Category: 1st Semester

Bioremediation of Oil

Introduction

Public awareness of oil spills has increased over the years as more publicity has been focusing on this subject and the massive harm it does to our environment. The problem of oil spillage has increased as well, and recent studies suggest that 5-10 million tons of oil are spilled into the ocean annually. There are six major sources of spilled oil. The first is cargo tanker washings at sea. These oil tankers use seawater to stabilize their craft after discharging their oil, and then, the contaminated water is discharged into the sea when the tanker is refilled. Another source is waste oil pumping at sea which contributes 500,000 tons of oil annually. Also in-port oil losses are caused by collisions at port and the procedures used during loading and unloading. This contributes over one million tons annually. Exploration losses occur because of accidental damage to offshore drilling rigs and the blowout of wells. The last source is motor oil. It is assumed that over 2 million tons of unaccounted for motor oil reach coastal waters every year.

The damage oil spills can create is often underestimated. A single gallon of oil can actually spread over four acres of ocean. Oil spills can have devastating effects on marine life. Oil that remains after evaporation goes through an emulsification process that creates a highly viscous, sticky material. This material becomes thick enough to sink to the bottom of the ocean. It sticks to anything it comes in counter with including marine wildlife. Oil left after emulsification is broken down by microorganisms and photo-oxidation. After 3 months only 15% of the original oil volume remains and it forms tarry clots that float to shore. However, if the oil spill is extensive or occurred close to the shore line, the oil would not have sufficient time for emulsification and evaporation so the oil would create a viscous film of oil over any solid surface that comes in contact with the oil.

The short-term effects of these types of oil spills include four main areas of the environment. One effect is reduced light transmission. Light intensity under an oil spill can be reduced by up to 90%. This thwarts marine plant and protist growth because they can no longer use photosynthesis. Another effect is reduced amounts of dissolved oxygen in the water. Since the surface of the water is covered by the filmy oil, the water can no longer uptake oxygen. Oil spills also affect marine birds. Oil covered birds can be drowned or become handicapped. It also reduces their ability to fly and float on water, and their feathers no longer serve as insulation leaving them to die from exposure. The last area of short-term effect is the toxic effect of oil on the marine environment. Compounds such as benzene, toluene, xylene, naphthalene, and phenanthrene are toxic to man and marine life. Within only days oil spills can do massive damage to the marine life.

In addition to short-term effects, there are also long-lasting more serious effects of oil spills. The chemicals found in oil can mimic the chemical messengers found in oceanic water. This can deceive many marine animals that depend on these messengers to find food, escape from predators, or locating habitats suitable for reproduction. More stable oil components can be consumed by smaller species and passed on to animals higher up on the food chain, including humans. Oil can also serve as a concentration medium for other toxins like pesticide allowing them to reach humans and marine animals in a more potent form.

To reduce these effects, clean-up efforts must be fast and effective. There are four main mechanical methods used when cleaning an oil spill. Skimmers can be used to pull a thin layer of water off the surface and then separating the oil and the water. The oil can then be disposed of and the water returned to the ocean; however, this is only effective in calm waters. Booms or barriers can be used to contain the oil spill that will later be skimmed so it does not spread any further. Absorbent materials such as cloth, straw, powdered clay, and pine bark are spread over the spill surface. Chemical dispersants, detergents, and solvents can also be used, but these substances are toxic to the open water marine life and shore-dwelling organisms. The last mechanical method is burning the oil off the surface. This method was ineffective because oil is not completely combustible and it left carcinogens and thick toxic smoke over the area.

Bioremediation is the newest method of oil spill clean up and far more effective than any of the mechanical methods used. Mechanical methods are costly and can only remove about 10-30% of the contamination. Bioremediation makes use of indigenous oil-consuming microorganisms, called petrophiles, by enhancing and fertilizing them in their natural habitats. This microbial clean-up method cleans the oil as well as a number of other harmful pollutants and is perhaps the best, most environmentally safe process used today.

Petrophiles are very unique organisms that can naturally degrade large hydrocarbons and utilize them as a food source. This makes them singularly qualified for cleaning oil spills and even tanker bottoms containing oil residue. In bioremediation several different types of these microorganisms are used on the oil slick. They are dispersed by boats or aircraft and may be mixed with nutrients. The oil slick begins to show visible signs of degradation after only days. The microbes can then be assimilated up through the food chain leaving having no adverse effects on the marine environment.

Hydrocarbon degradation is an aerobic process. It uses oxidation to break down the large hydrocarbons in exposed petroleum into acetates and then eventually carbon dioxide. This final and complete oxidation into carbon dioxide is performed by the Krebs cycle, which creates components needed for the synthesis of amino acids and other compounds the microorganism may need.

