by the
genus Rhodococcus, with approximately 49.8% relative abundance. e genus Paraburkholderia was also identied
from the enrichment culture supplemented with n-hexadecane, but its relative abundance was only 4.0%. ese
data suggest that the enrichments of naphthalene, BTEX, and hexadecane degraders were successfully accom-
plished and the bacterial group belonging to the genus Paraburkholderia may degrade naphthalene, BTEX, and
aliphatic hydrocarbons.
The genera Azotobacter, Ralstonia, Nitrobacter, Rhodanobacter, Thermomonas, Hydrogenophaga, and
Curvibacter were identied from the enrichment cultures supplemented with BTEX or n-hexadecane as minor
bacterial groups present.
Isolation of major bacteria from the enrichment cultures and their biodegradation abili-
ties. To isolate major bacteria with naphthalene-, BTEX-, and hexadecane-degrading abilities from the enrich-
ment cultures, the nal enrichment culture samples were spread on R2A agar and bacterial strains belonging to
the genera Paraburkholderia and Rhodococcus, the dominant bacterial genus groups in the enrichment cultures,
were isolated. Bacterial strains belonging to the genus Paraburkholderia were isolated from all three enrichment
cultures supplemented with naphthalene, BTEX, and n-hexadecane, while bacterial strains belonging to the genus
Rhodococcus were isolated from only the enrichment culture supplemented with n-hexadecane. Bacterial strains
belonging to the genera Ralstonia, Rhodanobacter, and Bacillus that were identied as minor bacterial groups
in the enrichment cultures were also successfully isolated, but the isolation of minor bacterial groups such as
Azotobacter, Nitrobacter, and ermomonas from the enrichment cultures failed.
e naphthalene-, BTEX-, and n-hexadecane-biodegrading abilities of bacterial isolates from the enrichment
cultures were evaluated in sterile 160 ml serum bottles. Interestingly, all Paraburkholderia strains regardless of
their isolation source had the ability to degrade naphthalene and all BTEX compounds, but they did not degrade
n-hexadecane despite the fact that they were also isolated from the enrichment culture supplemented with
n-hexadecane (data not shown). Rhodococcus strains isolated from the enrichment culture supplemented with
n-hexadecane clearly showed n-hexadecane degradation ability, but they did not degrade naphthalene and BTEX
compounds (data not shown). However, all other minor bacterial isolates did not exhibit the ability to degrade
naphthalene, BTEX, or n-hexadecane (data not shown). Although it was reported that some Paraburkholderia
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strains may have an biodegradation ability for naphthalene or BTEX compounds, Paraburkholderia strains
showing biodegradation abilities for naphthalene or BTEX compounds and moreover for both naphthalene and
all BTEX compounds have not yet been reported. erefore, in this study, we investigated the metabolic and
genomic features of a Paraburkholderia strain, strain BN5, which showed the ability to biodegrade naphthalene
and all BTEX compounds.
Biodegradation of naphthalene and BTEX by strain BN5 in minimal salt basal media (MSB)
and soil slurry systems. The ability of strain BN5 to degrade naphthalene, BTEX compounds, and
n-hexadecane was further evaluated over time in MSB (Fig.2). e degradation tests clearly showed that strain
BN5 had the ability to degrade naphthalene as well as all six BTEX compounds. However, strain BN5 did not
degrade n-hexadecane, even aer 14 days of incubation (data not shown). Naphthalene degradation occurred
more quickly than BTEX compound degradation by strain BN5, potentially because 30 mg/l of naphthalene was
singly present in the test serum bottles, while 30 mg/l of each six BTEX compounds (total 180 mg/l) were present
together in the test serum bottles.
Figure 1. Bacterial community compositions of the nal enrichment cultures supplemented with naphthalene,
BTEX mixture (benzene, toluene, ethyl benzene, and o-, m-, p-xylene xy1:1:1:1:1:1), and hexadecane. e
bacterial 16S rRNA gene sequences were classied at the genus level using the mothur soware against the
SILVA Gold reference database. “Others” represents taxa that comprised <1% of the total reads in three
samples.
Figure 2. Biodegradation of naphthalene and BTEX (benzene, toluene, ethyl benzene, and o-, m-, p-
xylene xy1:1:1:1:1:1) compounds by strain BN5 in serum bottles containing naphthalene (30 mg/l) or BTEX
mixture (initial concentration of each BTEX compounds, 30 mg/l) in MSB media. Serum bottles without the
inoculation of strain BN5 were used as negative controls; naphthalene and BTEX decreases in the negative
controls were negligible (not shown). e symbols represent averages of triplicate experiments and the error
bars indicate their corresponding standard deviations.
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e naphthalene and BTEX degradation by strain BN5 was also evaluated in soil slurry systems to better
mimic the contaminated soil environment (Fig.3). e degradation tests demonstrated that the degradation of
naphthalene and BTEX compounds in the soil slurry systems did not occur, or occurred at a very low rate, with-
out treatment with strain BN5 or nutrients (1 g/l of NH4Cl and 0.5 g/l of Na3PO4) during 7 days of incubation,
despite the fact that the soil slurry systems were established using unsterilized soil. However, the biodegradation
of naphthalene and BTEX compounds was signicantly improved by the addition of nutrients. When strain BN5
was inoculated into the soil slurry systems, the biodegradation of the tested compounds was also improved. In the
soil slurry systems that were treated with both nutrients and strain BN5 together, the biodegradation of naphtha-
lene and BTEX compounds was greatly improved. ese results suggest that strain BN5 was metabolically active
in the degradation of naphthalene and BTEX compounds in the slurry conditions mimicking (physiologically and
ecologically) the contaminated site. However, the results also suggest that there is a lack of nutrients for the bio-
degradation of naphthalene and BTEX compounds by bacteria in the contaminated site, and a supply of nutrients
containing nitrogen and phosphorus may be necessary to improve the biodegradation.
