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Isolation and characterization of 4-tert-butylphenol-utilizing Sphingobium fuliginis
strains from Phragmites australis rhizosphere sediment
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Running title: Degradation of 4-tert-butylphenol by S. fuliginis
TADASHI TOYAMA1*
, NAONORI MOMOTANI2, YUKA OGATA
2, YUJI MIYAMORI
3,
DAISUKE INOUE2, KAZUNARI SEI
2, KAZUHIRO MORI
1, SHINTARO KIKUCHI
3, and
MICHIHIKO IKE2
1Department of Research, Interdisciplinary Graduate School of Medicine and Engineering,
University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan
2Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1
Yamadaoka, Suita, Osaka 565-0871, Japan
3Division of Applied Sciences, Muroran Institute of Technology, 27-1 Mizumoto, Muroran
050-8585, Japan
*Corresponding author
Mailing address: Department of Research, Interdisciplinary Graduate School of Medicine and
Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan
Phone: +81-55-220-8346
Fax: +81-55-220-8346
E-mail: [email protected]
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Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00258-10 AEM Accepts, published online ahead of print on 27 August 2010
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ABSTRACT 24
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We isolated three Sphingobium fuliginis strains from Phragmites australis rhizosphere
sediment that were capable of utilizing 4-tert-butylphenol as a sole carbon and energy source.
These strains are the first 4-tert-butylphenol-utilizing bacteria. The strain designated TIK-1
completely degraded 1.0 mM 4-tert-butylphenol in basal salts medium within 12 h, with
concomitant cell growth. We identified 4-tert-butylcatechol and 3,3-dimethyl-2-butanone as
internal metabolites by gas chromatography – mass spectrometry. When 3-fluorocatechol was
used as an inactivator of meta-cleavage enzymes, strain TIK-1 could not degrade
4-tert-butylcatechol and 3,3-dimethyl-2-butanone was not detected. We concluded that
metabolism of 4-tert-butylphenol by strain TIK-1 is initiated by hydroxylation to
4-tert-butylcatechol, followed by a meta-cleavage pathway. Growth experiments with 20
other alkylphenols showed that 4-isopropylphenol, 4-sec-butylphenol, and 4-tert-pentylphenol,
which have alkyl side chains of three to five carbon atoms with g-quaternary or g-tertiary
carbons, supported cell growth, whereas 4-n-alkylphenols, 4-tert-octylphenol, technical
nonylphenol, 2-alkylphenols, and 3-alkylphenols did not. The growth rate on
4-tert-butylphenol was much higher than on the other alkylphenols. Degradation experiments
with various alkylphenols showed that strain TIK-1 cells grown on 4-tert-butylphenol could
degrade 4-alkylphenols with variously sized and branched side chains (ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl,
tert-octyl, n-nonyl, and branched nonyl) via a meta-cleavage pathway, but not 2- or
3-alkylphenols. Along with the degradation of these alkylphenols, we detected methyl alkyl
ketones that retained the structure of the original alkyl side chains. Strain TIK-1 may be useful
in the bioremediation of environments polluted by 4-tert-butylphenol and various other
4-alkylphenols.
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INTRODUCTION
4-tert-Butylphenol is an alkylphenol with a tertiary branched side chain of four carbon
atoms at the para position of phenol. It is an industrially important chemical and is
abundantly and widely used for the production of phenolic, polycarbonate, and epoxy resins.
Production of 4-tert-butylphenol in the European Union in 2001 was 25,251 tonnes (t) (9). In
Japan, according to the National Institute of Technology and Evaluation
(http://www.safe.nite.go.jp/english/sougou/view/ComprehensiveInfoDisplay_en.faces),
production of 4-tert-butylphenol amounted to 27,761 t in 2007. 4-tert-Butylphenol is widely
distributed in aquatic environments, including river waters (20), seawaters (17), river
sediments (17), marine sediments (23), and effluent samples from sewage treatment plants
and wastewater treatment plants (22). Furthermore, 4-tert-butylphenol interacts with estrogen
receptors (29, 30, 34, 35, 39) and exhibits other toxic effects on aquatic organisms and
humans (4, 15, 16, 25, 26, 42, 43). Therefore, it is necessary to study the biodegradation of
4-tert-butylphenol to understand its fate in the aquatic environment, to establish technologies
to treat the waters polluted by it, and to remove it from contaminated environments.
Studies of the biodegradation of alkylphenols have focused mainly on branched
4-nonylphenol. Several strains of sphingomonad bacteria, including Sphingomonas sp.
TTNP3 (38), Sphingobium xenophagum Bayram (11), and Sphingomonas cloacae S-3T (10),
have recently been isolated from activated sludge. These strains can degrade branched
4-nonylphenol and utilize it as a sole carbon source. In addition, several Pseudomonas strains
that can degrade medium-chain 4-n-alkylphenols (e.g. 4-n-butylphenol) and utilize them as
sole carbon sources have been isolated from activated sludge or contaminated soil; they
include Pseudomonas veronii INA06 (1), Pseudomonas sp. KL28 (21), and Pseudomonas
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putida MT4 (36). Biodegradation of branched 4-nonylphenol and 4-n-butylphenol has been
well studied, but little is known about the biodegradation of 4-tert-butylphenol, although this
compound has a similar structure to those of branched 4-nonylphenol and 4-n-butylphenol.
There is only one report on the biotransformation of 4-tert-butylphenol—by resting cells of S.
xenophagum strain Bayram grown on technical nonylphenol—but this strain cannot grow on
4-tert-butylphenol (11, 14). To our knowledge, there are no reports of bacteria that utilize
4-tert-butylphenol as the sole carbon source, and the biochemical pathway of
4-tert-butylphenol utilization has not been described.
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Here we characterize three Sphingobium fuliginis strains—TIK-1, TIK-2, and
TIK-3—isolated from rhizosphere sediment of the reed Phragmites australis. These strains
could use 4-tert-butylphenol as a sole carbon source. On the basis of additional tests of strain
TIK-1, we propose that it degrades 4-tert-butylphenol through 4-tert-butylcatechol along a
meta-cleavage pathway. We also show that strain TIK-1 cells grown on 4-tert-butylphenol can
degrade a wide range of 4-alkylphenols via a meta-cleavage pathway.
