Metabolism of nitrodiphenyl ether herbicides by dioxin-degrading bacterium Sphingomonas wittichii RW1

Article abstract of DOI:10.1021/jf801362k

Nitrodiphenyl ether herbicides, including chlomethoxyfen, nitrofen, and oxyfluorfen are potent herbicides. Some metabolites and parent compounds are considered as possible mutagens and endocrine disruptors. Both properties pose serious hygienic and environmental risks. Sphingomonas wittichii RW1 is a well-known degrader of polychlorinated dibenzo-p-dioxins, dibenzofurans, and diphenyl ethers. However, no detailed research of its metabolic activity has been performed against pesticides with a diphenyl ether scaffold. In this study, we report S. wittichii RW1 as a very potent diphenyl ether herbicide- metabolizing bacterium with broad substrate specificity. The structures of metabolites were determined by instrumental analysis and synthetic standards. Most pesticides were rapidly removed from the culture medium in the order of nitrofen > oxyfluorfen > chlomethoxyfen. In general, herbicides were degraded through the initial reduction and N-acetylation of nitro groups, followed by ether bond cleavage. Relatively low concentrations of phenolic and catecholic metabolites throughout the study suggested that these metabolites were rapidly metabolized and incorporated into primary metabolism. These results indicate that strain RW1 has very versatile metabolic activities over a wide range of environmental contaminants.

Full text of DOI:10.1021/jf801362k

9146 J. Agric. Food Chem. 2008, 56, 9146–9151
Metabolism of Nitrodiphenyl Ether Herbicides by
Dioxin-Degrading Bacterium Sphingomonas wittichii
RW1
YOUNG SOO KEUM, YOUNG JU LEE, AND JEONG-HAN KIM*
Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
Nitrodiphenyl ether herbicides, including chlomethoxyfen, nitrofen, and oxyfluorfen are potent her-
bicides. Some metabolites and parent compounds are considered as possible mutagens and endocrine
disruptors. Both properties pose serious hygienic and environmental risks. Sphingomonas wittichii
RW1 is a well-known degrader of polychlorinated dibenzo-p-dioxins, dibenzofurans, and diphenyl
ethers. However, no detailed research of its metabolic activity has been performed against pesticides
with a diphenyl ether scaffold. In this study, we report S. wittichii RW1 as a very potent diphenyl
ether herbicide-metabolizing bacterium with broad substrate specificity. The structures of metabolites
were determined by instrumental analysis and synthetic standards. Most pesticides were rapidly
removed from the culture medium in the order of nitrofen > oxyfluorfen > chlomethoxyfen. In general,
herbicides were degraded through the initial reduction and N-acetylation of nitro groups, followed by
ether bond cleavage. Relatively low concentrations of phenolic and catecholic metabolites throughout
the study suggested that these metabolites were rapidly metabolized and incorporated into primary
metabolism. These results indicate that strain RW1 has very versatile metabolic activities over a
wide range of environmental contaminants.
KEYWORDS: Diphenyl ether herbicide; mutagen; metabolism; Sphingomonas wittichii; reduction; acety-
lation
INTRODUCTION
Although diaryl ethers are relatively resistant to biological
degradation, several microorganisms, including some Sphin-
gomonad are able to metabolize or mineralize various substituted
diaryl ethers (13-17).
Nitrodiphenyl ether herbicides (e.g., nitrofen, oxyfluorfen, and
acifluorfen) are very potent herbicides (1, 2). From a toxicologi-
cal point of view, they can induce various physiological effects,
including master cell activation, organ morphogenesis, and
several endocrine disruptor activities (3-5). In addition, some
metabolites were known as possible mutagens (6, 7). Because
of these harmful effects, productions and use of them are of
regulatory concern in Korea and other countries.
Environmental dissipation of nitrodiphenyl ether herbicide
occurred through physical removal or photochemical and
biological degradation (e.g., refs 8-11). Among the environ-
mental transformation procedures of diphenyl ethers, photo-
chemical cyclization to dibenzofurans or dioxins is of particular
interest since the products are highly toxic to animals (12).
As structural constituents, diaryl ether groups are common
in numerous pesticides (e.g., aryloxyphenoxypropionates, ben-
zoylureas with aryloxy appendage, nitrodiphenyl ethers, pyre-
throids, and pyrimidinyloxybenzoates, strobilurins), flame re-
tardants (e.g., polybrominated diphenyl ethers), and in-house
hygienic materials (e.g., triclosan).
