Cryptosporidium and Bacillus anthracis Removal Potentials as Determined
by Bacillus Endospore Survival for the Des Moines Water Works Fleur and
McMullen Treatment Plants Dennis R. Hill – Microbiologist – Des Moines Water Works - 2010
An assessment of the pathogen removal capacity of a drinking water utility treatment
process is difficult to determine. Some of the best approaches study similar biological
agents as surrogate organisms, thus eliminating many variables that are inherent when
using physical, chemical, or pilot plant evaluations.
From these surrogate biological agents, I chose to use Bacillus endospores to
help determine the Cryptosporidium and Bacillus anthracis removal potentials of the Des
Moines Water Works Fleur and McMullen treatment plants.
Cryptosporidium is a pathogenic microorganism that causes a severe and prolonged intestinal
disease. It is the most difficult microbe for water utilities to remove during conventional
treatment. It is small and can challenge a treatment process in high numbers without warning or
historical precedence. Furthermore, it has near total resistance to disinfection by hypochlorite,
traditionally the final step used to ensure pathogen free drinking water.
Even one small incident of drinking water borne illness threatens the health and lives of
a utility’s customers, undermining confidence and potentially damaging
a utility’s reputation for many years afterwards.
Various microbiological studies performed at Des Moines
Water Works have revealed that a few nonpathogenic microbes evade
coagulation, lime softening flocculation, and sand filtration, thereby
entering the clear well where chlorination takes place. Some of these
cells persist in the distribution system for days, though their viability
has not been confirmed. If Cryptosporidium were similarly able to
evade chemical treatment and filtration, the microbe would surely
resist hypochlorite treatment and remain a true disease risk in the
distribution system.
There is no indication that DMWW
has had an incident of Cryptosporidium
treatment breakthrough, but numerous
studies have shown the common background
level of the microorganism in the Raccoon
River and Des Moines River is small,
suggesting that the treatment plants’
Cryptosporidium removal capacity might not
have been effectively challenged.
With the advent of large hog and
cattle confinement operations, paired with
the potential of a human wastewater system
failure, the possibility of a Cryptosporidium
challenge to the utility has become a growing
concern.
Effective sedimentation, flocculation,
coagulation, and filtration are theoretically
able to accomplish a combined total of at
least a 3 Log10 (1,000 fold) reduction of the
microbe as required by EPA for utilities in
LT2 Bin 1, 4 to 4.5 Log10 (10,000+ fold) for
those in Bin 2, 5 to 5.5 (100,000+ fold) for those in Bin 3, and 5.5 to 6 (310,000+ fold) for those in
Bin 4, yet how does one know for sure? Is the process of an aging treatment utility, or the
design of a new one, adequate? Educated predictions can be made based on physical and
chemical studies, such as turbidity or chlorine demand, of a treatment plant; however, none of
these assessments is a substitute for a biological determination.
Pilot plant studies can be run, but with dubious correlation to the actual plant
performance.
Some of the best methods use various naturally occurring surrogate microbes. They
allow an on-line assessment of the actual treatment process, using a biological entity.
Nevertheless, because surrogate microbes differ physically and physiologically to the target
pathogens, there still is a possibility of a study either over or under estimating a utility’s
pathogen removal potential.
Certain algal and cyanobacterial species that are significantly larger than
Cryptosporidium oocysts manage to reach the DMWW clear well as entities of the filter effluent
water. Yet, size alone does not represent an organism’s susceptibility to treatment, so these
organisms are not suitable surrogates.
Even a few heterotrophic
bacteria, including coliform bacteria, can
occasionally be recovered from filter
effluent water, but most of them have
been shown to correlate poorly with true
Cryptosporidium removal.
The best microbial surrogates
that naturally exist in soil and river water
in large numbers are the various
species of Bacillus.
Bacillus is a
heterotrophic bacterium whose species
produce
environmentally
and chemically
resistant
endospores,
physically smaller, yet similar to Cryptosporidium oocysts in their
durability. Their high numbers in river water allow the many-fold
reduction that must be accomplished to demonstrate sufficient Log10
removal.