MATERIALS AND METHODS
Chemicals. Alkylphenols (4-ethylphenol, 2-n-propylphenol, 2-isopropylphenol,
3-isopropylphenol, 4-n-propylphenol, 4-isopropylphenol, 2-sec-butylphenol,
2-tert-butylphenol, 3-tert-butylphenol, 4-n-butylphenol, 4-sec-butylphenol, 4-tert-butylphenol,
4-n-pentylphenol, 4-tert-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-n-octylphenol,
4-tert-octylphenol [4-(1,1,3,3-tetramethylbutyl)phenol], 4-n-nonylphenol, technical
nonylphenol, and 2,4-di-tert-butylphenol); 4-tert-butylcatechol; 3,5-di-tert-butylcatechol;
methyl alkyl ketones (2-pentanone, 2-hexanone, 3-methyl-2-pentanone,
3,3-dimethyl-2-butanone, 2-heptanone, 2-octanone, 2-nonanone, 2-decanone, and
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2-undecanone); and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were purchased from
Tokyo Chemical Industry (Tokyo, Japan). Catechol was purchased from Wako Pure Chemical
Industries (Osaka, Japan).
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Culture media. We used basal salts medium (BSM; pH 7.2) containing 1.0 g (NH4)2SO4,
1.0 g K2HPO4, 0.2 g NaH2PO4, 0.2 g MgSO4·7H2O, 0.05 g NaCl, 0.05 g CaCl2, 8.3 mg
FeCl3·6H2O, 1.4 mg MnCl2·4H2O, 1.17 mg Na2MoO4·2H2O, and 1 mg ZnCl2 per liter of water.
BSM containing 4-tert-butylphenol (4tBP-BSM) as the sole carbon source was used for
cultures of 4-tert-butylphenol-degrading bacteria. Agar solid medium was prepared with 2.0%
(wt:vol) agar.
Enrichment, isolation, and identification of 4-tert-butylphenol-degrading bacteria. A
sample of P. australis rhizosphere sediment was obtained from Inukai Pond at Osaka
University, Suita, Osaka, Japan. The rhizosphere sediment sample was collected from about
20 cm depth, around the P. australis roots. Detailed physicochemical analysis revealed that
the sediment sample had a pH of 6.9, a low organic carbon content (ignition loss, 2.2%), and
undetectable levels (<0.001 mg kg–1
) of alkylphenols (4-n-butylphenol, 4-tert-butylphenol,
4-tert-octylphenol, and 4-nonylphenol). For enrichment of 4-tert-butylphenol-degrading
bacteria from the rhizosphere sediment, about 1 g wet-weight of sediment sample was added
to 100 mL 4tBP-BSM (0.5 mM 4-tert-butylphenol). The mixture was incubated at 28 ºC on a
rotary shaker at 120 rpm for 14 days, and then 1 mL of this enrichment culture was transferred
to 100 mL fresh 4tBP-BSM (0.5 mM) and incubated for 14 days. After a third transfer, the
enriched culture was serially diluted and spread on 4tBP-BSM (0.5 mM) agar plates, and the
plates were incubated at 28 ºC. The isolated bacterial strains, designated TIK-1, TIK-2, and
TIK-3, were characterized and identified by physiological and phylogenetic analyses as
described previously. The strains were morphologically and physiologically characterized by
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using an API 20NE kit according to the instruction of the manufacturer (BioMérieux Japan,
Tokyo, Japan). A comparative 16S rRNA gene sequence analysis was performed as follows.
Partial 16S rRNA genes were amplified by PCR using primer 27F
(5ガ-AGAGTTTGATCCTGGCTCAG-3ガ) and 1392R (5ガ-ACGGGCGGTGTGTACA-3ガ) (3).
The determination of the amplified 16S rRNA gene sequence was entrusted to Takara Bio
(Shiga, Japan). The 16S rRNA gene sequences were compared to reference sequence by using
BLAST similarity searches (2), and closely related sequences were obtained from GenBank.
The sequences were aligned by using CLUSTAL W (8). A phylogenetic tree was produced by
njplotWIN95 software (31).
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Growth and degradation experiments. Each isolate was grown overnight in 4tBP-BSM
(1.0 mM). Cells were harvested by centrifugation (9600 × g, 4 ºC, 10 min), washed twice with
50 mM potassium phosphate buffer (pH 7.5), and inoculated to a cell density (as determined
by the optical density at a wavelength of 600 nm [OD600]) of 0.02 (i.e. OD600 = 0.02) in 100
mL 4tBP-BSM (1.0 mM) or in 100 mL BSM containing 1.0 mM 4-tert-butylcatechol as the
sole carbon source. Culture was carried out at 28 ºC and 120 rpm under dark conditions. The
experiments for each of the two types of BSM were conducted in triplicate. Cell densities,
substrate concentrations, and metabolic products were monitored over the 24-h experimental
period (see below). We also performed 4-tert-butylphenol degradation experiments using cells
of each isolate grown on glucose.
Inhibition experiments. To check for the degradation of 4-tert-butylphenol via a
meta-cleavage pathway, inhibition experiments were conducted with 3-fluorocatechol, which
is a suicide inactivator of meta-cleaving catechol 2,3-dioxygenase (5, 18). Whole cells of
strain TIK-1 grown on 4-tert-butylphenol, prepared as described above, were resuspended in
50 mM potassium phosphate buffer (pH 7.5) with or without 0.2 mM 3-fluorocatechol, and
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then the suspension was incubated for 30 min before the start of the experiment. Then, 5 mL
of one of the two types of cell suspension (i.e. whole cell suspensions with or without
3-fluorocatechol treatment) was added to 5 mL of 50 mM potassium phosphate buffer (pH
7.5) containing 1.0 mM 4-tert-butylphenol or 4-tert-butylcatechol. The reaction mixture
(approximately 0.1 mg dry cell mL&1
) was incubated at 25 ºC under dark conditions. The
inhibition experiments were conducted in triplicate. Substrate concentrations were monitored
over the 360-min experimental period (see below).
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Enzyme assays. Cells of strain TIK-1 grown on 4-tert-butylphenol, prepared as described
above, were resuspended in 50 mM potassium phosphate buffer (pH 7.5). The cell suspension
was kept on ice at all times and broken by ultrasonication (30 W, four cycles of 30 s with a
30-s interval). Each sample was centrifuged (12,000 × g, 4 ºC, 30 min) and the supernatant
was used as a crude cell extract for enzyme assays. Catechol 1,2-dioxygenase activity and
catechol 2,3-dioxygenase activity were assayed by methods described by Nakazawa and
Nakazawa (28) and Takeo et al. (37), respectively. The reaction was performed at 25 ºC in 1.5
mL of 50 mM potassium phosphate buffer (pH 7.5) containing the appropriate concentrations
(10 to 1000 たM) of catechol. In the catechol 1,2-dioxygenase assay, catechol 2,3-dioxygenase
was inactivated by the addition of H2O2 (0.01% vol:vol) for 5 min before the addition of
catechol. In the catechol 2,3-dioxygenase assay, acetone was added (10% vol:vol) to
preparations for catechol 2,3-dioxygenase measurement. Protein was measured by the Lowry
method with bovine serum albumin as the standard. We defined one unit of enzyme activity
(U) as the amount of enzyme that converted 1 µmol of substrate per minute.
