Drinking water and wastewater treatment are essential to the hygienic operation of modern cities. In the United States, the majority of the population is served by centralized drinking water distribution systems and centralized sewage systems. Water systems have long been recognized as potential transmission routes for infectious diseases, but two rapidly-evolving research areas will be discussed in this dissertation. First, wastewater systems can be monitored not only for their pathogen transmission potential related to waterborne illness, but also to gain insight more broadly into disease and behavior dynamics of the contributing community. This practice is referred to as wastewater-based surveillance (WBS), and candidate targets must be shed in feces or urine to be detected in wastewater but do not necessarily present a safety concern through exposure to wastewater. Second, advances in high-throughput sequencing and bioinformatics have enabled the characterization of microbial communities that naturally inhabit piped drinking water systems, beyond the detection of microbial contaminants. The development of flow cytometry methods to acquire counts of total and intact cells has enabled rapid and more comprehensive measurement of bacterial abundance in drinking water compared to existing methods. By using a combination of these techniques and other water quality measures to study drinking water and pipe biofilms, the importance of deterministic factors (e.g., changes in water use patterns) and stochastic factors (e.g., migration) on both microbial abundance and microbial community composition can be investigated.

Although WBS had been utilized prior to the pandemic, coronavirus disease 2019 (COVID-19) gave urgency to the need for additional sources of public health data and propelled the technique forward in research and practice. Wastewater is an inherently pooled sample that can be tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, providing community-scale data on disease trends at a much lower cost than widespread individual testing and while overcoming some of the biases of clinical testing. However, wastewater is a heterogenous matrix, with sources of target signal variability including inconsistencies in sample collection and processing, nucleic acid degradation based on travel time and conditions in the sewer, and signal dilution due to rainfall and diurnal flow changes. Laboratory and analysis strategies can be employed to improve the interpretability and comparability of wastewater signal. For example, target signal can be normalized to an endogenous biomarker to account for variability in fecal content between samples. However, more research is needed to apply and develop systematic methods to interpret the true SARS-CoV-2 signal from noise introduced in wastewater samples and to increase the comparability of wastewater data across sample sites and timepoints.

The COVID-19 pandemic also highlighted the importance of understanding how water age impacts drinking water quality and microbiota as social distancing measures led to population shifts and large-scale changes in water demand. Elevated water age and stagnation of drinking water can lead to degradation of chlorine residual, bacterial growth, and changes in microbial community composition that may favor opportunistic pathogens and nitrifiers. The risks of drinking water stagnation had been established but not over the extended duration of widespread building closures that occurred during the pandemic. In addition, it was still unclear how these changes in water use patterns would impact the composition of microbial communities within drinking water pipes. Finally, water management approaches, such as repeated restorative flushing, were being recommended and implemented, warranting testing of the efficacy of this approach in buildings. COVID-19 lockdowns created a unique opportunity to study the effects of prolonged stagnation in building plumbing on drinking water quality and take preventative measures.

As a response to the COVID-19 pandemic, the goals of this research were to monitor microbiota in engineered water systems to (i) optimize quantitative surveillance of COVID-19 through wastewater testing and (ii) evaluate changes in distribution system and building drinking water quality and microbial communities due to widespread changes in water demand. Three independent research studies were conducted to achieve these aims.

First, to improve the interpretation of SARS-CoV-2 signal in wastewater, raw wastewater was collected weekly from five sewersheds and one residential facility in the San Francisco Bay Area. The concentrations of the N1 gene of SARS-CoV-2 in wastewater samples were compared to geocoded COVID-19 clinical testing data. To adjust for variation in sample fecal content, four normalization biomarkers were evaluated: crAssphage, pepper mild mottle virus (PMMoV), Bacteroides ribosomal RNA (rRNA), and human 18S rRNA. Of these, crAssphage displayed the least spatial and temporal variability. Both unnormalized SARS-CoV-2 RNA signal and signal normalized to crAssphage had positive and significant correlation with clinical testing data (Kendall's Tau-b (τ)=0.43 and 0.38, respectively), but no normalization biomarker strengthened the correlation with clinical testing data. Of the biomarkers tested, crAssphage was the most promising due to low spatial and temporal variability and because crAssphage-normalized N1 concentration had the strongest correlation with clinical testing data. PMMoV also had low spatial and temporal variability and may be appropriate as a normalization biomarker in some contexts. Conversely, Bacteroides rRNA performed poorly as a biomarker due to its higher spatial and temporal variability. Human 18S rRNA was not suitable as a normalization biomarker due to its variability in sample concentrations, high degradation rate, and ubiquity as a laboratory contaminant. This study supports that trends in wastewater surveillance data reflect trends in COVID-19 disease occurrence and presents tools that could be applied to make wastewater signal more interpretable and comparable across studies.

