UNIVERSITY OF HOUSTON

Comparison between Total Fecal Coliform and E. coli Concentrations in Water

And Sediment Civil and Environmental Engineering Research Experience for Undergraduates

Becky Jimenez, REU Student

Dr. Hanadi Rifai, Faculty Advisor

Anuradha Desai, Graduate Mentor

7/28/2009

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Table of Contents

Introduction ....................................................................................................................................2

Background and Theory................................................................................................................3

Site Background .......................................................................................................................3

Characteristics of Pathogens in Samples ..................................................................................4

Method ............................................................................................................................................4

Analysis ...........................................................................................................................................6

Comparing Water and Sediment Samples ................................................................................7

Temporal Effects ......................................................................................................................7

Solar Effects ...........................................................................................................................10

Decay Rate………………………………………………………………………………….11

Conclusion ....................................................................................................................................14

References .....................................................................................................................................15

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Abstract:

Studies on fecal indicator bacteria (FIB) in sediment have shown that sediment contains

greater concentrations of fecal coliforms (FC) than the overlying water. (D. L. Craig 2004)

(LaLiberte1981) (Niewolak 1998) (Burton Jr. 1987) By only analyzing water, the amount of

bacterial and pathogenic concentration may be underestimated. Research has shown that

sediment provides a more suitable environment than water by offering protection from predators

and insolation and by providing higher concentrations of nutrients and organic carbon for

bacteria. (Lee 2006) (D. L. Craig 2002) Thus, the amount of bacteria in sediment gives more

accurate results regarding the safety of certain sites than only measuring bacteria concentrations

in water from the same areas.

Introduction:

The goal of this research will be to measure the fluctuation in concentrations of fecal

indicator bacteria (FIB) in sediment and water and to compare the concentration between the two

in Cole Creek. Fecal indicator bacteria are known to survive in water, but there is more research

concerning the presence of FIBs in water than in sediment. Most often, only FIB levels in water

are considered when monitoring water standards and that of the underlying sediment is not

measured despite the research that has shown that sediment can harbor a higher FIB

concentration because it provides a more protective environment for bacteria than water does.

(Niewolak 1998) By comparing the concentrations, one would be able to determine whether

using FIB levels in water as a standard for water quality is sufficient, or, if, only using those

values in water is underestimating possible bacterial contamination, and whether it would be

necessary to take into account FIB concentration in sediment.

Figure 1: Cole Creek

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Background and Theory:

Fecal indicator bacteria are known to survive in water, but there is an increasing amount

of research concerning the presence of FIBs in sediment. Studies on fecal indicator bacteria in

sediment have shown that sediment may contain greater concentrations of FIBs than the

overlying water. By incorporating concentration levels of FIBs in sediment into water quality

standards, safer guidelines can be established when monitoring the safety of water used for

recreational purposes.

Higher concentrations of bacteria are believed to exist in sediment, because it provides a

more suitable environment for bacteria than water by providing larger amounts of nutrients and

carbon on particles, decreasing sunlight inactivation, and providing protection against predators

such as flagellates which are unable to graze on bacteria when they are attached to sediment

because of the particle’s large size. (Friesa 2008) For this reason, the amount of bacteria in

sediment could give more accurate results regarding the safety of certain sites than only

measuring bacterial concentrations in water from the same areas. Examining the concentrations

of FIBs in sediments, allows an opportunity for analysis to be taken of sediment conditions that

may support FIB concentration.

Site Background

Cole Creek, part of the Whiteoak Bayou watershed, is also identified by segment number

1017B and Station ID 16593. The soils in the Whiteoak Bayou watershed have been classified by

the STATE Geographic Database (STATSGO) as Clodine and Katy soil series. Clodine soils are

characterized as very deep, somewhat poorly drained, and moderately permeable. Katy soils are

very deep, somewhat poorly drained, and very slowly permeable soils. See Table 1. (CDM 2008)

Figure

2: Cole Creek- Station ID 16593

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Table1: Soil Series in Whiteoak Bayou Watershed

Map Unit

ID:

Soil Series

Name:

Min Available

Water Capacity

(in/in):

Max Available Water Capacity

(in/in):

Min Bulk Density

(g/cmˆ3):

TX 100 Clodine 0.15 0.2 1.35

TX 248 Katy 0.15 0.2 1.3

Characteristics of Pathogens in Samples

Total coliforms are a type of harmless microorganisms that live within the intestines of

humans, warm- and cold-blooded animals. Total coliforms are used as indicators that signal the

presence of fecal pathogens. Fecal indicator bacteria (FIB) fall under this category of which

E.coli is a widely known member. The presence of E. coli is evidence of recent fecal

contamination, and this bacteria is known to survive longer in sediment than in water. (Nature

