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Applied and Environmental Microbiology, December 2000, p. 5329-5333, Vol. 66, No. 12
Institut für Biogeochemie und
Meereschemie, Universität Hamburg, D-20146
Hamburg,1 UFZ, Umweltforschungszentrum
Leipzig-Halle GmbH, D-04318 Leipzig,3 and
Fakultät für Biologie, Universität
Konstanz, D-78457 Konstanz,2 Germany
Received 26 June 2000/Accepted 29 September 2000
Anaerobic degradation of 2-methylnaphthalene was investigated with
a sulfate-reducing enrichment culture. Metabolite analyses revealed two
groups of degradation products. The first group comprised two succinic
acid adducts which were identified as naphthyl-2-methyl-succinic acid
and naphthyl-2-methylene-succinic acid by comparison with chemically
synthesized reference compounds. Naphthyl-2-methyl-succinic acid
accumulated to 0.5 µM in culture supernatants. Production of
naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with
dense cell suspensions. The conversion of 2-methylnaphthalene to
naphthyl-2-methyl-succinic acid was detected at a specific activity of
0.020 ± 0.003 nmol min Aromatic hydrocarbons were
considered to be recalcitrant in the environment under anoxic
conditions until the first evidence of anaerobic BTEX degradation was
reported in 1985 (17). Toluene turned out to be the most
easily degradable aromatic hydrocarbon, and various pure bacterial
strains have been isolated since then. Toluene can be degraded with
nitrate, ferric iron, or sulfate as the electron acceptor, under
fermentative conditions, and by phototrophic bacteria (7, 8, 20,
21, 25, 32). The pathway of toluene degradation has been
investigated in detail for denitrifying bacteria of the genera
Azoarcus and Thauera, and it was shown that the
first degradation step is the addition of fumarate to the toluene
methyl group (3, 4, 9, 13). The reaction is catalyzed by
benzylsuccinate synthase, which belongs to the glycyl radical enzyme
family. The benzylsuccinate synthase reaction appears to be
representative of the degradation pathways of a number of environmental
pollutants, such as m-xylene, o-xylene, and
m-cresol, which all exhibit the addition of fumarate to the methyl group as the initial activation reaction (2, 16, 24). Further steps in anaerobic toluene degradation include a coenzyme A
(CoA) transferase reaction of benzylsuccinate with succinyl-CoA, generating benzylsuccinyl-CoA and succinate (19). In the
following reactions, benzylsuccinyl-CoA is probably oxidized through
beta-oxidation to benzoyl-CoA, which enters the benzoyl-CoA degradation pathway.
Anaerobic degradation of polycyclic aromatic hydrocarbons has been
demonstrated in a few microcosm studies (5, 6, 18, 23, 27),
and recently, naphthalene-degrading denitrifying and sulfate-reducing
cultures were reported (1, 10, 22, 26, 33). In experiments
with one marine and one freshwater culture, 2-naphthoic acid was the
major metabolite of anaerobic naphthalene degradation and was generated
by incorporation of bicarbonate into the carboxyl group (22,
33). The further degradation pathway might proceed via reduction
of the aromatic ring system in analogy to the benzoyl-CoA degradation
pathway, as reduced 2-naphthoic acid derivatives have been identified
in culture supernatants (22).
Here we report on the anaerobic degradation of 2-methylnaphthalene by a
sulfate-reducing enrichment culture from a freshwater sediment.
Metabolites were extracted from culture supernatants and analyzed by
gas chromatography-mass spectrometry (GC-MS). The first enzyme reaction
in the anaerobic 2-methylnaphthalene degradation pathway, catalyzed by
naphthyl-2-methyl-succinate synthase, was identified in dense cell suspensions.
Cultivation of bacteria.
A 2-methylnaphthalene-degrading,
sulfate-reducing bacterial culture was enriched from a contaminated
aquifer as described earlier (22). Subcultures were
inoculated with 10% volume of the liquid phase in 100-ml serum bottles
half filled with carbonate-buffered, sulfide-reduced freshwater medium,
pH 7.4, with trace element solution SL10 (29, 30). Solid
2-methylnaphthalene crystals were added (2 to 4 mg/50 ml) together with
10 mM sulfate as the electron acceptor. The bottles were flushed with
N2-CO2 (80/20), closed with Viton rubber
stoppers (Maag Technik, Dübendorf, Switzerland), and incubated at
30°C in the dark.
Analysis of metabolites.
