The Sterilization of Water Through Ultraviolet Radiation

Without water, every living thing would perish, yet, for some people, the issue is

not access to water, but the quality of the water. Many microorganisms, such as

Escherichia coli, live within water, so it becomes necessary to sterilize water before it

can be utilized. The purpose of this experiment was to discover if a variation of

ultraviolet light radiation could be an effective way of purifying water.

The experiment was conducted through the use of a UVC light filter. Through this

filter, E. coli infected water was run. Using a spectrometer, the water was tested to

observe the changes, if any, to the amount of bacteria found in the water. The

spectrometer measures the amount of light that passes through a test tube. The bacteria

were grown in separate Petri dishes to ensure that one batch of infected water would not

infect another.

It was found that the exposure to the UVC light did have a significant effect on

the amount of E. coli found in a batch of water. Using the data collected from the trials, a

matched-pairs t-test was conducted, resulting in a p-value of 0.006894. The E. coli

numbers shrank because the ultraviolet light disrupted the bacterium’s ability to

reproduce by damaging its nucleic acid.

Table of Contents

Introduction..........................................................................................................................1

Review of Literature............................................................................................................3

Problem Statement...............................................................................................................6

Experimental Design............................................................................................................7

Data and Observations.........................................................................................................9

Data Analysis and Interpretation.......................................................................................12

Conclusion.........................................................................................................................17

Acknowledgements............................................................................................................21

Appendix A........................................................................................................................22

Works Cited.......................................................................................................................24

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Introduction

One of the leading problems in the world today is the lack of clean drinking water

(“Water”). Every day people are dying because of the lack of water, or the lack of clean

water. Dehydration can be a dangerous situation, causing people to have low blood

pressures and rapid heartbeat, or in more extreme cases: delirium, unconsciousness, and

death. The reason for this issue in many countries is because of the lack of money or

supplies to properly treat and clean the water. 

Because water is such an important aspect of life, it is crucial to understand how it

is prepared for the public to ingest safely. It also becomes important to cleanse the water

safely and in a cost effective manner. The purpose of this experiment was to help

determine if ultraviolet light could be used as an efficient means of water sterilization,

specifically through the eradication of Escherichia coli. The results of this experiment

could lead to a switch to a healthier and cheaper means of water purification, or it could

eliminate a possibility and lead to another means of preparing water.

This experiment was conducted through the use of a UVC light filter and E. coli.

The infected water was run through this filter for a period of one hour. Using a

spectrometer, the water was tested to observe the changes, if any, to the amount of

bacteria found in the water after treatment. The bacteria were grown in separate Petri

dishes to ensure that one batch of infected water would not infect another.

It is already known that UV light affects the nucleic acids of bacteria in such a

way that they can no longer reproduce, essentially killing them. This is why some water

treatment centers utilize a combination of both chemical and UV treatments, like the

Warren Wastewater Treatment Center. This is also why in some areas, like New York

City, are attempting to make the switch to a UV based water treatment (Greenemeier).

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The Environmental Protection Agency enacted the Safe Drinking Water Act, which

recommended that the use of chlorine be decreased for the treatment of water because of

the possible cancer-causing byproducts created, such as trihalomethanes and haloacetic

acids (Greenemeier). For this reason, it is important to find alternate means of water

disinfection.

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Review of Literature

E. coli, or Escherichia coli, is a rod shaped bacterium and usually grows in the

intestines of warm-blooded organisms (“E. coli”). E coli is facultatively anaerobic, which

means that it is an organism that makes adenosine triphosphate, or ATP, by an aerobic

respiration. ATP is used to transport chemical energy throughout a cell, providing help to

its working metabolism. This can only happen if oxygen is present. However, if oxygen

is not present, E. coli is able to produce ATP by switching processes to an anaerobic

respiration, where the oxygen is not present. E. coli is made up of almost all carbon

molecules, with the exception of a few other important elements that are bound to the

carbon. E. coli obtains almost all of the carbon it needs from glucose molecules, which

are obtained by its host organism, or the part of the organism that will provide a food

source and nourishment. 

