Investigation of the Effects of Geochemical Perturbation and Arsenic as a Co-contaminant on Dehalococcoides mccartyi-containing TCE Dechlorination Consortia

Trichloroethene (TCE) has emerged as a challenging groundwater contaminant to be remediated due to the formation of a slowly-dissolving dense non-aqueous phase liquid (DNAPL), and has caused persistent health risks to human health. In situ bioremediation of TCE using Dehalococcoides mccartyi (Dhc)-containing microbial communities provides a greener and more cost-effective alternative to the traditional pump and treat method. However, in situ dechlorinating microbial communities are susceptible to geochemical perturbations originating from the interplay of microbial activities and geological conditions at the contamination sites. Additionally, co-contaminants further complicate the bioremediation strategies due to their non-uniform toxicity and biodegradability. For instance, arsenic, a common co-contaminant to TCE, poses severe toxic effects on most microorganisms and has the potential to change its solubility and form due to naturally-occurring biotransformation. The main goal of this research was to proactively control the risks and uncertainty associated with geochemical perturbation and co-contamination by simulation in a lab setting using simplified microbial communities to acquire mechanistic understanding of the regulatory and metabolic interactions./nGroundwater and soil salinization caused by climate change, drought, and poor agricultural management can induce salinity stress to in situ bioremediation microbial communities. However, researchers have not characterized the overall salt stress response in Dhc, nor the impact of elevated salinity on the performance of Dhc-containing dechlorinating consortia. In this research, I studied the effects of salinity in an environmentally-relevant concentration range on Dhc strain 195 (Dhc195) axenic culture and a Dhc-containing defined consortium that also consists of Desulfovibrio vulgaris Hildenborough (DvH) and Pelosinus fermentans R7 (PfR7). I found that Dhc195 has intrinsically high salt tolerance, potentially due to its capability of biosynthesizing mannosylglycerate, a unique osmoprotectant. I described the overall salt stress response of Dhc195 using transcriptomics analysis and identified the key genes and metabolic pathways that were differentially expressed during osmo-adaptation. Subsequently, I analyzed the performance of the consortium in both batch reactors and completely mixed flow reactors (CMFRs), which represent and simulate different bioremediation sites. I also evaluated the effectiveness of amending glycine betaine and vitamin B12 to ameliorate the adverse effects caused by salinity upsurge as potential engineered solutions./nInterpretation of transcriptomics data in less-studied non-model organisms, such as the species in Dhc-containing consortia, faces the challenges of lack of gene annotations and mis-annotations. As a potential solution, functional genetics analysis, including gene silencing and heterologous gene expression, relies on the development of genetics tools in expression hosts. In this research, I developed the workflow of using CRISPR-Cas9-nickase to achieve homology-directed repair (HDR)-mediated genome editing in DvH through all-in-one vectors. The establishment of the workflow expedites the genetics study in this model sulfate-reducing bacterium and helps researchers to explore its potential to be used as an anaerobic heterologous expression host./nArsenic poses severe toxic effects on TCE-dechlorinating microbial communities and further complicates the bioremediation of TCE at co-contamination sites. Previous research demonstrated enhanced robustness of the growth of Dhc under As(III) perturbation when it’s in a syntrophic relationship with DvH. In this research, I further investigated the As(V) reduction capability of DvH and how arsenate reduction and sulfate reduction impacted arsenic sulfide mineral formation under different redox conditions. By incorporating Methanosarcina acetivorans C2A (MsA), a common archaea species present in naturally occurring Dhc-containing communities, into the co-culture of Dhc195/DvH, MsA further ameliorates the adverse effects posed by As(III), allowing robust TCE dechlorination under 20 M./nOverall, this research expands our current knowledge about Dhc-containing consortia under a common geochemical perturbation, i.e., elevated salinity, and the impact of a frequently-occurring co-contaminant, arsenic. The results provide environmental engineers valuable guidance on the bioremediation strategies and risk mitigation methods at in situ bioremediation sites.

Advisor: Alvarez-Cohen, Lisa