Novel Approaches to Monitor Virus Fate Through Water Treatment Processes

As population growth, climate change, and urbanization strain drinking water sources, the increasingly common use of diverse and impacted water supplies necessitates a better understanding of contaminant fate in this setting. Among the human health hazards found in water supplies, viral pathogens are of principal concern, because they can be present in elevated concentrations, are highly infectious, and are difficult to remove due to their small size. Effective viral pathogen removal is of particular importance in direct potable water reuse, in which wastewater is transformed into drinking water. A multibarrier approach to treatment is traditionally used for contaminant removal, where different treatment processes are placed in series and cumulatively reduce virus concentrations to levels that pose no significant public health risk. However, the persistence of several important waterborne viruses (e.g., human norovirus) through treatment processes is not well characterized due to difficulties in virus culturability. This raises questions about whether proposed reuse treatment schemes are sufficient to protect human health. In addition, monitoring strategies used to ensure treatment performance in real-time are not sufficiently sensitive to validate virus reductions, likely resulting in the design of overengineered treatment schemes for virus removal. This dissertation sheds light on alternative molecular and predictive modeling approaches for estimating virus fate through disinfection when traditional methods are not feasible and evaluates flow virometry as a novel approach to accurately validate virus reductions through treatment in real-time. Results demonstrate that alternative methods to accurately determine virus susceptibility to UV254 disinfection treatments can be applied effectively when culture-based approaches are not possible. Specifically, the UV254 sensitivity of human norovirus was established with these alternative approaches and confirmed through use of a novel culture system. The findings show that commonly used approaches to estimate infectious human norovirus levels overestimate norovirus survival through UV254 disinfection. Further, flow virometry, a high-throughput method for detecting and enumerating virus particles, was explored as a sensitive method to ensure virus reductions through treatment in real-time. Work revealed that flow virometry could effectively detect large dsDNA virus populations, while smaller RNA and DNA viruses were not reliably measured. Proof-of-concept experiments evaluating virus removal through ultrafiltration indicated that while flow virometry could detect particles in the same size range as viruses, little improvement over currently used monitoring approaches was observed due to limitations in the detection capabilities of current flow cytometers. Taken together, this dissertation research improves our understanding of human norovirus fate through treatment and provides novel methods that can be applied to monitor virus behavior through treatment. Ultimately, this research aids in the development of a regulatory framework that will make direct potable reuse more feasible, economical, and environmentally sustainable while still guaranteeing public health protection.PHDEnvironmental EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/168036/1/nrockey_1.pd

The utility of flow cytometry for potable reuse

Protecting public health from pathogens is critical when treating wastewater to drinking water standards (i.e., planned water reuse). Viruses are a principal concern, yet real-time monitoring strategies do not currently measure virus removal through reuse processes. Flow cytometry (FCM) has enabled rapid and sensitive bacteria monitoring in water treatment applications, but methods for virus and protozoa monitoring remain immature. We discuss recent advances in the FCM field and FCM applications for quantifying microorganisms in water. We focus on flow virometry (FVM) developments, as virus enumeration methods show promise for water reuse applications. Ultimately, we propose FVM for near real-time monitoring across treatment to more accurately validate virus particle removal and for pilot studies to characterize removal through understudied unit processes

White Paper on the Application of Molecular, Spectroscopic, and Other Novel Methods to Monitor Pathogens for Potable Reuse