Detection of 4-tert-butylcatechol cleavage products. For detecting the
4-tert-butylcatechol cleavage products, we conducted a 4-tert-butylcatechol degradation test
using whole cells of strain TIK-1 grown on 4-tert-butylphenol. Cells, prepared as described
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above, were resuspended in 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM
4-tert-butylcatechol. Eighteen identical 10-mL whole-cell reaction mixtures (approximately
0.1 mg dry cells mL&1
) were incubated in closed vials at 28 ºC. Three vials were sampled at
the start of the experiment and three more at each of 10, 20, 30, 40, and 60 min, and cells in
the vials were broken by ultrasonication (30 W, 4 cycles of 30 s at 30-s intervals). The
samples were then subjected to analysis for metabolites (see below).
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Utilization and degradation of other alkylphenols. Alkylphenols with variously
positioned, sized, and branched alkyl chains were used for growth experiments with strain
TIK-1 and for degradation tests using strain TIK-1 whole cells grown on 4-tert-butylphenol.
In the growth experiments, cells prepared as described above were added at an OD600 of 0.02
to 100 mL BSM supplemented with one of the following alkylphenols as the sole carbon
source at 0.5 mM: 4-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 3-isopropylphenol,
4-n-propylphenol, 4-isopropylphenol, 2-sec-butylphenol, 2-tert-butylphenol,
3-tert-butylphenol, 4-n-butylphenol, 4-sec-butylphenol, 4-tert-butylphenol, 4-n-pentylphenol,
4-tert-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-n-octylphenol, 4-tert-octylphenol,
4-n-nonylphenol, technical nonylphenol, or 2,4-di-tert-butylphenol. Cultivation was carried
out at 28 ºC and 120 rpm. The growth experiments were conducted in triplicate. Cell densities,
substrate concentrations, and metabolic products were monitored over the 120-h experimental
period (see below).
In the degradation tests, whole cells of strain TIK-1 grown on 4-tert-butylphenol were
suspended in 10 mL of 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM of
one of the above alkylphenols. The whole-cell reaction mixtures (approximately 0.1 mg dry
cells mL&1
) were incubated in closed vials at 28 ºC and 120 rpm. Three vials from each
treatment were sampled at the start of the experiment and at 3, 24, 48, and 72 h.
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Concentrations of substrates and metabolic products were monitored (see below). As
inhibition experiments, the degradation experiments were also performed in the presence of
0.2 mM 3-fluorocatechol.
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Analytical procedures. Bacterial growth was monitored by recording the increase in
OD600. The dry weights of cells were also determined at the end of the growth experiments.
For dry weight measurement, the cells were harvested by centrifugation (9600 × g, 4 ºC, 10
min), washed twice with 50 mM potassium phosphate buffer (pH 7.5), and then filtered
through a pre-weighed filter (pore size 0.2 µm, polycarbonate; Millipore, Tokyo, Japan). The
filter, together with the cells, was dried at 90 ºC for 3 h and then weighed.
Alkylphenols and 4-tert-butylcatechol concentrations were determined by
high-performance liquid chromatography (HPLC). The 3,3-dimethyl-2-butanone
concentration was determined by gas chromatography–mass spectrometry (GC-MS), and
metabolites of 4-tert-butylphenol and other alkylphenols were analyzed by GC-MS. For the
HPLC and GC-MS analyses, the culture sampled at each sampling point was acidified with 1
N HCl to pH 2 to 3, shaken for 3 min with an equal volume of 2:1 (vol:vol) ethyl
acetate:n-hexane, and centrifuged (3200 × g, 4 ºC, 10 min); the organic layer was then
collected. For HPLC analysis, the extract (200 たL) was dried under flowing nitrogen, and the
dry extract was dissolved in 200 たL acetonitrile before analysis. For GC-MS analysis, the
extract (2 to 10 mL) was dried under nitrogen flow, and the dry extract was dissolved in 100
たL acetonitrile before analysis. In addition, the extract (2 to 10 mL) was dried under nitrogen
flow and then trimethylsilylated (TMS) at 60 ºC for 1 h using 100 たL of BSTFA–acetonitrile
solution (1:1, vol:vol). The sample with TMS derivatization was analyzed by GC-MS.
HPLC analysis was conducted in a Shimadzu HPLC system with an UV/vis detector and a
Shim-pack VP-ODS column (150 mm · 4.6 mm, particle size 5 µm; Shimadzu, Kyoto, Japan).
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In the HPLC analysis, acetonitrile:water (8:2, vol:vol) was used as the mobile phase and
detection was at a wavelength of 277 nm. The GC-MS analysis was conducted with a
Shimadzu GC/MS system (GCMS-QP2010) and an Rxi-5ms capillary column (30 m, 0.25
mm ID, 1.00 µm df; Restek, Bellefonte, PA, USA). For the GC-MS analysis, two column
temperature programs were used. For the analysis of metabolites without TMS derivatization,
the temperature was held at 60 ºC for 2 min, increased to 300 ºC at 5 ºC min&1
, and then held
at 300 ºC for 5 min. For the analysis of metabolites with TMS derivatization, the column
temperature was held at 90 ºC for 1 min, increased to 150 ºC at a rate of 15 ºC min&1
,
increased to 300 ºC at 5 ºC min&1
, and then held at 300 ºC for 6 min. The injection, interface,
and ion-source temperatures were 280 ºC, 280 ºC, and 250 ºC, respectively. Helium
(99.995%) was used as the carrier gas at a flow rate of 1.0 mL min&1
. The degradation
products were identified by their electron impact mass spectrometry (EIMS) spectral data.
The National Institute of Standards and Technologies (NIST) MS fragment library (NIST08)
was used for the identifications.
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Nucleotide sequence accession numbers. The 16S rRNA gene sequence data of strains
TIK-1, TIK-2, and TIK-3 have been submitted to the DDBJ/EMBL/GenBank databases under
accession numbers AB491315, AB491316, and AB491317, respectively.
RESULTS
Isolation and identification of 4-tert-butylphenol-degrading bacteria. The P. australis
rhizosphere sediment sample was incubated in 4tBP-BSM (0.5 mM) to check for
4-tert-butylphenol-degrading ability. The 4-tert-butylphenol completely disappeared from the
rhizosphere sediment culture within 14 days. Enrichment culture of the rhizosphere sediment
resulted in the isolation of three bacterial strains—designated TIK-1, TIK-2, and TIK-3—that
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could be aerobically grown on 4-tert-butylphenol as a sole carbon and energy source. Strains
TIK-1 and TIK-3 formed creamy yellow colonies on 4tBP-BSM (0.5 mM) agar plates,
whereas strain TIK-2 formed bright yellow colonies on the same agar plates. The three
isolates were rod-shaped, gram-negative, and catalase- and oxidase-positive bacteria. They
utilized glucose, L-arabinose, and maltose as sole carbon sources, but not D-mannose,
D-mannitol, N-acetyl-D-glucosamine, gluconate, n-caprate, adipate, D,L-malate, citrate, or
phenylacetate. The partial 16S rRNA gene sequences of the isolates corresponded to each
other and showed the closest sequence identity (99.8%) to that of Sphingobium fuliginis TKPT.
