Genetic Catabolic Probes to Assess the Natural Attenuation of 1,4-Dioxane
Alvarez, Pedro J.
Doctor of Philosophy
Remediation of aquifers contaminated with 1,4-dioxane (dioxane) is a difficult task because dioxane can be recalcitrant to biodegradation, is not easily removed by volatilization or adsorption, and is highly mobile in groundwater. Monitored natural attenuation (MNA), which relies primarily on biodegradation, is often the most cost-effective approach to manage large and dilute groundwater plumes of priority pollutants, such as those formed by dioxane. However, the burden of proof that MNA is an appropriate solution lies on the proponent, which requires demonstration of the presence and expression of relevant biodegradation capabilities. Therefore, an innovative micro-extraction of aqueous samples coupled with gas chromatography/mass spectrometry (GC/MS) was developed to monitor dioxane attenuation with low part-per-billion detection sensitivity. Soluble di-iron monooxygenases (SDIMOs), especially group-5 SDIMOs (e.g., tetrahydrofuran [THF]/dioxane monooxygenases), are of significant interest due to their potential role in the initializing the cleavage of cyclic ethers. In this study, seven gene clusters encoding SDIMOs were annotated in the genome of Pseudonocardia dioxanivorans CB1190, a well-characterized bacterial dioxane degrader. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) results revealed that only the dxmA gene encoding the large hydroxylase of the putative dioxane monooxygenase was significantly up-regulated when fed with dioxane and its structural analog, THF, compared to controls fed with acetate. This is in accordance with previous evidence implicating the key role of dioxane/THF in initiating the oxidation of cyclic ethers. Additional efforts to evaluate the presence of SDIMO genes in groundwater samples were undertaken using advanced molecular tools, such as functional gene array (i.e. GeoChip) and PCR-DGGE. Both assays demonstrated the widespread distribution of various dioxane-degrading SDIMO genes at a dioxane-impacted site in Alaska. Notably, a group-5 thmA-like gene was enriched in source-zone samples with higher dioxane concentrations, suggesting selective pressure by dioxane. Microcosm assays with 14C-labeled dioxane showed that the highest mineralization capacity corresponded to the source area, which was presumably more acclimated and contained a higher abundance of SDIMO genes. Thus, converging lines of evidence based on both pure bacterial cultures and complex environmental samples corroborate the usefulness of THF/dioxane monooxygenases as biomarkers of dioxane natural attenuation. A primer/probe set was then developed to target bacterial genes encoding the large hydroxylase subunits of THF/dioxane monooxygenases using Taqman (5’-nuclease) chemistry. The probe targets conserved regions surrounding the active site, thus enabling detection of multiple dioxane degraders. Real-time PCR using reference strain genomic DNA demonstrated the high selectivity (no false positives) and sensitivity of this probe. Microcosm tests prepared with groundwater samples from 16 monitoring wells at five different dioxane-impacted sites showed that enrichment of this catabolic gene (up to 114-fold) was significantly correlated to the amount of dioxane degraded. A significant correlation was also found between biodegradation rates and the abundance of thmA/dxmA genes, suggesting them as reliable indicators of dioxane biodegradation activity. Furthermore, pyrosequencing-based metagenomics and 16S rDNA profiling was used to understand how dioxane contamination incidents along with chlorinated solvents and other hydrocarbons have affected the indigenous microbial communities and the microbes that are critical to carbon cycling at the site in Alaska. Actinobacteria and Proteobacteria were the dominant bacterial phyla. However, shifting of the microbial communities structures among various sampling locations was significantly related to the types and presence of external carbon sources (e.g., synthetic chemicals and naturally released gases) at the site. This indicates that the indigenous microbes have adapted to the local environment probably due to long term of acclimation, and may be contributing to the presence of natural attenuation. This study comprehensively evaluated the essential roles of THF/dioxane monooxygenases in dioxane degradation in dioxane-degrading bacterial strains and environment samples. This is the first report to elicit the impact from dioxane and other co-contaminants on shaping functional and phylogenetic structures of microbial communities at a dioxane-impacted site. The development of the novel catabolic biomarker (thmA/dxmA) is of great research and engineering value to unequivocally characterize both dioxane biodegradation potential and activity for enhanced MNA forensics.
1,4-Dioxane; Monitored natural attenuation; Biomarkers; Bioremediation; Monooxygenase