INTRODUCTION

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Heterocyclic aromatic compounds constitute an important fraction of petroleum- and coal-derived tar oils and account for approximately 5% of creosote (32). Among the sulfur heterocyclic compounds, benzothiophene and dibenzothiophene usually dominate and are the major sulfur-containing compounds in crude oil. Investigations of microbial degradation of benzothiophene and dibenzothiophene have focused on (i) the characterization of biochemical degradation reactions and microbial desulfurization processes to remove sulfur from petroleum and (ii) the inhibitory effects of thiophenes on the degradation of other compounds.

So far, only cometabolic degradation in the presence of an additional primary substrate has been reported for the microbial degradation of benzothiophene (6). Two primary aerobic transformation reactions have been described: dioxygenase-catalyzed diol formation followed by extradiol cleavage, analogous to aerobic naphthalene degradation by pseudomonads (11, 14, 15), and the oxidation of the sulfur atom, resulting in a sulfone (16, 18, 33).

Several environmental studies reported heterocyclic compounds to inhibit the aerobic and anaerobic degradation of other aromatic compounds, indicating their significance in mixed contaminations such as oil-tar (3, 12, 13, 24, 27, 30). On the other hand, effective degradation of benzothiophene in a mixture with other aromatic compounds was observed in a contaminated aquifer (7).

Fewer data are available on the anaerobic degradation of benzothiophene, although aquifers polluted with aromatic hydrocarbons usually become anoxic as a consequence of the high oxygen demand for the degradation of organic compounds. Some investigations gave evidence for the anaerobic degradation of benzothiophene and dibenzothiophene leading to the production of hydrogen sulfide (22, 26). Biphenyl was the major product of dibenzothiophene degradation by the sulfate-reducing bacterium Desulfovibrio desulfuricans strain M6 (21). In that study, dibenzothiophene probably served as an electron acceptor for anaerobic respiration, a process which has been described as well for other organosulfur compounds (23). In an aquifer-derived methanogenic microcosm, mononuclear aromatic and alicyclic transformation products of benzothiophene, such as p-hydroxybenzene sulfonic acid, phenylacetic acid, benzoic acid, phenol, cyclohexane carboxylic acid, 2-hydroxythiophene, and others, have been observed (17).

The present study focused on the cometabolic anaerobic conversion of benzothiophene with naphthalene as a primary substrate. The formation of metabolites by a sulfate-reducing enrichment culture is discussed, and laboratory results are compared with the fate of benzothiophene in a tar-oil-contaminated aquifer.

	MATERIALS AND METHODS

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Sampling site. The study site was a former gas plant area (Testfeld Süd) located in southwestern Germany. The gas manufactory was run between 1870 and 1970. Due to multiple spills of coal tar and its distillation products, contaminants are widespread over the site. Large amounts of nonaqueous-phase liquids and a contamination plume about 300 m long were found in the aquifer. The quaternary aquifer is highly heterogeneous, resulting in complicated groundwater flow paths (5). Groundwater was sampled in February 1998 at well B14, located in the source area of the contamination plume, and at well B42, positioned approximately 130 m further downstream. The groundwater was anoxic over the whole site and showed a negative redox potential of less than 300 mV in the source area, where significant sulfate depletion and the formation of hydrogen sulfide indicated sulfate reduction.

Sampling methods. Groundwater was sampled with submersible pumps (Conrad, Hamburg, Germany). After the well volume was exchanged at least once and a constant redox potential could be measured in the collected water, samples were placed in glass flasks (0.1 and 1 liter) closed with Teflon-sealed caps. Solid sodium hydroxide was added to stop biological activities (final concentration, 0.1 M).

Extraction procedures. (i) Aromatic hydrocarbons. For hydrocarbon analyses, 0.1 liter (B14) or 1 liter (B42) of groundwater was extracted in duplicate immediately after sampling with 10 ml (B14) or 5 ml (B42) of hexane containing an internal standard (10 mg of perdeuterated phenanthrene-d₁₀ liter¹). Extracts were stored at 4°C until analyses with gas chromatography (GC) and GC-mass spectrometry (MS).