Figure 3. Biodegradation of naphthalene (A), benzene (B), toluene (C), ethyl benzene (D), m-, p-xylene (E),
and o-xylene (F) by strain BN5 in soil slurry systems. Symbols in the gures are as follows: untreated (-○-),
treated with nutrients (-●-), treated with strain BN5 (-□-), and treated with nutrients and strain BN5 (-■-).
Unsterilized freshwater and soil were used for the setting of the soil slurry systems. e symbols represent
averages of triplicate experiments and the error bars indicate their corresponding standard deviations.
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General features of the whole genome of strain BN5. To investigate the genomic and metabolic fea-
tures of strain BN5, its whole genome was completely sequenced, and its general features summarized in Table1.
e complete genome of strains BN5 consisted of two circular chromosomes of 4,378.9 and 2,992.2 kb and six
circular plasmids of 665.0, 489.4, 162.8, 152.4, 39.5, and 27.5 kb with an average G wiC content of 62.9% (Table1
and Fig.S2). Most of the genomic features of strain BN5, such as the number of chromosomes, total chromo-
some sizes, total gene numbers, protein coding sequences, and total rRNA and tRNA gene numbers are relatively
similar with those of other Paraburkholderia strains39–41. A phylogenetic analysis based on the 16S rRNA gene
sequences indicated that strain BN5 formed a phyletic lineage within the genus Paraburkholderia (Fig.S3) and
was most closely related to P. phytormans PsJNT, known to be a plant colonizing bacterium, with a very high 16S
rRNA gene sequence similarity (99.4%). However, the average nucleotide identity (ANI) and in silico DNA-DNA
hybridization (DDH) values of strains BN5 and PsJNT were 88.5% and 36.5%, respectively, clearly lower than the
95% ANI and 70% DDH cut-o values generally accepted for species delineation42,43, suggesting that strain BN5 is
a new species of the genus Paraburkholderia44. e total genome size of strain BN5 (8.9 Mb) was marginally larger
than that of strain PsJNT (8.2 Mb), and while strain BN5 harbors six plasmids, P. phytormans PsJNT contains only
one plasmid of 121 kb40, indicating that strain BN5 may have more diverse and versatile metabolic capabilities.
CRISPR, clustered regularly interspaced short palindromic repeats, along with cas (CRISPR-associated)
genes, are a bacterial defense mechanism against bacteriophage predation and their presence has been gener-
ally accepted as an evidence for previous bacteriophage infections. Genomic analysis showed that the genome
of strain BN5 contains only one possible CRISPR (Table1), while 23 predicted genomic islands (GIs) and 171
predicted transposase genes, indicating lateral gene transfers, were identied. ese ndings suggests that the
genome of strain BN5 has experienced extensive and complex genetic alterations or exchanges by lateral gene
transfers, rather than from bacteriophage infections during its evolutionary history. Strain BN5 harbors genes
encoding 29 monooxygenases and 54 dioxygenases that may be related to the biodegradation of various organic
compounds including naphthalene, BTEX, and other hydrocarbons; among them, 6 monooxygenase and 14 diox-
ygenase genes are located on plasmids, indicating possible inow of the biodegradation genes. Interestingly, strain
BN5 also harbors chromosomal two alkane 1-monooxygenase (alkB) genes (CJU94_RS03555 on chromosome
1 and CJU94_RS23135 on chromosome 2). e presence of alkB genes suggests that strain BN5 may have the
potential to degrade aliphatic hydrocarbons with dierent chain lengths or under specic conditions, although
the strain did not show hexadecane degradation ability in the above degradation tests. erefore, the ability
of strain BN5 to degrade dierent aliphatic hydrocarbons with dierent chain lengths (heptane, nonane, and
n-hexadecane) was evaluated. e biodegradation tests showed that strain BN5 does not degrade n-hexadecane,
in agreement with our previous biodegradation tests, but does have the ability to degrade short chain aliphatic
hydrocarbons such as heptane (C7) and nonane (C9) (Fig.4). In particular, nonane had been completely degraded
within ve days of incubation.
Genome-based prediction of naphthalene and BTEX degradation pathways and their experi-
mental verication. Previous studies have shown that naphthalene and BTEX compounds are metabolized
through a variety of degradation pathways depending on the microorganism39. e catabolic genes and metabolic
pathways of naphthalene and BTEX compounds in strain BN5 were bioinformatically predicted and experimen-
tally veried through gas chromatography/mass spectrometry (GC/MS) analysis of the metabolic intermediates
of naphthalene and each BTEX compound. e catabolic genes of naphthalene and BTEX compounds in strain
BN5 are located on plasmid 2 of 489.4 kb (pBN2), suggesting that strain BN5 probably obtained its degradation
capabilities of naphthalene and BTEX compounds through plasmid transfer.