E. coli is more often harmless than it is harmful, but some strands of the bacteria

are known for carrying diseases. Most of the time, E. coli will live in the lower intestines

of a warm-blooded organism and works to digest the food it eats. It is also known for

causing digestive infections such as urinary tract infection, or UTI, and traveler’s

diarrhea. This happens because some strands of E. coli produce a powerful toxin, called

Shiga toxin and is known as STEC, or Shiga toxin-producing E. coli. This toxin works to

damage the lining of the small intestines, which causes diarrhea, and causes blood to be

present. This toxin is so powerful that it can cause renal failure (kidney failure), and can

lead to death. There are several other types of this strand of E.coli. E.coli can help cause a

variety of other infections such as meningitis and pneumonia. These diseases are usually

contracted from common contaminated foods, such as ground beef, milk, produce, and

water (“Basic Information About E. coli…”) 

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It is important to purify water because most of child diseases, 85%, and more than

half, 65%, of adult diseases are caused by dirty water that carries viruses and bacteria

(“Ultraviolet Light as a Method…”). Water treatment is the process of cleansing water by

ridding it of (potentially) harmful foreign objects and microbes. To do this, four main

steps must be followed. The first step is called coagulation and flocculation. During this

step, positively charged particles are introduced to the water. The particles bind with the

negatively charged dirt in the water and form clumps called floc. The floc settle down at

the bottom of the tank because of their weight. This is step two: sedimentation. The third

step of the water purification process is called filtration. During this step, the water is

passed through differently sized filters to catch the floc. The final step of the process is

the disinfection of the water supply. Currently the way to do this is to run the disinfectant,

like chlorine, through the water to kill any remaining bacteria (“Water Treatment”)

(Granburg). Some places use chlorine, UV light, ozone, or a combination thereof as a

means of disinfection ("Basic Information about E. Coli…”).

The exposure of bacteria to ultraviolet radiation destroys the bacteria. The light

penetrates the cell wall of the bacteria and rearranges their genetic material (Ultraviolet

Light as a Method…). When the genetic material is rearranged the bacteria lose their

ability to reproduce, thus “deactivating” the bacteria. At this point the bacteria are no

longer harmful to animals, because it can no longer reproduce and mutate to avoid

immune systems.

Ultraviolet radiation is a clean and effective means of sterilization of water,

however it does have drawbacks. After generations of exposure to the light, the bacteria

mutate and adapt to their environment (Alcántara-Díaz, Breña-Valle, Serment-Guerrero).

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After this adaptation occurs the bacteria are essentially immune to the effects of the

ultraviolet light radiation at that magnitude. To remedy this, the bacteria must be exposed

to the light for a longer period of time, at a different wavelength, or a combination of

both. If neither of those changes has an effect on the bacteria’s nucleic acid, then a new

means of sterilization would need to be found.

The experiment being conducted consisted of water, E. coli, and an ultraviolet

light. After the E. coli was cultivated using Petri dishes and auger, it was introduced in

the water, which was kept in small cup-like containers. After this, a sample of the water

was analyzed for E. coli content using a spectrometer, which shines light through the test

tube, illuminating the E. coli colonies. The water was then put back into its container and

exposed to the UV light for a half hour. The last step of the experiment was to take a

sample of the disinfected water and analyze it again using the spectrometer. The numbers

gathered from before and after disinfecting the water were compared using a matched

pairs t-test to see if there was a significant change in the number of E. coli colonies in the

water after being exposed to ultraviolet light for a set time.

In the experiment that was conducted, it was expected that the exposure of E. coli

to ultraviolet light over time would dramatically decrease the number of E. coli colonies.

This can be concluded because when E. coli is exposed to these lights over a period of

time, the E. coli will absorb the UV energy. This energy is absorbed by the reproductive

parts of the bacteria, and damages it the point where the bacteria itself cannot reproduce.

(Ultraviolet Light as a Method…). Previous experiments have shown that UV light is an

effective way of disinfecting water (Benami, Gillor, Gross).

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Problem Statement

Problem:

To determine if exposure to ultraviolet light will significantly decrease the

number of Escherichia coli colonies in water.