(ii) Metabolite analyses. Groundwater samples or cultures were stored under alkaline conditions (sodium hydroxide, 0.1 M) at 4°C until extraction. Samples were extracted twice with hexane to remove aromatic hydrocarbons. After acidification of the residual water phase to pH 2 with hydrochloric acid (6 M), metabolites were extracted three times with diethyl ether. The combined extracts were concentrated by vacuum evaporation and dried over anhydrous sodium sulfate. Residual solvent was removed by a gentle stream of nitrogen, and 80 µl of methanol and 10 µl of trimethylchlorosilane were added immediately for methylation of carboxylic acid groups. The solution was heated at 75°C for 1 h. After the solution was cooled to room temperature, 1 ml of demineralized and preextracted water (pH 2) was added; the water phase was extracted two times with 1 ml of hexane and once with 1 ml of diethyl ether. The combined extracts were dried over anhydrous sodium sulfate and purified over a silica gel column (70/230 mesh, 0.5 by 5 cm). The metabolites were eluted with diethyl ether and concentrated under a gentle stream of nitrogen, and an internal standard (5-cholestane; 10 mg liter¹ in hexane) was added.

Chemicals. Solvents and other chemicals of analytical grade were obtained from E. Merck AG (Darmstadt, Germany). Solvents were distilled before use. Reference compounds and internal standards for GC-MS analyses were obtained from Aldrich (Steinheim, Germany).

Organisms and growth conditions. The sulfate-reducing culture N47 was enriched from soil of the study site, Testfeld Süd, with naphthalene as the sole carbon and energy source in the presence of the solid adsorber resin Amberlite-XAD7 (Fluka, Buchs, Switzerland) (31). The organisms were grown in 100-ml serum bottles half filled with bicarbonate-buffered mineral medium (pH 7.3) (38). The medium was reduced with 1 mM sodium sulfide, and 10 mM sodium sulfate was added as an electron acceptor. The headspace was flushed with N₂-CO₂ (80:20), and the bottles were closed with butyl rubber stoppers (Maag Technik, Dübendorf, Switzerland). The substrate naphthalene (15 mg) and auxiliary substrates, such as benzothiophene (2 mg), were dissolved in 1 ml of heptamethylnonane and injected with a syringe through the stoppers.

GC and GC-MS. GC analyses were performed with a Carlo Erba Fractovap 4160 apparatus equipped with a 60-m capillary column (0.32-mm inner diameter, 0.25-µm film thickness, DB-5; J & W Scientific) and a flame ionization detector (FID). Hydrogen was used as the carrier gas. The temperature program for the analysis of hydrocarbons was 40°C (3 min isothermal), 40 to 220°C (4°C/min), 220 to 310°C (15°C/min), and 300°C (10 min isothermal). For metabolite analyses, the program was 80°C (3 min isothermal), 80 to 220°C (2°C/min), 220 to 300°C (15°C/min), and 300°C (10 min isothermal). The injection mode was on-column. An internal standard was applied for quantification (perdeuterated phenanthrene-d₁₀ or 5-cholestane at 10 mg liter¹ in hexane).

Synthesis of 5-carboxybenzo[b]thiophene (compound 3). 5-Carboxybenzo[b]thiophene (compound 3) was synthesized in a three-step procedure from p-bromothiophenol (Fig. 1).

View larger version (6K): [in this window] [in a new window]   FIG. 1.   Synthesis of 5-carboxybenzo[b]thiophene (compound 3) from p-bromothiophenol. Compound 1, 1-bromo-4-(2,2-dimethoxyethylsulfanyl)-benzene; compound 2, 5-bromobenzo[b]thiophene. NaOMe, sodium methoxide; MeOH, methanol; PPA, polyphosphoric acid; PhCl, monochlorobenzene; Mel, methyl iodide.

(i) Synthesis of 1-bromo-4-(2,2-dimethoxyethylsulfanyl)-benzene (compound 1). To a solution of 57 mmol of sodium methoxide in 50 ml of methanol, 10 g (53 mmol) of p-bromothiophenol and 6.7 g (58 mmol) of dimethoxy-2-bromoethane were added in one portion at room temperature. The reaction mixture was refluxed for 4 h. After evaporation of the solvent, the remaining liquid was distilled under reduced pressure to give 13.91 g (95%) of 1-bromo-4-(2,2-dimethoxyethylsulfanyl)-benzene (compound 1) as a colorless liquid boiling at 98°C (0.013 kPa).

