Use of 13C nuclear magnetic resonance to assess fossil fuel biodegradation: fate of [1-13C]acenaphthene in creosote polycyclic aromatic compound mixtures degraded by bacteria

Abstract

[1-13C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled 13C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows 13C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1-13C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs) and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From 13C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by naphthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain Pseudomonas sp. strain A2279 mitigated the accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of the substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.

FIG. 1

Representative ¹³C-NMR spectra of extractable compounds obtained in biodegradation experiments with Pseudomonas sp. strain BR after 2 days of incubation of [1-¹³C]acenaphthene (20% 1-¹³C, 10 mg/25 ml) and naphthalene (10 mg/25 ml) (A) and after 7 days of incubation of creosote PACs (25 mg) spiked with 1 mg of [1-¹³C]acenaphthene (B). For assignment of resonances A to G, see Fig. 2. T, tetramethylsilane; S, solvent (CDCl₃); N, signals due to the natural abundance of aromatic [¹³C]carbon atoms in acenaphthene and creosote PACs.

FIG. 2

Assignment of ¹³C-NMR signals to detected metabolites of the pathway for acenaphthene degradation by CREOMIX culture and individual strains BR and BC. •, ¹³C-labeled carbons.

FIG. 3

Representative ¹³C-NMR spectra (expanded baseline region) of extractable compounds obtained in biodegradation experiments with creosote PACs (25 mg) spiked with 1 mg of [1-¹³C]acenaphthene. (A) Day zero; (B) CREOMIX culture, 3 days of incubation; (C) CREOMIX culture, 14 days of incubation; (D) CREOMIX culture plus Pseudomonas sp. strain A2279, 3 days of incubation; (E) CREOMIX culture plus Pseudomonas sp. strain A2279, 14 days of incubation. For assignment of the signals, see Fig. 2 and the legend to Fig. 1.