Next, we aimed to evaluate the impacts of pandemic-related building occupancy reductions (>2 months) and weekly restorative flushing on bulk drinking water quality parameters, bacterial communities, and the occurrence of undesirable microorganisms in tap water. Water samples were collected from fixtures (showers, faucets, and drinking fountains) in three university buildings on the University of California, Berkeley campus, which is served by a drinking water treatment facility using protected surface water and chloramine as a residual disinfectant. Reduced occupancy led to diminished chloramine and elevated intact cell counts, but values remained stable after additional weeks of limited water use. Flushing temporarily improved water quality, with chlorine and cell counts remaining stable for at least one day but returning to levels measured prior to flushing within one week. Alpha diversity of microbial communities was lower under more stagnant conditions, and fixture identity, not flushing, was the most influential factor on bacterial community composition, suggesting a strong influence from local biofilm. Although the genera Mycobacterium, Legionella, Pseudomonas, Nitrosomonas, and Nitrospira were detected in samples via amplicon sequencing, concentrations measured via qPCR of the pathogenic species M. avium complex, L. pneumophila, and P. aeruginosa as well as amoA, a functional gene of ammonia-oxidizing bacteria, were very low or below the detection limit, supporting that stagnation alone did not lead to high occurrence of undesirable microorganisms. Under the conditions of this case study, repeated flushing on a weekly timescale during low occupancy periods was not sufficient to maintain chlorine residual and prevent bacterial growth in fixtures. Building managers need to weigh the temporary water quality benefits of flushing against the labor and water resources required considering local context.

Finally, using bench-scale simulated distribution systems fed with municipal tap water (treated surface water with chloramine as a residual disinfectant), we aimed to characterize the impacts of elevated water age on microorganisms in bulk water and pipe wall biofilms. The reactors allowed for more controlled conditions than a full-scale study and enabled collection of biofilm samples. Reactors were operated for six months prior to building closures and seven months after, providing an opportunity to study how elevated water age entering the reactors impacted microbial communities. During the months after building closures, chloramine levels in the reactors dropped below the detection limit, while cell counts and ATP concentrations increased over an order of magnitude in both the bulk water and biofilms. Microbial communities shifted after building closure, and key taxa were identified that influenced the shift. Many metagenome-assembled genomes that were enriched after building closures contained functional genes related to the degradation of histidine, leucine, phthalate, and pyrimidine, supporting that microorganisms with the functional potential to degrade amino and nucleic acids may be favored in the higher water age conditions. Throughout the experiment, the bacterial community in the bulk water of the reactors was dominated by microorganisms that grew in the reactor (i.e., growth in the bulk water or transfer from the biofilm) and not microorganisms from the influent tap water. Additionally, communities clustered by reactor, which were operated in parallel as replicates, supporting the strong influence of historical contingency in shaping drinking water microbial communities.

Large-scale outbreaks and disruptions to typical operations of society are not unique to the COVID-19 pandemic, and the spread of infectious diseases is expected to increase due to increasing global populations and connectivity, human activities, and human-induced changes to the environment. Drinking water supplies need to be resilient to these disruptions in typical operation. This research informs drinking water system management approaches that can lead to stable water microbiomes and water quality. Building on this research, future studies should go beyond characterization and investigate intentional engineering of drinking water microbiomes. Additionally, municipal wastewater systems need to be fully utilized to provide insight into managing outbreaks, increasing the resiliency of public health systems. Based on this research, approaches are recommended to increase the translatability of wastewater monitoring data that could be used for targets beyond SARS-CoV-2.

Sewage treatment is a pressing global problem, with both environmental as well as human health impacts. In areas where piped sewerage systems are viable, waste stabilization pond systems are one possible method for the treatment of wastewater. Pond systems are often advantageous due to ease of construction and operation as well as their low cost and high effectiveness. With regards to human health, the most important aspect of sewage treatment is the removal and inactivation of pathogens. Pathogen inactivation in pond systems has been attributed to multiple factors including sedimentation, predation, temperature, pH, and sunlight. This research investigates the sunlight-mediated inactivation of pathogens in surface waters, with a primary focus on wastewater treatment pond systems. Two common fecal bacterial indicator organisms were studied: the gram-positive Enterococcus faecalis and gram-negative Escherichia coli.

Laboratory experiments were conducted in 150-mL batch reactors that were filled with different aqueous solutions, including deionized water, D₂O, waste stabilization pond water, or combinations of these waters. These solutions were spiked with E. faecalis or E. coli, exposed to simulated sunlight, and loss of culturability was monitored over time. Temperature, pH, dissolved oxygen concentration, and irradiance were carefully controlled and monitored. Furthermore, some experiments involved the addition of photosensitizers or quenchers of radical oxygen species (ROS), while in others we measured the concentrations of ROS in the reactors.

