Biotransformation of the high-molecular weight polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene by Sphingobium sp. strain KK22 and identification of new products of non-alternant PAH biodegradation by liquid chromatography electrospray ionization tandem mass spectrometry

Abstract

A pathway for the biotransformation of the environmental pollutant and high-molecular weight polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene by a soil bacterium was constructed through analyses of results from liquid chromatography negative electrospray ionization tandem mass spectrometry (LC/ESI(-)-MS/MS). Exposure of Sphingobium sp. strain KK22 to benzo[k]fluoranthene resulted in transformation to four-, three- and two-aromatic ring products. The structurally similar four- and three-ring non-alternant PAHs fluoranthene and acenaphthylene were also biotransformed by strain KK22, and LC/ESI(-)-MS/MS analyses of these products confirmed the lower biotransformation pathway proposed for benzo[k]fluoranthene. In all, seven products from benzo[k]fluoranthene and seven products from fluoranthene were revealed and included previously unreported products from both PAHs. Benzo[k]fluoranthene biotransformation proceeded through ortho-cleavage of 8,9-dihydroxy-benzo[k]fluoranthene to 8-carboxyfluoranthenyl-9-propenic acid and 9-hydroxy-fluoranthene-8-carboxylic acid, and was followed by meta-cleavage to produce 3-(2-formylacenaphthylen-1-yl)-2-hydroxy-prop-2-enoic acid. The fluoranthene pathway converged with the benzo[k]fluoranthene pathway through detection of the three-ring product, 2-formylacenaphthylene-1-carboxylic acid. Production of key downstream metabolites, 1,8-naphthalic anhydride and 1-naphthoic acid from benzo[k]fluoranthene, fluoranthene and acenaphthylene biotransformations provided evidence for a common pathway by strain KK22 for all three PAHs through acenaphthoquinone. Quantitative analysis of benzo[k]fluoranthene biotransformation by strain KK22 confirmed biodegradation. This is the first pathway proposed for the biotransformation of benzo[k]fluoranthene by a bacterium.

Figure 1

Results of LC/ESI(–)-MS(/MS) analyses of extracts from strain KK22 exposure to benzo[k]fluoranthene.A. Absorbance at 254 nm.B. Fragmentation pattern acquired from product ion scan analysis of the deprotonated molecule [M – H]⁻ = 315.C. Extracted ion chromatograms from full scan analyses that compare the relative abundances of the biotransformation product that corresponded to the deprotonated molecule [M – H]⁻ = 261 as detected in exposed cells and abiotic and biotic controls.D. Fragmentation pattern acquired from product ion scan analysis of the deprotonated molecule [M – H]⁻ = 261 of exposed cells from C above. Molecular structures proposed for each product are shown. Details are given in Table 1.

Figure 2

Results of LC/ESI(–)-MS/MS product ion scan analyses of extracts from strain KK22 exposure to benzo[k]fluoranthene. Fragmentation patterns acquired from the deprotonated molecules: (A) [M – H]⁻ = 265; (B) [M – H]⁻ = 223; (C) [M – H]⁻ = 239; (D) [M – H]⁻ = 229; and (E) an authentic standard of 1,8-naphthalic anhydride, [M – H]⁻ = 229. Molecular structures proposed for each product are shown. Details for all acquisitions are given in the text and Table 1.

Figure 3

Pathways proposed for the biotransformation of the HMW PAHs benzo[k]fluoranthene and fluoranthene and the PAH acenaphthylene by Sphingobium sp. strain KK22. Products in brackets were not identified in the culture medium. Products in boxes were detected in extracts from benzo[k]fluoranthene and fluoranthene biodegradation, [M – H]⁻ = 223, or in extracts from benzo[k]fluoranthene, fluoranthene and acenaphthylene biodegradation, [M – H]⁻ = 229 and [M – H]⁻ = 171.

Figure 4

Results of LC/ESI(–)-MS/MS product ion scan analyses of extracts from strain KK22 exposure to fluoranthene. Fragmentation patterns acquired from the deprotonated molecules: (A) [M – H]⁻ = 265,; (B) [M – H]⁻ = 239; (C) [M – H]⁻ = 195; (D) [M – H]⁻ = 207 (parent deprotonated molecule is not visible); and (E) [M – H]⁻ = 223. Molecular structures proposed for each product are shown and details for all acquisitions are given in Table 2.