Fluorescence-based methods of virus estimation in wastewater: Current challenges and future perspectives

• Author / Creator

  Dlusskaya, Elena

• Increasing freshwater scarcity drives humankind to look for alternative sources of potable water as well as water for agricultural and industrial applications. It is estimated that by 2025 half of the world population will be living in water-scarce regions. This pressure has forced governments to explore alternative water sources and more sustainable approaches to manage water services. Municipal wastewater reuse is part of the solution to address these population, industrial, and environmental challenges. However, municipal wastewater with human excreta as its’ major constituent is the source of human pathogens by the mere nature of it. Of all microbial hazards present in municipal wastewater, human enteric viruses cause infection at the lowest doses while their concentration in untreated sewage can exceed 106 viruses per litre. Enteric viruses are also highly resistant to water treatment processes and persist in the environment. To ensure public health and safety, the wastewater industry regulations are moving toward virus testing in sewage-impacted waters. This shift requires a set of robust and well-validated methods. Despite the popularity of flow cytometric virus enumeration in marine research and some attempts to use it for engineered waters, there has been no thorough understanding of the basic mechanisms behind this method. This research focused on developing and validating a sensitive and accurate real-time method to monitor the total indigenous virus population during the treatment of municipal wastewater with intended reuse. Experiments using a set of pure-culture bacteriophages with various genome sizes demonstrated that human enteric viruses were generally below the limit of detection by current flow cytometry (flow virometry). SYBR® Green and other fluorescent nucleic acid binding dyes were shown to form auto-fluorescent particles that interfere with the targeted virus flow cytometric signal. In addition, the presence of surfactants in the sample enhanced this non-specific signal of fluorescent dye particles that obscure the true virus signal. Hence, replacing the organic wastewater background of wastewater samples with tris-EDTA buffer reduced the non-specific flow cytometric signal and improved virus resolution. Flow virometry, however, was demonstrated to be quantitative when suitable controls were used, including a set of serial dilutions of purified bacteriophage cultures and stained viral free diluent that allowed unambiguous proof of virus identity of the flow cytometric signals of interest. Alternatively, the excitation/emission fluorescence scanning-based assay for virus quantification was shown to be a more sensitive alternative to flow virometry for real-time virus monitoring scenarios, as it measures total fluorescence that is emitted by stained viral nucleic acids. Heating the virus sample to expose viral nucleic acid to a fluorescent dye and the addition of low concentrations of humic acid sodium salt into samples improved this fluorescence scanning assay sensitivity even further. Overall, this study describes and explains the fundamental colloid and fluorescence mechanisms in sample matrices impacting virus enumeration. These along with the development of more sensitive instruments for analysis could turn flow virometry into a useful tool for water quality monitoring.

• Subjects / Keywords

  • Wastewater
  • Virometry
  • Fluorescence-based methods

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-n4qx-jx22

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.