Transport of Viruses, Bacteria,
and Protozoa in Groundwater
Joe Ryan
Civil, Environmental, and Architectural Engineering Department
University of Colorado, Boulder
Environmental Engineering Seminar
October 11, 2000
Acknowledgments
Students
University of Colorado: Jon Loveland, Jeff Aronheim, Annie Pieper, Becky
Ard, Robin Magelky, Jon Larson, Theresa Navigato, Yvonne Bogatsu
UCLA/Yale University: Jun Long, Ning Sun, Chun-han Ko
Collaborators
Ron Harvey, U.S. Geological Survey
Menachem Elimelech, Yale University
Funding
National Water Research Institute
U.S. Environmental Protection Agency
Laboratory Assistance
Chuck Gerba, University of Arizona
Joan Rose, University of South Florida
Field Assistance
Denis LeBlanc & Kathy Hess, U.S. Geological Survey
Public Health Problem
Waterborne Disease Outbreaks
estimates for the United States
1 to 6 million illnesses per year
1000 to 10,000 deaths per year
only 630 documented outbreaks 1971-1994
Milwaukee, Wisconsin, 1993
Cryptosporidium, the “hidden germ”
about 400,000 illnesses, greater than 100 deaths
DNA evidence: human, not bovine, origin
Public Health Problem
Waterborne Disease Outbreaks
acute gastrointestinal illness
short duration, “self-resolving” for most people
chronic, severe, fatal for some
infants and elderly
pregnant women
immuno-compromised
more serious illnesses
heart disease, meningitis, diabetes (coxsackie
virus)
liver damage, death (hepatitus virus)
Public Health Problem
Microbial Perpetrators
viruses
bacteria
protozoa
Where are they coming from?
groundwater (58%), surface water
point source, non-point source
Viruses
Enteric
replicate only in
gut
Size
20 – 200 nm
Structure
protein capsid
RNA or DNA
virus
health effect
coxsackie
“hoof and
mouth”
echo,
adeno
respiratory
disease
Norwalk,
rota, calici,
astro
gastroenteritis
hepatitis A
hepatitis E
jaundice, liver
damage,
death
Viruses
Life Cycle
ingestion
drinking water
within the gut
adsorption
penetration
transcription
replication
assembly
host cell lysis
excretion from
gut
Bacteria
Enteric
grow in gut (only?)
Size
0.5 to 2 m
Structure
cell walls
proteins
phospholipids, fatty
acids
bacterium
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
motililty
flagellae
cilia
health effect
Bacteria
Life Cycle
ingestion
meat, vegetables,
drinking water
within the gut
adsorption
penetration
growth
release of toxins
excretion from gut
Vibrio Cholera adhering to rabbit villus
E. coli adhering to calf villus
Protozoa
Enteric
grow in gut only
Size
3 to 12 m
Cyst Structure
rugged protective
membrane
carries
trophozoites
protozoan
health effect
Cryptosporidium
parvum
diarrhea
Giardia lamblia
chronic
diarrhea
Protozoa
Life Cycle
ingestion
drinking water
within the gut
excystation
parasitic growth
cyst formation
excretion from
gut
Occurrence in Groundwater
Viruses
38% positive by PCR
7% positive by cell culture
Bacteria
40% positive for coliform bacteria
50-70% positive for enterococci
Protozoa
12% Giardia and/or Cryptosporidium
(5% in vertical wells)
Monitoring in Groundwater
Maximum Contaminant Level
coliform bacteria – 40 per liter
viruses – 2 per 107 L (proposed, GWDR)
Ground Water Disinfection Rule
will require disinfection unless “proof” of adequate
“natural disinfection”
viruses nominated as target microbe
Virus Transport Models
predictions of travel time
attachment and inactivation
Microbe Transport
Microbe Transport
Transport equation
c
c
 dc
 b s
 D 2  v   katt c  kdet s  c  
t
x
 dx
  t
2
dispersion
advection
kinetic
attachment/
release
growth or
inactivation/
“die-off”
equilibrium
attachment/
release
Attachment
kinetic
colloid filtration
collision frequency 
collision efficiency 
concentration
Microbe Attachment
tracer
microbe
release
equilibrium
distribution coefficient
linear, reversible
concentration
first-order (kdet)
much slower than
attachment
time
tracer
microbe
time
Microbe Attachment
Surface Chemistry
capsids, cell walls
carboxyl – RCOOamine – RNH3+
net surface charge
usually negative
pHpzc ~3-4
for viruses, pHpzc can be estimated from protein
content of capsid
Microbe Attachment
Porous Media Surface Chemistry
negative
quartz, feldspars, etc.