Characteristic Chromosome (Chr) or plasmid (pBN)
Type Chr1 Chr2 pBN1 pBN2 pBN3 pBN4 pBN5 pBN6
Size (Kb) 4,388.9 2,992.2 665.0 489.4 162.8 152.4 39.5 27.5
G + C content (%) 63.2 63.7 61.3 60.0 59.7 61.5 59.0 59.7
Total genes 3,965 2,632 619 533 166 145 39 32
Protein coding sequences 3,823 2,535 569 483 143 137 37 29
Pseudogenes 76 81 49 50 23 8 2 2
rRNA (16S, 23S, 5S) operons 3 3 — — — — — —
tRNA genes 54 7 1 — — — — 1
Other RNA genes 4 — — — — — — —
Genomic islands 18 5
CRISPRs (questionable)*– (1) — ——————
Predicted transposase genes 38 28 17 52 27 9 — —
Monooxygenase genes 14 9 1 4 1 — — —
Dioxygenase genes 17 23 4 10 — — — —
Alkane 1-monooxygenase genes 1 1 — — — — — —
GenBank accession numbers NZ_CP022989–96.1
Table 1. General features of the chromosomes and plasmids in P. aromaticivorans strain BN5. *CRISPRs with
fewer than three perfect repeats or nonidentical repeats are considered “questionable”.
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It has been generally reported that one of two catabolic pathways, the catechol pathway or the gentisate
pathway, is generally used by bacteria for the biodegradation of naphthalene45. Bioinformatic analysis of the
genome showed that the naphthalene degradation gene cluster in strain BN5 is located on plasmid pBN2 and
its operon structure is similar to those of Alteromonas naphthalenivorans46, Polaromonas naphthalenivorans47,48,
and Ralstonia sp. strain U249 known as the nag operon, involved in metabolizing naphthalene via the gentisate
biochemical degradation pathway (Fig.5A and TableS1). Figure5A and TableS1 show the physical map of the
naphthalene catabolic genes and their associated functions, respectively. Gene homology and putative functions
clearly show that the naphthalene degradation gene cluster contains naphthalene dioxygenase (nagAaAbAcAd),
salicylate-5-hydroxylase (nagGH), and gentisate 1,2-dioxygenase (nagI) genes as key naphthalene metabolic
enzymes that are regulated by a LysR-type regulatory gene (nagR), suggesting that strain BN5 metabolizes naph-
thalene through salicylate and gentisate to fumarate and pyruvate, as do Ralstonia sp. U249, P. naphthalenivorans
CJ248, and A. naphthalenivorans SN246 (Fig.5B). Because salicylate and gentisate are key metabolic intermediates
in the gentisate pathway for naphthalene biodegradation, the presence of these intermediates was investigated
using a gas chromatography-mass spectroscopy detector (GC-MSD) during naphthalene degradation by strain
BN5. GC/MS analysis showed that salicylate and gentisate were detected from naphthalene-grown cell suspen-
sions (Fig.S4), which veried that strain BN5 metabolizes naphthalene via the gentisate biochemical degradation
pathway.
Bioinformatic analysis showed that gene clusters probably responsible for benzene/toluene/xylene degrada-
tion were also found to be located on plasmid pBN2. e benzene/toluene/xylene catabolic genes were split into
two gene clusters with an approximate distance of 18.7 kb (Fig.6A) and their associated functions are described in
Figure 4. Biodegradation of heptane, nonane, and hexadecane by strain BN5 in serum bottles containing
heptane, nonane or hexadecane (100 mg/l) in MSB media. Serum bottles without the inoculation of strain BN5
were used as negative controls and the concentrations of heptane, nonane, and hexadecane were measured aer
ve days of incubation at 30 °C. e bars represent averages of triplicate experiments and the error bars indicate
their corresponding standard deviations.
Figure 5. Physical map of naphthalene degradation genes located on the plasmid pBN2 of strain BN5 (A) and
a proposed biochemical pathway of naphthalene degradation (B). e putative functions of the naphthalene
biodegradation genes were predicted and are presented in TableS1.
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TableS2. Previously, two primary benzene biodegradation pathways with the conversions of benzene to phenol or
benzene to cis-dihydrobenzenediol as the result of the rst oxidation have been proposed as being typical aerobic
benzene biochemical pathways50,51. No gene encoding benzene monooxygenase for the conversion of benzene to
phenol was identied from the genome of strain BN5. However, it has been reported that toluene monooxygen-
ase responsible for ring monooxidation of toluene and phenol 2-hydroxylase converting phenol to catechol are
also able to oxidize benzene to phenol50. e metabolic gene cluster analysis showed that strain BN5 harbored all
genes encoding toluene 4-monooxygenase (tmoABCDE) and phenol 2-hydroxylase (dmpKLMNOP) (Fig.6A and
TableS2). In addition, strain BN5 harbored genes coding for phenol 2-hydroxylase converting phenol to catechol
and catechol 2,3-dioxygenase (xylE) converting catechol to 2-hydroxymuconate semialdehyde in the gene cluster.
