The prevention of waterborne diseases requires rapid detection of pathogens in drinking water, with an ultimate goal of in-line monitoring in real time. Standard cultivation-based methods are too time-consuming and thus not suitable for this purpose. Many technologies were proposed to achieve this goal, such as ELISA, PCR, FISH, FTIR and flow cytometry. However, they still have limitations of non-specificity, complexity and high cost. Therefore, an optical system is proposed and developed towards the in-line monitoring of bacteria, which combines the advantages of FTIR and micro-flow cytometer for bacterial identification and precise quantification. The in-line use requires obtaining IR spectra of bacterial cells directly in water, which is achieved using a CaF2 liquid cell. The spectra of a series of bacterial samples are collected and analyzed using principal component analysis for their differentiation. A preliminary study on fabricating a CaF2 concentrator is conducted, in which a novel phenomenon on stress release of silicon nitride film on CaF2 substrate is discovered and studied. To determine the concentration of bacteria in drinking water, a micro-flow cytometer is built based on a micro-fabricated device that integrates on-chip beam-shaping optics and microfluidic channels. With this micro-flow cytometer and optimized data analysis for counting particles in real time, linearity with correlation coefficient of over 0.99 is achieved for the dependences of throughput on both volumetric flow rate and concentration of sample. With a one-dimensional hydrodynamic focusing, no degradation of the counting efficiency is demonstrated when the focused sample stream expands. The high accuracy of counting makes this micro-flow cytometer a promising candidate for low concentration applications. Counting of E. coli DH5α cell suspensions in phosphate buffered saline is performed using the micro-flow cytometer. Side-scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a haemocytometer. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2-4µm) as their scattering intensities are different. / Thesis / Doctor of Philosophy (PhD)

Conventional drinking water treatment consists of a coagulation, flocculation, gravity separation, filtration and disinfection processes each working individually but also as an interdependent system. One of the main reagents used for drinking water treatment are coagulants that destabilise the suspended particles which results in the formation of flocs. For many years, the coagulant of choice was aluminum sulphate, also know as alum. Alum has slowly been replaced by new coagulants, such as polyalumium sulphates and polyaluminum chlorides, because they yield more consistent plant performance than with alum over the wide temperature range experienced by Canadian treatment plants. Recent research has determined that the alum solubility envelop varied significantly in terms of pH range with temperature, thus cold temperature performance may be improved by adjusting the coagulation pH. Dissolved air flotation (DAF) is now used at some water treatment plants to replace sedimentation because it is much more compact than gravity settling, and it is somewhat better than sedimentation for the removal of algae, organics and operation in cold temperatures. The objective of this thesis is to help operators and managers of drinking water treatment plants incorporating DAF by: a) investigating the cold water turbidity removals of DAF systems using alum, the most economical coagulant; and b) investigating the impact of DAF saturator pressure on the bubble sizes produced and floc removal. This first initiative is based on fairly recent research on the impact of pH on the cold-temperature aluminum solubility. It uses this knowledge about the impact of pH to evaluate DAF treatment of Ottawa River water in cold-water conditions using DAF batch tests. The effect of pH against final turbidity at cold temperatures was first evaluated by increasing the pH of the coagulated water, the higher pH helped attain good turbidity removals. For the coagulant dose tested, good turbidity removals were observed for both warm and cold waters at nearly the same pOH conditions. At room temperature the turbidity removals increase with both increasing flocculation G and flocculation time. While at cold temperatures, when aluminum flocs are known to be much more fragile, the turbidity removals appear to be independent of G and GT. The second initiative studied the relationship between floc size and bubble size in DAF systems by changing the DAF saturator pressure. Increasing the saturator pressure did not significantly decrease the mean bubble size. The flocs attach to bubbles that were significantly larger than the bubbles. The assessment of DAF efficiency based on the unitized effluent floc distribution proved inconclusive, it may be possible that the conditions resulting with the larger mean effluent floc size has a greater removal efficiency since it began with a smaller fraction of small flocs entering the flotation stage.

