Chemosphere 165 (2016) 173e182

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Detoxification of hydroxylated polychlorobiphenyls by Sphingomonas

sp. strain N-9 isolated from forest soil
Satomi Mizukami-Murata a, b, Futa Sakakibara b, Katsuhide Fujita c, Makiko Fukuda d,
Masato Kuramata b, Kazuhiro Takagi b, *
a
The Japan Society for the Promotion of Science, Tokyo, Japan
b
Institute for Agro-Environmental Sciences, NARO, Tsukuba, Ibaraki, Japan
c
National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
d
Technology Research Association for Single Wall Carbon Nanotubes, Tsukuba, Ibaraki, Japan

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Sphingomonas sp. strain N-9 utilizes

chlorinated OH-PCBs and PCBs hav-
ing 1e4 Cl.

Strain N-9 degrades OH-PCBs and
PCBs at imbalanced positions of
chlorobiphenyl rings.

Strain N-9 converts OH-PCBs into
chloro-hydroxybenzoic acids having
lower toxicity.

a r t i c l e i n f o a b s t r a c t

Article history: To examine the biodegradation of hydroxylated polychlorobiphenyls (OH-PCBs), we isolated Sphingo-
Received 11 April 2016 monas sp. strain N-9 from forest soil using mineral salt medium containing 4-hydroxy-3-chlorobiphenyl
Received in revised form (4OH-3CB) at the concentration of 10 mg/L. Following incubation with strain N-9, the concentration of
26 August 2016
4OH-3CB decreased in inverse proportion to strain N-9 proliferation, and it was converted to 3-chloro-4-
Accepted 26 August 2016
hydroxybenzoic acid (4OH-3CBA) after 1 day. We observed that strain N-9 efficiently degraded lowly
chlorinated OH-PCBs (1e4 Cl), while highly chlorinated OH-PCBs (5e6 Cl) were less efficiently trans-
Handling Editor: T Cutright formed. Additionally, strain N-9 degraded PCBs and OH-PCBs with similar efficiencies, and the efficiency
of OH-PCB degradation was dependent upon the positional relationships between OH-PCB hydroxyl
Keywords: groups and chlorinated rings. OH-PCB biodegradation may result in highly toxic products, therefore, we
Biodegradation evaluated the cytotoxicity of two OH-PCBs [4OH-3CB and 4-hydroxy-3,5-dichlorobiphenyl (4OH-3,5CB)]
Hydroxylated PCBs and their metabolites [4OH-3CBA and 3,5-chloro-4-hydroxybenzoic acid (4OH-3,5CBA)] using PC12 rat
Sphingomonas pheochromocytoma cells. Our results revealed that both OH-PCBs induced cell membrane damage and
Toxicity evaluation
caused neuron-like elongations in a dose-dependent manner, while similar results were not observed for
PC12 cells

Abbreviations: CBA, chlorobenzoic acids; 4-CBA, 4-chlorobenzoic acid; 3,4-CBA, 3,4-chlorobenzoic acid; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl
sulfoxide; ECD-GC, electron capture-detector gas chromatograph; HPLC, high-pressure liquid chromatography; LC-ESI()-MS-MS, liquid chromatography electrospray
ionization-negative mass spectrometry; KC300, Kanechlor 300; LDH, lactate dehydrogenase; MM, mineral salt medium; NGF, nerve growth factor; OH-PCBs, hydroxylated
polychlorobiphenyls; OH-CBAs, chloro-hydroxybenzoic acids; 4OH-2CB, 4-hydroxy-2-dichlorobiphenyl; 4OH-3CB, 4-hydroxy-3-chlorobiphenyl; 4OH-40 CB, 4-hydroxy-40 -
chlorobiphenyl; 4OH-5CB, 4-hydroxy-5-chlorobiphenyl; 4OH-3,5CB, 4-hydroxy-3,5-dichlorobiphenyl; 4OH-20 ,3,5CB, 4-hydroxy-20 ,3,50 -trichlorobiphenyl; 4OH-20 ,40 ,60 CB, 4-
hydroxy-20 ,40 ,60 -trichlorobiphenyl; 4OH-20 ,3,40 ,60 CB, 4-hydroxy-20 ,3,40 ,60 -tetrachlorobiphenyl; 4OH-20 ,3,40 ,5,60 CB, 4-hydroxy-20 ,3,40 ,5,60 -pentachlorobiphenyl; 4OH-
20 ,3,30 40 ,5,50 CB, 4-hydroxy-20 ,3,30 40 ,5,50 -hexachlorobiphenyl; 4OH-3CBA, 3-chloro-4-hydroxybenzoic acid; 4OH-3,5CBA, 3,5-chloro-4-hydroxybenzoic acid; PCBs, poly-
chlorinated biphenyls; PCB#8, 2,40 -dichlorobiphenyl; PCB#18, 2,20 ,5-trichlorobiphenyl; PCB#33, 2,30 ,40 -trichlorobiphenyl; THs, thyroid hormones; UPLC, ultra-performance
liquid chromatography; YE, yeast extract.
* Corresponding author. NIAES, 3-1-3 Kannondai, Tsukuba, Ibaraki, Japan. Tel./fax: þ81 29 838 8325.
E-mail address: ktakagi@affrc.go.jp (K. Takagi).

