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

Abstract

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.

FIG. 1

Partially reconstructed GC-MS total ion chromatograms. Top, chemically synthesized naphthyl-2-methyl-succinic acid (V) and naphthyl-2-methylene-succinic acid (VI) (as dimethyl esters). Bottom, methylated extracts from supernatants of cultures grown with 2-methylnaphthalene. I, isomers of decahydro-2-naphthoic acid; II, isomers of octahydro-2-naphthoic acid; III, 2-naphthoic acid; IV, 5,6,7,8-tetrahydro-2-naphthoic acid. , fatty acid methyl ester. Compounds marked with an asterisk are tentatively identified according to their mass spectra.

FIG. 2

Mass spectra of the chemically synthesized reference compounds naphthyl-2-methyl-succinic acid (V) and naphthyl-2-methylene-succinic acid (VI) (as dimethyl esters).

FIG. 3

Analysis of the naphthyl-2-methyl-succinate synthase reaction in dense cell suspensions catalyzing the addition of fumarate to 2-methylnaphthalene. (A) Naphthyl-2-methyl-succinic acid production in the presence of cells and 1 mM fumarate (□), with fumarate in the absence of cells (○), and in the presence of cells without fumarate (▵). (B) Absorption spectra of the naphthyl-2-methyl-succinic acid fraction eluting from the HPLC reversed-phase column at 7.8 min. Solid line, absorption spectrum of the chemically synthesized naphthyl-2-methyl-succinic acid; dashed line, absorption spectrum of the compound produced in dense cell suspensions.

FIG. 4

Proposed scheme of the upper pathway of anaerobic 2-methylnaphthalene (1) degradation to the central intermediate 2-naphthoic acid (8). 2, fumaric acid; 3, naphthyl-2-methyl-succinic acid; 4, naphthyl-2-methyl-succinyl-CoA; 5, naphthyl-2-methylene-succinyl-CoA; 6, naphthyl-2-hydroxymethyl-succinyl CoA; 7, naphthyl-2-oxomethyl-succinyl-CoA. Compounds marked with an asterisk were identified in the present study as free acids.