Hypothesis

The oil in all three investigations will be visibly reduced by both oil-consuming microorganisms and this process can be measured by turbidity and the appearance of the oil on the surface. The sand in Investigation #3 may hinder or slow the degradation process.

Materials

Investigation #1: Visual Determination of Oil Degradation

The materials needed for Investigation #1 were 2 test tubes with caps, 60mL of distilled water, a density indicator strip, 1 test tube rack, 3 sterile pipettes, 8-10 drops of refined oil, .5 mL of the Pseudomonas culture, and .5 mL of the Penicillium culture.

Investigation #2: Simulated Oil Spill Clean Up in Water

The materials needed for Investigation #2 were 2 clear jars with caps, 400 mL of distilled water, a density indicator strip, 2g nutrient fertilizer, 30-40 drops of refined oil, 3 sterile pipettes, 1.25 mL of the Pseudomonas culture, and 1.25 mL of the Penicillium culture.

Investigation #3: Simulated Oil Spill along the Shore

The materials needed for Investigation #3 were 2 petri plates, 60 mL of distilled water, sand, 2 g nutrient fertilizer, 3 sterile pipettes, 30-40 drops of refined oil, 1.25 mL of the Pseudomonas culture, and 1.25 mL of the Penicillium culture.

Methods

Investigation #1: Visual Determination of Oil Degradation

Two tubes were labeled as follows: Tube #1: Pseudomonas species, Tube #2 Penicillium species. 5 mL of distilled water were added to each tube. Next, 4-5 drops of oil were added to each tube and allowed to form a thin layer over the water surface. Notes were taken on the oil’s general appearance. With a sterile pipette, Tube #1 was inoculated with 0.5 mL of the Pseudomonas culture, and Tube #2 was inoculated with 0.5 mL of the Penicillium culture. The cap was placed on both tubes and each was inverted several times allowing the microorganisms to mix with the oil. With caps loosened one-half turn, the tubes were incubated at 30° C. Each tube was observed and inverted every 24 hours for 4 days and each observation was recorded in the appropriate table.

Investigation #2: Simulated Oil Spill Clean Up in Water

The two jars were labeled as follows: Jar #1 Pseudomonas species, Jar #2 Penicillium species. Both jars were filled half way with distilled water. 15-20 drops of oil were added to each jar and allowed to spread over the entire water surface. An initial observation was taken of the oil appearance. Fertilizer was sprinkled over the entire oil layer in both jars. Using sterile pipettes, Jar #1 was inoculated with 1.25 mL of the Pseudomonas culture, and Jar #2 was inoculated with 1.25 mL of the Penicillium culture. The jars were then incubated at 30° C for four days with the caps loosened one-half turn. Observations were taken every 24 hours for the four days and a disposable pipette was used to blow bubbles into the culture after each observation.

Investigation #3: Simulated Oil Spill along the Shore

The lids of the petri plates were labeled as follows: Petri dish #1- Pseudomonas species, Petri dish #2- Penicillium culture. A layer of sand was spread 4-5 mm thick in each plate. The sand was then moistened with distilled water in both dishes. 15-20 drops of oil were added to each dish, and then fertilizer was sprinkled over the entire oil surface. Using sterile pipettes, Petri dish #1 was inoculated with 1.25 mL of the Pseudomonas culture, and Petri dish #2 was inoculated with 1.25 mL of the Penicillium culture. The petri dishes were then incubated at 30° C for four days. The plates were observed every 24 hours and recorded in the appropriate tables.

Results

Investigation #1: Visual Determination of Oil Degradation

Observations: Pseudomonas Species Application

Table 1

Observations: Penicillium Species Application

Table 2

Investigation #2: Simulated Oil Spill Clean Up in Water

Observations: Pseudomonas Species Application

Table 3

Observations: Penicillium Species Application

Table 4

Investigation #3: Simulated Oil Spill along the Shore

Observations: Pseudomonas Species Application

Table 5

Observations: Penicillium Species Application

Table 6

Investigation #1: Visual Determination of Oil Degradation Questions

Describe the physical characteristics and appearance of oil on Day 0.

The oil on Day 0 had not yet been degraded in any way. It was a transparent light amber that stayed completely separate from the water. It formed a thin, slick layer over the water surface.

Describe any changes in the physical characteristics and appearance of the oil on Day 1 and beyond, and discuss possible causes for such changes.

The oil began to get darker and lose its slick qualities. It no longer covered the entire surface of the water and was separated into sections.

Is there a difference in the rate of oil degradation between the bacterial and fungal cultures?