2004)

Fecal indicator bacteria are used to measure the quality standards of water used for

agricultural, industrial, and recreational purposes. A correlation is made between the survival of

FIBs and pathogens which cause diseases. If FIBs survive, then pathogens are assumed to

survive under the same environmental conditions. (Knee 2008) When exposed to certain

environmental elements, the bacteria eventually die-off. Sunlight exposure, pH levels, chemicals

placed in water to inhibit bacterial growth, predators and temperature are some environmental

conditions that affect their ability to survive. But still some bacteria have been known to survive

in sediment for months. (Nature 2004)

Method:

Prior to going to the sampling site a reconnaissance group goes out to the designated

location to determine where the best location would be to collect samples. Once the site is

chosen, the sampling can begin. Sampling occurred at Cole Creek, which is part of theWhiteoak

Bayou Watershed.

Samples of sediment and water were taken at relatively close distances to each other to

provide for a better comparison between sediment and water samples. At a later time, the

sampling site was moved to an area within a fifty-foot radius of the original site because of a

decrease in sediment availability. The samples were collected over a twelve hour period from six

in the morning until six in the evening to allow time to experience the effects of sunlight on the

bacteria.

At the location, the YSI 6820 Multi-Parameter Water Quality Sonde was used to measure

the physical conditions of the water, after having been previously calibrated for conductivity,

turbidity, and pH. The YSI readings were then checked and recorded every ten minutes for

temperature, pH, conductivity, and salinity.

A UV reader was also used to measure incoming UV radiation every ten minutes. These

readings and the times for which they were taken along with those for the YSI were collected in

a logbook.

Water samples were collected in previously sterilized buckets and poured into three four

ounce Nasco Whirl-Pak Bags to ensure that if an analysis needs to be repeated there is enough of

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the sample left over to replicate the process on the sample. The bags contain a tablet of sodium

thiosulfate to counteract any chlorine that may affect the bacteria count. The samples are then

stored in a dark cooler filled with ice to prevent bacteria die-off.

During the sampling period, field blanks are collected for every 10 to 20 samples to

confirm that none of the equipment that is used to analyze and collect the sample has been

contaminated. The field blanks are then analyzed for fecal indicator bacteria in the same way that

the samples are processed. If the field blank shows no growth of bacteria, then the equipment

used did not contaminate the samples that are collected.

Lab blanks were also taken to certify that the deionized (DI) water used to process the

samples was not contaminated by fecal indicator bacteria. Duplicates were collected at three

different times to ensure that the collection method is carried out consistently and accurately.

Sediment was collected from the banks of Cole Creek, because of a relatively rocky area

in the middle of the creek. The sediment was collected in a bucket and deposited into glass jars

that were then placed into an ice-filled cooler.

In the lab, the water samples were processed using the IDEXX-Colilert method. All the

samples collected at the site were labeled with the site location ID and the time of collection.

These were matched up with previously prepared and labeled bottles with dilutions of 1:1, 1:10,

and 1:100 of DI water. Thus, the 1:1 dilution is completely filled with a pure sample of water

collected at the site, the 1:10 contains 10 mL of bayou water, and the 1:100 dilution is filled with

1mL of sample water.

One packet of Colilert reagent is then poured into each bottle. The Colilert will detect

total coliforms and E. coli in the water which will be visible because total coliforms consume

and metabolize the reagent making the sample turn yellow, the E. coli can be identified because

the reagent causes the E.coli to become fluorescent. The bottles are then shaken until the reagent

dissolves. Duplicates of each dilution are processed as well. The dilutions are then placed into

the corresponding Quanti-Trays with large and small wells, which are then sealed.

The time and temperature-35±0.5°C- for when the trays are incubated is recorded. The

trays- including lab and field blanks- are then incubated for 24 hours and are then read for total

coliforms and E.coli. The number of large and small wells that are yellow are counted and

identified as positive for total coliforms. Those that are yellow and fluoresce are positive for E.

coli. The number of wells that are positive for total coliforms and E. coli correspond to the most

probable number per 100 mL (MPN/dL).

For the sediment samples, a balance was used to measure out approximately 10 grams of

sediment. The sediment was placed into a 100 mL bottle containing 90 mL of deionized water.

The sediment-water mixture was then sonicated for two minutes, to separate any bacteria that

could possibly be attached to the sediment particles. The sonic probe was inserted into the bottle

without touching the bottom of the bottle. Between sonications, the sonic probe was wiped down

with alcohol wipes to remove any bacteria that may have been left over from the previous

sample. This mixture was then used as the water sample that was poured into the three different

dilutions. The dilutions used to process the sediment samples were different and were changed to

dilutions of 1:10, 1:100, and 1:1000.