A count of Bacillus endospores recovered from filter effluent
water will provide an estimate of a treatment plant’s Cryptosporidium
removal capacity. This pre-chlorination step has been chosen to accurately represent
Cryptosporidium’s resistance to chlorine, considering that Bacillus endospores have sensitivity
to chlorination.
One species of Bacillus, Bacillus anthracis, is a bacterium that causes the deadly disease
anthrax. Because it makes environmentally resistant endospores, it is a disease agent that has
been selected for biological warfare, as well as for terrorist attacks. The addition of the
organism to a utility’s treatment system is not an effective or likely means of distribution of this
microbe, but the possibility still exists. Naturally, utility personnel might wonder what their
plant’s Bacillus anthracis removal capacity might be.
A count of Bacillus endospores recovered from finished water will provide an estimate of
a treatment plant’s Bacillus anthracis removal capacity.
Materials and Methods:
To determine the Cryptosporidium and Bacillus anthracis removal capacities of DMWW’s
drinking water plants, a study was performed, which employed Bacillus endospores as
surrogate entities of both organisms.
When assessing the Fleur plant endospore removal capacities, the flow ratios of the
Raccoon River, Des Moines River, and Gallery were calculated to determine the contribution of
each. Sample collection was timed so that each sample represented the same batch of water.
This usually meant there were several hours between the samples of each set, so to ensure
testing accuracy; the samples of each site were processed soon after collection.
Similarly, when assessing the McMullen plant endospore removal capacities, the flow
rates of Crystal Lake, and the Raccoon River-influenced radial collector wells were calculated.
Samples were subjected to heat treatment to kill all of the vegetative bacterial cells,
leaving only the heat-resistant Bacillus endospores, plus those of a few other endospore
forming genera of insignificant numbers. Sample aliquots of 200ml were added to capped
250ml Erlenmeyer flasks, which were placed within a
water bath. The flasks of water were rotated at 95rpm
in 75ºC water and held for 15 minutes, once reaching
temperature. Afterwards, they were rapidly cooled in
an ice bath.
A one-liter Millipore glass membrane filtration
unit, which received 85mm diameter sterile Millipore
filters, was used to filter the samples. The samples
were filtered with
dedicated
filtration
units, or they were
filtered from cleanest
to most soiled, with rinses of sterile water in between. When
a period exceeding an hour occurred between samples, the
funnel was autoclave sterilized.
The filters were laid atop tryptic soy agar containing
2% agar. They were incubated for 24 hours at 35ºC.
After incubation, all colonies of Bacillus growing on the
membrane filters were counted using magnification.
Bacillus colonies derived from filtration for endospores
had to be kept from developing confluent growth on the agarfed filters. This was accomplished by controlling the volume
of water filtered. One liter each of filter effluent and finished
water was used. Daily heterotrophic plate counts performed
on the rivers helped provide an estimate of the endospore
counts, which was roughly one-tenth that of the overall heterotrophic bacterial number.
Gram stains of all of the colonies from the initial cultures were performed to confirm that
the bacteria were endospore-forming species, ensuring that non-endospore forming bacteria
were not surviving the heat treatment. There were no exceptions. Catalase tests were also
randomly done to confirm Bacillus as the dominating or sole genera of endospore derived
colonies.
Multiple studies of each plant were performed.
They included water conditions spanning from cold clear
winter water to turbid warm
summer water.
Results and Discussion:
Sample
1
2
3
4
5
6
7
8
9
10
STD
AVE
%STD
To aid in determining the correct working volume for the river
water samples use in the study, a chart of the number endospores
recovered and the turbidity of the rivers was constructed. The
close correlation between the two parameters made the
determinations feasible.
The Raccoon River averaged 8212 endospores per NTU of
turbidity, with a Pearson’s correlation of 0.99 where 1.0 represents
complete correlation.
The Des Moines River averaged 9406 endospores per
NTU of turbidity. There were not enough samples to determine
the correlation.