Hypothesis:

The number of E. coli colonies will significantly decrease if the water is exposed

to ultraviolet light (UVC light specifically) for a period of one hour.

Data Measured:

The exposure time of the E. coli was the independent variable and the dependent

variable was the number of E. coli colonies. Using the spectrometer, the amount of light

that passed through the test tube was measured, and consequently, the amount of E. coli

was measured. Then, after the treatment, the amount of E. coli was found again and

recorded. Using a matched pairs t-test, the data was tested and compared to see if there

was a significant change in the amount of E. coli.

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Experimental Design

Materials:Water (160 L)E. coliUV Light Filter 2 Bottles (2L) Container (4L)30 Petri Dishes Agar (11.5 g)

2 Transfer Loops SpectrometerTI NspireErlenmeyer Flask (1L)Test Tubes (9 mL)Weigh Boat

Procedure:Agar Preparation

1. Measure out 500 mL of water in the flask. Place stirring magnet in flask.

2. Mass out 11.5 grams of nutrient agar using a weigh boat.

3. Place flask on hot plate and turn temperature to high.

4. Carefully pour nutrient agar into the flask using weigh boat.

5. Let water boil until the stirring magnet mixes in the agar completely. *Do not to let agar boil over.*

6. Turn off hot plate, remove flask from heat. Carefully pour the agar into the Petri dishes, just enough to cover the bottom of the dish.

7. Let agar cool and harden. Flip the Petri dishes and put in fridge overnight.

E. coli Transfer

1. Sterilize transfer loop and test tube rim using flame.

2. Place 1 mL of water in test tube.

3. Using transfer loop, transfer E. coli from container to test tube and mix.

4. Sterilize the transfer loop again using flame.

5. Pour water over nutrient agar in Petri dishes and spread around. Carefully pour out excess water.

Experiment

1. Place Petri dish in a 37 °C incubator to grow the bacteria. Growth will take 24 hours.

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2. Label Petri dishes 1-30. Randomize the Petri dishes using the TI Nspire.

3. Using the transfer loops, move the bacteria from the Petri dish to the tub of water. Let the bacteria grow for 24 hours.

4. Fill a test tube with E. coli infected water, and using the spectrometer, record the amount of E. coli in the water.

5. Empty the test tube back into the appropriate tub of water. Let E. coli grow for 24 hours.

6. Expose the batch of water to the UV light for one hour.

7. Fill a test tube with the treated water, and using the spectrometer, record the amount of bacteria in the water.

8. Repeat steps 3-7 for the other tubs of water/Petri dishes.

Diagram:

Figure 1. Experimental Setup

Figure 1 above displays the setup of the experiment conducted. Shown in the

picture is a 2 L bottle, which held E. coli infected water for 24 hours, a tub, which held E.

coli infected water being treated, and the submersible pump, which was used to treat the

water.

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Data and Observations

Table 1Data from Trials

Date Petri Dish

E. coli Before

E. coli After

26-Oct 1 93.6 96.027-Oct 28 97.8 98.428-Oct 5 97.6 98.729-Oct 16 96.8 98.52-Nov 13 97.4 96.42-Nov 23 98.0 98.64-Nov 2 97.6 98.44-Nov 11 98.0 98.15-Nov 30 97.2 96.85-Nov 7 97.3 98.26-Nov 24 97.2 98.26-Nov 12 97.4 99.6

Table 1 above shows the results of each trial. The table shows the percentage

recorded by the spectrometer before and after the Escherichia coli in the water was

treated by the UVC light filter. In the trials, the percentages increase in all but two trials,

which means that the amount of light shining through the water is increasing due to the

UVC light treatment. On Petri dish 30, there was a decrease in the percentage which

means that the light going through contaminated water decreased due to more

E. coli growth in the water. The highest percentage occurred on Petri dish 23 and the

lowest occurred on Petri dish 1. This outcome most likely occurred due to the lack of

cleaning done to the container of contaminated water between each trial. The first trial

has the lowest amount of E. coli growth due to the cleanliness of the container. The

average before percentage for the trials was 97.1583 and the average after percentage for

the trials was 97.9917.

