Identification of anthraquinone-degrading bacteria in soil contaminated with polycyclic aromatic hydrocarbons

Abstract

Quinones and other oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are toxic and/or genotoxic compounds observed to be cocontaminants at PAH-contaminated sites, but their formation and fate in contaminated environmental systems have not been well studied. Anthracene-9,10-dione (anthraquinone) has been found in most PAH-contaminated soils and sediments that have been analyzed for oxy-PAHs. However, little is known about the biodegradation of oxy-PAHs, and no bacterial isolates have been described that are capable of growing on or degrading anthraquinone. PAH-degrading Mycobacterium spp. are the only organisms that have been investigated to date for metabolism of a PAH quinone, 4,5-pyrenequinone. We utilized DNA-based stable-isotope probing (SIP) with [U-(13)C]anthraquinone to identify bacteria associated with anthraquinone degradation in PAH-contaminated soil from a former manufactured-gas plant site both before and after treatment in a laboratory-scale bioreactor. SIP with [U-(13)C]anthracene was also performed to assess whether bacteria capable of growing on anthracene are the same as those identified to grow on anthraquinone. Organisms closely related to Sphingomonas were the most predominant among the organisms associated with anthraquinone degradation in bioreactor-treated soil, while organisms in the genus Phenylobacterium comprised the majority of anthraquinone degraders in the untreated soil. Bacteria associated with anthracene degradation differed from those responsible for anthraquinone degradation. These results suggest that Sphingomonas and Phenylobacterium species are associated with anthraquinone degradation and that anthracene-degrading organisms may not possess mechanisms to grow on anthraquinone.

FIG 1

Example of DNA quantification in fractions obtained after ultracentrifugation of extracts from each duplicate incubation of feed soil with unlabeled anthraquinone (a) and [U-¹³C]anthraquinone (b). Fractions are numbered from the bottom of the centrifuge tube up; for both of the incubations with ¹³C-labeled anthraquinone, fractions 5 to 8 were selected as those containing heavy (¹³C-enriched) DNA. The data for the different duplicate incubations are represented by different symbols in each panel.

FIG 2

Quantification over time of major sequences identified in clone libraries of heavy DNA from incubations with [U-¹³C]anthracene in feed soil (a) and [U-¹³C]anthraquinone in feed soil (Phenylobacterium) and treated soil (Sphingomonas) (b). Error bars represent standard deviations from triplicate qPCRs; if not visible, the error bars are within the size of the symbol. AG1, anthracene group 1.