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Applied and Environmental Microbiology, September 1998, p. 3225-3231, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Biotransformation of the Major Fungal Metabolite
3,5-Dichloro- p-Anisyl Alcohol under Anaerobic Conditions
and Its Role in Formation of
Bis(3,5-Dichloro-4-Hydroxyphenyl)methane
Frank J. M.
Verhagen,1,*
Henk J.
Swarts,2
Joannes B. P. A.
Wijnberg,2 and
Jim A.
Field1
Division of Industrial Microbiology,
Department of Food Technology and Nutritional
Sciences,1 and
Laboratory of Organic
Chemistry,2 Wageningen Agricultural
University, Wageningen, The Netherlands
Received 10 March 1998/Accepted 15 June 1998
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ABSTRACT |
Higher fungi have a widespread capacity for biosynthesis of
organohalogens. Commonly occurring chloroaromatic fungal metabolites can end up in anaerobic microniches at the boundary of fungal colonies
and wetland soils. The aim of this study was to
investigate the environmental fate of a major fungal metabolite,
3,5-dichloro-p-anisyl alcohol, under anaerobic
conditions. This compound was incubated with methanogenic sludge
to study its biotransformation reactions. Initially,
3,5-dichloro-p-anisyl alcohol was readily demethylated in stoichiometric quantities to 3,5-dichloro-4-hydroxybenzyl
alcohol. The demethylated product was converted further via two routes: a biotic route leading to the formation of
3,5-dichloro-4-hydroxybenzoate and 2,6-dichlorophenol, as well as an
abiotic route leading to the formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane. In the first route, the
benzyl alcohol moiety on the aromatic ring was oxidized, giving
3,5-dichloro-4-hydroxybenzoate as a transient or accumulating product,
depending on the type of methanogenic sludge used. In sludge previously
adapted to low-molecular-weight lignin from straw, a part of the
3,5-dichloro-4-hydroxybenzoate was decarboxylated, yielding detectable
levels of 2,6-dichlorophenol. In the second route,
3,5-dichloro-4-hydroxybenzyl alcohol dimerized, leading to the
formation of a tetrachlorinated bisphenolic compound, which was
identified as bis(3,5-dichloro-4-hydroxyphenyl)methane. Since
formation of this dimer was also observed in incubations with
autoclaved sludge spiked with 3,5-dichloro-4-hydroxybenzyl alcohol, it
was concluded that its formation was due to an abiotic process.
However, demethylation of the fungal metabolite by biological processes
was a prerequisite for dimerization. The most probable reaction
mechanism leading to the formation of the tetrachlorinated dimer in the
absence of oxygen is presented, and the possible environmental
implications of its natural occurrence are discussed.
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INTRODUCTION |
Organohalogens are usually perceived
by the public as undesirable pollutants of anthropogenic origin.
However, 2,450 different naturally occurring halogenated compounds have
been identified so far (15). The higher fungi,
basidiomycetes, have a widespread capacity for biosynthesis of
organohalogens (39). Adsorbable organic halogens (AOX)
and/or low-molecular-weight halogenated compounds are produced by 68 genera of basidiomycetes from 20 different families (11).
Most of the 81 halogenated metabolites identified from basidiomycetes
to date are chlorinated, although brominated and iodated metabolites
have also been demonstrated.
The compound 3,5-dichloro-p-anisyl alcohol belongs to the
group of chlorinated anisyl metabolites (CAM) and is a major metabolite of fungi belonging to the genera Hypholoma,
Pholiota, Stropharia, Lepista,
Oudemansiella, Phellinus, Phylloporia,
and Bjerkandera (11). When species belonging to
these genera were grown in liquid culture media, the concentrations of
3,5-dichloro-p-anisyl alcohol ranged from 2.4 mg/liter in
cultures of Phellinus torulosus to 108.4 mg/liter in
cultures of Hypholoma elongatum (37).
H. elongatum (Pers. ex Fr.) Ricken is a fungal species
typical of wetlands, where it grows in moss (Sphagnum spp.
and Polytrichum spp.). The fungus is rather general in The
Netherlands, since it appears in 10 to 25% of the
5-km2-grid blocks investigated (3).
Hypholoma fasciculare is the most commonly occurring
species of the basidiomycetes in The Netherlands, since it was
observed in 69% of the 5-km2-grid blocks investigated and
showed the highest percentage (1.3%) of all mushroom sightings
(30). The 3,5-dichloro-p-anisyl alcohol concentrations of this fungus were up to 36.2 and 71.2 mg per liter in
nitrogen-limited and nitrogen-rich culture media, respectively (40). When cultivated on forest litter, concentrations of
3,5-dichloro-p-anisyl alcohol reached 204.9 mg per kg (dry
weight) of substrate after 84 days of incubation.
