|dc.description.abstract||Hydrocarbon contamination of Antarctica poses a threat to the unique and fragile ecosystems of this continent. Bioremediation, utilising indigenous hydrocarbon-degrading bacteria, has been proposed as an environmentally friendly method for clean up of contaminated terrestrial sites in Antarctica. Design and implementation of a successful bioremediation process requires detailed information of the degradative bacteria present at the contaminated site(s). This study focused on toluene and naphthalene-degrading Pseudomonas species isolated from JP8 jet-fuel contaminated sites at Scott Base, Antarctica.
Isolates were assessed for their ability to grow on toluene and meta-toluate or naphthalene as a sole carbon source on solid and in liquid media at 6°C and 15°C. All the toluene degrading isolates could grow on 5 mM meta-toluate in liquid and on solid media at 6°C and 15°C. Toluene degrading isolates, with the exception of 8/43 and 8/51, were able to grow on solid media supplemented with toluene vapour but none could grow on toluene in liquid media. Naphthalene degrading isolates could grow on solid media, with naphthalene vapour as a carbon source, at 15°C but at 6°C two of the six isolates, 8/47 and 7/38, could not grow on solid media supplemented with naphthalene vapour. None of the toluene and naphthalene degrading isolate could grow on carbon-free solid or liquid media without the addition of a growth substrate. Presumptive plasmids were isolated from the following toluene degrading isolates, 8/46, 7/163, 7/167, 7/22, and the following naphthalene degrading isolates, 7/156, 7/38, ant5, ant9. Two toluene degrading isolates, 7/167 and 8/46, were studied in more depth. Both isolates grew in liquid media, with meta-toluate supplied as a sole carbon source, over a range of temperatures, 6°C, 1 DOC, 15°C, 2DoC, and 25°C. The optimum growth temperature for isolate 7/167 is between 20°C and 25°C and the optimum growth temperature for isolate 8/46 is 15°C. Isolates were able to sustain growth on meta-toluate at 6°C, 15°C, and 25°C. Isolate 7/167 could not sustain growth on meta-toluate at 30°C. Isolate 8/46 could sustain growth on meta-toluate at 30°C but could not grow at 35°C. All of the Antarctic isolates are psychrotrophic.
Degradative plasmids were isolated from 7/167 and 8/46. Restriction enzyme digest fragment patterns of plasmid DNA generated with Xhol showed isolates 7/167 and 8/46 contain different sized plasmids, 61 kb and 95 kb, respectively. Regions of toluene catabolic genes were amplified by the Polymerase Chain Reaction. Sequence data of the PCR products produced using xylB primers revealed isolate 7/167 to be 100% homologous to the xylB gene region amplified from the archetypal TOL plasmid, pWWO, isolated from the Northern Hemisphere, and 8/46 to be 92% homologous.
Assays of four enzymes, involved in toluene degradation, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase, from isolates 7/167 and 8/46 showed catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and -benzaldehyde dehydrogenase to be present and active. No activity was detected for catechol 1,2-dioxygenase, indicating the presence of a meta cleavage pathway of toluene degradation, typical of that found on TOL type plasmids, and not ortho cleavage of catechol typical of chromosomal gene expression.
Isolates 7/167 and 8/46 contain large plasmids, similar to Northern Hemisphere isolated TOL degradative plasmids, and cleave meta-toluate via the meta cleavage pathway. This degradative mechanism is analogous to the toluene degradative pathway encoded by the archetypal TOL plasmid, pWWO, in the mesophilic bacterium P. putida mt-2. These indigenous psychrotrophic toluene degrading Antarctic bacteria have the ability to degrade meta-toluate at temperatures typical of the Antarctic soil at Scott Base in the summer months. These toluene degrading Antarctic bacteria have the potential to be used to develop a bioremediation programme to successfully clean up JP8 jet fuel contaminated soil at Scott Base, Antarctica.||