Sphingomonad bacteria are commonly found in highly
contaminated natural environments with pesticides and poly-
halogenated aromatic compounds (13, 18-21). Sphingomonas
wittichii RW1 is a well-known polychlorinated dibenzo-p-
dioxins (PCDD) degrading bacterium (22, 23). Because of its
promising xenobiotic degradation potential, genetic engineering
has been performed to improve environmental adaptation (24).
According to previous degradation studies, various xenobiotics
(PCDDs, dibenzofurans, hydroxylated and brominated diphenyl
ethers, and aromatic heterocycles), with a common structural
building block, namely, diaryl ether groups (20, 22, 23), were
readily metabolized by strain RW1. These findings indicate a
possible application of strain RW1 for bioremediation of toxic
pesticides with similar structural groups (e.g., nitrodiphenyl
ethers and aryloxyphenoxylpropionates). However, no related
research has been published so far.
In this study, metabolism of selected nitrodiphenyl ether
herbicides has been investigated with strain RW1. Metabolites
were determined with instrumental analysis and synthetic
standards. Comparative analysis of metabolite profiles from
structurally diverse herbicides gave several interesting aspects
of metabolisms.
* To whom correspondence should be addressed. College of
Agriculture and Life Sciences, Seoul National University, San 56-1,
Sillim-dong, Gwanak-gu, Seoul 151-742, Korea. Tel: 82-2-880-4644.
Fax: 82-2-873-4415. E-mail: kjh2404@snu.ac.kr.
10.1021/jf801362k CCC: $40.75  2008 American Chemical Society
Published on Web 09/09/2008

Diphenyl Ether Herbicide Metabolism of Sphingomonas wittichii
J. Agric. Food Chem., Vol. 56, No. 19, 2008 9147
Table 1. Extraction Efficiencies of Parent Pesticides and Selected
Metabolites
IDa
compounds
recovery ( SDb
1
2
3
4
5
6
7
8
9
nitrofen
oxyfluorfen
chlomethoxyfen
aminonitrofen
aminooxyfluorfen
aminochlomethoxyfen
N-acetylaminonitrofen
N-acetylaminooxyfluorfen
N-acetylaminochlomethoxyfen
95.5 ( 7.2
99.3 ( 4.3
97.2 ( 9.5
78.2 ( 10.4
82.5 ( 5.9
75.2 ( 10.1
89.2 ( 5.9
94.3 ( 5.2
90.4 ( 9.5
a Structures were described in Figure 1. b Standard deviations of three replicates.
Figure 1. Structures of nitrodiphenyl ether herbicides and selected
metabolites in this study.
layer was dried over anhydrous sodium sulfate. After the removal of
solvent, the residue was dissolved in ethyl acetate (2 mL).
To determine the chemical structures of metabolites (4-9), an aliquot
amount of extracts from the batch culture was dried under nitrogen
atmosphere, and the residue was derivatized with BSTFA-TMCS (200
µL) in dry pyridine (800 µL) for 2 h at 80 °C. Derivatized samples
were analyzed with GC-MS, and the results were compared with those
of nonderivatized extracts. Phenolic metabolites (10-14) from kinetic
experiments were analyzed after the same derivatization, while 1-9
were analyzed without derivatization.
Instrumental Analysis. Pesticides and their metabolite profiles were
analyzed with GC-MS (Shimadzu GCMS QP-2000 and GC-2010),
equipped with a DB-1 column (60 m, 0.25 µm film thickness, 0.2 mm
i.d.; Agilent Technologies, USA). Helium was a carrier gas at a flow
rate of 1 mL/min. The column temperature was programmed as follows:
95 °C (10 min) and raised to 295 °C at a rate of 2 °C/min, and held for
20 min. Temperatures of injection port and interface were set at 270
and 254 °C, respectively. The mass spectrometer was operated at
electron impact (EI) mode at 70 eV.
MATERIALS AND METHODS
Chemical. Chemical structures of pesticides and selected metabolites
were described in Figure 1. Nitrofen and oxyfluorfen were obtained
from Sigma-Aldrich (Sigma-Aldrich Korea, Korea). Chlomethoxyfen
was kindly provided from Dongbang Agro Inc. (Korea). Other reagents
for chemical synthesis and bacterial cultures were obtained from Aldrich
or TCI (Tokyo Chemical Industries Inc., Japan). Ethyl acetate and other
solvents were of HPLC or higher grade. Concentrated hydrochloric
acid was from Duksan Chemical Co (Korea). Aminonitrofen, ami-
nochlomethoxyfen, and aminooxyfluorfen were prepared by literature
method with modification (6). Briefly, pesticides were reduced with
aqueous suspension zinc powder for 4 days under room temperature.
