Anaerobic Cometabolic Conversion of Benzothiophene by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer

Abstract

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [¹³C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the ¹³C content of the carboxyl group were formed, indicating the addition of a C₁ unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.

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

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 liter⁻¹). 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 (5α-cholestane; 10 mg liter⁻¹ 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.

For growth experiments with ¹³C-labeled bicarbonate, 10 ml of freshwater medium was supplemented with 20 mM sodium ¹³C-bicarbonate (Sigma, St. Louis, Mo.). Due to carryover of ¹²C-bicarbonate from the inoculum and from the gas phase, the resulting buffer consisted of a mixture of 50% ¹²C-bicarbonate and 50% ¹³C-bicarbonate. The medium was further treated as described above.

The cultures were incubated without shaking at 30°C in the dark, and microbial activity was monitored by measuring an increase in the sulfide concentration (9). Samples were removed with a syringe through the stoppers. Biological activity was stopped with 0.1 M NaOH in the samples used for metabolite analyses.

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 5α-cholestane at 10 mg liter⁻¹ in hexane).

For detection of sulfur-containing organic compounds, a sulfur-selective flame photometric detector (FPD) was used simultaneously with a FID on a stream splitter (1:1) connected to the capillary column.

GC-MS measurements were performed with a Hewlett Packard 6890 gas chromatograph coupled with a Quattro II mass spectrometer (Micromass, Manchester, United Kingdom). The gas chromatograph was equipped with a 30-m capillary column (0.32-mm inner diameter, 0.25-μm film thickness, DB-5; J & W Scientific), and helium was used as the carrier gas. Temperature programs were the same as for GC analyses.

The following MS conditions were applied: ionization mode, electron impact ionization energy, 70 eV; emission current, 200 μA; source temperature, 180°C; and mass range, m/z 50 to 400.

For identification of metabolites, instrumental library searches of the NIST/NIH/EPA mass spectral database (National Institute of Standards and Technology, National Institutes of Health, and U.S. Environmental Protection Agency), comparison with published mass spectra, and coinjection with commercially available and chemically synthesized authentic reference compounds were used.

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).

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

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).

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.

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.

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.

All microbially produced carboxybenzothiophene isomers accumulated during cometabolism experiments with naphthalene as a growth substrate and benzothiophene as an auxiliary substrate. After 37, 79, and 86 days, extracts revealed a continuous increase in the concentrations of the carboxybenzothiophenes relative to the naphthalene metabolites 2-naphthoic acid and 5,6,7,8-tetrahydro-2-naphthoic acid. The traces of metabolites in the culture supernatants at day 1 were derived from carryover from the inoculum. At day 86, approximately 3% of the initially applied benzothiophene (2 mg) was oxidized to carboxybenzothiophene isomers, as calculated by the concentrations of the carboxythiophenes produced.

As a further transformation product, dihydrocarboxybenzothiophene was tentatively identified by the mass spectrum and by evidence of the sulfur heteroatom in GC-FPD analyses (Fig. 4C). The reduced derivative was found in many but not in all analyzed extracts. The mass spectrum of the methyl ester of dihydrocarboxybenzothiophene was indicative of an aromatic acid, as assumed from the strong intensity of the molecular ion (M⁺) and the fragment at M⁺ − OCH₃ (m/z 194 and m/z 163). Thus, an isomer with a carboxylic group at a benzylic position is expected. In contrast to the three nonreduced aromatic carboxybenzothiophene isomers, only one dihydrocarboxybenzothiophene isomer was found, provided the separation of putative isomers under the applied GC conditions.

The formation of carboxybenzothiophenes was investigated by using a [¹³C]bicarbonate buffer in the growth medium. MS analyses demonstrated the incorporation of the ¹³C label from bicarbonate into the carboxylic group of the carboxybenzothiophenes, as shown by the mass shift of 1 mass unit in fragments containing the carboxylic group of 2-carboxybenzothiophene (m/z 161 to 162 and m/z 192 to 193) (Fig. 4D). Due to the carryover of significant amounts of nonlabeled bicarbonate from the inoculum, the mass spectrum represents a mixture of nonlabeled and ¹³C-labeled acids. The mass fragment representing the mere benzothiophene core after splitting off of the carboxylic group (m/z 133) did not show this mass shift.

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).

TABLE 1.

Concentrations of selected contaminants and corresponding aromatic acids in highly contaminated groundwater (well B14) of Testfeld Süd and in less contaminated groundwater (well B42) sampled ca. 130 m downstream of the contamination source

Compound	Concn (μg liter−1) in well:
	B14	B42a
Toluene	230	1
o-Xylene	400	1
m,p-Xylene	530	2
Indane	1210	1
Indene	2260	9
Naphthalene	5880	12
Benzothiophene	430	2
Benzoic acid	3	+
2-Methylbenzoic acid	5	+
3-Methylbenzoic acid	9	−
4-Methylbenzoic acid	5	−
5-Indanoic acid	470	+
2-Naphthoic acid	5	+
1-Naphthoic acid	5	+
Carboxybenzothiophene isomersb	54	1
Dihydrocarboxybenzothiophene	220	−

^a+, detected by MS analyses in the single-ion mode, but the concentration was <1 μg liter⁻¹; −, not detected by MS analyses in the single-ion mode. 

^bSum of the concentrations of compounds I, II, and III in Fig. 3. 

In addition to well B14, groundwater was sampled approximately 130 m downstream of the groundwater flow path, in well B42, which was located within the contamination plume, as indicated by tracer experiments (5). Well B42 contained significantly lower concentrations of naphthalene (5,880 to12 μg liter⁻¹) and benzothiophene (430 to 2 μg liter⁻¹) than well B14. Traces of acidic metabolites, e.g., benzoic acid, naphthoic acid, and indanoic acid isomers, were found in the downstream well when single-ion-mode GC-MS was used, but the concentrations of these compounds were too low for quantification (<1 μg liter⁻¹). Also, the concentrations of 5-indanoic acid and dihydrocarboxybenzothiophene, which were apparently enriched in well B14 compared to metabolites such as 2-naphthoic acid, were not enhanced in downstream well B42. The concentration of the tentatively identified dihydrocarboxybenzothiophene was below the detection limit, whereas traces of nonreduced carboxybenzothiophene isomers were determined at up to 1 μg liter⁻¹.

DISCUSSION

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.