Therefore, we identified strains TIK-1, TIK-2, and TIK-3 as S. fuliginis. We used the
neighbor-joining method to establish the phylogenetic relationships of strains TIK-1, TIK-2,
and TIK-3 on the basis of their 16S rRNA gene sequences, and also from their relationships to
closely related type strains and previously isolated alkylphenol-degrading bacteria (Fig. 1).
All three isolates were phylogenetically related to 4-nonylphenol-degrading and
4-tert-octylphenol-degrading bacteria and distant from medium-chain
4-n-alkylphenol-degrading bacteria.
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Degradation of 4-tert-butylphenol and 4-tert-butylcatechol by strain TIK-1. All three
isolates were able to rapidly degrade 4-tert-butylphenol at almost the same rate, and we
selected strain TIK-1 for further studies. Strain TIK-1 grown on 4-tert-butylphenol completely
degraded 1.0 mM 4-tert-butylphenol within 12 h, and the bacterial cell density (OD600)
increased in parallel with 4-tert-butylphenol degradation (Fig. 2A). One metabolite peak was
detected by HPLC analysis at a retention time (RT) of 2.3 min after 3 h of incubation, along
with a decrease in 4-tert-butylphenol at an RT of 4.4 min. The metabolite peak was identified
as that of 4-tert-butylcatechol, because the RT in HPLC and the MS spectrum of the isolated
peak corresponded to those of authentic 4-tert-butylcatechol. The concentration of
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4-tert-butylcatechol in the culture medium increased over the period from 3 to 6 h, when it
reached a maximum of 0.06 mM, and it subsequently decreased to an undetectable level
within 18 h (Fig. 2A). Furthermore, another notable metabolite peak was detected by GC-MS
analysis without TMS derivatization after 3 h of incubation at an RT of 4.6 min. The RT and
MS spectrum of this peak corresponded to those of authentic 3,3-dimethyl-2-butanone, which
retains the tert-butyl chain structure. The concentration of 3,3-dimethyl-2-butanone in the
culture medium increased over the period from 3 to 9 h, when it reached 0.12 mM, and it then
gradually decreased to 0.009 mM within 24 h (Fig. 2A).
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In addition, strain TIK-1 grown on 4-tert-butylphenol completely degraded 1.0 mM
4-tert-butylcatechol within 9 h, with concomitant cell growth (Fig. 2B).
3,3-Dimethyl-2-butanone was formed after 3 h of incubation, along with the decrease in
4-tert-butylcatechol. The concentration of 3,3-dimethyl-2-butanone in the culture medium
increased between 3 and 6 h to 0.13 mM, and then gradually decreased to 0.008 mM within
24 h (Fig. 2B).
The dry weights of cells were determined after a 24-h incubation of strain TIK-1 with
1.0 mM 4-tert-butylphenol or 1.0 mM 4-tert-butylcatechol. Yield coefficients of the cells
were 75.3 ± 6.99 mg dry weight/mmol 4-tert-butylphenol and 74.9 ± 1.85 mg dry
weight/mmol 4-tert-butylcatechol.
These results indicated that 4-tert-butylphenol was initially transformed to
4-tert-butylcatechol by strain TIK-1, and then 4-tert-butylcatechol was further metabolized
and served as the growth substrate.
In 4-tert-butylphenol degradation experiments using strain TIK-1 cells grown on glucose,
degradation of 4-tert-butylphenol was observed after a lag period of 42 h, and 1.0 mM
4-tert-butylphenol disappeared within 60 h (data not shown). The results indicated that
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4-tert-butylphenol-degrading activity was inducible in strain TIK-1. 288
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Inhibition of 4- tert-butylphenol and 4-tert-butylcatechol degradation in strain
TIK-1. 3-Fluorocatechol, a meta-cleavage inhibitor, substantially inhibited the degradation of
4-tert-butylphenol and 4-tert-butylcatechol by whole cells of strain TIK-1 grown on
4-tert-butylphenol (Fig. 3). Whole cells without 3-fluorocatechol treatment rapidly degraded
0.5 mM 4-tert-butylphenol within 80 min. 4-tert-Butylcatechol was formed and accumulated
transiently during the first 60 min. When the 4-tert-butylphenol was depleted, the
4-tert-butylcatechol also disappeared (Fig. 3A). Whole cells without 3-fluorocatechol
treatment also completely degraded 0.5 mM 4-tert-butylcatechol within 60 min (Fig. 3B). In
contrast, whole cells with 3-fluorocatechol treatment degraded 4-tert-butylphenol slightly, and
1.86 たmol of 4-tert-butylphenol was removed within 360 min. Over the experimental period,
4-tert-butylcatechol was formed and accumulated, with the corresponding stoichiometric
degradation of 4-tert-butylphenol. By the end of the 360-min experiment, 1.74 たmol of
4-tert-butylcatechol had been formed (Fig. 3C). Whole cells with 3-fluorocatechol treatment
degraded 4-tert-butylcatechol slightly, and 0.41 たmol 4-tert-butylcatehol was removed (Fig.
3D). Hollender et al. (18) observed similar inhibition by a 3-halocatechol of 4-chlorophenol
biodegradation via a meta-cleavage pathway and the accumulation of 4-chlorocatechol.
Murdoch and Hay (27) demonstrated that isobutylcatechol can be metabolized by
Sphingomonas sp. strain Ibu-2 via a meta-cleavage pathway and that this metabolism is
inhibited by the addition of 3-fluorocatechol. Thus, these results suggest the involvement of a
meta-cleavage pathway in 4-tert-butylphenol degradation by strain TIK-1.
Identification of enzyme involved in degradation of 4-tert-butylphenol by strain
TIK-1. To investigate whether degradation of 4-tert-butylphenol proceeds via an ortho- or a
meta-cleavage pathway, we tested for the presence of catechol 1,2-dioxygenase and catechol
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2,3-dioxygenase (Table 1). We detected the activity of the meta-cleavage pathway enzyme
catechol 2,3-dioxygenase in a cell extract of strain TIK-1 grown on 4-tert-butylphenol,
whereas we did not detect catechol 1,2-dioxygenase activity. These results also support the
conclusion that a meta-cleavage pathway is involved in 4-tert-butylphenol degradation by
strain TIK-1.