After extraction with ethyl acetate, the organic residue was purified
with silica gel column chromatography. N-Acetyl derivatives of
aminonitrofen, aminochlomethoxyfen, and aminooxyfluorfen were
prepared by acetylation with acetyl chloride. Acetyl chloride (150
µmole) was added to a solution of amino metabolite (100 µmole) and
triethylamine (40 µmole) in chloroform (10 mL) with stirring. The
mixture was further stirred for 2 h at 50 °C. The reaction mixture was
diluted with distilled water (100 mL) and extracted with diethyl ether
(100 mL). The organic layer was washed with distilled water (100 mL
× 2). The organic residue was further purified with silica gel TLC.
Culture of Bacterium. Sphingomonas wittichii RW1 obtained from
Korean Agricultural Culture Collection (KACC, #12172) and was
maintained in nutrient agar. The seed culture was prepared in M9
medium (25), supplemented with dibenzo-p-dioxin (100 mg/L) at 150
rpm and 30 °C for 3 days. For the kinetic experiment, the seed culture
(1 mL) was inoculated to M9 medium (100 mL) for nitrofen or M9
medium, supplemented with nutrient broth (NBM, 100 mL, 100 mg
nutrient broth/100 mL of M9 medium) for nitrofen, oxyfluorfen, and
chlomethoxyfen. Sterilized control was prepared from 2-day old cultures
in nutrient broth and sterilized for 30 min at 120 °C. Pesticides (1000
mg/L, 100 µL in dimethyl sulfoxide) were fortified to the above
medium. Cultures were maintained at 150 rpm and 30 °C. Three
replicate experiments were performed for each treatment. For the
preparation of metabolites, the strain was cultured in NBM (1 L) with
crystalline pesticide (100 mg) at the same conditions.
RESULTS
Chemical Syntheses of Metabolites. Among several deg-
radation products of nitrodiphenyl ether herbicides, six metabo-
lites (4-9, Figure 1), commonly found in soil, plants, or
animals, were prepared by literature methods with some
modification (6). Although reduced metabolites (4-6, Figure
1) can be easily prepared by literature methods, substituted
chlorine was also labile to reduction. For example, only trace
amounts of 4 and 6 were obtained after prolonged reflux (2 h),
where monochlorinated aminodiphenyl ethers were dominant
products (data not shown). In comparison with the literature
method, reduction under room temperature proceeded slowly.
However, the reaction yield was reasonable (35-65%), and no
trace of monochlorinated byproduct was observed. Acetamide
metabolites (7-9) were prepared in almost quantitative yield
without any trace of byproduct.
Extraction Efficiencies of Pesticides and Selected Metabo-
lites. Efficiencies of extraction method were determined with
Extraction and Derivatization of Metabolites. For degradation
kinetics, aliquot amount of culture (50 mL) was diluted with saturated
NaCl solution (50 mL) and extracted with ethyl acetate (50 mL × 2)
without the separation of the bacterial cell. The organic layer was dried
over anhydrous sodium sulfate. After the removal of solvent, the residue
was dissolved in ethyl acetate (1 mL) and analyzed with gas
chromatography-mass spectrometry (GC-MS).
Extraction efficiencies of pesticides and metabolites (1-9) were
determined as follows: To a heat-sterilized control culture (100 mL,
precultured for 3 days in NBM), stock solutions (100 µL, 1000 mg/L
in dimethyl sulfoxide) were added and extracted as described above.
1-9. Overall, the recoveries were in the range of 75.2-97.2%
(Table 1). Parent pesticide and N-acetylamino metabolites gave
better recoveries than those of amino metabolites (89.2-97.2
vs 75.2-82.5%).
Growth of Bacterium and Kinetics of Herbicide Degrada-
tion. Both nitrofen and nutrient broth can support bacterial
growth, where strain RW1 grew much faster in NB than in the
mineral medium with nitrofen (Figure 2). In NB-supplimented
M9 medium (NB), bacterial growth was initiated without the
lag phase and reached the stationary phase after 4 days.
However, strain RW1 grew more slowly in nitrofen-suppli-
mented M9 medium and did not reach the stationary phase even
after 7 days. The presence of nitrofen did not produce any
difference in bacterial growth, cultured in NB alone and in
nitrofen-supplimented NB.