http://dx.doi.org/10.1016/j.chemosphere.2016.08.127
0045-6535/© 2016 Published by Elsevier Ltd.
174 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182
their metabolites. These results indicated that strain N-9 can convert OH-PCBs into chloro-
hydroxybenzoic acids having lower toxicity.
1. Introduction
PCBs have been recognized as universally distributed pollutants
resistant to degradation. Due to their high chemical and physical
stability, PCBs have been widely used for a variety of industrial
applications, including lubricants and plasticizers (Tehrani and
Aken, 2014). Although their production ended in the 1970s, PCBs
still persist in the environment and represent a serious environ-
mental problem (Tehrani and Aken, 2014).
In addition to PCBs, OH-PCBs were detected in rain, snow, and
surface water samples from different sites in Ontario, Canada, and
in sediment samples from Osaka Bay, Japan (Kawano et al., 2005;
Ueno et al., 2007). Total OH-PCB concentrations were detected at
ranges of 0.87e130 pg/L in water, and 230e990 pg/g in particulate
organic matter (Ueno et al., 2007). Specifically, higher OH-PCB
concentrations have been detected in surface waters from sites
near sewage treatment plants (Ueno et al., 2007). Chlorinated OH-
PCBs, which have 3e5 Cl groups, were identified in sediment
samples from Osaka Bay (Kawano et al., 2005).
Recently, it was shown that some PCB isomers are converted to
hydroxylated products through a variety of mechanisms, including
metabolic transformation in living organisms and abiotic reactions
with hydroxyl radicals (Anderson and Hites, 1996; Passatore et al.,
2014). In living organisms, OH-PCBs are generated from metabolic
PCB transformation in various life forms, including mammals,
plants, fungi, and bacteria (Passatore et al., 2014). Mammals
transform PCBs to OH-PCBs in the liver through the cytochrome
P450 enzymatic pathway, and some congeners seem to accumulate
preferentially in the blood prior to their excretion from the body
(Bergman et al., 1982; Quinete et al., 2014). Plants also transform
PCBs by following a sequence of reactions similar to those involved
in mammalian PCB metabolism (Aken et al., 2010). Plants uptake
PCBs and oxidize them to various hydroxylated products by using
cytochrome P450 monooxygenase and peroxidase (Zhai et al.,
2010). White-rot fungi, including Phanerochaete chrysosporium
and Paecilomyces lilacinus, were examined for their ability to
degrade PCBs and generate monohydroxylated intermediates from
4,40 -dichlorobiphenyl (Kamei et al., 2006; Sietmann et al., 2006).
PCBs are degraded by many types of aerobic bacteria, including
species of the genera Pseudomonas, Burkholderia, Achromobacter,
Comamonas, Ralstonia, Acinetobacter, Rhodococcus, Sphingomonas,
and Bacillus (Pieper, 2005), which degrade PCBs using a biphenyl-
related pathway to generate different metabolic intermediates,
such as dihydroxylated derivatives (Pieper, 2005). Alcaligenes sp.
strain Y42 and P6 generated both monohydroxyl and dihydroxyl
intermediates during the process of PCB degradation (Furukawa
et al., 1979). In abiotic reactions, PCBs undergo chemical reactions
involving OH radicals to produce OH-PCBs (Anderson and Hites,
1996). Anderson and Hites (1996) estimated the total global loss
rate of PCBs from the atmosphere due to removal by OH radicals
formed by photochemical reactions at 8300 t/year.
OH-PCBs may have higher toxicity than PCBs due to their being
highly soluble, with toxic effects resulting in inhibition of mito-
chondrial respiration, generation of reactive oxygen species, and
DNA damage (Dreiem et al., 2009; Ptak et al., 2010). OH-PCBs have
been shown to disrupt endocrine functions, such as THs and es-
trogens (Meerts et al., 2004; Ptak et al., 2006). While OH-PCBs may
© 2016 Published by Elsevier Ltd.
exhibit higher toxicity levels, regulations for OH-PCBs do not exist,
unlike those for other classes of environmental pollutants, such as
PCBs and dioxins (Tehrani and Aken, 2014).
Few reports have been published describing bacterial degrada-
tion of OH-PCBs. The anaerobic bacteria Desulfitobacterium dehalo-
genans dechlorinates 3,30 ,5,50 -tetrachloro-4,40 -dihydroxybihpenyl
(Wiegel et al., 1999), while the aerobic bacteria Burkholderia xen-
ovorans LB400 and Comamonas testosteroni (Pseudomonas testoster-
oni) B-356 are capable of OH-PCB degradation (Sondossi et al., 1991;
Tehrani et al., 2012, 2013). Strain B-356 degrades 2-, 3-, and 5-
chloro-4-hydroxybiphenyl via a biphenyl/chlorobiphenyl pathway
(Sondossi et al., 1991). Additionally, strain LB400 efficiently trans-
formed hydroxylated derivatives of 4-chlorobiphenyl and 2,5-
dichlorobiphenyl, while those of 2,4,6-trichlorobiphenyl were not
efficiently transformed (Tehrani et al., 2012, 2013). However, these
reports focused on bacteria that were originally isolated for PCB
degradation, and which were capable of degrading lowly chlori-
nated OH-PCBs with only 1e2 Cl groups.
Here, we isolated Sphingomonas sp. strain N-9 from forest soil
using mineral medium containing 4OH-3CB, and investigated its
capacity to degrade OH-PCBs. The biochemical problems associated
with microbial degradation of these compounds include the pos-
sibility that less toxic compounds may be converted into products
exhibiting increased toxicity. Given that there are few reports
evaluating the toxicities of OH-PCB metabolites, this work also
evaluated these products using PC12 rat pheochromocytoma cells.
2. Materials and methods
2.1. Chemicals
Nine types of 4OH-PCBs, three types of PCBs, and KC300 were
examined in this study. 4OH-3CB and 4OH-40 CB were purchased
from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Other OH-
PCBs (4OH-2CB, 4OH-3,5CB, 4OH-20 ,3,5CB, 4OH-20 ,40 ,60 CB, 4OH-
20 ,3,40 ,60 CB, 4OH-20 ,3,40 ,5,60 CB, and 4OH-20 ,3,30 40 ,5,50 CB) were
purchased from AccuStandard Chemicals (New Haven, CT, USA).
KC300 and PCBs (PCB#8, PCB#18, and PCB#33) were also pur-
chased from AccuStandard Chemicals. For the study of metabolism,
4OH-3CBA and 4OH-3,5CBA were purchased from Tokyo Chemical
Industry Co., Ltd. 4-CBA and 3,4-CBA were purchased from Sigma-
Aldrich Co., Ltd. (St. Louis, MO, USA). Stock solutions of the inves-
tigated chemicals were prepared as follows: OH-PCB and CBA were
prepared at 10 mg/mL in DMSO, each PCB was prepared at 5 mg/mL
in DMSO, and KC300 was prepared at 0.5 mg/mL in acetone. All
solutions were stored in glass vials at 20  C.
2.2. Growth media
The MM contained 1.2 g/L Na2HPO4$12H2O, 0.5 g/L KH2PO4, and
0.5 g/L NH4NO3. The medium was autoclaved and then supple-
mented with sterilized mixed vitamins (1 mL) and a trace element
solution (10 mL) in a total volume of 1 L (Takagi et al., 2009). All
chemicals were purchased from Wako Pure Chemical Industries
(Osaka, Japan). Strain N-9 was cultured in MM with 0.05% YE
(Becton Dickinson, Franklin Lakes, NJ, USA), and on agar medium
(MM with 0.05% YE and 1.5% agar).
 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182
2.3. Extraction and analysis of OH-PCBs, PCBs, and their
metabolites
