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Ly

Acknowledgements
I would like to thank the Niles North STEM teachers, Richard Thielsen, Christine Camel, and
Susan Posnock for their guidance throughout this project. I am especially thankful to my
mentor, Dr. Daniel Nelson, from University of Maryland. Not only did he did he suggest the idea
for my current project, but he also provided numerous suggestions that proved to be very
helpful. Lastly, I would like to thank my family and friends for their support. Thank you to
everyone who helped me complete my project.

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Purpose:
The purpose of this experiment is to increase the longevity of the LifeStraw water
filter using an enzyme known as lysozyme. Like other filters, the LifeStraw becomes clogged
with bacteria and other particular matter. Once clogged, the LifeStraw can no longer be used.
Lysozyme is known for its ability to degrade bacterial cell walls; thus, if applied to the LifeStraw,
it could potentially unclog the filter, increasing its efficiency.

Hypothesis:
If lysozyme and EDTA are combined into a solution then the solution will be able to
successfully lyse the
E.coli
clogging the LifeStraw.

Rationale:
Acting as a permeabilizer, the EDTA will disintegrate the outer membrane of the cell
wall allowing for increased permeability. The lysozyme will then be able to access the glycan
backbone located in the second layer of the cell wall. Lysozyme specifically breaks down the
-1,4-glycosidic bonds formed between N-acetylglucosamine (GlcNAc) and N-acetylmuramic
acid (MurNAc), which are the main components of the gram-negative cell walls like
E.coli.

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Review of Literature
Water covers approximately 70% of the earth but unfortunately, less than 1% is
available for consumption. In total, 780 million people, worldwide, do not have access to clean
water (WHO & UNICEF 2012). Majority of these people live in developing countries that do not
have the resources to adequately treat water; thus, approximately 3.4 million people die each
year from water or hygiene related diseases such as diarrhea (WHO 2008). The United Nations
Development Program estimates that the water and sanitation crisis [has claimed] more lives
through disease than any war [has claimed] through guns (2006). As the world population
continues to grow exponentially the question of water and sanitation grows more pressing.
Scientist have already developed numerous solutions in order to purify water. Each
solution is unique in its own way. Despite their differences, all solutions have one thing in
common: they strive to rid water of harmful bacteria and substances. Water is contaminated
with a variety of substances such as bacteria, pathogens, chemicals etc, all of which could be
potentially fatal. This experiment will be focusing on bacteria specifically
Escherichia coli
(
E.coli
),
a type of fecal coliform as well as one of the most commonly found bacteria in water (New York
Department of Health 2011).
E.coli
and other types of fecal coliform can be found in the intestinal tracts of
warm-blooded animals including humans. In the intestines,
E.coli
aid the digestion process by
breaking down food. In addition,
E.coli
also play a vital role in the production of vitamin K
(Jacques & Ngo 2014). While most strains are harmless, there are some strains that can lead to
serious health issues including death. More importantly, the presence of
E.coli
is used as an

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indicator for more serious organisms known as pathogens (Washington State Department of
Health n.d.).
Pathogens are disease causing organisms that can come in a variety of forms: bacteria,
viruses, protozoa etc. The concentration of a specific pathogen is typically small, but the variety
of different pathogens is relatively large; therefore, it is inefficient and impractical to test for
each one individually. Instead, a total coliform test is used. Total coliform and pathogens come
from the same source, but unlike pathogens, the concentration of total coliform in water is
plentiful making them easy to identify (New York Department of Health 2011).
Bacterial contaminants in water are not necessarily fatal but they can lead to diarrhea
and abdominal cramps which can be easily treated in most cases. Although treatment is
available, diarrheal diseases are not to be taken lightly (Washington State Department of
Health n.d). 1.5 million people each year die from diarrheal diseases, a common symptom from
ingesting contaminated water (Vestergaard n.d.). Parasites like
cryptosporidium (crypto)
and
giardia
, all of which can be found in water, are the leading cause of diarrheal disease. Both
cryptosporidium
and
giardia
are surrounded by an outer shell. This outer shell protects the
parasites and allow them to remain independent for long periods of time. It also enables the
parasite to be very tolerant against different types of disinfectants specifically chlorine (CDC
2013, CDC 2011). Although disinfectants are ineffective, point-of-use (POU) treatment devices
have been known to successfully filter out these contaminants.
POU treatment devices rely on the same treatment technologies that are implemented
in central or public treatment plants; however, central treatment plants aim to treat all the
water distributed to consumers while POU treatment devices only treat a portion of the total