One packet of Colilert reagent to was added to each dilution. The bottles were closed and

shaken until the bottles showed no trace of the reagent. Labeled Quanti-Trays with the same

information as the diluted bottles were sealed once the entire contents of each diluted bottle were

carefully poured into the corresponding Quanti-Tray.

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The trays were incubated at 35°C for 24 hours. A record of the date, time, and

temperature when the incubation began and ended was kept in a log sheet. To read the Quanti-

trays, all the wells that had a distinct yellow color were counted. The number of small and large

yellow wells was recorded in a log book.

Then, the tray was placed inside a dark box with a UV light. The UV light was used to

identify the number of fluorescent wells. The large and small fluorescent wells were then

marked. The number of wells that were both yellow and fluorescent (non-yellow, fluorescent

wells were not counted) were recorded in the log book. These numbers would then provide the

most probable number (MPN) per 100 mL of sample.

Analysis:

Based on the test used to evaluate the sample, bacteria concentrations are measured as

either colony forming units (CFU) or most probable number (MPN) per 100 milliliters (100 mL).

Both are considered equal. The most probable number is based on a statistical approximation of

the actual number of colony forming units in a water sample. Contact recreation with water is

supported when the minimum sample requirements are met; that is, if the geometric mean for

samples that are tested for E.coli do not exceed 126 cfu or MPN per 100 mL; and/or individual

samples do not exceed 394 CFU or MPN per 100 mL more than twenty-five percent of the time.

(Chief Engineer’s Office 2008)

From previously gathered data for Cole Creek, the minimum E. coli concentration was

150 and the maximum 240,000(MPN/dL). The data was collected beginning December 4th

, 2001

through February 21, 2005, the number of samples taken was 38 and the percentage of samples

exceeding 394 MPN/dL was 95% and the geometric mean 2,845 MPN/dL. (CDM 2008)

The geometric means of E. coli concentration in water samples for the years 2002, 2003,

2004, 2005, where calculated to be 970, 672, 2561, and 1200 (MPN/dL). While the geometric

mean for water samples collected gave a value of 441 (MPN/dL).

0

500

1000

1500

2000

2500

3000

2002 2003 2004 2005 2009E.

co

li C

on

cen

trat

ion

(M

PN

/dL)

Years

Geometric Mean

Figure 3: Geometric mean of E. coli concentrations for different years

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When testing whether the samples met the minimum standards for contact recreation, it

was found that 15 out of 24 samples exceeded the allowable E. coli concentration limits, and the

geometric mean for all samples was over the allowable limit. For sediment, all the samples were

over the 394 MPN/ 100mL limit; the geometric mean for the sediment samples was 110470. See

Table 2.

Table 2: Percent Exceedance and Geometric Mean

Cole Creek

Samples

Collected

Samples > 394

MPN/__ % Exceeding Standard

Geometric

Mean

Water

(MPN/dL) 24 15 62.5 441

Sediment

(MPN/kg) 24 24 100 110470

Percent exceeding standard was calculated using the following formula:

The acceptable E. coli concentration limits were over both requirements for water and sediment

samples collected at Cole Creek.

Comparing Water and Sediment Samples

In comparing sediment and water samples, milliliters of water and grams of sediment

were assumed to be equal for theoretical analysis. (Byappanahalli, 2003) When compared to the

total coliform concentration in water, the concentration in sediment exhibits the same trend, but

the measured concentration of total coliforms in sediment was much larger than that in water.

E. coli concentrations in sediment were one order of magnitude higher than those in

water when the maximum concentrations of E. coli in sediment and water were compared. When

comparing the total coliform concentrations, the maximum concentration was three order of

magnitude higher than the maximum total coliform concentration in water. See Table 3.

Table 3: Maximum and minimum E. coli and total coliform concentrations

CONCENTRATION IN: TOTAL COLIFORM E. coli

SEDIMENT (MPN/kg)

MINIMUM 238200 7100

MAXIMUM 24196000 1670000

WATER (MPN/dL)

MINIMUM 19177 147

MAXIMUM 61410 2894

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Temporal Effects

Total coliform and E. coli concentration as measured throughout the sampling period was

seen to be influenced by the temperature. As the day progressed and the temperature increased,

the concentration of total coliforms and E. coli decreased for water and sediment samples. See

Figures 1-4.