The bacterial culture degraded the oil slightly faster possibly because bacteria can reproduce at a faster rate asexually.

Can you identify which is the bacteria and which is the fungus on agar slants? Describe the growth and appearance characteristics of both types of microbes. Can you think of any advantages in using bacteria over fungus to degrade oil?

The fungal colonies are larger than that of bacteria, but the bacteria was spread over a larger area and had more numerous smaller colonies. Bacteria would be more advantageous because it could spread over a larger area faster because it reproduces quickly and abundantly.

What does an increase in turbidity indicate?

An increase in turbidity indicates the break down of oil and the growth of colonies.

What is the turbidity level of your cultures after four days of incubation? How long do you think your cultures will continue to grow?

The turbidity of our culture was 1 after four days of incubation. The colonies will most likely grow until all the available oil has been degraded.

What is the limiting growth factor in your test tubes?

The amount of oil available limits growth in our test tubes and also possibly the amount of surface area.

Suggest optimum conditions that promote growth of bacteria and fungi. Suggest optimum conditions to culture bacteria and fungi.

Bacteria and fungi grow in any damp, warm area where there is any kind of available food source however optimum growth conditions are probably at about 30° C.

Investigation #2: Simulated Oil Spill Clean Up in Water

Describe what happens to oil after several days of microbial degradation. Are the microbes breaking up the oil? Can you detect an increase in microbial growth?

Oil degraded by microorganisms begins to appear darker and less slick and no longer covers the entire surface of the water. It is also less transparent and less oil appears on the surface. Microbial growth and oil breakdown can be detected by measuring the turbidity of the water.

Is the oil over the surface completely degraded? Can you still see any oil remaining on the surface? If so, explain.

The oil on the surface is not completely degraded and remaining oil is still visible on the surface. Four days did not allow the degradation process to complete and the smaller area may have caused the oil to be more concentrated than in an actual spill.

What happens to oil when it is biologically degraded in the ocean?

Oil that remains after evaporation goes through an emulsification process that creates a highly viscous, sticky material. This material becomes thick enough to sink to the bottom of the ocean. It sticks to anything it comes in counter with including marine wildlife. Oil left after emulsification is broken down by microorganisms and photo-oxidation. After 3 months only 15% of the original oil volume remains and it forms tarry clots that float to shore.

What is the purpose of the nutrient fertilizer used over the oil spill?

Nutrient fertilizer is used to enhance and speed the degradation of oil by increasing the bacterial and fungal growth.

Are there any adverse effects of using a fertilizer over an actual oil spill to enhance indigenous microbial growth?

Bioremediation is perhaps the most environmentally safe process of oil removal used today. It uses indigenous microbial growth so it does not introduce any new species that may damage the strict ecosystem. An increase in this one type of organism may cause a slight unbalance in this ecosystem but compared to other methods it has virtually no effect on the environment.

Did you observe an increase in turbidity over time? Which of the two simulations is more turbid? Explain.

An increase in turbidity over time was observed. The second simulation was the more turbid because the fertilizer increased microbial growth and enhanced oil degradation.

Did you observe more fungal and bacterial growth in the test tubes or in the jars? Explain.

The jars showed more fungal and bacterial growth because of the nutrient fertilizer and there was more space for growth in the jar.

Bases on the information provided, do you think that the microorganisms would be affected by water temperatures? Would they follow the floating oil or be dissipated by shifting winds or currents? And if they did eat the oil, would the residue damage marine life?

The microorganisms would definitely be affected by changes in temperature. These organisms grow best at fairly warm temperatures, and growth would be minimal in colder conditions. The microorganisms grow directly on the oil and would therefore not be dissipated by shifting. No harmful residue has been found after using bioremediation. All that is left after this method is masses of food and non-toxic living cells.

Based on the physical characteristics of oil and water discuss possible resulting problems associated with oil spills.

Because oil and water do not mix, oil can be spread over vast areas of oceanic surface if it is not contained. After emulsification, oil forms a thick, viscous substance that adheres to anything it comes in contact with. Also oil prevents light from reaching the lower areas of the ocean and reduces plant growth.

In this investigation, we evaluated the ability of microbes to degrade oil under optimum conditions. Based on your findings, discuss possible environmental limitations in using such a method over an actual oil spill in the ocean.

The temperature at an actual oil spill site could not be controlled and may drop below the microorganisms’ range of temperature that it can live. Also lower temperatures may reduce the rate of degradation. The oil may be further spread and dispersed by shifting winds making it harder for the microorganisms to cover its surface.

If you had to decide which clean up method to use in an actual spill, would you use such a bioremediation method or use a mechanical method described in the introduction? Explain your decision.

