Antibiotic resistance (AR) is a critical and looming threat to human health that requires action across the One Health continuum (humans, animals, environment). Coordinated surveillance within the environmental sector is largely underdeveloped in current National Action Plans to combat the spread of AR, and a lack of effective study approaches and standard analytical methods have led to a dearth of impactful environmental monitoring data on the prevalence and risk of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments. In this dissertation, integrated surveillance approaches of surface water and wastewater systems are demonstrated, and efforts are made towards standardizing both metagenomic- and culture-based techniques for globally comparable environmental monitoring. A field study of differentially-impacted watersheds on the island of Puerto Rico post-Hurricane Maria demonstrated the effectiveness of metagenomics in defining direct impact of anthropogenic stress and human fecal contamination on the proliferation of ARGs in riverine systems. The contribution of treated wastewater effluents to the dissemination of highly mobile and clinically-relevant ARGs and their connection to local clinical settings was also revealed. At the international scale, a transect of conventional activated sludge wastewater treatment plants (WWTPs), representing both US/European and Asian regions, were found to significantly attenuate ARG abundance through the removal of total bacterial load and human fecal indicators, regardless of influent ARG compositions. Strong structural symmetry between microbiome and ARG compositions through successional treatment stages suggested that horizontal gene transfer plays a relatively minor role in actively shaping resistomes during treatment. Risk assessment models, however, indicated high-priority plasmid-borne ARGs in final treated effluents discharged around the world, indicating potentially increased transmission risks in downstream environments. Advancements were also made toward standardizing methods for the generation of globally representative and comparable metagenomic- and culture-based AR monitoring data via two comprehensive and critical literature reviews. The first review provides guidance in next-generation sequencing (NGS) studies of environmental AR, proposing a framework for experimental controls, adequate sequencing depths, appropriate use of public databases, and the derivation of datatypes that are conducive for risk assessment. The second review focuses on antibiotic-resistant Enterococcus spp. as robust monitoring targets and an attractive alternative to more widely adopted Gram-negative organisms, while proposing workflows that generate universally equivalent datatypes. Finally, quantitative metagenomic (qMeta) techniques were benchmarked using internal reference standards for high-throughput quantification of ARGs with statistical reproducibility. / Doctor of Philosophy / Antimicrobials have contributed to the reduction of infectious diseases in humans and animals since the early 20th century, increasing productivity and saving countless lives. However, their industrial-scale application across human, animal, and agricultural sectors over the last several decades, especially the use of antibiotics, have engendered the proliferation of antibiotic resistance (AR). AR occurs when changes in bacteria cause the drugs used to treat infections to become less effective and has become one of the leading public health threats of the 21st century. The global spread of AR through the transmission and evolution of antibiotic resistant bacteria (ARB; known colloquially as "superbugs") and antibiotic resistance genes (ARGs) across the One Health continuum (i.e., humans, animals, and the environment) is resulting in increased hospitalization, length of hospital stays, suffering, death, and overall health-care associated costs globally. This dissertation demonstrates the use of metagenomics, the sequencing of all genetic material (e.g., DNA) recovered from a microbial community, for the comprehensive monitoring of ARB and ARGs in aquatic environments, a key pathway for the dissemination of AR into and out of human populations. In order to impede the proliferation of AR, surveillance systems are currently in place to track the spread and evolution of ARB and ARGs in humans and livestock, as well as agri-food sectors. However, the surveillance in natural and built environments (i.e., rivers and domestic sewage) has significantly lagged due to the lack of standard monitoring targets and methodologies. It is also a goal of this dissertation to suggest guidance for the collection of metagenomic- and culture-based AR monitoring data to generate universally comparable results that can be included in centralized databases. Riverine systems are ideal models for tracking input of antibiotic resistance to the natural environment by human activity. After Hurricane-Maria, many of Puerto Rico's wastewater treatment plants (WWTPs) went offline, discharging raw sewage to local surface waters. In a cross-sectional study of watersheds impacted by WWTPs, the abundance of ARGs was directly correlated to increases in local population density. Also, highly mobile and clinically-relevant ARGs were found directly downstream of WWTPs across the island. We found that many of these ARGs corresponded well to forms AR endemic to the region. WWTPs are the primary engineering controls put in place to curb the spread of human and animal waste streams and can help to reduce AR. An international transect of conventional activated sludge WWTPs representing US/Europe and Asia were sampled to garner a mechanistic understanding of the fate or ARGs through treatment. Although WWTPs remove total bacteria, human fecal indicators, and much of the abundance of ARGs, mobile and clinically-relevant ARGs are discharged around the world in large quantities. Consideration is needed in certain regions of iv the world where the managing of human waste streams is the first line of defense against the dissemination of resistance to local communities. Two comprehensive critical literature reviews were conducted to evaluate the various methodologies for generating and analyzing metagenomic- and culture-based AR monitoring data. These reviews address the need for experimental rigor and disclosure of extensive metadata for inclusion in future, centralized databases. The articles further provide guidance with respect to universally comparable datatypes and efficient workflows that will aid in the scale-up of the collection of environmental monitoring data within a global surveillance framework. Finally, a study was conducted to benchmark the use of internal DNA reference standards for the absolute quantification of ARGs (i.e., on a ARG copy per volume of sample basis). The statistical framework for ARG detection and its implications for wastewater-based surveillance systems of AR are also discussed.