<p dir="ltr">Interactions between drinking water quality and delivery systems, pipe materials, and attitudes</p>

Opportunistic premise (i.e., building) plumbing pathogens (OPPPs) and antibiotic resistant bacteria are emerging microbial concerns in drinking water. OPPPs, such as Legionella pneumophila, are the leading cause of drinking water disease in many developed countries. Contributing factors include the relative success in controlling fecal pathogens, the presence of complex building plumbing systems that create habitats for OPPPs, and the relative resistance of OPPPs to disinfectants, and aging populations that are susceptible to infection. Concurrently, drinking water is increasingly being scrutinized as a potential environment that is conducive to horizontal gene transfer of antibiotic resistance genes (ARGs), selection pressure for enhanced survival of resistant bacteria, and a route of transmission of antibiotic resistant pathogens. While maintaining a disinfectant residual is an established approach to controlling OPPPs in premise plumbing, some studies have indicated that co-resistance and cross-resistance to disinfectants can increase the relative abundances of resistant bacteria and ARGs. Thus, there may be trade-offs to controlling both OPPPs and antibiotic resistance in premise plumbing that call for controlled study aimed at optimizing residual disinfection application for this purpose. A critical review of the scientific literature in Chapter 2 revealed that premise plumbing is a biologically and chemically complex environment, in which the choice of pipe material has cascading effects on water chemistry and the corresponding premise plumbing microbiome. This, in turn, has broad implications for the control of OPPPs, which need to be elucidated through controlled experiments in which worst case premise plumbing conditions are held constant (e.g., warm temperature), while other variables are manipulated. Chapter 3 introduces the convectively-mixed pipe reactors (CMPRs) as a novel low-cost, small footprint approach to replicably conduct such experiments. The CMPRs were demonstrated to effectively simulate key chemical and biological phenomena that occur in distal reaches of premise plumbing. In Chapter 4, the CMPRs were leveraged to study the interactive effects of four disinfectants (chlorine, monochloramine, chlorine dioxide, and copper-silver ionization) and three pipe materials (PVC copper, and iron). The CMPRs were inoculated with two antibiotic-resistant OPPPs: Pseudomonas aeruginosa and Acinetobacter baumannii. It was found that pipe-material (PVC or PVC combined with iron or copper) profoundly impacted the water chemistry in a manner that dictated disinfection efficacy. In Chapter 5, we applied shotgun metagenomic shotgun sequencing to evaluate effects of the combination of pipe material and disinfectant type on the wider microbial community, especially their ability to select for or reduce ARGs. In Chapter 6, we used CMPRs and metagenomic sequencing in a study comparing Dutch drinking water practices to our prior testing in an American system. Dutch drinking water is of interest because of lack of historical use of disinfectants was hypothesized to result in a microbial community that is relatively depleted of ARGs or mobile genetic elements, which can enhance spread of ARGs as disinfectants are applied. Generally, it was found that OPPPs required higher doses of disinfectants for inactivation than the general microbial community, sometimes concentrations approaching the regulatory limits in the US (e.g., 4 mg/L of total chlorine). Even successful reductions were modest, typically ~1-log, and failed to eliminate either P. aeruginosa or A. baumannii. Moreover P. aeruginosa, A. baumannii, and non-tuberculous mycobacteria varied substantially in their preference for pipe material and susceptibility to disinfectants. We found that disinfectants tended to increase the relative abundance of OPPPs, ARGs, and mobile genetic elements. Disinfectants were sometimes associated with net increases in levels of these pathogens and genes when applied at low levels (e.g., 0.1 mg/L of monochloramine), which effectively acted to reduce competition from less resistant and non-pathogenic taxa. When a low dose of monochloramine was applied to PVC CMPRs in the US, we estimated from metagenomic sequencing data that this water contained roughly 100,000 cells per milliliter of taxa known to contain pathogenic members. The Dutch drinking water exhibited more diverse microbial communities and lower relative abundances of taxa containing pathogens. ARGs were two times proportionally more abundant in CMPRs operated in the US without disinfectant than in the corresponding CMPRs operated in the Netherlands. The findings of this dissertation can help to optimize the application of in-building disinfectant addition for addressing concerns related both to OPPPs and antibiotic resistance. The studies herein highlight the necessity of developing comprehensive OPPP and antibiotic resistance control strategies that emphasize not just disinfectant dose, but other key control parameters such as contact time, hydraulics, and