In an actual oil spill situation, a combination of bioremediation and mechanical methods would probably be most effective. Absorbent materials such as cloth, straw, powdered clay, or pine bark, could be used to contain the oil spill, while bioremediation is allowed to break down the oil into its harmless components.

Assuming that you need 10-6 lbs. of highly concentrated cell mass mixed with the nutrient fertilizer for the degradation of oil covering 0.022 sq. ft, as in the simulated oil spill, estimate the amount of cell mass and fertilizer mixture in pounds that would be needed to degrade the oil covering 1 square mile of an ocean. Assume there is the same amount of oil relative to the area.

12,672 lbs. would be needed to degrade one square mile of ocean.

Investigation #3: Simulated Oil Spill along the Shore

Describe the physical and chemical changes of the oil after several days of microbial degradation.

The oil began to get darker and lose its slick qualities. It no longer covered the entire surface of the water and was separated into sections. The chemical changes that occur, are that the carbon bonds are oxidized and broken into smaller hydrocarbon chains.

How effective do you think such a method is when used in an actual oil spill on the shore?

The dryer, sandier areas would slow the degradation process and inhibit the microorganisms from reaching parts of the oil spill, however, although slower, the method would still be fairly effective.

Discuss the physical limitations of using a mechanical method to clean up an oil spill on the shore. What are the limitations of using a bioremediation method? Which of the two methods would be most efficient and economical to clean up oil spills?

Mechanical methods would be virtually impossible to use in shoreline cleanup especially without further damaging the local wildlife. Rocks and other physical barriers would prevent skimming or absorbing the oil. Bioremediation would also be slightly hindered by these physical barriers, but it would still be the most efficient and economical way to cleanup a shoreline area.

Discuss the effect an oil spill on the shore would have on plant life along shorelines, protists and other larger animals.

Oil components are usually toxic to most organisms. Marine birds and mammals are especially effected by oil spills. They lose their ability to float on water and their insulation against cold, which can leave them severely handicapped or dead.

In the Alaskan oil spill, chemical detergents were not used. Why? Explain the use of detergents.

Chemical detergents were not used in the Alaskan oil spill because most of these chemicals are toxic to benthic, littoral, and open water marine life.

Discuss the potential of bioremediation procedures in detoxifying the air, water, soil, and waste materials.

Bioremediation has great potential for cleaning oil out of any type of medium as long as the microorganisms are able to grow in that environment. The microorganisms may be able to be genetically modified to grow on other mediums and even become airborne, however genetic engineering is still very controversial at this time.

What can be done to prevent oil spills.

Oil spills could be prevented by strict government regulation of all oil transportation and general public concern for the subject. Carefulness is the main key in preventing oil spills since most are caused by human error. Other methods of prevention could include new fuel sources or other means of transportation such as aircraft or truck.

How are we affected by oil spills? Discuss the physical, environmental, and economic consequences of oil spills.

Oil spills cause severe damage to the environment which can in turn effect humans. Oil spills are extremely wasteful and can cost millions of dollars to clean. Oil is also a non-renewable resource, which means that every time an oil spill occurs tons of oil are lost forever.

Research other energy sources that can be used to cut our dependence on oil?

Hydroelectricity, solar power, and wind power are all alternative energy sources that could reduce our dependence on oil. However, none of these sources are efficient enough to replace our need for oil. Nuclear power plants have began to grow, but these plants are highly expensive and dangerous. A last alternative to oil, could be gasohol, which is an alcohol made from corn that can be burned for fuel.

Error Analysis

Errors in this lab could be caused by incorrect measurements, insufficient oxygenating of the specimens, or mishandling of the sterile equipment. The tubes in Investigation #1 were not consistently inverted. The potency of the Penicillium culture was also questionable and there was no visible fungal growth in the original Luria broth culture.

Discussion and Conclusion

Investigation #1 acted as a control for the next two simulations. It showed that microbial growth and degradation did occur in a controlled environment. It could be used as a comparison to the amount of oil degraded in the other simulations to see if the variables had any effect on oil degradation. Investigation #2 was a simulation of an actual ocean oil spill with the use of a nutrient fertilizer. The nutrient fertilizer did speed oil degradation and a large amount of the oil was completely degraded in both jars. In Investigation #3, a shoreline cleanup was simulated with the addition of sand to the petri dish. The sand slightly hindered the effectiveness of the microbial degradation, but it was still effective in the partial breaking down of the oil.

All three investigations showed that the Pseudomonas culture was slightly more effective than the Penicillium culture in oil degradation. This may have been caused by a higher rate of reproduction in bacteria than in fungi.