We began by identifying and describing the role of oxygen, exogenous sensitizers, light intensity and light wavelength upon the sunlight-mediated inactivation of E. faecalis and E. coli. We found that, while in DI water the presence of UVB wavelengths increased inactivation of both E. faecalis and E. coli, in pond water, the presence of the UVB wavelengths did not increase the rate of E. faecalis inactivation. Pond water constituents played a dual role, as either photosensitizers increasing inactivation rates (E. faecalis) or as light-attenuators, decreasing inactivation rates (E. coli). Inactivation rates of both E. faecalis and E. coli were correlated to dissolved oxygen concentrations, though differently. Decreasing dissolved oxygen below air saturation always decreased inactivation rates, however increasing above air saturation was not as straightforward. In waste stabilization pond water, where oxygen increases during the day due to algal photosynthesis, raising dissolved oxygen above air saturation increased inactivation of E. coli slightly, however E. faecalis inactivation remained constant.

Next we characterized the effects of pH on the sunlight-mediated inactivation of E. faecalis and E. coli, studying inactivation in the absence and presence of sunlight. E. coli inactivation in pond and DI water had the same trend, with small changes in inactivation rates between more moderate pH, but above and below extremes (pH<4 or pH>9) sharp increases in inactivation rates. E. faecalis, however, was more sensitive to small changes in pH, particularly in pond water.

We then investigated which ROS were involved in inactivation of E. faecalis and E. coli. Photosensitizers produce reactive oxygen species following the absorption of light and the transfer of light-energy to oxygen through a number of different pathways. Although our focus was upon exogenous mechanisms, endogenous mechanisms could not be excluded because in the presence of exogenous sensitizers damage by both exogenous and endogenous mechanisms can occur simultaneously. While we found evidence for the involvement of ¹O₂ in endogenous inactivation of both E. faecalis and E. coli, the importance of other species, while likely, remains unclear. As expected, no evidence was found to support a role for ROS produced exogenously in the inactivation of E. coli. A combination of quencher and D₂O experiments, together with ROS measurements in pond water, provided strong evidence for the importance of exogenous ¹O₂ in E. faecalis inactivation. In addition, E. faecalis was significantly more sensitive than E. coli to inactivation by ¹O₂ produced by the synthetic sensitizer rose bengal, which followed the same pattern as their sensitivity to pond water constituents.

To apply our results from laboratory microcosms to actual treatment systems, we developed a simple model based on our empirical data. The model takes into account the effects of dissolved oxygen concentration, pH, and pond water sensitizers as well as the effects of light attenuation on sunlight mediated inactivation. We applied the model to scenarios that might occur in natural settings, and discuss the strengths and limitations of our approach.

Three mechanisms have been proposed to describe sunlight-mediated inactivation: direct UVB damage to DNA, indirect endogenous inactivation caused by UVB light, and indirect exogenous inactivation involving all wavelengths of sunlight up to 550 nm. While the first mechanism is a likely part of sunlight-mediated inactivation, it did not dominate the inactivation of either bacteria in this study. The second mechanism, while previously attributed to UVB light, should be expanded to include UVA and visible wavelengths, as these caused E. faecalis inactivation in our DI water wavelength experiments. The third mechanism dominated E. faecalis inactivation and was driven by the UVA and visible wavelengths, though E. faecalis inactivation occurred through all three mechanisms. E. coli, on the other hand, was not subject to the third, exogenous mechanism in our microcosms. Instead E. coli inactivation was dominated by endogenous mechanisms, and these mechanisms were driven by the UVB and UVA wavelengths. A final mechanism was described that does not fit into the three mechanisms above; exogenous production of H₂O₂, which then crosses into cells and causes endogenous microbial damage. This mechanism was not important in our pond water microcosms because H₂O₂ concentrations were low due to high scavenging by pond water constituents, but it may be important in other waters. Only E. coli appears susceptible to such a mechanism.

Enteric pathogens are a leading cause of diarrheal illness in low-and middle-income countries and are transmitted through the fecal-oral route. Exposure routes can be blocked through adequate drinking water treatment, sanitation facilities and hygiene practices (WaSH). While these pathways are well understood conceptually, few studies have explored household transmission and the impacts of WaSH interventions with empirical measurements of enteric pathogens. In this dissertation, indicators and pathogens were measured in household reservoirs (stored drinking water, soil, and mothers and child hands) in rural Bangladesh to quantify the impact of a sanitation intervention on household level fecal contamination. Additionally, the reliability of indicator Escherichia coli to suggest risk of enteric pathogen infection was evaluated in this context. Fecal indicator organisms were also used to assess the importance of animal fecal sources in these households. Lastly, the association between bacterial communities in household reservoirs was quantified using 16S rRNA gene sequencing. Through this work I showed that the sanitation intervention in place had limited impact on household fecal contamination. In later chapters, indicator E. coli is shown to be a useful tool to indicate risk in this context as higher concentrations of indicator E. coli were associated with higher prevalence of pathogenic E. coli genes. I also determined that animals were potential sources of Giardia and pathogenic E. coli genes on mother hands. In Chapter 4, shared bacteria between mother and child hands, hands and soil, and mother hands and stored water suggest the potential for transmission of pathogens.

