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Cara Broshkevitch and Anne Richards
Statistics
In the following experiment, the change in mass of the HDPE plastic will be measured. The
original mass of the pretreated plastic will be measured with an analytical balance after
exposure to UV radiation and then again after thermal radiation. After exposure to the
organisms, Phanerochaete chrysosporium fungus and the bacteria Pseudomonas putida
and Sphingomonas macrogoltabidus, the final mass will be measured, using the same
balance. The final mass will be measured after rinsing off any organismal material.
The mass change of the plastic in different situations will be compared. Originally, the plastic
will be exposed to the each organism separately. This data will be compared. Then, the
plastic will be exposed to different ratios of the organisms. This data will be compared
together and with the data found earlier.
The control groups are the amount of plastic degradation when the plastic is exposed to no
pretreatment or biodegradation, when exposed to only UV radiation, when exposed to
only thermal radiation, and when exposed to both UV and thermal radiation. Therefore,
the controls will be built into the comparison of the degradation amounts with the
organisms.
The data will be organized in a series of data tables. The data tables will be organized into a
variety of graphs.
Three data points will be needed to start analysis: each of the three trials for each of the three
organisms will be averaged together.
We will first conduct a “Before and After.” Paired comparison tests will compare plastic masses
before and after exposure to the individual organisms to determine if their degradation of
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Cara Broshkevitch and Anne Richards
the HDPE is statistically significant. We will then perform paired comparison tests on
the combinations of organisms to determine if their degradation of the HDPE is
statistically significant. An ANOVA test will then be used to evaluate which
combination of organisms results in the greatest amount of degradation of the HDPE
plastic.
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Cara Broshkevitch and Anne Richards
Experimental Design
Title: Effect of Phanerochaete chrysosporium fungus and the bacteria Pseudomonas putida and
Sphingomonas macrogoltabidus on the degradation of pretreated HDPE plastic
Problem: The purpose of this investigation will be to determine the best combination of
Phanerochaete chrysosporium fungus and the bacteria Pseudomonas putida and
Sphingomonas macrogoltabidus to degrade a maximum amount of UV and thermally
treated HDPE plastic.
Hypothesis: If the ratios of P. chrysosporium fungus, P. putida bacteria, and S. macrogoltabidus
bacteria are varied, then the maximum change in HDPE mass will be obtained.
Independent variables – Ratios of P. chrysosporium fungus, P. putida bacteria, and S.
macrogoltabidus bacteria (see pages 10-11 for ratios)
Dependent variable – Change in mass of the HDPE plastic
Constants – Type of plastic (HDPE), initial mass of plastic (1 gram), exposure time to UV
radiation (48 hours), wavelength of UV radiation (UVA-365 nm), exposure time to
thermal radiation (48 hours), temperature of thermal radiation (150°C), amount of
minimal media, containers used (plastic Petri dishes, glass Erlenmeyer flask),
temperature of culture environment (room temperature (20°C)), initial amount of
organisms placed on plastic (optical density of 1 for P. putida and S. macrogoltabidus, 1
cm by 1 cm square for P. chrysosporium)
Controls– Mass of the HDPE plastic without pretreatment or biodegradation, mass after UV
radiation, mass after thermal radiation, and mass after both UV and thermal radiation
Repeated Trials – Three trials for each combination of organisms
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Cara Broshkevitch and Anne Richards
Materials –

Organisms:
o P. chrysosporium fungal strain (comes freeze dried)

Can be obtained from ATCC (Catalog Number: 32629)

Biosafety Level: 1
o P. putida bacterial strain (comes freeze dried)

Can be obtained from ATCC (Catalog Number: 4359)

Biosafety Level: 1
o S. macrogoltabidus bacterial strain (comes freeze dried)


Can be obtained from ATCC (Catalog Number: 51380)

Biosafety Level: 1
Culturing bacteria and fungi
o Disposable, plastic, sterile, Petri dishes
o Culture test tubes
o Erlenmeyer flask
o Minimal media (M63)
o Nutrient broth
o Potato dextrose broth
o Agar
o 10 % Bleach (for sterilization)
o Paper towels
o Inoculating loop
o 95 % Alcohol (for sterilization)
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Cara Broshkevitch and Anne Richards
o Bunsen burner (for sterilization)
o Parafilm
o Incubator
o Sterile Pipette
o Optical spectrometer
o Knife