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flow making them ideal for home use. Selective treatment allows POU devices to be extremely
cost effective; thus, POU devices are ideal candidate for public water systems (PWS) in areas
where central treatment is not affordable (EPA 2006). In addition, POU devices offer a high
degree of flexibility because so many different treatment technologies are available.
The most commonly used POU devices implement reverse osmosis or granular activated
carbon, a type of filtration; although other options such as distillers, adsorptive media, and ion
exchange are also available (EPA 2006, MDH n.d.). Reverse osmosis is a process in which
dissolved inorganic solids are removed from solutions. In the case of water treatment, tap
water is pushed through a semipermeable membrane that only allows water to pass through.
Contaminants that are 0.001 micron or larger are too big to cross through the membrane.
Instead, these contaminants are flushed down the drain. Water treated through reverse
osmosis are improved ascetically: better taste, odor, and appearance. In addition, little energy
is consumed during this process which allows the device to produce gallons of clean water for
only pennies a day (ESP Water Products 2009).
Reverse osmosis devices my seem ideal, but these advantages are offset by the
inefficiencies these devices offer. While the membranes are successful in removing salts,
natural minerals, bacteria, and pathogens, dangerous chemicals like pesticides can cross
through the membrane because of their small molecular composition. To prevent this,
complementary carbon filters are installed to improve its efficiency and effectivity (Binnie et al
2002). In addition, in comparison to other treatment alternatives, reverse osmosis devices are
slow and wasteful. For every one gallon of water produced, at least two to three gallons are
wasted (MDH n.d.).

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Unlike reverse osmosis, granular activated carbon, an alternative treatment option,

does not waste water. These devices use a carbon source such as coal or wood to reduce the
concentration of organic chemicals (MDH n.d.). Organic chemicals and other contaminants are
attracted to the surface of the carbon source. This attraction allows the activated carbon to
draw contaminants out of the water and into its pores. Over time, the pores become saturated
or filled. Once saturated, rather than purifying water, the filter will do the opposite.
Contaminants trapped inside the pores of the carbon will flow back into the solution. To
prevent this, some granular activated carbon devices shutdown once the filter has reached the
breakthrough point. The filter can then be reactivated by changing the filter cartridge (Lemley
et al 1995).
Although most filters can be reactivated by
changing the filter, not all filters have disposable
cartridges, for example, the LifeStraw. The LifeStraw
is POU filter created by Vestergaard. It focuses on the
transformation of microbially contaminated water.
Using hollow fibre material, the LifeStraw can filter
up to one thousand liters water while removing
99.99% of bacteria and protozoan parasites including
cryptosporidium
and
giardia
. The LifeStraw also
reduces turbidity or muddiness by filtering out
particulate matter that is larger than 0.2 microns
(Vestergaard n.d.).

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True to its name, the LifeStraw works like an actual straw. Suction is used to power the
filter, thus, it requires no batteries or replacement parts. The LifeStraw works by allowing water
to enter through the bottom. Once inside of the LifeStraw, the water is forced up through the
hollow fibre due to the suction power generated. The hollow fibre traps the contaminants while
clean water exits through the tiny pores (Vestergaard n.d.).
There are three main components in the LifeStraw that aid the reduction of bacteria.
The first is the 15 micron plastic mesh screen located on the base of the straw. This screen
provides the first step to filtration. It filters out relatively large contaminants and organic
matter. After passing the screen, the water enters a compartment of halogenated ion exchange
(Walters 2008). This compartment consists of iodinated resin in the form of small polymer
beads. As water passes through the beads, iodine is released into the water. Negatively charged
particles then surround the resin and displace the iodine. The leftover free iodine attaches itself
to the cell wall or membrane of microbes which are later absorbed into a silver impregnated
carbon block (Edison 2002). When the microbes come into contact with the silver ions, the
sulphurhydryl group in the cell reacts with the silver to create a silver-sulphur molecule. The
silver-sulphur combination inhibits the cells respiratory abilities by preventing the transfer of
proton, leading to the cells death (Bayatti 1997).
In many ways the LifeStraw is the ideal filter for large-scale disasters and day to day
filtering requirements, but there also some drawbacks. The LifeStraw cannot be used for large
volumes of water, it is not able to produce water to be stored, and residual iodine can leave an
unpleasant taste in the effluent water (Walters 2008). Additionally, the LifeStraw has a short
life expectancy. After a year, the filter is expected to be clogged with bacteria and other matter.