(ii) Synthesis of 5-bromobenzo[b]thiophene (compound 2). For the synthesis of compound 2 (36), 13.91 g (50.4 mmol) of p-1-bromo-4-(2,2-dimethoxyethylsulfanyl)-benzene (compound 1) was added to a refluxing mixture of 20 ml of polyphosphoric acid in 250 ml of monochlorobenzene. After 27 h of reflux, the brownish liquid was decanted from the polyphosphoric acid and washed twice with water, and the aqueous phase was extracted twice with dichloromethane. After the sample was dried, the solvent was removed under reduced pressure and the remaining dark oil was distilled at 1.3 kPa to yield 7.44 g (70%) of 5-bromobenzo[b]thiophene (compound 2), which crystallized upon standing at room temperature.

(iii) Synthesis of 5-carboxybenzo[b]thiophene (compound 3). A mixture of 0.74 ml (11.9 mmol) of methyl iodide and 1.26 g (5.9 mmol) of 5-bromobenzo[b]thiophene (compound 2) was slowly added to 0.41 g (19.3 mmol) of magnesium turnings in 20 ml of anhydrous diethyl ether; gentle boiling of the solvent was maintained. The mixture was refluxed for 30 min, cooled to room temperature, treated with a large excess of pulverized carbon dioxide, and allowed to stand for 12 h. The remaining solid was treated with 50 ml of hydrochloric acid (1 M) and extracted three times with diethyl ether. After solvent removal, 1.1 g of crude 5-carboxybenzo[b]thiophene (compound 3) was furnished as a pale yellow powder. Recrystallization from a water-ethanol (3:1) mixture gave 518 mg of analytically pure 5-carboxybenzo[b]thiophene (compound 3) with a melting point of 210°C. The identity of the compound was proven with ¹H nuclear magnetic resonance analysis.

	RESULTS

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Cometabolic conversion of benzothiophene. The sulfate-reducing culture N47 was grown in parallel with the same amount of naphthalene as a primary substrate and the polycyclic or heterocyclic aromatic compounds acenaphthene, phenanthrene, anthracene, fluorene, fluoranthene, benzothiophene, and dibenzothiophene as auxiliary substrates. The primary substrate, naphthalene, was oxidized in all cases, as indicated by the production of significant amounts of sulfide (Fig. 2). Inhibitory effects on sulfide production or growth with naphthalene in the parallel cultures were observed only in the benzothiophene-containing culture. However, neither the analysis of produced sulfide nor the analysis of aqueous concentrations of auxiliary substrates would be accurate enough to identify potential oxidation of minor amounts of auxiliary substrates in the range of a few percentage points. Therefore, the cultures were analyzed for possible metabolites of cometabolic conversion of the different auxiliary substrates, but only with benzothiophene could a number of degradation products be identified. Within the observation period of 100 days, the enrichment culture N47 could not grow with benzothiophene as the only carbon source (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Sulfide production from naphthalene oxidation by enrichment culture N47. A representative growth experiment is shown for parallel cultures containing as a substrate(s) naphthalene only (closed squares) and naphthalene plus one of the auxiliary substrates, phenanthrene (circles), anthracene (triangles), fluorene (inverted triangles), acenaphthene (diamonds), fluoranthene (stars), benzothiophene (open squares), and dibenzothiophene (crosses).

Identification of metabolites in sulfate-reducing enrichment cultures. Extracts of the sulfate-reducing enrichment culture N47 grown with naphthalene and benzothiophene as an auxiliary substrate exhibited the same naphthalene metabolites as those described in an earlier study on anaerobic naphthalene degradation (2, 29). 2-Naphthoic acid and reduced compounds, such as tetrahydro-, octahydro-, and decahydro-2-naphthoic acid isomers, were identified. In addition to the naphthalene-derived metabolites, three isomeric benzothiophene transformation products identified as carboxybenzothiophenes were observed (Fig. 3). GC analyses with a sulfur-specific detector (FPD) verified the presence of the sulfur heteroatom in these transformation products. Two isomers eluted close together under the applied GC conditions (compounds II and III; Fig. 3B). The mass spectra of the three carboxybenzothiophene methyl esters were almost identical, differing only in the relative intensities of their mass fragments (Fig. 4A and B). Comparison with the methyl esters of commercially available 2-carboxybenzothiophene and chemically synthesized 5-carboxybenzothiophene revealed that compound I was identical to 2-carboxybenzothiophene and that compound II was identical to the 5-carboxy isomer, as verified by identical mass spectra and GC retention times in coelution experiments.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Partial gas chromatogram from cultures grown on naphthalene with benzothiophene as an auxiliary substrate and extracted after 0 (A), 37 (B), 79 (C), and 86 (D) days. I, II, and III, isomers of methyl esters of carboxybenzothiophenes (black peaks); IV, 2-naphthoic acid methyl ester; V, 5,6,7,8-tetrahydro-2-naphthoic acid methyl ester. For more structures, see Fig. 4.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Mass spectra of benzothiophene derivatives extracted from a sulfate-reducing culture grown with naphthalene as a carbon source and benzothiophene as an auxiliary substrate. (A) Methyl ester of 2-carboxybenzothiophene (compound I in Fig. 3). (B) Methyl ester of 5-carboxybenzothiophene (compound II in Fig. 3). (C) Methyl ester of tentatively identified dihydrocarboxybenzothiophene (not shown in Fig. 3). (D) Mass spectrum of 2-carboxybenzothiophene (compound I) extracted from a culture grown on naphthalene with benzothiophene as an auxiliary substrate in a medium buffered with [13C]bicarbonate.

