Date of Award


Publication Type

Master Thesis

Degree Name



Civil and Environmental Engineering

First Advisor

Rajesh Seth





Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.


The increasing levels of fecal pollution in recreational waters is becoming a major concern since it poses a health risk and has negative impacts on aquatic life and the economy. The major threats to water quality come from point sources such as combined sewer overflows (CSO) and sanitary sewer overflows (SSO). The most sustainable solution is to stop the problem at the source, therefore, treatment of the overflows using ozonation is the subject of study in this thesis. Culture-dependent methods (IDEXX Colilert) are currently being used by regulatory agencies but due to several disadvantages of these methods culture-independent methods are gaining more popularity. The objective of this research was to develop a culture-independent method using quantitative real time polymerase chain reaction (qRT-PCR) that is known for detecting only viable cells for RNA analysis and compare it to other culture-independent methods (DNA and PMA treated DNA (pDNA)) as well as culture-depend methods on control and ozone treated samples. The developed methods were validated with E. coli by targeting the 23S rRNA gene. Further, these methods are tested on raw and primary wastewater before and after ozone treatment with multiple indicator markers (fecal indicator bacteria, microbial source tracking markers, and pathogens) using a nanofluidic open array. Overall, pDNA and RNA methods were better correlated with culture-based methods compared to DNA when tested on ozone treated samples and therefore are better for quantifying viable cells. The nanofluidic open array proved to be a faster tool for quantifying disinfection efficiencies based on DNA, pDNA, and RNA. Overall, ozone disinfection is an effective treatment for reducing microbial contamination.

Available for download on Friday, December 18, 2020