Biological nutrient removal (BNR) systems employ engineered biological processes—including nitrification, denitrification, and biological phosphorus removal—to remove nutrients from wastewater. Since their original implementation, BNR systems have adapted to challenges, such as the presence of inhibitory compounds and demands for more energy- and resource-efficient wastewater treatment. Advancements in alternative BNR technologies made in response to these demands have highlighted the metabolic versatility of microbial communities present in BNR systems. This versatility is also observed in the expanded capacity of BNR systems to remove not only human-derived carbon, but also complex trace organic emerging contaminants (ECs). Based on conventional monitoring alone, the roles of specific bacteria and metabolic mechanisms in the removal of nutrients and ECs remain unclear. A detailed understanding of the actors and mechanisms in BNR systems can be attained through application of molecular biology tools, including those targeting community (a) structure and potential function through DNA analysis and (b) extant function through RNA analysis. This dissertation encompasses three objectives, which seek to link detailed molecular-level information to the performance and metabolic versatility of several nutrient-removing communities. The first objective was to assess the utility of gene expression assays to indicate and predict nitrification inhibition by toxic heavy metals based on functional responses of nitrifying bacteria. Through this assessment, it was found that genes related to both catabolic and anabolic pathways could be used as indicators of nitrification inhibition. The second objective was to investigate the effects of reactor operating conditions on simultaneous nitrogen and phosphorus removal by examining the microbial community structure and metabolism of a survey of full-scale BNR systems. A variety of BNR configurations and operating conditions, all capable of sustained nutrient removal, selected for different nitrogen- and phosphorus-removing communities. The activity of these communities was also dependent on configuration and operating conditions, as indicated by analysis of gene expression. Finally, the third objective was to examine the expanded capacity of BNR systems to attenuate ECs by investigating the removal of the EC bisphenol A (BPA) by microbial communities involved in nutrient removal. Communities derived from both full-scale and lab-scale systems were capable of biodegrading BPA, though each community was uniquely influenced by reactor processes and BPA exposure conditions. Results from this work also offered insights into the utility of assessed genes as biomarkers for metabolic activity and the importance of accurately characterizing in-situ responses of BNR systems. In both lab-scale and full-scale studies, certain genes demonstrated increased sensitivity to nutrient-removing activity. At lab-scale, observed differences between inhibition of ammonia oxidation through discrete and continuous Cu(II) exposure indicated that conventional short-term, ex-situ batch assays may underestimate inhibition in a parent reactor of interest. The benefit of gene expression assays to accurately reflect in-situ responses was also examined in full-scale BNR systems removing both nitrogen and phosphorus. Findings from full-scale BNR systems revealed the long-term effects of changes to process configurations on microbial community structure and activity. Despite differences in operating conditions and the resulting nitrogen- and phosphorus-removing communities, a variety of configurations sustained nutrient removal. Long-term effects were also characterized in the context of EC removal. Differences in BPA degradation rates and microbial community profiles in lab-scale mixed culture communities after extended BPA exposure showed the lasting influence of both reactor processes and BPA exposure conditions. Assessment of microbial community structure was also used to identify BPA-degrading bacteria. Results from each of the three objectives could be used in the development of biomarkers to assess and predict (1) process upsets or inhibition, (2) nutrient removal process performance, or (3) capacity for EC removal. Integrating analysis of microbial community structure and function with reactor performance monitoring and mechanistic modeling that includes such advanced knowledge holds the potential to not only guide effective operation of BNR systems, but also identify opportunities for more efficient and even concomitant nitrogen, phosphorus, and EC removal.