Raccoon River Turbidity vs. Endospores per
Liter
Endospores per Liter
1200000
1000000
Endospores
recovered
28
27
30
29
21
29
35
30
31
25
4
29
13
180
Endospores per liter
160
Turbidity of RR
140
800000
120
100
600000
80
400000
60
40
200000
20
0
0
1
2
3
4
5
6
7
River Turbidity in NTU's
The
membrane
filtration
procedure used is theoretically
able to capture one bacterial
endospore per liter of water
filtered. An endospore is one
five-hundred-trillionth (1 x 10-15)
of a liter of water, so the
sensitivity of the test is extremely
high. To confirm the reproducibility of the test, ten one-liter
samples spiked with Raccoon River water were consecutively
filtered and the percent relative standard deviation was calculated.
The average number of colonies that arose from the endospores
recovered was twenty-nine. A standard deviation of four and a
relative standard deviation of 13% were accomplished, proving the
method has good reproducibility, even when a moderately low
number of endospores are recovered.
Individual Plant Assessments:
The following chart presents the Fleur plant data collected on different sampling days. The left
half of the chart contains the technical collection data, and the right half shows the results.
The first three sets of samples were collected in February and March, during periods of
cold water and low pumpage. The first, second, and fourth sets were performed when we were
using Raccoon River water, and the third when we were using De Moines River water.
Furthermore, the third set was performed during near flood conditions.
Infiltration gallery water is always used as part or all of DMWW source water. When this
bank filtered water mixes with the river water, it represents our first step of
endospore/Cryptosporidium oocyst reduction, as shown in the first column of the results. The
Log10 values for just bank filtration and for combined bank filtration and river water are both
shown.
Next is shown the combined endospore/Cryptosporidium oocyst reduction accomplished
by the ferric chloride sedimentation basins and the lime softening basins, followed by a column
of data showing the effectiveness of sand filtration.
At this point, the relative Cryptosporidium removal potential was calculated, which had
an overall range of 3.5 to 3.8 Log10. The combined basin and filter removal which EPA
concentrates upon ranged from 3.1 to 3.5 Log10.
The final step of chlorination to produce finished water exerts a killing effect on
endospores, so the Bacillus anthracis removal potential was calculated at this step, ranging
from 4.0 to 4.9 Log10.
The next chart contains the data from the McMullen treatment plant. The first four sets
of samples were collected in February and March, during periods of cold water and low
pumpage.
McMullen water is collected by radial collector wells underneath the Raccoon River, and
therefore is bank filtered. The only exception to this is when the well water is supplemented
with water from Crystal Lake. When the lake is used, its level is maintained with water from the
Raccoon River. Because of the rain this year, its use was not required, so its effect on
treatment was not included in this study.
Once again I studied the step by step reduction of the endospores as the water was
treated through the plant. Results are shown for bank filtration, the clarifiers, filtration, and
chlorination. The overall relative Cryptosporidium removal potential ranged from 3.8 to 5.4
Log10. The combined clarifier and filter removal that EPA concentrates upon ranged from
1.6 to 3.1 Log10. The relative Bacillus anthracis removal potential ranged from 4.8 to greater
than 6.0 Log10.
EPA Log10 Cryptosporidium Removal Credits:
The following chart presents a list of log credits that EPA grants for treatment steps specific to
DMWW operations.
Treatment Process
EPA Cryptosporidium log credit
Bank
filtration
gallery)
0.5 log credit for 7.5 m setback; 1.0 log credit for 15 m setback.
(infiltration
Conventional Treatment:
Sedimentation, flocculation
coagulation, filtration
3.0 log credits
Coagulation, direct filtration
2.5 log credits
Combined filter
performance
0.5 log credit for combined filter effluent turbidity≤0.15 NTU in 95% of samples
each month.
Membranes
Credit equivalent to removal efficiency demonstrated in challenge test for device if
supported by direct integrity testing. Potentially, 5 or more log removals.
DMWW Plant Comparisons to EPA Log10 Credits
The Fleur infiltration gallery and the McMullen radial collector wells are not set back far enough
to give DMWW any Cryptosporidium removal credits. Nevertheless, the infiltration gallery bank
filtration of the Fleur plant averaged an impressive 3.2 Log10 reduction of
endospores/Cryptosporidium oocysts when assessed without being mixed with river water. The
radial collector well alluvial bank filtration of the McMullen plant averaged a 2.2 Log10 reduction
of the organisms, when assessed without being mixed with Crystal Lake water.