Direct measurements in the environment have also demonstrated that
3,5-dichloro-p-anisyl alcohol is an important fungal
compound. The concentrations of 3,5-dichloro-p-anisyl
alcohol and 3,5-dichloro-p-anisaldehyde in wood samples
colonized by Hypholoma spp. at forested sites ranged from 24 to 180 mg of CAM per kg (dry weight) of sample (10).
Estimations showed that the yearly production of AOX by H. fasciculare was 110 g of AOX per hectare of forest
(40). Most of the AOX is due to CAM compounds.
In wetlands with H. elongatum and wet forest soils with
H. fasciculare, 3,5-dichloro-p-anisyl
alcohol can end up in anaerobic microniches at the boundary of the
fungal colonies and soil. Except for the studies examining the
anaerobic biodegradability of 2,4-dibromophenol and other brominated
phenols produced by marine hemichordates (20, 35), nothing
is known about the environmental fate of natural organohalogens under
anaerobic conditions. The purpose of this study was to study the
environmental fate of the major fungal metabolite
3,5-dichloro-p-anisyl alcohol under anaerobic conditions.
This was done by incubating the compound with methanogenic sludge,
which was cultivated on either straw extracts or volatile fatty acids,
and studying its biotransformation reactions.
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MATERIALS AND METHODS |
Methanogenic sludge and culture conditions.
Granular
methanogenic sludge, previously cultivated on alkaline extracts of
wheat straw (WS-sludge), was obtained from S. Kortekaas, Department of
Environmental Technology, Wageningen Agricultural University,
Waginengen, The Netherlands. WS-sludge was grown for more than 1 year
in a 1-liter upward flow anaerobic sludge bed (UASB) reactor fed with a
filtered (cheesecloth) black liquor extract diluted to 8 g of
chemical oxygen demand (COD) per liter. The load of the reactor was
23 g of COD per liter per day. The black liquor extract was
prepared from 100 g of wheat straw and 10 g of
Na2CO3 in 1 liter of tap water (120°C for
2 h). The diluted black liquor extract was supplemented as
follows: yeast extract, 0.1 g/liter (Oxoid, Ltd., Basingstoke,
Hampshire, United Kingdom); NH4Cl, 0.28 g/liter;
KH2PO4, 0.25 g/liter;
MgSO4 · 7H2O, 0.1 g/liter;
CaCl2, 0.008 g/liter; trace elements solution, 1 ml; and
the antifoam agent Klaraid 4039, 0.05 ml (Grace Dearborn, Mijdrecht,
The Netherlands). The composition of the trace elements solution was
described by Zehnder et al. (43). WS-sludge was washed
with tap water and centrifuged (30,000 × g, 15 min) before use. One gram of fresh WS-sludge contained 0.0919 g of
volatile suspended solids (VSS). The ash content of the dry sludge
solids was 8.1%.
Granular methanogenic sludge, previously grown on volatile fatty acids
(VFA-sludge), was obtained from M. van Eekert, Department of
Environmental Technology, Wageningen Agricultural University, Wageningen, The Netherlands. VFA-sludge was grown for more than 3 years
in a 10-liter UASB reactor on a mixture of sodium acetate (18.6 mM),
sodium propionate (14.2 mM), and sodium butyrate (13 mM), supplemented
with minerals and trace elements according to the method of Alphenaar
(1). VFA-sludge was washed with tap water and centrifuged
(30,000 × g, 15 min) before use. One gram of fresh
VFA-sludge contained 0.1587 g of VSS, and the ash content of the dry
solids was 20.6%.
Biotransformation experiments.
3,5-Dichloro-p-anisyl alcohol was used as a model substrate.
The composition of the medium was as follows:
3,5-dichloro-p-anisyl alcohol, 0.35 g/liter;
NaHCO3, 2.50 g/liter;
(NH4)2HPO4, 0.07 g/liter;
MgSO4 · 7H2O, 0.04 g/liter; and sodium
acetate, 0.25 g/liter. The pH was adjusted to 7.2. Medium (25 ml in
100-ml serum bottles) was inoculated with 0.25 g of wet WS-sludge
or VFA-sludge and stoppered with n-butyl stoppers. Bottles
were made anaerobic by flushing with 70/30
N2-CO2 and were incubated on a rotary shaker (100 rpm) at 30°C in the dark. Bottles with substrate but lacking sludge and with sludge but lacking 3,5-dichloro-p-anisyl
alcohol were incubated in parallel to the biotransformation bottles and were harvested as controls. The biotransformation experiment was conducted in triplicate. Samples were taken daily. At the time of
sampling, separately incubated bottles were sacrificed for analysis.