Sample preparation, gas
chromatographic analysis, and GC-MS measurements were performed as
described previously (22). Culture growth was stopped with
100 mM NaOH, and samples were stored at
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Anaerobic Degradation of 2-Methylnaphthalene by a
Sulfate-Reducing Enrichment Culture
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 mg of
protein1 only in the presence of cells and fumarate. We
conclude that under anaerobic conditions 2-methylnaphthalene is
activated by fumarate addition to the methyl group, as is the case in
anaerobic toluene degradation. The second group of metabolites
comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives,
including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic
acid, and decahydro-2-naphthoic acid. These compounds were also
identified in an earlier study as products of anaerobic naphthalene
degradation with the same enrichment culture. A pathway for anaerobic
degradation of 2-methylnaphthalene analogous to that for anaerobic
toluene degradation is proposed.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C until metabolite
analysis. Naphthalene was extracted with hexane from the alkaline
sample. After acidification to pH 2.0 with 6 M hydrochloric acid, the
water phase was extracted three times with dichloromethane to isolate
carboxylic acids and aromatic alcohols. The combined dichloromethane
extracts were concentrated by vacuum evaporation, dried over anhydrous
sodium sulfate, and derivatized with ethereal diazomethane. The solvent
was removed by a gentle stream of nitrogen, and products were
transferred to hexane and analyzed by GC-MS.
Synthesis of naphthyl-2-methylene-succinic acid.
Synthesis
of naphthyl-2-methylene-succinic acid (compound VI in Fig.
1 and 2)
was performed according to references 11 and 28. Sodium metal (2.2 g of sodium, 96 mmol) was
dissolved in absolute methanol (70 ml of methanol) at 0°C under
nitrogen, followed by addition of diethylsuccinate (22.4 g, 128 mmol).
Naphthalene-2-carbaldehyde (10 g, 64 mmol) (Fluka) was dissolved in
absolute methanol (40 ml) and added dropwise within 40 min to the
diethylsuccinate-methanol solution, which was heated under reflux
cooling. After 2 h, NaOH (2 M, 160 ml) was added, and the mixture
was heated for a further 6 h. The solution was concentrated by
evaporation, HCl (37%, 40 ml) was added, and the aqueous phase was
extracted three times with ethyl acetate. The combined organic layers
were washed with saturated NaCl (twice, 50 ml each time), dried over
MgSO4, and concentrated under vacuum. After the addition of
hexane (30 ml) and benzene (30 ml), yellowish crystals precipitated.
They were collected by filtration and recrystallized with 30 ml of
ethanol (yield, 14.1 g [86%]; melting point, 188 to 189°C).
The product was identified with 1H nuclear magnetic
resonance (dimethyl sulfoxide [(NMR)
DMSO]-d6): 
3.5 (s, 2H), 7.5-7.6 (m, 2H),
7.9-8.0 (m, 6H), 12.6 (br s, 2H). NMR spectra were collected on a
Bruker AC250 instrument (Bruker Analytik, Rheinstetten, Germany).
Elemental analysis gave a C value of 69.9% and an H value of 4.7%.
According to the formula C15H12O4,
a C value of 70.3% and an H value of 4.7% were expected. GC-MS
analysis revealed two peaks (relative amounts, 1:34) with identical
mass spectra which are attributed to the E and Z isomers of
naphthyl-2-methylene-succinic acid (Fig. 1).
|
, fatty
acid methyl ester. Compounds marked with an asterisk are tentatively
identified according to their mass spectra.
|
Synthesis of naphthyl-2-methyl-succinic acid.
Naphthyl-2-methyl-succinic acid (compound V in Fig. 1 and 2) was formed
by catalytic reduction of naphthyl-2-methylene-succinic acid (7 g, 27.3 mmol) with palladium on activated carbon (0.7 g, 10% Pd) (Fluka), at a
hydrogen pressure of 100 kPa for 40 h (11). The
reaction mixture was filtered to remove the catalyst, and the filtrate
was concentrated under vacuum. Addition of hexane (1 ml) resulted in
precipitation of yellowish white crystals (6.3 g, 90%; melting point,
165 to 167°C). The product was identified with 1H NMR
(DMSO-d6): 2.2-2.3 (m, 1H), 2.4-2.5 (m,
1H), 2.9-3.1 (m, 3H), 7.3-7.4 (m, 1H), 7.4-7.5 (m, 2H), 7.7 (s, 1H),
7.8-7.9 (m, 3H), 12.3 (s, 2H). Elemental analysis gave a C value of
69.3% and an H value of 5.6%. According to the formula
C15H14O4, a C value of 69.8% and
an H value of 5.4% were expected. GC-MS analysis revealed only one
single peak (Fig. 1).
