Grantee Research Project Results
2022 Progress Report: Novel Quantitative Methods for Indigenous Viruses in Wastewater, Improving the Assessment of Water Reuse Treatment Performance
EPA Grant Number: R840258Title: Novel Quantitative Methods for Indigenous Viruses in Wastewater, Improving the Assessment of Water Reuse Treatment Performance
Investigators: Gim Aw, Tiong , Rose, Joan B. , Shen, Yun
Institution: Tulane University , University of California Riverside , Michigan State University
EPA Project Officer: Ludwig-Monty, Sarah
Project Period: August 1, 2021 through July 31, 2024 (Extended to July 31, 2026)
Project Period Covered by this Report: August 1, 2021 through July 31,2022
Project Amount: $1,239,241
RFA: Viral Pathogen and Surrogate Approaches for Assessing Treatment Performance in Water Reuse (2021) RFA Text | Recipients Lists
Research Category: Water Treatment
Objective:
Project objectives are to: (1) characterize the composition of indigenous viruses in wastewater (including viruses with three different forms: free, particle-associated, and vesicle-cloaked viruses) using a metagenomic approach, with the purpose of identification of viral surrogates for assessing treatment performance; (2) design and develop individual based quantitative assays for new viral surrogates using new instrumentation and molecular viability methods; and (3) validate new surrogates and new quantitative detection assays (from Objective 1 & 2) for estimating reduction of viruses through full-scale advanced water treatment processes (with some bench-scale work).
Progress Summary:
We have sampled wastewater from five water reclamation facilities in California, Florida, and Ohio. We have developed a sequential membrane filtration to isolate particle-associated viruses in wastewater. A series of filters was used to capture viruses associated with particles of different size ranges. Preliminary results indicate that adenoviruses and crAssphage tend to be associated with different sizes of particles in secondary effluent. These samples will be used for virus metagenomic analysis.
We conducted a particle size distribution analysis of wastewater samples collected from a full treatment train of one of the participated utilities in order to understand a range of particle sizes in wastewater. For influent, most of the particles were in the range of 100 to 300 µm. For the microfiltration effluent, most of the particles larger than 200 µm were removed. The dominant particle size was from 50 to 200 µm. No particles were found after reverse osmosis (RO) and ultraviolet light (UV) irradiation.
We have optimized the method of isolating viral vesicles from wastewater. We compared six different vesicle isolation methods by testing the yield and purity of vesicles isolated from a murine norovirus culture through qPCR and tandem mass spectrometry (MS/MS).
We have developed and validated digital PCR assays for viral fecal indicator crAsspahge as well as enteric viruses such as enterovirus, norovirus, hepatitis A, and adenovirus. Using new digital PCR method, the limit of detection for viruses was as low as 0.34 gene copies per reaction and 1554 gene copies per L in environmental water.
Future Activities:
We will continue wastewater sampling from full-scale water reuse systems to isolate free, particle-associated, and vesicle-cloaked viruses for metagenomic sequencing. We will optimize metagenomic sequencing workflow for viruses including bioinformatic approaches.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this projectSupplemental Keywords:
Pathogens, effluent, discharge, innovative technology, analytical, measurement methods, monitoring, geneticsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.