sample 2 bioremediation

Bioremediation of Oil

Introduction:
Throughout the years, people have become more aware of oil spills. The media has taken care of that by taking pictures of the disastrous effects that oil spills cause. Oil spills have become an increasing problem because drilling has moved offshore and huge tankers are used to transport the oil. Every year, about 5 to 10 million tons of oil is spilled into the ocean annually. Sources of sea contamination include the use of seawater to stabilize the ship after it has docked, the dirty water is dumped back out to sea. Normal ships, other than tankers, dump out about 500,000 tons of oil into the ocean every year. Another source of ocean contamination is when ships collide with each other in ports and oil is released. Loading and unloading oil into the ships usually causes some of the oil to be spilled into the ocean. Both of these accidents cause about 1 million tons of oil to contaminate the ocean. Tanker accidents are among the major sources of oil spills. The largest spill involved losing 3,000,000 barrels of oil! Offshore drilling or oil wells on shore often break or leak exposing oil directly to the ocean or to streams that eventually reach the ocean. Motor oil is also a threat, around 2 million tons of used motor oil reaches water.

When oil is spilled, around 25% of it is evaporated within days. The remaining oil attaches to almost anything it touches including organisms such as birds. Oil that gets on birds causes their feathers to stick together and they can’t fly, float, insulate themselves, or obtain food. Birds that come in contact with oil die. Most of the spilled oil sinks to the bottom of the ocean or is eaten by microorganisms. After 3 months, 15% of the original oil remains. That oil is in the form of dense, black, tarry lumps that end up on shore. Oil that is spilled far away from shore is less of a threat to organisms that live on shore because the oil has enough time to disappear. Oil that is spilled close to shore often washes up and sticks to everything, this is what makes everything die. However, oil has an effect on the marine life no matter where is it spilled. The amount of light that enters the water is reduced by 90% causing many marine plant and protist growth to decrease because of the reduced rate of photosynthesis. Oil also decreases the amount of oxygen that dissolves in water, which is bad for marine organisms. Oil is a toxin, and many marine organisms die from the contact with it. Benzene, toluene, xylene, naphthalene, and phenanthrene are some of the toxins that are in oil. Organisms that get killed because of these toxins involve fish, shellfish, worms, crabs, mircocrustaceans, and other invertebrates. Oil also interferes with chemical messengers. Chemical messengers mediate important biological processes that are important for organisms to survive. Oil blocks taste receptors on marine organisms or it mimics natural stimuli, which causes bad effects on some marine organisms. Oil that is eaten by small organisms gets passed along the food chain until, eventually, humans eat it. Oil also serves as a pesticide that can reach marine organisms and humans in very high, dangerous concentrations.

The main cause for oil spills is human error. Countermeasures to prevent oil spills are concentrated towards prevention. Skimmers are used to get the oil from the top layer of the ocean. Skimmers are attached to ships and once the oil has been picked up, it is separated from the water inside the ship. The water is then dumped back out and the oil is disposed of or reclaimed. Materials, such as straw, powdered clay, sawdust, chopped corncobs stuffed in cloth "sausages", and other organic and inorganic absorbents are used to contain the oil in areas where they can be skimmed. Chemical dispersants such as detergents and solvents are used to get rid of the oil, but these chemicals are toxic to bottom-dwelling organisms, shore-dwelling organisms, and open water marine life. The attempt to burn oil has failed. It doesn’t undergo complete combustion and unburned black smoke contains toxic stuff. Incomplete combustible oil fractions contain carcinogens like phenathrene and anthracenes.

Oil is made up of hydrocarbons. Hydrocarbons are organic compounds consisting of only hydrogen and carbon. If oil is heated to certain degrees, the hydrocarbons will fall apart and the oil will evaporate, unfortunately, this option is not possible when oil is in the ocean because you can’t heat the entire ocean. The chief use of all the forms of petroleum is as fuel. Natural gas is used for heating, gasoline is used in cars and other combustion engines, kerosene is used in tractor and jet engines, and gas oil in Diesel engines. Kerosene and gas oil are also used for heat.

