Molecular monitoring of dehalogenating bacteria in contaminated aquifers

Chlorinated organic compounds (COC) are chemicals of anthropic origin that find very broad applications in both urban and industrial settings. They are recalcitrant organic pollutants which have toxic and carcinogenic properties. Additionally, because of their physical and chemical characteristics, COC tend to bioaccumulate and biomagnify through the food chain. This class of environmental pollutants is extremely widespread making them the most common contaminants in groundwater sites. By leeching into aquifers, they diffuse and form a contamination plume, which constitutes a dense non-aqueous phase liquid (DNAPL). This Thesis work focuses on monitoring the biodegradation of those compounds in contaminated aquifers by applying molecular tools to assess and monitor the microbial biomass as well as the presence of functional genes (RDases) related to the degradation of COC, and bacterial genera mostly associated with the degradation of those chlorinated compounds (i.e. Dehalococcoides and Dehalobacter). The analyses were performed on water samples that were filtered to gather the microbial biomass, subsequently the filters were cut, and DNA extraction was performed using the PowerSoil kit produced by Qiagen. The quantification of the functional genes, bacterial biomass and bacterial genera of interest was then performed by quantitative Real-Time PCR. Data obtained were then examined with Excel to produce heatmaps describing the behaviours of the different sites allowing for the monitoring of the performance of the bioremediation process. Data obtained shows a good bioremediation efficacy in site 1 where a good diffusion and abundance of genes specific for the degradation of COCs could be identified. Site 2 is instead proceeding at a lower level. It was also possible to detect several genes involved in COC metabolism, some of which were present in high copy numbers. However, these genes were not consistently distributed across all samples and were completely absent in others. This difference in the genetic potential between Site 1 and Site 2 was likely attributable to either the earlier initiation of the bioremediation process at Site 1 or the different bioremediation strategy adopted.