Stable-isotope probing of the polycyclic aromatic hydrocarbon-degrading bacterial guild in a contaminated soil

The bacteria responsible for the degradation of naphthalene, phenanthrene, pyrene, fluoranthene or benz[a]anthracene in a polycyclic aromatic hydrocarbon (PAH)-contaminated soil were investigated by DNA-based stable-isotope probing (SIP). Clone libraries of 16S rRNA genes were generated from the (13) C-enriched ('heavy') DNA recovered from each SIP experiment, and quantitative PCR primers targeting the 16S rRNA gene were developed to measure the abundances of many of the SIP-identified sequences. Clone libraries from the SIP experiments with naphthalene, phenanthrene and fluoranthene primarily contained sequences related to bacteria previously associated with the degradation of those compounds. However, Pigmentiphaga-related sequences were newly associated with naphthalene and phenanthrene degradation, and sequences from a group of uncultivated γ-Proteobacteria known as Pyrene Group 2 were newly associated with fluoranthene and benz[a]anthracene degradation. Pyrene Group 2-related sequences were the only sequences recovered from the clone library generated from SIP with pyrene, and they were 82% of the sequences recovered from the clone library generated from SIP with benz[a]anthracene. In time-course experiments with each substrate in unlabelled form, the abundance of each of the measured groups increased in response to the corresponding substrate. These results provide a comprehensive description of the microbial ecology of a PAH-contaminated soil as it relates to the biodegradation of PAHs from two to four rings, and they underscore that bacteria in Pyrene Group 2 are well-suited for the degradation of four-ring PAHs.