Direct potable reuse (DPR) is gaining interest in water-scarce and water-conscious regions of the world as a technology capable of treating domestic wastewater to potable water standards. Because wastewater is the source water of such treatment schemes, the removal of harmful biological and chemical contaminants present in wastewater is necessary. One of the primary challenges for water reuse applications is the effective removal of pathogens, including viruses and protozoa. A major limitation for the acceptance of DPR is the inability to actively monitor pathogen levels through potable reuse treatment trains. Although various methods to measure pathogen concentrations exist, the required reduction of these levels through treatment processes means pathogen quantification in finished water is not currently feasible in real-time. The lack of rapid, reliable, and inexpensive methods for monitoring through DPR has also hindered the ability of utilities to confirm sufficient pathogen removal through reuse. In this poster presentation, we will present findings from our collaborative project funded by the Water Environment and Reuse Foundation. WE&RF project 14-17 involves an extensive literature review on the current state of microbiological monitoring methods that have potential relevance in water reuse or application in the future. The white paper is intended to inform the DPR field on how to best monitor for pathogens through water reuse treatment trains. The presentation will present the major topics and conclusions from our collected research. In particular, we will discuss the challenges posed by extremely low pathogen concentrations in most of the DPR treatment train and the potential of concentration techniques coupled with detection methods to detect specific pathogens at various stages of DPR treatment trains. In addition, surrogate monitoring capabilities will be evaluated to determine methods for confirming adequate pathogen removal through treatment. Much of our white paper focuses on novel quantification, concentration, and surrogate monitoring methods that we find most promising for future water reuse applications. Specifically, recent developments in human norovirus (HuNoV) culture methods will surely aid in informing the ability of treatment trains to achieve adequate virus removal for the mitigation of public health risk. Of the many viral pathogens of interest in DPR, HuNoV is of principal concern because of its high concentration in raw wastewater and its large burden of disease. Flow cytometry (FCM) is another technique that will be highlighted in the poster presentation because of its real-time monitoring capabilities. FCM is a high-throughput technology that has been used effectively in real-time bacterial monitoring of environmental matrices. We believe FCM will become an important approach to near real-time virus monitoring in the future. New advances in quantitative PCR techniques also have promise in reuse applications to indicate pathogen contamination. Although not real-time, PCR-based methods are more rapid than culture-based methods. The potential ability of PCR-based methods to differentiate infective and non-infective microorganisms will also be reviewed. The poster presentation will conclude with a review of the most important research topics that are necessary to improve microbiological monitoring and pathogen credit maintenance in DPR applications

UV disinfection of human norovirus: evaluating infectivity using a genome-wide PCR-based approach

The removal and inactivation of infectious human norovirus is a major focus in water purification, but its fate through disinfection treatment processes is largely unknown owing to the lack of a readily available infectivity assay. In particular, norovirus behavior through unit processes may be over- or underestimated using current approaches for assessing human norovirus infectivity (e.g., surrogates, molecular methods). Here we fill a critical knowledge gap by estimating inactivation data for human norovirus after exposure to UV254, a commonly used disinfection process in the water industry. Specifically, we used a PCR-based approach that accurately tracks positive-sense single-stranded RNA virus inactivation without relying on culturing methods. We first confirmed that the approach is valid with a culturable positive-sense single-stranded RNA human virus, coxsackievirus B5, by applying both qPCR- and culture-based methods to measure inactivation kinetics with UV254 treatment. We then applied the qPCR-based method to establish a UV254 inactivation curve for human norovirus (inactivation rate constant = 0.27 cm2 mJ -1). Based on a comparison with previously published data, human norovirus exhibited similar UV254 susceptibility compared with other enteric single-stranded RNA viruses (e.g., Echovirus 12, feline calicivirus), but degraded much faster than MS2 (inactivation rate constant = 0.14 cm2 mJ-1). In addition to establishing a human norovirus inactivation rate constant, we developed an approach using a single qPCR assay that can be applied to estimate human norovirus inactivation in UV254 disinfection systems

Biofilms in Full-Scale Drinking Water Ozone Contactors Contribute Viable Bacteria to Ozonated Water

Concentrations of viable microbial cells were monitored using culture-based and culture-independent methods across multichamber ozone contactors in a full-scale drinking water treatment plant. Membrane-intact and culturable cell concentrations in ozone contactor effluents ranged from 1200 to 3750 cells/mL and from 200 to 3850 colony forming units/mL, respectively. Viable cell concentrations decreased significantly in the first ozone contact chamber, but rose, even as ozone exposure increased, in subsequent chambers. Our results implicate microbial detachment from biofilms on contactor surfaces, and from biomass present within lime softening sediments in a hydraulic dead zone, as a possible reason for increasing cell concentrations in water samples from sequential ozone chambers. Biofilm community structures on baffle walls upstream and downstream from the dead zone were significantly different from each other (<i>p</i> = 0.017). The biofilms downstream of the dead zone contained a significantly (<i>p</i> = 0.036) higher relative abundance of bacteria of the genera <i>Mycobacterium</i> and <i>Legionella</i> than the upstream biofilms. These results have important implications as the effluent from ozone contactors is often treated further in biologically active filters and bacteria in ozonated water continuously seed filter microbial communities