GOLD

Bioremediation of Controlled Oil Spill with Microbes

In my project I will test if changing amounts of motor oil and nutrients in a controlled fresh water oil spill with added microbes will affect the amount and speed the oil will be bioremediated.
Keira Van Vliet
Grade 7

Hypothesis

Testable Question

How does varying the amount of nutrients and motor oil in a controlled fresh water oil spill with added microbes affect bioremediation of the oil?

Hypothesis

Phase #1 – Group 1 – Varying Amounts of Motor Oil

  • Lowest Motor Oil (2.5mL): I think this amount of motor oil will be completely bioremediated within the two-week period. From my research, bioremediation is most successful when as much source contaminant and free oil is removed.  
  • Medium Motor Oil (5mL): I think this amount of motor oil will only be partially bioremediated by the time the two-week period is up. The thicker oil column will mean the bioremediation will take longer because there is more oil to consume and because it is harder for the microbes to access the top of the oil layer.
  • Highest Motor Oil (8mL): I think this amount of motor oil will also only be partially bioremediated and even more oil will be remaining than in the medium test.

Phase #1 – Group 2 – Varying Amounts of Nutrients

  • Lowest Nutrients (1g): I think that this amount of nutrients will not do much to stimulate microbial growth and contaminant removal and would have similar results to the controls with zero nutrients.
  • Medium Nutrients (2g): I think that this amount of nutrients will have the most bioremediation because I think this amount will not be toxic to the microbes but will still stimulate bioremediation and microbe reproduction.
  • Highest Nutrients (3g): From my research, I know that high amounts of nutrients may kill the microbes but I am not sure what ratio will be toxic. This nutrient is the highest of my tests so I think it is most likely to have toxic effects.

Phase #2 – Pond Water – Varying Amounts of Nutrients

  • No Nutrients (0g): I think that no nutrients will still have a high level of bioremediation based on my phase 1 test.
  • Medium Nutrients (1.5g): I think that this amount of nutrients will not have the least amount of bioremediation based on results from my phase 1 test.
  • Highest Nutrients (3g): I think that this amount of nutrients will have the highest level of bioremediation. Phase 1 showed that this amount of nutrients is not toxic and stimulates microbial reproduction and bioremediation. 

Research

This is only a summary of research completed before and during this experiment. More research details are in the logbook and science fair binder.

Hydrocarbons

Crude oil is unrefined petroleum, "Crude oil, also known as petroleum, is an energy-rich liquid consisting mainly of hydrocarbons." (Government of Canada)

"Petroleum, also called crude oil, is a fossil fuel. Like coal and natural gas, petroleum was formed from the remains of ancient marine organisms, such as plants, algae, and bacteria. Over millions of years of intense heat and pressure, these organic remains (fossils) transformed into carbon-rich substances we rely on as raw materials for fuel and a wide variety of products." (National Geographic)

Oil Spills

It is important that when oil spills happen that they are cleaned up in the most effective way that is done with consideration to the environment. According to the EPA “Oil spills occurring in freshwater bodies are less publicized than spills into the ocean even though freshwater oil spills are more frequent and often more destructive to the environment.” It is important to properly clean the site of oil as we source our drinking water from many freshwater bodies of water, the water bodies and their surroundings are used as nesting grounds and food sources for many biotic freshwater organisms. All freshwater organisms and vegetation are vulnerable to the deadly and toxic effects oil spills can have on them and their environment. According to the EPA “Standing water such as marshes or swamps with little water movement are likely to incur more severe impacts than flowing water because spilled oil tends to "pool" in the water and can remain there for long periods of time.” It is even more important to properly clean the areas because it could save lives for all of the creatures living in the area.

“Spilled oil can harm living things because its chemical constituents are poisonous. This can affect organisms both from internal exposure to oil through ingestion or inhalation and from external exposure through skin and eye irritation. Oil can also smother some small species of fish or invertebrates and coat feathers and fur, reducing birds' and mammals' ability to maintain their body temperatures.” (Office of Response and Restoration)

The Process of Oil Breakdown

Biodegradation is a natural process that occurs in the environment.

