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Experimenting with microbial degradation of petroleum

Experimenting with microbial degradation of petroleum

Introduction: (Initial Observation)

Petroleum based pollutants are widely distributed and have polluted many oceans, soils and underground waters. Because of long term health and environmental hazards that such pollutants can produce, many scientists are studying on methods of removing or destroying such pollutants.

In this project you study the effect of microbes in degradation and break down of petroleum products. You may also measure the rate of such effects on different petroleum products.


If you have any questions, click on the help button at the top of this page to send me your questions. I may respond by email, but often I update this page with the information that you need.

Project Advisor

Information Gathering:

Find out about oil spills and oil spill treatment methods. Read books, magazines or ask professionals who might know in order to learn about the effect or area of study. Keep track of where you got your information from.

Oil is the most common pollutant in the oceans. More than 3 million metric tons 1 of oil contaminate the sea every year.

Oil spills occur in oceans, lakes, and rivers all over the world. Some of these spills are large and very damaging to nature while some are small and occur regularly.

When an oil spill occurs, the oil floats on top of the water. This is because the oil is less dense than the water. Density is a measure of how much mass an object contains for a specific volume.

The extent of damage a spill does to the environment is related to the oil type, size of the spill, weather conditions, location of the spill, and the timing of the spill.

Winds, tides, and waves will all have an effect on where the oil goes and how much damage it does.

The oil from a spill can effect the marine environment by:

  • coating the substrate and organisms living on nearby shorelines with oil.
  • evaporating into the atmosphere to become an aerosol that is transported by the wind.
  • breaking into smaller particles in the water that are ingested by zooplankton and small fishes and enters the food chain.
  • coating the fur and feathers of mammals and birds, destroying their insulating ability reducing buoyancy, and harming the animals when the oil is ingested as they clean themselves. Animals die from exposure, drowning, suffocation, or from ingestion of oil.

The majority of oil pollution in the oceans comes from land. Runoff and waste from cities, industry, and rivers carries oil into the ocean. Ships cause about a third of the oil pollution in the oceans when they wash out their tanks or dump their bilge water.

Oil spills account for less than 15% of the total oil in the oceans but are probably the most obvious form of oil pollution. The damage caused by oil spills is certainly seen right away.

We’ve all seen images of the water’s surface and shoreline covered with oil and dying animals and plants. Oil spills will continue to be problem and source of pollution as long as ships and barges move most of our petroleum products around the world.

When oil leaks or spills into water it floats on the surface of both freshwater and saltwater. Oil floats because it is less dense than water. Density is a property of every liquid, solid, and gas. Density tells how much mass is in a specific volume (mass divided by the volume) of a material.

You may have seen this written as an equation: Density = mass / volume

It may help to think of density as the relative heaviness of a material or how compact or crowded the molecules are in the material. Denser materials have larger number values than less dense materials. For example, oil has a density of 0.85 g/cm3 and seawater has a density of 1.02 g/cm 3 .

When these two liquids are mixed together, the denser seawater forms a layer underneath the less dense oil.

It’s much easier to clean-up an oil spill because of oil’s lower density. You can imagine how difficult it would be to clean-up a spill if oil was denser than water and formed a layer along the bottom instead of the surface. We humans have come up with some pretty creative ways to clean-up spilled oil and we’ve described some of the major methods below.


Americans primarily use mechanical methods to clean-up oil spills. Listed here are the three categories of mechanical tools used to contain and recovery spilled oil.

4. Booms– It’s easier to clean-up oil if it’s all in one spot, so equipment called containment booms act like a fence to keep the oil from spreading or floating away. Booms float on the surface and have three parts: a ‘freeboard’ or part that rises above the water surface and contains the oil and prevents it from splashing over the top, a ‘skirt’ that rides below the surface and prevents the oil from being pushed under the booms and escaping, and some kind of cable or chain that connects, strengthens, and stabilizes the boom. Connected sections of boom are placed around the oil spill until it is totally surrounded and contained.

5. Skimmers– Once you’ve contained the oil, you need to remove it from the water surface. Skimmers are machines that suck the oil up like a vacuum cleaner, blot the oil from the surface with oil-attracting materials, or physically separate the oil from the water so that it spills over a dam into a tank. Much of the spilled oil can be recovered with skimmers. The recovered oil has to be stored somewhere though, so storage tanks or barges have to be brought to the spill to hold the collected oil. Skimmers get clogged easily and don’t work well on large oil spills or when the water is rough.