For the analysis of OH-PCBs and the metabolites of OH-PCBs and
PCBs, samples (3 mL) were extracted from culture media with the
same volume (3 mL) of acetonitrile. Samples were vortexed for
1 min, and supernatants were used for analysis after being centri-
fuged at 15, 500  g for 1 min. Concentrations of these chemicals
were analyzed using a Quattro-Micro API mass spectrometer
(Waters, Milford, MA, USA) in an ACQUITY UPLC system (Waters)
under LC-ESI ()-MS-MS (UPLC-MS) (Letcher et al., 2005). Mas-
slynx NT software v4.0 (Waters) controlled the instrument, data
acquisition, and evaluation. An InertSustain Phenyl HP Column
[150 mm  2.1 mm (inner diameter); GL Science, Tokyo, Japan] was
used with a mobile phase comprised of 60% acetonitrile and 40%
acetic acid aqueous solution (0.2%). The injection volume was 10 ml.
To assess the full-scan mass spectra of these compounds, each
chemical (5 ng/L) was first individually infused at a flow rate of
10 mL/min into the ESI()-MS, and then scanned over a nominal
mass-to-charge ratio range of 65e500 amu. ESI()-MS parameters
were optimized for the maximum ion current of the [M-H] iso-
topic culture for each chemical. The mass spectrometer was used in
single-ion recording mode to monitor the (M-H) ions of interest
(m/z 203, 237, 273, 307, 341, 375, and 409). Other mass spectrom-
eter settings included the source temperature (120  C), desolvation
gas temperature (350  C), capillary voltage (3.5 kV), cone voltage
(30 V and 40 V), cone gas flow (50 L/h), and desolvation gas flow
(800 L/h).
For the analysis of PCBs, samples were extracted from culture
with the same volume (3 mL) of acetonitrile. Samples were vor-
texed for 1 min, and supernatants were used for analysis after being
centrifuged at 15,500  g for 1 min. PCBs were monitored by HPLC
(Series 1100, Hewlett-Packard, Palo Alto, CA, USA) equipped with
an ultraviolet detector set to detect wavelengths at 220 nm. A
Wakosil II 5C18 RS column [250 mm  4.6 mm (inner diameter);
Wako Pure Chemical Industries] was used in these analyses. The
injection volume was 50 ml. Mobile phases of acetonitrile in 0.1%
phosphoric acid aqueous solution (1.0 mL/min flow rate at 40  C)
were used in the analysis of PCB. PCB#8 used a mobile phase of 90%,
and PCB#18 and PCB#33 used a mobile phase of 95%.
2.4. KC300 extraction and analysis
Culture media samples (3 mL) were extracted with three vol-
umes of n-hexane (9 mL), and KC300 concentrations were analyzed
with an ECD-GC (Shimadzu, Kyoto, Japan). A capillary-type column
with a splitless injector (HP-50 þ with 50% PhMe silicone gum) and
an inside diameter of 0.53 mm, a length of 15 m, and film thickness
of 1 mm was used. The oven, injector port, and detector were
maintained at temperatures of 160  C, 240  C, and 280  C, respec-
tively. The injection volume was 2 ml. Peak KC300 assignments were
based on relative retention times published for all 209 PCB con-
geners (http://www.env.go.jp/recycle/poly/manual/sim_method-
io.pdf).
2.5. Enrichment and isolation of 4OH-3CB-degrading bacteria
Samples were taken from the lower 0e5 cm of forested soil
(uncontaminated by PCBs or OH-PCBs) in Tsukuba (Ibaraki, Japan).
Enrichment of 4OH-3CB-degrading bacteria was performed using
the original soil-charcoal perfusion method (Takagi et al., 2009),
and the enrichment culture was carried out under dark conditions
at 25  C by circulating 250 mL MM containing 10 mg/L 4OH-3CB.
The culture medium supernatant was sampled every two or three
days, and extracted with the same volume (1 mL) of acetonitrile.
175
The concentration of 4OH-3CB in each sample was determined by
UPLC-MS. The medium was replaced when 4OH-3CB was degraded
completely in the culture medium. After a 2-month incubation, the
enriched charcoal and soil (~1 g) were crushed and suspended in
50 mM phosphate buffer (pH 7.0), and the same buffer was added to
the suspension to derive a 107-fold dilution. The diluted suspen-
sion was then inoculated on agar media containing 50 mg/L 4OH-
3CB. After successive incubation at 25  C for 3 days, colonies were
isolated into 17-mL sterile glass tubes containing 7 mL MM sup-
plemented with 0.05% YE and 10 mg/L 4OH-3CB. The tube cultures
were shaken at 180 rpm and 30  C for 7 days, and tube cultures
successfully degrading the 4OH-3CB were identified as 4OH-3CB-
degrading bacteria.
2.6. Identification of strain N-9
Morphologic and physiologic characterizations of cells from
strain N-9 were outsourced to Techno Suruga Laboratory (Shizuoka,
Japan). Tests of carbon-source utilization were performed using
GN2 MicroPlates (BioLOG, Hayward, CA, USA) with incubation
times of 7 days. The 16S ribosomal RNA sequencing was performed
according to Kuramata et al. (2015). The 16S rRNA sequences ob-
tained were compared with other bacterial sequences in the NCBI
database (http://www.ncbi.nlm.nih.gov/). A phylogenetic tree was
constructed using ClustalW through the web server of the DNA
Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp/top-j.html), and
depicted using the Dendroscope program (http://www-ab.
informatik.uni-tuebingen.de/software/dendroscope/welcome.
html).
2.7. Experimental designs
Cells were preincubated for 2 days in MM with YE 0.05% and
4OH-3CB (10 mg/L) with shaking (180 rpm) at 30  C. Cells were
harvested by centrifugation at 6000  g at 25  C for 10 min. Su-
pernatant was removed and resuspended in MM to an OD600 of 2.0.
In all experiments, cells (starting concentration: OD600 ¼ 0.02)
were incubated in 3 mL MM containing 3 ml of the stock solution of
each chemical in a 10-mL sealed test tube at 30  C with agitation by
shaking at 180 rpm for 7 days. For tests of 4OH-3CB effects, cells
were cultured in MM with 0.05% YE and 4OH-3CB (0e30 mg/L). For
tests of YE effects, cells were cultured in MM with 4OH-3CB
(10 mg/L) and YE (0%e0.5%). For biodegradation tests, cells were
cultured in MM with 0.05% YE and supplemented with each
chemical (final concentration: 10 mg/L OH-PCB, 5 mg/L PCB, or
0.5 mg/L KC300). As a control, cells were deactivated by auto-
claving at 121  C for 20 min and then added to each medium (final
concentration: OD600 ¼ 0.3). They were cultured under the same
conditions as described above. Growth was monitored by OD600
measurements using a V-630 spectrophotometer (JASCO, Tokyo,
Japan). Concentrations of OH-PCB and its metabolites were
determined by UPLC-MS, and concentrations of PCBs and KC300
were determined by HPLC and ECD-GC, respectively. All experi-
ments were conducted in triplicate.
2.8. Culture and observation of PC12 cells
PC12 cells were purchased from the RIKEN BioResource Center
(Ibaraki, Japan). PC12 cells were cultured in DMEM (Gibco, Thermo
Fisher Scientific, Billerica, MA, USA) supplemented with 10% heat-
inactivated fetal bovine serum (Gibco), 10% human serum (Gibco),
100 U/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher
Scientific). The DMEM cultures were placed in a 75-cm2 flask
(Corning; Corning, NY, USA) and incubated at 37  C in a 5% CO2
atmosphere.
176 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182

