Molecular Examination of the Identity and Activity of Polycyclic Aromatic Hydrocarbon-degrading Microorganisms
Project Leader: Eugene L. Madsen Grant Number: R21ES012834 Funding Period: 2003-2004
Polycyclic aromatic hydrocarbons (PAHs) are chemicals highly relevant to agencies concerned with human health (Superfund, the Environmental Protection Agency, and the Agency for Toxic Substances and Disease Registry) because members of this calss of compounds are toxic, carcinogenic, and ubiquitous. Naturally occurring microbial communities offer promise as agents that can diminish the threat of PAHs to human health, but there is an immense amount of information yet to be discovered before this promise can be realized. PAH biodegradation (detoxification) has been studied for decades in laboratory settings and with isolated pure cultures of microorganisms. Despite these efforts, the identity of microorganisms actually responsible for PAH biodegradation in contaminated field sites has never been discerned. The goal of this project is to use laboratory and field studies to discover fundamental facts and principles about the identity, diversity, and activity of naturally occurring microorganisms involved in the metabolism of PAHs. This goal will be achieved by five major aims integrated around new, novel molecular and analytical chemistry procedures that rely on 13C-labeling techniques. The five major aims are (i) identifying active populations using a stable isotope probing (SIP) technique that traces the flow of 13C-labeled PAHs into the DNA of soil microbial populations-the procedure supplies 13C-labeled PAHs to soil, extracts microbial community DNA, separates 13C- from 12C-DNA via CsCl ultracentrifugation, and then the 16S rRNA genes present in the 13C-DNA fraction are amplified by PCR, cloned, and sequenced; (ii) verifying the PAHs are metabolized using gas chromatography/mass spectrometry to monitor the conversion of 13C-PAHs to 13CO2; (iii) ascertaining the bioavailability of PAHs in soil by distinguishing sequestered from non-sequestered forms of PAHs using Pyrolysis-Gas Chromatography/Mass Spectrometric (PY-GC/MS) analysis of 13C- and 12C-labeled PAH pools; (iv) cataloguing the presence and diversity of genes in the PAH-degrading microbial community via molecular profiling of 16S rRNA and dioxygenase genes; and (v) working to purify and isolate the microorganisms involved in PAH biodegradation so that their physiological and genetic properties can augment our ability to understand and manage the fate of PAHs in contaminated sites.