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Once this occurs, the bacteria can no longer be removed. To improve the LifeStraw, this
experiment will be focusing on cleaning the filter by lysing the bacteria in the hollow fibre
through the use of an enzyme.
In order to lyse the bacteria in the filter, the chosen enzyme must be able to penetrate
the bacterial cell wall. The cell wall acts as shield by protecting the cells protoplast from
mechanical damage and from osmotic rupture otherwise known as lysis. In order to protect the
more sensitive plasma membrane, the outer wall must be made of a porous and rigid material
that has a high tensile strength; thus, the ideal material is murein. Murein is type of
peptidoglycan, a polymer of glycan cross linked with short chains of amino acids. The built of
peptidoglycan is dependent on the bacteria but all bacterial peptidoglycan contains
N-acetylmuramic acid (Todar n.d.).
Bacterial cell walls can be categorized into two groups: gram-positive and
gram-negative. Gram-positive cell walls consists of several thick layers of peptidoglycan. In
general, the chemical composition and structure of gram-negative bacteria are very similar
except the peptidoglycan layer is much thinner because of the addition of the outer membrane.
The outer membrane consists of a combination of lipopolysaccharides and phospholipids
(Salazar & Asenjo 2007, Todar n.d.).
Certain enzymes like lysozyme can digest peptidoglycan located in the cell wall. These
enzymes are known as murein hydrolases. Murein hydrolases can be categorized into three
groups: glycosidases, endopeptidases, and amidases. Glycosidases such as lysozyme split the
polysaccharide chain while endopeptidase split the polypeptide chain. Amidases cleave the
junction between polysaccharides and peptides (Salazar & Asenjo 2007).

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The most well studied muramidase is lysozyme. Lysozyme specifically splits the
-1,4-glycosidic bonds between N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid
(MurNAc). In
E.coli
, the GlcNAc and MurNAc construct the glycan backbone, but due to the
outer membrane, the lysozyme effectivity is decreased (Sekiguchi & Yamamoto 2012). To
degrade the outer membrane, a permeabilizer could be added. Permeabilizers disintegrate the
LPS layer which increases the permeability of the outer membrane. A commonly used
permeabilizer is ethylenediaminetetraacetic acid (EDTA)(Vaara 1999). EDTA works by providing
electrostatic interactions between the protein and the LPS. These interactions withdraw
divalent (having the electron valence of two) cations which undermines the stability of the
outer membrane. Through EDTA treatment, the amount of LPS is greatly removed which opens
new pathways for different agents (Vaara 1999).
The lysozyme and EDTA combination was first tested by Repaske in 1956. In his
experiment,
Lysis of Gram-negative bacteria by lysozyme
, Repaske developed a new method of
lysozyme treatment that allowed lysozyme to penetrate gram-negative cell walls at the pH of
7.5 to 8.0. Prior to this methods, researchers applied the Nakamura technique in which cells
were exposed to lysozyme at the pH of 3.5 and then incubated for long period of time. Once
lysis finally occurred, the pH would have risen to at least 9.6. Repaske was able to avoid this
jump in pH through the addition of EDTA which allowed to lysozyme to function at a more
neutral level (Repaske 1956).
More recently, scientist have been testing the lysozyme-EDTA combination in food
preservation. In one experiment different combinations of EDTA, nisin, and lysozyme was
tested to determine its ability to reduce microbial contamination of food. Observations showed

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that the lysozyme and EDTA combination was able to successfully interact with
Enterococcus
faecalis
and
Weissella viridescens
. In contrast to previous experiments, the EDTA and nisin
combination did not enhance antimicrobial interactions against gram-negative bacteria nor did
the lysozyme and nisin combination against gram-positive bacteria (GIll et al 2003).
Like other experiments, this experiment will be focusing on the interactions between
lysozyme-EDTA and gram-negative bacteria. If the lysozyme and EDTA duo are able to
effectively degrade the gram-negative
E.coli
clogging the filter, then the LifeStraw will be able
to function for a longer period of time making it more cost effective. For those living in
developing countries, the cost of the LifeStraw is still too expensive; thus, yearly replacements
are impossible. Because lysozyme is an enzyme, it can be quickly massed produced at a low
cost. If the solution is successful, rather than disposing the LifeStraw, the LifeStraw can be
continuously reused making it a more practical solution.

