Many cities face growing water scarcity. In response, these cities are developing innovative strategies to tap into alternative local sources of drinking water. One promising local source is treated wastewater that can be purified through advanced treatment processes. Like desalinated seawater, this purified water is also missing a natural community of bacteria that is present in all other drinking water sources. A lack of natural bacteria in purified water could provide an opportunity for unhealthy bacteria to grow. This study will compare the stability of bacteria in purified drinking water from seawater desalination to bacteria in water from traditional water sources. Additionally, the study will conduct laboratory tests to determine whether deliberately adding benign bacteria to purified wastewater can prevent instability of bacterial communities like those found in water infrastructure. The findings of this work will help engineers plan for the safe distribution of highly purified water to protect the public and help secure the Nation's water supply. The research will broaden participation in engineering by providing opportunities for research and career advancement for underrepresented students. <br/><br/>The advanced treatment of wastewater for direct potable reuse (DPR) generates extremely pure water. While the chemical conditioning of DPR water to prevent pipe corrosion is already under study, little work has investigated the potential impacts of DPR on the microbiology of drinking water distribution systems (DWDS). All DWDS harbor biofilms, and the addition of DPR water containing few cells may create biological instability and the potential for growth of opportunistic pathogens. Inoculating water with benign microorganisms via seeding may be effective for decreasing microbial risks in the DWDS. This study will examine whether deliberately seeding advanced treated water with a filter media-derived microbial community results in a more stable distribution system microbiome. The scientific objectives are to: 1) survey full-scale distribution systems in the U.S. to examine current biostability; 2) compare the microbial communities of two current systems, one conventional and one that uses RO-treated water, during distribution; 3) characterize changes to the microbial communities of mock-distribution systems during the introduction of blended conventional water and DPR water that is either microbially seeded or unseeded; and 4) examine the effect of disinfectant residual concentrations on the microbiome in seeded and unseeded mock-distribution systems. This study will use flow cytometry-based cell counting coupled to molecular methods, including amplicon and metagenomic sequencing, to observe microbiological changes in the full-scale and mock drinking water distribution systems. This study will provide insight into theoretical microbial ecology, the water microbiome, and produce valuable information to improve the design and operation of advanced treatment plants for direct potable reuse.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.