 “Bioremediation is an engineered technique applied by humans to clean the environment.” (The Biology Notes)

  • The final by-products of bioremediated oil are carbon dioxide, water, trace carbon, and lipids (which are white fatty acids). The lipids are a source of food for larger organisms to survive. (UltraTech)

Aerobic biodegradation is done by aerobic microorganisms when an adequate supply of oxygen is available for their activity.

  • It is a rapid method that degrades the contaminants completely when compared to anaerobic biodegradation.

Anaerobic biodegradation takes place in the absence of oxygen. Its pathway has four major steps:

  • Hydrolysis
  • Acidogenesis
  • Acetogenesis
  • Methanogenesis

Organic substances are subjected to anaerobic digestion and converted into carbon dioxide and methane.   

Oil Eating Microbes

 “Oleophilic microbes (Oil Eating Microbes -OEM's) are normally found in marine environments.” (Edvotek)

OEMs eat hydrocarbons (compounds that make up crude oil) Microbes separate hydrocarbons into fatty acids which are split into carbon atoms that have been reused by the microbes in metabolism. Oil is broken down into metabolites, carbon dioxide, trace carbon, lipids and water.    

The 6 conditions that affect microbe growth.

  • Reservoir. Environment where most microbes grow.
  • Food. Water and nourishment.
  • Oxygen. Most need oxygen to survive.
  • Darkness. Warm and dark environments is needed.
  • Temperature. Most grow best at body temperature.
  • Moisture. Grow well in moist places.

Some scientific studies have been done proving that when you add fertilizer in the right ration it has effective effects to stimulate microbial growth as well as bioremediation. Although in some tests there have been toxic effects on the microorganisms observed when there is to much fertilizer added. This is one of the main reasons why I wanted to test fertilizer because I wanted to see how much fertilizer the microbes can handle as well as if the fertilizer stimulates microbial growth and bioremediation.

Definitions 

  • Biodegradation: Biodegradation is the process by which microorganisms break down/decompose substances (mainly organic matter) in simpler substances such as water and carbon dioxide. Bioremediation is an important process because it replenishes the area the microbes are in with nutrients. Microorganisms degrade the matter for their growth and metabolism. In the example of Oil Eating Microbes degrading the motor oil in my experiment, the microbes bioremediated many compounds of the crude oil straight chain, branched-chain, and six-membered ring hydrocarbons. When the microbes break down the crude oil, they separate the hydrocarbons into lipids (fatty acids). The carbon atoms are then removed from the lipids, and the microbes use them in their metabolism. The remaining components of the lipids are consumed by other small organisms in the site. 
  • Bioremediation:  Bioremediation is very similar to biodegradation this table which I summarized is from the Biology Notes it clearly summarizes the differences between biodegradation and bioremediation.
  • Lipids: Fatty Acids or by-products that are insoluble in water but soluble in organic solvents. (Oxford Languages)
  •  Microbes: Microbes are minuscule organisms that are found everywhere. Most are to small to be seen with the unaided eye. Microbes live in the air, water, and soil. 
  • Oxidization: Oxidization is when a substance/state of matter becomes combined or chemically with oxygen.

Biodegradation vs Bioremediation

   

Biodegradation is the process of decomposing organic materials in the environment by microorganisms.

Bioremediation is a waste management technique that uses biological agents to clean the contaminants in the environment.

   
   
   
   
   
   
   

Nature of the Process

   

It is a natural process that happens without human intervention.

It is an engineered process that happens with human intervention.

   
   
   
   
   

Speed

   

Slower Process

Faster Process

   

Control

   

Controlled by Nature

Controlled Process

   
   

Effects

   

It is both beneficial and harmful.

It always has beneficial effects.