Table 2

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Observations From TrialsTrial Observations

1 Trial went as expected28 Ran smoothly5 Went as expected16 Ran smoothly13 The tub was not cleaned23 Treated for longer than an hour2 Trial went well11 Treated for less than an hour30 Treated for less than an hour7 Ran smoothly24 Treated for longer than an hour12 Trial went well

Table 2 above shows the observations for the trials. For trials with Petri dish

numbers 11 and 30, the contaminated water was not treated for an entire hour. For the

trials with Petri dish numbers 23 and 24, the contaminated water was treated for longer

than an hour. Because of this, the trials could have differed from those whose timing was

more accurate. In the trial with Petri dish number 13, the bin holding the water should

have been cleaned, but was not due to time restrictions. This could cause there to be a

change in the results. 

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Figures 2 and 3. Transfer of Escherichia coli

Figure 4. Testing of WaterFigures 2, 3, and 4 above show the process by which this research was conducted.

Prior to the occurrence of Figure 2, the E. coli was inoculated on a Petri dish. In Figures 2

and 3, which took place 24 hours later than growing the E. coli in the Petri dishes, a

transfer loop is being sterilized so the E. coli can be transferred into the container of

water, where it grew for another 24 hours. Figure 4 shows the testing of the water using

the spectrometer.

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Data Analysis and Interpretation

The data collected from the experiment can be considered reliable because the

trials were randomized which helps eliminate bias and unknown interactions between the

trials. The trials were randomized by assigning a number to the Petri dishes. Then, using

the random integer function on the TI-Nspire, the trials were randomized for the order in

which the Petri dishes were inoculated. The trials were repeated many times which helps

to reduce variability and allowed the effect of the ultraviolet light to be the only variable

tested. This ensures consistent outcomes throughout the experiment and allows any

outliers to become apparent. Before using the spectrometer, which was used to measure

the amount of Escherichia coli in the water, it was “zeroed” out, meaning the

spectrometer was set to 100% transmittance for clear, fresh water between each trial to

serve as a control for the experiment, keeping the trials from affecting one another.

Figure 5. Box Plot

Figure 5 above shows the box plot created of the data collected. The box plot does

not have any outliers and is normally distributed. The median for the box plot is 0.85 and

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the mean is at 0.8333. The median and mean of the box plot are nearly the same,

indicating that the data is normally distributed.

To test if the ultraviolet light had an effect on the amount of E. coli, a matched

pairs t-test was used. This test was used because the means from one population before

treatment were being compared to the means of the same population after treatment and

the standard deviation was unknown.

In order to run the matched pairs t-test, some assumptions had to be met. These

assumptions are that a simple random sample (SRS) was conducted, the samples are

pulled from a single population, and the samples come from a normal population or at

least 30 trials be conducted. The first assumption, the SRS, was met with the

randomization of the trials. The second assumption was met because the water samples

for a trial were pulled from a single bucket of water, thus the paired samples came from a

single population. The last assumption was met as well, even though thirty trials were not

run, because the data is normally distributed with no outliers, as can be seen in the box

plot in Figure 5.

H 0 :µ=0

H a : μ1>µ0

Figure 6. t-test Hypotheses

Figure 6 above shows the hypotheses of the matched pairs t-test that was

conducted. The null hypothesis states that the mean difference between samples will be

zero, thus indicating no significant change in the water, and therefore the treatment did

not work as expected. The alternate hypothesis states that the mean difference between

samples will be greater than zero, thus indicating a possible significant change in the

water samples.

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Figure 7. t-test Results

In Figure 7, shown above, the results from the matched pairs t-test are displayed

as well as the corresponding p-graph. The t-value, p-value, and degrees of freedom are

shown, as well as the sample mean, sample standard deviation, and the sample size. The

t-value of 2.92603 yielded a p-value of 0.006894. The null hypothesis is rejected because

the p-value is less than the alpha level of 0.05. There is significant evidence that the

amount of E. coli in the water sample was decreased after being treated with ultraviolet

light. There is a 0.69% chance of getting these results by chance alone if the null

hypothesis is assumed to be true. A sample calculation for finding the t-value is shown in

Appendix A.1. The shaded portion of the p-graph represents the probability that the null

hypothesis would fail to be rejected. This too supports the hypothesis that the treatment

would have a significant effect on the amount of E. coli found in the water sample.