For structural identification of metabolites, batch cultures (1 L) were
centrifuged (4000g, 30 min). The supernatant was saturated with NaCl
and extracted with ethyl acetate (250 mL × 2). After adjusting the pH
to 2.0 with concentrated hydrochloric acid, the supernatant was extracted
with additional ethyl acetate (200 mL × 2). The combined organic

9148 J. Agric. Food Chem., Vol. 56, No. 19, 2008
Keum et al.
Figure 2. Degradation of nitrofen in sterilized control, viable cells in M9,
and nutrient broth-supplimented M9 medium by Sphingomonas wittichii
RW1 (A) and the growth of the bacterium (B). Error bar indicates the
standard deviations of three replicate experiments.
Figure 4. GC-MS total ion chromatogram of ethyl acetate extracts of
culture supernatants, grown for 4 days with nitrofen (A), oxyfluorfen (B),
and chlomethoxyfen (C). Samples for chlomethoxyfen was not derivatized.
Figure 3. Degradation of oxyfluorfen and chlomethoxyfen by Sphingomo-
nas wittichii RW1, grown in nutrient broth-supplimented M9 medium.
Symbols: circles for chlomethoxyfen, triangles for oxyfluorfen, blank symbol
for the control, and filled symbol for viable culture. Error bar indicates the
standard deviations of three replicate experiments.
Table 2. List of Metabolites of Nitrofen, Oxyfluorfen, and Chlomethoxyfen
by Sphingomonas wittichii RW1
All three herbicides were rapidly degraded by strain RW1
(Figures 2 and 3). After 7 days, complete degradation of nitrofen
was observed in NB, while 26% of pesticide remained in M9
medium (Figure 2). Oxyfluorfen and chlomethoxyfen were
much more persistent than nitrofen. Approximately, 25 and 42%
of residual oxyfluorfen and chlomethoxyfen still persisted in
the culture medium (Figure 3).
ID
4
name
aminonitrofen
Rt, min
MS fragmentation
65.350 325(M+,99), 327(64), 310(41)
312(28), 149(100), 134(25)
60.592a 253(M+,92), 255(60), 183(11),
108(100)
5
aminooxyfluorfen
62.092 403(M+,100), 405(42), 374(53),
376(21), 359(83), 361(31)
Metabolisms of Herbicides. Several metabolites were found
in the culture supernatant. Approximately, 3-5 metabolites were
found from culture supernatants for each pesticide (Figure 4
and Table 2). At the initial incubation period (2-3 days), the
highly retained metabolites (retention times (Rts), 59-74 min)
were rapidly accumulated. Mass/charge ratio of molecular ions
of nonderivatized 4, 5, and 6 were lower than those of parent
pesticides (m/z of parent-30), and all of them can give mono-
TMS derivatives (m/z of parent -30 + 72). Their mass spectral
fragmentations and retention times were exactly the same as
those of synthetic amino pesticides, of which the nitro group in
parents was reduced to amine group (Figure 5). These
metabolites were identified as aminonitrofen (4), aminooxy-
fluorfen (5), and aminochlomethoxyfen (6). Metabolites 7, 8,
and 9 retained much longer than parent pesticides (Table 2).
Their molecular weights were higher than parents (m/z of parent
+ 12) and could not be derivatized with BSTFA-TMCS. Mass
spectral patterns and retention times were well-correlated with
synthetic N-acetyl derivatives of 4, 5, and 6 (Figure 5). These
metabolites were confirmed as N-acetyl derivative of 4, 5, and
6. 2,4-Dichlorophenol (13) was detected in culture supernatants
of nitrofen and chlomethoxyfen. N-acetylaminophenol (12)
metabolite was found only in nitrofen-treated cultures. No traces
of corresponding analogues were found in oxyfluorfen and
chlomethoxyfen cultures. During the metabolism of oxyfluorfen,
additional metabolites were observed. Metabolites 10 and 14
(Rts, 66.367 and 48.117 min) were tentatively identified as
59.217a 331(M+,100), 333(33), 302(76),
304(30), 274(56)
6
7
8
9
aminochlomethoxyfen
N-acetylaminonitrofen
N-acetylaminooxyfluorfen
65.183a 283(M+,100), 285(63), 268(64),
270(39), 240(30), 242(5)
72.033 295(M+,51), 297(36), 253(100),
255(67), 183(9), 108(68)
67.892 373(M+,100), 375(40), 331(94),
302(79), 274(34)
N-acetylaminochlomethoxyfen 74.525 325(M+,73), 327(49), 283(100),
285(61), 268(52) 270(32),
240(25), 242(16)
66.367 417(M+,100), 419(40), 402(57),
375(87), 360(37)
10 desethyl N-acetyl-
aminooxyfluorfen
11 4-trifluoromethylcatecholb
47.017 322(M+,95), 307(64), 276(10),
249 (7), 232(100), 217(49),
143(15)
12 4-acetamidophenol
13 2,4-dichlorophenol
14 3-ethoxy-4-nitrophenol
47.658 223(M+,84), 208(25), 192(4),
181(100), 166(79)
31.400 234(M+,43), 236(29), 219(100),
221(71), 183(46), 185(19)
48.117 255(M+,53), 240(25), 227(7),
212(100), 194(41)
a Retention times of non-derivatized metabolite. b Tentative identification.