To determine the effects of OH-PCBs and their metabolites on strain B1. This degree of 16S rRNA relatedness suggested that strain
neurite outgrowth, PC12 cells were observed using an optical mi- N-9 represented a novel Sphingomonas species.
croscope (Carl Zeiss, Baden-Württemberg, Oberkochen, Germany)
after a 2- or 3-day cultivation. 3.3. Transformation of 4OH-3CB by strain N-9

2.9. Cytotoxicity evaluation Time-dependent 4OH-3CB transformation by strain N-9 was

To examine the cytotoxicity of OH-PCBs (4OH-3CB and 4OH-
3,5CB) and their metabolites (4OH-3CBA and 4OH-3,5CBA), cell
membrane damage was detected by LDH assay. Cells (1  105 cells/
well) were seeded in 96-well plates in DMEM for 1 day, and DMEM
was replaced with NGF (50 ng/L) and exposed to each chemical
(10 mg/L) for 2 or 3 days. The culture supernatant was collected by
centrifugation at 510g for 10 min at 25  C and used for the LDH
assay. Cells exposed to only 1% (v/v) DMSO in NGF were used as
controls. Analysis of LDH was measured with tetrazolium salt using
a Cytotoxicity Detection KitPLUS (Roche Diagnostics GmbH, Man-
nheim, Germany) according to the manufacturer's protocol. The
amount of formazan salt formed was measured at 490 nm using a
SpectraMax 190 absorbance microplate reader (Molecular Devices,
LLC, Sunnyvale, CA, USA) (Decker and Lohmann-Matthes, 1988). All
experiments were conducted in triplicate.
3. Results
3.1. Enrichment and isolation of 4OH-3CB-degrading bacteria
At the beginning of enrichment, the degradation of 4OH-3CB
(10 mg/L) in the perfusion apparatus was observed after 5 days of
circulation. After 2 months of circulation and six exchanges of the
medium, 4OH-3CB was completely degraded within 3 days, and the
enriched charcoal and soil were harvested in order to carry out
subsequent colony isolation. Several types of colonies exhibiting
examined in MM with 0.05% YE (Fig. 1). Growth of strain N-9
increased over time, and peaked after a 20-h culture (OD600 ¼ 0.2).
The concentration of 4OH-3CB (9.7 mg/L) decreased in inverse
proportion to the growth. A novel peak was generated by UPLC-MS
detection of strain N-9 transformation of 4OH-3CB (Fig. 2A and B).
The mass spectra indicated molecular-ion peaks at m/z 171 and 127
(Fig. 2D), and comparison of the mass spectra allowed identifica-
tion of the intermediates as 4OH-3CBA, based on the UPLC-MS
retention time and fragmentation patterns matching those of
4OH-3CBA standards. The 4OH-3CBA (9.2 mg/L) intermediates
were measured at concentrations suggesting almost quantitative
conversion of 4OH-3CB (Fig. 1). Strain N-9 did not decrease levels of
this metabolite after 21 days of incubation (data not shown).
3.4. Effects of 4OH-3CB on strain N-9
The potential inhibitory ability of 4OH-3CB on strain N-9 was
examined by growing cells in the presence of increasing concen-
trations of the compound and monitoring the OD600 (Fig. 3A).
Exposure to 20 mg/L 4OH-3CB did not result in significant inhibi-
tion of growth, and the 4OH-3CB was completely degraded. How-
ever, 4OH-3CB at concentrations >30 mg/L inhibited strain N-9
growth, with no significant transformation of 4OH-3CB.
0.25
A
Growth (OD600nm)

