The Effects of Essential Elements on Bioremediation
E. Harder, Little Flower Academy

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Background Research
A.  The Properties of Oil
B.   Bioremediation
C.   Biostimulation
D.   Bacteria
E.   Elements in a Bacterial Cell
F.   Bibliography

A.  The Properties of Oil
    Oil is a term applied to liquid fats derived from various sources including plant seeds, animal fats, and mineral deposits.  Petroleum, also known as crude oil, is naturally found in sedimentary rock.  It is made of hydrocarbons, which are organic compounds of hydrogen and carbon.  Sulfur and oxygen are also found in petroleum, but it is usually a smaller amount, from 0.1 to 5%.  These molecules can be divided into three classes: aliphatics, alicyclics, and aromatics.  Aliphatics have carbon atoms arranged in long, open chains; alicyclics have circles of carbon; and aromatics have six carbons arranged in a ring, bonded three times.
    Scientists have been able to see the toxicity of oil by its effect on marine life.  Crude oil is very dangerous, not only to marine ecosystems, but to humans too.  While humans can digest vegetable oil, they cannot break down crude and motor oil.   According to the National Ocean Service, oil spills can have serious long-term impacts to the environment.

    “The long-term impacts to birds and mammals include lower reproduction rates and physical mutations in offspring. Harmful oil components can contaminate fish that are in turn eaten by other fish, seabirds, and humans, thus passing these harmful components up the food chain. Once oil is trapped in sediments, it can be recirculated into the water and remain in the food chain for many years. Some research indicates that oil can remain in sediments for hundreds of years.” (NOAA/NOS)

    Spilled oil is extremely difficult to clean up.  It spreads rapidly, and with a fast current and wind can form a slick within minutes.  After spreading, the lighter parts of the oil immediately begin evaporating.  The benefits of this, however, are often offset by wave action, which mixes the water into the oil to form a heavy and sticky water-in-oil emulsion, called “chocolate mousse”.  The mousse can form in as little as ten to twenty hours after a spill.  It is important, therefore, that rescue workers arrive at an oil spill quickly. 
    In addition to a short response time, a successful clean up operation requires equipment, such as skimmers and pumps, which can handle emulsified oil and debris or dispersants, manpower, and a place to dispose of the oil.  No major oil spill has ever had such essential equipment on site in a short time frame.  Without them, unfortunately, the oil spreads, eventually becoming as thin as paint, even more difficult to clean up than the thick, sticky mousse.   Return to top.

B.  Bioremediation
    Bioremediation is the process of using living micro-organisms to clean up a contaminated site.  Micro-organisms do this by removing toxins from materials.  They decompose these compounds by using enzymes, specific proteins that control reactions in living cells.  Organisms that produce enzymes capable of degrading petroleum are useful in cleaning up oil spills.  Some common ones that break down oil are pseudomonas, flavobacterium, arthrobacter, and azotobacter.  Bioremediation accounts for 5 to 10 percent of all pollution treatment and has been used successfully in cleaning up leaking underground gasoline storage tanks.  Other toxic substances that have been successfully bioremediated include the solvent toluene, the moth repellent naphthalene (mothballs), the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), and the fungicide and wood preservative pentachlorophenol.
    Bioremediation has many applications, from the ordinary garden compost to the removal of selenium and other toxic metals from waste.  The best agents for bioremediation are the ones that can break down contaminants without becoming contaminated or harmful themselves.   
    There are several advantages to bioremediation.  One is minimal cleanup.  The adverse reactions on the environment seem to be minimal.  “Also, bioremedial agents often accomplish their work in site, meaning that they act directly within the contaminated site, thereby avoiding the need for costly physical removal and cleanup of wastes.” (Smith: 200).   The Exxon Valdez was the first major oil spill which bioremediation was used on a wide scale.  Return to top.
   
C.  Biostimulation
    Biostimulation is the process of adding nutrients to help oil-eating bacteria grow and reproduce faster, but it does not work in all conditions.  While temperature, pH, oxygen, and ocean salinity are only four of the factors that can affect a successful biostimulation process, overall, it has been used successfully.  “In summary, laboratory studies have shown that biostimulation…can enhance the rates of oil biodegradation, particularly in marine environments.”  (Zhu et al: 8).  One of the first places biostimulation was used on a large scale was on the shoreline after the Exxon Valdez disaster. 
    In spite of the fact that biostimulation on the open sea is difficult because of tides, it ha advantages. It is inexpensive and environmentally friendly.  On the other hand, it takes time for the treatment to work, making it unpractical for oil spills that require immediate cleanup.  Another issue is that adding nutrients can result in undesirable side effects such as algae blooms.   Return to top.