Formation of bis(3,5-dichloro-4-hydroxyphenyl)methane. (i)
Experiment I.
In order to study the formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane, 3,5-dichloro-4-hydroxybenzyl
alcohol (0.1 g/liter) was incubated with deuterated 2,6-dichlorophenol.
Otherwise the composition of the medium was identical to that described
for the biotransformation experiments. The incubations of 100 ml of medium in 250-ml serum bottles were inoculated with 1.00 g of wet
WS-sludge, stoppered, flushed, and incubated as referred to above. Two
times per week, a concentrated solution of deuterated 2,6-dichlorophenol was added to the incubations, providing a final concentration of 5 mg per liter. Triplicate incubated bottles were
sacrificed for analysis two times per week and analyzed for concentrations of 3,5-dichloro-4-hydroxybenzyl alcohol, deuterated 2,6-dichlorophenol, and adduct with and without incorporated deuterium label.
(ii) Experiment II.
In a subsequent experiment,
3,5-dichloro-p-anisyl alcohol (0.25 g per liter) and
3,5-dichloro-4-hydroxybenzyl alcohol (0.25 g per liter) were separately
incubated with living and autoclaved WS-sludge under anaerobic and
aerobic conditions as well as sequential anaerobic-aerobic conditions.
The composition of the medium was the same as that in the
biotransformation experiments. The medium (100 ml in 250-ml serum
bottles) was inoculated with 1.00 g of wet WS-sludge, stoppered
with n-butyl stoppers, and made anaerobic by flushing with
70/30 N2-CO2. Half of the bottles were
autoclaved two times on subsequent days for 1 h at 121°C. All
bottles were incubated on a rotary shaker (100 rpm) at 30°C in the
dark. The bottles that were incubated aerobically were injected with 50 ml of pure O2. Control bottles with test compound but
lacking sludge were incubated in parallel and were harvested as
controls. After 10 days of incubation, half of the bottles initially
incubated anaerobically with living and autoclaved sludge were aerated
by injection of 50 ml of pure O2 per bottle, after which,
bottles were incubated again for 6 weeks. At the time of sampling,
bottles were sacrificed in duplicate for analyses of all chlorinated
compounds present.
Extraction procedure and compound identification.
Medium
with sludge was filtered over a paper filter (type V259; Schut BV,
Heelsum, The Netherlands), after which the filtrate and the filter with
the sludge were separated. The filtrate, after adjustment to pH 2 with
4 M H2SO4, was extracted three times with 10 ml
of freshly distilled ethyl acetate, and organic layers were combined. A
filter with sludge was subjected to overnight soxhlet extraction with
freshly distilled ethyl acetate. The filtrate and the filter-sludge
extracts were washed with water and concentrated under reduced pressure
at ambient temperature. The concentrate was filtered over silica gel 60 (230 to 400 mesh; Merck, Darmstadt, Germany) with ethyl acetate as the
eluent. After removal of the solvent under reduced pressure, the
residue was redissolved in 1 ml of ethyl acetate containing 410 µg of
4-bromoanisole as the internal standard. The substrate and derived
compounds were identified by gas chromatography (GC) analyses and,
incidentally, GC-mass spectrometry (MS). GC analyses were performed
with a Varian 3600 gas chromatograph equipped with a fused-silica
capillary column (DB17; 30 m by 0.25-mm internal diameter film
thickness, 0.25 µm) and a 1:1 end splitter with each split leading to
separate detectors. Parallel detection was carried out by flame
ionization detection (FID) and electron capture detection (ECD). The
carrier gas and flow were N2 at 1.2 ml per min. The
injector temperature was 220°C, the FID temperature was 230°C, the
ECD temperature was 275°C, and the temperature program was 70 to
250°C at 7°C per min and then hold for 20 min. The injection
volume was 10 µl. The split ratio was 1:100. GC-MS analyses were
performed on a HP5970B quadrupole mass spectrometer coupled to a
HP5890 gas chromatograph equipped with a fused-silica capillary
column (DB17; internal diameter; 30 m by 0.25-mm film
thickness, 0.25 µm). The carrier gas and flow were He at 1.1 ml per
min. The injector temperature was 220°C. The temperature program was
identical to that used for GC. The injection volume was 10 µl, and
the split ratio was 1:100. Electron impact-MS data were obtained at 70 eV. Identification of compounds was achieved by comparison of retention
times and mass spectra with data of synthetic reference compounds.
