DECIPHERING BACTERIAL CATABOLIC GENES FOR PAH-DEGRADATION

Abstract

Polyaromatic hydrocarbons (PAHs) are considered as hazardous organic priority pollutants. PAHs are immense public concern and critical environmental challenge around the globe due to their toxic, carcinogenic, and mutagenic properties, and their ubiquitous distribution, and persistence in the environment. The knowledge about the harmful effects of PAHs on the ecosystem along with human health has resulted in the interest of researchers on the degradation of these compounds. Whereas physico-chemical treatment of PAHs is cost and energy-prohibitive, bioremediation i.e. degradation of PAHs using microbes is becoming an efficient and sustainable approach. A broad range of microbes including bacteria, fungi, and algae has been found to have the capability to transform/degrade PAHs under both aerobic and anaerobic conditions. Microbial genetic makeup containing genes encoding catabolic enzymes is responsible for the PAH-degradation mechanism. The degradation capacity of microbes may be induced by exposing them to higher PAH concentration, resulting in genetic adaptation or changes responsible for high efficiency towards removal/degradation. In order to devise efficient bioremediation strategies for PAH degradation, the identification and study of the metabolic potential of microbial species are essential. The goal of this study was to isolate PAH-degrading bacterial strains from petroleum-contaminated soil that can utilize PAHs (three-ring (phenanthrene, anthracene, and fluorene) and four-ring (pyrene)) as their sole carbon source. PAH-utilizing bacterial strains were isolated from petroleum-contaminated soil from the siding area, Bijwasan supply location of Bharat Petroleum Corporation Limited in Delhi, India. Seven bacterial strains with different morphology were isolated and acclimatized under a mixture of four PAH compounds in a concentration range of 50mg/L to 10,000mg/L. Two Gram-positive bacterial strains (DTU-1Y and DTU-7P) were found to be resistant to high PAH (10,000mg/L) exposure. The 16S rRNA gene sequencing of these two strains identified DTU-1Y as Kocuria flava and DTU-7P as Rhodococcus pyridinivorans. Both the isolated strains belonged to the actinobacteria phylum, a group of gram-positive bacteria. Most of the species belonged to the actinobacteria phylum representing a role in the degradation of xenobiotic compounds such as polychlorinated biphenyls, pesticides, dioxins, PAHs, etc. The strain K. flava DTU-1Y could degrade 53-63% of 10 mg/l of phenanthrene, anthracene, fluorene, and pyrene in 15 days of incubation period. whereas R. pyridinivorans had degradation efficiency in the range of 56-66%. The consortium of both strains exhibited almost similar degradation efficiency as vi observed individually indicating that there was no inhibitory or synergistic effect of these strains over each other. Catechol 2,3-dioxygenase (C23O), dehydrogenase, and peroxidase enzyme activities during PAH-degradation coincided with degradation of PAHs, highlighting the role of these enzymes in catabolizing PAHs. This is the first investigation confirming the participation of C23O, dehydrogenase, and peroxidases enzyme of these two strains in PAH degradation. The bacterial strains reported in this study were also examined for catabolic gene expression during PAH-degradation and the evolutionary relationship of isolated bacterial strains with known PAH-degrading bacterial strains having PAH-catabolic genes/enzymes involved in PAH-bioremediation was also confirmed. The degradation efficiency of both strains (monoculture and consortium) was more than 53% for three-ring and four-ring PAHs (10 mg/L) when used individual PAH compound or in a mixture of PAHs. The degradation efficiency of isolated bacterial strains for a PAH compound was not affected by the presence of other PAH compounds. It was observed that the degradation efficiency for individual PHE, ANT, and PYR of the selected bacterial strains i.e. K. flava DTU-1Y, R. pyridinivorans-DTU-7P, and consortium was almost similar to the degradation efficiency for respective PAHs in the mixture of the PAHs (PHE, ANT, FLU and PYR). In the case of FLU, degradation by K. flava DTU-1Y, R. pyridinivorans-DTU-7P, and consortium was found slightly lower in the mixture of PAHs (PHE, ANT, FLU, and PYR) as compared to degradation of FLU individually. Both the isolated strains having the ability to degrade three-ring and four-ring PAHs showed C23O, peroxidase, and dehydrogenase activity during PAH-degradation when present as a single PAH compound or a mixture of PAHs. The C23O enzyme may be responsible for the initiation of hydrolysis through meta cleavage of benzene ring; whereas dehydrogenases and peroxidases might catalyze further oxidation of PAHs into simpler non-toxic forms. The presence of C23O, which catalyzes PAH-degradation via meta-cleavage, indicated C23O involvement in the initial ring cleavage of pyrene and based on enzyme activity a probable degradation pathway of pyrene was suggested. The catabolic gene expression for naphthalene dioxygenase (NAH) and catechol 2,3-dioxygenase (C23O) was confirmed in isolated strains in the present study and significant catabolic gene expression during degradation of PAHs concluded that K. flava DTU-1Y and R. pyridinivorans DTU-7P are efficient PAH-degraders and can be used for the development of an efficient bioremediation method for cleaning of PAH-contaminated environmental matrix.