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Continuous increases in the interest and implementation of wastewater reuse due to intensified water stress has escalated the concerns of emerging contaminants. Among emerging contaminants there are microbial (antibiotic resistance) and chemical (pharmaceuticals) elements which have been shown to survive wastewater treatment. This dissertation aims to mitigate emerging contaminants by means of understanding and/or developing the appropriate disinfection strategies, with the intention to provide knowledge that would facilitate towards safe and sustainable water reuse. The first part of this thesis explored microbial risk component of antibiotic resistance. Antibiotic resistance genes are abundant in treated wastewater, and only pose a risk if taken up by potential pathogens through natural transformation. Our results showed that solar irradiation can double natural transformation rates, mediated by reactive oxygen species generation, which led to upregulation in DNA repair and competence genes in Acinetobacter baylyi ADP1. Treatment with UV-C254 nm irradiation also resulted in upregulation in DNA repair genes, nevertheless we observed a decrease in natural transformation rates. These results imply that direct damage of antibiotic resistance genes (ARG) could inhibit their spread and therefore risk, despite other factors contributing to the contrary. The next chapter in this dissertation postulated that the UV/H2O2 combination would be ideal to treat microbial and chemical emerging contaminants in effluent generated from an anaerobic membrane bioreactor. We demonstrated that at an optimal UV intensity and H2O2 concentration, we were able to achieve a 2 and 6-log reduction of the two antibiotic resistance genes and bacteria and used in this study, respectively, and more than 90% removal of the three pharmaceutical compounds. These observations suggest that UV/H2O2 has great potential in treating effluent with high nitrogen concentrations, preserving the fertilization benefit of AnMBR effluent. Overall, this dissertation revealed the potential of UV-based treatments for treated wastewater intended for reuse. Post-membrane processes effluent allows one to deploy UV-C254 nm to selectively target DNA and therefore ARB and ARG that may be still present in the treated wastewater. At the same time, coupling chemical oxidants with UV-C (i.e., UV AOP) would further enhance the means to simultaneously oxidize and degrade potentially harmful chemical contaminants.

Microplastics are present in municipal wastewater treatment plant (WWTP) effluents; however, it is unclear whether these contaminants are ingested by biota living downstream of these outfalls. This study examined whether microplastic levels in caged biota, resident fish, and environmental samples were elevated near the Waterloo and Kitchener WWTP outfalls along the Grand River in the fall of 2019. Amphipods (Hyalella azteca), fluted-shell mussels (Lasmigona costata), and rainbow trout (Oncorhynchus mykiss) were caged at one upstream reference site and two impacted sites downstream of the Kitchener WWTP for 14 (amphipods and trout) or 28 (mussels) days. Rainbow darter (Etheostoma caeruleum) were collected using a backpack electrofisher from 10 sites up and downstream of both the Kitchener and Waterloo WWTPs, along with surface water and sediment samples. Whole body Hyalella, fish digestive tracts, and fluted-shell mussel tissues (hemolymph, digestive glands, and gills) were digested in 20% potassium hydroxide. Environmental samples were processed using filtration and density separation, then visual identification of microplastics was done. Elevated particle counts were found in rainbow trout digestive tracts at the Kitchener outfall site, compared to the upstream reference and downstream farfield sites. Additionally, particle concentrations in sediment were significantly higher at the Waterloo outfall, compared to all other sites (except for one upstream location). However, whole Hyalella, fluted-shell mussel tissues (hemolymph, digestive glands, and gills), digestive tracts of rainbow darter, and surface waters did not show elevated counts downstream of these discharges. Across all samples, fibers were the most common morphology, and blue and clear particles were prevalent in samples collected near WWTPs. Overall, these findings suggest that the Kitchener and Waterloo WWTPs could be important sources of particles to the Grand River, adding to our understanding of the fate of this contaminant in freshwater ecosystems. / Thesis / Master of Science (MSc)

Despite claims of nearly 100% access to potable drinking water in the US, issues of drinking water quality, accessibility, and equity persist in many regions of the country. Drinking water is a common health concern in rural communities, where social, geographic, and economic challenges can inhibit the provision of reliable municipal water. Households without access to municipal water often rely on private wells, which are solely the responsibility of the homeowner to test, treat, and maintain, or roadside springs. These water sources often do not employ water treatment and users can therefore be uniquely susceptible to environmental contaminants. The goal of this research was to examine point-of-use (POU) treatment options that can be used by individuals to improve their drinking water quality and reduce exposure to common contaminants prior to consumption. Two drops (~0.10 mL) of unscented, household bleach in one gallon of spring water is a simple, low-cost treatment option that successfully inactivates total coliform and E. coli and provides an appropriate free chlorine residual (> 0.5 mg/L) over a 1-month