Field studies. To assess in situ transformation processes, the contaminated groundwater was analyzed for benzothiophene and possible metabolites. Major groundwater pollutants near the contamination source (well B14) were monoaromatic hydrocarbons and low-molecular-weight polycyclic aromatic hydrocarbons, such as benzene, toluene, ethylbenzene, xylene, indane, indene, naphthalene, and benzothiophene, constituting a typical contaminant pattern for former gas plant sites (Table 1). Polar compounds were dominated by aromatic acids structurally related to the observed aromatic hydrocarbons, such as benzoic acid, methylbenzoic acids, and naphthoic acids. Aromatic acids were found in significantly lower concentrations than hydrocarbons, except for the two major components, 5-indanoic acid and a sulfur heterocyclic compound tentatively identified as dihydrocarboxybenzothiophene, which reached significant concentrations of several hundred micrograms per liter. Coinjection experiments and identical mass spectra identified the same dihydrocarboxybenzothiophene in the groundwater extracts as in the laboratory experiments. In addition, the corresponding nonreduced carboxybenzothiophene isomers that accumulated in culture supernatants during the laboratory cometabolism experiments were identified in the groundwater extracts (Table 1).

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	DISCUSSION

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Aromatic acids are well-known by-products of anaerobic degradation of unsubstituted or alkylated aromatic hydrocarbons (10, 19, 39). The sulfate-reducing enrichment culture N47 used in this study produced 2-naphthoic acid as a central intermediate during the degradation of naphthalene and 2-methylnaphthalene, together with a number of reduced bicyclic carboxylic acids deriving from 2-naphthoic acid (2, 29, 31). In addition, three gas chromatographically separable carboxybenzothiophene isomers accumulated when culture N47 was grown with naphthalene as a carbon and energy source and benzothiophene as an auxiliary substrate. Two isomers were identified, 2-carboxybenzothiophene and 5-carboxybenzothiophene. In addition, reduced dihydrocarboxybenzothiophene was tentatively identified on the basis of the mass spectrum.

It was shown in earlier studies on anaerobic naphthalene degradation with [¹³C]bicarbonate-buffered growth medium that the carboxyl group of 2-naphthoic acid is generated by the incorporation of a C₁ unit from bicarbonate or carbon dioxide (29, 39). Here, we observed that when benzothiophene was presented as an auxiliary substrate in such labeling experiments with [¹³C]bicarbonate, the carboxyl group of the generated carboxybenzothiophenes was also labelled. Thus, cometabolic conversion of benzothiophene by the addition of a C₁ unit, analogous to the transformation of naphthalene to 2-naphthoic acid, is likely. However, the first enzymatic reaction in anaerobic naphthalene degradation has neither been measured nor been investigated in detail, and it is not clear yet by which enzyme mechanism the first activating step proceeds. The occurrence of 2- and 5-carboxybenzothiophenes indicates a rather nonspecific reaction for benzothiophene conversion, involving an attack at the benzene as well as the thiophene ring.

