Abstract of PhD Thesis

Physiology and Genetics of polycyclic Aromatic Hydrocarbon Degradation by Pseudomonas alcaligenes PA-10 and Potential in-situ Remediation of Contaminated Soil

Mark O'Mahony (2007) - University College Cork

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, with some known to have toxic, mutagenic and carcinogenic properties. Therefore there is considerable interest in the remediation of PAH contaminated sites, with both chemical and biological techniques investigated for this process. Here we examine the potential of using ozone to remove phenanthrene from spiked soil samples, both alone and in combination with a microbial inoculant, Pseudomonas alcaligenes PA-10, a strain which is capable of degrading a number of PAHs. The results indicated that the greater the water content of the soil the less effective the ozone treatment, with air-dried soils showing the greatest removal of phenanthrene; while soils with higher levels of clay and organic carbon may possibly also reduce the effectiveness of the ozone treatments. P. alcaligenes PA-10 has potential to transform PAHs in soil, especially in situations where the indigenous microbial population is unable to transform the pollutant. Under the conditions used in our experiments, however, pre-ozonation did not enhance subsequent biodegradation of phenanthrene in the soils, and may even have an inhibitory effect in some soils possibly due to the release of toxic products during ozonation. These results indicate that the physical, chemical and biological properties of the soil need to be carefully considered before implementing remediation techniques. Subsequently, a number of putative open reading frames were identified in P. alcaligenes PA-10, some of which may have a role in PAH metabolism in this strain. We appear to have identified a novel operon, designated ido, which may be involved in the initial attack of the PAH ring. Two genes, idoA and idoC, appear to encode the components of a flavin dependent oxygenase, which is unlike the ferredoxin dependent dioxygenases typically associated with aerobic metabolism of PAHs by bacteria, while the third gene, idoB,  may be involved in dehydrogenation after oxygenation of the aromatic ring. Again, this gene is not similar to the cis-dihydrodiol dehydrogenases typically involved in PAH metabolism. Expression studies indicated increased activity of IdoA in the presence of the IdoC protein, which strongly suggests an association between the two proteins, with IdoC proposed to supply reduced flavin to the oxygenase component. A number of genes potentially involved in further metabolism of PAHs via catechol or protocatechuate were also identified, as well as a number of other adjacent open reading frame, whose role in PAH metabolism has yet to be determined. One such open reading frame appears to encode an O-methyltransferase gene, which may be involved in additional detoxification of PAHs under certain conditions. Analysis of the promoter region of the ido operon indicated the presence of potential sigma 70 type promoters, but the use of promoter probe vectors to further analyse the regulation of this novel operon were unsuccessful. Overall, ozonation has enormous potential to remediate PAH contaminated sites, both alone and in combination with biodegradation, and P. alcaligenes PA-10 has potential for use as a microbial inoculant and contains a novel operon involved in PAH metabolism.