Based on these results, a potential benzene degradation pathway, which has phenol and catechol as intermediates,
in strain BN5 was proposed (Fig.6B). To conrm the benzene biodegradation pathway, the metabolic inter-
mediates of benzene were investigated using a GC-MSD during benzene biodegradation: phenol and catechol
were detected as metabolic intermediates from benzene-grown suspensions (Fig.S5). e detection of phenol
and catechol as metabolic intermediates and the metabolic gene analysis suggest that strain BN5 metabolizes
benzene through a metabolic pathway with rst benzene monooxidation by toluene monooxygenase or phenol
2-hydroxylase and phenol and catechol as metabolic intermediates.
It has been proposed that aerobic toluene biodegradation is typically initiated by ve dierent oxidations,
the methyl group oxidation, ring monooxidation at position 2, 3, or 4, or ring 2,3-dioxidation of toluene, as
the rst step52. e bioinformatic analysis of the benzene/toluene/xylene catabolic genes shows that strain BN5
harbors genes encoding a complete toluene 4-monooxygenase (tmoABCDE), 4-cresol dehydrogenase (pchCF),
and 4-hydroxybenzaldehyde dehydrogenase (pchA) as putative toluene biodegradation genes (Fig.6A and
TableS2), possibly indicating that toluene degradation in strain BN5 is initiated by ring monooxidation at the
para position of toluene to form 4-hydroxytoluene (p-cresol) by TmoABCDE, followed by the conversion of
4-hydroxytoluene to 4-hydroxybenzaldehyde and 4-hydroxybenzoate by PchCF and PchA subsequently. Based on
Figure 6. Physical maps of the metabolic genes probably responsible for benzene, toluene, and xylene
degradation located on the plasmid pBN2 of strain BN5 (A). e metabolic genes are split into two gene
clusters with a distance of approximately 18.7 kb and their putative functions were predicted and presented in
Supplementary TableS2. Based on the predicted functions of the metabolic genes and the conrmation through
GC/MS analysis of the metabolites, the biochemical pathways of benzene (B), toluene (C), and xylene (D)
degradation in strain BN5 were proposed. Genes encoding the enzymes with asterisks were not identied in
the genome of strain BN5 by bioinformatics analysis. e putative functions of the naphthalene biodegradation
genes were predicted and presented in TableS2.
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the gene annotations, a potential toluene biodegradation pathway of strain BN5 was proposed (Fig.6C). However,
the TmoABCDE- and PchCF-encoding genes were present in dierent gene clusters, indicating that they may be
separately and independently regulated. To conrm the proposed toluene biodegradation pathway, the toluene
biodegradation intermediates were investigated using a GC-MSD: 4-hydroxybenzoate was detected as a meta-
bolic intermediate from toluene-grown cell suspensions (Fig.S6). Because 4-hydroxybenzoate is not a metabolic
intermediate in four other toluene biodegradation pathways, it was concluded that strain BN5 metabolizes tolu-
ene through the toluene 4-monooxygenase pathway.
Figure2 showed that strain BN5 has the ability to degrade all three xylene compounds, which sug-
gests that strain BN5 harbors all xylene degradation genes. However, genes related to the xylene biodegrada-
tion, except for genes encoding benzoate 1,2-dioxygenase (XylXY) converting methyl benzoate (toluate) to
1,2-dihydroxy-methyl-cyclohexa-3,5-diene-carboxylate were not identied in the genome of strain BN5 by bio-
informatic analysis. Generally, it has been reported that biodegradation of xylenes including three structural
isomers of dimethyl benzene (o-, m-, and p-xylenes) is initiated through the oxidation of a methyl substituent
of xylene to 2-methylbenzyl alcohol by xylene monooxygenase or the direct oxidation of the aromatic ring by
xylene dioxygenase53–55. In the xylene monooxygenase pathway, 2-methylbenzyl alcohol is subsequently con-
verted to methyl benzaldehyde, methyl benzoate, and 2-dihydroxy-methyl-cyclohexa-3,5-diene-carboxylate by
benzylalcohol dehydrogenase, benzylaldehyde dehydrogenase, and benzoate 1,2-dioxygenase (XylXY), respec-
tively. erefore, based on the presence of XylXY-encoding genes (Fig.6A and TableS2), a possible xylene biodeg-
radation pathway in strain BN5 was proposed (Fig.6D). To conrm the proposed xylene biodegradation pathway,
xylene biodegradation intermediates from xylene-grown cell suspensions using each o-, m-, and p-xylene as a
sole carbon source were investigated through GC/MS analysis. e results showed that methylbenzyl alcohol
and 1,2-dihydroxy-methyl-cyclohexa-3,5-diene-carboxylate were detected as metabolic intermediates from
xylene-grown cell suspensions (Fig.S7), which suggests that strain BN5 may metabolize xylene through the oxi-
dation of a methyl substituent of xylene to 2-methylbenzyl alcohol by xylene monooxygenase. However, the
genome annotation of strain BN5 failed to uncover the genes encoding xylene monooxygenase, benzylalcohol
dehydrogenase, and benzylaldehyde dehydrogenase, which might be caused by their very low sequence homolo-
gies with known corresponding genes.