Mechanical methods to clean up oil only clean 10% to 30%. Also it takes along time and problems accumulate like limited manpower, no money, machines breaking down etc. Bioremediation only takes about 10-20 days to break down the oil. It involves spreading microbes called petrophiles over the oil. The petrophiles then degrade the oil biologically and convert it into food and non-toxic living cells. Boats, airplanes, or other vehicles are used to put the microbes over the oil. A fertilizer is also added to the mix to speed up the indigenous microbial action. The oil begins to break up and turn into a yellowish color. The oil is made up of remaining oil fractions and microbial masses, which get eaten by larger marine life. The process has to be aerobic, other wise, the microbes have little effect on the oil. So when the oil sinks down under the water, nothing happens to it, but when it is brought up out of the water, the microbes can break it down. The final products are acetate units, which are then broken into carbon dioxide by the Krebs cycle.

Hypothesis:
This experiment is a simulation of an oil spill in the ocean and on shore. It will prove that microbes can break down oil in a marine environment.

Materials:
#1- Materials used in this part of the lab included: 2 test tubes, 60 mL of distilled water, 1 density indicator strip, 1 test tube rack, oil, Pseudomonas culture, Penicillium culture, gloves, and an apron.

#2- Materials used in this part of the lab included: 2 plastic jars with caps, 400 mL of distilled water, 1 density indicator strip, 2 g of nutrient fertilizer, Pseudomonas culture, Penicillium culture, gloves, and an apron.

#3- Materials used in this part of the lab included: 2 petri dishes, 60 mL of distilled water, 2 g of nutrient fertilizer, Pseudomonas culture, Penicillium culture, gloves, and an apron.

Methods:
#1- First off, wash your hands for about 30 seconds before putting on your gloves and aprons. Label test tube #1 with Pseudomonas and test tube #2 with Penicillium with a marker. Add 5 mL of distilled water to each of the test tubes. Next, add 4-5 drops of oil to the test tubes until there is a fine layer of oil on the surface. Record the observations of the oil in the charts on Day 0. Insert .5 mL of Pseudomonas culture into tube #1 and .5 mL of the Penicillium culture in tube #2. Invert the test tubes several times and put them in an incubator with their caps loosened. Set the incubator at 30o C. Observe the tubes once every 24 hours for 3 days, recording your observations. Be sure to wash your hands before and after the observations for 30 seconds. Put any contaminated materials into an autoclave bag every time you observe the tubes.

 

#2- Wash your hands for about 30 seconds before putting on your gloves and aprons. Label jar #1 with Pseudomonas and jar #2 with Penicillium using a marker. Fill both of the jars half way with distilled water. Add 15-20 drops of oil into each of the jars until a thin layer has formed on the surface of the water. Observe the oil and record the observations under Day 0 on the charts. Sprinkle some nutrient fertilizer over the entire oil layer to increase the microbial growth and degradation process. Next, add 1.25 mL of Pseudomonas culture into jar #1 and 1.25 mL of the Penicillium culture in jar #2. Put the jars into the incubator at 30o C with their tops loosened. Observe the jars once every 24 hours for 3 days, recording your observations. To increase the dissolved oxygen level in the water, use a disposable pipette to blow oxygen bubbles into the water. Be sure to wash your hands before and after the observations for 30 seconds. Put any contaminated materials into an autoclave bag every time you observe the jars.

#3- Wash your hands for about 30 seconds before putting on your gloves and aprons. Label dish #1 with Pseudomonas and dish #2 with Penicillium using a marker on the lids of the petri dishes. Spread some sand into the dishes until there is about a 4-5 mm layer of sand. Spread the sand evenly on the dishes. Pour water over the sand so that a layer of water is covering the sand. Add 15-20 drops of oil into the water until there is oil spread evenly across the surface. Make observations of the oil and mark them under Day 0. Sprinkle some nutrient fertilizer over the entire oil layer to increase the microbial growth and degradation process. Next, add 1.25 mL of Pseudomonas culture into dish #1 and 1.25 mL of the Penicillium culture in dish #2. Put the jars into the incubator at 30o C. Observe the dishes once every 24 hours for 3 days, recording your observations in the tables. Be sure to wash your hands before and after the observations for 30 seconds. Put any contaminated materials into an autoclave bag every time you observe the dishes.