Enzyme tests. Naphthyl-2-methyl-succinate synthase was measured in dense cell suspensions which were prepared in the absence of dioxygen. Cells (150 ml) grown with 2-methylnaphthalene were collected by centrifugation under anoxic conditions (30 min, 16,000 × g) and resuspended in 1 ml of potassium phosphate buffer, 20 mM, pH 7.0, in a glove box. The buffer was reduced with 1 mM titanium(III) citrate (31). The cell suspension was injected into a 4-ml glass vial which was closed with a silicon rubber stopper and flushed with N2. 2-Methylnaphthalene (200 µM) was solubilized in the same titanium(III)-reduced potassium phosphate buffer, pH 7.0, with or without 2 mM fumarate, and incubated for 2 days at 30°C in half-filled closed glass vials under N2 to achieve equilibrium between the water and gas phases. The 2-methylnaphthalene-containing buffer (1 ml) was added to the cell suspension, and 2 ml of the gas phase was withdrawn with a syringe and exchanged with 2 ml of the gas phase from the 2-methylnaphthalene-containing buffer vial. The reaction vial was incubated at 30°C in the dark. Samples of 150 µl were taken with a syringe through the stopper, and 100 µl was mixed with 400 µl of ethanol (99.8%) and subjected to high-performance liquid chromatography (HPLC) analysis after removal of precipitates by centrifugation (5 min, 15,000 × g). Naphthyl-2-methyl-succinic acid concentrations were determined by HPLC analysis on a Beckman System Gold equipped with a C18 reversed-phase column and UV detection at 206 nm. Eluent was isocratic acetonitrile-100 mM ammonium phosphate buffer, pH 3.5 (40/60).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Growth with 2-methylnaphthalene. A sulfate-reducing culture that was enriched with naphthalene as the sole carbon and energy source was able to grow with crystalline 2-methylnaphthalene but not with 1-methylnaphthalene (22). The culture did not grow with 300 µM toluene as the sole carbon and energy source within 100 days of observation, either in the presence or in the absence of the solid adsorber resin Amberlite XAD7. XAD7 was used to provide the cells with toluene at a continuously low concentration, below toxic levels (30 to 50 µM). The identified metabolites naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid (see below) could also not be used as carbon sources at a concentration of 40 mg/liter.
Identification of metabolites. GC-MS analysis of metabolites from supernatants of 2-methylnaphthalene-grown cultures revealed two groups of metabolites. The first group consisted of the major metabolite naphthyl-2-methyl-succinic acid and of naphthyl-2-methylene-succinic acid (Fig. 1). Both compounds were identified by GC coinjection and comparison of the mass spectra with chemically synthesized reference substances (Fig. 2). The chromatogram of chemically synthesized naphthyl-2-methylene-succinic acid revealed two separate peaks with identical mass spectra probably representing the E and Z isomers. Both peaks were identified in culture supernatants (Fig. 1). The absolute configuration of the compounds was not determined. Naphthyl-2-methyl-succinic acid accumulated in culture supernatants up to 0.5 µM, as determined by HPLC.
The second group of metabolites was identified as 2-naphthoic acid and a series of reduced derivatives (Fig. 1) (for mass spectra see reference 22). 5,6,7,8-Tetrahydro-2-naphthoic acid and two isomers of octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The most reduced metabolites were two decahydro-2-naphthoic acid isomers (decalin-2-carboxylic acid). The compounds were identified by their mass spectra and by coelution with the chemically synthesized reference compounds (22).Naphthyl-2-methyl-succinate synthase activity.
The first
reaction step in anaerobic 2-methylnaphthalene degradation was analyzed
in dense cell suspensions. In the presence of 2-methylnaphthalene and
fumarate, a continuous production of naphthyl-2-methyl-succinate was
observed (Fig. 3A).
Naphthyl-2-methyl-succinate was identified by HPLC with a diode array
detector, by coelution with the chemically synthesized reference
compound, and by its UV-visible-light absorption spectrum (Fig. 3B).
Production of naphthyl-2-methyl-succinate was observed neither in the
absence of cells nor in the absence of fumarate, indicating that
fumarate is added to the methyl group of 2-methylnaphthalene as the
first reaction step. The specific naphthyl-2-methyl-succinate synthase activity of three independent cell suspension experiments was 0.020 ± 0.003 nmol min1 mg of
protein1. This represents 2.5% of the substrate turnover
rate in the growing culture.
|
), with fumarate in the
absence of cells (
), and in the presence of cells without fumarate
(
). (B) Absorption spectra of the naphthyl-2-methyl-succinic acid
fraction eluting from the HPLC reversed-phase column at 7.8 min. Solid
line, absorption spectrum of the chemically synthesized
naphthyl-2-methyl-succinic acid; dashed line, absorption spectrum of
the compound produced in dense cell suspensions.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study we report on the anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture and the identification of the first enzyme in the pathway, naphthyl-2-methyl-succinate synthase.
The 2-methylnaphthalene-degrading culture was enriched with naphthalene as sole carbon and electron source and sulfate as the electron acceptor (22). Subsequent substrate utilization tests revealed that the culture could also utilize 2-methylnaphthalene as a growth substrate.