Drinking water in low-income countries and stormwater in the U.S. is often contaminated with pathogens, posing health risks to local communities. In response, distributed filters are being used to provide the needed protection from exposure and infectious disease. Two examples of these distributed filters include point-of-use (POU) devices at the household scale for drinking water treatment and bioretention basins at the community scale for stormwater treatment. Sand is a common media used in distributed filters, but pathogen removal is often low due to unfavorable interactions between the sand surface and microorganisms. Removal may be increased, however, if certain amendments are added that create favorable interactions between microorganisms and the filter media, either through electrostatic attraction or hydrophobic interactions. This dissertation focused on the use of three media amendments, including a quaternary ammonium silane (QAS), zero valent iron (ZVI), and biochar, in distributed filters to improve removal of indicator bacteria and viruses from drinking water and stormwater.

In the first part of this dissertation, a QAS-coated sand was evaluated for drinking water treatment in POU filters. Varying influent water matrices were introduced to QAS-coated sand columns at representative flow rates, and the effluent was evaluated for indicator bacteria, indicator virus, and human virus removal. In basic water matrices with 1 mM NaCl and Escherichia coli (E. coli) or MS2 coliphage (MS2), removal was observed to increase with depth and decrease with increasing ionic strength, flow velocity, and experiment length. E. coli attached to QAS-coated sand were observed to have permeable membranes, providing evidence of inactivation. Major limitations, however, were observed when the influent contained humic acid or other viruses. Rapid fouling in the presence of humic acid was likely caused by competition for positively charged binding sites, and low PRD1 and Adenovirus 2 removal may have stemmed from steric interference between viral capsid features and the media surface.

In the second part of the dissertation, ZVI and biochar amendments were evaluated for stormwater treatment in bioretention basins in two long-term experiments. In the first experiment, stormwater columns were operated intermittently to examine E. coli and MS2 removal in conventional bioretention media (CBM) and ZVI-amended media. A 1-year storm (3.2 cm) for Berkeley, CA was introduced to the columns once per week for 5.15 h. Afterwards, the columns were drained and remained in this state until the next week’s storm. This pattern was repeated for 46 weeks. In CBM, E. coli removal increased as the columns aged and correlated with a decrease in hydraulic conductivity. Some evidence that biological activity contributed to E. coli removal was observed in a sodium azide experiment. In a ZVI-amended silica sand, E. coli and MS2 removal was above 1 log through week 11 but decreased quickly afterwards to levels observed in silica sand. The presence of other adsorbates, including natural organic matter (NOM), phosphate (PO43-), and other organisms, likely contributed to the observed decrease in removal. In ZVI-amended CBM, E. coli and MS2 removal remained low throughout the experiment, likely due to the NOM, PO43-, and organisms leached by the compost in CBM. Iron leaching was also observed in ZVI-amended CBM. In both ZVI-amended media types, large hydraulic conductivity reductions and cementation issues raised concerns over long-term permeability in the field.

In the second experiment, columns were operated as a batch system where stormwater was injected into a column and allowed to rest in the media for 24 h. After this rest period, fresh influent was injected into the column again, and the expelled effluent was evaluated for E. coli and MS2 removal. This pattern was repeated daily for 75 d. In biochar-amended silica sand, E. coli removal above 4 log was observed throughout the experiment. In iron-amended silica sand, MS2 removal above 2 log and PO43- removal around 90% was observed after 75 d, and hydraulic conductivity did not decrease as observed in unsaturated columns.

Results from this dissertation provided insights into the limits of media amendments for pathogen removal, identified possible removal mechanisms of indicator organisms in mature bioretention basins, and demonstrated the promise of a batch configuration with increased rest periods and media amendments for long-term removal of indicator organisms.

Urine is a “waste” stream that contains the majority of nitrogen, phosphorus, and potassium present in domestic wastewater. Separate collection and treatment of urine can potentially reduce treatment costs, environmental pollution due to nutrient discharges, and dependence on energy-intensive synthetic fertilizers. Closing the nitrogen loop between conventional fertilizer production and wastewater treatment can improve system efficiencies in industrialized settings and generate revenue that can be used to enhance sanitation access in developing communities. Harnessing the benefits of urine separation requires selective separation of nitrogen from other urine constituents. In this dissertation, two unit processes were characterized to recover nitrogen as ammonium from urine: ion exchange and electrochemical stripping. Observations were made at several scales, from molecular-scale mechanisms to process performance metrics and systems-level evaluation. In sum, this dissertation furthers understanding of two processes for recovering nitrogen as ammonium from urine as well as the overall source separation and resource recovery paradigm.