Streams and rivers are a vital part of our ecosystem. They are imperiled by human ecological activities such as urbanization, industrialization, and agriculture which discharge excess nitrate and other pollutants into our waterways. Here, this dissertation seeks to understand the physical and biogeochemical processes which attenuate pollutants in stream corridors. The focus is hyporheic zones which form the interface between surface water and groundwater below and adjacent to stream channels, and riparian zones which form the interface between channels and adjacent uplands, both of which can attenuate pollutants. In this context, soil-pipes can dominate subsurface hydraulics. This research first employed MODFLOW and MT3D-USGS to model transient hyporheic hydraulics and nitrate transport in a length of riparian/riverbank soil to probe the effects of soil pipes on hydraulics and denitrification due to peak flow events in the channel. Findings showed that inserting just one soil pipe 1.5 m in length caused a ~75% increase in both hyporheic exchange and denitrification. A rough upscaling showed soil pipes could remove up to ~3% of nitrate along a 1-km reach. Next, the ability of soil pipes to bypass the often championed ability of riparian buffers to remove nitrate migrating from uplands to the channel was evaluated. This effort also employed MODFLOW and MT3D-USGS to simulated hydraulics and nitrate removal along a length of riparian soil. Findings showed that soil pipes increased flow of nitrate to the banks by five orders of magnitude in some cases. We posited a non-dimension parameter which governs when nitrate bypass is significant. In addition to soil pipes, dune bedforms can also enhance hyporheic exchange, primarily in the stream/riverbed. Again employing MODFLOW but now pairing with the transport code SEAM3D to simulate microbially-mediated aerobic metabolism of dissolved organic carbon and dissolved oxygen, the combined effects of dune translation and microbial growth and death were explored. Major findings include that neglecting microbial growth can lead to inaccurate modeling of biogeochemistry, and that aerobic metabolism increased with celerity. The results herein bolster knowledge of natural pollutant attenuation in stream and river corridors, and have implications for pollutant mitigation strategy and stream credit allocation. / Doctor of Philosophy / Streams are a vital part of our ecosystem. They are imperiled by human ecological activities such as urbanization, industrialization, and agriculture which discharge nitrate and other pollutants into our waterways. Here, this dissertation seeks to understand the physical and biological processes which attenuate pollutants. The hyporheic zone is the interface between surface water and groundwater below the bed and adjacent to stream banks, and can attenuate pollutants. Transient peak flow events such as a storm or snow melt raise the stream water levels, causing the water pressure in the stream channel to temporarily outweigh the water pressure in the soil pore spaces adjacent to the stream channel. This drives water into the banks subjecting it to pollutant attenuation processes. Soil pipes (long cylindrical void spaces created by decayed plant roots) are prevalent along stream banks, and they dominate subsurface hydraulics. This dissertation implemented a numerical study on a chunk of riparian soil to probe the effects of soil pipes on hydraulics and denitrification. Findings showed that inserting just one – 1.5 m soil pipe caused a ~75% increase in both water flow volume into the bank and nitrate removal. Riparian buffers are the vegetated strips adjacent to stream channels and have long been championed as stalwarts of pollutant removal. Soil pipes undermine this by acting as a bypass mechanism. A numerical study was again performed on a chunk of riparian soil to quantify the effects soil pipes on riparian bypass of nitrate. Findings showed that soil pipes increased flow of nitrate to the banks by five orders of magnitude in some cases. This means that a buffer enhancement strip with fine roots that prevent the formation of soil pipes should be installed along riparian buffers. In addition to soil pipes, dune bedforms can increase flowrate of water into the hyporheic zone. This dissertation modeled the combined effects of dune translation and microbial growth and death. Major findings include that neglecting microbial growth can lead to inaccurate modeling of biogeochemistry, and that biodegradation increases with increased dune velocity. The results herein bolster knowledge on natural pollutant attenuation in streams, and have implications in terms of pollutant mitigation strategy and stream credit allocation.

