Ecology Research Project:
The Effect
of Untreated Sewage on
Escherichia coli Population Levels in
Leah Doughty and Brianna Clark
BI 341
Fall 2004
Abstract
In
this experiment we tested to see whether or not sewage
dumping into
Keywords: sewage dumping,
Introduction
Contamination
of aquatic ecosystems originating from domestic sewage is readily indicated by
the presence of elevated Escherichia coli (E. coli) levels
(Whitman et al. 2003). Drastically high quantities of this bacterium, which
tends to grow in a pH range of 5.5 to 7.5, have been a continual problem to the
southern portion of
In
the
Overall,
many of these beach closures have been speculated by Kinzelman et al. (2003) and
Whitman et al. (2003) to be directly associated with local untreated sewage
releases by wastewater treatment plants. In southeastern
As
an environmental agency, the Milwaukee Metropolitan Sewerage District has a
strong commitment to pollution prevention and improving the quality of local
waters. Thus, MMSD’s mission clearly states that they work to cost-effectively protect public health and the environment, prevent
pollution and enhance the quality of area waterways (MMSD 2004). The city's
sewer system was so overwhelmed by torrential rains this past spring that MMSD
ended up dumping 4.6 billion gallons of untreated, raw sewage into
Overall,
recent studies have reported that elevated counts of Escherichia coli
indicate the presence of sewage due mainly to local wastewater treatment plant
dumping (Whitman et al. 2003). This experiment was designed in order to test whether or not sewage
dumping into
Materials
- Petrifilm E. coli / Coliform Count Plate
(30)
- Test tube rack
- Large test tubes with screw tops (12)
- Water blanks (distilled) (1)
- Bacterial colony counter
- Distilled water
- Bunsen burner
- Fume hood
- Safety gloves
- Goggles
- Difco Manual 9th and 10th Edition
- Wader boots and shoes
- Sterile pipettes (30)
- Syringes (2)
- pHydrion
- Incubator (37ºC)
- Label tape
- Open type reel tape measure (m)
- Sharpie markers
- Cleaning supplies
- Map of
Methods
Our
project began by us writing up a research proposal. Shortly thereafter, our
idea was approved to be both a practical and ethical experimental study by the
Assistant Professor of the Biology Department at
On
this second day of experimental work, one mL from each of the ten samples was
used to inoculate one 3M Microbiology Products Petrifilm E. coli /
Coliform Count Plate #2006-01KC. We then followed the procedures that were
outlined in the
After
inoculation from the water samples, the Petrifilm plates were incubated at 37°C and were read on experimental day three. pH readings of the lake water samples were also taken
on this day using the pHydrion
On
Monday, November 2, 2004 (third day of research), we viewed our Petrifilm plates
in order to count E. coli and other fecal coliform colonies. At this time, we evaluated whether or not any
Petrifilm plates should be rerun. This decision was mainly based on the colony
growth similarity between sample pair plates. If a set had similar numbers of
bacterial growth, we did not re-plate. New Petrifilm plates were only created
if a pair had very different quantities of bacterial colony growth on them.
Thus, we decided that new Petrifilm plates needed to be made from test tubes 2,
5, 6, 8, and 10. Plates that were not being rerun were stored in the
refrigerator to minimize further growth.
In
addition to being re-plated, sample number six first was diluted 1:10 in a
separate sterile tube, with sterile, distilled water, and then used to inoculate
a new pair of plates. This was done because the first pair of Petrifilm plates
for sample number six had bacterial growth that was too numerous to count.
After inoculation from the five water samples mentioned above, the Petrifilm
plates were incubated at 37°C
and were read on experimental day four.
On
the fourth day of our experiment (Wednesday, November 03, 2004), the final
bacteria growth (E. coli and other fecal coliforms) for the five
re-plated Petrifilm plates was counted and recorded. Due to the 1:10 dilution
of water sample number six, the bacteria colony counts of this sample’s plates
were multiplied by ten to get the actual total colony count.
Overall,
this experiment took one week to complete. We made sure to record all of our
data and observations in our laboratory notebooks. Lastly, we used Microsoft
Excel to make a few tables, graph our results, generate a R2 value
by means of a linear trendline equation and determine a correlation value
between increased E. coli levels and close proximity to MMSD’s Jones
Island Plant. These statistics will additionally
demonstrate to us whether or not untreated sewage dumping into
(a)
(b)
Figure 1:
Table 1: Specific
locations of
|
Sample Location # |
Distance South of (Along |
|
1 |
805 meters |
|
2 |
955 meters |
|
3 |
1105 meters |
|
4 |
1255 meters |
|
5 |
1405 meters |
|
6 |
1555 meters |
|
7 |
3165 meters |
|
8 |
3315 meters |
|
9 |
3465 meters |
|
10 |
3615 meters |
Figure 2: Pictures of
the unstable
Figure 3: Pictures of the fenced-off
Results/Data
First, we determined that each of the ten water
samples collected had a pH of 7.5. Next, we implemented XY scatter plots for
both E. coli and other fecal coliform bacteria plate averages (Table 2)
and discovered that a correlation was present (Figure 4 and Figure 5). An equation of: y = -0.0003x + 1.0858, and an r-value
that was approximately equal to -0.193 existed for E. coli. While we
obtained an equation of: y = -0.0029x + 52.504, and an r-value that was
approximately equal to -0.070 for other fecal coliform bacteria. These r-values
indicate negative correlations between bacterial growth and distance
from the water treatment plant (Figure 4 and Figure 5). However, they are not very strong. Instead, they are
fairly weak correlations.