DMWW qualifies for 3.0 Log10 credits by having sedimentation, flocculation coagulation,
and filtration for its complete treatment process. If it were assumed that 0.5 Log10 credits were
for rapid sand filtration, the remaining 2.5 Log10 credits would be for sedimentation, and
flocculation coagulation (the sum of all of the Fleur plant basin chemical treatment, or of the
McMullen plant ferric chloride mixing chamber and the chemical treatment in the upflow
clarifiers).
The Fleur plant sedimentation and flocculation coagulation processes averaged an
endospore/Cryptosporidium oocyst reduction of 2.4 Log10, which
would fall a little short of the EPA credit grant of 2.5 Log10 relative to
the assumption made above. The McMullen plant’s combined ferric
chloride and lime softening in the upflow clarifiers averaged an
endospore/Cryptosporidium oocyst reduction of 1.3 Log10, which
would fall significantly short EPA’s credit grant of 2.5 Log10 relative to
the assumption made above.
Neither plant’s rapid sand filters produce filter effluent
turbidities of ≤0.15 NTU’s in 95% of the samples each month, so
EPA grants DMWW no credits. Nevertheless, in this endospore
study, the filters of both plants met or exceeded the 0.5 Log10 credits that EPA would have
otherwise granted.
The Fleur plant successfully removed greater than 3.0 Log10 with its conventional
treatment each time it was tested.
The McMullen plant’s convention treatment
succeeded in removing greater than 3.0 Log10 with its convention treatment only once
out of the five times those steps were studied.
Plant Comparisons:
The following graphs show the Log10 removal for each step of the two plants.
On average, the bank filtration of the infiltration gallery at the Fleur plant was ten times
more effective in its endospore/Cryptosporidium oocyst reduction than was the alluvium of the
radial collector wells at the McMullen plant. The Fleur bank filtration was also less variable than
was the McMullen bank filtration. These differences are likely reflective of the soil and depth of
the two. On average, the large old basins at the Fleur plant were fifteen times more effective
than were the upflow clarifiers at the McMullen plant. The filters were comparable in their
endospore/Cryptosporidium removal efficiency, and for Bacillus anthracis removal, the chlorine
step was also comparable.
EPA grants its log credits on the premise that the treatment steps are working at an
optimal level. In this light, the sample test that shows the lowest performance for each step is
significant. Whereas the bank filtration and sand filtration at each site maintain a good log
removal at both plants, the Fleur basins and McMullen clarifiers do not. The lowest Fleur basin
value falls short by 0.4 Log10, and the lowest McMullen clarifier value falls short by 1.8 Log10.
The source water for both plants is the Raccoon River, nevertheless, in these 2010
studies the McMullen plant overall performance exceeded the Fleur plant by tenfold, because it
used only bank filtered water, when the Fleur plant was using unfiltered river water along with
infiltration gallery water. If the Fleur plant were to use only infiltration gallery water, and the
McMullen plant were to use from Crystal Lake, the results might reverse.
When only the basin/clarifier and filtration steps that EPA rates are assessed, the
Fleur plant’s performance exceeded the McMullen plant by tenfold.
Average Log10 Endospore/Cryptosporidium
Removal Values
6
5
Log10 Removal
4
3
2
1
0
Bank
Fleur Bank
Filtration - Filtration
alone
w/river
Fleur
3.2
McMullen
2.2
0.3
Basin/
Clarifier
Filters
Chlorine
for B.
anthracis
Total for
Crypto.
Total for B.
anthracis
2.4
1
0.9
3.6
4.6
1.3
1.1
0.9
4.9
5.3
Individual Log10 Endospore/Cryptosporidium
Removal Values
Fleur
7
Fleur
6
Fleur
Fleur
Log10 Removal
5
McMullen
4
McMullen
3
McMullen
McMullen
2
McMullen
1
McMullen
EPA Log Credit
0
Bank Fleur Bank Basin/
Filtration - Filtration - Clarifier
alone
w/river
Filters
Chlorine
for B.
anthracis
Total for Total for B.