Ion exchange is an established technique for separating charged species from solutions and has recently been applied to urine. Household cartridges could be employed to concentrate nitrogen at the source of urine production. Four adsorbents were compared using equilibrium isotherms and fixed-bed columns; Dowex Mac 3, a synthetic ammonium-specific resin, exhibited the highest adsorption density and regeneration efficiency. Based on comparing synthetic urine solutions of increasing complexity and similarity to real urine, competition from sodium and potassium did not significantly decrease ammonium adsorption. Because urine composition varies, several models were compared to experimental data to predict adsorption isotherms. Flow rate did not significantly affect ammonium adsorption nor regeneration; higher ammonium concentration in urine and proton activity in regenerants increased adsorption and regeneration efficiencies. Only trace organics that were positively charged at urine pH values (~9) adsorbed, and only two of ten compounds studied were detected in the ammonium sulfate product. Potassium recovery via cation exchange and phosphate recovery via struvite precipitation and anion exchange did not significantly affect ammonium adsorption, indicating potential implementation of complete nutrient recovery treatment trains.

Electrochemical stripping was shown to recover nitrogen as ammonium sulfate from urine based on the charge and volatility of ammonia. This novel approach combines electrodialysis with membrane stripping and selectively recovered 93% of nitrogen in batch studies at lower energy consumption than conventional ammonia stripping. Based on continuous-flow nitrogen fluxes, transport from the cathode to the trap chamber was identified as the rate-limiting step of electrochemical stripping. Synthetic urine solutions were used to determine the effects of urine composition and operating parameters on nitrogen flux, recovery efficiencies, and competing reactions. Electrochemically produced chlorine was found to be a loss mechanism for nitrogen but was mitigated by organic compounds present in urine. Nitrogen was selectively separated from other urine constituents, including sodium, potassium, inorganic anions, trace organic compounds, and metals.

Building on the molecular and process-scale insights in the laboratory, ion exchange for nitrogen recovery was evaluated in a container-based sanitation system in Nairobi, Kenya. No losses in performance metrics were observed over ten adsorption-regeneration cycles or with locally-produced columns ten times larger than lab scale. Parameters that can be economically and regularly measured to indicate process performance, such as absorbance and electrical conductivity, were identified as useful proxies for urine and ammonium breakthrough. While ammonium sulfate produced from urine was slightly more expensive than trucking urine to a local waste treatment facility, urine-derived fertilizers could be produced for less than conventional fertilizers in Kenya. Producing fertilizer from waste could reduce costs of sanitation provision and increase access to fertilizer.

Insights from this dissertation are useful to researchers and practitioners interested in closing loops to generate revenue from “waste” streams and reduce environmental impacts of excreta treatment. Adding fundamental understanding of two nitrogen recovery processes informs the design of pilot and demonstration scale installations to separately collect and treat urine at household and building scales. Beyond urine treatment, ion exchange and electrochemical stripping can be applied to other nitrogen-rich waste streams such as anaerobic digester effluent, landfill leachate, and food processing wastes.

Over three hundred million people throughout the world receive supply from piped drinking water distribution networks that operate intermittently. This dissertation evaluates the effects of intermittent supply on water quality, pipe damage and service reliability in four study zones (one continuous and three intermittent) in a peri-urban drinking water distribution network in Arraiján, Panama. Normal water quality in all zones was good, with 97% of routine water quality grab samples from the distribution system and household taps having turbidity < 1 NTU, total coliform and E. coli bacteria concentrations < 1 MPN / 100 mL, and ≥ 0.3 mg/L free chlorine residual. However, negative pressures that represent a risk for contaminant intrusion and backflow were detected in three of the four study zones, and water quality during the first flush when supply resumed after an outage was sometimes degraded. High and transient pressures that could cause pipe damage were detected in study zones with intermittent pumping, but filling and emptying of distribution pipes due to intermittent supply was not associated with transient or extreme pressures. Operational challenges, including frequent infrastructure failures, difficulty monitoring the network, and a lack of system information, resulted in unreliable supply in the intermittent zones. Continuous pressure and flow monitoring methods used in this research could be helpful tools for operators of intermittent distribution networks to provide more reliable service and identify hydraulic conditions that could lead to contaminant intrusion or pipe breaks.

Regions facing water scarcity are expanding their water portfolios to augment supplies and meet future demand. Direct potable reuse, the deliberate introduction of advanced treated municipal wastewater into a drinking water treatment facility or distribution system, has gained considerable traction in the United States in recent years. The high concentrations of pathogens and chemical contaminants in municipal wastewater necessitates a high degree of treatment to meet drinking water standards and protect public health. Therefore, in potable reuse systems, municipal wastewater is typically treated by conventional wastewater treatment processes (e.g., sedimentation and activated sludge) followed by “advanced” treatment processes (e.g., microfiltration, reverse osmosis, and advanced oxidation). To date, the main research focus on microbial water quality in potable reuse systems has been quantifying the removal of pathogens (primarily enteric viruses and protozoa) present in municipal wastewater. While essential, this research does not provide insight into how advanced treatment alters the entire microbial community or the potential for microbial growth in treated water. Moreover, the management of microbial risk and water quality in distribution systems remains poorly studied in the context of direct potable reuse.