10

S.Ly

Materials:
Goggles

Escherichia coli
(
E.coli)

Gloves

Nutrient broth

Lysozyme

Electronic balance

Ethylenediaminetetraacetic acid (EDTA)

500 mL flask

LifeStraw by Vestergaard

Autoclave

Funnel

Sterile loops

Parafilm

Pipettes

Vacuum flask and tube

Incubator

Graduated cylinder

Stir Rod

Distilled water

Magnetic stirrer

Stopwatch and Timer

Air Regulator

Building the Vacuum:

1. Attach one end of the tube to the vacuum flask
and other to the vacuum nozzle
2. Using parafilm, wrap the mouthpiece [refer to
page 4 for diagram of LifeStraw] of the LifeStraw
until it fits tightly into the opening of the flask
3. Create a funnel to fit around the other end of
the LifeStraw
4. Secure funnel using parafilm
5. Turn on vacuum and pour distilled water to test
if vacuum is correctly set up
11

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EDTA Solution Preparation:

1. Using an electronic balance, mass out 2.29 grams of EDTA
2. Pour EDTA into a one liter volumetric flask
3. Fill flask with distilled water
4. Drop a stir rod at the bottom into the flask and place on a magnetic stirrer
5. When the solution is homogenous, cover with parafilm
Lysozyme Solution Preparation:
1. Pour 5 grams of lysozyme into a 500 mL flask
2. Pour 300 mL of distilled water into the flask
3. Drop a stir rod into the flask and place on a magnetic stirrer
4. Cover with parafilm and place in fridge for storage
E.coli
Broth Solution Culture Procedure:
1. Light the burner
2. Agitate stock tube by gently tapping or stroking the end of the tube with the other hand
3. Flame the mouths of the sterile medium and stock tube
4. Draw about 0.1 mL of the bacteria-containing suspension into a pipet
5. Insert the end of the pipet into the sterile tube and release the contents into the broth
6. Remove the pipet from the tube and flame the mouths of both tubes
7. Replace the caps
8. Gently agitate the inoculated tube and label it

12

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Experimental Procedure:
1. Turn on vacuum
2. Use a graduated cylinder to measure out 100 mL of distilled water
3. Pour into the LifeStraw
4. Measure the amount of time it takes for 100 mL of water to filter through the LifeStraw.
It is important to be as consistent as possible
5. Turn vacuum off
6. Release vacuum by pulling the LifeStraw out
7. Dab the mouthpiece of the LifeStraw with a paper towel to remove excess water
8. Place LifeStraw back into the flask
9. Repeat steps 1-8 three times to calculate the flow rate
10. Use graduated cylinder to measure out 10 mL of the
E.coli
broth solution and pour into
the filter
11. Follow steps 1-9
12. Continue to pour 10 mL of E.coli into the filter until the filter is clogged. The filter will be
clogged once the flow rate is zero.
13. Turn on vacuum
14. Use a graduated cylinder to measure out 15 mL of the EDTA solution and pour into filter
15. As soon as the solution begins to filter, turn off the vacuum and pull out the LifeStraw
16. Lay the filter on its side for 10 minutes
17. Place filter back into the flask and turn on filter so the EDTA can be filtered out
18. Unhook the vacuum tube from the vacuum nozzle

13

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19. Attach the vacuum tube to an air regulator. The tube should now be attached to the
vacuum flask and air regulator.
20. Turn on air regulator for 30 seconds to blow out any of the dead
E.coli
21. Repeat steps 1-9 to determine the flow rate after the filter is treated with EDTA
22. Use a graduated cylinder to measure out 15 mL of the lysozyme solution and pour into
the filter
23. As soon as the solution begins to filter, turn off the vacuum and pull out filter
24. Lay the filter on its side for 15 minutes
25. Place filter back into the flask and turn on filter so the lysozyme solution can be filtered
out
26. Unhook the vacuum tube from the vacuum nozzle
27. Attach the vacuum tube to an air regulator. The tube should now be attached to the
vacuum flask and air regulator.
28. Repeat steps 1-9 to test for flow rate after the lysozyme solution has be added
29. Repeat steps 1-29 for a total of three trials.
30. Repeat steps 1-30 to test the effectiveness of the lysozyme solution at 25 and 35
minutes

Variables
Independent Variable:
Amount of time the lysozyme has to degrade the bacteria
Dependent Variable:
The flow rate after the lysozyme solution has been added
Control Group:
Initial flow rate and flow rate before the EDTA and lysozyme solution is added

14

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Constants:
Concentration of EDTA and lysozyme solution, time EDTA solution is in the filter,
volume of EDTA and lysozyme solution, flow rate timing method, and the amount of time the
air regulator was used

Results
*All flow rates are calculated with 100 mL of water per second
15 MINUTES LYSOZYME TREATMENT

