Isolation and Molecular Characterisation of Polycyclic Aromatic Hydrocarbons (PAHs) Degrading Bacteria from Petrochemical Contaminated Soil

Abstract

Because of their ubiquitous nature, polycyclic aromatic hydrocarbons (PAHs) are widely dispersed in the environment as a result of both natural and human processes. One of the ways to deal with the harmful effects of these chemicals is through the use of microorganisms capable of degrading the pollutants. A petrochemical contaminated site was searched for these microbes. Eleven bacterial strains were obtained in this work using the culture enrichment technique on Bushnell Hass medium supplemented with (naphthalene, anthracene, and phenanthrene) as the only source of energy. Enumeration utilizing the spread-plate technique and liquid media were used to examine the PAH breakdown capacities of bacterial strains. The isolates were identified using standard methods of morphological and biochemical identifications. Furthermore, 16sRNA was utilized in order to classify the isolates at molecular level. The presence of PAHs degrading genes was also analysed in the isolates. Four isolates (G1, G2, G5, and G6) out of a total of eleven were able to tolerate and degrade the test PAH's up to 600 mg/l in liquid media. Isolate G1 showed the highest growth during screening followed by G6 while there were no differences between the other two isolates as demonstrated by an increase in their optical densities after 120 hours of incubation. Based on 16S rRNA gene sequences and molecular phylogenetic analysis, the isolate was identified as Pseudomonas stutzeri, Stenetrophomonas sp, Pseudomonas lactis, and Achromobacterxylosoxidans with the accession numbers OM039162, OM52851, OM52852, and OM52853 respectively. Fragments of 350 bp, 350 bp, and 867 bp for ring hydroxylating dioxygenase (RHD), hydratase-aldolase, and catechol 2, 3-dioxygenase were obtained from partial PCR amplification of catabolic genes, demonstrating the presence of a PAH degradation pathway in the organisms. These isolates have great potential for application in the bioremediation of PAHs-contaminated sites.