Evaluation of the sources and cycles of water quality contaminants in watersheds is critical for effective surface water resource management. In particular, iron (Fe) and manganese (Mn), commonly found in rocks and sediments, have adverse impacts on water quality. However, controlling Fe and Mn in surface water systems is often complex and requires careful consideration of the hydrologic and biogeochemical factors that influence the speciation and mobility of these metals. This dissertation investigates the sources and cycles of Fe and Mn in surface waters designated for human use. Here, I present the findings from three field- and laboratory-based studies conducted at sites in western Virginia, United States. The first study examines the impacts of reservoir-derived and watershed-derived metals on water quality along the 180 km reach of the Roanoke River downgradient from Leesville Dam. The results from this study showed strong temporal influences on river water quality immediately downgradient of the dam, resulting from seasonal reservoir dynamics. Further downgradient in the Roanoke River, water quality was strongly tied to hydrologic conditions resulting from influences generated in the watershed. The second study investigated the effects of increasing dissolved oxygen (DO) concentrations in the hypolimnion of stratified drinking water reservoir on Fe and Mn oxidation and removal. Results from a whole-ecosystem experiment showed that increasing DO concentrations through hypolimnetic oxygenation was effective for preventing the accumulation of soluble Fe in the water column. Although Mn oxidation increased under well-oxygenated conditions, soluble Mn still accumulated in the hypolimnion. Results from a laboratory experiment demonstrated that the oxidation of Mn was strongly tied to the activity of Mn oxidizing microbes. The third study examined the relative contribution of external and internal metal loadings to the exchange of metals between sediments and the water column and the source/sink behavior of a seasonally stratified reservoir under varying hydrologic conditions in the inflows and outflows and redox conditions in the reservoir hypolimnion. Results from this study showed that redox conditions strongly influenced the exchange of metals between the sediment and aqueous phase, but had little effect on the source/sink behavior of the reservoir, while external tributary loadings had little effect on internal redox cycles, but was a strong indicator for whether the reservoir behaved as a net metal source or sink. Overall, the findings from these studies exemplify the value of characterizing the hydrologic and biogeochemical drivers of Fe and Mn cycles for managing the water quality effects of these metals in surface water supplies. / Ph. D.

Increases in reactive nitrogen from human actions have led to negative impacts on surface water (SW) and groundwater (GW) quality, and it is important to better understand denitrification processes in aquatic systems. The hyporheic zone has unique biogeochemical conditions, and is known to attenuate contaminants originating from SW and traveling through the hyporheic zone, together with necessary reactants. However, the ability of the hyporheic zone to attenuate contaminants from deeper upwelling GW plumes as they exit to SW is less understood. I used MODFLOW and SEAM3D to simulate hyporheic flow cells induced by riverbed dunes and upwelling GW together with mixing dependent denitrification of an upwelling nitrate (NO3-) plume. My basecase model scenario entailed dissolved organic carbon (DOC) and dissolved oxygen (DO) advecting from SW and DO and NO3- advecting from GW, which is typical of water in agricultural land uses. I conducted a sensitivity analysis to determine controls on mixing dependent denitrification. Mixing dependent denitrification increased with increasing hydraulic conductivity, decreasing lower bottom flux, as well as increasing DOC in SW and NO3- in GW. Non-mixing dependent denitrification also occurred when there was SW NO3-, and I found its magnitude was much greater than mixing dependent denitrification. Nevertheless, potential for hyporheic zones to attenuate upwelling NO3- plumes seems to be substantial, though highly variable depending on biogeochemical reaction rates as well as geomorphic, hydraulic and biogeochemical conditions. Stream and river restoration efforts may be able to increase both mixing and non-mixing dependent reactions by increasing hyporheic zone residence times. / Master of Science