   
   

Time and Location

   

It happens everywhere in the environment.

It happens at the contaminated site or in a laboratory for experiments.

   
   
   

 

Variables

Control 

  • Type and size of test tubes 
  • Type of microbes (except controls) 
  • Same type and size of plastic covered magnetic stir stick (Phase 1: 4cm/Phase 2: 3.25cm)
  • Same process to culture microbes
  • The same type of un-used motor oil
  • Same temperature of room / heat mat
  • The same number of rotations of test tubes per day (Phase 1: 5 clockwise rotations / Phase 2: 15 seconds)
  • Same amount and type of distilled water (30mL)
  • Phase 2: Same type and amount of pond water collected from clean end of pond (30mL)

Manipulated

  • Phase #1: Group 1: Amount of motor oil (2.5mL, 5mL, 8mL)
  • Phase #1: Group 2: Amount of nutrients (fertilizer 1g, 2g, 3g)
  • Phase #2: Pond Water: Amount of nutrients (fertilizer 0g, 1.5g, 3g)

Dependent 

  • Increase/decrease of oil bioremediation (height/colour of oil column)

Procedure

Materials List

Procedure

Phase #1

Activate Microbes

  1. Calibrate scale.
  2. Wash Hands.
  3. Wash equipment with soap and water and triple rinse.
  4. Put on nitrile gloves, goggles, apron, and tie hair back.
  5. Add 2g of Oil Eating Microbe (OEM) powder to 1,000mL of cold tap water into a 1,000mL beaker.
  6. Place 4cm plastic-coated magnetic stir bar inside of the beaker.
  7. Cover opening of the beaker with tin foil.
  8. Place flask on a magnetic stirrer set at 200rpm (rotations per minute) and let stir for 24 hours or until cloudy Source of error because my stirrer does not show the speed, so it is approximately 200rpm.

After 24 hours Initiate Phase #1

  1. Place 30 test tubes into stands.
  2. Label each test tube. 1A-1O and 2A-2O.
  3. Pour 30mL of distilled water into 30 test tubes.
  4. Dissolve 2g of nutrients into 15 test tubes – fertilizer 20-8-8 (1A through 1O).
  5. Pour 5mL of motor oil into the other 15 test tubes (2A through 20).
  6. Pour 2.5 mL of motor oil into test tubes 1A, 1G, 1H, and 1I.
  7. Pour 5mL of motor oil into test tubes 1B, 1J, 1K, and 1L.
  8. Pour 8mL of motor oil into test tubes 1C, 1M, 1N, and 1O.
  9. Dissolve 1g nutrients (approximately 1/4 tsp) into test tubes 2A, 2G. 2H, and 2I.
  10. Dissolve 2g nutrients (approximately ½ tsp) into test tubes 2B, 2J, 2K, and 2L.
  11. Dissolve 3g nutrients (approximately ¾ tsp) into test tubes 2C, 2M, 2N, 2O.
  12. To test tubes 2D – 2O and 1D-1O add 25mL of microbe mixture (do not stop the stirring process until ready to pour inside test tubes).

*Test tubes 1A-1C should only have 30mL distilled water, 2g nutrients, 25mL microbe mixture, and variations of motor oil (2.5 1A, 5 1B, and 8 1C), and test tubes 2A, 2B, and 2C. should only have 30mL distilled water, 5mL motor oil, 25mL microbe mixture, and variations of fertilizer (1 2A, 2 2B, and 3 2C).

  1. Using an mm ruler measure the amount of motor oil per test tube and right down in the chart, does not include cloudy white bioremediated oil.
  2. Place the test tubes on a seed-starting mat set at approximately 26 degrees C.
  3. Each day for 14 days measure the amount of oil and any observations that you may have.
  4. Each day shake the test tubes for 15 seconds in all directions to represent the water current.
  5. On the 14th day after finishing your observations soak the microbes in 10% bleach and 90% water for 30 minutes then dispose of in the garbage.
  6. Disinfect all surfaces and equipment.
  7. Dispose of gloves after each use and thoroughly wash hands.

