Quantification of Naphthalene Dioxygenase (NahAC) and Catechol Dioxygenase (C23O) Catabolic Genes Produced by Phenanthrene-Degrading Pseudomonas fluorescens AH-40

Abstract

Background: Petroleum polycyclic aromatic hydrocarbons (PAHs) are known to be toxic and carcinogenic for humans and their contamination of soils and water is of great environmental concern. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies.

Objective: Detection of the ability of Pseudomonas fluorescens AH-40 to consume phenanthrene as a sole carbon source and determining the variation in the concentration of both nahAC and C23O catabolic genes during 15 days of the incubation period.

Methods: In the current study, a bacterial strain AH-40 was isolated from crude oil polluted soil by enrichment technique in mineral basal salts (MBS) medium supplemented with phenanthrene (PAH) as a sole carbon and energy source. The isolated strain was genetically identified based on 16S rDNA sequence analysis. The degradation of PAHs by this strain was confirmed by HPLC analysis. The detection and quantification of naphthalene dioxygenase (nahAc) and catechol 2,3-dioxygenase (C23O) genes, which play a critical role during the mineralization of PAHs in the liquid bacterial culture were achieved by quantitative PCR.

Results: Strain AH-40 was identified as pseudomonas fluorescens. It degraded 97% of 150 mg phenanthrene L^-1 within 15 days, which is faster than previously reported pure cultures. The copy numbers of chromosomal encoding catabolic genes nahAc and C23O increased during the process of phenanthrene degradation.

Conclusion: nahAc and C23O genes are the main marker genes for phenanthrene degradation by strain AH-40. P. fluorescence AH-40 could be recommended for bioremediation of phenanthrene contaminated site.

Keywords: 16S rDNA; Bacteria; PAHs pollutant; Pseudomonas fluorescens; catabolic genes; isolation.

Fig. (1)

Phylogenetic relationship between strain AH-40 and other 16S rDNA sequences of published strains belonging to Pseudomonas sp. In the phylogenetic tree, AH-40 and Pseudomonas fluoresences were clustered together as one clade. GenBank accession numbers are given in parentheses.

Fig. (2)

The growth profile of strain AH-40 (Log CFU/ml) (), the remaining concentration of phenanthrene (mg/L) () during the incubation period of 15 days at 30ºC. Phenanthrene concentration in negative control () Error bars represent the standard deviation (SD±) of three replications.

Fig. (3)

Agarose gel electrophoresis of PCR products amplified with Nah-AC specific primer sets using the genomic DNAs from strain AH-40. The copy number nahAC gene for the 5 samples during phenanthrene degradation in 15 days incubation period. Error bars indicate the standard deviation of the two independent PCR runs.

Fig. (4)

Agarose gel electrophoresis of PCR products amplified with Nah-AC specific primer sets using the genomic DNAs from strain AH-40. The copy number C23O gene for the 5 samples during phenanthrene degradation in 15 days incubation period. Error bars indicate standard deviation (SD±) of the two independent PCR runs.