time period, without exceeding free chlorine taste thresholds (< 2 mg/L). Efforts to distribute information on this disinfection protocol to spring users in southern West Virginia and southwestern Virginia were well-received; however, only 60% of surveyed spring users report that they plan to implement the protocol. POU faucet filters have been successfully implemented in homes reliant on municipal water to reduce metal contaminant levels in drinking water. Few studies have assessed the effectiveness of these filters in improving water quality in homes reliant on private wells. Faucet-mounted POU filters distributed to homes reliant on private wells in Virginia and southern West Virginia statistically significantly lowered levels of Ba, Cd, Cr, Total Coliform, U, Cu, Pb, Al, Fe, Mn, Zn, and Sr in tap water. However, levels of many contaminants of interest still exceeded at least one Safe Drinking Water Act regulation/recommendation in several filtered samples. Additionally, less than half of study participants reported that they liked using the filters with several citing issues with flowrate. Faucet-mounted POU filters can also be a useful tool in assessing exposure to contaminants at the tap. The acid flow-through method of metals recovery has previously proven to be successful in recovering dissolved Pb from dosed filters. In this study, the acid flow-through extraction method was applied to water spiked with high or low levels of Pb, Fe, or Cu. While faucet-mounted activated carbon filters successfully removed Pb and Cu from dosed influent (>91% removal), filter behavior under influent Fe concentrations of greater than 300 ppb was extremely variable. The acid flow-through method of metals extraction provided some recovery from filters dosed with high and low concentrations of Pb (38.9-70.4%). Recovery of Cu and Fe was variable, likely in part due to Fe and Cu leaching from filter media, suggesting that alternative methods of metals extraction and recovery from POU faucet filters dosed with Fe and Cu, or other common water contaminants (e.g., As, Ba, Cd), must be explored. While POU treatment can be useful in improving drinking water quality in rural households, limitations to adoption persist and must be addressed along with efforts to protect drinking water quality in homes in a more permanent, sustainable way. / Doctor of Philosophy / Drinking water quality is a common health concern in rural communities, where social, geographic, and economic challenges can make municipal water quality unreliable. Households without access to municipal water often use private wells and sometimes roadside springs for drinking water. These water sources are often untreated which can expose users to environmental contaminants such as bacteria or metals. The goal of this research was to study point-of-use (POU) treatment options that can be used by individuals looking to improve their drinking water quality and reduce their exposure to common contaminants, perhaps while waiting for more permanent improvements and upgrades. Household bleach is a simple, low-cost way of lowering levels of bacteria in roadside spring water that is being used as drinking water. Two drops of unscented, household bleach in one gallon of spring water successfully kills total coliform and E. coli bacteria and provides an enough leftover chlorine to continue to disinfect the water for 1-month. This information was given to spring users in southern West Virginia and southwestern Virginia and, while most people who provided feedback found the information useful, only 60% of surveyed spring users report that they plan to implement this protocol. Point-of-use faucet filters have been found to successfully reduce metals contaminant levels in drinking water in homes that use municipal water. However, few studies have tested the effectiveness of POU faucet-mounted filters in lowering contaminant levels in water in homes reliant on private wells. Faucet-mounted POU filters given to homes reliant on private wells in Virginia and southern West Virginia lowered levels of many contaminants of interest in tap water, including lead, copper, iron, and total coliform bacteria. However, in some of the filtered samples, levels of many of these contaminants were higher than at least one Safe Drinking Water Act regulation. Less than half of study participants reported that they liked using the filters with several stating that they had issues with low flowrate. Faucet-mounted POU filters can also be a useful tool in better understanding exposure to contaminants at the tap. The acid flow-through method of metals recovery has previously proven to be successful in recovering lead, and other metals, that are collected inside the filters during water treatment. In this study, an extraction method using acid was tested on filters that treated water with high or low levels of lead, iron, or copper. The filters were successful in removing lead and copper from test water, but filters were not as consistently successful in removing iron from test water. The extraction method using acid provided some recovery from filters dosed with high and low concentrations of lead (38.9-70.4%). However, recovery of copper and iron was more inconsistent, suggesting that a different method of metals recovery may be necessary. While POU treatment can be useful in improving drinking water quality in rural households, there are limits to how useful it is in certain situations, such as when treating water with extreme water quality. In order to make sure rural households have access to safe drinking water, these limits need to be addressed and efforts need to be made to figure out a way to protect and supply drinking water in a more permanent way.