different morphologies were observed on agar plates containing 0.2

4OH-3CB, and 4OH-3CB-degrading abilities of individual isolates
were examined in tube cultures. Three of the 20 isolates were able 0.15
to transform 4OH-3CB; however, the 4OH-3CB transformation ef-
ficiency was low for all isolates except one strain, which was
0.1
designated as strain N-9 and submitted to the National Institute of
Agrobiological Sciences Genebank (https://www.gene.affrc.go.jp/
about_en.php; MAFF 311684). 0.05

3.2. Characteristics of strain N-9 0

0 5 10 15 20 25
Detailed physiological properties are shown in Table S1. Strain 10
N-9 consisted of rod-shaped (0.7e0.8  0.9e1.0 mm), gram-
negative, motile bacteria (Fig. S1A), with circular colonies that
measured 1 mm after a 1-day incubation, and were yellow in color 8 B
Concentration (mg/L)

on agar medium in the presence of 4OH-3Cl (Fig. S1B). The agar

plate on which strain N-9 was grown in the presence of 4OH-3Cl
resembled an iridescent oil slick (Fig. S1C). The temperature range 6
for growth was 20e37  C, and many substrates, including -glucose
and -galactose, were assimilated.
The 16S ribosomal RNA sequence of strain N-9 (1481 nt; Gen-
4
Bank accession no. LC101917) was compared with other bacterial 4OH-3CB
nucleotide sequences. Strain N-9 exhibited high sequence similar-
ity with Sphingomonas spp., with the highest similarity (99%) 4OH-3CBA
observed with Sphingomonas sp. strain PF-K (DQ202286). Notably, 2
Sphingomonas sp. strain PF-K, which was isolated from caverns in
Arizona, does not have any reported OH-PCB- or PCB-degradation
ability (Ikner et al., 2007). We constructed a phylogenetic dendro- 0
gram using the 16S rRNA sequence associated with strain N-9 and 0 5 10 15 20 25
those of representative strains of Sphingomonas exhibiting PCB- Incubation time (h)
degradation capacities (Fig. S2). The 16S rRNA of strain N-9
exhibited 98.0% sequence identity to Sphingomonas yanoikuyae Fig. 1. (A) Growth and (B) mass balance of 4OH-3CB by Sphingomonas sp. strain N-9.
 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182 177

1003 N1 phenyl 802015:27:23

141106_008 2: Scan ES-
TIC
1003 N phenyl 6040
100 100
5.88e6

100
C
150629_022 154 (4.048)
203
A HO 3: Scan ES-
1.13e6

4OH-3CB

(%)
50

%
(%)

%
205 0 -0
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
Time

1 2 3 4
206
201
Time (min)
102 117 131 133 149 163 209 235 237 238 258 265 280 450
180 188 293 302 306 323 344 346 348 367 376 388 405 407 421 445 467 488 489
0 m/z
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Acquired range (m/z)

1003 1 phenyl 802015:21:15

D 141106_007
100
2: Scan ES-
TIC
Cl 5.45e6
1003 phenyl 5050
150307_013 105 (2.504) Cm (101:121) B HO
OH
3: Scan ES-
100
127 4.42e5
O

%
171
%

-0 Time
129 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
173

106 117 131 174

241 261 315
136 147 165 195 199 201 217 233 255 279 291 294 310 335 337 353 365 368 391 395 411 413 431 440 449 459 473 489 491
0 m/z
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Fig. 2. Identification of the 4OH-3CB metabolite by Sphingomonas sp. strain N-9. (A) UPLC chromatograph of the control reaction. (B) UPLC chromatograph of the product after a 1-
day incubation. (C) Mass spectrum of the 4OH-3CB peak at 2.7 min (arrow). (D) Mass spectrum of the metabolite (4OH-3CBA) peak at 2.1 min (arrow).

3.5. Effects of YE on strain N-9

We evaluated the effects of YE on strain N-9 with respect to 0.3

4OH-3CB degradation and strain N-9 proliferation (Fig. 3B). In the (A)
absence of YE, almost no 4OH-3CB transformation or strain N-9 100% 100%
proliferation was observed. In the presence of 0.05% YE, 100% of the
Growth (OD600)

0.2
10 mg/L 4OH-3CB was transformed, and the OD600 of strain N-9 was
0.25 ± 0.05. In the presence of 0.5% YE, only 17.30 ± 1.34% 4OH-3CB
was transformed over the duration of the experiment, while
growth measured by OD600 increased to 1.03 ± 0.1. 0.1