Pretreatment of plastic
o HDPE plastic (5 grocery bags)
o Distilled water
o Scissors
o Oven (capable of 150°C) (for thermal radiation)
o UVA lamp (for 365 nm UV radiation)

Measuring mass
o Analytical balance
o 0.22 filter
o Tap water
o Pipette
o Tweezers
o Autoclave
o Olympus (possibly SEM) microscope

Measuring CO2
o Vernier CO2 probe
o Enclosed container
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Cara Broshkevitch and Anne Richards
o Computer
Procedure 1.
Nine Petri dishes will be labeled: three for the P. chrysosporium fungal strain, three for
the P. putida bacterial strain, and three for the S. macrogoltabidus bacterial strain.
2.
The lab bench will be cleaned using 10% bleach and paper towels (This is where all work
will be done throughout the investigation. The Bunsen burner will be kept on throughout
all work on the lab bench. Whenever the Bunsen burner is used, a lab coat and goggles
will be needed for safety.).
3.
240 mL of nutrient broth and 250 mL of potato dextrose broth will be prepared (boiled
and then autoclaved). Agar will be added to the potato dextrose broth in a ratio of 35
grams/L (therefore 8.75 grams will be added). 10 mL of nutrient broth will be poured
into 6 culture test tubes. A small amount of potato dextrose agar will be poured into 3
Petri dishes so that it just covers the bottom of the dishes. While pouring, the lids of the
Petri dishes will be held at an angle to avoid contamination.
4.
Directions will be followed for rehydrating the freeze-dried organisms.
5.
An inoculating loop will be dipped in 95% alcohol, sterilized over a Bunsen burner, and
then allowed to cool briefly.
6.
This inoculating loop will be used to add the rehydrated bacteria to the test tube cultures.
The inoculating loop holding bacteria will be swirled in the culture test tubes 1-6. The
loop will sterilized in between samples. A 1 cm by 1 cm square of fungi will be placed
into Petri dishes 7-9. Placement of the organisms will be as follows: P. putida, tubes 1-3;
S. macrogoltabidus, tubes 4-6; and P. chrysosporium, dishes 7-9.
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Cara Broshkevitch and Anne Richards
The culture test tubes will be sealed with screw-on caps. The caps will be only loosely
tightened since the bacteria are aerobes. The Petri dishes will be closed and sealed with
Parafilm.
8.
The organisms will be left to grow in an incubator for 2 days. The P. chrysosporium
fungus will be left at 24°C, the P. putida bacteria at 26°C, and the S. macrogoltabidus
bacteria at 30°C.
9.
The three organisms will then be subcultured throughout the investigation. Every 2
weeks, new cultures will be established. After the new cultures have been established,
10% bleach will be added to old aqueous broth-organism solutions overnight and then
poured down the drain. Old agar-organism mixtures will be autoclaved at 121°C and a
psi of 15 for 15 minutes.
10.
After two days, a sterile pipette will be used to move the contents of culture test tube 1
(holding nutrient broth and P. putida bacteria) into the spectrophotometer for the optical
spectrometer (this will be done on the lab bench sterilized with 10% bleach and paper
towels). An optical density reading will be taken.
11.
The contents of culture test tube 1 will be diluted with nutrient broth until an optical
density of 1 is obtained.
12.
Step 11 will be repeated for culture test tubes 2-6 (holding nutrient broth and bacteria).
13.
A knife will be dipped in 95% alcohol, sterilized over a Bunsen burner, and then allowed
to cool briefly.
14.
A 1 cm by 1 cm square will be cut from Petri dishes 7-9 (containing potato dextrose agar
and P. chrysosporium fungi). For the rest of the investigations trials, fungi in a 1 cm by 1
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Cara Broshkevitch and Anne Richards
cm square will be assumed to keep a relatively constant mass as long as a square with
dense fungi but no spores is chosen.
15.
Meanwhile, nine grams of HDPE plastic will be cut from a grocery bag in 1-gram
increments. These 1-gram samples of HDPE plastic will be measured using the
