Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3, 4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.     

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture

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 microM 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(-1) mg of protein(-1) 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.     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.     

Anaerobic degradation of p-xylene in sediment-free sulfate-reducing enrichment culture

Anaerobic degradation of p-xylene was studied with sulfate-reducing enrichment culture. The enrichment culture was established with sediment-free sulfate-reducing consortium on crude oil. The crude oil-degrading consortium prepared with marine sediment revealed that toluene, and xylenes among the fraction of alkylbenzene in the crude oil were consumed during the incubation. The PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene for the p-xylene degrading sulfate-reducing enrichment culture showed the presence of the single dominant DGGE band pXy-K-13 coupled with p-xylene consumption and sulfide production. Sequence analysis of the DGGE band revealed a close relationship between DGGE band pXy-K-13 and the previously described marine sulfate-reducing strain oXyS1 (similarity value, 99%), which grow anaerobically with o-xylene. These results suggest that microorganism corresponding to pXy-K-13 is an important sulfate-reducing bacterium to degrade p-xylene in the enrichment culture.     

Anaerobic oxidation of n-dodecane by an addition reaction in a sulfate-reducing bacterial enrichment culture

We identified trace metabolites produced during the anaerobic biodegradation of H(26)- and D(26)-n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C---H or C---D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H(26)- and D(26)-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated n-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H(26)-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.     

The role of iron in enhancing anaerobic toluene degradation in sulfate-reducing enrichment cultures

Ferrous iron enhanced the toluene degradation rate of sulfidogenic enrichment cultures inoculated with contaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Ferrous iron had an analogous effect on the degradation rate of benzoic acid, a transient metabolite of anaerobic toluene degradation in these cultures, when benzoic acid was used as a sole carbon and energy source. Two hypotheses were proposed to explain iron's effect: (a) Iron may have prevented sulfide toxicity via precipitation of sulfide as FeS, and (b) iron might have been a limiting nutrient required for degradation (i.e., amendments of iron could have compensated for iron removed from solution by precipitation as FeS). To test these hypotheses, substrate degradation rates were compared in the presence of FeSO₄ (a sulfate source that both precipitates sulfide species and precludes iron limitation) versus ZnSO₄ (a sulfate source that precipitates sulfide species but does not preclude iron limitation) versus MgSO₄ (a sulfate source that neither precipitates sulfide nor precludes iron limitation). For both toluene and benzoic acid, FeSO₄ and ZnSO₄ were comparable in their enhancement of substrate degradation rates and were superior to MgSO₄ in that respect. Thus, iron appears to ameliorate sulfide toxicity, not nutritional iron limitation, in these cultures. The observation that ethylenediaminetetraacetic acid, a chelating agent capable of retaining iron in solution in the presence of sulfide, did not stimulate the cultures is consistent with this conclusion. The implications of these results for bioremediation of fuel-contaminated aquifers that contain sulfate-reducing bacteria are discussed. Correspondence to: H.R. Beller.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

Enzymatic reactions in anaerobic 2-methylnaphthalene degradation by the sulphate-reducing enrichment culture N 47

HscC, a DnaK homolog in Escherichia coli, consists of adenosine triphosphatase (ATPase), substrate-binding and C-terminal domains. Overexpression of HscC markedly inhibits growth of host cell and reduces the sigma(70)-dependent promoter activity presumably by forming a complex with sigma(70). To identify the region(s) of HscC responsible for growth inhibition and complex formation with sigma(70), domain swapping experiments were carried out between DnaK and HscC. Thus the chimeric proteins carrying the ATPase domain of HscC and substrate-binding domains of either HscC or DnaK were found to inhibit the growth of the cell, reduce the sigma(70)-dependent promoter activity and form a complex with sigma(70). These results indicate that the ATPase domain of HscC rather than the substrate-binding domain is important for determining its functional specificity.     

Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype

The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the 'candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation.     

Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture

Anaerobic benzene degradation is an important process in contaminated aquifers but is poorly understood due to the scarcity of microbial cultures for study. We have enriched a ferric iron-reducing culture that completely mineralizes benzene to CO₂. With ¹³C₆-labelled benzene as the growth substrate, ring-labelled benzoate was identified as a major intermediate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of culture supernatants. With increasing incubation time, ¹³C₇-labelled benzoate appeared, indicating that the carboxyl group of benzoate derived from CO₂ that was produced from mineralization of labelled benzene. This was confirmed by growing the culture in ¹³C-bicarbonate-buffered medium with unlabelled benzene as the substrate, as the label appeared in the carboxyl group of benzoate produced. Phenol was also identified as an intermediate at high concentration. However, it was clearly shown that phenol was formed abiotically by autoxidation of benzene during the sampling and analysis procedure as a result of exposure to air. The results suggest that, in our culture, anaerobic benzene degradation proceeds via carboxylation and that caution should be exercised in interpreting hydroxylated benzene derivatives as metabolic intermediates of anaerobic benzene degradation.     