desethyl N-acetylaminooxyfluorfen and 3-ethoxy-4-nitrophenol,
respectively. The mass spectrum of metabolite 11 (Rt, 47.107
min; M+, 322) showed the presence of trifluoromethylphenyl
(m/z 143), TMS-derivatized hydroxy trifluoromethylphenyl (m/z

Diphenyl Ether Herbicide Metabolism of Sphingomonas wittichii
J. Agric. Food Chem., Vol. 56, No. 19, 2008 9149
various easy-to-use substrates in the natural environment, other
than pesticides, this phenomenon is one of the most serious
drawbacks of the corresponding bacterium for use in bioreme-
diation. However, the experimental results indicate that at least
in selected conditions, nitrofen degradation by strain RW1 was
not suppressed through such a phenomenon.
Among the selected herbicides, nitrofen was the most labile
to biodegradation, followed by oxyfluorfen and chlomethoxyfen.
Various factors of substrates and microorganisms can change
the biodegradation rate of xenobiotics. For example, hydropho-
bicities (e.g., log P) of pesticides and their structural fitness on
degradative enzymes may determine the degradation rate of
selected pesticides. However, a detailed explanation of the
different metabolic rates in this study is yet to be studied.
The most interesting metabolic aspects of nitrodiphenyl ethers
by this strain were the initial reduction of the nitro group,
followed by N-acetylation. These reactions have never been
reported with this strain. Rapid accumulation of 4-9 revealed
that reduction of the nitro group is a much more dominant
reaction. For example, 6 and 9 were attributable to most fractions
of chlomethoxyfen metabolites. Although specific enzymes for
these reductions were not identified from this organism, various
redox enzymes have been reported from other bacterial species
(27). N-Acetylation of metabolites 4-6 was also enzymatic since
no trace of 7-9 was observed in amino metabolite-supplemented
sterilized culture (data not shown).
Figure 5. Mass spectrum of selected metabolites of oxyfluorfen from
culture supernatant and synthetic metabolite standards. Aminooxyfluorfen
from culture supernatant (A) and synthetic standard (B); N-acetylami-
nooxyfluorfen from supernatant (C) and standard (D).
As in other Sphingomonads (13, 20, 30), dissociation of the
diaryl ether bond was also found in strain RW1 during
nitrodiphenyl ether metabolism (Figure 7). Catecholic metabo-
lite 14 implies the dissociation of diaryl ether groups. Although
ether bond cleavage is a common metabolic pathway of diaryl
ethers in animal, plant, fungi, and bacteria (24, 28-31),
corresponding enzymes of eukaryotes and prokaryotes are
different. Bacterial degradation of diphenyl ethers and related
environmental contaminants are usually catalyzed by multi-
component dioxygenase system (14, 18, 28), while eukaryotic
biodegradation is generally catalyzed by monooxygenase or
peroxidase (e.g., refs 10, 15, 17, and 32. These differences
result in the differential metabolite profiles. For example,
both catechol and phenol are common metabolites from
bacteria, while no catechol is usually observed in eukaryotic
metabolism (10, 15, 17, 30-32).
In addition to diaryl ether, other types of ether (aryl alkyl
ether) may also be labile to degradation in strain RW1.
Oxyfluorfen (2) contains two different ethers, namely, diaryl
ether and aryl alkyl ether. Metabolite 10 is an example of aryl
alkyl ether cleavage (Figure 7). Although several bacteria can
catalyze this reaction (e.g., refs 13 and 33, similar types of
cleavage and corresponding enzymes have not been reported
from this strain. Close inspection of the recently released draft
genome of this strain will give more detailed information.
Figure 6. Kinetics of metabolites with the diphenyl ether group during
the culture period. Error bar indicates the standard deviations of three
replicate experiments.
