Prelab STUDENT GUIDE

Name Gabriela Agripino Date April 21 2021

Phenomenon
Materials

Credit: NOAA
Per group:
culture tubes and caps, 4
pipets, 5
pencil
test tube rack or beaker
microbial suspension
distilled water
oil
Shared:
tetrazolium

Deepwater Horizon site

Safety
Wear goggles while
performing the procedure.
Wash your hands when
finished.
On April 20, 2010, Deepwater Horizon, an oil rig drilling in the Gulf of
Mexico, exploded and eventually sank. In addition to the tragic loss of human
life, this disaster is considered the largest accidental marine oil spill in the
history of the petroleum industry. Oil flowed for 87 days until crews finally
succeeded in capping the well, which was located approximately 5,000 feet
beneath the surface. By then, approximately 4.9 million barrels of crude
oil had been discharged into the Gulf, creating a spill area of 68,000 square
miles—roughly the size of the state of Oklahoma. As you can imagine, the
cleanup response operation was immense. Amazingly, nature itself helped
with about 50 percent of the cleanup! How is this possible?
Some microorganisms living in the ocean have specialized metabolic
pathways that enable them to use oil as food and convert it into energy.
These microbes, mostly bacteria and some fungi, biodegrade the long-chain
hydrocarbons of petroleum and chemically convert them into energy and
nutrients for their own biological processes. In doing so, they also transform
the complex structure of oil into simpler, nontoxic by-products.

Driving Question
How can microbes be used to clean up an oil spill?

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STUDENT GUIDE SG Prelab
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Background

Microscopic Oil Eaters

Scientists recognize great potential in using biodegradation to speed up the breakdown of harmful oil from spills.
The process of using living things to clean up environmental pollution is called bioremediation. Bioremediation
using oil-degrading microbes causes minimal physical disruption to the environment, permanently removes
harmful oil, is cost effective, and causes little damage to the ecosystem.
There are hundreds of species of bacteria that can degrade oil. In this simulation, you will use a microbial
suspension prepared from a product that is used as a septic system additive. When added to a septic system, the
microbes degrade oil, fats, and other carbon compounds that can clog drains and holding tanks. By setting up and
investigating biodegradation with these microbes, you can explore the general nature of oil spill bioremediation.

Stop, Read, and Think

Read the entire Procedure. Then, respond to the following questions.
1. What substance will you use to simulate an oil spill?
Distilled water and oil.
2. What is the source of the microbes you will use?
The source of the microbes will be the microbial suspension.
3. How will you know if biodegradation is taking place?
If biodegradation is taking place, the microbes will begin breaking down the tetrazolium, causing the color
of the indicator to turn pink depending on intensity.
4. What makes this activity a simulation rather than an actual environmental oil spill bioremediation experiment?
This activity is a simulation rather than an actual environmental oil spill experiment because the oil being
used is not actually crude oil.
5. Use the space below to create and fill in a graphic that shows the chemicals to be added to each tube. Refer to
your graphic while performing the procedure.

Microbial Microbial
Water Water
Suspension Suspension

Oil Oil Oil Oil

Tetrazolium Tetrazolium Water Water

Tube 1 Tube 2 Tube 3 Tube 4

6. Based on the contents of each tube, which of the tubes do you expect to turn pink, and why?
Based on the tube contents. I expect Tube 2 to turn pink because it has the source of microbes, or the suspension, and
the tetrazolium indicator, which will make it turn pink when the microbes begin biodegrading.

©2018 Carolina Biological Supply Company S-2

S-? Carolina EcoKits® Oil Spill Bioremediation
STUDENT GUIDE Prelab (continued)

Prelab Questions
1. For tubes 1 and 2, what was the experimental variable? Which tube served as the control? Why is a control
necessary?

In experiment A, tubes 1 and 2 had a 0.02% tetrazolium indicator as a variable - and its change of color would
represent microbial degradation. The purpose of tube 1 was to show microbial activity in bioremediating the oil spill
without suspension to show it.

2. Describe and interpret the results seen in tubes 1 and 2.

Experiment A resulted in Tube 1 having essentially no change - while tube 2 turned very pink in color as a
consequence of the microbial suspension, which served as a source for the microbes and caused the tetrazolium
substance breakdown

3. For tubes 3 and 4, what was the experimental variable? Which tube served as the control? Why is a control
necessary?

Experiment B, however, did not use the tetrazolium indicator at all - just the microbial suspension in tube 4, and
tube 3 with nothing but water and oil. Tube 3 was the control, and the microbial suspension was the variable
being tested.

4. Describe and interpret the results seen in tubes 3 and 4.

The result of Experiment B consisted of the control tube, 3, remaining unharmed due to its simple makeup of
just oil and water - tube 4, on the other hand, had visible changes in the composition of the oil without visual
interference of the tetrazolium, which made it turn cloudy rather than change in colors in the absence of the
indicator.

5. Based on the results of your simulation, describe two challenges that researches might face when using
biodegradation to help clean up an oil spill.

When using biodegradation to clean up an oil spill, researchers can find trouble when attempting to properly
source microbes in a proportional manner, enough to get the job done - and also when trying to clean up quickly
before the oil becomes too dispersed in the water.

©2018 Carolina Biological Supply Company S-4 Carolina EcoKits® Oil Spill Bioremediation
Laboratory Investigation
Name
Materials
culture tubes and caps, 3
pipets, 4
test tube rack or beaker
tetrazolium indicator, 6 mL
microbial suspension, 6 mL
distilled water, 6 mL
oil, 6 mL
Carolina EcoKits® Oil Spill Bioremediation
STUDENT GUIDE
Date
Now, your group will pose questions about biodegradation and test an
experimental variable to see if you can make the oil spill bioremediation
simulation even more realistic.
Stop and Think
a. Work together in your group to come up with a question that you can
test experimentally, to see how changing a physical condition of the
biodegradation experiment might affect the ability of the microbes to
degrade oil.
Does heavily mixing oil spills with large quantities of water impact the manner in
which microbes biodegrade it?
b. What is your hypothesis?
My hypothesis would be that allowing the oil to become too disperse in water
would cause these working microbes to have a harder time biodegrading it in
the large presence of another substance.
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