Phase #2

Collect the Pond Water

  1. Have parent drive out to pond site. In this instance the pond was located on my Grandparent’s acreage in West Bragg Creek.
  2. Make sure ice is thick enough to walk on before going on ice.
  3. Use ice auger to auger down through ice.
  4. Clean out snow and ice using a soup ladle with strainer.
  5. Let pond water settle prior to collecting the sample.
  6. Use a clean yogurt container to scoop pond water up through ice hole and into larger bucket for storage. Collect multiple ice cream buckets full of water.
  7. Let water sit in house to warm up to room temperature prior to conducting experiment.

Activate Microbes

  1. Calibrate scale.
  2. Wash Hands.
  3. Wash equipment with soap and water and triple rinse.
  4. Put on nitrile gloves, goggles, apron, and tie hair back.
  5. Add 1g of Oil Eating Microbe (OEM) powder to 500mL of cold tap water (that has been set out to sit for a couple of hours) into a 1,000mL beaker.
  6. Place 4cm plastic-coated magnetic stir bar inside of the beaker.
  7. Cover opening of the beaker with tin foil.
  8. Place the beaker on a magnetic stirrer set at 200rpm (rotations per minute) and let stir for 24 hours or until cloudy *Source of error because my stirrer does not show the speed, so it is approximately 200rpm.

After 24 hours Initiate Phase #2

  1. Place 13 test tubes into stands.
  2. Label each test tube: 3A to 3M.
  3. Pour 30mL of pond water into 13 test tubes.
  4. Dissolve 1.5g of nutrients (fertilizer 20-8-8) into test tubes 3H, 3I & 3J.
  5. Dissolve 3g nutrients (approximately ¾ tsp) into test tubes 3K, 3l & 3M.
  6. Add 25mL of microbe mixture to all test tubes except 3A & 3B. (do not stop the stirring process until ready to pour inside test tubes).
  7. Using a pipette measure out and add 2.5mL of motor oil to all test tubes except 3C & 3D.

** Here is what the test tubes should have in them up to this point:

  • All: 30mL pond water (except control #1), 25ml microbes (except control #1), 2.5mL motor oil (except control #2)
  • Control #1 x2 (3A and 3B): No Microbes, 55mL pond water, 2.5mL oil
  • Control #2 x2 (3C and 3D): No Oil, 25mL microbes, 30mL pond water
  • Test #1 (3E, 3F & 3G): 0g nutrients
  • Test #2 (3H, 3I, & 3J): 1.5g nutrients
  • Test #3 (3K, 3L, & 3M): 3g nutrients

  1. Using an mm ruler measure the amount of motor oil per test tube and right down in the chart, does not include cloudy white bioremediated oil.
  2. Place the test tubes on a seed-starting mat set at approximately 26 degrees C.
  3. Each day for 7 days measure the amount of remaining motor oil and any observations that you may have.
  4. Each day swirl the test tubes for 15 seconds in all directions to represent the water current.
  5. On the 14th day after finishing your observations soak the microbes in 10% bleach and 90% water for 30 minutes then dispose of in the garbage.
  6. Disinfect all surfaces and equipment.
  7. Dispose of gloves after each use and thoroughly wash hands.

 

Observations

Observations and Raw Data

Experiment Raw Data

The following measurements were collected during the different phases of the experiment.

Experiment Observations

* More detailed written observations are included in the logbook.

Phase 1 Group 1 Pictures

 

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Analysis

Data Analysis

* Additional data analysis details are included in the logbook.

Phase #1 Data Analysis

In Table 1 the oil column height measurement data was averaged for the three test tubes in each group. The data from Table 1 was used to create Graph 1 (Group 1) and Graph 2 (Group 2). These graphs show how the amount of non-bioremediated oil column height changed over the 14-day period. In Group 1 the amount of motor oil was varied so there were various starting oil column heights. In Group 2 the oil column height was the same for all tests at the start. It is easier to identify which test tube groupings had the most bioremediation occur.