Crypto. anthracis
EPA total credit
To determine the variability of each process step for each plant, I plotted the standard deviation
values against each other. The data sets are very small and therefore the relative performance
of each plant might change once more data is generated, nevertheless, the general variability of
each step for either plant alone is valid. For each step, there was less variability at the Fleur
plant than at the McMullen plant.
Standard Deviation to show variability of each plant's process
(The lower the value, the less variablility for the treatment step.)
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
Bank
Fleur Bank
Filtration - Filtration alone
w/river
Fleur STDV
0.29
McMullen STDV
0.58
0.10
Basin/
Clarifier
Filters
Chlorine
for B.
anthracis
Total for
Crypto.
Total for
B.
anthracis
0.10
0.21
0.36
0.38
0.38
0.38
0.33
0.53
0.39
0.50
When the basin/clarifier lime and carbon dioxide pH values were compared for each plant, the
differences were small. The Fleur plant lime values were higher than McMullen’s for the first
three tests and lower for the fourth test, but no discernable improvement or depreciation of the
Log10 removal values occurred.
Differences in the carbon dioxide values were also
inconsequential. The disparity in the two plant performances apparently is due to their many
design differences.
Lime pH Values
11.6
CO2 pH Values
9.9
11.4
9.85
11.2
9.8
11
9.75
10.8
9.7
10.6
9.65
9.6
10.4
9.55
10.2
9.5
10
9.45
9.8
1
Fleur Lime pH
2
3
4
5
9.4
6
11.19 11 11.37 10.47
McMullen Lime pH 10.87 10.80 10.88 10.74 10.37 10.35
1
Fleur CO2 pH
2
3
4
5
6
9.87 9.62 9.57 9.67
McMullen CO2 pH 9.67 9.61 9.71 9.72 9.57 9.79
Low and High Pumpage:
Doing several studies allows a comparison of plant effectiveness during low and high pumpage
episodes. However, there were numerous episodes of rain in 2010, which kept pumpage
demand low at the Fleur plant. This resulted in a lack of data for the year where the plant was
being run at its pumpage capacity limit.
The McMullen plant was run and tested during moderate to high pumpage conditions.
Its average overall treatment effectiveness was not reduced with an increase to maximum of
pumpage. However, its average clarifier and filter combination performance was reduced by 0.6
Log10, or fourfold.
Fleur Plant
w/ River
McMullen Plant
Overall performance
McMullen Plant
Clarifier & filters
Low Pumpage
3.6 Log10
4.4 Log10
2.5 Log10
High Pumpage
???
4.6 Log10
1.9 Log10
The following graph depicts the percentage of maximum pumpage at which both plants were
operated throughout the testing period. The line markers show when the endospore tests were
performed.
Waterborne Outbreak Potential:
The average overall Fleur plant reduction in endospores as determined by the 2010
assessments is 3.6 Log10, representing a 4000-fold reduction in Cryptosporidium.
The average overall McMullen plant reduction in endospores as determined by the 2010
assessments is 4.5 Log10, representing a 31,500-fold reduction in Cryptosporidium.
These are impressive endospore/Cryptosporidium oocyst removal values. However, it is
important to reiterate that the McMullen plant was not using any Crystal Lake water during the
year, meaning all of the treated water was bank-filtered water.
Nevertheless, despite the impressive endospore/Cryptosporidium removal potentials of both
plants, there still remains the possibility of disease agents surviving the treatment processes
and infecting drinking water customers. Because of the agriculture and towns along the
Raccoon and Des Moines rivers, contamination of the water with 200 Cryptosporidium oocysts
per liter (a moderate amount) is easily possible. A river contaminated with this much
Cryptosporidium would result in a potential of 22,500 customers becoming ill from Fleur plant
water, and a potential 2,900 customers becoming ill from McMullen plant water, (accepting
various mathematical assumptions, and treatment variables).
This emphasizes the value of assessing a treatment plant’s pathogen removal capacity, and
Bacillus endospores are presently the best way of determining the removal of the most
challenging of pathogens, Cryptosporidium.
Have we already experienced a pathogen breakthrough? Before a drinking waterborne
outbreak is detected:
1. A critical number of those people sickened must visit the hospital before the doctors
recognize a problem. Many people with intestinal illness do not go to the hospital.