temperature. The functional diversity of OPPPs, antibiotic resistant bacteria, and the background premise plumbing microbiome further necessitates broad, holistic programs for monitoring and control. / Doctor of Philosophy / Efforts to provide safe drinking water face two emerging threats: the rise of pathogens that thrive in the plumbing environment that delivers water to the tap and the rise of antibiotic resistance. In the US and many other parts of the world, opportunistic pathogens are the predominant agents responsible for disease spread by tap water. Opportunistic pathogens tend to infect aged or immunocompromised individuals (hence, 'opportunistic') and grow well in in-building plumbing. Globally, antibiotic resistance is on the rise and becoming a fundamental threat to modern medicine. Pathogenic bacteria become resistant to antibiotics used to treat infections when they acquire antibiotic resistance genes (ARGs), which can happen either by mutation or from other resistant bacteria sharing ARGs. Overuse or misuse of antibiotics can impose selection pressure that stimulates horizontal gene transfer and enhance survival of bacteria that are resistant. Prior studies have suggested that under some circumstances, disinfectants used to control pathogens in drinking water can also select for antibiotic resistant bacteria. Thus, the overarching goal of this research was to optimize the type and dose of disinfectant used, depending on building-level factors such as pipe material, for effectively controlling proliferation of both opportunistic pathogens and antibiotic resistance. This dissertation largely focuses on in-building plumbing systems, which are home to potentially tens of thousands of bacterial cells per milliliter of water or per square centimeter of internal pipe surfaces. These bacteria interact not only with each other and other microbes, but also with features of the plumbing environment, such as the water chemistry or the pipe materials. Building plumbing systems are highly intricate ecosystems that can undermine the effectiveness of disinfectants provided by utilities. One major contribution of this research is the development of the convectively-mixed pipe reactors (CMPRs) as a simple and easy-to-use test system that recreates combinations of features of interest encountered in in-building plumbing. We applied the CMPRs to study two common residual disinfectants (chlorine and monochloramine) supplied by water utilities, and two other disinfectants (chlorine dioxide and copper-silver ionization) which are commonly dosed by building operators, especially in hospitals and other buildings housing individuals susceptible to infection. These four disinfectants were applied to CMPRs consisting of PVC, copper, and iron pipe. Chemical, culture, and DNA methods were used to understand how these disinfectants affected the microbes and their ecology. We then took the opportunity to set up CMPRs in the Netherlands, where there has been no historical exposure to chlorine because their water quality regulations emphasize limiting nutrients in the water and elevating the hot water line temperatures as means to control microbial growth. The CMPRs effectively produced worst-case plumbing scenarios, where opportunistic pathogens were especially difficult to control through residual disinfection. Dosed disinfectants tended to be no longer measurable in the water after five hours. The CMPRs also showed that the disinfectant most effective for one pathogen could be the least effective for another. If doses were applied near regulatory limits, the concentrations of pathogens and antibiotic resistance genes decreased. However, opportunistic pathogens tended to survive better than background populations of bacteria. Bacteria carrying ARGs also survived some disinfectant conditions better as well. Thus, if doses were applied at levels that could inactivate some microbes, but not the opportunistic pathogens, pathogen abundances sometimes increased. These results were largely confirmed in the experiment with Dutch drinking water. Here, chlorine appeared to be more problematic than monochloramine in terms of enriching pathogens and antibiotic resistance. We also noted that Dutch waters garnered more diverse microbial communities, with fewer DNA markers for pathogens and antibiotic resistance. In general, this research takes a key step towards optimizing application of residual disinfectants for control of both opportunistic pathogens and antibiotic resistance. Because disinfectants can have negative impacts on drinking water microbial communities when supplied insufficiently, it is important that the other features of in-building plumbing, such as the selection of pipe material or the hydraulics, facilitate disinfectants reaching all portions of plumbing and at the necessary concentrations. It is recommended that the selection process for disinfectant type and dose considers the plumbing materials and other conditions such that disinfection can be aimed towards controlling multiple opportunistic pathogens, which can vary in their susceptibility, and antibiotic resistance.

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