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

GroundwaterGroundwater

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

Environmental Engineering Seminar October 11, 2000

Acknowledgments

StudentsUniversity 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 Problem

Waterborne Disease Outbreaks estimates for the United States

1 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 Problem

Waterborne Disease Outbreaks acute gastrointestinal illness

short 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 Problem

Microbial Perpetratorsvirusesbacteriaprotozoa

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

Viruses

Entericreplicate only in gut

Size20 – 200 nm

Structureprotein capsidRNA or DNA

virushealth effect

coxsackie“hoof and mouth”

echo, adeno

respiratory disease

Norwalk, rota, calici, astro

gastroenteritis

hepatitis Ahepatitis E

jaundice, liver damage, death

Viruses

Life Cycleingestion

drinking water

within the gutadsorptionpenetrationtranscriptionreplicationassemblyhost cell lysis

excretion from gut

Bacteria

Entericgrow in gut (only?)

Size0.5 to 2 m

Structurecell walls

proteinsphospholipids, fatty acids

motililtyflagellaecilia

bacteriumhealth 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

Bacteria

Life Cycleingestion

meat, vegetables, drinking water

within the gutadsorptionpenetrationgrowthrelease of toxins

excretion from gut

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

Protozoa

Entericgrow in gut only

Size3 to 12 m

Cyst Structurerugged protective membranecarries trophozoites

protozoanhealth effect

Cryptosporidium parvum

diarrhea

Giardia lambliachronic diarrhea

Protozoa

Life Cycleingestion

drinking water

within the gutexcystationparasitic growthcyst formation

excretion from gut

Occurrence in Groundwater

Viruses38% 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 Groundwater

Maximum Contaminant Levelcoliform 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 Transport

Transport equation2

2b

att det

c c dc sD v k c k s c

t x dx t

dispersion

advection kinetic attachmen

t/release

equilibrium attachmen

t/release

growth or inactivatio

n/“die-off”

Microbe Attachment

Attachmentkinetic

colloid filtrationcollision frequency collision efficiency

releasefirst-order (kdet)

much slower than attachment

equilibriumdistribution coefficientlinear, reversible

time

con

cen

trati

on

tracer

microbe

time

con

cen

trati

on

tracer

microbe

Microbe Attachment

Surface Chemistrycapsids, cell walls

carboxyl – RCOO-

amine – RNH3+

net surface chargeusually negativepHpzc ~3-4

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

Microbe Attachment

Porous Media Surface Chemistrynegative

quartz, feldspars, etc.clay faces

positiveiron, aluminum oxidesclay edges

electrostatic interactionsfavorable deposition sitesunfavorable deposition sites

Microbe Attachment

Microbe Sizesmall

collisions caused by Brownian motion

largecollisions caused by settling

Microbe DensityRange 1.01 to 1.05 g cm-3

collisions caused by settling

Microbe Attachment

Optimal 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)

co

llis

ion

fre

qu

en

cy

Brow nian

Interception

Settling

Total

Microbe Attachment

Target Organismcollision efficiency

about the same for all microbesvariation in comes from porous media

collision frequencyfavors bacteria

BACTERIA, but…adhesion favored for growthbiofilms

Virus Attachment

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

Virus Attachment

Transport 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/Inactivation

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

Microbe Growth/Inactivation

Inactivationviruses – mainly temperature-dependent

bacteria – lysis? predation?

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

Virus Inactivation

Virusesinactivation in solution

first-order decay

inactivation on surfaces?effect of strong attachment forces

1 2 3 4

ln C

/Cat

t

-8

-6

-4

poliovirus

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

-2aluminum metal

pfu

3H RNA14C protein

Virus Inactivation

Bacteriophage 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

Summary

Predicting microbe transportless difficult for viruses, protozoa cysts

no 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