2. Those ill often are not adequately tested for specific pathogens that might build a
suspicious scenario. Cryptosporidium is not routinely tested by clinical laboratories
unless the doctor specifically asks for it, which is not common.
3. Once the cases are reported to the state health department, the epidemiologists must
amass a convincing amount of data before officially acting.
4. When the incidents are investigated, there might not be enough data correlation to
incriminate a specific source of the disease.
The unlikelihood of the detection of small waterborne outbreaks might give utilities a
reprieve from enduring the undesirable consequences, but a false sense of security can evolve
into a disastrous circumstance if a large breakthrough eventually occurs.
Des Moines Water Works could enhance its water treatment by further optimizing its sand
filters. It could also optimize its sedimentation and coagulation flocculation processes to better
treat for microorganisms, especially at the McMullen plant. Otherwise, it could add membrane
filtration, ozonation, or ultra violet light radiation processes; or wean the plants off of river water
through various technologies such
as aquifer storage and recovery
wells.
However, how far do we
go? Is it reasonable to try to be
perfect? Do our customers want
to pay the extra cost for these
enhancements?
If a drinking water plant is
operating
beyond
EPA’s
expectations, should some or all
of the responsibility of a drinking
waterborne disease outbreak be
placed on the shoulders of the
polluters?
Unfortunately, what
might be just, does not often
succeed politically.
It is important to educate our customers as to how well our utilities are operating, and as
to how significantly some watersheds are being polluted. Having convincing data that supports
a utility’s treatment effectiveness is also important, and Bacillus endospore assessments would
represent a valuable part of that data.
Cryptosporidium outbreak information:
Comprehensive data was assembled, critiqued, and published by various researchers of:
• Gunther F. Craun & Associates,
• Centers for Disease Control and Prevention,
• Atlanta Research and Education Foundation,
• Atlanta Veterans Affairs Medical Center; and
• The U.S. Environmental Protection Agency.
The following information covers thirty-five years, and was taken from their 2010 publication:
Causes of Outbreaks Associated with Drinking Water in the United States from 1971 to 2006.
This chart shows the percentage of each
etiological agent involved in waterborne
outbreaks for the time studied.
Parasites, most of which were likely
Cryptosporidium, account for 18% of the
characterized outbreaks.
The 44% of
unknown
etiology
likely
includes
Cryptosporidium cases, also.
This chart is an assessment of the type of
utility involved in the outbreaks studied.
The surface water categories add up to
19% of the total, and the ground water
categories add up to 52% of the total.
This illustrates that, despite the relative
purity of ground water, such systems are
most easily breached because of the lack
of multiple barriers.
This chart shows how the
outbreaks
were
distributed
throughout the year for each
general type of system.
The
systems are classified as being
individual, non-community and
community.
The individual are
private wells, non-community are
small systems in parks and similar
sites, and the community systems
are public utilities.
All three categories have the most outbreaks in the summer, with the non-community systems
increasing the most, likely due to an increase in recreational park activity during that time.
This chart shows the
outbreaks as they
were distributed over
the 35-year period,
dominating during the
late 70’s and early
80’s. There is a trend
downwards
for
community and noncommunity
systems,
and a small trend
upwards for individual
systems,
as
emphasized by the
next two graphs.
77% Reduction
22% Increase
The following chart illustrates the percentage of system deficiencies charted by utility type,
over time grouped in sets of 11-years, to show changes in operation.
The red bar represents surface water system deficiencies. Notice that the deficiencies
drop from around 21% to 5%, following the 1993 Milwaukee Cryptosporidium incident.
The green bar represents ground water system deficiencies. There has been no change
in their average of 25%.
Summary of Study
Both the Fleur and the McMullen plants showed an overall reduction in the endospore counts
beyond EPA’s bin-1 requirement, each time they were tested. However, when their
sedimentation, flocculation, coagulation, and filtration steps were assessed, the Fleur plant
averaged a 3.4 Log10 removal, and the McMullen plant fell short by only averaging a 2.4 Log10
removal. Nevertheless, the overall performances provide good empirical data indicating that,
when operating optimally, the Des Moines Water Works treatment plants will be able to
effectively reduce significant numbers of Cryptosporidium and Bacillus anthracis from their
source water. The Fleur plant was not tested during its maximum pumpage capacity, and the
McMullen plant was not tested when using Raccoon River fed Crystal Lake water. In 2011,
weather and water demand might allow testing during these two conditions.