Reference compounds.
3,5-Dichloro-p-anisyl
alcohol was prepared as described by de Jong et al. (10).
3,5-Dichloro-4-hydroxybenzyl alcohol was prepared by reduction of
methyl 3,5-dichloro-4-hydroxybenzoate (Lancaster, Mühlheim am
Main, Germany) with LiAlH4 in ether. 3,5-Dichloro-4-hydroxybenzoic acid was purchased from Lancaster. 2,6-Dichlorophenol was purchased from Aldrich (Aldrich-Chemie, Steinheim, Germany). 2,6-Dichlorophenol-d2 was
prepared by carboxylating commercially available (Acros Organics, Geel,
Belgium) deuterated phenol-d5 in the
para position according to Komiyama and Hirai (21). The obtained deuterated 4-hydroxy benzoate was
chlorinated and subsequently decarboxylated, yielding
2,6-dichlorophenol-d2 as a white solid
(38).
The adduct bis(3,5-dichloro-4-hydroxyphenyl)methane was prepared by
purging a solution of 1.0 g (5.0 mmol) of
bis(4-hydroxyphenyl)methane in 30 ml of acetic acid with
Cl2 at room temperature. The mixture, which contained a
white precipitate, was stirred for 30 min and then filtered. The
residue was recrystallized from acetic acid to give 0.65 g (39%)
of the adduct as a white solid. The melting point, was 194 to 195°C
(184 to 185°C [6]). For 1H NMR (200 MHz,
aceton-d6), 
(ppm) was as follows: 3.85 (s, 2H, CH2) and 7.26 (s, 4H, C-2, C-2', C-6 and C-6'). For
13C NMR (aceton-d6), (ppm) were as follows:
39.2 (t, CH2), 122.6 (4s, C-3, C-3', C-5 and C-5'), 129.6 (4d, C-2, C-2', C-6, and C-6'), 134.9 (2s, C-1 and C-1'), and 148.4 (2s, C-4 and C-4'). For high-resolution MS, the value calculated for
C13H8Cl4O2
(M+) was 335.9278; that found was 335.9278. For MS,
m/e (relative intensities) were as follows [M + 4]+ (28); [M + 2]+ (58); and
[M]+ (46); 301 (100); 265 (16); 231 (37); 202 (17); 175 (29); 139 (23); 115 (26); 101 (41); 87 (13); and 75 (25).
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RESULTS |
Biotransformation experiment.
Biotransformation of
3,5-dichloro-p-anisyl alcohol by WS-sludge started
after 1 day of incubation (Fig.
1A). Initially, the methoxy-group was
demethylated quantitatively, yielding 3,5-dichloro-4-hydroxybenzyl alcohol as a product after 6 days of incubation. Thereafter, the benzyl
alcohol group was slowly oxidized, yielding
3,5-dichloro-4-hydroxybenzoic acid as a product. After 20 days of
incubation, most of the 3,5-dichloro-4-hydroxybenzyl alcohol formed was
eliminated. Part of the 3,5-dichloro-4-hydroxybenzoic acid formed was
subsequently decarboxylated, resulting in the formation of
2,6-dichlorophenol as a detectable product (Fig. 1B). The final
concentration of 2,6-dichlorophenol after 20 days of incubation
amounted to 7.5 mg/liter. A part of the initial substrate is
unaccounted for as metabolites produced at the end of incubation, which
might be due to minor metabolites not detected by the analytical
procedures applied. Moreover, further in this section, the occurrence
of a new tetrachlorinated bisphenolic compound in the incubation
bottles will be reported.
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FIG. 1.
Biotransformation of 3,5-dichloro-p-anisyl
alcohol by WS-sludge under anaerobic conditions. (A)
3,5-Dichloro-p-anisyl alcohol ( ),
3,5-dichloro- 4-hydroxybenzyl alcohol ( ), and
3,5-dichloro-4-hydroxybenzoic acid ( ). (B) 2,6-Dichlorophenol ( )
and bis(3,5-dichloro-4-hydroxyphenyl)methane in the aqueous phase
( ).
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In the biotransformation experiments with VFA-sludge, the conversions
of 3,5-dichloro-
p-anisyl alcohol were similar to those
found
with the WS-sludge (Fig.