Table 2 shows the average reduction in oil column height over the 14-day period. Graph 3 (Group 1) and Graph 4 (Group 2) show this data visually. This was done to visually see the oil reduction over time since some of the test started at different oil heights at the start.

Table 3 shows the average oil column height difference from Day 1 to 14, the percentage of oil remaining and the percentage of oil bioremediated. This is visually represented in Graph 5 and 6. Graph 5 shows the total oil column height reduction over the 14 days for both Group 1 and 2. Graph 6 shows the oil column height percentage reduction which is the percent of oil column that was bioremediated over two weeks for both groups.

The Phase #1 Group 1 (varying oil column – all 2g nutrients at start) data shows:

  • Test 1J/K/L with the medium oil column (10mm) had the most oil column reduction (7mm) which was also the biggest percent reduction (70%) for Group 1.
  • Test 1M/N/O with the biggest oil column (14mm) had the second largest oil column reduction (5mm) but this only represents 36% oil column reduction  which was the lowest out of the non-control tests in Group 1.
  • Test 1G/H/I with the smallest oil column (6mm) had the smallest oil column reduction (4mm) outside of the controls. This is 67% of oil column reduction which is the second largest percent reduction but only 3% lower than the 10mm start test.
  • The Control tests with no bugs showed 14-20% oil column reduction. This is possibly oxidized oil rather than bioremediated oil but when measurements were collected in Phase #1 oxidized oil was not yet understood.
  • The time from 7 to 14 days increased the amount of bioremediated oil for all non-control test tubes. This is important because in Phase 2 the experiment was only 7 days due to time restriction.

The Phase #1 Group 2 (varying nutrients – all 8mm oil column at start) data shows:

  • Test 2M/N/O with the most nutrients (3g nutrients) and Control 2D/E/F (no nutrients) had the largest oil column reduction after 14 days. They both reduced by 6 mm (75%). This percent reduction is 5% higher than the highest percent reduction from Group 1.
  • Test 2G/H/I (1g nutrients) and Test 2J/K/L (2g nutrients) had the second largest oil column reduction after 14 days. They both reduced by 5 mm (63% reduction).
  • The other Control 2A/B/C with no microbes and variations of nutrients (1g/2g/3g) all decreased by 3mm after 14 days. This is most likely oxidized oil.  
  • Some measurements went up and down which is most likely from human error or the shaking/stirring process.

Phase #2 (Pond Water) Data Analysis

In Table 4 the oil column height measurement data was averaged for the three test tubes in each group. The data from Table 4 was used to create Graph 7 which show how the amount of non-bioremediated oil column height changed over the 7 day time period.

Table 5 shows the average oil column height difference from Day 1 to 7, the percentage of oil remaining and the percentage of oil bioremediated. This is visually represented in Graph 8 and 9. Graph 8 shows the total oil column height reduction over the 7 days. Graph 6 shows the oil column height percentage reduction which is the percent of oil column that was bioremediated over one week.

The data from Phase #2 (Pond water; varying nutrients; 5mm oil column at start) shows:

  • Test 3K/L/M with the most nutrients (3g) showed an oil column decrease of 5mm (100%) over the course of 7 days.
  • Test 3E/F/G with no nutrients showed an oil column decrease of 4.5mm (90%) over the course of 7 days.
  • Test 3H/I/J with the middle amount of nutrients (1.5g) showed an oil column decrease of  4mm (80%) over 7 days.
  • All of these tests were within 1mm of reduction after 7 days.