6. Sorbents– These are materials that soak up liquids by either absorption or adsorption. Oil will coat some materials by forming a liquid layer on their surface (adsorption). This property makes removing the oil from the water much easier. This is why hay is put on beaches near an oil spill or why materials like vermiculite are spread over spilled oil. One problem with using this method is that once the material is coated with oil, it may then be heavier than water. Then you have the problem of the oil-coated material sinking to the bottom where it could harm animals living there. Absorbent materials, very much like paper towels, are used to soak up oil from the water’s surface or even from rocks and animal life on shore that becomes coated with oil.


Chemicals, such as detergents, break apart floating oil into small particles or drops so that the oil is no longer in a layer on the water’s surface. These chemicals break up a layer of oil into small droplets. These small droplets of oil then disperse or mix with the water. The problem with this method is that dispersants often harm marine life and the dispersed oil remains in the body of water where it is toxic to marine life.


7. Burning– Burning of oil can actually remove up to 98% of an oil spill. The spill must be a minimum of three millimeters thick and it must be relatively fresh for this method to work. There has been some success with this technique in Canada. The burning of oil during the Gulf War was found not as large a problem as first thought because the amount of pollution in the atmosphere did not reach the expected high levels. Field-testing is needed to check the feasibility of this technology.


1. Bioremediation– There are bacteria and fungi that naturally break down oil. This process is usually very slow- it would take years for oil to be removed by microorganisms. Adding either fertilizer or microorganisms to the water where the spill is located can speed up the breakdown process. The fertilizer gives the bacteria and fungi the nutrients they need to grow and reproduce quicker. Adding microorganisms increases the population that is available to degrade the oil. A drawback to adding fertilizers is that it also increases the growth of algae. When the large numbers of algae die they use up much of the oxygen so that there isn’t enough oxygen in the water for animals like fish.

Over time, a number of things can happen to oil that has been spilled. The oil may evaporate, reach the shore and cover beaches, remain suspended in the water for long periods, or sink into ocean sediments. The problem of cleaning up oil often becomes more difficult the longer the oil is in the water.

Oil spills can happen anywhere and anytime. The paragraph below tells about an oil spill that happened in New England when a tanker ran aground.


Pollutant: Any substance that contaminates or makes the environment impure. Pollutants are commonly man-made wastes.

Density: The mass or quantity of a substance per unit of volume (m/V). The relative heaviness of a material or how compact or crowded the molecules are in the material.

Absorption: The process of taking in another substance, in the same manner that a sponge would.

Adsorption: When a liquid or solid takes up a substance and holds it on its surface, so that the substance coats the molecules of the solid or liquid.

Dispersant: A chemical or material that when added to some other substance causes it to break apart and scatter about.

Bioremediation: Using natural biological processes to correct or counteract an environmental hazard or ecological disaster. An example of bioremediation is adding fertilizer or bacteria to the water to help clean-up an oil spill.

Ecosystem: An ecological unit of all the living organisms plus the nonliving, physical environment and how they function together.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

Can soil bacteria break down oil? How long does it take for bacteria to break down or digest oil? What is the rate of degradation?

The purpose of this project is to determine the effect of chemicals and soil bacteria in reducing the concentration of oil in a polluted water.

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

The independent variable is the period of time that oil is exposed to bacteria.

The dependent variable is the rate of degradation or reduction in oil.

Controlled variables are temperature, type and amount of bacteria, type and concentration of oil in water.


Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.

Some soil bacteria are able to degrade oil and other large molecule pollutants. My hypothesis is based on my gathered information and previous observation that oil spots on the soil disappear after a few months.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Following is a sample experiment to determine the effect of bacteria on oil degradation.

Experiment 1:


Oil released into the environment is a well-recognized problem in today’s world. Oil spills affect many species of plants and animals in the environment, as well as humans. The search for effective and efficient methods of oil removal from contaminated sites has intensified in recent years, in part due to the enormous publicity of the Exxon Valdez spill. One promising method that has been researched is the biological degradation of oil by bacteria. The bacteria metabolize the oil in much the same way humans convert food into energy. Like food, oil is a compound rich in carbon. The following experiment can be used to demonstrate that some types of bacteria can degrade oil. Also, other variables can be selected and incorporated into the lesson plan to allow additional experimentation.