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Figure 8. Confidence Interval

Figure 8 above shows the results from the confidence interval at a 95%

confidence level. The lower bound of the confidence interval is 0.206492 and the upper

bound is 1.46017. It can be said, with 95% confidence, that the true population difference

of the amount of E. coli in the water lies between the boundaries of the confidence

interval. The difference of about 0.8333 falls between the boundaries of 0.206492 and

1.46017, thus supporting the alternate hypothesis of the test and the hypothesis of the

experiment by showing that there is a significant difference in the amount of E. coli in the

water before and after treatment. A sample calculation for the confidence interval can be

found in Appendix A.2.

Based on the results of the data, the boxplot, and the tests conducted, it was

concluded that the ultraviolet light did have an effect on the amount of E. coli found in a

water sample. The t-test conducted showed that the null hypothesis should be rejected,

thus indicating that there was a significant change in the amount of E. coli in the water

before and after the treatment. The confidence interval also supported this conclusion by

showing, with 95% confidence, that the true mean difference is greater than zero, thus

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showing that there is an actual difference between water samples after treatment. The

data collected also supported this conclusion because the values received after treatment

showed a decrease in E. coli for most individual trials.

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Conclusion

The purpose of this experiment was to see if exposing a batch of Escherichia coli

infected water to an ultraviolet light for a period of time would have an effect on the E.

coli, decreasing it, found in the water sample; this was found to be true, accepting the

hypothesis. In order to do this, the E. coli first had to be grown, which was done using

nutrient agar and an incubator. Then, after a 24-hour growth period, the bacteria were

transferred into a container of water. This was then left to grow for another 24 hours,

after which the water was transferred to another bucket containing the submersible UV

filter pump used for treating the water. The water was left with the pump running for a

period of one hour. A sample of water was tested, using a spectrometer, to find the initial

amount of E. coli and another sample was collected after the treatment was over. These

two values were compared using a matched-pairs t-test.

This experiment was based on the knowledge that ultraviolet light kills bacteria,

as well as previous experiments conducted. The experiment was also based on the current

treatment performed at water treatment centers. This experiment differed from current

treatments in the fact that it sought only to see the effect UV light has on bacteria, as

opposed to a combination of UV light and chlorine.

Based on prior knowledge, it was hypothesized that the accumulation of E. coli

would be decreased by a significant amount if the water was exposed to ultraviolet C

radiation. The analysis performed agreed with this hypothesis. A p-value of 0.006894

meant that the amount of bacteria found in the water would decrease when exposed to the

UV light. The data collected also supported this hypothesis by yielding an average before

percentage of 97.1583 and an average after percentage of 97.9917. These percentages

represent the amount of light that would pass through a test tube full of water. Clean

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water has a percentage of 100, therefore anything lower than that would represent some

object in the water; the lower the percentage, the higher the amount of bacteria in the

water.

Based on scientific findings, these results appear to be accurate. It is known that

the exposure to ultraviolet light damages the bacteria’s nucleic acid, thus destroying its

ability to reproduce ("Ultraviolet Light as a Method of Water Purification."). While the

radiation does not actually kill the bacteria in the water it does cause its “death” as a

bacterium’s purpose is to reproduce. If enough of the bacteria lose their ability to

reproduce, eventually the population will decline. This decline was the observed increase

in percentage of light in the test tubes in the experiment conducted. Since this decline was

observed, the results concur with those in the accepted scientific community.

When paired with other experiments, the one conducted can lead to an exact

amount of light and time needed to rid a particular amount of water of harmful bacteria

completely. With this knowledge, ultraviolet radiation as a means of water purification

can be put into effect anywhere that has access to electricity. As a result, the use of

chlorine in most water treatments can be reduced or eliminated, lowering the health risk

associated with the overuse of chemicals.