Phase #1 Tables and Graphs 

Table 1
Table 1
Graph 1
Graph 2
Graph 2
Table 2
Graph 3
Graph 4
Table 3
Graph 5
Graph 6

Phase #2 Tables and Graphs

Table 4
Graph 7
Table 5
Graph 8
Graph 9

 

Phase #1 – Groups 1 – Varying Amounts of Nutrients

  • Lowest Motor Oil (2.5mL): I think this amount of motor oil will be completely bioremediated within the two-week period. From my research, bioremediation is most successful when as much source contaminant and free oil is removed.  This hypothesis was incorrect because there was still 2mm of motor oil remaining after the two-week experiment.
  • Medium Motor Oil (5mL): I think this amount of motor oil will only be partially bioremediated by the time the two-week period is up. The thicker oil column will mean the bioremediation will take longer because there is more oil to consume and because it is harder for the microbes to access the top of the oil layer. This hypothesis was correct because although not all of the motor oil was bioremediated all except for 3mm were bioremediated and/or oxidized.
  • Highest Motor Oil (8mL): I think this amount of motor oil will also only be partially bioremediated and even more oil will be remaining than in the medium test. This hypothesis was also correct because only 5mm of the 14mm were bioremediated.

Phase #1 – Group 2 – Varying Amounts of Nutrients

  • Lowest Nutrients (1g): I think that this amount of nutrients will not do much to stimulate microbial growth and contaminant removal and would have similar results to the controls with zero nutrients. This hypothesis was incorrect because the fertilizer might have stimulated bioremediation these test tubes started with 8mm of motor oil and dropped to 3mm over the course of 2 weeks.
  • Medium Nutrients (2g): I think that this amount of nutrients will have the most bioremediation because I think this amount will not be toxic to the microbes but will still stimulate bioremediation and microbe reproduction. This hypothesis was also incorrect because this test tube did not have the most bioremediation. These test tubes also went from 8mm to 3mm over the course of 2 weeks.
  • Highest Nutrients (3g): From my research I know that high amounts of nutrients may kill the microbes but I am not sure what ratio will be toxic. This nutrient is the highest of my tests so I think it is most likely to have toxic effects. My hypothesis was also incorrect because this amount of nutrients did not prove to be toxic to the microbes. These test tubes also had the most bioremediation occur and went from 8mm to 2mm over the course of 2 weeks.

Phase #2 – Pond Water – Varying Amounts of Nutrients

  • No Nutrients (0g): I think that no nutrients will still have a high level of bioremediation based on my phase 1 test. My hypothesis was correct because these test tubes went from 5mm to 0.5mm in 7 days which was the second most amount of bioremediation.
  • Medium Nutrients (1.5g): I think that this amount of nutrients will not have the least amount of bioremediation based on results from my phase 1 test. This hypothesis was correct because these test tubes did have the least amount of bioremediation occur, they went form 5mm to 1mm in 7 days.
  • Highest Nutrients (3g): I think that this amount of nutrients will have the highest level of bioremediation. Phase 1 showed that this amount of nutrients is not toxic and stimulates microbial reproduction and bioremediation.  Please refer to data analysis tab for details on data and how it was analyzed. This hypothesis was correct because this amount of fertilizer was not toxic and stimulated microbial reproduction and bioremediation. These test tubes had the most bioremediation occur and went form 5mm to 0mm in 7 days.

 

 

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Conclusion

Experiment Conclusions

Please refer to data analysis tab for details on data and how it was analyzed.

General project conclusions are:

  • The highest amounts of oil had a lower percent reduction. This may be because the microbes needed more time and/or because they could not reach the surface of the oil column easily. This may indicate large oil volumes require a longer time period, may require source removal to have further success and/or may require additional mixing.  
  • With medium and small amounts of oil present a significant amount of bioremediation can occur in 7-14 days.
  • Nutrient addition of 3g into pond water with a 5mm oil column does not have a significant effect on the amount of bioremediation over 7 days. The type and ratio of fertilizer used may have affected these results. This may also indicate the pond water has nutrients present.  
  • Some degradation of oil occurs even without the addition of microbes to the samples, particularly in pond water.  
  • It was challenging to visually assess the difference between non-bioremediated oil, bioremediated oil, oxidized oil and/or lipids in the test tube experiment.