3.6. Transformation of OH-PCBs by strain N-9 0%

0
The capability of strain N-9 to metabolize OH-PCBs was tested 0 10 20 30
by growth in MM supplemented with 0.05% YE and incubation with
10 mg/L of each target compound for 7 days (Fig. 4). Among mono- 4OH-3CB concentration (mg/L)
OH-PCBs, 100% of 4OH-3CB was transformed, while 32.8 ± 0.2% and
23.0 ± 1.5% of 4OH-2CB and 4OH-40 CB were transformed, respec-
tively. Strain N-9 was capable of transforming lowly chlorinated 120 1.2
(B) Degradation Growth
OH-PCBs (1e4 Cl). Remarkably, 4OH-3CB, 4OH-3,5CB, and 4OH-
4OH-3CB degradation (%)

20 ,3,40 ,60 CB, which have chlorobiphenyl rings substituted at posi- 100 1.0
Growth (OD600)

tion 3, were apparently transformed more easily by strain N-9, with

100% transformation rates observed for 4OH-3CB and 4OH-3,5CB, 80 0.8
and 46.2 ± 1.3% for 4OH-20 ,3,40 ,60 CB. However, more highly chlo-
rinated OH-PCBs (5e6 Cl) resulted in lower transformation per- 60 0.6
centages, specifically 11.0± 3.1% for 4OH-20 ,3,40 ,5,60 CB and 0% for
40 0.4
4OH-20 ,3,30 40 ,5,50 CB.
Three metabolites from nine OH-PCBs, including 4OH-3CB, were
20 0.2
detected by UPLC-MS. The mass spectra revealed molecular-ion
peaks at m/z 205 following strain N-9 incubation with 4OH-3,5CB
0 0
(Fig. S3AeD), indicating an intermediate of 4OH-3,5CBA. Molecular-
0 0.05 0.5
ion peaks at m/z 321 following strain N-9 incubation with 4OH-
20 ,3,40 ,60 CB (Fig. S4AeD) revealed an intermediate compound hav- YE concentration (%)
ing a diol structure (4OH-20 ,3,40 ,60 chlorobiphenyldiol), though this Fig. 3. Physiological characteristics of Sphingomonas sp. strain N-9. (A) Effects of 4OH-
was not identified. No other metabolites were detected from the 3CB on Sphingomonas sp. strain N-9. The numbers (%) on the symbols (C) show the
other OH-PCBs. rates of 4OH-3CB degradation. (B) Effects of YE on Sphingomonas sp. strain N-9.
178 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182

HO HO

100

OH-PCBs degradation (%)

80

60 HO

HO HO
40 HO
HO
HO

20
HO

Fig. 4. Transformation of OH-PCBs by Sphingomonas sp. strain N-9.

3.7. Transformation of PCBs by strain N-9
Strain N-9 was assayed for its ability to degrade KC300 PCB
mixtures. (Fig. S5). The components of the peaks resolved by ECD-
GC, and the degradation associated with these components, were
calculated for each peak (Table 1). Ten of the 42 KC300 congeners
were degraded, which included most of the di-, tri-, and tetra-
chlorobiphenyl peaks from the KC300 compounds. The capabil-
ities of strain N-9 to metabolize the standards associated with
PCB#8, PCB#18, and PCB#33 were tested and the degradation rates
and their metabolites determined (Table 2). PCB#8 and PCB#33
were 61.6 ± 4.0% and 100% transformed, respectively, while lower
transformation levels were recorded for PCB#18 (14.1 ± 10.2%).
Novel molecular-ion peaks at m/z 155 and 189 were detected by
UPLC-MS following strain N-9 incubation with PCB#8 and PCB#33,
respectively (Figs. S6 and S7, Table 2), indicating intermediates
identified as 4-CBA and 3,4-CBA, and measured at concentrations
suggesting almost quantitative conversion of PCB#8 and PCB#33,
respectively (Table 2).
3.8. Cytotoxicity evaluation of OH-PCBs and their metabolites using
PC12 cells
To examine the cytotoxicity of OH-PCBs (4OH-3CB and 4OH-
3,5CB) and their metabolites (4OH-3CBA and 4OH-3,5CBA) trans-
formed by strain N-9, the effects of each chemical on PC12 cells
incubated in NGF were examined by LDH assay and observed by
microscopy (Figs. 5 and 6). Compared to the control, LDH activity in
the PC12 culture increased in proportion to the concentration of
4OH-3CB after a 2-day instillation, and 33.1 ± 5.5% cytotoxicity was
detected at 100 mg/L 4OH-3CB (Fig. 5A). Induction of neurite
outgrowth was observed at 50 mg/L 4OH-3CB (Fig. 6A). LDH activity
and induction of neurite outgrowth in PC12 cells treated with 4OH-
3CBA were not significantly different from those observed in con-
trol cells after a 2-day instillation (Figs. 5C and 6B).
Following exposure to 4OH-3,5CB, LDH activity increased after a
3-day instillation, and neurite outgrowth began after a 2-day
instillation (Figs. 5B and 6D). We detected 49.3 ± 4.7% cytotox-
icity at 75 mg/L of 4OH-3,5CB following a 3-day instillation

Table 1
Analysis of KC300 degradation by Sphingomonas sp. strain N-9.

Peak No. Retention time (mn) Congener identification Degradation (%)*

2 10.6 2,4-dichlorobiphenyl (PCB#7), 100.0

2,5-dichlorobiphenyl (PCB#9)
3 11.3 2,30 -dichlorobiphenyl (PCB#6) 100.0
4 11.6 2,3-dichlorobiphenyl (PCB#5), 91.5 ± 1.5
2,40 -dichlorobiphenyl (PCB#8)
6 13.7 2,20 ,5-trichlorobiphenyl (PCB#18), 41.7 ± 0.6
4,40 -dichlorobiphenyl (PCB#15),
2,20 ,4-trichlorobiphenyl (PCB#17)
9 15.0 2,30 ,5-trichlorobiphenyl (PCB#26) 58.5 ± 4.0
10 15.2 2,30 ,4-trichlorobiphenyl (PCB#25) 23.3 ± 4.3
11 15.6 2,4,40 -trichlorobiphenyl (PCB#28), 11.4 ± 1.5
2,40 ,5-trichlorobiphenyl (PCB#31)
12 16.5 2,3,30 -trichlorobiphenyl (PCB#20), 91.7 ± 0.3
2,30 ,40 -trichlorobiphenyl (PCB#33), 2,20 ,5,60 -tetrachlorobiphenyl (PCB#53)
13 17.2 2,20 ,3,6-tetrachlorobiphenyl (PCB#45) 83.8 ± 2.5
16 18.0 2,20 ,4,40 -tetrachlorobiphenyl (PCB#47), 100.0
2,20 ,4,5-tetrachlorobiphenyl (PCB#48)