2). However,
some differences were
observed. Demethylation started after 7 days and
was completed
after 13 days of incubation. Oxidation of the benzyl
alcohol group
was slower with the VFA-sludge than with WS-sludge. After
38 days
of incubation, 49% of the 3,5-dichloro-4-hydroxybenzyl alcohol
formed was still present in the incubation medium. In contrast
to the
incubations with WS-sludge, very little accumulation of
3,5-dichloro-4-hydroxybenzoic acid was observed in the incubations
with
the VFA-sludge. Concentrations of 3,5-dichloro-4-hydroxybenzoic
acid
were constant after 2 weeks of incubation and amounted to
approximately
9 mg/liter. Also in contrast to the incubations
with the WS-sludge, the
occurrence of 2,6-dichlorophenol was not
detected in the incubations
with VFA-sludge during the entire
experiment (Fig.
2B).
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FIG. 2.
Biotransformation of 3,5-dichloro-p-anisyl
alcohol by VFA-grown methanogenic sludge under anaerobic conditions.
(A) 3,5-Dichloro-p-anisyl alcohol (),
3,5-dichloro-4-hydroxybenzyl alcohol (), and
3,5-dichloro-4-hydroxybenzoic acid (). (B) 2,6-Dichlorophenol ()
and bis(3,5-dichloro-4-hydroxyphenyl)methane in the aqueous phase
().
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Formation of bis(3,5-dichloro-4-hydroxyphenyl)methane.
During
the biotransformation experiments with both sludges, a new
tetrachlorinated bisphenolic compound was observed in the aqueous
phase, which was identified as bis(3,5-dichloro-4-hydroxyphenyl)methane (Fig. 1B and 2B). The MS spectrum of this compound is given in Fig.
3. However, most of the compound was
found to be adsorbed to the sludge and was isolated by means of soxhlet
extraction with freshly distilled ethyl-acetate. The distributions of
the tetrachlorinated compound over the aqueous and solid phases towards the end of the incubations were comparable between the two sludges used
and were as follows: WS-sludge incubations, solid phase, 84.9 ± 3.1 mg/liter; aqueous phase, 12.0 ± 2.7 mg/liter;
and VFA-sludge incubations, solid phase, 78.9 ± 2.5 mg/liter; aqueous phase, 13.8 ± 1.8 mg/liter.
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FIG. 3.
MS spectrum of
bis(3,5-dichloro-4-hydroxyphenyl)methane, formed by incubation of
3,5-dichloro-4-hydroxybenzyl alcohol with
2,6-dichlorophenol-d2. The spectrum lacks ion
peaks, illustrating the incorporation of the deuterium label of
2,6-dichlorophenol-d2 into the product.
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In order to study the formation of the tetrachlorinated compound in
more detail, 3,5-dichloro-4-hydroxybenzyl alcohol was
incubated with
2,6-dichlorophenol-
d2 in the presence of
WS-sludge.
Incubations containing autoclaved WS-sludge were
treated similarly
to the bottles with living sludge and were
incubated in parallel
as controls. After 17 days of incubation, the
total amounts of
bis(3,5-dichloro-4-hydroxyphenyl)methane in the
aqueous and solid
phases were 6.7 and 50.4 mg/liter in the living and
autoclaved
sludge incubations, respectively. The
bis(3,5-dichloro-4-hydroxyphenyl)methane
formed did not contain the
deuterium label (Fig.
3), indicating
that 2,6-dichlorophenol was
not a building block of the tetrachlorinated
compound. Moreover,
deuterated 2,6-dichlorophenol was recovered
quantitatively. This result
also indicated that the formation
of
bis(3,5-dichloro-4-hydroxyphenyl)methane was due to an abiotic
process.
In a subsequent experiment, 3,5-dichloro-
p-anisyl alcohol
and 3,5-dichloro-4-hydroxybenzyl alcohol were incubated with living
and
autoclaved WS-sludge under anaerobic, aerobic, and successive
anaerobic
(first 10 days) and aerobic (6 weeks) conditions. The
results of these
incubations are given in Table
1. The
bis(3,5-dichloro-4-hydroxyphenyl)methane
was formed under all
incubation conditions with 3,5-dichloro-4-hydroxybenzyl
alcohol
as the substrate, including both living and autoclaved
sludge. On the
other hand, if the fungal metabolite
3,5-dichloro-
p-anisyl
alcohol was used directly, the
tetrachlorinated compound was only
formed in the incubations with
living sludges, which were incubated
anaerobically. With
3,5-dichloro-4-hydroxybenzyl alcohol as the
substrate, the yields of
bis(3,5-dichloro-4-hydroxyphenyl)methane
were higher if the sludge was
autoclaved and if oxygen was present.
With
3,5-dichloro-
p-anisyl alcohol as the substrate, the
succession
of anaerobic and aerobic incubation conditions provided a
higher
yield of the tetrachlorinated compound than was provided by the
completely anaerobic incubations.
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TABLE 1.
