Grantee Research Project Results
2023 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, 2022 through July 31,2023
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 characterized particle size distribution of wastewater using the dynamic light scattering method. The majority (> 90%) of particles in the secondary effluent were smaller than 1 µm which suggest effective particle removal during secondary treatment processes.
We conducted secondary effluent sampling from August 2022 to February 2023 (n=30) from five full-scale wastewater reclamation facilities (WRFs) in California, Florida, and Ohio. Particle-associated viruses in wastewater were isolated using a sequential filtration and quantified by digital polymerase chain reaction (dPCR). Significant differences were observed between free viruses (detected in the 0.45 µm filtrate) and particle-associated viruses (retained on the filters) for pepper mild mottle virus (PMMoV), noroviruses and enteroviruses. Adenoviruses were found to be significantly less abundant in colloid particles (on the 0.45 µm filters) than in other particle sizes and crAssphage was found to be highly associated with small suspensible particle (on the 3 µm filters). By analyzing the isolated viral vesicles, we demonstrated the broad presence of both human norovirus genotypes GI/GII vesicles in wastewater and the vesicles contributed to 17-45% of total human noroviruses. We also performed the metagenomic analysis for both total and vesicle viral DNA/RNA, and the total and vesicle viruses showed distinct compositions. Notably, an array of human viral pathogens was found to be associated with vesicles, such as norovirus, rotavirus, and astrovirus, whereas vesicle-associated bacteriophages and algal, plant, and animal viruses were also extensively identified. Our results regarding particle-associated viruses fill the knowledge gap of the prevalence of viruses in wastewater for water reuse.
We developed an optimized protocol for viral metagenomics analysis including sequencing library preparation procedures and bioinformatics.
We evaluated the efficiency of intercalating azo dyes propidium monoazide (PMA) for the quantification of viable viruses in wastewater and water samples using digital PCR. Our preliminary results showed promising applications for quantifying viable viruses in wastewater with digital PCR.
A bench scale activated sludge reactor has been established to evaluate the hydraulic retention time (HRT) on removal of particle-associated viruses in wastewater.
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 to determine changes in virus diversity after each treatment step. We will develop a systematic bioinformatic approaches for viral metagenomics. We will apply a capsid integrity PCR with the intercalating azo dyes for quantifying virus removal during full-scale advanced water treatment processes. We will conduct bench-top experiments to study the mechanisms and removal efficiencies of particle-associated viruses and viral vesicles through disinfection.
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.