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Detection of 4-tert-butylcatechol meta-cleavage products. To detect 4-tert-butylcatechol
meta-cleavage products, we conducted a 4-tert-butylcatechol degradation test using whole
cells of strain TIK-1 grown on 4-tert-butylphenol. Ethyl acetate:n-hexane extracts with TMS
derivatization were analyzed by GC-MS. The peaks of four metabolites were detected by
GC-MS analysis at RTs of 12.6, 18.0, 19.6, and 19.9 min after 10 min of incubation, along
with a decrease in 4-tert-butylcatechol at an RT of 14.6 min. The EIMS spectral data and ion
peak patterns of these metabolites are shown in Figure S1 and Table S1 in the supplemental
material. The relative abundances of the molecular ion (M)+ in these EIMS spectra were low.
The ion peaks showing loss of CH3, loss of CH3 + CO, and loss of C4H9O2Si had relatively
high intensities. These EIMS spectral characteristics have been observed for different
meta-cleavage products (24, 33, 40). Also, in the inhibition experiment with 3-fluorocatechol,
these metabolite peaks were not detected. Therefore, these metabolites were tentatively
identified as meta-cleavage products of 4-tert-butylcatechol. The meta-cleavage of
4-alkylcatechol yields two possible products, depending on the ring-cleavage styles, namely
proximal cleavage (2,3-cleavage) and distal cleavage (1,6-cleavage) (37). However, we did
not identify the ring-cleavage style of strain TIK-1 and meta-cleavage products, because we
could not isolate an adequate amount of each metabolite and get authentic compounds.
Utilization and degradability of various alkylphenols. The ability of strain TIK-1 to
utilize and degrade a variety of alkylphenols is summarized in Table 2. Among the
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alkylphenols tested, 4-isopropylphenol, 4-sec-butylphenol, and 4-tert-pentylphenol were
utilized by strain TIK-1 for growth, in addition to 4-tert-butylphenol. Rates of cell growth on
4-isopropylphenol (specific growth rate, 0.091 h–1
), 4-sec-butylphenol (0.15 h–1
), and
4-tert-pentylphenol (0.094 h–1
) were substantially lower than on 4-tert-butylphenol (0.22 h–1
).
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Whole cells of strain TIK-1 grown on 4-tert-butylphenol degraded the following
alkylphenols: 4-ethylphenol, 4-n-propylphenol, 4-isopropylphenol, 2-tert-butylphenol,
4-n-butylphenol, 4-sec-butylphenol, 4-tert-butylphenol, 4-n-pentylphenol, 4-tert-pentylphenol,
4-n-hexylphenol, 4-n-heptylphenol, 4-n-octylphenol, 4-tert-octylphenol, 4-n-nonylphenol,
technical nonylphenol, and 2,4-di-tert-butylphenol. Methyl alkyl ketones (except in the cases
of 4-ethylphenol, 2-tert-butylphenol, and 2,4-di-tert-butylphenol) were detected as the main
degradation products at 3 h of incubation (Table 2). The GC-MS RTs and EIMS spectra of
2-pentanone, 2-hexanone, 3-methyl-2-pentanone, 3,3-dimethyl-2-butanone, 2-heptanone,
2-octanone, 2-nonanone, 2-decanone, and 2-undecanone corresponded to those of authentic
compounds (see Figure S2 and Table S2 in the supplemental material). Other methyl alkyl
ketones detected were tentatively identified by analysis of their EIMS spectral data (see
Figure S2 and Table S2 in the supplemental material). These methyl alkyl ketones were
probably derived from the alkyl side chains of alkylphenols, because they had the same
structures as the original alkyl side chains. 3,3,5,5-Tetramethyl-2-hexanone formed by
4-tert-octylphenol degradation and methyl branched nonyl ketones formed by technical
nonylphenol degradation persisted in each closed vial after the end of the experiments.
However, the concentrations of the other methyl alkyl ketones in the closed vials decreased to
undetectable levels within 24 h. In the 2,4-di-tert-butylphenol degradation test,
3,5-di-tert-butylcatechol was accumulated as the dead-end degradation product. The RT and
EIMS spectrum of this GC-MS peak corresponded to those of authentic
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3,5-di-tert-butylcatechol (see Figure S2 and Table S2 in the supplemental material). The
amount of 2,4-di-tert-butylphenol lost (0.186 mM) was approximately stoichiometrically
equal to the amount of 3,5-di-tert-butylcatechol (0.178 mM) formed by the end of the
experiment, indicating that the former was transformed to the latter. In the inhibition
experiments in the presence of 3-fluorocatechol there was no substantial degradation of the
alkylphenols or production of the corresponding methyl alkyl ketones.
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2-n-Propylphenol, 2-isopropylphenol, 3-isopropylphenol, 2-sec-butylphenol, and
3-tert-butylphenol were neither utilized for growth nor degraded by strain TIK-1.
DISCUSSION
Three aerobic bacteria (Sphingobium fuliginis strains TIK-1, TIK-2, and TIK-3) capable
of utilizing 4-tert-butylphenol as the sole carbon and energy source were isolated from P.
australis rhizosphere sediment in a pond. All three strains were phylogenetically related to
branched 4-nonylphenol-degrading sphingomonad strains rather than to
4-n-butylphenol-degrading Pseudomonas strains (Fig. 1).
Previous studies of the biodegradation of alkylphenols have clearly demonstrated that
branched 4-nonylphenol and 4-tert-octylphenol, which have long alkyl side chains with
g-quaternary carbons, can be degraded by sphingomonad strains TTNP3 (6, 7), Bayram
(11–14), and PWE1 (32) by an ipso-substitution mechanism; in contrast, 4-n-butylphenol,
which has a shorter alkyl side chain with an g-secondary carbon, can be degraded by
Pseudomonas strains KL28 (21) and MT4 (36) via a meta-cleavage pathway. Strains KL 28
and MT4 cannot degrade 4-tert-butylphenol. Resting cells of strain Bayram grown on
technical nonylphenol show ipso-hydroxylating activity for 4-tert-butylphenol, but this strain
cannot utilize 4-tert-butylphenol as a sole carbon source (11, 14). Until now, nothing has been
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known about the bacteria capable of utilizing 4-tert-butylphenol, which has a shorter alkyl
side chain with an g-quaternary carbon, or the mechanisms of this biodegradation. Strain
TIK-1 is thus, to our knowledge, the first 4-tert-butylphenol-utilizing bacterium to be
identified.