Review of My Hypothesis Compared to Results

Phase #1 – Groups 1 – Varying Amounts of Nutrients

  • Lowest Motor Oil (2.5mL): I think this amount of motor oil will be completely bioremediated within the two-week period. From my research, bioremediation is most successful when as much source contaminant and free oil is removed.  This hypothesis was incorrect because there was still 2mm of motor oil remaining after the two-week experiment.
  • Medium Motor Oil (5mL): I think this amount of motor oil will only be partially bioremediated by the time the two-week period is up. The thicker oil column will mean the bioremediation will take longer because there is more oil to consume and because it is harder for the microbes to access the top of the oil layer. This hypothesis was correct because although not all of the motor oil was bioremediated all except for 3mm were bioremediated and/or oxidized.
  • Highest Motor Oil (8mL): I think this amount of motor oil will also only be partially bioremediated and even more oil will be remaining than in the medium test. This hypothesis was also correct because only 5mm of the 14mm were bioremediated.

Phase #1 – Group 2 – Varying Amounts of Nutrients

  • Lowest Nutrients (1g): I think that this amount of nutrients will not do much to stimulate microbial growth and contaminant removal and would have similar results to the controls with zero nutrients. This hypothesis was incorrect because the fertilizer might have stimulated bioremediation these test tubes started with 8mm of motor oil and dropped to 3mm over the course of 2 weeks.
  • Medium Nutrients (2g): I think that this amount of nutrients will have the most bioremediation because I think this amount will not be toxic to the microbes but will still stimulate bioremediation and microbe reproduction. This hypothesis was also incorrect because this test tube did not have the most bioremediation. These test tubes also went from 8mm to 3mm over the course of 2 weeks.
  • Highest Nutrients (3g): From my research I know that high amounts of nutrients may kill the microbes but I am not sure what ratio will be toxic. This nutrient is the highest of my tests so I think it is most likely to have toxic effects. My hypothesis was also incorrect because this amount of nutrients did not prove to be toxic to the microbes. These test tubes also had the most bioremediation occur and went from 8mm to 2mm over the course of 2 weeks.

Phase #2 – Pond Water – Varying Amounts of Nutrients

  • No Nutrients (0g): I think that no nutrients will still have a high level of bioremediation based on my phase 1 test. My hypothesis was correct because these test tubes went from 5mm to 0.5mm in 7 days which was the second most amount of bioremediation.
  • Medium Nutrients (1.5g): I think that this amount of nutrients will not have the least amount of bioremediation based on results from my phase 1 test. This hypothesis was correct because these test tubes did have the least amount of bioremediation occur, they went form 5mm to 1mm in 7 days.
  • Highest Nutrients (3g): I think that this amount of nutrients will have the highest level of bioremediation. Phase 1 showed that this amount of nutrients is not toxic and stimulates microbial reproduction and bioremediation.  Please refer to data analysis tab for details on data and how it was analyzed. This hypothesis was correct because this amount of fertilizer was not toxic and stimulated microbial reproduction and bioremediation. These test tubes had the most bioremediation occur and went form 5mm to 0mm in 7 days.

 

 

Application

Next Steps and Application to the Real World

Next Steps and General Application

 