Figure 4: Total Coliform Concentration Change with Time in water

Figure 5: E. Coli Concentration Change with time in water

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Figure 6: Fluctuation of Total Coliform Concentration in Sediment

Figure 7: Fluctuation of E. coli Concentration in Sediment

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Solar effects

The effects of sunlight did show some correlation with the total coliform and E. coli

concentrations, although not as much as in other experiments which support the theory that

sunlight is one of the most powerful factors in bacterial inactivation. (Whitman 2004) But

temperature was a better indicator of E. coli concentration than UV exposure, because the effects

of UV light can vary considerably and quickly because of sudden changes in cloud cover.

Additionally, as the UV measurement changes, reading the UV value is dependent on the

estimation of the person who is recording the value.

Figure 8: Effect of UV radiation on Total Coliform Concentration in Water

Figure 9: Effect of UV radiation on E. coli Concentration in Water

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Figure 10: UV Radiation effect on E. coli Concentration in Sediment

Figure 11: Affect of UV radiation on E. coli Concentration in Water

Decay Rate

When measuring the rate of decay for bacteria, the form of the inactivation curve

followed the exponential decay law: N= N₀ , where N₀ was the initial concentration of

E.coli, N was the concentration at time t, and k ( ) was the rate at which the bacteria decayed,

which gave a negative coefficient.

From other prior experiments done that monitored the decay rate of bacteria- specifically-

E. coli- in sediment, the fell within range of those values. Where one experiment gave a

values of 0.95, 0.99, and 0.96 for sediment samples taken at three different locations, the data

gave a value of 0.7479. (D. L. Craig 2004)

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Figure 12: Decay rate of E. coli in sediment

The form of the decay rate for water gave an of 0.1833 when the bacteria

concentration at 12:30 P.M. and 4:00 P.M. (observations 14 and 21, respectively) were used, but

when running a regression on all the values during the sampling period, those two concentrations

were seen as outliers, going over the allowable value of two. (D. L. Craig 2004)

Table 4: Residual Output

Observation Predicted Y Residuals Standard Residuals

1 6.537576654 -0.33504114 -0.577567728

2 6.500241762 -0.21052619 -0.362920013

3 6.462906869 -0.29539038 -0.509214933

4 6.425571977 -0.01375371 -0.023709622

5 6.388237085 -0.03560769 -0.061383064

6 6.350902192 0.113686111 0.1959802

7 6.3135673 0.141631263 0.24415404

8 6.276232408 0.091954779 0.158518184

9 6.238897515 -0.27531817 -0.474613036

10 6.201562623 0.002995139 0.005163234

11 6.164227731 0.121770364 0.209916409

12 6.126892839 0.061371285 0.105796183

13 6.089557946 -0.38577547 -0.665027181

14 6.052223054 1.918171853 3.306680997

15 6.014888162 0.199719937 0.344291425

16 5.977553269 0.33418154 0.576085894

17 5.940218377 -0.05968539 -0.102889919

18 5.902883485 -0.32315366 -0.557075249

19 5.865548592 -0.68376504 -1.178722786

20 5.8282137 -0.38579599 -0.665062552

21 5.790878808 1.480129731 2.551552847

22 5.753543915 -0.76311133 -1.315505556

23 5.716209023 -0.07430195 -0.128086987

24 5.678874131 -0.21928862 -0.378025306

25 5.641539239 -0.40509728 -0.698335481

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Figure 13: Decay rate of E. coli in water, prior to removal of residuals

After removing the two outliers at times 12:30 P.M. and 4:00 P.M, the rate of decay in

water gave a value of 0.666. Values for E. coli decay in water have been measured to be

0.84, 0.89, 0.89, and 0.95 at four other locations. Thus, the value calculated here fell within a

reasonable value for bacteria decay rates in water. Generally, the decay rate and total coliform

and E. coli concentration began with high values and then showed a decline.

Figure 14: Decay rate of E. coli in water

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Conclusion:

Most studies only consider the concentrations of fecal indicator bacteria in water and

overlook the concentration of bacteria in sediments. By examining the concentrations of FIBs in

sediments, this experiment opens new ground for research. Because the data shows that sediment

does contain greater concentrations of bacteria, one can look into what conditions are favorable

for bacteria growth in sediment than in water. When considering potential health hazards from

water quality, the levels of bacteria in sediment should not be ignored but rather incorporated

into deciding if water quality is safe for use.

Acknowledgements

The research study described herein was sponsored by the National Science Foundation under the

Award No. EEC-0649163. The opinions expressed in this study are those of the authors and do not

necessarily reflect the views of the sponsor. I would also like to thank Dr. Hanadi Rifai, Anu

Desai, Norma Moreno, Sean Carbonaro, Scott Rauschhuber, Yaa Amoah, Sharon Wells, Nathan

Howell, Jenni McFarland, Lisa Grecho, Maria Modelska, Stephen Ray, Zack Van Brunt, Matt

Feaga and Divagar Lakshmanan.

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