analytical balance and their amount adjusted to be exactly 1-gram each.
16.
Each of the nine HDPE samples will then be exposed to UV radiation with a wavelength
of 365 nm for 48 hours using a UVA lamp.
17.
Each of the nine samples of HDPE will be massed using the analytical balance.
18.
Each of the nine HDPE samples will then be exposed to thermal radiation at 150°C for 48
hours using an oven.
19.
Each of the nine HDPE samples of plastic pretreated with both UV and thermal radiation
will be massed using the analytic balance.
20.
As a control, step 15 will be repeated. Then step 18 will be repeated without any
previous exposure to UV radiation. Each sample will be massed using the analytical
balance to determine if thermal radiation causes further plastic degradation.
21.
250 mL of M63 minimal media will be prepared (boiled and then autoclaved) on the lab
bench cleaned with 10% bleach and paper towels) and a small amount poured into 9 Petri
dishes so that it just covers the bottom of the dishes. Agar will be added at a ratio of 35
g/mL (therefore 8.75 grams will be added) to the M63 before being poured into dishes 79 (for the fungus to grow on). While pouring, the lids of the Petri dishes will be held at
an angle to avoid contamination. Whenever handling the minimal media, it is necessary
to wear gloves, goggles, and a lab coat to reduce the risk of skin and eye irritation.
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Cara Broshkevitch and Anne Richards
An inoculating loop will be dipped in 95% alcohol, sterilized over a Bunsen burner, and
then allowed to cool briefly.
23.
The bacteria will be swirled in the M63 contained in Petri dishes 1-6. The 1 cm by 1 cm
samples of potato dextrose agar and fungi will be placed in Petri dishes 7-9. The P.
putida bacteria and S. macrogoltabidus bacteria will have an optical density of 1, and the
P. chrysosporium fungus will be in 1 cm by 1 cm samples. Placement of the organisms
will be as follows: P. putida, dishes 1-3; S. macrogoltabidus, dishes 4-6; and P.
chrysosporium, dishes 7-9.
24.
Each of the nine HDPE samples will be added to one of the nine Petri dishes (P. putida,
dishes 1-3; S. macrogoltabidus, dishes 4-6; and P. chrysosporium, dishes 7-9).
25.
The Petri dishes will be closed and sealed with Parafilm.
26.
The HDPE plastic samples will be exposed to the organisms for 14 days. After 14 days,
we will examine the plastic samples under the Olympus (possibly SEM) microscope to
look for visible damage to the plastic. If there is damage, we will proceed with the
following steps. If not, we will continue plastic exposure to organisms, checking for
damage once a week.
27.
The Olympus microscope will be used to take pictures of the HDPE biodegraded
samples.
28.
After this time, a pipette will be used to gently rinse the HDPE plastic samples with tap
water over a 0.22 filter. The plastic samples will be held over the filter using tweezers.
29.
A spatula will be used to scrape off as much bacteria as possible adhering to the plastic
samples.
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Cara Broshkevitch and Anne Richards
The bacteria and fungus will be caught using this filter, as they are too large, and
disposed of in an Autoclave at 121°C and a psi of 15 for 15 minutes. 10% bleach will be
added to the M63 overnight and then poured down the drain. The plastic will be labeled
and saved, but treated as if it is bacteria (since not all of the bacteria probably will
probably be scraped off the HDPE). Eventually, the plastic will be autoclaved at 121°C
and a psi of 15 for 15 minutes.
31.
The plastic will then be dried and the mass of each HDPE sample measured using the
analytical balance. To find the change in mass, the final mass of the plastic will be
subtracted from the mass of the plastic measured after pretreatment. Each of these
changes in mass will be averaged from the three trials for each strain (P. putida, dishes 13; S. macrogoltabidus, dishes 4-6; and P. chrysosporium, dishes 7-9) to obtain the
experiment’s controls.
32.
Steps 1-8, 10-19, 21-31 will then be repeated using different combinations of P.