Formation of bis(3,5-dichloro-4-hydroxyphenyl)methane
from 3,5-dichloro-p-anisyl alcohol and
3,5-dichloro-4-hydroxybenzyl alcohol by wheat straw-grown methanogenic
sludge after 7.5 weeks of incubation
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DISCUSSION |
The environmental fate of CAM has not been the subject of any
study so far. Nevertheless, they are important organohalogen compounds
occurring in significant concentrations in the natural environment
(10). In this study, the anaerobic biotransformation of the
major fungal metabolite 3,5-dichloro-p-anisyl alcohol was evaluated. This metabolite is produced de novo up to concentrations of
108 mg per liter by H. elongatum (37). This
is an important fungus occurring in wetlands, where anaerobic
microniches are expected to adjoin the fungal colonies. The results
taken as a whole indicate that 3,5-dichloro-p-anisyl alcohol
is initially biotransformed in anaerobic environments via a
demethylation reaction to 3,5-dichloro-4-hydroxybenzyl alcohol. This
demethylated product is further converted via two routes. The first is
a biotic route leading to the formation of
3,5-dichloro-4-hydroxybenzoate and 2,6-dichlorophenol. The second is an
abiotic route leading to the formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane. The proposed overall
pathway is given in Fig. 4.
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FIG. 4.
Biotransformation pathway of
3,5-dichloro-p-anisyl alcohol by methanogenic sludge under
anaerobic conditions and the formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane.
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Demethylation.
The key step in the biotransformation of
3,5-dichloro-p-anisyl alcohol is the demethylation of the
methoxy moiety at the 4-position of the aromatic ring. The
demethylation occurred without any lag phase in the case of
WS-sludge and after 6 days of incubation, demethylation was
complete, quantitatively yielding 3,5-dichloro-4-hydroxybenzyl alcohol
as a product. The anaerobic bacteria of this sludge were precultivated
on a substrate containing low-molecular-weight lignin compounds from
wheat straw with similar methoxy moieties. This might explain the lack
of any lag phase. However, it was remarkable that even in the
incubations with VFA-sludge, which was grown on defined VFA for
more than 3 years, demethylation started after 1 week and was completed
after 2 weeks of incubation. Apparently, microorganisms with the
capacity to demethylate methoxy aromatics were still present and viable
in the VFA-sludge, since adaptation to the completely new substrate
occurred rapidly.
There are some reports of demethylation of chlorinated methoxy
aromatics in the literature. Phenyl methyl ethers such as vanillic
and
syringic acids are examples of low-molecular-weight products
derived
from fungal lignin depolymerization (
9,
22). These
compounds
were demethylated by acetogenic bacteria belonging to
the genera
Acetobacterium,
Eubacterium, and
Clostridium (
4,
7,
8).
Clostridium
thermoaceticum as well as other anaerobic
bacteria were shown to
be able to grow by using the methoxyl group
as an energy
source (
13,
29,
36). Also, chlorinated methoxy
aromatics can
be demethylated by anaerobic bacteria. Häggblom
et al.
(
16) reported the anaerobic O demethylation of
chlorinated
guaiacols by the acetogenic bacteria
Acetobacterium
woodii and
Eubacterium limosum. Anaerobic cell
suspensions of both
E. limosum and
A. woodii were
able to O demethylate di-, tri-, and tetrachloroguaiacols
to the
corresponding catechols, which accumulated and were not
further
metabolized. Liu and Jones (
24) reported that 2,3- and
3,5-dichloroanisole were initially demethylated by freshwater
sediment
slurries, producing 2,3- and 3,5-dichlorophenol.
The results from the literature therefore support the ubiquitous
occurrence of acetogenic bacteria in anaerobic environments,
which
could catalyze the rapid demethylation of
3,5-dichloro-
p-anisyl
alcohol. Once the original fungal
compound has been demethylated,
3,5-dichloro-4-hydroxybenzyl alcohol
was converted further via
two routes.
Route I. (i) Oxidation of benzyl alcohol.
In route I, a biotic
transformation of 3,5-dichloro-4-hydroxybenzyl alcohol
occurred, leading to 3,5-dichloro-4-hydroxybenzoate and
ultimately to 2,6-dichlorophenol. Similar anaerobic oxidations of
the benzyl alcohol moiety to benzoate have been studied in relation to
the anaerobic degradation of toluene. For the degradation of
toluene in denitrifying bacteria, several pathways have been proposed.
One of the proposed pathways, although disputed and contested, starts
with an oxidation of the methyl group via benzyl alcohol and
benzaldehyde to benzoate and further to benzoyl-coenzyme A (2,
23). Similar findings have also been reported under Fe(III)
reducing and methanogenic conditions (14, 25). Methyl group
oxidation by anaerobic bacteria has also been demonstrated with
phenolic compounds such as p-cresol (5, 18).
