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In between 1970 and 2007 nearly 6,062,712 tons 5.5 million metric tons of Oil spilled around the world. When oil spill occurs liquid petroleum is released into the environment and the black sticky substance covers everything it touches: soil, water and wildlife8. Recently largest oil spill occurred in Gulf of Mexico in US, is causing substantial loss of life and releasing 35,000 to 60,000 barrels of oil per day 4. In the environment, there are billions of tiny "workers" busy ridding the water and beaches of thick black oil. These tiny microscopic organisms are called Oleophilic or oil eating microbes (OEMs). These are bacteria which use oils in the environment as their food source; this process is known as bioremediation5.
What is Bioremediation?
"Remediate" means solve a problem and "Bioremediate" means using biological organisms to solve environmental problems such as contaminated soil or water 1. Harmful chemicals found in gasoline and oil spill are eaten up by microscopic "bugs" or microbes present in soil or water. Microbes completely digest these chemicals into water and harmless gases such as carbon dioxide 2.
Oil eating Bacteria:
Hydrocarbon degrading bacteria are ubiquitously present in the environment. There are more than 20 genera of marine hydrocarbon degrading bacteria are distributed over several (sub) phyla (Î±, Î², Î³ proteobacteria; Gram positives; Flexibacter-Cytophage Bacteroides). Single bacterial species are capable to degrade only a limited number of the compounds in crude oil. Combination of different bacterial species is usually involved in oil degradation 6. Mr. Mohan chakrabarty patented the first version of genetically modified oil eating bacteria in 1981. He removed the plasmids from four species of pseudomonas oil eating bacteria, combined and exposed them to ultra violet rays to create single stable species3.
Design of Bioremediation process:
A team of scientists investigated in situ crude oil degradation after prestige oil spill in November 2002. Their goal was to access the bacterial population after the oil spill to detect the evidences of crude oil degradation taking place at the contaminated site. The team used stable isotopes (13C/12C) to determine the origin of dissolved inorganic carbon (DIC) in control and contaminated marine water samples. As crude oil originates from biological process, crude oil is depleted in 13 C. So their biodegradation product CO2 will also be more depleted 13C when compared with the typical marine dissolved inorganic carbon and atmospheric dissolved carbon dioxide.
The dissolved inorganic carbon isotopic composition of samples taken from along the shore of a contaminated site showed degradation of a depleted 13C source of prestige crude oil. These results showed that natural population oxidizing this carbon source to CO2. These results can be reproduced in the laboratory when water samples from contaminated site are used, although these process requires nitrogen and phosphorous amendments, these elements are limited in the marine ecosystem. Results obtained from the experiment showed the presence of microbiota readily able to degrade contaminants in water samples7.
Laboratory design of bioremediation:
This experiment is used to understand the optimal conditions requires for the conversion of oil into hydrocarbons. Bacterial bioremediation is used to clean up the contaminated site of water or land. Oil eating bacteria are isolated from nature or modified by scientists to improve oil degradation.
This experiment will take several days to obtain results. The steps requires for the experiment are:
Setup the cells to grow in a flask
Take 250 ml flasks, to each clean flask add 125 ml tap water.
To each flask add 1.3 g of oil eating powder.
Stir the flasks to mix oil and water.
Keep the flaks overnight to allow the growth of cells.
When milky suspension is evident then the experiment is ready for design. Experiment can be done by several ways like:
Altering the pH of the cells:
Add one or two drops of HCl to one flask and NaOH to other flasks. Cells pH can be monitored by using pH paper. Different levels of pH of flasks are maintained to observe the effect of pH on cells growth.
Altering the temperature:
Keep flasks at different temperatures like room temperature, 30oC, 37oC and 45oC.
Add different food oils such as vegetable oil, olive oil, peanut oil etc to flasks and provide stirring or shaking to observe which oil(s) gives better results of growth.
Bacterial cells are plated before and after incubation with oil, to observe which parameters favors cells growth.