Two aerobic ethylbenzene metabolic pathways using either the aromatic ring oxidation of ethylbenzene
by ethylbenzene dioxygenase to cis-1,2-dihydroxy-2,3-dihydroethylbenzene56 or the ethyl group oxidation of
ethylbenzene by naphthalene dioxygenase to 1-phenethyl alcohol15,57 have been proposed. Bioinformatic analysis
showed that ethylbenzene dioxygenase or genes homologous with ethylbenzene dioxygenase were not identied
from in the genome of strain BN5 although it carries 29 monooxygenases and 54 dioxygenases, suggesting that
strain BN5 may metabolize ethylbenzene through the ethyl group oxidation of ethylbenzene by naphthalene
dioxygenase. In the ethyl group degradation pathway that uses naphthalene dioxygenase, 1-phenethyl alcohol is
typically converted into 2-hydroxy acetophenone by naphthalene dioxygenase or into benzoylacetate by acetophe-
none carboxylase57,58. erefore, metabolic intermediates of ethyl benzene biodegradation were investigated using
a GC-MSD: during ethylbenzene degradation, 2-hydroxy-acetophenone was detected from ethylbenzene-grown
cell suspensions (Fig.S8). Moreover, a gene encoding acetophenone carboxylase was not identied from the
genome analysis of strain BN5. Based on the analysis of the genome and intermediates of strain BN5, we propose
a possible ethyl benzene biodegradation pathway in strain BN5 that naphthalene dioxygenase catalyzes in multi-
ple steps, which was in common with those in previous reports15,57 (Fig.S9).
Discussion
Bioremediation using bacterial metabolic processes is considered to be one of the most ecient and eco-friendly
clean-up techniques of contaminated environments11–14. e microbial catabolic capability for organic pollutants
is a prerequisite for bioremediation practices and thus the isolation and characterization of microorganisms that
have the ability to metabolize a wide range of organic pollutants has been extensively studied11,45,53,59–61. To date,
articial synthetic basal media containing a sole organic compound have mostly been used to enrich or isolate
microorganisms that have organic substance-degradation ability on25,26,29,31.
However, the use of articial synthetic basal media as an enrichment medium has oen resulted in the enrich-
ment of microorganisms that only grow successfully in laboratory conditions, not in contaminated sites32,62. In
addition, although the isolated microorganisms have the ability to degrade organic pollutants in laboratory con-
ditions, they oen show a low degradation ability in eld conditions due to various unfavorable factors21,23,63.
erefore, the use of microorganisms that can actively degrade organic substances at contaminated sites is nec-
essary for the successful clean-up of contaminated sites. To identify and isolate microorganisms that eectively
degrade organic pollutants at contaminated sites, various approaches such as enrichment culture33, metagenom-
ics64, stable isotope probing27,65 and uorescence in situ hybridization (FISH)66 have been used. Among them, an
enrichment culture mimicking contaminated environments is one of the easiest and most promising approaches
to identify and isolate microorganisms that have pollutant degradation ability at contaminated sites33,35,67–69.
Therefore, to isolate microorganisms responsible for the degradation of BTEX, naphthalene, and aliphatic
hydrocarbons in petroleum (gasoline and diesel)-contaminated soil, soil extract, not an articial synthetic basal
medium, was used as an enrichment medium.
ree enrichment cultures for the isolation of naphthalene-, BTEX-, and aliphatic hydrocarbon-degrading
bacteria from gasoline and diesel fuel-contaminated soil were established. A bacterial group belonging to
Paraburkholderia was present, with 0.9% relative abundance in the contaminated soil, and predominantly
enriched in the enrichment cultures using soil extract supplemented with naphthalene and BTEX (Figs1 and S1).
e degradation tests showed that Paraburkholderia strain BN5 that was isolated from the enrichment cultures
had the ability to degrade naphthalene as well as all BTEX compounds in a slurry system designed to mimic
the contaminated soil conditions (Fig.3), again reinforcing the idea that Paraburkholderia may be responsible
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SCIENTIFIC REPORTS | (2019) 9:860 | https://doi.org/10.1038/s41598-018-36165-x
for the biodegradation of naphthalene and BTEX compounds in the contaminated soil. However, many other
biotic and abiotic factors aecting biodegradation activity of Paraburkholderia that we are not able to mimic in
the slurry system are possibly present in contaminated soils21,70. In addition, it is not thought that the growth of
Paraburkholderia with 0.9% of relative abundance in the contaminated soil entirely relies on only the biodegra-
dation of naphthalene and BTEX compounds because contaminated soils contain many other organic carbon
sources. erefore, in situ biodegradation test may be necessary to assess more clearly biodegradation activity of
Paraburkholderia in the contaminated soil.
Paraburkholderia species were also identied from the enrichment culture supplemented with n-hexadecane,
although their relative abundance was low (Fig.1) and Paraburkholderia strains isolated from the enrichment
cultures had no degradation activity for n-hexadecane. However, bioinformatic analysis of the genome of
Paraburkholderia strain BN5 showed that the strain harbors genes encoding two alkane 1-monooxygenases, a key
enzyme in the aliphatic hydrocarbon biodegradation (Table1). e biodegradation test of strain BN5 for aliphatic
hydrocarbons with dierent chain lengths showed that strain BN5 had the ability to biodegrade short chain ali-
phatic hydrocarbons such as heptane and nonane (Fig.4), which might be the reason that Paraburkholderia was
only found in minor abundance in the hexadecane enrichment culture.
