Transport of Viruses, Transport of Viruses, Bacteria, and Protozoa in Bacteria, and Protozoa in

GroundwaterGroundwaterJoe RyanCivil, Environmental, and Architectural Engineering DepartmentUniversity of Colorado, Boulder

Environmental Engineering Seminar October 11, 2000

AcknowledgmentsStudents

University of Colorado: Jon Loveland, Jeff Aronheim, Annie Pieper, Becky Ard, Robin Magelky, Jon Larson, Theresa Navigato, Yvonne BogatsuUCLA/Yale University: Jun Long, Ning Sun, Chun-han Ko

CollaboratorsRon Harvey, U.S. Geological SurveyMenachem Elimelech, Yale University

FundingNational Water Research InstituteU.S. Environmental Protection Agency

Laboratory AssistanceChuck Gerba, University of ArizonaJoan Rose, University of South Florida

Field AssistanceDenis LeBlanc & Kathy Hess, U.S. Geological Survey

Public Health ProblemWaterborne Disease Outbreaks

estimates for the United States1 to 6 million illnesses per year1000 to 10,000 deaths per yearonly 630 documented outbreaks 1971-1994

Milwaukee, Wisconsin, 1993Cryptosporidium, the “hidden germ”about 400,000 illnesses, greater than 100 deathsDNA evidence: human, not bovine, origin

Public Health ProblemWaterborne Disease Outbreaks

acute gastrointestinal illnessshort duration, “self-resolving” for most peoplechronic, severe, fatal for some

infants and elderlypregnant womenimmuno-compromised

more serious illnessesheart disease, meningitis, diabetes (coxsackie virus)liver damage, death (hepatitus virus)

Public Health ProblemMicrobial Perpetrators

virusesbacteriaprotozoa

Where are they coming from?groundwater (58%), surface waterpoint source, non-point source

VirusesEnteric

replicate only in gut

Size20 – 200 nm

Structureprotein capsidRNA or DNA

virus health effect

coxsackie “hoof and mouth”

echo, adeno

respiratory disease

Norwalk, rota, calici, astro

gastroenteritis

hepatitis Ahepatitis E

jaundice, liver damage, death

VirusesLife Cycle

ingestiondrinking water

within the gutadsorptionpenetrationtranscriptionreplicationassemblyhost cell lysis

excretion from gut

BacteriaEnteric

grow in gut (only?)Size

0.5 to 2 mStructure

cell wallsproteinsphospholipids, fatty acids

motililtyflagellaecilia

bacterium health effect

Escherichia coli, Shigella spp., Camplylobacter jejuni, Yersinia spp.

gastroenteritis (arthritis, pneumonia, Guillain-Barre syndrome)

Salmonella spp.enterocolitis (heart disease, meningitis, arthritis, pneumonia)

Legionella spp.Legionnaire’s disease, Pontiac fever, death

Vibrio cholera diarrhea, vomiting, death

BacteriaLife Cycle

ingestionmeat, vegetables, drinking water

within the gutadsorptionpenetrationgrowthrelease of toxins

excretion from gut

Vibrio Cholera adhering to rabbit villus E. coli adhering to calf villus

ProtozoaEnteric

grow in gut onlySize

3 to 12 mCyst Structure

rugged protective membranecarries trophozoites

protozoan health effect

Cryptosporidium parvum diarrhea

Giardia lamblia chronic diarrhea

Protozoa

Life Cycleingestion

drinking waterwithin the gut

excystationparasitic growthcyst formation

excretion from gut

Occurrence in GroundwaterViruses

38% positive by PCR7% positive by cell culture

Bacteria40% positive for coliform bacteria50-70% positive for enterococci

Protozoa12% Giardia and/or Cryptosporidium(5% in vertical wells)

Monitoring in GroundwaterMaximum Contaminant Level

coliform bacteria – 40 per literviruses – 2 per 107 L (proposed, GWDR)

Ground Water Disinfection Rulewill require disinfection unless “proof” of adequate “natural disinfection”viruses nominated as target microbe