Results:
#1- Pseudomonas Table 1

 

General appearance characteristics of oil

Color of oil

Turbidity of water (number of bars disappearing)

Day 0

It’s lying on top of the water. The water is clear

Yellowish/ gold

0

Day 1

Yellow ring around the surface of the water. It is clear in the middle of the ring, the water is cloudy

Yellow

0

Day 2

Oil still appears as a ring on the top but it is breaking apart, the water is cloudy

Cloudy yellow

1

Day 3

Oil has degraded more, water still cloudy

Light-cloudy yellow

0

Day 4

It appears to be dispersing and running down the sides of the tube, the water is cloudy

Light to white amber

1

Penicillium Table 2

 

General appearance characteristics of oil

Color of oil

Turbidity of water (number of shade bars disappearing)

Day 0

It’s lying on top of the water. The water is clear

Yellowish/ gold

0

Day 1

Yellow ring around the surface of the water. It is clear in the middle of the ring, the water is cloudy

Yellow

0

Day 2

The oil formed a thin layer over the top, but a greater concentration of oil lies on the sides, the water is clear

Cloudy yellow

0

Day 3

Pretty much the same, water crystal clear.

Cloudy yellowish white

0

Day 4

Same as before, water clear

Light yellow amber

0

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

– The oil laid on the water surface and the water was clear.

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

– The oil formed a ring around the side of the tube on top of the water. It also started to seep down the sides of the tube. As time passes, oil gets heavy and sinks.

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

– The bacterial culture had a faster degradation rate than the fungal culture.

4. On an agar slant, can you identify which is the bacteria and which is the fungus? Describe the growth and appearance of both types of microbes. Can you think of any advantages in using the bacteria over the fungus to degrade oil?

– The bacteria grows in small colonies while the fungus grows largely everywhere. The bacteria also grows faster than the fungus. With this information, bacteria can quickly degrade the oil.

5. What does an increase in turbidity indicate?

– The break down of oil.

6. What is the turbidity level of your cultures after 4 days of incubation? How long do you think your cultures will continue to grow?

– 1 and 0. The cultures will continue to grow until the oil is all gone.

7. What is the limiting factor in your test tubes?

– The amount of oil and the size of the test tubes.

8. Research the nutritional requirements and environmental conditions that promote growth of bacteria and fungi. Suggest optimum conditions to culture bacteria and fungi.

– They grow in damp warm air and grow in stuff that they consume and survive.

#2- Pseudomonas Table 3

 

General appearance characteristics of oil

Color of oil

Turbidity of water (number of shaded bars disappearing)

Day 0

On top of water, water clear

Amber

0

Day 1

Oil appears as a ring on the top of the water, water is amber colored.

Cloudy amber

2

Day 2

Oil is cloudy, darker in color

Pale orangish-yellow

5

Day 3

Oil cloudy, darker in color, surface is less slick

Cloudy orange yellow

5

Day 4

Oil is compacted on the sides and small clumps on surface

Cloudy yellow-orange

5

Penicillium Table 4

 

General appearance characteristics of oil

Color of oil

Turbidity of water (number of shaded bars disappearing)

Day 0

On top of water

Amber

0

Day 1

Oil is still on top of the water

Amber

1

Day 2

Oil not as slick on surface

Pale, clear orangish yellow

1

Day 3

Oil on top layer in globs on water but thick on the sides. The water is orange color

Cloudy orange

1

Day 4

On sides of jar, formed a thin layer of surface

Cloudy orange

5

 

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

– The oil is less on the surface and doesn’t appear as slick, it appears as small clumps on the surface. The microbes are breaking up the oil. Yes.

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

– No. Yes. The microbes haven’t completely degraded the oil it takes more time.

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

– It gets broken down into products that can be eaten and not harmful to the environment.

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

– To speed up the growth and speed of the microbes while they degrade the oil.

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

– An in-balance in the environment will occur as more microbes develop, and the oxygen level will decrease as it becomes dissolved into the microbial growth.

6. Did you observe an increase in turbidity over time? Which of the 2 simulations is more turbid? Explain.

– Yes. The bacterial simulation is more turbid because the bacteria allows more space for the fertilizer to sink down into the water.

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

– In the jars because there was more space and more fertilizer.

8. Based 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?

– Yes because the temperature affects the growth and speed of the microorganisms. I think they would follow the floating oil because the microorganisms would be stuck to the oil. The residue wouldn’t damage marine life because it isn’t toxic and marine organisms can eat it.