In this dissertation, improved microbial monitoring methods were applied to two advanced wastewater treatment facilities and a lab-scale simulated direct potable reuse distribution systems to evaluate the impact of advanced treatment on microbial numbers, viability, and growth potential in direct potable reuse systems. Microbial water quality through the treatment and distribution systems was evaluated by measuring changes in total and potentially viable prokaryotic cells, total and intracellular ATP, organic carbon, several primary and opportunistic pathogens, and two antibiotic resistance genes. Enhanced microbial analysis methods such as flow cytometry for cell counts, ATP analysis, and dead-end ultrafiltration for quantitative PCR analyses were applied and evaluated as research tools and/or routine monitoring methods for potable reuse systems.

Through this work I showed that the introduction of advanced treated water into a drinking water distribution system holds the potential for improving microbial water quality, but growth during the distribution of advanced treated water must still be properly managed. The microbial water quality of drinking water distribution systems may improve following the introduction of advanced treated water by a lower potential for microbial growth, lower microbial viability, and greater stability of a chlorine residual during simulated distribution. Specifically, chlorine residual decay and the numbers of potentially viable cells (i.e., intact cell counts measured by flow cytometry) were lower in distribution systems fed with advanced-treated water as compared to those fed with 100% conventional water. The prevalence of the opportunistic pathogens Legionella pnuemophila and Mycobacterium avium complex were unaffected by the use of advanced treated water. Furthermore, assimilable organic carbon concentrations (i.e., a common metric for microbial growth potential) in advanced treated water was similar to or less than values reported for finished and distributed drinking waters in the United States. The transportation, conditioning, and storage of reverse osmosis permeate without a chlorine residual resulted in large increases in microbial numbers, but no more than those typically seen in drinking water with a degraded residual. The relative abundance of sul1 remained unchanged by the handling of RO permeate, leading to high increases in absolute sul1 gene counts. Regardless, the application and maintenance of a chlorine residual substantially suppressed microbial numbers during distribution of advanced treated water; however, the passage of ammonia through advanced treatment could cause significant variability in the performance of chlorine disinfection.

I also demonstrated the utility of flow cytometry, ATP analysis, and dead-end ultrafiltration (to concentrate water for quantitative PCR analyses) as informative research and monitoring methods for potable reuse systems. In particular, these tools were used to successfully quantify microbial cells in matrices with low biomass (e.g., reverse osmosis permeate) with relatively good precision. Furthermore, flow cytometry and ATP analysis showed promise as routine/continuous tools to monitor the performance of treatment processes such as microfiltration, reverse osmosis, and chlorination. These tools also provided additional insights into how the chlorine residual impacted microbial viability (e.g., changes in intact cell counts) in simulated distribution systems.

Overall, this body of work contributes to a better understanding of engineered water systems and will ultimately inform more comprehensive water quality monitoring strategies for water treatment systems. Further work is needed to characterize microbial water quality in different types of advanced treatment trains, and utilities that implement direct potable reuse should use conventional and improved monitoring methods to evaluate full-scale distribution systems before and after the introduction of advanced-treated water.

Constructed wetlands harness natural processes to provide a low cost and energy technology for wastewater treatment. Although not the primary goal of wetland design, wetlands may remove and inactivate pathogens via a number of mechanisms including sunlight inactivation, predation, and sorption coupled to sedimentation. The contribution of sunlight inactivation is generally insignificant in vegetated wetlands due to light screening by emergent macrophytes and deep water. A novel wetland design, termed open-water wetlands, utilizes geotextile fabrics on the wetland bottom to prevent the growth of emergent vegetation. This allows sunlight to penetrate the shallow water column (<30 cm), thereby enhancing sunlight inactivation of pathogens. The goal of this research was to evaluate the potential of open-water wetlands to inactivate pathogens in secondary municipal wastewater effluent. To this end, indicator organism inactivation was monitored in a pilot-scale open-water wetland. In addition, to gain insight into inactivation mechanisms (e.g., endogenous versus exogenous inactivation), laboratory experiments were conducted using photosensitizer-free water, wetland water and simulated sunlight. These studies resulted in a descriptive model that predicts inactivation rates of bacteria in open-water wetlands and other sunlit water bodies.

As a first step towards developing a complete photoinactivation model for bacteria, a model was developed for a simpler organism (MS2 coliphage) considering only endogenous inactivation (Chapter 2; published in Environmental Science and Technology, 2014). The objective of the model was to account for wavelength specificity of photoinactivation, using a photoaction spectrum (PAS), and accounting for changes in the light spectrum due to season and attenuation in the water column. Experiments were set up to measure inactivation rates of MS2 in photosensitizer-free water in under natural sunlight in different seasons (one summer and one winter day), and in a wetland water column under simulated sunlight. The PAS model successfully predicted MS2 inactivation rates based on natural sunlight irradiances throughout the year, as well as in different water column depths. One of the challenges in developing and applying the PAS model was the uncertainty in measuring and predicting (using a radiative transfer model) sunlight irradiance in the range of 280 - 300 nm. The possibility of using total UVB as a simplified model parameter for estimating rates of endogenous inactivation was also tested using the experimental results. Although the total UVB model has a major limitation (it values each wavelength in the range of 280 - 320 nm equally), this simplified model can be used for estimating endogenous inactivation rates of organisms whose photoaction spectra have not yet been measured.