Most treatment plants can be breached by microorganisms if the source water is heavily
contaminated. This supports intense watershed education and care by IDNR, agricultural
organizations, state colleges, environmental organizations, and water utilities, especially by
minimizing cattle waste contamination, which is the most likely of animal waste to carry the
species of Cryptosporidium that infects humans. It also raises the important questions of “To
what degree should a drinking water utility invest in expensive technology to maximize its water
purification ability?’, ‘Who becomes responsible for a waterborne outbreak, when a treatment
plant is being optimally operated beyond EPA’s expectations?’ and ‘When will the watershed
polluters be held responsible for their infractions, instead of the water utility?’
Slogans:
Des Moines Water Works: “Water you can trust for life”
IDNR: “Leading Iowans in caring for our natural resources”
Iowa Farm Bureau: “People, Progress, Pride”
Iowa Soybean Association: “Expanding opportunities. Delivering results”
Iowa Environmental Council: “A safe, healthy environment and sustainable future for Iowa”
References
Gertjan Medema, Peter Teunis, Mirjam Blokker, Daniel Deere, Annette Davison, Philippe Charles, JeanFrançois Loret, Risk Assessment of Cryptosporidium in Drinking Water, 2009 World Health Organization
Rice, Eugene W., Fox, Kim R., Mlitner, Richard J., Lytle, Darren A., Johnson, Clifford H., Evaluating Plant
Performance using endospores. 1996, Journal AWWA, American Water Works Association.
Nieminski, Eva C., Bellamy, William D., Moss, Linda R., Using Surrogates to Improve Plant Performance,
March 2000, Journal AWWA, American Water Works Association
EPA The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) Implementation Guide,
August 2007
D. G. Korich, J. R. Mead, M.S. Madore, N. A. Sinclair, C. R. Sterling, Effects of ozone, chlorine dioxide,
chlorine, and monochloramine on Cryptosporidium parvum oocyst viability, May 1990 Applied
Environmental Microbiology, p.1423-1428
L. V. Venczel, M. Arrowwood, M. Hurd, M. D. Sobsey, Inactivation of Cryptosporidum parvum oocysts and
Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine, April 1997, Applied
Environmental Microbiology, p.1598-1601
CETA Application guide for the use of Surface Decontaminants in Biosafety Cabinets, CAG-004-2007,
January 30, 2007
Nicolson, Wayne L.; Munakata, Nobuo; Horneck, Gerdo; Melosh, Henry J.; Setlow, Peter. Resistance of
Bacillus Endospores to Extreme Terrestial and Extraterrestial Environments, September 2000, Vol. 64
No. 3, Microbiology and Molecular Biology Reviews, American society for Microbiology
Hongbin Liu, Nicholas H. Bergman, Brendan Thomason, Shamira Shallom, Alyson Hazen, Joseph
Crossno, David A. Rasko, Jacques Ravel, Timothy D. Read, Scott N. Peterson, John Yates III, Philip C.
Hanna. Formation and composition of the Bacillus anthracis Endospore, January 2004, p. 164-178 No. 1,
Journal of Applied Microbiology, American society for Microbiology
Jessica L. Dang, Karen Heroux, John Kearney, Ameneh Arasteh, Mark Gostomski, and Peter A.
Emanuel. Bacillus Spore Inactivation Methods Affect Detection Assays, August 2001, p. 3665-3670, Vol.
67, No. 8, Journal of Applied Microbiology, American Society for Microbiology
`
Discussion of Method Details and Adjunctal Studies:
Suitability of Bacillus endospores as Cryptosporidium surrogates:
Despite DMWW having ample plate count data of heterotrophic bacteria throughout
many
years
with
which
treatment
effectiveness could be estimated, Bacillus
endospores were selected. Endospores
have similar chemical and environmental
resistance as Cryptosporidium oocysts,
as previously mentioned, but they also
are less likely to colonize sample faucet
nozzles as do the vegetative cells of
biofilm bacteria. Furthermore, despite the
habit of some Bacillus species to form
tenacious chains of vegetative cells,
resulting in individual colonies arising
Vegetative Bacillus cells
Vegetative Bacillus cells
from clusters of bacteria instead of
forming tenacious chains
transforming into individual
individual bacteria, Bacillus endospores
endospores
normally separate into individual entities,
thus they are more likely to form single
colonies from single endospores. This greatly increases the accuracy of the plate colony
counts.