Photodegradation is a major degradation mechanism for many environmental contaminants, both in indoor environments as well as in outdoor environments, and many of the contaminants are persistent and toxic. However, there is a lack of knowledge about the exact degradation process for many of them. It is important to not only monitor and study environmental contaminants that are commonly found. But it is also important to be able to identify their degradation products, however, due to lack of standards there is a difficulty to conduct identification. There is also the drawback with most spectral libraries used for identification being recorded at low resolution, when new instruments are being developed for using high resolution. This makes it challenging to identify compounds with spectral peaks that differ only slightly in their mass-to-charge ratio (m/z). Thus, the aim of this study was to determine potential degradation products from different compounds commonly found in indoor environments and then add these findings to a spectral library for high resolution mass spectrometry. This was conducted by first exposing the standards to ultraviolet (UV) light, and then the chemical analysis was performed using a gas chromatography coupled with a high resolution mass spectrometer. All data was then processed in different software in order to determine potential products. Many of the compounds did not indicate any degradation using the experimental settings, and for some not even the parent compound could be identified. In total, four potential degradation products were found in this study; degradation products were identified for hexabromobenzene (HBB) with penta-, tetra-, and tribromobenzene, as well as one potential product for 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB).

Pharmaceuticals are an important class of pollutants in aquatic ecosystems. Detected concentration are typically in the range 1 ng/L – 1 μg/L. Traditional wastewater treatment does not provide a complete removal of these contaminants; hence, they may have a negative impact on the environment. In addition, microalgae are an ecologically-meaningful target group of species for bioindication purposes as well as primary production and oxygen supply. The present work aimed to investigate the effect of Ibuprofen on the green alga Pseudokirchneriella subcapitata. Algal cultures were exposed to five different concentrations of the drug (5, 15, 45, 135, 405 mg/L) for four days. Absorbance measured at 680 nm was determined every day and obtained data were transformed into cell concentration (cells/mL) by a previously prepared calibration curve. Specific growth rate, generation time, percent inhibition and effective concentration were calculated. Moreover, one way ANOVA with Tukey’s test were applied to observe differences between groups and time periods. Based on this study, all the cultures treated with Ibuprofen had a growth inhibition as well as presenting a lag phase. Increasing the Non-Steroidal Anti-Inflammatory drug (NSAID) concentration reduced the growth rate and consequently, increased the percent inhibition in a concentration-dependent manner. According to this report, new research should be focused on the development of hybrid systems for degradation and removal of pharmaceuticals. NSAID pollution may lead to a reduction in the diversity and number of functional groups of eukaryotic algae. Finally, more research should be devoted to the toxicity of drugs in a variety of test organisms and development of reliable methods for toxicity test at low and chronic exposures to achieve more realistic conclusions.