Bottle of Ready pour media is heated by any of two methods listed below:
Microwave method: Media bottle is heated on high for two 30 seconds intervals. Mix and heat on high for an additional 25 seconds or more until all media dissolved. Occasionally spin the media for further melting.
Hot plate or burner method: Bottle is placed in a beaker partially filled with water. Beaker is boiled on a hot plate or burner. Occasionally spin the media for further melting.
Melted media is allowed to cool approximately 55oC.
Place these bottles in a 55oC water bath to avoid solidification and transfer 5 ml of media to each Petri plate5.
Mode of action of oil degrading bacteria:
Oil is made up of hydrocarbons, which are consumed by oil eating bacteria as food source. The basic step of oil cleaning involves the applying of oil eating microbe's solution to the spill. This solution increases the surface area and increases oxygen supply to microbes.
There are three basic types of hydrocarbons, straight chains, branched chains and 6-member rings. All these hydrocarbons are broken down by oil eating bacteria. Oil eating bacteria breaks hydrocarbons into fatty acids or carbolic acids; these are further broken down for energy and carbon atoms, which are further used in citric cycle to generate energy. In this way the oil is broken down into basic, non toxic elements as carbon, carbon dioxide and water.
Oil eating microbes require air, water and a source of nutrients such as oil for survival. For successful bioremediation process oil eating microbes needs an environment with a temperature of -2 to 60 o C and a pH of 5.5-10. Oil eating microbe's survival in bioremediation is inhibited by other factors like lack of oxygen, moisture or mineral nutrients and unfavorable concentrations of waste. Oil eating microbes begin their work once all these factors are corrected5.
Is bioremediation safe?
Bioremediation is very safe because it uses naturally occurring microbes for degrading harmful chemicals. This method will not affect people working at the site or community. No dangerous chemicals used for bioremediation. Fertilizers added for the growth of microbes are the same that used for lawns and gardens. As Bioremediation changes the harmful chemicals into harmless water and gases, harmful chemicals are completely destroyed. EPA (Environmental Protection Agency) test sample of water to make sure that bioremediation works2.
How long will it take?
Time for bioremediation of a site depends on the following factors:
Size and depth of the polluted area.
Types and amount of harmful chemicals present.
Whether cleanup occurs above ground or underground.
All these factors vary from site to site. Microbes may take several months to years to clean up a site by eating harmful chemicals2.
Why Bioremediation is necessary?
Petroleum is made up of hydrocarbons. Many of these hydrocarbons dissolve slowly in water. Aromatic hydrocarbons like benzene are more soluble in water and these are toxic to living cells. These aromatic hydrocarbons attack fat-like membranes surrounding cells and badly affect their normal functioning. Many species of microorganisms like bacteria, yeast and fungi feed upon the wide variety of compounds found in petroleum. They obtain both energy and tissue building materials from petroleum.
By Genetic engineering scientists can improve the ability of bacteria to metabolize petroleum. There has been research going on to develop oil eating super bugs. Even without oil eating bacteria oceans have high capacity of petroleum degradation. Scientist found active hydrocarbon degradation in the Arctic Ocean even during cold and darkness of the winter.
The problem with oil spill is the potential short-term environmental damage. Although it is not considered a major waste, crude oil coats and kills sea life and alters the neighboring beaches, rocks, trees etc. If left to nature oil spills will continue to plate local ecosystem indefinitely; however, oil eating microbes are useful in degrading hydrocarbons and clean up the spill to restore the environment5.
Several thousands of dollars are spent to recover oil spills. Bioremediation has lot of advantages to recover oil spill because bacteria used in this method breaks harmful chemical compounds present in oil into harmless gases like carbon dioxide and water. Adding bioluminescence and color coding to bacteria we get bacteria that not only eats oil but also helps alert us to the presence of oil spills and other environmental pollutants oozing from pipes or storage tanks into water or soil8. So in future we may expect more genetically modified bacteria which help in protecting environment from oil spills.