Improving The Ability Of The Pseudomonas Putida To Metabolize Petroleum & Its Derivatives MAG

By Unknown, Unknown, Unknown

   Problem Description

As petroleum and its derivatives have become a major necessity to the developed nations of the world, the amount of environmental pollution caused by petroleum products has significantly increased, much to the detriment of our ecosystem. Contaminated ground water supplies, sludge on beaches, and the death of wildlife are only a few results of the pollution caused by petroleum products. Every year, millions and millions of dollars are spent on cleaning up oil spills. Industries which allow petroleum pollutants to seep into the soil and water are contaminating our environment. The most devastating form of petroleum pollution to our environment is caused by oil spills, like have occurred in: (1) the Prince William Sound off the coast of Alaska by the Exxon Valdez, (2) the Gulf of Mexico by the Mega Borg tanker which spilled almost four million gallons of crude oil, and (3) the Persian Gulf during the Gulf War, all of which ultimately killed countless numbers of aquatic and wildlife.

How alkanes are


by bacteria

Microorganisms isolated based on their ability to degrade hydrocarbons often belong to the genus Pseudosmonas. These bacteria have several catabolic pathways and are easily handled and grown in the laboratory. Cometabolism of organic substrates by diverse microbial populations plays a major role in the environment.

Petroleum is a complex mixture of alkanes. It is generally known that simple alkanes (methane, ethane, short and/or unbranched chains) are more easily degraded than complex alkanes (petroleum, long and/or branched chains). However, the degradation is the same. The initial bacterial degradation of alkanes begins when the terminal methyl group of the alkane is oxidized forming an alcohol. The alcohol is oxidized by dehydrogenase to a carboxylic acid which is metabolized in the -oxidation pathway of fatty acids).

Factors Affecting Rate of Biodegradation

There are several factors that can affect the biodegradation of alkanes. One of the most important factors is the presence of oxygen. Oxygen supplies are usually limited in calm waters as the oxygen is not readily circulated. Inorganic nutrients are necessary for the efficient biodegradation of petroleum products. Inorganic nutrients such as nitrogen and phosphorus increase the efficiency of biodegradation by microbes. The molecular composition of the alkane is another important factor affecting its biodegradation. Another factor is the physical state of the alkane. In water, when there is wind and wave action, oil-in water or water-in-oil emulsions may form. An oil-in-water emulsion spreads the oil over a large surface area providing the bacteria with a larger surface area to act upon. However, a water-in-oil emulsion tends to form tarballs with a comparatively smaller surface area for the bacteria to act on. Temperature can also affect the rate at which petroleum can be catabolized by bacteria. The degradation process slows at lower temperatures.

We propose to use simple genetic manipulation techniques to alter the promoter and enhancer regions on the genes that control the ability of P. putida to metabolize oil. By doing this, we intend for the oil metabolizing ability to be enhanced.

Field Studies

The field studies would be used to determine the metabolic rate of the bacteria on the petroleum in different environments. The metabolic rates of the bacteria would be tested in several types of land and water environments under controlled laboratory conditions. We would make three cultures for each environment. The first culture would be the basic bacteria without alkane metabolizing abilities, P. syncyanea. The second culture would consist of the original, unaltered P. putida. The third culture would consist of the altered P. putida. These cultures would be studied for a selected period of time, during which we would measure the amount of substrate consumed through enzymatic activity, and the metabolites produced.

Detecting Enzymatic Activity

To determine if the altered bacteria are effectively metabolizing the substrate, petroleum, we would compare the enzymatic activity of the altered and unaltered bacteria. We would accomplish this by using gas-liquid chromatography and mass spectrometry, which will measure the amount of remaining substrate. Differences in residual substrate would indicate the effectiveness of the altered gene regions. A determination of the actual metabolites could be done with thin-layer chromatography.


The use of bacteria in bioremediation efforts necessitates that we consider the possible hazardous effects that bacteria can have on other living organisms. Fortunately, there are more non-pathogenic bacteria than pathogenic types, but when large numbers of bacteria are placed in an environment where they will come into contact with indigenous organisms, care must be taken to ensure the safety of the ecosystem. Generally, bacteria naturally involved in oil degradation are self-limiting based on the amount of substrate available, thus when the oil is gone the numbers of bacteria are greatly reduced and negative effects on the environment do not occur. Scientists must ensure that genetically altered bacteria placed at sites in need of bioremediation are also reproductively limited by substrate availability. Ideally, bacteria would be genetically altered making them dependent solely on petroleum products for their energy source.

There is a relationship between economic development and environmental problems. The tendency has been to prioritize economic development at the expense of the environment. Blazej (1991) claims that in today's industrialized world, man's undue power over the environment has reached such a level that "nature loses its self-regulating ability." Industrial biotechnology has the ability to solve many environmental problems and is becoming an integral part of the world economy. The future holds great promise with the proper and controlled use of recombinant DNA techniques in biotechnology.

We must recognize that bacteria can mutate on their own, therefore genetic alterations that we impose may not necessarily be permanent. Sometimes in the fields of molecular biology and genetics, researchers discover that "nature knows best." If we find that the mutations fail to enhance the oil-eating ability of P. putida, we can still consider our work successful in that we have gained knowledge and experience in molecular biology and genetics. u

Editor's Note: In order to fit all the finalists, these have been edited for publication.

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