These studies showed that a Achromobacter sp. oxidized
p-cresol to p-hydroxybenzyl alcohol with
p-cresol methylhydroxylase, after which the latter compound
was oxidized further to p-hydroxybenzoate.
Neilson et al. (
31) studied the transformation of
3,5-dichloro-4-hydroxybenzaldehyde by metabolically stable anaerobic
enrichment
cultures. They reported that a small part of the
3,5-dichloro-4-hydroxybenzaldehyde
was reduced to
3,5-dichloro-4-hydroxybenzyl alcohol, but that
the majority was
oxidized to 3,5-dichloro-4-hydroxybenzoate after
70 days of
incubation. The results from Neilson et al. (
31)
indicate that perhaps 3,5-dichloro-4-hydroxybenzaldehyde was a
transient intermediate in the oxidation of
3,5-dichloro-4-hydroxybenzyl
alcohol to 3,5-dichloro-4-hydroxybenzoate,
although the aldehyde
was never detected in our studies.
(ii) Decarboxylation.
The second step in the formation of
2,6-dichlorophenol is decarboxylation of
3,5-dichloro-4-hydroxybenzoate. Zhang and Wiegel (45)
studied the anaerobic degradation of 3-chloro-4-hydroxybenzoate in
freshwater sediments and found that the degradation of the substrate
proceeds via either 2-chlorophenol or 4-hydroxybenzoate to phenol and
subsequently to benzoate. In both possible degradation routes, a
decarboxylation step is involved. In the study by Neilson et al.
(31), 3,5-dichloro-4-hydroxybenzoate, which accumulated from
the anaerobic oxidation of 3,5-dichloro-4-hydroxybenzaldehyde, was also
decarboxylated and converted to 2,6-dichlorophenol, in a similar
fashion to that observed in the incubations with WS-sludge in our
study. A Clostridium species, which was able to transform 4-hydroxy-benzoate and 3,4-dihydroxybenzoate and produced phenols as
the final transformation product, was isolated and characterized (44). Later, Zhang et al. (46) isolated the amino
acid-utilizing, hydroxybenzoate-decarboxylating bacterium
Clostridium hydroxybenzoicum from methanogenic
freshwater pond sediment, from which an oxygen-sensitive reversible 4-hydroxybenzoate decarboxylase was purified and
characterized (17).
(iii) Dechlorination.
Surprisingly, dechlorination of
2,6-dichlorophenol was not observed in these studies, although this
compound is readily dechlorinated under methanogenic conditions.
Possibly the incubation time of the experiments was too short for
activating and building up a dehalogenating microbial population.
Route II. Formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane.
In route II,
an abiotic dimerization of 3,5-dichloro-4-hydroxybenzyl
alcohol occurred, leading to the formation
of the adduct bis(3,5-dichloro-4-hydroxyphenyl)methane. Based on the chemical synthesis of bis(3-chloro-4-hydroxyphenyl) methane by a condensation reaction of 3-chloro-4-hydroxybenzyl alcohol and
2-chlorophenol (6), it was expected that 2,6-dichlorophenol was involved in the formation of this adduct. Therefore,
deuterium-labelled 2,6-dichlorophenol was added to
3,5-dichloro-4-hydroxybenzyl alcohol in the presence of living and
autoclaved WS-sludge. However, it was observed that the label of
2,6-dichlorophenol was not incorporated into the adduct. This indicated
that the adduct was most likely formed by a dimerization reaction of
3,5-dichloro-4-hydroxybenzyl alcohol. Next, it was observed that the
concentration of adduct in incubations spiked with
3,5-dichloro-4-hydroxybenzyl alcohol were much higher in the bottles
containing autoclaved sludge than in those containing living sludge,
which indicated that formation of the adduct was an abiotic process. In
the bottles containing living sludge, a competition between the biotic
and abiotic routes probably occurred, leading to a mixture of
biotransformation products and adduct. Although the dimerization
reaction itself was due to an abiotic process, the biological process
of 3,5-dichloro-p-anisyl alcohol demethylation was a
prerequisite for adduct formation. The presence of oxygen prevented the
formation of the adduct from 3,5-dichloro-p-anisyl alcohol
in living sludge, probably because the acetogenic bacteria involved in
the demethylation step are strict anaerobes (4). However,
oxygen was stimulatory for the dimerization reaction in bottles spiked
with 3,5-dichloro-4-hydroxybenzyl alcohol or if
3,5-dichloro-p-anisyl alcohol was first allowed to become
demethylated by prior anaerobic incubation.
