The chemical fate of biological pollutants in treatment processes

Transcription

1 The chemical fate of biological pollutants in treatment processes 92a Source: Centers for Disease Control and Prevention's Public Health Image Library Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #4814 Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #4814 Krista Rule Wigginton Assistant Professor of Environmental Engineering University of Michigan

2 The Wigginton team aims to provide mechanistic descriptions of biomolecule pollutant fate in engineered and natural environments. To wastewater treatment plant Wastewater treatment plant Drinking water treatment plant

3 Digester Lagoon Nonthermal Plasma

4 What is a virus? Genome RNA or DNA Capsid Proteins Lipid Envelope

5 Infective viruses are detected by adding sample to complimentary cells. When complimentary cells aren t available, virus genes detected by PCR.

6 The 2014/2015 Ebola outbreak highlights the potential value of a mechanistic understanding of virus fate in the environment. Source: Centers for Disease Control and Prevention's Public Health Image Library At that time, there had been very few published studies on the environmental fate of ebolavirus.

7 We often look to the behavior of surrogate viruses in the environment and in treatment processes to guess how new viruses might behave. (+)ssrna ~19kb genome Capsid ~800 nm long, 80 nm in diameter Lipid envelope Source: Centers for Disease Control and Prevention's Public Health Image Library 92a MS2 Poliovirus Hepatitis A Virus Mouse hepatitis virus Transmissible gastroenteritis coronavirus Source: Centers for Disease Control and Prevention's Public Health Image Library Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #4814 Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #4814 (-)ssrna ~3.5kb genome 25 nm diameter No lipid envelope (+)ssrna ~7.5kb genome 30 nm diameter No lipid envelope (+)ssrna ~7.5kb genome 27 nm diameter No lipid envelope (+)ssrna ~32 kb genome 100 nm diameter Enveloped (+)ssrna ~29kb genome 90 nm diameter Enveloped

8 Log Inactivation We have been studying the impact of the lipid bilayer in untreated wastewater. Survival Partitioning MHV Phi 6 MS2 T3 Detection Enveloped viruses lose infectivity faster than nonenveloped viruses in raw wastewater. Inactivation of enveloped viruses in wastewater is highly dependent on temperature. Enveloped viruses sorb to a greater extent to wastewater solids (~25%) than nonenveloped viruses (<5%) Time (h) Ye, Yinyin, et al. "Survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater." Environmental Science & Technology (2016).

9 But surrogates with similar genomes and structures aren t always appropriate Fresh Urine Human Polyomavirus Excreted in urine (up to gc/l) Nonenveloped dsdna Storage (>3 months) Fertilizer Is urine storage effective at removing infective human polyomaviruses from the urine? PCR suggested the polyomaviruses were present after long storage times

10 Double strand DNA surrogates for Polyomavirus survived for long periods of time in stored urine samples. Surrogate dsdna virus T3 virus lasted months in stored urine Surrogate dsdna virus T4 virus persisted in stored urine Found a lab with special strain of polyomavirus that can be cultured in vitro. Michael Imperiale, Ph.D. Additional experiments lead us to believe disulfide bonds in virus capsids make viruses susceptible to the chemical conditions in stored urine.

11 We re particularly interested in why certain enveloped viruses are so much more stable in aqueous environments. T 90 (days) Human Coronavirus SARS 9 Human Coronavirus 229E <1 Swine Coronavirus TGEV 11 Murine Coronavirus MHV 9 Avian influenza virus H5N Hantavirus 3 HIV <1 Poliovirus (non-enveloped) 56 **Distilled water or culture media matrix and temperatures ranging from C. Wigginton, Ye, Ellenberg, 2015, Emerging investigators series: the source and fate of pandemic viruses in the urban water cycle, Environmental Science: Water Research & Technology,.

12 We previously developed a mechanistic description of bacteriophage MS2 inactivation during disinfecting treatments. Heat Free Chlorine / UV 254 ClO 2 1 O2 / Genome A protein Capsid proteins Wigginton, K.R. et al. (2012) ES&T, Vol. 46, pp

13 We are currently developing a mechanistic description of how enveloped viruses lose infectivity in treatment processes. Infectivity assays Binding assays Genome entry assays Virus particle MALDI MS Orbitrap-MS Virus lipids genome qpcr MALDI MS Orbitrap-MS MALDI MS Orbitrap-MS Virus proteins oligomers MALDI MS Orbitrap-MS v v v v v Peptides v v

14 We often use PCR and qpcr to detect viruses and ARGs in water and track their decay through various water treatment processes. Wastewater treatment plant What are we detecting with qpcr? What are we trying to remove in our treatment processes?

15 Antibiotic resistant genes (ARG) are very diverse in structure, mechanisms, and potential impact on human health. Sequence of DNA Within bacterial cell Within bacteriophage (virus that infects bacteria) In extracellular DNA Conjugation Transduction Transformation

16 Our project seeks to identify the damage required to inactivate ARGs in an extracellular plasmid. pwh1266 plasmid (8900 bp) Acinetobacter baylyi Tet A (1200 bp) amp R (860 bp) Transformation efficiencies ~10-4

17 Plasmid pwh1266 treated with various doses of UV 254 and DNA reaction kinetics measured with transformation assays, qpcr, and (soon) mass spectrometry. UV 254 Tet gene and amp gene quantified by transformation assay Tet and amp genes quantified by short amplicon and long amplicon RT-qPCR

18 The rates of decay measured with transformation assays and qpcr assays were compared %!0.500% 8900 bp 860 bp Log C/Co!1.000%!1.500% Damage outside of gene can inactivate the gene!2.000%!2.500% amp%qpcr%(209%bp)% amp%qpcr%(861%bp)% amp%transforma<on% DNA cleavage is not responsible for inactivation by UV. Plasmid damage may be repaired in the host cells.!3.000% 0.000%!3.000% 0.050% 0.100% 0.150% 0.200% 0.250% UV Dose (J/cm2) UV Dose (J/cm2)

19 This is the same approach taken with synthetic other organic contaminants. How do these molecules react? How do these molecules partition? At what point have they reacted enough to not be a threat? Can we predict how new molecules will behave based on their chemistry?

20 Acknowledgements to my research team: Acknowledgements to my funding sources: Project #s and Project RD And the Environmental Biotech research teams: Project R996610