Microbial Interactions with Water Contaminants and Surfaces: Applications Ranging from Bioremediation to Biofouling Prevention

While microorganisms drive nearly all of the Earth’s major biogeochemical cycles, a subset of those microorganisms exert a spectrum of deleterious effects on health, environmental, and industrial processes. This broad range of potential benefits and pitfalls, makes it critical to properly characterize and manage this microbial world. This dissertation describes original research on the treatment of contaminated water using biodegradation by bacterial cultures as well as visualizing and quantifying pathogenic biofilms on surfaces.Solvent stabilizers, such as 1,4-dioxane, are frequently detected in water resources and often co-occur with chlorinated volatile organic compounds (CVOCs). Both classes of trace organic compounds are persistent in the environment and have carcinogenic properties. First, a microbial community comprised of the anaerobic chlorinated ethene-degrading consortium (KB-1) and aerobic bacterial strain, Pseudonocardia dioxanivorans CB1190, was formulated in a defined medium and verified to biodegrade mixtures of chlorinated ethenes and 1,4-dioxane under varying redox conditions. Further, CB1190 was shown to survive 100 continuous days of anaerobic incubation and multiple anaerobic-aerobic cycles. After aeration, it biodegraded 1,4-dioxane rapidly because minimal loss or lag occurred in the induction of the genes, dxmB and aldH, which code for key enzymes in the 1,4-dioxane degradation pathway. While vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE) are inhibitors of 1,4-dioxane biodegradation, surprisingly both compounds were degraded by CB1190. Increasing concentrations of VC decreased 1,4-dioxane biodegradation rates, whereas increasing 1,4-dioxane did not have as significant of an effect on VC biodegradation. VC was found to be the strongest inhibitory CVOC with respect to 1,4-dioxane biodegradation, but it was also utilized as a source of carbon and energy to support CB1190’s growth. Metabolic flux analyses confirmed that VC-derived intermediates were incorporated into CB1190’s central metabolism. A pilot-scale project utilizing in situ aerobic biostimulation and bioaugmentation with CB1190 demonstrated successful removal of CVOCs and 1,4-dioxane from groundwater. Biostimulation (e.g., air sparging) resulted in a significant CVOC removal, but limited 1,4-dioxane removal. When CB1190 culture was added along with air sparging, the concentrations of 1,4-dioxane and CVOCs, such as cDCE and VC, substantially decreased. This signifies the importance of establishing appropriate microorganisms in the subsurface for the remediation of contaminated environments. Contrastingly, microbial adhesion to medical tubing surfaces is undesirable due to adverse patient health outcomes. To mitigate biofilm formation, polysiloxane materials coated with a hydrophilic, zwitterionic polysulfobetaine polymer were designed. These samples were systematically tested against bacterial and fungal agents in static and flow conditions. Additionally, a non-strain-specific protocol combining four microbiological assays was developed to accurately quantify cellular and extracellular components attached to catheter surfaces. The efficacy of this multipronged approach was demonstrated using four pathogenic, clinical isolates, two types of silicone catheters in vitro, and indwelling patient catheters. A quantitative understanding of microbial interactions with chemicals and materials will be valuable for improved environmental bioremediation systems as well as limiting biofilms on medically relevant surfaces

Sequential Anaerobic-Aerobic Biodegradation of Trichloroethylene and 1,4-Dioxane

Chlorinated ethenes, such as trichloroethylene (TCE) and their stabilizers, such as 1,4- dioxane, are widespread groundwater contaminants. Bioremediation can be an effective approach, but opposing redox conditions favored by chlorinated ethane-and 1,4-dioxane- degrading bacteria pose a challenge for their concurrent bioremediation. We engineered a microbial community composed of the anaerobic chlorinated ethene-degrading consortium (KB-1�) and aerobic (Pseudonocardia dioxanivorans CB1190 (CB1190)) bacterial strain, which uses 1,4-dioxane. After anaerobic incubation and TCE reduction, CB1190 + KB-1� coculture was viable and rapidly biodegraded 1,4-dioxane in the presence of oxygen. Aerobic biodegradation of cis-1,2-dichloroethylene (cDCE) by CB1190 was also described. As a plume disperses downgradient, the redox conditions change from anaerobic (source zone) to aerobic (leading edge). The results from this study demonstrate that the engineered microbial community can survive redox changes and biodegrade chlorinated ethenes and 1,4-dioxane. This approach could reduce the cost, energy, and substrates required for in-situ bioremediation of contaminant mixtures

Sequential Anaerobic-Aerobic Biodegradation of Trichloroethylene and 1,4-Dioxane

Chlorinated ethenes, such as trichloroethylene (TCE) and their stabilizers, such as 1,4- dioxane, are widespread groundwater contaminants. Bioremediation can be an effective approach, but opposing redox conditions favored by chlorinated ethane-and 1,4-dioxane- degrading bacteria pose a challenge for their concurrent bioremediation. We engineered a microbial community composed of the anaerobic chlorinated ethene-degrading consortium (KB-1�) and aerobic (Pseudonocardia dioxanivorans CB1190 (CB1190)) bacterial strain, which uses 1,4-dioxane. After anaerobic incubation and TCE reduction, CB1190 + KB-1� coculture was viable and rapidly biodegraded 1,4-dioxane in the presence of oxygen. Aerobic biodegradation of cis-1,2-dichloroethylene (cDCE) by CB1190 was also described. As a plume disperses downgradient, the redox conditions change from anaerobic (source zone) to aerobic (leading edge). The results from this study demonstrate that the engineered microbial community can survive redox changes and biodegrade chlorinated ethenes and 1,4-dioxane. This approach could reduce the cost, energy, and substrates required for in-situ bioremediation of contaminant mixtures

Identification of Naegleria fowleri in Domestic Water Sources by Nested PCR

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home