Trial 1: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

6.151338585

100

0.012176

10

2.98495512

48.52529378

0.124407

20

2.282332304

37.10301868

0.030481

30

1.542680495

25.07877714

0.056765

40

1.352888483

21.99339972

0.020017

50

1.083785759

17.61869785

0.022866

60

1.2146798

19.74659308

0.084978

Flow Rate with the Addition of EDTA

1.248431273

20.2952781

0.013348

Flow Rate with the Addition of Lysozyme Solution

1.686389298

27.41499715

0.126673

Table 1

15

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Graph 1A

Graph 1B

16

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Trial 2 : Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

13.48430293

100

0.153462

10

6.310612434

46.79969345

0.117148

20

4.29035946

31.81743604

0.149489

30

2.590455709

19.2108982

0.029865

40

2.101965779

15.58824205

0.051423

50

1.641019552

12.16985083

0.012282

60

0.981329893

7.277572285

0.003372

Flow Rate with the Addition of EDTA

1.238220634

9.182681826

0.008055

Flow Rate with the Addition of Lysozyme Solution

1.453438494

10.77874401

0.123128

Table 2

17

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Graph 2A

Graph 2B

18

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Trial 3: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

21.46220051

100

1.276338

10

9.545258655

44.47474363

0.519928

20

5.531713091

25.77421215

0.078618

30

4.253419031

19.818187

0.057424

40

3.687069024

17.17936156

0.138072

50

2.190590003

10.20673534

0.023576

60

1.94156207

9.04642592

0.01387

70

1.323316181

6.165799172

0.017563

80

1.128155041

5.256474241

0.008083

Flow Rate with the Addition of EDTA

1.60199043

7.464241281

0.314324673

Flow Rate with the Addition of Lysozyme Solution

3.490682364

16.2643265

0.314324673

Table 3

19

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Graph 3A

Graph 3B
20

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15 Minutes Lysozyme Treatment: Percent Change of Flow Rate

Trial Number

EDTA Percent
Change

Lysozyme Percent
Change

Total Change

0.54869

7.11972

7.66841

1.905109541

1.596062184

3.501171725

2.207767041

8.800085214

11.00785225

Average

1.553855527

5.838622466

7.392477993

Standard Deviation

0.721419293

3.07737931

3.07079436

Table 4

Graph 4

21

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25 MINUTES LYSOZYME TREATMENT

Trial 1: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

13.43713659

100

0.412493

10

9.790487159

72.86140981

0.810363

20

4.304207709

32.03217947

0.177564

30

2.444568206

18.19262749

0.060154

40

1.439147827

10.71022697

0.014061

50

1.409192348

10.48729644

0.017529

60

0.998639362

7.431935778

0.007501

Flow Rate with the Addition of EDTA

1.134961011

8.446449904

0.005222

Flow Rate with the Addition of Lysozyme Solution

1.277660538

9.508428593

0.012839

Table 5

22

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Graph 5A

Graph 5B
23

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Trial 2: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

11.72789499

100

0.282721

10

7.936824573

67.67475815

0.539564

20

3.896292924

33.2224404

0.084292

30

2.649236518

22.58919029

0.08672

40

2.440421331

20.80869015

0.027587

50

2.097065849

17.88100806

0.03246

60

1.946116126

16.59390817

0.015868

70

1.524591287

12.99970103

0.010283

80

1.381357957

11.77839636

0.005629

90

1.152522221

9.827187414

0.003192355

Flow Rate with the Addition of EDTA

1.37131759

11.69278537

0.012579369

Flow Rate with the Addition of Lysozyme Solution

1.552701239

13.23938558

0.012467634

Table 6

24

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Graph 6A

Graph 6B

25

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Trial 3: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

16.7560407

100

0.374224

10

10.00569614

59.71396417

0.431369

20

6.85920203

40.93569687

0.071184

30

5.941540337

35.45909468

0.171113

40

4.780580261

28.53048848

0.157513

50

2.3319178

13.91687835

0.036174

60

1.79391463

10.70607706

0.015176

70

1.479497648

8.829637466

0.011697

80

1.014007171

6.051591713

0.004619

Flow Rate with the Addition of EDTA

1.094288284

6.530709156

0.007782801

Flow Rate with the Addition of Lysozyme Solution

1.244415693

7.426669078

0.00537145

Table 7

26

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Graph 7A

Graph 7B

27

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25 Minutes Lysozyme Treatment: Percent Change of Flow Rate

