Biodegradation of pyrene and catabolic genes in contaminated soils cultivated with Lolium multiflorum L

In the soil environment, polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) are of great environmental and human health concerns due to their widespread occurrence, persistence, and carcinogenic properties. Bioremediation of contaminated soil is a cost-effective, environmentally friendly, and publicly acceptable approach to address the removal of environmental contaminants. However, bioremediation of contaminants depends on plant–microbe interactions in the rhizosphere. The microorganisms that can mineralize various PAHs have PAH dioxygenase genes like nahAc, phnAc, and pdo1. To understand the fate of pyrene in rhizospheric and non-rhizospheric soils in the presence or absence of Pb, pyrene biodegradation, bacterial community structure, and dioxygenase genes were investigated in a pot experiment. Soil was amended with Pb (a representative heavy metal), pyrene, and a Pb/pyrene mixture. After 8 weeks of aging, one set of pot microcosms was cultivated with rye grass (Lolium multiflorum L) seedlings, while another set was not cultivated for the purpose of comparing rhizosphere and non-rhizosphere pyrene degradation. Pyrene was extracted from freeze-dried soil and plant samples using a Soxhlet extraction method and the extracts were dried to 1 mL under gentle nitrogen flow and analyzed using gas chromatograph mass spectrometry (Agilent 6890, USA). Soil DNA was extracted from triplicate samples and the DGGE was performed using a Bio-Rad Dcode™ Universal Mutation Detection System (Bio-Rad, USA). PAH dioxygenase genes, including nahAc, phnAc, and pdo1, were detected using PCR amplification. Similarly, pyrene degraders were also investigated using plate counting technique. Biodegradation rates recorded over an 18-week period showed that rye grass promoted significant (P < 0.05) pyrene degradation. Pyrene removal efficiency from rhizospheric soils was 59.1 ± 2.1% and 68.7 ± 2.3% in pyrene- and Pb/pyrene-amended soils, respectively. The results indicate that pyrene dissipation was significantly (P < 0.05) higher in Pb/pyrene-amended soils than only-pyrene-amended soils. The plant growth promoted the degradation of pyrene and accounted for 12.1% to 17.0% of dissipation enhancement in the rhizospheric soils. In this study, the DGGE profiles revealed a shift in soil bacterial community structure in all amended soils, with a higher number and greater complexity of banding patterns in Pb/pyrene-amended samples than in either Pb- or pyrene-amended samples. In the control and Pb-amended soil, pdo1 and nahAc genes were not detected throughout the incubation period but were detected in the pyrene- and Pb/pyrene-amended soils. However, phnAc genes were not detected in either amended or non-amended soils throughout the incubation period. The addition of pyrene had a dramatic effect on the number of pyrene degraders. Plants contribute to the degradation of PAHs by increasing the size of microbial population, promoting microbial activity, and modifying microbial community diversity in the rhizosphere. In this study, the presence of plants significantly promoted the degradation of pyrene in the soil due to enhanced bacterial community size and increased the number of pyrene degraders. Similarly, the results of this study have also clearly shown that pyrene remaining in soils only accounted for about 1/3 of the total pyrene addition, suggesting that most of the pyrene added could be removed by plant and/or microbial degradation. HMs and PAHs interaction towards degradation of PAHs can be both negative and positive depending on type and concentration of both HMs and PAHs. In both rhizospheric and non-rhizospheric soils, the pyrene degradation was in line with the changes of bacterial structure, increasing number of pyrene degraders, and the prevalence of dioxygenase genes (nahAc and pdo1). This work represents the first report that Pb can affect the dissipation of pyrene and functional genes were detected in both rhizospheric and non-rhizospheric soils amended with pyrene and Pb/pyrene. Pyrene removal efficiency for rhizospheric soils was higher than for non-rhizospheric soils and pyrene dissipation was accelerated in the presence of Pb in both rhizospheric and non-rhizospheric soils. The bacterial community structure was changed and the addition of pyrene indicated dramatic effects on the number of pyrene degraders. The catabolic genes, including nahAc and pdo1, which are responsible for HMW-PAH degradation, were confirmed in both rhizosphere and non-rhizosphere soils amended with pyrene or Pb/pyrene. Our findings suggest that the interaction between bacterial community and plant roots could influence the PAH degradation both in the presence and absence of HMs in the contaminated soils.