D.  Bacteria
    Some of the types of bacteria that can degrade hydrocarbons are achromobacter, bacillus, flavobacterium, and pseudomonas.  These bacteria belong to the Kingdom Monera and are heterotrophic, eating carbon as their major food source.  In an untouched environment, they make up less than 0.1% of the bacterial population.   
    Bacteria are prokaryotic cells, which means they lack a membrane-bounded nucleus.  They are smaller than most eukaryotic (cells containing a membrane-bounded nucleus) cells.  Most range in size from 0.2 to 3.0 micrometers.  They exist in three basic shapes: spherical, rod-shaped, and spiral shaped.  Bacteria can be aerobic, meaning needing oxygen; anaerobic, meaning oxygen is deadly; or facultative anaerobes, which means they can use oxygen or survive without it. 
    Many of the bacteria capable of degrading oil are Gram negative and rod-shaped.  These include Pseudomonas, Acinetobacter, Flavobacterium, Corynebacterium, and Alcaligenes.  Pseudomonas exist in the Phylum Pseudomonads.  They are straight, or slightly curved rod-shaped, motile by one or several flagella, and aerobic.  They reproduce asexually, and in an ideal environment, can divide every twenty minutes. 
    Usually their growth is shown in four stages: Lag Phase, Log Phase, Stationary Phase, and Death Phase.   The Lag Phase is when bacteria acclimate to a new environment.  Once they are acclimated, the Log Phase begins.  This is when they begin multiplying logarithmically.  The Stationary Phase is when the bacteria begin competing for a limited supply of food.  The Death Phase takes place when lack of food and increased toxic waste causes them to die.  Return to top.

E.  Elements in a Bacterial Cell
    A bacterial cell is made up of 50% carbon, 20% oxygen, 14% nitrogen; 8% hydrogen, 3% phosphorus; 1% potassium; 1% sulfur; 0.2% iron, and 0.5% each of calcium and magnesium.  (VanDemark, Batzing: 134).  In addition, bacteria require other nutrients, such as sodium, zinc, manganese, molybdenum, copper, nickel, tungsten, selenium, and cobalt.  (Brock et al: 119).
    Elements of a bacterial cell
Carbon
•Principal source of cellular  materials.
• Pseudomonas can use over 100 different molecules as carbon sources, including     
 proteins, fats, carbohydrates, and hydrocarbons.
•Found in carbon dioxide and organic compounds.
Hydrogen
•Together with oxygen,  essential to all aspects of cell life.
•    Used in cellular water and material.
•    Found in organic compounds, H2, and H2O
Oxygen
•    Main ingredient in cellular water and material.
•    Important for aerobic respiration.
•    Found in H2O, organic compounds, CO2, and  O2.
Nitrogen
•    Major ingredient in proteins and nucleic acids. 
•    Present in the cell wall.
•    Found in decay of dead organisms.  Inorganic forms are ammonia or nitrate.          
     Most bacteria can use ammonia as their sole nitrogen source.  Not all can use nitrate.
Phosphorus
•    Important to energy metabolism.
•    Used in nucleic acids and phospholipids.
•    Found as phosphate salts.
Sulfur
•    Used in amino acids, such as cysteine, and methionine.
•    Found from inorganic sources, such as sulfate or sulfide.
Potassium
•    Activates enzymes used in protein synthesis.
Magnesium
•    Stabilizes ribosomes.
•    Used in formation of cell walls.
Calcium
•    Buffers electrical charges within a cell.
•    Stabilizes cell walls.
Iron
•    Important for respiration.
•    Found in many cellular proteins.  
Other
Includes such elements as:
   Sodium:  Requirement based on the location of the species.
    Chloride
    Zinc: Important in enzymes, such as carbonic anhydrase, alcohol dehydrogenase,                         RNA, and DNA binding proteins.
    Selenium:  Important for some metabolic functions.
    Chromium
   Cobalt:  Important in formation of Vitamin B12.
    Molybdenum:  Important for enzyme activity.
    Copper:  Important to enzymes involved in respiration.

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Eileen Harder, 2004