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The distribution and diversity of PAC-degrading bacteria and key degradative genes
Long, Rachel May
Thesis or dissertation
University of Exeter
Petroleum hydrocarbons are the most widespread contaminants in the environment. Interest in the biodegradation of polycyclic aromatic hydrocarbons and compounds (PAHs/PACs) is motivated by their ubiquitous distribution, their low bioavailability, high persistence in soils and their potentially deleterious effects to human health. Identifying the diversity of microorganisms that degrade PAHs/PACs can be utilised in the development of bioremediation techniques. Understanding the mechanisms of bacterial populations to adapt to the presence of pollutants and the extent that lateral transfer of key functional genes occurs, will allow the exploitation of microbial PAC/PAH-degradative capabilities and therefore enhance the successful application of bioremediation strategies. A key aim of this study was to isolate and identify PAC-degrading bacteria for potential use in future bioremediation programmes. A series of PAC enrichments were established under the same experimental conditions from a single sediment sample taken from a highly polluted estuarine site. Distinct microbial community shifts were directly attributable to enrichment with different PAC substrates. The findings of this study demonstrate that five divisions of the Proteobacteria and Actinobacteria can degrade PACs. By determining the precise identity of the PAC-degrading bacteria isolated, and by comparing these with previously published research, this study showed how bacteria with similar PAC degrading capabilities and 16S rRNA signatures are found in similarly polluted environments in geographically very distant locations e.g. China, Italy, Japan and Hawaii. Such a finding suggests that geographical barriers do not limit the distribution of key PAC-degrading bacteria. This is significant when considering the diversity and global distribution of microbes with PAC-degradative capabilities and the potential for utilising these microbial populations in future bioremediation strategies. In the laboratory, enrichment of bacteria able to utilise PAHs has commonly been performed in liquid media, with the PAH dissolved in a carrier solvent. This study found the presence of a carrier solvent significantly affects the resultant microbial population. Although the same sediment sample was used as the bacterial source in all enrichments, different bacterial strains were obtained depending upon the presence of the carrier solvent and the PAH. This is important when considering appropriate methodology for the isolation of PAH-degrading bacteria for future bioremediation programmes. Additionally, the species comprising the resultant population of the enrichment when a carrier solvent was present were similar to previously reported PAH-degrading species. Such a finding necessitates review of previously reported PAH-degrading bacterial species that have been isolated and identified from enrichments using a carrier solvent. Understanding how bacteria acclimatise to environmental pollutants is vital for exploiting these mechanisms within clear up strategies of contaminated sites. Two major lineages of the α subunit of PAH dioxygenases were identified: Actinobacteria and Proteobacteria. Comparison of the α subunit phylogeny with the 16S rRNA phylogeny implies that the PAH-dioxygenases evolved prior to the separation of these phyla or that lateral transfer occurred in the very distant past. No evidence for lateral transfer of the α subunit between the Actinobacteria and Proteobacteria was found in the phylogenetic analyses of this research. Multiple lateral transfer events were inferred between the species of the Actinobacteria and between the classes of the Proteobacteria. The clustering of the taxa within the α subunit phylogeny indicates that lateral transfer of the α subunit gene occurred after the separation of the classes of Proteobacteria and also after the speciation of the γ-Proteobacteria. These findings reveal how bacteria have acclimatised to PAH pollutants through multiple lateral transfer events of a key PAH-degradative gene. This knowledge of the transfer of genetic material will broaden our prospects of exploiting microbial populations.
PhD in Biological Sciences