Endospore chlorine sensitivity experiment:
Research on the chlorine sensitivity of Cryptosporidium oocysts and Clostridium perfringens
endospores (site reference) confirmed Cryptosporidium’s strong resistance to chlorine and the
greater sensitivity of clostridial endospores to the chemical.
However, research performed on the relative chemical sensitivity of Cryptosporidium oocysts
and Bacillus endospores by CETA (Controlled Environment Testing Association) concluded that
Bacillus endospores can actually be the less sensitive of the two.
Prions
Chemically resistant
Bacterial endospores
Protozoan oocysts
Non-enveloped viruses
Fungal spores
Vegetative bacteria
Enveloped viruses
Chemically sensitive
Since Cryptosporidium oocysts have been shown by many research groups to have near total
resistance to chlorine, and since heterotrophic plate counts and preliminary endospore cultures
indicate that the endospores prevalent in our rivers are reduced in number as they pass through
our chlorination clear wells, I conducted a study to decipher this apparent discrepancy with the
CETA results.
Three
over a period
water.
•
•
•
one-liter beakers of heat-treated raw water were studied
of five hours, the average residence time of our clear well
Beaker one water was continually mixed.
Beaker two water was left undisturbed to determine if the
endospores might settle or float, thus giving an alternate
reason why there are low numbers of endospores in our
finished water.
Beaker three water was adjusted to have a 1.2 mg/L
chlorine concentration to simulate our clear well
disinfectant levels. It was continually mixed.
Three samples of 300ml were filtered and cultured from each
beaker. The water samples of beakers one and three were transferred
to separate flasks and thoroughly mixed prior to filtration. The water of
beaker two was drawn-off with a large syringe and added to three
separate flasks, where it was mixed and then filtered. The cultures of
beaker two were numbered in succession, making possible counts of the
upper, middle, and lower layers of water.
After incubation, the endospore-borne colony counts were as
follows:
Culture
number
1
2
3
Mixed
Water
84
89
84
Undisturbed
Water
87
70
75
Chlorinated
Water
3
3
3
The mixed water samples yielded a uniform recovery of endospores. The undisturbed
water yielded a less uniform recovery of endospores, yet there appeared to be no significant
settling or floating effect experienced by the endospores. The chlorinated water yielded a
uniform and significant decrease in the samples’ endospore numbers (1.5 Log10); roughly
equaling the Fleur and McMullen treatment plants’ clear well reductions, thus confirming the
initial observation that the Bacillus endospores of our river water are affected by chlorination.
With this information, I continued the water treatment effectiveness study with my initial
hypothesis intact.
Ruling-out other endospore forming bacteria:
Of the ten bacterial genera that produce endospores, Bacillus is by far the most common. The
endospores of all the endospore-forming bacteria are suitable entities for use as surrogates for
Cryptosporidium, yet most of the others are ruled-out as potential colony formers in the method
used, by one or more unmet growth requirement. In the end, the only possible interfering genus
is Amphibacillus, which is catalase negative versus catalase positive Bacillus. This simple test
was performed on many randomly selected colonies and showed that Bacillus was primarily if
not solely the only genus propagated in this study.
Table of Endospore-Forming Bacteria
Key characteristics
Genus
Bacillus
Gram positive rods, aerobic, catalase positive
Characteristics that rule-out the bacterium from this study
Amphibacillus
Catalase negative, short rod
Clostridium
Anaerobic
Desulfotomaculum Anaerobic, catalase negative
Oscillospira
Unculturable, large rods
Sporohalobacter
Anaerobic, gram negative
Sporolactobacillus Anaerobic, catalase negative
Sporosarcina
Coccal morphology, chemolithotrophic
Sulfobacillus
Chemolithotrophic
Syntrophospora
Anaerobic