Naphthalene degradation and incorporation of naphthalene-derived carbon into biomass by the thermophile Bacillus thermoleovorans

Abstract

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60 degrees 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 chromatography-mass 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.

FIG. 1

Growth of B. thermoleovorans Hamburg 2 on naphthalene. Growth was performed aerobically in a batch reactor with 3 mM naphthalene in a mineral medium at 60°C.

FIG. 2

IRMGC-MS chromatograms of the hydrolyzable amino acid fraction of an experiment with nonlabeled naphthalene compared to a degradation study with ¹³C-labeled naphthalene. ALA, alanine; GLY, glycine; THR, threonine; VAL, valine; LEU, leucine; ILE, isoleucine; PRO, proline; ASP, aspartic acid; GLU, glutamic acid; PHE, phenylalanine; LYS, lysine.

FIG. 3

(Top panel) Partial reconstructed total ion chromatogram of the fatty acid fraction (as methyl esters). C₁₅ to C₁₈ indicate the number of carbon atoms. i, iso (ω2-methyl alkanoic acid); a, ante-iso (ω3-methyl alkanoic acid); n, linear alkanoic acid. (Bottom panel) Mass spectrum of the iso C_17:0 fatty acid methyl ester highly enriched in ¹³C. The frame shows the comparison with an experiment with unlabeled naphthalene.

FIG. 4

Mass spectrum of ¹³C-labeled 2-carboxycinnamic acid. Compared to the unlabeled compound, the masses of the molecular peak and the respective fragment peaks are increased by one mass unit. y axis, intensity; x axis, m/z.

FIG. 5

Metabolites of naphthalene formed by mesophilic and thermophilic microorganisms (hypothetical scheme of precursor product relationship).