232), and TMS-derivatized dihydroxy trifluoromethylphenyl
(m/z 249) groups, while no characteristic isotope patterns of
chlorine atoms were observed. This metabolite was tentatively
identified as 4-trifluoromethylcatechol.
In general, the concentration of metabolites 4 to 9 accounts
for 40-90% of the parent pesticide degradation (Figure 6). Two
initial metabolites (6 and 9) of chlomethoxyfen were gradually
accumulated throughout the experimental period with concomi-
tant increase of other metabolites (e.g., 2,4-dichlorophenol).
However, similar types of metabolites (4 and 7) in the nitrofen-
treated culture were accumulated up to 4 days and then rapidly
decreased (Figure 6). The amounts of aminonitrofen and
aminooxyfluorfen (4 and 5) were higher at initial incubation
period (2-3 days) than those of N-acetyl derivatives (7 and 8),
but the latter were more persistent to degradation; larger amount
of metabolite were found at 4 to 7 days.
The presence of 12 and 13 during nitrofen metabolism
indicates that the initial reaction (dioxygen addition, Figure 7)
can occur both in the halogenated phenyl and nitrophenyl sides.
However, only the phenolic metabolite from the halogenated
phenyl side (13) was observed during chlomethoxyfen metabo-
lism. These findings suggest that the initial reaction by dioxy-
genase or other equivalent enzymes in strain RW1 may have
very complex regioselectivity.
DISCUSSION
In relation to biomass production, nitrofen showed no
difference with that of nutrient broth alone, indicating no
appreciable effects of this pesticide on bacterial growth (Figure
2). However, slow growth in nitrofen-supplimented M9 medium
suggested that the pesticide is a less favorable carbon source
than natural nutrients. It is noteworthy that both biomass
production and nitrofen degradation were enhanced by NB
supplementation. In some Gram-negative bacteria, the degrada-
tion of xenobiotics is repressed by the presence of readily
metabolized natural carbon sources (26). Because there are
Slow but definitive growth of strain RW1 in nitrofen-
supplimented minimal salt medium suggested that organic
carbons from nitrodiphenyl ethers can be incorporated into
primary metabolism to support bacterial growth. In consideration
of mass balance, metabolites 4-14 can only account for

9150 J. Agric. Food Chem., Vol. 56, No. 19, 2008
Keum et al.
(2) Hallahan, B. J.; Camilleri, P.; Smith, A.; Bowyer, J. R. Mode of
action studies on a chiral diphenyl ether peroxidizing herbicide.
Plant Physiol. 1992, 100, 1211–1216.
(3) Mastorakos, G.; Karoutsou, E. I.; Mizamtsidi, M.; Creatsas, G.
The menace of endocrine disruptors on thyroid hormone physiol-
ogy and their impact on intrauterine development. Endocr. 2007,
31, 219–237.
(4) Teshima, R.; Nakamura, R.; Nakajima, O.; Hachisuka, A.; Sawada,
J. Effect of two nitrogenous diphenyl ether pesticides on mast
cell activation. Toxicol. Lett. 2004, 150, 277–283.
(5) Takayasu, H.; Nakazawa, N.; Montedonico, S.; Puri, P. Down-
regulation of Wnt signal pathway in nitrofen-induced hypoplastic
lung. J. Pediatr. Surg. 2007, 42, 426–430.
(6) Draper, W. M.; Casida, J. E. Diphenyl ether herbicides: mutagenic
metabolites and photoproducts of nitrofen. J. Agric. Food Chem.
1983, 31, 227–231.
(7) Tanaka, Y.; Iwasaki, H.; Kitamori, S. Biodegradation of herbicide
chlornitrofen (CNP) and mutagenicity of its degradation products.
Water Sci. Technol. 1996, 34, 15–20.
(8) Andreoni, V.; Colombo, M.; Gennari, M.; Nègre, M.; Ambrosoli,
R. Cometabolic degradation of acifluorfen by a mixed microbial
culture. J. EnViron. Sci. Health B. 1994, 29, 963–987.
(9) Gennari, M.; Ne`gre, M.; Raimondo, E. Effect of soil properties
on adsorption and desorption of acifluorfen. J. Agric. Food Chem.
1994, 42, 2329–2332.
(10) Frear, D. S.; Swanson, H. R.; Mansager, E. R. Acifluorfen
metabolism in soybean: diphenylether bond cleavage and the
formation of homoglutathione, cysteine, and glucose conjugates.
Pestic. Biochem. Physiol. 1983, 20, 299–310.