The pollution of streams and rivers is a growing concern, and environmental guidance increasingly suggests stream restoration to improve water quality. �Solute retention in off channel storage zones such as hyporheic zones and floodplains is typically necessary for significant reaction to occur. �Yet the effects of two common restoration techniques, in stream structures and inset floodplains, on solute retention have not been rigorously compared. �We used MIKE SHE to model hydraulics and solute transport in the channel, inset floodplain, and hyporheic zone of a 2nd order stream. �We varied hydraulic conditions (winter baseflow, summer baseflow, and storm flow), geology (hydraulic conductivity), and stream restoration design parameters (inset floodplain length, and presence of in stream structures). �In stream structures induced hyporheic exchange during summer baseflow with a low groundwater table (~20% of the year), while floodplains only retained solutes during storm flow conditions (~1% of the year). �Flow through the hyporheic zone increased linearly with hydraulic conductivity, while residence times decreased linearly. �Flow through inset floodplains and residence times in both the channel and floodplains increased non linearly with the fraction of bank with floodplains installed. �The fraction of stream flow that entered inset floodplains was one to three orders of magnitude higher than that through the hyporheic zone, while the residence time and mass storage in the hyporheic zone was one to five orders of magnitude larger than that in floodplain segments. �Our model results suggest that in stream structures and inset floodplains are complementary practices. / Master of Science

Increased reactive nitrogen from human activities negatively affects surface water (SW) quality. The hyporheic zone, where SW and groundwater interact, possesses unique biogeochemical conditions that can attenuate contaminants (e.g., denitrification), including mixing-dependent reactions that require components from both water sources. Previous research has explored mixing-dependent denitrification in the hyporheic zone but did not address the effects of varying SW depth as would occur from storms, tides, dam operation, and varying seasons. We simulated steady and unsteady hyporheic flow and transport through a riverbed dune using MODFLOW and SEAM3D, and varied SW depth, degree of sediment heterogeneity, amplitude and frequency of sinusoidal fluctuations, among others to determine these effects. We found that increasing steady state surface water depth from 0.1 to 1.0 m increased non-mixing dependent aerobic respiration by 270% and mixing-dependent denitrification by 78% in homogeneous sediment. Heterogeneous hydraulic conductivity fields yielded similar results, with increases in consumption due to variation in correlation length and variance of less than 5%. Daily SW fluctuation, including variation of amplitude, period, and sinusoidal versus instantaneous changes had significantly less impact than longer-term trends in SW depth. There is potential for the hyporheic zone to attenuate NO3- in upwelling groundwater plumes. Restoration efforts may be able to maximize the potential for mixing-dependent reactions in the hyporheic zone by increasing residence times. / Master of Science / Increased nitrogen in runoff from human activities negatively affects surface water quality. The hyporheic zone is where surface water and groundwater interact, and the mixing between the waters can help to this nitrogen to undergo reaction (denitrification), potentially stopping the contaminant from spreading. Previous research has explored this idea, but has not addressed the impact of varying surface water depth, as would realistically occur due to storms, tides, dam operation, and varying seasons. We simulated both constant and fluctuating surface water conditions on a riverbed dune to see the effects on hyporheic flow and denitrification. Test variables included the surface water depth, the degree of sediment heterogeneity, the amplitude and frequency of surface water fluctuations. We found that increasing the steady-state surface water depth had the most dramatic increase on the amount of reaction undergone. This trend was also seen in heterogeneous sediment. Any daily-scale surface water fluctuations, including runs that varied the amplitude, period, and sinusoidal vs instantaneous changes in surface water depth, had significantly less impact than longer-term trends in surface water depth.