Many members belonging to the genera Pseudomonas, Rhodococcus, Mycobacterium, Sphingomonas, and
Burkholderia have been considered to probably possess a broad range of catabolic potentials toward diverse aro-
matic compounds30,39,71–73. In particular, it has been largely assumed through the genomic analysis that many
members of Alcaligenaceae, Burkholderiaceae, and Comamonadaceae families have biodegradation potentials
towards a vast array of aromatic compounds, including several priority pollutants39. Auret and colleagues
reported that they successfully isolated Rhodococcus strains showing broad degradation capabilities toward vari-
ous compounds, including naphthalene, BTEX, aliphatic hydrocarbons, and more recalcitrant compounds such
as methyl tert-butyl ether and ethyl tert-butyl ether, but the genomic and biochemical features of the strains for
the biodegradation of the organic compounds were not investigated38. is study is the rst report for the enrich-
ment, isolation, and genomic and biochemical analysis of Paraburkholderia with biodegradation abilities for all of
naphthalene, BTEX, and short chain aliphatic hydrocarbons although there is a report that P. phytormans PsJN,
a close relative of strain BN5, may have a potential degrading-activity for organic compounds30,40,71.
e degradation tests clearly showed that strain BN5 had a biodegradation ability for all of naphthalene,
BTEX compounds, and short chain aliphatic hydrocarbons, but almost biochemical degradation gene clusters,
except for the naphthalene degradation gene cluster, are not well organized (Figs5 and 6 and TablesS1 and S2).
In addition, most of these degradation genes were encoded on a plasmid (plasmid pBN2). ese analyses suggest
that strain BN5 may have recently acquired its biodegradation abilities via horizontal gene transfer (HGT) or the
gain of a plasmid with degradation genes, resulting in successful adaptation to gasoline and diesel-contaminated
soil. Phylogenetic analysis indicated that toluene monooxygenase and alkane 1-monooxygenase genes were not
homologous with those of the genera Paraburkholderia and Burkholderia, indicating that they might have been
introduced via HGT (Fig. S10). Although strain BN5 was able to biodegrade naphthalene, all BTEX compounds,
and short chain aliphatic hydrocarbons, some key degradation genes such as xylene degradation genes were not
identied in the genome of strain BN5.
In this study, we isolated Paraburkholderia strain BN5, capable of degrading naphthalene, BTEX, and short
chain aliphatic hydrocarbons from gasoline and diesel fuel-contaminated soil. e genomic analysis of strain
BN5 suggests that strain BN5 may have acquired its biodegradation capabilities via plasmid gain, HGT, and/or
gene changes under the contaminated soil conditions, resulting in enhanced ecological tness by enabling strain
BN5 to degrade all of naphthalene, BTEX, and short chain aliphatic hydrocarbons. We predicted the degradation
pathways of naphthalene and all BTEX compounds in strain BN5 by analyzing the naphthalene and BTEX degra-
dation gene clusters, and the identied biochemical degradation pathways were experimentally veried through
the analysis of their metabolic intermediates. e availability of strain BN5 and its complete genomic information
will provide us with insights into better understanding of the physiological and genomic features of strain BN5
and its naphthalene-, BTEX-, and aliphatic hydrocarbon-degrading ability in soil environments contaminated
with gasoline and diesel fuels. However, further studies on gene regulation of the biodegradation pathways in
strain BN5 are necessary to understand the simultaneous biodegradation features of naphthalene-, BTEX-, and
aliphatic hydrocarbons by strain BN5 in contaminated soil more clearly.
Methods
Soil samples and enrichment cultures. Soil extract was used as an enrichment medium to isolate
bacteria responsible for degrading pollutants in a contaminated site. For the preparation of soil extract and
the enrichment of naphthalene-, BTEX (benzene, toluene, ethyl benzene, and o-, m-, p-xylene)-, and aliphatic
hydrocarbon-degrading bacteria, soil samples were obtained from a gasoline and diesel fuel-contaminated site
(37°50′21.5″N 126°59′37.2″E) of a gas station located in Yangju, Gyeonggi Province, South Korea. Only freshly
collected soil was used for the preparation of the soil extracts, which were prepared according to the procedure
described previously67. In brief, 800 g of the contaminated soil was sieved through a 2 mm mesh and then mixed
with 2 liters of distilled water. e mixture was vigorously agitated for 2 h at room temperature (approximately
25 °C) and centrifuged (5,000 rpm, 25 °C, 10 min). e supernatant was ltered through a 0.45 µm membrane
lter (Millipore, USA) and then supplemented with NH4Cl (1 g/l) and Na3PO4 (0.5 g/l) for nutrients. ree cot-
ton-plugged 500 ml Erlenmeyer asks containing 100 ml of the soil extract and 10 g of the contaminated soil
were prepared, and 0.5 g of naphthalene pellets, 1.0 ml of BTEX mixture (benzene, toluene, ethyl benzene, and
o-, m-, p-xylene yl1:1:1:1:1:1), and 1.0 ml of n-hexadecane were directly added into each of three asks. e three
enrichment cultures were incubated at 25 °C with shaking (180 rpm) and subcultured (1:20) into fresh soil extract
containing the same amount of each compound three times every two weeks.