clay faces
positive
iron, aluminum oxides
clay edges
electrostatic interactions
favorable deposition sites
unfavorable deposition sites
Microbe Attachment
Microbe Size
small
collisions caused by Brownian
motion
large
collisions caused by settling
Microbe Density
Range 1.01 to 1.05 g cm-3
collisions caused by settling
Microbe Attachment
Optimal Size for
Transport
Brow nian
Interception
Settling
Total
collision frequency
about 1-2 m
bacteria
viruses collide by
diffusion
protozoa collide by
settling
protozoa also
removed by
straining
1.00E+02
1.00E+00
1.00E-02
1.00E-04
1.00E-06
1.E-09
1.E-08
1.E-07
1.E-06
1.E-05
particle diameter (m)
1.E-04
Microbe Attachment
Target Organism
collision efficiency
about the same for
all microbes
variation in  comes
from porous media
collision frequency
favors bacteria
BACTERIA, but…
adhesion favored for
growth
biofilms
Virus Attachment
Bacteriophage
PRD1
Cape Cod field
experiments
sewagecontaminated zone
uncontaminated
zone
100 L injections
multi-level samplers
Virus Attachment
bromide
32
P - PRD1
0.8
0.15
UNCONTAMINATED
0.6
0.10
0.4
0.2
0.05
0.0
0.00
0
2
4
6
8
10
12
14
0.8
CONTAMINATED
0.6
0.4
0.2
0.0
0
2
4
6
8
time (d)
10
12
14
PRD1 C/C0
bromide C/C0
0.20
PRD1 C/C0
bromide C/C0
Transport
favored in
contaminated
zone
PRD1
attachment
sites blocked by
sewage organic
matter
collision
efficiency 
fraction of
favorable
deposition sites
Microbe Growth/Inactivation
Growth
viruses – no
replication outside
gut
bacteria – growth
possible, but
unlikely
protozoa – no
growth outside gut
Microbe Growth/Inactivation
Inactivation
viruses – mainly
temperaturedependent
bacteria – lysis?
predation?
protozoa –
generally resistant
to disinfection, so
inactivation is slow?
Virus Inactivation
Viruses
inactivation on
surfaces?
effect of strong
attachment forces
ln C/Catt
first-order decay
aluminum oxide
3
-6
H RNA
14
C protein
pfu
-8
1
2
3
-2
ln C/Catt
inactivation in
solution
poliovirus
-4
4
-4
aluminum metal
-6
3
-8
14
H RNA
C protein
-10
-12
-14
pfu
-16
-18
1
2
3
4
number of extractions (24 h)
Virus Inactivation
100
10-1
32
P PRD1
10-2
C/C0
Bacteriophage
MS2
Cape Cod
sediment
32P DNA
35S protein
capsid
rapid loss of
infectivity
release of
radiolabels
10-3
35
S PRD1
10-4
10-5
10-6
inf PRD1
10-7
10-8
0
5
10
15
20
time (days)
25
30
Summary
Predicting microbe transport
less difficult for viruses, protozoa cysts
no growth, inactivation simpler
more difficult for bacteria
motility
adhesion behavior motivated by growth, nutrients
growth, die-off more complicated
Bacteria should be target organism (?)
least frequent collisions, motility
may be complicated by longer-term adhesion
strategies