Of the factors that determine development potential in a given geographic area, the availability of water for residential, commercial, and industrial purposes is a primary indication of prospective growth. Governmental bodies at the regional, state and federal levels often need to identify water supply availability in order to identify growth potential. To address this need, a procedure for the preliminary assessment of water supply availability has been developed that can potentially be applied to any geographic area in the United States. The procedure uses the USGS demand cataloging unit as the basic planning area, with supply estimates from streamflow parameters at USGS gage locations and demand estimates from USGS demand reports. By comparing known supply and demand estimates in a base year, an overview of water supply availability in the region can be determined. With supply and demand data in a base year, projections of future water supply availability can then be made. Detailed projection of future water demand must account for changes in the amount of water use activities and the rates of water use within those activities, but a simplified procedure is applied here. Total offstream water use is averaged over the population in the base year to determine per-capita offstream use, which is assumed to remain constant in the future in this preliminary assessment procedure. Population is then projected and demand is forecast as a function of the projected population. The supply quantity is projected assuming each flow parameter derived from the historical record will remain constant in the future year. By comparing projected supply and demand estimates, water supply availability in future years can be anticipated in the planning area. / Master of Science

Heating, Ventilating and Air Conditioning Systems (HVAC) are not only one of the most energy consuming components in buildings but also contribute to green house gas emissions. As a result often environmental design strategies are focused on the performance of these systems. New HVAC technologies such as Geothermal Heat Pump systems have relatively high performance efficiencies when compared to typical systems and therefore could be part of whole-building performance design strategies. In collaboration with the Virginia Tech Corporate Research Center, Inc., this research studies the energy consumption and cost benefits of the Geothermal Heat Pump System that has been integrated and operated in the KnowledgeWorks I and II buildings located on the Virginia Tech campus. The purpose of this thesis is to understand the energy and cost benefits of the Geothermal Heat Pumps System when compared to the conventional package variable air volume (VAV) with hot water coil heating and air-source heat pump systems using computer simulation and statistical models. The quantitative methods of building energy performance and life-cycle cost analyses are applied to evaluate the results of simulation models, the in-situ monitoring data, and the associated documents. This understanding can be expanded to the higher level of architectural systems integration. / Master of Science

Yes / There is a need for an inexpensive, reliable and fast monitoring tool to detect contaminants in a short time, for quick mitigation of pollution sources and site remediation, and for characterization of natural dissolved organic matter (DOM). Fluorescence spectroscopy has proven to be an excellent technique in quantifying aquatic DOM, from autochthonous, allochthonous or anthropogenic sources. This paper reviews the advances in in situ fluorescence measurements of DOM and pollutants in various water environments. Studies have demonstrated, using high temporal-frequency DOM fluorescence data, that marine autochthonous production of DOM is highly complex and that the allochthonous input of DOM from freshwater to marine water can be predicted. Furthermore, river measurement studies found a delayed fluorescence response of DOM following precipitation compared to turbidity and discharge, with various lags, depending on season, site and input of dissolved organic carbon (DOC) concentration. In addition, research has shown that blue light fluorescence (λemission = 430–500 nm) can be a good proxy for DOC, in environments with terrestrial inputs, and ultraviolet fluorescence (λemission = UVA–320–400 nm) for biochemical oxygen demand, and also E. coli in environments with sanitation issues. The correction of raw fluorescence data improves the relationship between fluorescence intensity and these parameters. This review also presents the specific steps and parameters that must be considered before and during in situ fluorescence measurement session for a harmonized qualitative and quantitative protocol. Finally, the strengths and weaknesses of the research on in situ fluorescence are identified. / Authors, E.M. Carstea and C.L. Popa, acknowledge the support of the Ministry of Research and Innovation, CNCS-UEFISCDI, project number PN-III-P1-1.1-TE-2016-0646, within PNCDI III, project number 18N/2019, under the Core Program OPTRONICA VI, project number 19PFE/17.10.2018 and project number 152/2016, SMIS 108109.

Source water protection is important to maintain public health by keeping harmful pathogens out of drinking water. Non-point source pollution is often times a major contributor of pollution to surface waters, and this form of pollution can be difficult to quantify. This study examined physical, chemical, and microbiological water quality parameters that may indicate pollution and may help to identify sources of pollution. These included measures of organic matter, particles, and indicator organisms (fecal coliforms and E. coli). The parameters were quantified in the West Boylston Brook, which serves as a tributary to the Wachusett Reservoir and is part of the drinking water supply for the Metropolitan Boston area. Water quality was determined over four seasons at seven locations in the brook that were selected to isolate specific land uses. The water quality parameters were first analyzed for trends by site and by season. Then, a correlation analysis was performed to determine relationships among the water quality parameters. Lastly, ANOVA analyses were used to determine statistically significant variations in water quality along the tributary.