Identical ring cleavage products during anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin indicate a new metabolic pathway

Abstract

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 beta-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-bicarbonate-buffered 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 benzoyl-coenzyme 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.

FIG. 1.

Mass spectra of tetrahydro-2-naphthoic acids (as methyl esters) identified in supernatants of the sulfate-reducing enrichment culture N47 grown on different substrates. (A) Mass spectrum of commercially available 1,2,3,4-tetrahydro-2-naphthoic acid. (B to D) Mass spectra of the tentatively identified 5,6,7,8-tetrahydro-2-naphthoic acid produced in anaerobic degradation experiments with nonlabeled tetralin (B), [1-¹³C]naphthalene (C), and nonlabeled naphthalene in a [¹³C]bicarbonate-buffered medium (D).

FIG. 2.

Mass spectra of the dimethyl ester of the C₁₁H₁₆O₄-diacid metabolite produced during anaerobic degradation of nonlabeled naphthalene (A), per-deuterated naphthalene-d₈ (B), and per-deuterated naphthalene-d₈ in a [¹³C]bicarbonate-buffered medium (C). For chemical structure, see Fig. 5 (compound VII). MW, molecular weight.

FIG. 3.

Mass spectra of the dimethyl esters of 2-carboxycyclohexylacetic acid produced in anaerobic degradation experiments with nonlabeled naphthalene (A), [1-¹³C]naphthalene (B), per-deuterated naphthalene-d₈ (C), and nonlabeled naphthalene in a [¹³C]bicarbonate-buffered medium (D) used as substrates. MW, molecular weight.

FIG. 4.

Mass spectrum of the dimethyl ester of the chemically synthesized reference compound cis-2-carboxycyclohexylacetic acid (11). MW, molecular weight.

FIG. 5.

Proposed reductive 2-naphthoic acid pathway of anaerobic PAH degradation. Compounds A to E depict substances of the upper degradation pathways leading to either 2-naphthoic acid or 5,6,7,8-tetrahydro-2-naphthoic acid (for tetralin degradation). Compounds labeled with I to VIII depict metabolites of the reductive 2-naphthoic acid degradation pathway. Compounds: A, naphthalene; B, 2-methylnaphthalene; C, tetralin; D, fumaric acid; E, naphthyl-2-methyl-succinic acid; I, 2-naphthoic acid; II, 5,6,7,8-tetrahydro-2-naphthoic acid; III, octahydro-2-naphthoic acid (position of the double bond is unknown); IV, decahydro-2-naphthoic acid; V, hydroxydecahydro-2-naphthoic acid; IV, β-oxo-decahydro-2-naphthoic acid; VII, C₁₁H₁₆O₄-diacid (cis/trans configuration not determined); VIII, cis-2-carboxycyclohexylacetic acid.