  1. This experiment includes 4 treatments, each done in duplicate. Therefore, label the jars 1A, 1B, 2A, 2B, etc., up to 4B. The jars with the same numerical markings (for example 1A and 1B) will contain identical treatments. The jars should be clean.
  2. Put 150 ml distilled water and 2 grams of machine oil into each of the 8 jars.
  3. The first set of 2 jars (1A and 1B) will contain only distilled water and oil and will serve as controls. Set them aside.
  4. Add into the second set of jars the following mixture of inorganic nutrients: 0.25 g ammonium phosphate; 0.05 g magnesium sulfate; 0.25 g potassium phosphate; 1.25 g non-iodinated sodium chloride. The inorganic nutrients provide nitrogen and minerals to the organisms.
  5. Add into the third set of jars the soil sample. The best place to collect soil is from an area already contaminated with oil. That way it is likely that the surviving bacteria in the sample may be representative of oil-degrading species. One suggestion is to collect bacteria from where engine oil has repeatedly leaked on the ground for several years (service stations, dirt parking lots, etc.). If you don’t have access to such a place, soil taken from an organically rich area may be substituted, since oil-degrading bacteria are generally present in most soils. Approximately 5 g of the soil from your chosen area should be added to each of the two jars for this treatment.
  6. Into the fourth and final set of two jars, add both the inorganic nutrients and the soil sample as described in steps 4 and 5.
  7. Cover the top of the jars loosely with inverted Petri dish halves in order to reduce evaporation of the water and oil in the treatments (aluminum foil can be substituted). The dish halves should have a hole drilled in them to allow a piece of tubing to reach into the water and bubble up air from a small aquarium pump. (If you use 4-way splitters, only 2 pumps will be needed for the 8 jars.)
  8. Results may be recorded from each jar every 3 days or weekly for up to 30 days. Take results by performing a “greasy spot” test. To perform this test, cut a brown paper bag into a 16 x 16-inch square. Then, with a ruler and pencil, divide the large square into eight 2 x 2-inch squares. In the corner of each small square, put the number of each different treatment jar (1A, 1B, etc.), one per square. This way, there should be one 2 x 2-inch square for each jar.
    Using a pipette or dropper, draw a small quantity of liquid from just under the top of the water level of one jar. Deposit three drops of this liquid onto the center of the correct square of paper (if the sample was from jar 1A, place the three drops on the square for 1A, etc.). Take samples from the same place in each jar each time (just under the surface of the water. After a few hours, the water will evaporate, leaving a greasy spot in each small square. Circle the circumference of each greasy spot with a pencil and measure and record the diameter of each spot. Average the results from jars containing identical treatments. During the course of the experiment, the spots from the treatments containing the bacteria and inorganic nutrients should be smaller since the oil is being degraded.
  9. Place your cumulative data into graphs and compare. What trends do you find, if any? Where did activity plateau? Why were the soil sample, inorganic nutrients, oxygen, etc. necessary for the oil concentration to be decreased?



As previously stated, the soil sample provides bacteria to digest the oil. However, oil is composed only of hydrogen and carbon, and the bacteria need additional nutrients to grow. The inorganic nutrient mixture provides nitrogen and several essential minerals. The bacteria also require oxygen, provided by the air from the aquarium pumps.

The 1A and 1B jars will show if oil in presence of water may decompose by itself.

The 2A and 2B jars will show if nutrients alone are able to decompose oil and reduce the amount of oil.

The 3A and 3B jars will show if bacteria alone (without nutrients) can grow and decompose oil.

The 4A and 4B jars will show if bacteria with nutrients are able to grow and decompose oil.

Other experiments:

The above experiment is just an example. You can propose other questions, define other variables and state a different hypothesis.

One good experiment is to determine the effect of air in biodegradation of oil. In this case your experiment will be very similar to the above experiment. Time period will be a controlled variable. In other words you check all your samples after 10 days. Air will be an independent variable. In other words two of the samples get no air, two others get little air, two others get more air.

Another good experiment is finding the effect of temperature. We know that most bacteria grow best in warm temperatures (around body temperature). So you will need to test different temperatures from 5ºC up to 50ºC.

Finally you may study the effect of different soil types, different bacteria or different enzymes in breaking or reducing oil molecules.

Materials and Equipment:

  • eight 1-pint Mason jars
  • aquarium pumps and tubing
  • inorganic nutrients: ammonium phosphate, magnesium sulfate, potassium phosphate, and non-iodinated sodium chloride
  • lightweight machine oil (not motor oil; available at hardware stores)
  • pipettes that can deliver drops or reproducible small volumes
  • distilled water
  • brown paper bags
  • laboratory balance
  • soil sample (preferably collected from an oil-contaminated site); will contain soil bacteria

Where to get the materials?

If you do not have local contacts who can provide you with small amounts of free samples, then you will have to purchase them locally or order them online. To purchase locally contact a local scientific supplier. To order online you may search the Internet or try the following websites.

1. www.ChemicalStore.com

2. www.ScienceKitStore.com

3. www.ScienceProjectStore.com

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.


You will need to calculate the average

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.


Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.