(11) Scrano, L.; Bufo, S. A.; D’Auriab, M.; Meallier, P.; Behechti,
A.; Shramm, K. W. Photochemistry and photoinduced toxicity
of acifluorfen, a diphenyl-ether herbicide. J. EnViron. Qual. 2002,
31, 268–274.
(12) Mezcua, M.; Go´mez, M. J.; Ferrer, I.; Aguera, A.; Hernando,
M. D.; Ferna´ndez-Alba, A. R. Evidence of 2,7/2,8-dibenzod-
ichloro-p-dioxin as a photodegradation product of triclosan in
water and wastewater samples. Anal. Chim. Acta 2004, 524, 241–
247.
Figure 7. Metabolic pathways of nitrodiphenyl ether herbicides by
Sphingomonas wittichii RW1. Metabolites within brackets are threoretical
intermediates, not detected in this study.
(13) Adkins, A. Degradation of the phenoxy acid herbicide diclofop-
methyl by Sphingomonas paucimobilis isolated from a Cana-
dian prairie soil. J. Ind. Microbiol. Biotechnol. 1999, 23, 332–
335.
(14) Halden, R. U.; Peters, E. G.; Halden, B. G.; Dwyer, D. F.
Transformation of mono- and dichlorinated phenoxybenzoates by
phenoxybenzoate-dioxygenase in Pseudomonas pseudoalcaligenes
POB310 and a modified diaryl ether-metabolizing bacterium.
Biotechnol. Bioeng. 2000, 69, 107–112.
(15) Hiratsuka, N.; Wariishi, H.; Tanaka, H. Degradation of diphenyl
ether herbicides by the lignin-degrading basidiomycete Coriolus
Versicolor. Appl. Microbiol. Biotechnol. 2001, 57, 563–571.
(16) Hoagland, R. E.; Zablotowicz, R. M. Biotransformation of
fenoxaprop-ethyl by fluorescent Pseudomonas strains. J. Agric.
Food Chem. 1998, 46, 4759–4765.
(17) Hundt, K.; Jonas, U.; Hammer, E.; Schauer, F. Transformation
of diphenyl ethers by Trametes Versicolor and characterization
of ring cleavage products. Biodegradation 1999, 10, 279–286.
(18) Engesser, K. H.; Fietz, W.; Fischer, P.; Schulte, P.; Knackmuss,
H.-J. Dioxygenolytic cleavage of aryl ether bonds: 1,2-dihydro-
1,2-dihydroxy-4-carboxybenzophenone as evidence for initial 1,2-
dioxygenation in 3- and 4-carboxy biphenyl ether degradation.
FEMS Microbiol. Lett. 1990, 69, 317–322.
30-75% of parent pesticide degradation, which reveals further
degradation or incorporation into primary metabolism. It is well
known that bacterial degradation of substituted phenols proceeds
to muconate (34, 35). Because catechols and phenols from
nitrodiphenyl ether herbicide can also be metabolized into
muconate or its analogues, it can be postulated that strain RW1
may be able to assimilate selected pesticides through similar
pathways.
In summary, strain RW1 can utilize not only PCDD/Fs but
also several nitrodiphenyl ether herbicides as a sole carbon
source for growth. Reduction of the nitro group with consecutive
N-acetylation was the most dominant initial reaction, followed
by diaryl ether bond cleavage. Detailed analysis of metabolite
profiles suggests that degradative enzyme(s) in RW1 may have
complex regio-selectivities. Kinetic analysis indicates that
identified metabolites may be further degraded to support
bacterial growth. These findings suggest extremely versatile
metabolic activities of this strain in relation to possible
application in bioremediation.
ACKNOWLEDGMENT
(19) Keim, T.; Francke, W.; Schmidt, S.; Fortnagel, P. Catabolism of
2,7-dichloro- and 2,4,8-trichlorodibenzofuran by Sphingomonas
sp. RW1. J. Ind. Microbiol. Biotechnol. 1999, 23, 359–363.
(20) Kim, Y.-M.; Nam, I.-H.; Murugesan, K.; Schmidt, S.; Crowley,
D. E.; Chang, Y.-S. Biodegradation of diphenyl ether and
transformation of selected brominated congeners by Sphingomonas
sp. PH-07. Appl. Microbiol. Biotechnol. 2007, 77, 187–194.
(21) Sasaki, M.; Maki, J.-I.; Oshiman, K.-I.; Mstsumura, Y.; Tsuchido,
T. Biodegradation of bisphenol A by cells and cell lysates from
We thank Dr. Ho-Gil Hur for precious discussions.