To evaluate the performance of open-water wetlands in removing pathogens, fecal indicator bacteria concentrations were monitored in a pilot-scale open-water wetland over a one- year period (Chapter 3). The pilot-scale wetland provided effective inactivation of fecal indicator bacteria (up to 3-log and 2-log removal of E. coli and enterococci, respectively), primarily via sunlight inactivation. A novel model to predict endogenous and exogenous inactivation rates of fecal indicator bacteria in the wetland was developed and validated using data from laboratory experiments and field data. Endogenous inactivation was predicted using a total UV (sum of UVA and UVB irradiance) model. Exogenous inactivation was significant only for enterococci, and was modeled as a function of steady-state concentration of singlet oxygen in the bulk aqueous phase. The model was also applied to predict the land area required to achieve 3-log removal of fecal indicator bacteria throughout the year. Results suggested that during summer conditions in central California, open-water cells can provide greater than 3-log removal of non-pigmented fecal indicator bacteria in an area comparable to existing full-scale wetland systems.

To provide further insight into the role of dissolved organic matter (DOM) in the exogenous inactivation of indicator organisms, wetland DOM isolates from different wetland types (open-water, bulrush, and cattail) were prepared using solid-phase extraction and were tested for inactivation potential (Chapter 4). Inactivation of MS2 was enhanced by all wetland DOM isolates, as well as by two standard DOM isolates [Suwannee River Fulvic Acid (SRFA) and Pony Lake Fulvic Acid (PLFA)]. The steady-state concentration of singlet oxygen in the bulk phase of the DOM isolate solutions was positively correlated with the MS2 exogenous inactivation rate. For Ent. faecalis, inactivation was enhanced in solutions of wetland DOM isolates and PLFA; however, for the SRFA solution light screening dominated any photosensitizing effect. The degree of association of Ent. faecalis with DOM varied among the isolates, and a positive trend was observed between greater association and inactivation rate. Similar inactivation rates were observed in solutions of DOM isolates and their parent whole wetland water samples for Ent. faecalis, whereas for MS2, much lower inactivation rates were observed with the isolates than in the whole wetland water samples.

To quantify the effect of photosensitizer association on exogenous inactivation of fecal indicator bacteria, experiments were conducted measuring inactivation of Ent. faecalis (gram-positive) and E. coli (gram-negative) in the presence of a natural DOM and a model photosensitizer [Rose Bengal (RB)] at different Mg2+ concentrations (Chapter 5). Due to differences in cell wall structure, Ent. faecalis associated more strongly with DOM than E. coli. Inactivation of Ent. faecalis was enhanced by photosensitizer adsorption onto bacterial cells. Despite RB and EfOM association with E. coli cells, E. coli inactivation rates were not enhanced by adsorbed photosensitizers, likely due to the protection offered by the outer membrane. Inactivation of Ent. faecalis was not enhanced in irradiated solutions of polymer beads coated with RB (compared to sensitizer-free solutions), which were used to generate singlet oxygen in the absence of bacteria-sensitizer association, suggesting that association with photosensitizers is a prerequisite requirement for exogenous inactivation of Ent. faecalis.

A significant output of this research is the descriptive model to predict sunlight inactivation of fecal indicator bacteria, which can be used to evaluate designs for future applications of open-water wetlands. This research also produced several important outcomes. First, the monitoring data of the pilot-scale open-water provided evidence that open-water unit process wetlands can achieve significant inactivation of fecal indicator bacteria. This finding can be used to promote the adoption of the novel open-water wetland design for pathogen removal, as a component of multi-barrier approaches to control pathogen transmission, or to reduce the use of chemical disinfectants. Second, insight into the exogenous mechanism of fecal indicator bacteria inactivation suggested that the association between photosensitizers and bacterial cells plays a key role in exogenous inactivation for the gram positive model species Ent. faecalis. Third, lab-cultured bacteria were inactivated significantly more rapidly than indigenous wastewater indicator bacteria, emphasizing that lab-cultured indicators should not be used to determine inactivation rate constants for indigenous bacteria.