Enhanced anaerobic degradation of benzene by enrichment of mixed microbial culture and optimization of the culture medium

A heterogeneous mixed culture, originally collected from two different sources, namely cow-drug and sludge from the mineral medium containing 1% glucose and then adapted on benzene as the carbon and energy source. Under anaerobic conditions benzene was degraded via benzoic acid as a major intermediate in the benzene degradation pathway. The degradation rate of benzene was improved stepwise by the number of enrichments and optimization of the culture medium. The effects of microaerobic conditions and/or physicochemical treatment with H₂O₂ prior to anaerobic degradation were studied with respect to variations in benzene degradation rate, growth of biomass and gas produced is less than the theoretical value expected and the percentage of methane in the product gas was very small (3%–3.5%). The reason for this is not well understood but it is presumed that the major group of benzene-degrading bacteria present in the culture medium are sulphate reducers and the mixed consortium is unable to degrade certain complex aromatic intermediates in the benzene degradation pathway under the experimental conditions. For an actual explanation of the situation arising in this study, further investigations must be carrie out. However, the mixed culture is capable of oxidizing benzene more rapidly to intermediate compounds and also partly into gas under the culture conditions, compared to the published data for the anaerobic degradation of benzene.     

Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia.

The anaerobic biodegradation of naphthalene (NAP) and phenanthrene (PHE) was investigated by using sediment collected from the Arthur Kill in New York/New Jersey harbor. The initial cultures were composed of 10% sediment and 90% mineral medium containing 20 mM sulfate. Complete loss of NAP and PHE (150 to 200 muM) was observed after 150 days of incubation. Upon refeeding, NAP and PHE were utilized within 14 days. The utilization of both compounds was inhibited in the presence of 20 mM molybdate. [14C]NAP and [14C]PHE were mineralized to 14CO2. The activities could be maintained and propagated by subculturing in mineral medium. In the presence of halogenated analogs, 2-naphthoate was detected in NAP-utilizing enrichments. The mass spectrum of the derivatized 2-napththoate from the enrichment supplemented with both [13C]bicarbonate and NAP indicates the incorporation of 13CO2 into NAP. In the PHE-utilizing enrichment, a metabolite was detected by both high-pressure liquid chromatography and gas chromatography-mass spectrometry analyses. The molecular ion and fragmentation pattern of its mass spectrum indicate that it was phenanthrenecarboxylic acid. The results obtained with [13C] bicarbonate indicate that 13CO2 was incorporated into PHE. It appears, therefore, that carboxylation is an initial key reaction for the anaerobic metabolism and NAP and PHE. To our knowledge, this is the first report providing evidence for intermediates of PAH degradation under anaerobic conditions.     

Stable isotopic studies of n-alkane metabolism by a sulfate-reducing bacterial enrichment culture

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C6 to C12) and 1-13C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d14-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-13C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13C nuclei from 1-13C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.     

Acidophilic degradation of methanol by a methanogenic enrichment culture

Abstract An acidophilic methanogenic enrichment culture was obtained in a continuous up-flow anaerobic sludge blanket reactor operated at pH 4.2 with methanol as the sole carbon source. The specific methylotrophic methanogenic activity of the enriched reactor sludge at pH 5 was 3.57 g COD g⁻¹ volatile suspended solids day⁻¹ and the apparent doubling time of the biomass was 15.8 h. Acidic conditions were obligatory, since the enrichment culture was not able to produce methane or to grow at pH 7. Based on morphological characteristics, the dominant methanogenic species in the enrichment culture was a Methanosarcina .     

Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.     

Reduction of perchlorate by an anaerobic enrichment culture

Summary A mixed bacterial culture capable of reducing perchlorate stoichiometrically to chloride under naerobic conditions was enriched from municipal digester sludge. The reduction of 10 mM perchlorate resulted in oxidation of the medium and cessation of perchlorate reduction. The activity was recovered on addition of a reducing agent. Addition of air to the culture during perchlorate reduction immediately terminated the process and aeration for 12 h permanently destroyed the ability of the culture to reduce perchlorate. The culture also reduced nitrite, nitrate, chlorite, chlorate and sulfate. The presence of 10 mM nitrite or chlorite completely inhibited perchlorate reduction, whereas the same concentration of chlorate decreased the reduction rate. Nitrate or sulfate did not affect perchlorate reduction. Chlorate and chlorite, suspected intermediates in the reduction of perchlorate to chloride, were not detected in any cultures during reduction of perchlorate.     

Anaerobic cometabolic conversion of benzothiophene by a sulfate-reducing enrichment culture and in a tar-oil-contaminated aquifer.

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 [(13)C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the (13)C content of the carboxyl group were formed, indicating the addition of a C(1) 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.