Although the actual mechanism is not known, the formation of the adduct
most likely proceeds as outlined in Fig.
5. Under
the influence of acids or metal
salts present in the autoclaved
sludge, heterolysis of
3,5-dichloro-4-hydroxybenzyl alcohol to
a highly stabilized benzylic
carbocation can take place. Attack
of this electrophilic species on
another molecule of 3,5-dichloro-4-hydroxybenzyl
alcohol will then
result in the formation of a dimeric intermediate
which upon loss of
formaldehyde gives the adduct. The adduct formation
promoted by
oxygen can also be explained by this process, but
then radicals
are involved. From the incubations, it is clear
that the
presence of autoclaved sludge was essential for the formation
of
adduct, since no dimerization occurred in sterile medium alone.
It is
not known which organic compound or metal in the sludge
is responsible
for the adduct formation.
View larger version (15K):
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|
FIG. 5.
Proposed reaction mechanism for the formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane by dimerization of
3,5-dichloro-4-hydroxybenzyl alcohol.
|
|
Environmental implications.
Assuming that
bis(3,5-dichloro-4-hydroxyphenyl)methane is also formed
in natural environments, its formation might have some important
environmental implications. Wendel (41) tested the biological activity of the chemically produced adduct for use as
pesticide and found that it had strong bactericidal activities against
Staphylococcus aureus, Streptomyces albus, and
Streptococcus sp. The tetrachlorinated compound also had
antifungal activities (26). The adduct has a close
resemblance to some widely used pesticides.
Bis(5-chloro-2-hydroxyphenyl)methane (DCP) is a fungicide. This
compound is used on wooden structures in industrial facilities and was
reported to be very effective against many celluloytic fungi, which
included Penicillium, Aspergillus,
Cladosporium, and a Trichoderma sp.
(28). Hexachlorophene is
bis(3,4,6-trichloro-2-hydroxyphenyl)methane and is a
pesticide with many applications in animal health care. Hexachlorophene
was found to be very effective against Fasciola spp., which
are parasitic flatworms living in the livers of cows and sheep
and causing serious cattle diseases (33, 42).
Hexachlorophene was demonstrated in cow milk at about 8 ng/ml
in samples taken 24 h after oral administration of
hexachlorophene to cows (19). Matsumura et al.
(27) described that hexachlorophene hemolyzed washed
human erythrocytes and inhibited acetylcholinesterase activities in
erythrocyte membranes.
Bis(3,5-dichloro-4-hydroxyphenyl)methane also has a close
resemblance to nonchlorinated compounds with a bisphenolalkyl
structure.
Bisphenols, in particular
2,2-bis(4-hydroxy-phenyl)propane (bisphenol
A), are monomers of
various plastics, including polycarbonates
and epoxy resins, which are
used in numerous consumer products.
The release of bisphenol A
from some of these materials into the
food has recently been
reported (
32). Bisphenol A is an environmental
estrogen
(xenoestrogen) which belongs to a diverse group of estrogen-like
chemicals that mimic estrogenic actions. Bisphenol A has estrogenic
activity in vitro, probably caused by adverse effects on the
neuroendocrine
axis in susceptible human subpopulations
(
34). Gaido et al.
(
12) described how bisphenol A
can interact directly with steroid
hormone receptors in humans. In this
way, bisphenol A and related
compounds can bring disorder to the
hormone balance of the human
body, resulting in, among other effects,
declining fertility.
| |
ACKNOWLEDGMENTS |
This project was financially supported by the Technology
Foundation STW, Utrecht, The Netherlands, under project no. WLM33.3127, entitled "Fungal chlorinated aromatic metabolites: natural priority pollutants and dioxin precursors in the environment."
We thank Sjon Kortekaas and Miriam van Eekert, Department of
Environmental Technology, Wageningen Agricultural University, Wageningen, The Netherlands, for supplying wheat
straw-grown and VFA-grown methanogenic sludges. M. C. R. Franssen, Laboratory of Organic Chemistry, Wageningen Agricultural
University, Wageningen, The Netherlands, is acknowledged for valuable
suggestions and discussions concerning the mechanism of formation of
bis(3,5-dichloro-4-hydroxyphenyl)methane.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Industrial Microbiology, Department of Food Technology and Nutritional Sciences, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31 317 484976. Fax: 31 317 484978. E-mail:
Frank.Verhagen{at}imb.ftns.wau.nl.
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Applied and Environmental Microbiology, September 1998, p. 3225-3231, Vol. 64, No. 9
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Abstract
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