A concern with the use of ultraviolet radiation is the possibility of the bacteria

treated lying dormant within the water (Lockwood). It is possible, if the bacteria lie

dormant in the water, after many generations have passed for the bacteria that they could

eventually develop a mutation that could render the ultraviolet light useless in damaging

the bacteria. If the bacteria do develop an immunity to the radiation, the damage to the

nucleic acids can be reversed, thus allowing the population of E. coli to increase in the

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water instead of decrease (Zimmer and Slawson). A simple fix to this would be changing

the intensity and time of exposure, as different amounts of exposure are shown to have

different effects on the bacterium (Djurdjevic-Milosevic, Solaja, Topalic-Trivunovic, et.

al). Also, it has been shown that even in mutated strains on the bacterium, nucleic acid

synthesis can be slowed down, until a new method of water sterilization can be found

(Kantor).

During the testing of this experiment, many errors and experimental design flaws

became present. One of the problems while running the experiment was the lack of

cleaning done to the bin that was used to filter the water and the bottles that held the

water for the 24 hours while the E. coli grew. The containers were not properly cleaned,

meaning left over E. coli could have continued to grow, affecting the amount of

E. coli  tested in further trials. Because of this, the data collected from this experiment

could have been affected. In addition, the bacteria sometimes was not left to grow for the

24 hours planned. This could have caused more or less bacteria to grow in the Petri

dishes and containers, which could have further affected data from the trials. Another

error in the experiment was that for trials, the water often was not run through the filter

for an hour. This will affect the trials because this differs the time each trials’ water was

exposed to the UVC lights. This would affect the water that was used when reading the

spectrometer, which would affect the data collected from these trials. 

A way to expand this experiment would be changing the amount of radiation a

batch of water receives. That is, varying the time the water is exposed to the water in

addition to changing the type of light, i.e. UVA versus UVC. By changing the amount of

time exposure, an accurate measurement can be made for the amount of time required to

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remove a specific amount of bacteria from a specific amount of water. This would

decrease overexposure, which could lead to mutation in the bacterium, as well as limit the

amount of energy that is wasted. Testing different light types would find which light, if

any, has the most effect on the E. coli.

The value of this research comes in its real-life applications. Understanding that

ultraviolet light can be used as a method of decontaminating water can change the way

water is currently treated, leading to more efficient ways of cleansing water, as well as

lowering health risks associated with using chemicals in the water treatment process.

Also, this method is cheaper because less materials will be needed to clean the water. In

addition to being the economically and ecologically better alternative to chlorine, the use

of ultraviolet light can be instituted anywhere that has access to electricity or battery

powered lighting systems. This becomes important in areas where safe, clean water can

be difficult to find. Ultraviolet radiation is a better solution than boiling water in these

places because it will cause less evaporation of the water than boiling would. Water

treatment through the use of ultraviolet radiation is a better, and safer, alternative to

current means of water purification.

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Acknowledgements

The help from the following people is greatly appreciated, without them this

experiment would not be as great as it was:

Mr. Mark Estapa

Mrs. Kimberly Gravel

Mrs. Christine Tallman

Mr. Robert Granburg of the Warren Waste Water Treatment Center

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Appendix A: Sample Calculations

1.

t=x̄1−µ

S√N

t=0.8333−00.9866√12

t=2.9260

Figure 1. Sample Calculation

Figure 1 shows how the t value was found for the entire experiment. To solve for

t, the mean difference between the Escherichia coli populations (x̄1) was compared to

zero (µ), as that was the number expected if the treatment had no effect. This was then

divided by the sample standard deviation (S) over the square root of the population (N).

The t value was found to be 2.9260.

2.

CI= x̄1± t¿√ Sn

CI=¿(0.8333¿±1.796∗√ 0.986612

CI=0.206492¿1.460174

Figure 2. 95% Confidence Interval Sample Calculation

Figure 2 above shows the sample calculation for the 95% confidence interval. To

find the lower boundary, the square root of the sample standard deviation of the

population (S) divided by the number of trials was multiplied by the t star (t ¿¿ value and

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subtracted from the mean difference (x̄1). To find the upper boundary, the whole square

root times the t star (t ¿¿ value was added to mean difference.

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