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Bacterial community analysis of enriched cultures. Genomic DNA was extracted from the contam-
inated soil and the nal enrichment cultures using a FastDNA SPIN Kit for Soil (MPBio, USA), according to the
manufacturer’s instructions. e 16S rRNA genes of the genomic DNA were PCR-amplied using the barcoded
universal bacterial primers 27F (5′-X-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-X-TAC GGY TAC
CTT GTT ACG ACT T-3′), where X denotes the 16-nucleotide long barcode. PCR conditions were as follows:
94 °C for 5 min, followed by 32 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min, and 1 nal cycle of 72 °C
for 10 min. e PCR products were puried using a PCR purication kit (Solgent, Korea). A pooled composite
was prepared by mixing equal amounts of the puried PCR products and sequenced on a PacBio RS II instrument
using P6-C4 chemistry (Pacic Biosciences, USA) aer SMRTbell adapter ligation at Macrogen (Korea). e
PacBio raw data generated by the PacBio RS II single-molecule real-time (SMRT) sequencing were assembled in
circular consensus sequences (CCSs) using the RS_ReadsOfInsert protocol in the SMRT Analysis Soware (ver.
2.3) with the parameters “minFullPasses 5” and “minPredictedAccuracy 90” aer removing SMRTbell adapters,
and the CCSs were demultiplexed based on their PCR barcodes using the SMRT Portal72. e demultiplexed
sequencing reads were processed using the mothur soware73 as follows: sequences of 1,400–1,600 nucleo-
tides with fewer than two ambiguous nucleotides were selected and chimera sequences were removed using the
UCHIME program of the mothur soware74 against the SILVA Gold reference database (ver. 123). e resulting
high-quality sequences were classied using the naïve Bayesian classier based on the SILVA reference taxonomy,
with an 80% cuto.
Isolation of major bacteria from the enrichment cultures and biodegradation tests. To
isolate major bacteria from the enrichment cultures, the final enrichment cultures were serially diluted in
phosphate-buered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.2) and spread
onto R2A agar (BD, USA) and the agar plates were incubated at 25 °C. irty colonies from each enrichment were
randomly selected and their 16S rRNA genes were PCR-amplied using the 27F and 1492R primers as described
previously75. e PCR amplicons were double-digested with a mixture of HaeIII and HhaI restriction enzymes
and representative PCR products with unique restriction fragment length polymorphism (RFLP) patterns were
sequenced, as described previously75. e resulting 16S rRNA gene sequences were compared with those of all
reported type strains using the Nucleotide Similarity Search program in the EzTaxon-e server (https://www.
ezbiocloud.net/)76.
e naphthalene-, BTEX-, and hexadecane-biodegrading abilities of the isolates with unique RFLP patterns
were tested in triplicate in sterile 160 ml serum bottles containing 10 ml of minimal salt basal media (MSB)77 sup-
plemented with 30 ppm of naphthalene, 180 ppm of BTEX mixture, and 100 ppm of n-hexadecane, respectively,
as described previously33,75. In brief, cells of the isolates grown in R2A broth at 25 °C were inoculated into the
serum bottles at a density of approximately 107 cells/ml. e serum bottles were sealed with Teon-coated rubber
septa and incubated at 25 °C with shaking (180 rpm) for a week. e incubated media were extracted with meth-
ylene chloride (for BTEX and n-hexadecane) or ethyl acetate (for naphthalene) and the remaining BTEX, naph-
thalene, and n-hexadecane concentrations were measured using gas chromatography, as described previously33,75.
To infer the phylogenetic relationship of strain BN5, which shows naphthalene- and BTEX-biodegrading
ability, the 16S rRNA gene sequences of BN5 and some closely related type strains were aligned using the fast
secondary structure-aware Infernal aligner in the Ribosomal Database Project (RDP) (http://pyro.cme.msu.
edu/spring/align.spr)78, and a phylogenetic tree was constructed using the neighbor-joining algorithm with the
Kimura two-parameter model in the PHYLIP soware79.
Biodegradation of naphthalene and BTEX by strain BN5 in MSB and soil slurry systems. e
biodegradation ability of strain BN5 was evaluated with naphthalene and BTEX in sterile 160 ml serum bottles
containing 10 ml of MSB medium supplemented with naphthalene (30 mg/l) or BTEX mixture (180 mg/l). BN5
cells grown in R2A broth at 30 °C were inoculated at a density of approximately 2 × 107 cells/ml and incubated at
30 °C with shaking (180 rpm). ree bottles per sample were sacriced periodically and the compound concen-
trations were analyzed using HP 7890B gas chromatography (GC) coupled to a ame ionization detector (Agilent,
USA) with an HP-5 column (30 m length, 0.32 µm inner diameter, 0.25 µm lm thickness; J & W Scientic). e
GC oven temperature was held at 40 °C for 0.50 min and increased at a rate of 10 °C per min to 150 °C and then
at a rate of 15 °C per min to a nal temperature of 260 °C, which was held for 5 min. Uninoculated serum bottles
were used as negative controls.
To evaluate the biodegradation ability of strain BN5 with naphthalene and BTEX in soil slurry systems, four
sets of serum bottles (untreated; treated with 1 g/l of NH4Cl and 0.5 g/l of Na3PO4 as nutrients; treated with strain
BN5; and treated with nutrients and strain BN5) were prepared. ree grams of soil obtained from the gasoline
and diesel fuel-contaminated site were transferred into 160 ml serum bottles containing 10 ml of freshwater (col-
lected from the Han River, Korea, July 2017) (supplemented, or unsupplemented with nutrients) with naphtha-
lene (30 mg/l) or BTEX mixture (180 mg/l). BN5 cells were inoculated into the serum bottles at approximately
2 × 107 cells/ml. e serum bottles were incubated at 30 °C with shaking (180 rpm, with periodic inversion) and
three bottles per experimental set were sacriced periodically and the concentrations of naphthalene and BTEX
were analyzed using GC, as described above.