  • Generally, this project is important because oil spills happen in both salt and fresh water although saltwater seems to have more studies done on it. It is important that ideal quantities of different items that help speed up bioremediation in freshwater are understood.
  • Additional research into the type and ratio of nutrients is needed. Based on conclusions so far, the fertilizer selected does not have a significant effect on bioremediation. Fertilizer can affect the flora and fauna of the site so the risk to the site with adding chemical fertilizer may outway the benefits to the bioremediation process of adding fertilizer. More research and testing of natural nutrient sources would be helpful for freshwater use in the real world.
  • Tests with larger amounts of oil indicate that longer time periods eventually have success. It would be interesting to test for longer periods of time.
  • It would be interesting to do another test using pond water but in larger containers such as buckets to see how the larger sample size changes the test. Thinking about mixing and making sure oxygen is being provided to the microbes is also important.
  • Further tests could study how other variables may affect the rate of biodegradation (such as pH and temperature) although there is quite a lot of information available indicating the best range for these variables. The type and amount of nutrients has less research available, particularly for freshwater, which is why it was selected for this experiment.
  • The test showed the importance of removing excess oil prior to starting bioremediation of freshwater. Some ways to remove excess oil on freshwater before starting bioremediation include skimmers, boom arms, absorbent material, and even burning.
  • It would be interesting to source products that are sold commercially for real-world biodegradation application and test the products on freshwater oil spills as the microbes that I used were sold in small quantities for laboratory use. I was able to find a product sold by Acklands Granger in Calgary that apparently contains oil-eating microbes stored in a dried bentonite clay mix (Ultra-Archaea sold by Ultratech International). The directions for this product indicate it can be applied to oil-contaminated areas and the microbes will digest the pollutants.
Oil Eating Microbes in Bentonite Powder - Sold by Ultratech at Acklands Granger in Calgary

Bragg Creek Pond Application

The reason I became interested in bioremediation of hydrocarbons in freshwater is because my Grandparent’s had a bobcat spill diesel in their pond in Bragg Creek over Christmas this year. We helped research ways to clean up the spill and this led to my project idea. I could not test diesel specifically as it is too smelly to have in our house which is why I picked motor oil. Once the ice melts on the pond in the spring I would like to go out and study the spill area to see if there is any indication of remaining diesel. If there is still diesel present I am hoping I can take what I learned and help clean up the pond.  

Bobcat in Bragg Creek Pond Caused Diesel Spill

Sources Of Error

Sources of Error

Major Sources

The major sources of error for this project include:

  • The angle of measurement, the location of measurement of the oil column and human error affected the height observed in mm. 
  • Difficult differentiating between bioremediated motor oil, oxidized oil, lipids, and non-bioremediated oil. 
  • The microbes may not have been getting enough oxygen. As I did this test in my home I had to leave test tube lids on although I did mix (shake) the test tubes daily. This may not have been enough or represent real-life conditions. 
  • Phase #2 was only 7 days long (due to time restrictions). Results from Phase #1 show that the 7-14 day period was important for the larger oil column tests. 

Some ways to reduce these sources of error if I completed this project again:

  • Measuring at the exact same location and angle for each test tube. Possibly mark on each test tube the point of measurement and ensure the observer is looking directly at the oil column (not above or below).
  • Research and use methods that indicate or confirm bioremediation is occurring or has occurred. I was able to source a small amount of tetrazolium powder which changes colour when microbes are active. I could not source enough and at home, I didn't have the resources to include this as an indicator in my experiments. This would be very interesting to use in future tests. 
  • To help with the oxygen limitation I would recommend doing this experiment in a laboratory with proper ventilation and ensuring mixing occurs regularly. My research shows that oxygen is vital for this type of bioremedation to occur. 

Other Sources

Other sources of error for this project may include:

  • I cannot sterilize the air so there may be other contaminants in the test tubes. 
  • I cannot get every drop out of my pipettes and beakers when pouring the motor oil into the test tubes.
  • I cannot control the rpm (rotations per minute) for activating the microbes in the magnetic stirrer.
  • I cannot control the exact temperature of the seed starting mat which the test tubes were placed on for the last two weeks (approximately 28°C). 
  • I did the measurements every day in the evenings but they were at slightly different times. 

Citations

Useful Websites:

Acknowledgement

Thank you to:

  • Edvotek for supplying the freeze-dried microbes for this experiment. 
  • My family for helping me to source my materials, taking pictures during the experiments, helping ice auger and for the technology support.
  • Mr. Krawchuk and the Science Fair Club.