In culture supernatants two groups of metabolites could be identified
by GC-MS analysis. The first series consisted of
naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid.
Both compounds are structural analogs of the first metabolites in
anaerobic toluene degradation, benzylsuccinic acid and phenylitaconic
acid (13). Thus, it is very likely that
naphthyl-2-methyl-succinate is generated by the addition of fumarate to
the methyl group of 2-methylnaphthalene, a mechanism similar to the
benzylsuccinate synthase reaction. Indeed, a fumarate addition to the
methyl group of 2-methylnaphthalene to yield naphthyl-2-methyl-succinic
acid could be confirmed in cell suspension experiments. The reaction
with 2-methylnaphthalene depended on the presence of cells and
fumarate. In the presence of all reactants, the specific activity was
0.020 ± 0.003 nmol min1 mg of
protein1. The naphthyl-2-methyl-succinate synthase
activity in the enzyme test represented 2.5% of the substrate turnover
rate in the growing culture, which is comparable to data for the
similar enzyme benzylsuccinate synthase obtained by other authors
(4). As the culture was not able to grow with toluene as the
sole carbon and energy source the measured enzyme reaction is not only
a side reaction of a toluene-degrading capacity of the culture but an
activity specifically related to 2-methylnaphthalene degradation. The
second identified metabolite was naphthyl-2-methylene-succinic acid,
which is probably generated by beta-oxidation of
naphthyl-2-methyl-succinic acid. A first step therein may be a
CoA-dependent activation by a succinyl-CoA transferase. This type of
reaction has been shown for anaerobic toluene degradation by the
denitrifying bacterium Thauera aromatica (19).
The second group of metabolites consisted of 2-naphthoic acid and reduced derivatives such as 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were identified in an earlier study as metabolites of anaerobic naphthalene degradation by the same culture (22). In addition, some of these metabolites have been identified from a marine sulfate-reducing culture growing with naphthalene as the carbon source (33). As the two cultures were able to grow with 2-naphthoic acid as the sole source of carbon and energy, 2-naphthoic acid is likely to be a central intermediate in anaerobic degradation of naphthalene and 2-methylnaphthalene. The presence of the reduced derivatives indicates that the further degradation pathway of 2-naphthoic acid is probably initiated by ring reduction, in analogy to the anaerobic benzoyl-CoA pathway, perhaps after CoA-dependent activation (12, 14, 15). However, in the anaerobic benzoyl-CoA degradation pathway the aromatic ring is not completely reduced before water addition to initiate ring cleavage. Likewise, ring fission of the bicyclic system must not necessarily proceed via decahydro-2-naphthoic acid. The more reduced compounds may as well be dead-end metabolites. Nevertheless, the identification of common metabolites of both substrates suggests that anaerobic degradation of naphthalene and that of 2-methylnaphthalene share a common degradation pathway which is initiated by a reduction of the polycyclic aromatic ring system. At present, we do not know if ring cleavage is initiated from a monoaromatic ring system or if both rings are reduced before water addition.
Based on the present data, we propose an upper pathway for anaerobic
degradation of 2-methylnaphthalene which is analogous to anaerobic
degradation of toluene and other methylbenzenes (Fig. 4) (13, 19). In a first
activation step, fumarate is added to the methyl group of
2-methylnaphthalene by naphthyl-2-methyl-succinate synthase, probably
through a radical mechanism, in analogy to anaerobic toluene
degradation. Naphthyl-2-methyl-succinic acid is likely to be activated
by a succinyl-CoA-dependent CoA transferase and subsequent oxidation to
yield naphthyl-2-methylene-succinyl-CoA. The following sequence of
reactions proceeds via beta-oxidation and leads to the central
intermediate 2-naphthoic acid CoA-ester.
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ACKNOWLEDGMENTS |
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We are grateful to Bernhard Schink for continuous support.
Financial support by the Deutsche Forschungsgemeinschaft for parts of this work is gratefully acknowledged (grants Mi 157/11-3 and Schi 180/7-3).
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FOOTNOTES |
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* Corresponding author. Mailing address: Fakultät für Biologie, Universität Konstanz, Universitätsstr. 10, D-78457 Konstanz, Germany. Phone: 49-7531-884541. Fax: 49-7531-882966. E-mail: rainer.meckenstock{at}uni-konstanz.de.
Publication 101 of the Deutsche Forschungsgemeinschaft priority
program 546, Geochemical Processes with Long-Term Effects in
Anthropogenically Affected Seepage and Groundwater.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, December 2000, p. 5329-5333, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
Villanueva, L., Haveman, S. A., Summers, Z. M., Lovley, D. R.
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