Complete genome sequencing and bioinformatic analyses. For the whole genome sequencing of
strain BN5, genomic DNA was extracted using a Promega Wizard Genomic DNA purication kit (Promega,
USA), according to the manufacturer’s instructions. e genome of strain BN5 was sequenced using a combi-
nation of PacBio RS II SMRT sequencing with a 10-kb library and Illumina Hiseq2500 sequencing at Macrogen
(Korea). De novo assembly of the PacBio sequencing reads was performed through the hierarchical genome
assembly process. Paired-end reads (101-bp) derived from Illumina sequencing were mapped on the complete
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SCIENTIFIC REPORTS | (2019) 9:860 | https://doi.org/10.1038/s41598-018-36165-x
genome obtained by the PacBio sequencing for error corrections. e NCBI (http://www.ncbi.nlm.nih.gov/) and
Integrated Microbial Genomes (IMG; https://img.jgi.doe.gov/cgi-bin/er/main.cgi) servers were primarily used
for genome analysis.
The ANI and in silico DDH values between BN5 and its closely related strains were calculated using a
stand-alone soware (http://www.ezbiocloud.net/sw/oat)80 and the server-based genome-to-genome distance
calculator ver. 2.1 (http://ggdc.dsmz.de/distcalc2.php)81, respectively. Circular maps of the chromosomes and
plasmids of strain BN5 were generated using the web-based CGview tool (http://stothard.afns.ualberta.ca/
cgview_server/index.html)82. Clustered regularly interspaced short palindromic repeat (CRISPR) gene sequences
were identied using an online web service (http://crispr.i2bc.paris-saclay.fr/Server/). Genomic islands (GIs) were
predicted using the SIGI-HMM tool with Island Viewer 4 (http://www.pathogenomics.sfu.ca/islandviewer/)83.
e annotation and putative functions of genes associated with the biodegradation of naphthalene, BTEX,
and aliphatic hydrocarbons were bioinformatically analyzed based on data from the NCBI genomic database,
the Integrated Microbial Genomes (IMG) database, UniProt database (http://www.uniprot.org/), and BLAST
searches. e catabolic pathways of naphthalene and each BTEX compound were predicted based on the annota-
tion results of the metabolic genes.
Biodegradation tests of strain BN5 on heptane, nonane, and hexadecane in MSB. e bio-
degradation of heptane, nonane, and n-hexadecane by strain BN5 was evaluated in sterile 160 ml serum bottles
containing 10 ml of MSB medium supplemented with 100 mg/l of each of heptane, nonane, and n-hexadecane,
as described above. BN5 cells were inoculated at a density of approximately 2 × 107 cells/ml and the serum bot-
tles were incubated at 30 °C with shaking (180 rpm) for 5 days. e concentrations of heptane, nonane, and
n-hexadecane were analyzed using GC aer methylene chloride extraction, as described above.
GC/MS detection of naphthalene and BTEX metabolites. e catabolic pathways that were predicted
based on bioinformatic analyses were conrmed through the detection of metabolic intermediates of naphthalene
and BTEX compounds using a GC-MSD, as previously described15,33, with some modication. Triplicate serum
bottles containing 10 ml of MSB media supplemented with naphthalene or each of the individual BTEX com-
pounds (300 ppm) were prepared and incubated at 30 °C with shaking (150 rpm) for 2 days. Triplicate suspen-
sions were combined and acidied with HCl to approximately pH 2.0 and metabolic intermediates were extracted
with 10 ml of ethyl acetate. e ethyl acetate fractions were dried over anhydrous Na2SO4 and concentrated under
an atmosphere of N2 to a volume of 500 µL. Metabolic intermediates were derivatized with 50 µL bis(trimethyl-
silyl) triuoroacetamide (BSTFA) for 30 min and analyzed using a HP 7820 A gas chromatography-5977E mass
spectroscopy detector (GC-MSD) (Agilent, USA) equipped with an HP-5 capillary column. e GC oven tem-
perature was programmed to increase from 50 °C to 260 °C at 5 °C/min and then was held at 260 °C for 5 min. e
peaks were identied by matching query mass spectra to those in a mass spectral reference library.
Phylogenetic analysis of catabolic genes. Homologous amino acid sequences of naphthalene
1,2-dioxygenase large oxygenase component (NagAc), toluene-4-monooxygenase system protein A (TmoA), and
alkane 1-monooxygenase (AlkB) were identied using the UniProt database and maximum-likelihood (ML)
algorithm-based trees showing their phylogenetic relationships were constructed using the MEGA7 program and
the tree topologies were evaluated through bootstrap tests with 1,000 iterations84.
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Acknowledgements
is work was supported by the National Research Foundation (2017R1A2B4004888, 2018R1A5A1025077) of
the Ministry of Science, ICT and Future Planning, Republic of Korea.
Author Contributions
C.O.J. conceived the project. Yunho Lee and Yunhee Lee performed the experiments and conducted the
bioinformatics analysis. Yunho Lee and C.O.J. wrote the paper. All authors read and approved the final
manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36165-x.
Competing Interests: e authors declare no competing interests.
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