Trial Number

EDTA Percent
Change

Lysozyme Percent
Change

Total Change

1.014514126

1.061978688

2.076492814

1.865597957

1.546600213

3.41219817

0.479117444

0.895959921

1.375077365

Average

1.119743176

1.168179607

2.287922783

Standard Deviation

0.570898075

0.276034024

0.844982156

Table 8

Graph 8

28

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35 MINUTES LYSOZYME TREATMENT

Trial 1: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

9.671228087

100

0.426673

10

3.83743992

39.678931

0.122977

20

2.927644726

30.27169559

0.05952

30

2.034963136

21.04141395

0.017397

40

1.860494283

19.23741501

0.009025

50

1.259295607

13.02105168

0.003976

60

1.084002573

11.20853074

0.013338

Flow Rate with the Addition of EDTA

1.136408083

11.75040101

0.00871

Flow Rate with the Addition of Lysozyme Solution

1.332059771

13.77342938

0.013251

Table 9

29

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Graph 9A

Graph 9B

30

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Trial 2: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

22.48572102

100

0.681573

10

9.947315001

44.23836351

0.879769

20

6.531818372

29.04873883

0.124033

30

4.756276727

21.15243146

0.116228

40

3.803320469

16.91438076

0.056177

50

2.701565025

12.01458038

0.065263

60

2.238841175

9.956723975

0.026064

70

1.504461338

6.690740924

0.007243

80

1.3644621

6.068126965

0.006322

Flow Rate with the Addition of EDTA

1.641243473

7.29904757

0.002152

Flow Rate with the Addition of Lysozyme Solution

1.93909867

8.623689088

0.169822

Table 10

31

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Graph 10A

Graph 10B

32

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Trial 3: Flow Rate with E.coli Only

Amount of E.coli
(mL)

Flow Rate (mL/s)

Efficiency (%)

Standard Deviation

22.21218682

100

2.865894

10

14.55476232

65.52602151

1.429379

20

8.357070994

37.62381012

0.819307

30

4.280101321

19.26915776

0.084026

40

2.809460484

12.64828405

0.04458

50

1.674480755

7.538567762

0.015301

60

1.397946189

6.293599995

0.01135

Flow Rate with the Addition of EDTA

1.480704844

6.666182198

0.007834

Flow Rate with the Addition of Lysozyme Solution

1.64630866

7.411736058

0.023117

Table 11

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Graph 11A

Graph 11B

34

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35 Minutes Lysozyme Treatment: Percent Change of Flow Rate

Trial Number

EDTA Percent
Change

Lysozyme Percent
Change

Total Change

0.541870272

2.023028372

2.564898644

1.230920605

1.324641518

2.555562123

0.372582203

0.74555386

1.118136063

Average

0.71512436

1.364407917

2.079532277

Standard Deviation

0.37121327

0.522284282

0.679820468

Table 12

Graph 12

35

S.Ly

Percent Change of Flow Rate

Time

EDTA Percent
Change

Lysozyme Percent
Change

Total Change

15 Minutes

1.553855527

5.838622466

7.392477993

25 Minutes

1.119743176

1.168179607

2.287922783

35 Minutes

0.71512436

1.364407917

2.079532277

Table 13

Graph 13

36

S.Ly

Data Analysis
The objective of this experiment is to improve the longevity of the LifeStraw by treating
it with a combination of EDTA and lysozyme. To test for the effectiveness of this combination, a
total of nine trials were completed. The flow rate and efficiency before and after the lysozyme
treatment was graphed and then fitted with a trendline. Using percent change, the most
effective time for lysozyme was determined.
Time was manipulated to test for the effectiveness of the lysozyme solution. In the first
three trials, the lysozyme solution was allowed to remain in the LifeStraw for 15 minutes before
it was flushed out. Even though 15 minutes was the shortest allotted time, it proved to be the
most effective. On average, after 10 minutes of being treated with EDTA, the flow rate
increased by 1.554%. After the lysozyme solution had been introduced for 15 minutes, the flow
rate increased by an additional 5.839%, making the total change 7.391%. Improvements in the
flow rate due to the 15 minutes lysozyme treatment ranged from 3.500% (total change) to
11.007%. At its most effective, the lysozyme solution accounted for as much as 8.800% of the
increased flow rate. Even at its least effective, 1.596%, the increase in the flow rate after 15
minutes of treatment was still greater than all of the lysozyme percent change values after 25
minutes of treatment.
Although not as effective as the 15 minutes lysozyme treatment, the 25 minutes
lysozyme treatment still improved the flow rate. After 10 minutes of being treated with EDTA,
the flow rate increased by 1.119%. With the additional 25 minutes lysozyme treatment, the
flow rate increased by another 1.168%; thus total change was equal to 2.287%. In comparison
to the 15 minutes lysozyme treatment, the total change after 25 minutes of lysozyme