The recovered carboxybenzothiophenes accounted for 3% of the added benzothiophene and for only 0.4% of the supplied growth substrate, naphthalene. Thus, benzothiophene appears to be a rather poor cometabolic substrate for the naphthalene-degrading enzymes. The enzymes later in the pathway, which are responsible for the further degradation of the structural analogue 2-naphthoic acid, could at least reduce carboxythiophene, but the total oxidation of benzothiophene to CO₂ is unlikely, as carboxybenzothiophenes accumulated and the culture could not grow with benzothiophene as the primary substrate. On the other hand, benzothiophene appears to be a potent inhibitor of naphthalene-degrading cultures, as sulfide development upon naphthalene degradation and growth of the culture were significantly reduced. These results could be due to either reversible or irreversible inhibition of the involved enzymes or to toxic effects of benzothiophene or one of the metabolites produced. An action of benzothiophene as a competitive inhibitor, together with occasional conversion to produce carboxybenzothiophene, could account for the delayed growth but would not explain the drastically reduced sulfide development. In this case, the culture should produce the same amount of sulfide as the control after reaching the stationary growth phase.

More data are available for the anaerobic degradation of nitrogen heterocyclic compounds than for the anaerobic degradation of sulfur heterocyclic compounds. The successful isolation of the sulfate-reducing bacterium Desulfobacterium indolicum showed that nitrogen heterocyclic compounds can serve as a carbon source for growth under anoxic conditions (4). The activation of heterocyclic compounds, such as indole or quinoline, and their methylated isomers has been shown to proceed via anaerobic hydroxylation as the initial transformation step. The hydroxylation of indole to 2-oxoindole has been reported for cultures with nitrate (28), sulfate (20, 35), or CO₂ (37) as an electron acceptor. Accordingly, the hydroxylation of quinoline led to 2-hydroxyquinolinone with sulfate and CO₂ as electron acceptors (25, 34). Johansen et al. (20) described further hydrogenation of 2-hydroxyquinolinone to 3,4-dihydroquinolinone.

In the study reported here, hydroxylation products of the sulfur heterocyclic compound benzothiophene could not be detected. In addition to the two carboxybenzothiophene isomers, only reduced dihydrocarboxybenzothiophene was identified. No other transformation products, e.g., a further reduced derivative in analogy to anaerobic naphthalene degradation or ring cleavage products, could be identified in the culture supernatants.

Abiotic oxidation of methylbenzothiophene to the corresponding carboxybenzothiophene isomers has been reported for methylbenzothiophenes exposed to oxygen and light (1, 8). In the present study, abiotic oxidation can be assumed neither under the applied laboratory conditions nor in groundwater from the contaminated site, because in both cases the conditions were anoxic, strongly reducing, and dark. In addition, abiotic oxidation to carboxybenzothiophenes could be shown only for methylbenzothiophenes and not for unsubstituted benzothiophene.

The fate of benzothiophene in contaminated aquifers and anoxic environments, in particular, has been investigated only poorly. In the study reported here, the tar-oil-contaminated aquifer showed the same carboxybenzothiophene species as those identified in the benzothiophene degradation experiments with the sulfate-reducing enrichment culture N47, suggesting microbial conversion of benzothiophene in the aquifer. The intrinsic formation of the same carboxybenzothiophene isomers as in the laboratory experiments suggests that the same degradation mechanisms were responsible for benzothiophene transformation in situ and in vitro. If benzothiophene was present in the aquifer in high enough concentrations, then it is likely that naphthalene degradation was inhibited.

The distribution pattern for aromatic acids in the contaminated groundwater (well B14) indicated a significant accumulation of carboxybenzothiophenes in this zone of the aquifer. Together with 5-indanoic acid, dihydrocarboxybenzothiophene was enriched by about 2 orders of magnitude compared to the other aromatic acids, and its concentration was in the same range as that of the parent compound, benzothiophene. Although the identified aromatic acids, such as methylbenzoic acid, are likely to be degradation products of the corresponding aromatic hydrocarbons, such as xylene, they could just as well be derived from other sources and their appearance could be coincidental. Such could also be the case for the carboxybenzothiophenes detected, although the present knowledge of the pathways of anaerobic degradation of aromatic hydrocarbons suggests that the aromatic acids were generated from the corresponding parent compounds in situ.

So far, acidic metabolites are not part of common benzene, toluene, ethylbenzene, or xylene or polycyclic aromatic hydrocarbon monitoring programs, but the significant accumulation of carboxybenzothiophenes in the studied aquifer emphasizes their ecological relevance and the need to characterize their fate in the environment.