LITERATURE CITED
(1) Jacobs, J. M.; Jacobs, N. J.; Sherman, T. D.; Duke, S. O. Effect
of diphenyl ether herbicides on oxidation of protoporphyrinogen
to protoporphyrin in organellar and plasma membrane enriched
fractions of barley. Plant Physiol. 1991, 97, 197–203.

Diphenyl Ether Herbicide Metabolism of Sphingomonas wittichii
J. Agric. Food Chem., Vol. 56, No. 19, 2008 9151
Sphingomonas sp. strain AO1. Biodegradation 2005, 16, 449–
459.
(29) Yamazoe, A.; Yagi, O.; Oyaizu, H. Biotransformation of fluorene,
diphenyl ether, dibenzo-p-dioxin by Janibacter sp. Biotechnol. Lett.
2004, 26, 479–486.
(30) Schmidt, S.; Wittichii, R. M.; Erdman, D.; Wilkes, H.; Francke,
W.; Fortnagel, P. Biodegradation of diphenyl ether and its
monohalogenated derivatives by Sphingomonas sp. strain SS3.
Appl. EnViron. Microbiol. 1992, 58, 2744–2750.
(31) Wittichii, R.-M.; Schmidt, S.; Fortnagel, P. Bacterial degradation
of 3- and 4-carboxybiphenyl ether by Pseudomonas sp. NSS2.
FEMS Microbiol. Lett. 1990, 67, 157–160.
(32) Leather, G. R.; Foy, C. L. Metabolism of bifenox in soil and plants.
Pestic. Biochem. Physiol. 1977, 7, 437–442.
(33) Kohler, H. P. Sphingomonas herbicidoVorans MH: a versatile
phenoxyalkanoic acid herbicide degrader. J. Ind. Microbiol.
Biotechnol. 1999, 23, 336–340.
(34) El-Sayed, W. S.; Ibrahim, M. K.; Abu-Shady, M.; El-Beith, F.;
Ohmura, N.; Saiki, H.; Ando, A. Isolation and characterization
of phenol-catabolizing bacteria from a coking plant. Biosci.
Biotechnol. Biochem. 2003, 67, 2026–2029.
(35) Nordin, K.; Unell, M.; Jansson, J. K. Novel 4-chlorophenol
degradation gene cluster and degradation route via hydroxyquinol
in Arthrobacter chlorophenolicus A6. Appl. EnViron. Microbiol.
2005, 71, 6538–6544.
(22) Wittich, R. M.; Wilkes, H.; Sinnwell, V.; Francke, W.; Fortnagel,
P. Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain
RW1. Appl. EnViron. Microbiol. 1992, 58, 1005–1010.
(23) Nam, I. H.; Kim, Y. M.; Schmidt, S.; Chang, Y. S. Biotransfor-
mation of 1,2,3-tri- and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin
by Sphingomonas wittichii strain RW1. Appl. EnViron. Microbiol.
2006, 72, 112–116.
(24) Aso, Y.; Miyamoto, Y.; Harada, K. M.; Momma, K.; Kawai, S.;
Hashimoto, W.; Mikami, B.; Murata, K. Engineered membrane
superchannel improves bioremediation potential of dioxin-degrad-
ing bacteria. Nat. Biotechnol. 2006, 24, 188–189.
(25) Halden, R. U.; Colquhoun, D. R.; Wisniewski, E. S. Identification
and phenotypic characterization of Sphingomonas wittichii strain
RW1 by peptide mass fingerprinting using matrix-assisted laser
desorption ionization-time of flight mass spectrometry. Appl.
EnViron. Microbiol. 2005, 71, 2442–2451.
(26) Aranda-Olmedo, I.; Marin, P.; Ramos, J. L.; Marques, S. Role of
the ptsN gene product in catabolite repression of the Pseudomonas
putida TOL toluene degradation pathway in chemostat cultures.
Appl. EnViron. Microbiol. 20, 72, 7418–7421.
(27) Riefler, R. G.; Smets, B. F. Enzymatic reduction of 2,4,6-
trinitrotoluene and related a nitroarenes: Kinetics linked to one-
electron redox potentials. EnViron. Sci. Technol. 2000, 34, 3900–
3906.
(28) Nojiri, H.; Habe, H.; Omori, T. Bacterial degradation of aromatic
compounds via angular dioxygenation. J. Gen. Appl. Microbiol.
2001, 47, 270–305.
Received for review May 1, 2008. Revised manuscript received July 4,
2008. Accepted July 11, 2008.
JF801362K