Virus Transport Modelspredictions of travel timeattachment and inactivation

Microbe Transport

Microbe TransportTransport equation

2

2b

att detc c dc sD v k c k s ct x dx t

dispersion

advection kinetic attachmen

t/release

equilibrium attachmen

t/release

growth or inactivatio

n/“die-off”

Microbe AttachmentAttachment

kineticcolloid filtration

collision frequency collision efficiency

releasefirst-order (kdet)much slower than attachment

equilibriumdistribution coefficientlinear, reversible

time

conc

entra

tion

tracer

microbe

time

conc

entra

tion

tracer

microbe

Microbe AttachmentSurface Chemistry

capsids, cell wallscarboxyl – RCOO-

amine – RNH3+

net surface chargeusually negativepHpzc ~3-4

for viruses, pHpzc can be estimated from protein content of capsid

Microbe AttachmentPorous Media Surface Chemistry

negativequartz, feldspars, etc.clay faces

positiveiron, aluminum oxidesclay edges

electrostatic interactionsfavorable deposition sitesunfavorable deposition sites

Microbe AttachmentMicrobe Size

smallcollisions caused by Brownian motion

largecollisions caused by settling

Microbe DensityRange 1.01 to 1.05 g cm-3

collisions caused by settling

Microbe AttachmentOptimal Size for Transport

about 1-2 mbacteriaviruses collide by diffusionprotozoa collide by settlingprotozoa also removed by straining

1.00E-06

1.00E-04

1.00E-02

1.00E+00

1.00E+02

1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04

particle diameter (m)

colli

sion

freq

uenc

y

Brow nian

Interception

Settling

Total

Microbe AttachmentTarget Organism

collision efficiencyabout the same for all microbesvariation in comes from porous media

collision frequencyfavors bacteria

BACTERIA, but…adhesion favored for growthbiofilms

Virus AttachmentBacteriophage PRD1Cape Cod field experimentssewage-contaminated zoneuncontaminated zone100 L injectionsmulti-level samplers

Virus AttachmentTransport favored in contaminated zonePRD1 attachment sites blocked by sewage organic mattercollision efficiency fraction of favorable deposition sites

0 2 4 6 8 10 12 14

brom

ide

C/C

00.0

0.2

0.4

0.6

0.8

PR

D1

C/C

0

0.00

0.05

0.10

0.15

0.20

Col 21 vs Col 22 Col 21 vs Col 24

time (d)

0 2 4 6 8 10 12 14

PR

D1

C/C

0

brom

ide

C/C

0

0.0

0.2

0.4

0.6

0.8

bromide32P - PRD1

CONTAMINATED

UNCONTAMINATED

Microbe Growth/InactivationGrowth

viruses – no replication outside gutbacteria – growth possible, but unlikelyprotozoa – no growth outside gut

Microbe Growth/InactivationInactivation

viruses – mainly temperature-dependent

bacteria – lysis? predation?

protozoa – generally resistant to disinfection, so inactivation is slow?

Virus InactivationViruses

inactivation in solution

first-order decayinactivation on surfaces?effect of strong attachment forces

1 2 3 4

ln C

/Cat

t

-8

-6

-4poliovirus

aluminum oxide

pfu

3H RNA14C protein

number of extractions (24 h)1 2 3 4

ln C

/Cat

t

-18-16-14-12-10

-8-6-4-2

aluminum metal

pfu

3H RNA14C protein

Virus InactivationBacteriophage MS2Cape Cod sediment32P DNA35S protein capsidrapid loss of infectivityrelease of radiolabels time (days)

0 5 10 15 20 25 30

C/C

0

10-8

10-7

10-6

10-5

10-4

10-3

10-2

10-1

100

32P PRD1

35S PRD1

inf PRD1

SummaryPredicting microbe transport

less difficult for viruses, protozoa cystsno growth, inactivation simpler

more difficult for bacteriamotilityadhesion behavior motivated by growth, nutrientsgrowth, die-off more complicated

Bacteria should be target organism (?)least frequent collisions, motilitymay be complicated by longer-term adhesion strategies