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

– The marine plants wouldn’t be able to grow because of the absence of light caused by the oil. Birds would get hurt and would die and fish would die because of the decrease in dissolved oxygen entering the water from the surface and from oil getting into their gills, clogging them.

10. In this investigation, we evaluated the ability of microorganisms 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.

– Temperature, waves/shifting of the water, winds. The temperature can cause the bacteria to grow either slowly or not at all. Waves move everything around and so do winds.

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

– I would use the bioremediation method because it is cheaper and less manpower is needed. It is also a lot faster and gets the job done.

 

#3 Pseudomonas Table 5

 

General appearance characteristics of oil

Color of oil

Turbidity of water (number of shaded bars disappearing)

Day 0

Clumps on water surface

Light amber

0

Day 1

Spread evenly except for 2 large puddles

Light amber

0

Day 2

Spread evenly, less oil on surface

Light amber

0

Day 3

It disappeared, probably soaked in sand

Not any

0

Day 4

Same as yesterday

Not any visible

0

Penicillium Table 6

 

General appearance characteristics of oil

Color of oil

Turbidity of water (number of shaded bars disappearing)

Day 0

Clumps on water surface

Light amber

0

Day 1

Spread throughout the surface but still in yellow clumps

Light amber

0

Day 2

Spread out throughout the surface

Light amber

1

Day 3

Oil is in a ring around the sides of the dish.

Light amber

1

Day 4

Disappeared in the sand, there is a small foam buildup on the lid of the dish

Not visible

0

 

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

– The oil would soak into the sand.

2. How effective do you think such a method is when used in an actual oil spill on the shore? What happens to the degraded oil and the resultant microbial mass?

– The method is pretty effective because the oil eventually gets degraded and the residue sinks into the sand, out of harms way.

3. 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 2 methods would be the most efficient and economical to clean up oil spills?

– The amount of rocks and other physical barriers. The amount of fertilizer and the condition of the sand. The bioremediation method would most efficient for the reasons that I have already mentioned.

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

– Everything would be soaked in oil. The plants would probably all die as well as protests and large animals that come into contact with the oil. It is very sticky and prevents organisms from using their senses to find food.

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

– Detergents are toxins to the environment including bottom dwelling organisms and land animals. Detergents are used to clean clothes and other non-living materials.

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

– The procedures already go well in the water. In the air is a different story, the bacteria would have to be in the air, which would be dangerous for humans that live in that area. The same goes for soil, plants would get hurt if any of the bacteria was in the area. I think that it would do ok with waste materials in the water, nowhere else.

7. What can be done to prevent oil spills?

– The large tankers need to keep their oil protected from leaking out. Also the captains of the boats need to be very alert and watch out for any hazards that may cause a leak. Or we could just move all oil drilling on land and forget about all the tankers and other stuff.

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

– Oil can act as a pesticide towards humans and if we are near a spill for too long, we could have some problems, just like with any other pesticide. Oil spills do a lot of damage to the environment. Many animals and other marine life are killed and whole sections of wilderness are destroyed. When there is an oil spill, money is needed to clean it up. So governments have to spend a lot of money to get it cleaned up.

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

– Electricity has already begun to evolve into cars and other modes of transportation. This could cut down on our use of gasoline a lot. Wind and water generators also provide us with energy as well as nuclear power plants. Once the market for these resources has gone down a bit, everyone will be dependent on oil and there won’t be very many disasters like oil spills in the world.

Error Analysis:
If the wrong amounts of any of the substances that we put in the containers were wrong, the whole experiment could have failed. Especially if there wasn’t the correct amount of cultures inserted into the containers. It would have taken longer for the bacteria and the fungi to grow and the daily observations would have not been possible because nothing would have been visible.

Conclusions:
According to the results, bioremediation is a successful technique to clean up oil spills. In the first investigation, fertilizer wasn’t used and the oil wasn’t degraded that quick. Fertilizer was used in investigations 2 and 3, the oil was degraded much faster. Pseudomonas was the ideal microorganism for cleaning up the oil because it can reproduce faster and consume more of the oil than the Penicillium. In on shore spills, either microorganism is ideal because the oil was degraded and sank into the sand.