Genetic analysis of high molecular weight polycyclic aromatic hydrocarbon biodegradation

Many plant species enhance the biodegradation of polycyclic aromatic hydrocarbons (PAHs), but there is little understanding of the mechanisms by which this occurs. This research identified phytochemicals that stimulate pyrene degradation using crushed root tissues from 43 plants that were screened in soil spiked with 100 ppm pyrene. Among the plants tested, root tissues from Apium graveolens (celery), Raphanus sativus (radish), Solanum tuberosum (potato) and Daucus carota (carrot) were most effective for promoting disappearance of pyrene within 40 days. Experiments with A. graveolens showed that plant culture in soil contaminated with pyrene or benzo[a] pyrene was as effective as addition of crushed root tissues. Comparison of the chemical compositions of the effective plants suggested that linoleic acid was the major substance that stimulated PAH degradation. This hypothesis was supported in experiments examining degradation of pyrene and benzo[a]pyrene in soil amended with linoleate, whereas linolenic and palmitic acids did not stimulate degradation within a 20 d period. Antibiotic inhibitor studies implicated mycobacteria as the major microbial group responding to linoleic acid.; A second component of this research examined gene expression patterns for mycobacterial nidA-related dioxygenase genes after augmentation of PAH contaminated soils with plant tissues or pure linoleic acid. Results showing that mycobacteria were the predominant HMW-PAH degraders were corroborated by expression profiles for mycobacterial dioxygenase genes. To investigate nidA gene diversity, a degenerate primer set was designed and used to construct clone libraries from 6 soils. A total of 51 RFLP patterns were observed from 476 clones, indicating a high diversity of nidA related genes. The 51 gene groups were further sorted into five major groups for which specific probes were designed. Using Northern hybridization and real-time PCR methods, RNA expression for three of the five groups was shown to be upregulated in a similar fashion by addition of either sodium linoleate or celery root crushates. The results suggest that linoleic acid and plant tissues containing high amounts of linoleic acid both stimulate mycobacterial populations, but that there are differences at the subpopulation level in which groups carrying different variants of the dioxygenase gene respond to linoleic acid and plant tissues containing high amounts of this biostimulant. The development of degenerate primers for nidA genes and identification of mycobacteria as a prominent group responding to linoleic acid provides insight into the mechanisms by which plants may enhance PAH degradation, and tools for monitoring HMW-PAH degrader activity in soils.