37

S.Ly

treatment was significantly less. Even at its best, the lysozyme solution only accounted for as
much as 1.547% of the increased flow rate which is still less than the least effective trial for the
15 minutes treatment.
The 35 minutes lysozyme treatment proved to be the least effective when addressing
total change. On average, after the EDTA and lysozyme treatment, the flow rate increased by
2.079%. Despite having the lowest total change, the average percent change for lysozyme
treatment was higher than the average in the 25 minutes treatment. After being treated with
the lysozyme solution, the flow rate increased by 1.364% which is 0.196% greater than the
average in the 25 minutes lysozyme treatment. Although the 35 minutes average percent
change is not significantly greater than the average in the 25 minutes, it still demonstrates that
the lysozyme was more active during the 35 minutes time period.
All nine trials demonstrated that EDTA and lysozyme could effectively degrade the
E.coli
clogging the filter and increase the flow rate. Not only did all trials show that this combination
was effective but all nine trials also followed a polynomial trend when the flow rate was
2
graphed. Graph 1B, for example, had a correlation factor (R
) of 0.9164, meaning that the

polynomial trendline and the given equation (see graph) could be a good potential model.
Other graphs labeled with a B also exhibited similar characteristics.
Minor statistical analysis was performed on all flow rate averages. As can be seen from
the data tables, the standard deviation for most of the flow rates, at any point, were less than
one. In most cases, the standard deviation was practically zero, meaning that the data points
were very close to the calculated mean. Standard deviation was also calculated for the average
total change for each of the times. Both the 25 minutes and 35 minutes lysozyme treatments

38

S.Ly

had low standard deviations: 0.845 and 0.680, respectively. In contrast, the standard deviation
for the 15 minute lysozyme treatment, 3.071, was significantly greater. This does not mean that
the mean is completely inaccurate. It suggests that there is more variance in the data points.

Conclusion
The goal of this experiment was to successfully modify the LifeStraw water filter using
an enzyme known as lysozyme in order to increase the longevity of the filter. Because the
LifeStraw is a disposable filter, there is no cartridge that can be changed once it becomes
clogged; thus, after one year of daily use, the LifeStraw will no longer function. By introducing
lysozyme, a cell wall penetrating enzyme, the filter could potentially be cleaned and work
once again.
To test the effectiveness of lysozyme, a vacuum had to be built to replicate the
conditions necessary for the LifeStraw to function. Once a vacuum was assembled, the initial
flow rate was determined by calculating the amount of time it took for 100 mL of distilled water
to filter through. The filter was then clogged with
E.coli
poured in increments of 10 mL. When
the filter was clogged, 15 mL of the EDTA solution was introduced to the filter for 10 minutes.
After calculating the flow rate, the filter was treated with 15 mL of the lysozyme solution for 15,
25, or 35 minutes, depending on the trial. Once the lysozyme solution was flushed out of the
filter, the final flow rate was calculated.
All nine trials demonstrated the EDTA and lysozyme effectively degraded E.coli by
increasing the flow rate; thus, the hypothesis was supported. The introduction of EDTA to the
filter hindered the stability of the outer membrane of the cell wall. By degrading the LPS layer,

39

S.Ly

the main component of the outer membrane, the permeability of the wall increased. The
addition of EDTA has literally opened new passageways for lysozyme to enter. With increased
permeability, the lysozyme can access the inner layers constructed of peptidoglycan. More
importantly, the lysozymes gained access to the glycan backbone where it can break the
-1,4-glycosidic bonds formed between GlcNAc and MurNAc. By breaking these bonds and
destroying the cell wall, the
E.coli
is left with little protection and begins to die. Using the air
regulator, the dead
E.coli
was blown out of the filter; thus, increasing the flow rate.
Of the three times tested in this experiment, the 15 minute lysozyme treatment was the
most effective. Based on the results, after being treated with EDTA and the lysozyme solution
for 15 minutes, the flow rate increased by 7.391% which is significantly greater than 2.288%
and 2.079% increase from the 25 minutes and 35 minutes lysozyme treatment. The results
suggest that as time increases, the percent change decreases as does the enzyme activity.
Determining why the enzyme activity decreased over time is difficult as there are many possible
explanations.
One possibility is that there are inhibitors present that hinder the effectivity of the
enzyme. Inhibitors alter the catalytic abilities of the enzyme resulting in a decrease in the
reaction velocity. There are three types of inhibitors: competitive, noncompetitive, and
substrate inhibition. Competitive inhibition occurs when a substrate resembling the substrate is
present. Enzymes and substrates are similar to a lock and key. A false substrate is unable to
react with the enzyme; thus, enzyme activity is decreased. In noncompetitive inhibition, the
enzyme is altered in a way that it cannot accept the substrate, meaning that the reaction
cannot be completed. The last type of inhibition occurs when excessive amounts of the