chrysosporium fungus, P. putida bacteria, and S. macrogoltabidus bacteria with three
trials each. Combinations of the three organisms will include the following:
i.
(1/2) P. chrysosporium, (1/2) P. putida
ii. (1/2) P. chrysosporium, (1/2) S. macrogoltabidus
iii. (1/2) P. putida, (1/2) S. macrogoltabidus
iv. (1/4) P. chrysosporium, (3/4) P. putida
v. (3/4) P. chrysosporium, (1/4) P. putida
vi. (1/4) P. chrysosporium, (3/4) S. macrogoltabidus
vii. (3/4) P. chrysosporium, (1/4) S. macrogoltabidus
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Cara Broshkevitch and Anne Richards
viii. (1/4) P. putida, (3/4) S. macrogoltabidus
ix. (3/4) P. putida, (1/4) S. macrogoltabidus
x. (1/3) P. chrysosporium, (1/3) P. putida, (1/3) S. macrogoltabidus
33.
Further combinations may then be added based on any observed patterns of symbiosis
between the organisms.
34.
A mini side investigation will then be conducted to measure the amount of CO2 released
by the organisms and determine if they actually grow and flourish with HDPE as their
carbon source. First, directions will be followed for rehydrating the freeze dried
organisms.
35.
250 mL of M63 media will be boiled and autoclaved. Then on a lab bench, M63 minimal
media will be poured into 2 Erlenmeyer flasks so that it just covers the bottom.
36.
1 cm by 1cm squares of HDPE plastic pretreated with UV and thermal radiation will be
placed into both Erlenmeyer flasks.
37.
P. putida with an optical density of 1 will be added to the second Erlenmeyer flask.
38.
A Vernier CO2 probe will be placed in each of the Erlenmeyer flasks and attached to a
computer.
39.
The CO2 level in the enclosed container will be measured for 2 days.
40.
After this time, a pipette will be used to gently rinse the HDPE plastic samples with tap
water over a 0.22 filter. The plastic samples will be held over the filter using tweezers.
41.
A spatula will be used to scrape off as much bacteria as possible adhering to the plastic
samples.
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Cara Broshkevitch and Anne Richards
The bacteria or fungus will be caught using this filter and disposed of in an Autoclave at
121°C and a psi of 15 for 15 minutes. 10% bleach will be added to the M63 overnight
and then poured down the drain. The plastic will be labeled and saved, but treated as if it
is bacteria (since not all of the bacteria probably will probably be scraped off the HDPE).
Eventually, the plastic will be autoclaved at 121°C and a psi of 15 for 15 minutes.
43.
Steps 35-42 will be repeated for S. macrogoltabidus and P. chrysosporium. The S.
macrogoltabidus will have an optical density of 1. A 1 cm by 1 cm square of fungi will
be used and agar will be added at a ratio of 35 grams/L to the M63. The first trial will be
only HDPE plastic and minimal media and the second trial would be HDPE plastic and
minimal media exposed to the organisms.
References –
Ammala, A., Bateman, S., Dean, K., Petinakis, E., Sangwan, P., Wong, S., Yuan, Q., Yu, L.,
Patrick, C., & Leong, K.H. (2011). An overview of degradable and biodegradable
polyolefins. Progress in Polymer Science, 36, 1015-1049.
doi:10.1016/j.progpolymsci.2010.12.002
Arkatkar, A., Arutchelvi, J., Sudhakar, M., Bhaduri, S., Uppara, P.V., & Doble, M. (2009).
Approaches to enhance the biodegradation of polyolefins. The Open Environmental
Engineering Journal, 2, 68-80.
CDC/Office of safety, health, and environment. (2010, November 10). Biosafety in
microbiological and biomedical laboratories (BMBL) 5th edition. Retrieved from
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
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Shah, A., Hasan, F., Hameed, A., & Ahmed, S. (2008). Biological degradation of plastics: A
comprehensive review. Biotechnology Advances, 26, 246-265.
doi:10.1016/j.biotechadv.2007.12.005
Sivan, A. (2011). New perspectives in plastic biodegradation. Science Direct. doi:
10.1016/j.copbio.2011.01.013
Thraves, P., & Packer, R. (April 2009). Material safety data sheet. Retrieved from
http://www.hpacultures.org.uk/media/DF9/A0/Growing_Cultures_MSDS.pdf