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Our results here show that strain TIK-1 metabolizes 4-tert-butylphenol via a
meta-cleavage pathway (Fig. 4). That is, 4-tert-butylphenol is initially hydroxylated to
4-tert-butylcatechol; 4-tert-butylcatechol is then further metabolized along a meta-cleavage
pathway, eventually forming 3,3-dimethyl-2-butanone with retention of the structure of the
original tert-butyl side chain (Fig. 4). 3,3-Dimethyl-2-butanone was not the dead-end
degradation product, because this compound was degraded in closed vials by whole cells of
strain TIK-1 grown on 4-tert-butylphenol. In addition, we suspect that pyruvic acid is formed
through a meta-cleavage pathway, although it was not detected in our analysis. Pyruvic acid
(1.0 mM) supported cell growth of strain TIK-1 (data not shown). The proposed degradation
pathway of 4-tert-butylphenol by strain TIK-1 resembles that of 4-n-butylphenol degradation
by strains KL28 and MT4. n-Hexanal, an alkyl aldehyde with the original n-butyl side chain,
is formed in 4-n-butylphenol degradation via the distal meta-cleavage pathway by strains
KL28 (21) and MT4 (36). Interestingly, however, we did not detect alkyl aldehyde with the
original 4-tert-butyl side chain in the degradation of 4-tert-butylphenol by strain TIK-1; nor
did we detect n-hexanal in the degradation of 4-n-butylphenol. 3,3-Dimethyl-2-butanone and
2-hexanone, which are methyl alkyl ketones with the original butyl side chains, were formed
in the metabolism of 4-tert- and 4-n-butylphenol, respectively, by strain TIK-1. Therefore, the
pathway of degradation of 4-tert-butylphenol by strain TIK-1 almost certainly is not
completely the same as that of 4-n-butylphenol by strains KL28 and MT4. There may be a
difference in meta-cleavage styles—namely, proximal cleavage (2,3-cleavage) and distal
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cleavage (1,6-cleavage)—between strain TIK and strains KL28 and MT4. Our group is
undertaking further study to gain a more detailed understanding of the
4-tert-butylphenol-degrading enzymes in strain TIK-1.
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Strain TIK-1 also utilized several 4-alkylphenols but did not utilize 2- and
3-alkylphenols. An important factor determining whether 4-alkylphenol is utilized by strain
TIK-1 seems to be the presence of an g-quaternary or g-tertiary carbon, as shown by the
finding that strain TIK-1 utilized 4-tert-butylphenol, with an g-quaternary carbon, and
4-sec-butylphenol, with an g-tertiary carbon, as the sole carbon source, but not
4-n-butylphenol, with an g-secondary carbon. Also, strain TIK-1 utilized 4-tert-butylphenol at
a much faster rate than 4-sec-butylphenol. Alkylphenols with branched alkyl side
chains—especially with an g-quaternary carbon structure—are known to inhibit biological
attack and biotransformation of the alkyl group (7, 41), and they are much more estrogenic
than those with an g-tertiary or g-secondary carbon (34). Therefore, the growth-substrate
specificity of strain TIK-1 is a notable feature and is very different from that of previously
reported 4-n-butylphenol-degrading Pseudomonas strains. In addition, among the
alkylphenols tested in this study, the growth-substrate range of strain TIK-1 was limited to
4-alkylphenols with alkyl side chains of three to five carbon atoms (4-isopropylphenol,
4-sec-butylphenol, 4-tert-butylphenol, and 4-tert-pentylphenol). This indicates that the size of
the alkyl side chain is also crucial for the utilization of 4-alkylphenol by strain TIK-1.
In contrast to their growth-substrate specificity, strain TIK-1 cells grown on
4-tert-butylphenol showed degradative activity for a surprisingly wide range of
4-alkylphenols with variously sized and branched alkyl side chains (ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, tert-octyl,
n-nonyl, and branched nonyl) (Table 2). Although technical nonylphenol is a complex mixture
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of more than 100 nonylphenol isomers (19), a substantial amount (64%) of 0.5 mM technical
nonylphenol was degraded by whole cells of strain TIK-1 (Table 2). Thus, strain TIK-1
probably has degrading activity for various 4-nonylphenol isomers in technical nonylphenol.
Our results indicate that such alkylphenols are degraded by whole cells of strain TIK-1 grown
on 4-tert-butylphenol via a meta-cleavage pathway, and that methyl alkyl ketones retaining
the structure of the original alkyl side chains are formed by the aromatic ring cleavage.
Furthermore, cells of strain TIK-1 grown on 4-tert-butylphenol transformed a dialkylphenolic
compound (namely, 2,4-di-tert-butylphenol) to 3,5-di-tert-butylcatechol, but evidence for the
aromatic ring cleavage of this compound was not observed.
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The aerobic biodegradation of many aromatic compounds occurs via catechols as key
metabolites. The breakdown of catechols in general proceeds via an ortho- or meta-cleavage
pathway. Previous studies have demonstrated that a meta-cleavage pathway is involved in the
degradation of short- and medium-chain 4-n-alkylphenols (4-methylphenol, 4-ethylphenol,
4-n-propylphenol, 4-n-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol, and
4-n-heptylphenol) (21, 36, 37) and 2-alkylphenols (2-n-propylphenol, 2-isopropylphenol, and
2-sec-butylphenol) (24, 33, 40). To our knowledge, this is the first study to demonstrate that a
single bacterial strain can degrade 4-alkylphenols with various sized and branched side chains
of two to nine carbon atoms, including 4-tert-octylphenol and technical nonylphenol, via a
meta-cleavage pathway. Strain TIK-1 probably has meta-cleavage pathway enzymes with an
unprecedentedly wide substrate specificity.
In conclusion, our results provide evidence that S. fuliginis strain TIK-1 utilizes
4-tert-butylphenol via a meta-cleavage pathway. Another noteworthy finding is that the
substrate range of degradative activity in strain TIK-1 includes a wide range of 4-alkylphenols.
Strain TIK-1 is potentially useful for removal of 4-tert-butylphenol and other 4-alkylphenols
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from polluted environments. 456
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ACKNOWLEDGMENTS
This research was supported partly by a Grant-in-Aid for Encouragement of Young
Scientists (A) (no. 21681010) and Young Scientists (B) (no. 19710060) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan.
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565
566
568
569
570
572
573
575
577
578
579
581
33. Reichlin, F., and H.-P. E. Kohler. 1994. Pseudomonas sp. strain HBP1 Prp degrades 559
2-isopropylphenol (ortho-cumenol) via meta cleavage. Appl. Environ. Microbiol.
60:4587–4591.
34. Routledge, E. J., and J. P. Sumpter. 1997. Structural features of alkylphenolic chemicals 562
associated with estrogenic activity. J. Biol. Chem. 272:3280–3288.