40

S.Ly

substrate are present. Because so many substrate molecules are competing with one another,
the active site of the enzyme is blocked. If no substrate occupies the active site, the reaction
will not occur (Abboud et al n.d).
Inhibitors are one of the many explanations to why the enzyme activity decreased.
Another possibility is that as the reaction occurs, concentration of the substrate decreases until
only the enzyme is remaining. An enzyme acts as a catalyst by decreasing the activation time
required for the reaction to occur which increases the reaction velocity. A general enzyme
reaction can be represented as
S+EP+E
where S represents the substrate, E the enzyme, and P the product of the reaction. As can be
seen from the equation, the enzyme does not undergo any permanent change unlike the
substrate. As the time increases, more of the substrate will be used up until none is available
(Worthington 2010).
In this experiment the substrate was the -1,4-glycosidic bonds formed between
GlcNAc and MurNAc. Access to these bonds was extremely limited as it depended on the
effectivity of the EDTA treatment that was applied prior to the lysozyme treatment. As can be
seen in Graph 13, the effectivity of the EDTA decreased with each group of time trials; thus, the
lysozymes in the 25 minutes and 35 minutes trials most likely had limited interactions with the
substrate which resulted in a decrease in enzyme activity.
Despite the success of the experiment, experimental errors still occurred. These errors
led to inconsistencies in the data. One of the most important constants in this experiment was
the timing method. The timer began as soon as the water was poured into the funnel. Once the

41

S.Ly

water began to filter out in droplets rather than a continuous stream, the timer was stopped.
Determining the exact moment to stop the timer was difficult because of the variety of droplets
the LifeStraw formed. Sometimes the water would fall in long, fast droplets that appeared more
like a stream while other times each drop would fall individually. When timing in the
experiment, the timer was stopped once the drops fell individually but that was not always the
case. Minor inconsistencies in timing may have resulted in flow rates that are slightly faster or
slower. To prevent this, prior to actual experimentation, practice timing the flow rate. This
should minimize the inconsistencies later during actual experimentation, and hopefully, reduce
the error.
Other inconsistencies in the data may be a result of the volume of the EDTA and
lysozyme solution being poured into the filter. 15 mL of the EDTA and lysozyme solution was
poured into the filter during each trial. To trap the solution in the filter, not only did the vacuum
have to be turned off, but the filter had to be dislodged from the flask. Even when the vacuum
is turned off through the nozzle, the filter still feels the suction; thus it allows water to pass
through. To avoid this, the vacuum has to be released; the easiest way is dislodging it.
Dislodging the filter and laying it on its side also has another benefit. If the filter was to remain
upright, even after the vacuum is off and released, gravity would allow the solutions to pass
through. By laying the vacuum on its side, the solutions can remain trapped. Despite these
precautions, some of the 15 mL of the EDTA and lysozyme solution was still lost which may
have decreased the effectivity of the lysozyme-EDTA combination. Like when testing the flow
rate, this error can be reduced by performing practice rounds to prior to experimentation.
This experiment demonstrated that a combination of EDTA and lysozyme could help

42

S.Ly

revert the LifeStraw back to its original state. For this combination to be significant, further
experimentation must be conducted. The next step to this experiment would be to hold the
lysozyme treatment time constant and alter the EDTA treatment time. Rather than altering
time, the concentration of the enzyme or the EDTA solution could also be changed. By testing
different independent variables, hopefully an optimal combination of EDTA and lysozyme can
be discovered.
Although the current combination of EDTA and lysozyme cannot completely revert the
LifeStraw back to its original condition, it can improve the condition after an extended period of
use; therefore increase the longevity and practicality. For those living in developing countries,
the cost of the LifeStraw is still too expensive; thus, yearly replacements are impossible.
Because lysozyme is an enzyme, it can be quickly massed produced at a low cost. Rather than
replacing the LifeStraw, this solution can be used to extend its use making it more cost
effective.

43

S.Ly

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