Stable-isotope probing-based investigations of polycyclic aromatic hydrocarbon-degrading bacteria in contaminated soil

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic contaminants that are a global environmental problem. These compounds become more recalcitrant to remediation and increase in carcinogenic potential with increasing molecular weight. Engine exhaust and industrial process waste, like that from the sites of former manufactured-gas plants, contain high concentrations of PAHs and are major sources of benzo[a]pyrene contamination in the environment. Bioremediation, the use of microorganisms to remove PAH contamination, is the dominant strategy for removing PAH contamination from soil because many microorganisms can grow on PAHs. Stable-isotope probing (SIP) is a cultivation-independent technique used to identify microorganism able to grow on specific chemicals, such as PAHs. SIP was used to identify bacteria in soil from the site of a former manufactured-gas plant that are capable of degrading naphthalene, phenanthrene, anthracene, pyrene, fluoranthene, or benz[a]anthracene. Group-specific quantitative PCR primers were developed to determine whether the bacteria identified were capable of growth on the respective PAH. SIP with naphthalene, phenanthrene, and fluoranthene selected bacteria previously associated with the degradation of those compounds, and Pigmentiphaga was newly associated with naphthalene, phenanthrene, and anthracene degradation. A group of uncultivated Gammaproteobacteria known as Pyrene Group 2 was newly associated with fluoranthene and benz[a]anthracene degradation, and it was the only group of bacteria associated with pyrene degradation. A group of uncultivated Alphaproteobacteria was the primary anthracene-degrading group and was designated Anthracene Group 1; Herminiimonas was also newly associated with anthracene degradation. In experiments to evaluate the biases associated with using a commercial DNA extraction kit, performing multiple DNA extractions on the same anthracene-enriched soil sample did not affect qualitative results; however, shifts in the relative abundances of anthracene-degrading bacteria were observed between extracts. Since no microorganisms are known to grow on benzo[a]pyrene, a carcinogenic PAH, mineralization experiments and the results of the SIP investigations were used to obtain indirect evidence suggesting that bacteria capable of growth on other PAHs might participate in benzo[a]pyrene metabolism. None of the major SIP-identified bacteria were associated with benzo[a]pyrene mineralization, but members of the genera Cupriavidus, Luteimonas, and Rhizobium may be associated with benzo[a]pyrene mineralization