Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation


Sunil Khanna, Department of Biotechnology and Bioinformatics, NIIT University, Neemrana, Rajasthan, India. E-mail: Khanna85@yahoo.com


Aims:  In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation-independent communities is available.

Methods and Results:  Coal-tar-contaminated soil was collected, which consisted of 122·5 mg g−1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal-tar-contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α-subunit) common to all PAH dioxygenase enzymes and (iii) β-subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha-, Epsilon- and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α-subunit and β-subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe-2S] cluster binding site suggested that these gene fragments encode for α-subunit of dioxygenase gene.

Conclusions:  Sequencing of the cloned libraries representing α-subunit gene fragments (Rf1) and β-subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal-tar-contaminated soil.

Significance and Impact of the Study:  The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.