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.Polycyclic aromatic hydrocarbons (PAH) are hazardous compounds which are found on various contaminated sites such as former gas plant sites or mineral oil refineries. Due to their obvious persistence in anoxic environments, PAH have been considered to be recalcitrant under anoxic conditions. However, anaerobic degradation of naphthalene, methylnaphthalene, phenanthrene, and a few more PAH has been demonstrated in microcosm experiments converting trace amounts of radioactively labeled substrates to CO 2 (8,9,20,23,26). Nitrate, sulfate, or ferric iron served as the terminal electron acceptor. Whereas anaerobic degradation of toluene and ethylbenzene has been investigated with several pure cultures (1,4,10,11,15,21,22,24), attempts to cultivate anaerobic PAHdegrading bacteria have failed for a long time (18). A sulfatereducing enrichment culture of marine origin growing with naphthalene as the sole carbon and energy source has been reported recently (31). 2-Naphthoic acid and phenanthroic acid were identified as metabolites of naphthalene and phenanthrene degradation, respectively. [ 13 C]bicarbonate was incorporated into the carboxylic group of 2-naphthoic acid, and it was assumed that a carboxylation reaction was the initial step. Since the culture could also use this compound as a carbon source, it was suggested to be the first intermediate in anaerobic naphthalene degradation...

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 M 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.

The influence of microbial degradation on the 13C/12C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors alphaC for toluene degradation by Pseudomonas putida (1.0026 +/- 0.00017), Thauera aromatica (1.0017 +/- 0.00015), Geobacter metallireducens (1.0018 +/- 0.00029) and the sulphate-reducing strain TRM1 (1.0017 +/- 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar 13C/12C isotope fractionation factor (alphaC = 1.0015 +/- 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil-hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a 13C/12C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions.

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Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C 1 unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C 11 H 16 O 4 (C 11 H 16 O 4 -diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by ␤-oxidation of one of the side chains of the C 11 H 16 O 4 -diacid. Stable isotope-labeling growth experiments with either 13 C-labeled naphthalene, per-deuterated naphthalene-d 8 , or a 13 C-bicarbonatebuffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoylcoenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO 2 , was determined by the application of [1-13 C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatographymass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.The naphthalene metabolism of mesophilic microorganisms under aerobic conditions has been intensely investigated, and detailed information has been presented on degradation rates, metabolic pathways, and the involved enzymes (7,8,11,25). In contrast, little is known about the metabolism of naphthalene or other polycyclic aromatic hydrocarbons (PAH) by thermophilic bacteria. Several studies on the growth of thermophilic microorganisms on aromatic compounds such as benzoic acid, cresols, or phenols have been carried out; however, respective degradation pathways are largely unresearched (1,4,19,20). The degradation of xenobiotics by thermophilic microorganisms provides crucial advantages compared to mesophilic organisms, especially when they are applied in biotechnological processes. Limited bioavailability as a result of the low water solubility of hydrophobic contaminants may be overcome due to a higher water solubility at elevated temperatures. The water solubility of naphthalene, for example, rises from 30 mg liter Ϫ1 at 20°C to 130 mg liter Ϫ1 at 60°C (26). Moreover, diffusion rates increase at higher temperatures with an additional positive impact on bioavailability.The bacteria applied in this degradation study were isolated from a compost consisting of wooden ties treated with lignite tar. They were able to utilize naphthalene as a sole source of carbon and energy. Stable isotope labeled [1-13 C]naphthalene was used as a model contaminant. The fate of naphthalene was traced by means of the technique of 13 C isotope analysis, which has been successfully applied to trace metabolic pathways (3,18,22). Stable isotope labeling enabled us to trace quantitatively the transformation of the xenobiotic carbon into specific fractions such as CO 2 , biomass, and metabolites. Moreover, the incorporation of the xenobiotic carbon into the bacterial fatty and amino acid fraction was determined on a molecular level. We describe here str...

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.

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