Association of Growth Substrates and Bacterial Genera with Benzo[ a]pyrene Mineralization in Contaminated Soil

Abstract

Benzo[a]pyrene (BaP) is a carcinogenic polycyclic aromatic hydrocarbon (PAH) that is not known to be a bacterial growth substrate. Organisms capable of cometabolizing BaP in complex field-contaminated systems have not previously been identified. We evaluated BaP mineralization by a bacterial community from a bioreactor treating PAH-contaminated soil during coincubation with or after pre-enrichment on various PAHs as growth substrates. Pyrosequence libraries of 16S rRNA genes were used to identify bacteria that were enriched on the added growth substrate as a means of associating specific organisms with BaP mineralization. Coincubating the bioreactor-treated soil with naphthalene, phenanthrene, or pyrene inhibited BaP mineralization, whereas pre-enriching the soil on the same three PAHs enhanced BaP mineralization. Combined, these results suggest that bacteria in the bioreactor community that are capable of growing on naphthalene, phenanthrene, and/or pyrene can metabolize BaP, with coincubation competitively inhibiting BaP metabolism. Anthracene, fluoranthene, and benz[a]anthracene had little effect on BaP mineralization compared to incubations without an added growth substrate under either coincubation or pre-enrichment conditions. Substantial increases in relative abundance after pre-enrichment with phenanthrene, naphthalene, or pyrene, but not the other PAHs, suggest that members of the genera Cupriavidus and Luteimonas may have been associated with BaP mineralization.

Keywords: PAHs; benzo[a]pyrene; bioremediation; cometabolism; pyrosequencing; soil.

FIG. 1.

Initial ¹⁴C evolved as ¹⁴CO₂ after 24 h in the presence of ¹⁴C-BaP (mean and standard deviation, n=3). Open and closed bars are data from coincubation and pre-enrichment experiments, respectively, with the indicated growth substrate or in the absence of an added growth substrate (None). Data represent means and standard deviations of triplicate flasks for each condition. Asterisks indicate that the value is significantly different from the respective incubation without exogenous substrate. Abbreviations are as in Table 1.

FIG. 2.

Weighted and normalized principal coordinates analysis of pyrosequence libraries. Clusters of libraries referred to in the text are circled and numbered. Libraries are defined in Table 1. Clusters beginning with the letter C are from coincubation experiments and with the letter P are from the pre-enrichment experiments; BRS, bioreactor slurry.

FIG. 3.

Responses of selected bacterial genera (as % of the respective pyrosequence library) to preincubation with each of the polycyclic aromatic hydrocarbon (PAH) substrates or in the absence of added substrate (None) in comparison to the abundance in the inoculum (BRS); abbreviations are as in Table 1. Solid bars represent data from incubations in the absence of BaP and hatched bars represent incubations in the presence of BaP. No bar is shown if the genus was not in the library or was present at <1%. (a, b) Genera whose relative abundances increased in response to PAHs that enhanced BaP mineralization, but with negligible or no response to other PAHs. (c, d) Genera whose relative abundances increased in response to all PAHs. (e–h) Genera whose relative abundances increased in response to naphthalene and/or phenanthrene in at least one incubation condition (with or without BaP), but also increased in response to one or more PAHs that did not enhance BaP mineralization.