A primary goal of microbial monitoring in piped water systems is to ensure an acceptably low risk of ingesting enteric pathogens from contact with treated water. Microbial monitoring strategies in piped water systems are generally based on the type of water being treated and the intended use, but strategies that are developed with a narrow focus on each type of water system could disincentivize some beneficial monitoring targets. For example, raw wastewater contains genetic material from pathogens that does not pose a risk public health from the perspective of water treatment but could be monitored to assess the disease burden in the contributing population (e.g., severe acute respiratory syndrome coronavirus 2; SARS-CoV-2). Developing integrated microbial monitoring strategies in water systems could produce data that are beneficial for drinking water providers, wastewater treatment service providers, and public health departments.

The overall goal of this work was to apply enhanced methods of microbial assessment in piped water systems and to identify integrated microbial monitoring strategies with the potential to benefit public health. Enhanced methods of microbial assessment include methods to assess microbial abundance (e.g., intact cell counts, total cell counts, intracellular ATP, and total ATP), microbial community composition (e.g., 16S rRNA gene amplicon sequencing), and specific microbial targets (e.g., quantitative polymerase chain reaction; qPCR). Applications for enhanced microbial assessment include (i) routine assessment such as monitoring impacts of local-scale water quality conditions on microbial abundance in drinking water distribution systems (e.g., disinfectant concentration); (ii.) diagnostic or preventative assessment such as monitoring impacts of system-scale changes on microbial communities in drinking water distribution systems (e.g., transition to direct potable reuse); and (iii) public health surveillance such as monitoring pathogens in community sewersheds during disease outbreaks.

First, five measures of microbial abundance were applied in six chlorinated and chloraminated drinking water distribution systems and enhanced methods of microbial assessment (i.e., total and intact cell counts and total and intracellular ATP concentrations), were compared with heterotrophic plate counts. Flow cytometry-based intact cell counts were the least variable (intraassay coefficient of variation = 16.9%) and most quantifiable (97.6%) of the viability assays tested. Therefore, intact cell counts may be promising monitoring targets for diagnostic or preventative monitoring in drinking water distribution systems and in low microbial abundance conditions (i.e., monitoring advanced-treated wastewater). In one chloraminated distribution system, a generalized linear mixed model was used to asssess the effect of physicochemical water quality conditions on intact cell counts, and total chlorine had the greatest inverse effect on intact cells with a greater positive effect of temperature at lower levels of total chlorine.

Next, preventative monitoring of simulated distribution systems was completed during augmentation of conventional drinking water with advanced-treated wastewater. The five pipe loop rigs used to simulate this event were sampled over 21 weeks using 16S rRNA gene amplicon sequencing and total cell counts. While this simulation study may not accurately represent a full-scale direct potable reuse system, the experiments demonstrate the enhanced microbial monitoring that could be completed during full-scale implementation. It was observed that despite advanced-treated wastewater having high water quality (e.g., low concentrations of organic carbon and nutrients) and low cell counts, microorganisms were seeded and grew in reverse osmosis permeate. Furthermore, the pipe loop bulk water and biofilm bacterial community profiles shifted with introduction of the advanced-feedwater that had been seeded with microorganisms and nutrients.

Finally, the value of wastewater as a source of information to assess the health of the contributing population was investigated. Raw wastewater was collected from five locations in the San Francisco Bay Area during the coronavirus infectious disease 2019 (COVID-19) pandemic. For these samples, SARS-CoV-2 wastewater testing results were compared to geocoded COVID-19 clinical testing results. Findings include that SARS-CoV-2 was reliably detected (95% positivity) in frozen wastewater samples when reported daily new COVID-19 cases were 2.4 or more per 100,000 people. Additionally, spatio-temporal trends in wastewater SARS-CoV-2 signal and daily per capita COVID-19 cases were generally consistent with each other, with a few exceptions that could indicate clinical undertesting at some locations.

This research is useful to academics as well as practitioners considering or implementing management practices for integrated water systems. This dissertation includes one of very few studies on drinking water distribution system microbial impacts from direct potable reuse, which also provides recommendations for future microbial assessments in simulated direct potable reuse distribution systems. More work is needed applying DNA sequencing methods in full-scale systems as part of diagnostic or preventative monitoring and applying integrated monitoring methods in full-scale water systems. Additionally, while SARS-CoV-2 wastewater testing is a promising public health surveillance strategy, it is a relatively new application that requires more research and development. With these advancements to the field of wastewater-based epidemiology, pathogen targets in raw wastewater could be expanded to include enteric pathogens. Particularly as more full-scale direct potable reuse systems come online, these methods with other public health surveillance campaigns can be used to verify that consumption of advanced-treated wastewater does not result in transmission of pathogens back to the contributing population.