*Percent degradation was determined by direct comparison with a control sample. Degradation.
<10% was not considered significant and was not reported.
 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182 179

Table 2
Analysis of PCB degradation by Sphingomonas sp. strain N-9.

PCB Degradation, mg/L (%) Metabolite (mg/L) (min) (m/z) Metabolite

3.15 ± 0.11 3.2 ± 0.05 3.6 156.6

(61.6± 4.0%)

1.4 ND ND ND ND*
(14.1± 10.2%)

7.06 ± 0.18 6.6 ± 0.6 4.1 191

(100%)

*ND, not detected

(Fig. 5B). LDH activity and neurite outgrowth in PC12 cells treated
with 4OH-3,5CBA were not significantly different from that
observed in control cells after a 3-day instillation (Figs. 5D and 6E).
4. Discussion
In this study, OH-PCBs and their transformation metabolites
were analyzed using UPLC-MS-based methods. Methods for the
detection of OH-PCBs using HPLC-MS were recently reported by a
study that identified OH-PCBs in polar plants (Letcher et al., 2005).
It is generally necessary to derivatize OH-PCBs and their trans-
formation metabolites using diazomethane, acetyl, trifluoroacetyl,
or pentafluoropropinyl analogues before detection by gas chro-
matography (Letcher et al., 2005); however, these methods are
complicated and time consuming. Given that UPLC-MS-based
methods are capable of detecting OH-PCBs and their trans-
formation metabolites under the same analytical conditions, we
used this method for the experiments presented here.
We successfully isolated Sphingomonas sp. strain N-9 from forest
soil and subsequently confirmed its ability to degrade OH-PCBs.
Only two strains isolated from PCB-contaminated soil were re-
ported as OH-PCB-degrading bacteria (Sondossi et al., 1991; Tehrani
et al., 2012, 2013). Our results suggested that OH-PCB-degrading
bacteria inhabit native soil not contaminated by PCBs or OH-PCBs.

40 40
A 4OH-3CB C 4OH3CBA
30 30
Cytotoxicity (%)

Cytotoxicity (%)

20 20

10 10

0 0
0 25 50 75 100 0 25 50 75 100
-10 Concentration (mg/L) -10 Concentration (mg/L)

60 60
B 4OH-3,5CB D 4OH-3,5CBA
50 50
2 days
Cytotoxicity (%)

Cytotoxicity (%)

40 40
3 days
30 30

20 20

10 10

0 0
0 25 50 75 100 0 25 50 75 100
Concentration (mg/L) Concentration (mg/L)

Fig. 5. Cytotoxicity of the OH-PCBs (4OH-3CB and 4OH-3,5CB) and their metabolites (4OH-3CBA and 4OH-3,5CBA).
180 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182

Fig. 6. Effects of OH-PCBs (4OH-3CB and 4OH-3,5CB) and their metabolites (4OH-3CBA and 4OH-3,5CBA) on in vitro NGF-dependent neurite outgrowth in PC12 cells. These
photographs show representative results from triplicate experiments.