35. Sun, H., X.-L. Xu, J.-H. Qu, X. Hong, Y.-B. Wang, L.-C. Xu, and X.-R. Wang. 2007. 564
4-Alkylphenols and related chemicals show similar effect on the function of human and
rat estrogen receptor g in reporter gene assay. Chemosphere 71:582–588.
36. Takeo, M., S. K. Prabu, C. Kitamura, M. Hirai, H. Takahashi, D. Kato, and S. 567
Negoro. 2006. Characterization of alkylphenol degradation gene cluster in Pseudomonas
putida MT4 and evidence of oxidation of alkylphenols and alkylcatechols with
medium-length alkyl chain. J. Biosci. Bioeng. 102:352–361.
37. Takeo, M., M. Nishimura, H. Takahashi, C. Kitamura, D. Kato, and S. Negoro. 2007. 571
Purification and characterization of alkylphenol 2,3-dioxygenase from butylphenol
degradation pathway of Pseudomonas putida MT4. J. Biosci. Bioeng. 104:309–314.
38. Tanghe, T., W. Dhooge, and W. Verstraete. 1999. Isolation of a bacterial strain able to 574
degrade branched nonylphenol. Appl. Environ. Microbiol. 65:746–751.
39. Tollefsen, K.-E., S. Eikvar, E. F. Finne, O. Fogelberg, and I. K. Gregersen. 2008. 576
Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout
(Oncorhynchus mykiss) primary hepatocyte culture. Ecotoxicol. Environ. Saf.
71:370–383.
40. van der Maarel, M. J. E. C., and H.-P. E. Kohler. 1993. Degradation of 580
2-sec-butylphenol: 3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid,
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and 2-methylbutyric acid as intermediates. Biodegradation 4:81–89. 582
584
586
587
589
590
41. van Ginkel, C. G. 1996. Complete degradation of xenobiotic surfactants by consortia of 583
aerobic microorganisms. Biodegradation 7:151–164.
42. Yang, F., Z. Abdel-Malek, and R. E. Boissy. 1999. Effects of commonly used mitogens 585
on the cytotoxicity of 4-tertiary butylphenol to human melanocytes. In Vitro Cell. Dev.
Biol. Anim. 35:566–570.
43. Yang, F., R. Sarangarajan, I. C. L. Poole, E. E. Medrano, and R. E. Boissy. 2000. The 588
cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are
independent of tyrosinase activity. J. Invest. Dermatol. 114:157–64.
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Table 1. Specific activities of enzymes in crude cell-extract of S. fuliginis strain TIK-1 grown
on 4-tert-btuylphenol.
591
592
Enzyme Specific activity (mU mg–1
protein)
Catechol 1,2-dioxygenase
Catechol 2,3-dioxygenase
BDa
16.9
aBD, below limit of detection. Limit of detection in enzyme assays: 1 mU mg
–1 protein.593
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Table 2. Utilization and degradability of various alkylphenols by S. fuliginis strain TIK-1. 594
Substrate Growtha Transformation
ratio (%)b
Main degradation productc
4-Ethylphenol
2-n-Propylphenol
2-Isopropylphenol
3-Isopropylphenol
4-n-Propylphenol
4-Isopropylphenol
2-sec-Butylphenol
2-tert-Butylphenol
3-tert-Butylphenol
4-n-Butylphenol
4-sec-Butylphenol
4-tert-Butylphenol
4-n-Pentylphenol
4-tert-Pentylphenol
4-n-Hexylphenol
4-n-Heptylphenol
4-n-Octylphenol
4-tert-Octylphenol
4-n-Nonylphenol
Technical nonylphenol
2,4-Di-tert-butylphenol
&
&
&
&
&
+
&
&
&
&
+
+
&
+
&
&
&
&
&
&
&
100
0
0
0
100
100
0
9.7
0
100
100
100
100
100
100
100
100
98.0
100
64.0
37.2
N.D.d
N.D.d
N.D.d
N.D.d
2-Pentanonee
3-Methyl-2-butanonef
N.D.d
N.D.d
N.D.d
2-Hexanonee
3-Methyl-2-pentanonee
3,3-Dimethyl-2-butanonee
2-Heptanonee
3,3-Dimethyl-2-pentanonef
2-Octanonee
2-Nonanonee
2-Decanonee
3,3,5,5-Tetramethyl-2-hexanonef
2-Undecanonee
Methyl branched nonyl ketonesf
3,5-Di-tert-butylcatechole
a “+” indicates a substantial increase in cell density (OD600) from the initial 0.02 to OD600 >
0.05; “&” indicates no substantial increase in cell density.
595
596
597
598
599
600
bTransformation ratios (TR) were calculated from HPLC chromatograms obtained from 72-h
cultures of strain TIK-1 whole cells and autoclaved sterile controls as follows: TR (%) = [1 &
(substrate peak area from whole-cell culture)/(substrate peak area from sterile control)] · 100
cThe main degradation products were detected by GC-MS analysis.
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dN.D., not detected 601
602
603
604
605
eCompound identified by comparison of EIMS spectral data with those of commercial
authentic compounds.
fCompound identified by analysis of its EIMS spectral data.