Sunlight has long been known to inactivate microorganisms in natural waters and engineered systems. However, the mechanisms of inactivation are not yet fully understood. Solar disinfection (SODIS) is a treatment technology that relies on the germicidal effects of sunlight to inactivate pathogens in drinking water at the point of use. The objective of this work was to explore the roles of wavelengths, transition metals, and reactive oxygen species in the inactivation of indicator microorganisms in water, and discuss the implications of these findings for solar water disinfection. Alternative container materials and hydrogen-peroxide-producing additives were found to accelerate the sunlight inactivation of MS2 bacteriophage as well as E. coli and Enterococcus bacteria during field trials. Furthermore, it was observed that the inactivation of E. coli and Enterococcus derived from local wastewater was significantly slower than the inactivation of laboratory-cultures of the same organisms, while the inactivation of MS2 was slowest of all. The inactivation of all organisms appeared to be heavily dependent on the UVB-transparency of the container material used. To investigate these apparent wavelength effects in more depth, sunlight action spectra were measured in clear water for bacteriophage and E. coli. Both UVA (320 - 400 nm) and UVB (280 - 320 nm) light were found to contribute to the inactivation of PRD1 bacteriophage, while only UVB inactivated MS2. The inactivation of three laboratory E. coli strains and three E. coli strains isolated from wastewater was also studied. Both UVB and UVA wavelengths contributed to the inactivation of all strains, which exhibited strong similarities in their inactivation characteristics, while E. coli naturally present in fresh wastewater was found to be less sensitive to UVA than a cultured laboratory strain. A computational model was developed for interpreting the action spectra of these viruses and bacteria with 3-nm resolution. Studies were also conducted to investigate the roles of iron and reactive oxygen species in the photoinactivation of E. coli. Mutants lacking peroxidase and superoxide dismutase enzymes were found to be more sensitive to polychromatic simulated sunlight, while cells grown with low iron concentrations were more resistant to photoinactivation. Furthermore, prior exposure to light sensitized E. coli to subsequent exposure to hydrogen peroxide in the dark, an effect which was diminished for cells grown on low-iron media. Collectively, these results provide further evidence for the involvement of both UVA and UVB wavelengths in driving E. coli photoinactivation through a mechanism that appears to be consistent with intracellular photoFenton chemistry. These findings also reinforce the critical role of UVB wavelengths in the sunlight inactivation of viruses and, to a lesser extent, wastewater-derived bacteria. The approach to measuring photoaction spectra used in this work may be applicable to investigations of a variety of photobiological and photochemical systems. Finally, the fieldwork results suggest that additives and alternative container materials may be able to greatly accelerate the photoinactivation of microorganisms in drinking water.

Climate change and increasing patterns of drought throughout the world are challenging the effectiveness of conventional water systems. A growing population in conjunction with more extreme weather events, threatens water supply infrastructure and increases uncertainty about how utilities will meet demand without sacrificing water quality. This issue has recently manifested in California, prompting utilities to invest in alternative water sources as a means of ensuring that water infrastructure is resilient to climate change scenarios.

Decentralized wastewater treatment is a promising option for increasing the sustainability of water infrastructure as it spatially merges supply and demand, minimizing large conveyance requirements. Decentralization can also promote nutrient management and recovery as it enables the source separation of the different wastewater sources. Specifically closing the nitrogen loop, by capturing it and reusing it to generate valuable high-end products can potentially improve the efficiency of the system and create revenue streams.

However, smaller decentralized water treatment units are potentially more energy intensive and costly than their centralized alternatives per unit of water treated. Due to these efficiency tradeoffs, planning tools and frameworks for holistically assessing decentralized water treatment systems need to be developed to optimally manage the new urban water supply paradigm. Better data management and data-driven decision support tools can provide valuable insight on the benefits and impacts of implementing future water systems.

This research assesses the technical performance of emerging decentralized technologies and implementation scenarios for residential uses, by assessing the feasibility of integrating decentralized facilities in cities with existing wastewater infrastructure. This work aims to create algorithmic models that integrate the spatial design of a wastewater treatment and distribution network with a life-cycle assessment to determine the associated environmental impacts. This dissertation utilizes spatial modeling to contextually evaluate the implementation and distribution potential and uses a life-cycle assessment approach to provide an extensive analysis of all the life-cycle impacts. By incorporating environmental indicators and metrics, a planning support framework can be created to help guide the water industry towards smart investments for a less energy-intensive future. Specifically, this work will 1) investigate how spatial terrain variability, demographics and distribution affect the performance of decentralized water treatment systems, 2) analyze the major parameters that affect the energy intensity, cost and greenhouse gas emissions of these systems, 3) quantify the unit processes that mostly impact the prementioned metrics and 4) identify the optimal system scale for decentralized infrastructure implementation under various spatial and demographic conditions.

The insights from this dissertation can help wastewater researchers and practitioners understand the complex relationships between the system scale and system performance. By evaluating the potential benefits and tradeoffs, this work can lead to management tools that will help transition away from traditional water management and create a water supply that (1) is resilient to changes in climate, (2) uses local water sources, and (3) leaves more water in natural ecosystems. This dissertation further adds to the growing body of literature on decentralized wastewater treatment assessing optimal scales, reuse potential, resource recovery and sewerage connections to investigate key factors affecting future implementation.