Due to the fact that -0.193 and -0.070 are negative
values (which indicate negative associations between the variables), E. coli
and other fecal coliform levels in the lake did decrease as the distance away
from the Milwaukee Metropolitan Sewerage District’s (MMSD) Jones Island
Wastewater Treatment Plant increased. Thus, bacterial growth in
Table 2: Average results of bacteria colony
counts.
|
Sample
#’s |
Distance
South of (Along |
Average
of E. coli |
Average
of other fecal coliforms |
|
1a and 1b |
805 meters |
0.50 |
36.50 |
|
2a and 2b |
955 meters |
0.00 |
45.25 |
|
3a and 3b |
1105 meters |
0.00 |
1.00 |
|
4a and 4b |
1255 meters |
0.00 |
5.00 |
|
5a and 5b |
1405 meters |
0.00 |
70.25 |
|
6a and 6b |
1555 meters |
5.00 |
163.75 |
|
7a and 7b |
3165 meters |
0.00 |
14.00 |
|
8a and 8b |
3315 meters |
0.00 |
52.25 |
|
9a and 9b |
3465 meters |
0.00 |
28.50 |
|
10a and 10b |
3615 meters |
0.00 |
47.75 |
Figure 4: Correlation between distance from
Figure 5: Correlation between distance from
Discussion
Although
weak, the negative correlation values that we obtained from this experiment do support
our hypothesis that water samples which are
collected closer to the Milwaukee Metropolitan Sewage District’s (MMSD) Jones
Island Wastewater Treatment Plant will have higher E.
coli population levels than water samples that are taken further south
along Lake Michigan’s shoreline and away from this sewage dumping site.
These results do follow/relate to previously known
findings such as, the notion that contamination of aquatic ecosystems originating
from domestic sewage is readily indicated by the presence of elevated Escherichia
coli (E. coli) levels (Whitman et al. 2003).
Recent research has reported that elevated counts of E. coli
indicate the presence of sewage due mainly to local wastewater treatment plant
dumping (i.e. Jones Island Wastewater Treatment Plant dumping in the Spring of
2004) (Schultze and Rohde 2004, Whitman et al. 2003). Thus,
I feel that our evidence and data collected did support our hypothesis.
However, our results were of weak support due to the fact that the only
significant data points were for samples that were taken within the first 1500
meters south of
Another possible explanation for the weak results gained
deals with the time of year that we gathered our water samples. They were
collected during the fall season. Although, prior studies indicated that E.
coli levels are most prevalent and noticeable in
Regarding pH testing in this study, we discovered that the
pH of the ten lake samples was consistently 7.5. Since E. coli grows in
the pH range of 5.5 to 7.5, it was definitely possible for the bacteria to grow
and colonize in
If we were to repeat this experiment in the future, there
are a few aspects that we would want to alter before beginning. Sample size
would be one of them. Yes, we did collect ten different water samples from a
few different places along the shores of
Another suggestion for future studies would be to include
other directions in which the samples are collected. In addition to going along
the shoreline (both north and south), it would be interesting to gather samples
in intervals going further east and into
Since E. coli tends to grow best in warm conditions, it would be better to collect the samples during the warm summer months than in the crisp autumn.
The last facet that we would change would be to attempt to decrease human error. Since we were crunched for time, we both helped out where and when we could. Instead we would prefer that in possible future studies, specific tasks would be assigned and carried out at every sample site and in the lab by the same individual they were given to. By task delegation we could possibly lower the human error of us reading more or less measurements than our partner and in turn shifting the statistical results obtained.
Literature
Cited
Kinzelman, J.; Ng, C.;
Jackson, E.; S. Gradus, and R. Bagley.
2003. Enterococci as indicators of
Kirakosyan,
G.; K. Bagramyan, and A. Trchounian.
(2004, December). Redox sensing by Escherichia coli: effects of dithiothreitol, a redox reagent reducing
disulphides, on bacterial growth. Biochemical and biophysical research communications, 325, 803-806. Retrieved
on November 26, 2004 from EBSCOhost database.
MMSD:
Milwaukee Metropolitan Sewerage District (2004).
Agency and Facility Information. Retrieved on October 3, 2004 from the World
Wide Web: http://www.mmsd.com/home/index.cfm.
Schultze, S., and M.
Rohde. “Residents talk trash about
sewage.” JS Online 24 September 2004. Retrieved
on October 3, 2004 from the World Wide Web:
http://www.jsonline.com/news/metro/sep04/261546.asp.
Whitman, R.L., and M.B.
Nevers. 2003. Foreshore sand as a source of Escherichia coli
in nearshore water of
Whitman, R.L.;