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606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
Novosphingobium stygium SMCC B0712T (U20775)Novosphingobium taihuense DSM 17507T (AY500142)
Novosphingobium subarcticum DSM 10700T (X94102)
802
Sphingopyxis terrae NBRC 15098T (D13727)
995
Sphingomonas trueperi ATCC 12417T (X97776)Sphingomonas paucimobilis GIFU 2395T (D16144)
Sphingomonas sp. TTNP3 (EF514925 )*
1000
TIK-1TIK-2TIK-3
922
Sphingobium fuliginis TKPT (DQ092757)
1000
Sphingobium xenophagum DSM 6383T (X94098)
1000
Sphingobium cloacae S-3T (AB040739)*Sphingomonas sp. PWE1 (EU004850)*
Sphingobium chlorophenolicum ATCC 33790T (X87161)Sphingobium amiense YTT (AB047364)*
Sphingobium yanoikuyae NBRC 15102T (D13728)343
1000
564
690
697
977
996
756
980
Pseudomonas aeruginosa DSM 50071T (Z76672)Pseudomonas alcaligenes LMG 1224T ( Z76653)
Pseudomonas nitroreducens IAM 1439T (AM088473)
544
Pseudomonas stutzeri ICMP 12561T (AJ308315)
850
Pseudomonas putida DSM 291T (Z76667)Pseudomonas sp. KL28 (AY324319)**
Pseudomonas sp. CF600 (DQ777732)**
543
Pseudomonas putida MT4 (AB180734)**
348
886
991
Pseudomonas veronii INA06 (AB056120)**Pseudomonas fluorescens ICMP 3512T (AJ308308)
Pseudomonas chlororaphis DSM 50083T (Z76673)
631
Pseudomonas syringae LMG 1247t1T (Z76669)
613
721
983
Escherichia coli ATCC 11775T (X80725 )
1000
1000
0.02
Novosphingobium stygium SMCC B0712T (U20775)Novosphingobium taihuense DSM 17507T (AY500142)
Novosphingobium subarcticum DSM 10700T (X94102)
802
Sphingopyxis terrae NBRC 15098T (D13727)
995
Sphingomonas trueperi ATCC 12417T (X97776)Sphingomonas paucimobilis GIFU 2395T (D16144)
Sphingomonas sp. TTNP3 (EF514925 )*
1000
TIK-1TIK-2TIK-3
922
Sphingobium fuliginis TKPT (DQ092757)
1000
Sphingobium xenophagum DSM 6383T (X94098)
1000
Sphingobium cloacae S-3T (AB040739)*Sphingomonas sp. PWE1 (EU004850)*
Sphingobium chlorophenolicum ATCC 33790T (X87161)Sphingobium amiense YTT (AB047364)*
Sphingobium yanoikuyae NBRC 15102T (D13728)343
1000
564
690
697
977
996
756
980
Pseudomonas aeruginosa DSM 50071T (Z76672)Pseudomonas alcaligenes LMG 1224T ( Z76653)
Pseudomonas nitroreducens IAM 1439T (AM088473)
544
Pseudomonas stutzeri ICMP 12561T (AJ308315)
850
Pseudomonas putida DSM 291T (Z76667)Pseudomonas sp. KL28 (AY324319)**
Pseudomonas sp. CF600 (DQ777732)**
543
Pseudomonas putida MT4 (AB180734)**
348
886
991
Pseudomonas veronii INA06 (AB056120)**Pseudomonas fluorescens ICMP 3512T (AJ308308)
Pseudomonas chlororaphis DSM 50083T (Z76673)
631
Pseudomonas syringae LMG 1247t1T (Z76669)
613
721
983
Escherichia coli ATCC 11775T (X80725 )
1000
1000
0.02
FIG. 1. Phylogenetic relationships between strains TIK-1, TIK-2, and TIK-3, type strains of
Sphingomonadaceae and Pseudomonas sp., and previously isolated 4-nonylphenol-degrading
and 4-octylphenol-degrading (*), and medium-chain 4-n-alkylphenol-degrading (**) bacterial
strains, established by the neighbor-joining method on the basis of 16S rRNA gene sequences.
Numbers on branches indicate bootstrap confidence estimates obtained with 1000 replicates.
Scale bar represents an evolutionary distance (Knuc) of 0.02.
FIG. 1
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624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.204
-ter
t-B
uty
lph
en
ol
(mM
)
4-t
ert-
Bu
tylc
atec
hol
(mM
)
3,3
-Dim
eth
yl-
2-b
uta
no
ne
(mM
)
A
Time (h)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.20
Cel
l de
nsi
ty (
OD
600
)
B
Time (h)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.204
-ter
t-B
uty
lph
en
ol
(mM
)
4-t
ert-
Bu
tylc
atec
hol
(mM
)
3,3
-Dim
eth
yl-
2-b
uta
no
ne
(mM
)
A
Time (h)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.20
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.204
-ter
t-B
uty
lph
en
ol
(mM
)
4-t
ert-
Bu
tylc
atec
hol
(mM
)
3,3
-Dim
eth
yl-
2-b
uta
no
ne
(mM
)
A
Time (h)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.20
Cel
l de
nsi
ty (
OD
600
)
B
Time (h)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.20
0
0.2
0.4
0.6
0.8
1.0
1.2
0 3 6 9 12 15 18 21 240
0.05
0.10
0.15
0.20
Cel
l de
nsi
ty (
OD
600
)
B
Time (h)
FIG. 2. Degradation of 4-tert-butylphenol and cell growth of S. fuliginis strain TIK-1 in basal
salt medium (BSM) containing 1.0 mM 4-tert-butylphenol (A), and degradation of
4-tert-butylcatechol and cell growth of strain TIK-1 in BSM containing 1.0 mM
4-tert-butylcatechol (B). The concentrations of 4-tert-butylphenol (closed squares),
4-tert-butylcatechol (closed diamonds), and 3,3-dimethyl-2-butanone (open triangles), and the
cell densities (optical density at 600 nm [OD600]; open circles) were monitored over 24 h.
Data points represent the means of triplicate experiments and error bars indicate 95%
confidence intervals.
FIG. 2
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640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
4-t
ert-
Bu
tylp
hen
ol
(mM
)
4-t
ert-
Bu
tylc
ate
chol
(mM
)
A
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
B
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
4-t
ert-
Buty
lphen
ol (m
M)
4-t
ert-
Bu
tylc
atec
ho
l (m
M)
C
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
D
Time (min) Time (min)
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
4-t
ert-
Bu
tylp
hen
ol
(mM
)
4-t
ert-
Bu
tylc
ate
chol
(mM
)
A
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 4000
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
B
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 4000
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
4-t
ert-
Buty
lphen
ol (m
M)
4-t
ert-
Bu
tylc
atec
ho
l (m
M)
C
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 4000
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
D
Time (min) Time (min)
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 4000
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400
FIG. 3. Influence of 3-fluorocatechol on degradation of 4-tert-butylphenol and
4-tert-butylcatechol by whole cells of S. fuliginis strain TIK-1 grown on 4-tert-butylphenol.
Whole cells without 0.2 mM 3-fluorocatechol treatment were incubated in 50 mM potassium
phosphate buffer (pH 7.5) containing 0.5 mM 4-tert-butylphenol (A) or 0.5 mM
4-tert-butylcatechol (B). In addition, whole cells subjected to 0.2 mM 3-fluorocatechol
treatment for 30 min before the degradation tests were incubated in 50 mM potassium
phosphate buffer (pH 7.5) containing 0.5 mM 4-tert-butylphenol (C) or 0.5 mM
4-tert-butylcatechol (D). Concentrations of 4-tert-butylphenol (closed squares) and
4-tert-butylcatechol (open diamonds) were monitored over 360 min. Data points represent the
means of triplicate experiments and error bars indicate 95% confidence intervals.
FIG. 3
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33
662
663
664
665
666
667
668
669
670
671
672
.
OH OHOH
C
O
CH3
CO
COOHCH3
I I I
III
IV
meta-cleavage
pathway
OH OHOH
C
O
CH3
CO
COOHCH3
I I I
III
IV
meta-cleavage
pathway
FIG. 4. Proposed pathway for the metabolism of 4-tert-butylphenol by S. fuliginis strain
TIK-1.
(I) 4-tert-butylphenol; (II) 4-tert-butylcatechol; (III) 3,3-dimethyl-2-butanone; (IV) pyruvic
acid.
FIG. 4
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