Strain N-9, which was originally isolated by growth in the presence
of 4OH-3CB, was capable of transforming wide range of OH-PCB
congeners. Strain N-9 successfully degraded OH-PCBs (1e4 Cl)
under our experimental conditions, while highly chlorinated OH-
PCBs (5e6 Cl) were not transformed. P. testosteroni B-356 was re-
ported to degrade monohydroxylated PCBs, including 4OH-2CB,
4OH-3CB, and 4OH-5CB (Sondossi et al., 1991). B. xenovorans LB400
was also capable of transforming hydroxylated derivatives of 4-
chlorobiphenyl and 2,5-dichlorobiphenyl, while it was less
capable of transforming those of 2,4,6-trichlorobiphenyl (Tehrani
and Aken, 2014; Tehran et al., 2012). Our results suggested that
the transformation activities of strain N-9 were superior or equiv-
alent to those of previously reported OH-PCB-degrading bacteria,
though the experimental conditions differed.
Among mono-OH-PCBs, 4OH-3CB was more completely trans-
formed as compared to 4OH-2CB and 4OH-40 CB. Tehrani et al.
(2012) reported that 20 -hydroxy-4-chlorobiphenyl was more
completely degraded as compared to 30 -hydroxy- or 40 -hydroxy-4-
chlorobiphenyl by B. xenovorans LB400 growing on biphenyl
(Tehrani et al., 2012). These results may reflect that the efficiencies
associated with OH-PCB degradation depend upon positional re-
lationships between hydroxyl groups and chlorinates. In 12 types of
OH-PCB and PCB isomers, four isomers were metabolized to OH-
CBAs and CBAs as final products. Specifically, 4OH-3CB, 4OH-
3,5CB, and PCB#33 (20 ,3,4-CB) were apparently oxidized more
easily by strain N-9. These results suggested that strain N-9
degraded OH-PCBs and PCBs based on imbalanced positions of
chlorobiphenyl rings, indicating that preferential ring-based mo-
lecular fission may have occurred with non-chlorinated or lesser-
chlorinated rings. Our results are similar to those observed by
Furukawa et al. (1979) involving transformation of PCB derivatives
by Alcaligenes sp. strain Y42 and Acinetobacter sp. strain P6.
 S. Mizukami-Murata et al. / Chemosphere 165 (2016) 173e182
Strain N-9 completely degraded 20 mg/L 4OH-3CB.
B. xenovorans strain LB400 was inhibited by three hydroxylated
derivatives of 4-chlorobiphenyl at concentrations <10 mg/L when
cells were growing on succinate, and <2 mg/L when cells were
growing on biphenyl (Tehrani et al., 2012). Strain N-9 was isolated
as OH-PCB-degrading bacteria and enriched using 4OH-3CB, while
B. xenovorans strain LB400 was isolated as a PCB-degrading bacteria
using biphenyl. OH-PCBs may be more toxic than PCBs, as they have
been found to exhibit toxic effects not shown in PCBs (Tehrani and
Aken, 2014). Monohydroxy biphenyls and dihydroxy biphenyls
exhibit more toxic effects on bacteria, including Escherichia coli and
C. testosteroni strain B-356 relative to their parent PCBs (Sondossi
et al., 1991; Ca mara et al., 2004). These enrichment conditions
may reflect OH-PCB tolerance.
Compared to the culture in MM supplemented with 0.05% YE, a
reduction in 4OH-3CB degradation and an increase in cell growth
were observed in the presence of 0.5% YE. Similar phenomena were
observed in the degradation of other OH-PCBs (4OH-3,5CB and
4OH-20 ,3,40 ,60 CB) and PCBs (PCB#8 and PCB#33; data not shown). It
was reported that growth of B. xenovorans LB400 exposed to
monohydroxy biphenyl in K1 medium with succinate (1180 mg/L)
was superior to that observed with biphenyl (770 mg/L) (Tehrani
et al., 2012). However, the degradation rates of these OH-PCBs in
the presence of succinate were lower relative to those in the
presence of biphenyl, because the metabolism occurred through a
biphenyl-related pathway (Tehrani et al., 2012). The key genes
associated with the biphenyl pathway related to PCB degradation
include bph genes (bphA and bphD), which were detected in strain
N-9 by polymerase chain reaction (data not shown). Our results
may suggest that strain N-9 degraded OH-PCBs in a co-metabolic
system associated with a biphenyl-related pathway.
Our ultimate goal was bioremediation of OH-PCBs using strain
N-9, however, OH-PCBs may be converted into products with
increased toxicity using this strain. Therefore, we also evaluated the
toxicity of OH-PCB metabolites. Given that the two OH-PCBs (4OH-
3CB and 4OH-3,5CB) were completely degraded by strain N-9, we
examined these OH-PCBs and their metabolites (4OH-3CBA and
4OH-3,5CBA). There is less information concerning OH-PCB toxicity
as compared to PCBs. Among OH-PCBs and their metabolites (OH-
CBAs) used in our experiments, information on the median lethal
dose (LD50) has been reported for only one (4OH-3,5CBA; ipr-mus:
650 mg/kg, http://www.tcichemicals.com/eshop/ja/jp/catalog/
msds/). OH-PCBs affect endocrine function, especially as THs
(triiodothyronine and thyroxine), because the chemical structures
of OH-PCB molecules are similar to those of THs. THs also affect
brain and neuron development, with some OH-PCBs affecting
neuronal development of cerebellar Purkinje cells (Kuroda et al.,
2005, 2007). Exposure to OH-PCBs causes a decrease in thyroxine
levels in the brain of rats (Meerts et al., 2004). Additionally, OH-
PCBs interfere with c-Jun expression, which is essential for suc-
cessful peripheral nerve regeneration in PC12 cells (Shimokawa
et al., 2006). Thus, we evaluated the toxicity of OH-PCBs and their
metabolites using PC12 cells, which are used as a model of nerve-
cell differentiation (Ren et al., 2014).
In PC12 cells, administration of 4OH-3CB and 4OH-3,5CB caused
a dose-dependent increase in cytotoxicity (LDH release) and
observed neuron elongation. However, 4OH-3CBA and 4OH-3,5CBA
did not induce cell membrane damage or neuron elongation in
PC12 cells, indicating that the toxicity of 4OH-3CB and 4OH-3,5CB
can be lowered by strain N-9 transformation to 4OH-3CBA and
4OH-3,5CBA, respectively. Induction of LDH release was observed at
100 mg/L 4OH-3CB, though the neuronal elongation in PC12 cells
was observed at 50 mg/L following a 2-day incubation. Induction of
LDH release in PC12 cells was observed at 75 mg/L 4OH-3,5CB
following a 3-day incubation, though neuronal elongation was
181
observed after a 2-day incubation. These results suggested that
toxic mechanisms exerted on PC12 cells may differ between 4OH-
3CB and 4OH-3,5CB. Further studies may provide additional in-
sights into the mechanisms associated with OH-PCB effects on
PC12 cells.
5. Conclusions
Our results suggested that cytotoxicity evaluations of OH-PCBs
and their metabolites are required to perform bioremediation,
and not only in the case of Sphingomonas sp. strain N-9. Here, we
demonstrated that strain N-9 plays an important role in biodeg-
radation of OH-PCBs in liquid culture. Strain N-9 was able to
transform lowly chlorinated OH-PCBs (1e4 Cl), and strain N-9
degradation efficiencies associated with OH-PCBs were dependent
upon positional relationships between hydroxyl groups and chlo-
rinates on OH-PCB ring structures. Furthermore, we observed that
strain N-9 was capable of lowering OH-PCBs toxicity by their
transformation to OH-CBAs. Our results supported that bioreme-
diation methods may be established through cytotoxicity
evaluation.
Acknowledgments
This work was supported by a grant from the Research Fellow of
Japan Society for the Promotion of Science (JSPS KAKENHI Grant
Number 2640107).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2016.08.127.
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