Soil type-dependent responses to phenanthrene as revealed by determining the diversity and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system

A novel PCR primer system that targets a wide range of polycystic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD_α) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD _aα gene amplicons from soll DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD_α genes of Gram-positive and Gramnegative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD_α genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD_α gene-carrying soil bacteria to phenanthrene spiking.