Bioremediation of Marine Oil Spill : The Results of Extensive Lab Research Using Mutagensis
Authors: Nour M.H. , Thompson I. 
Published: December 2007

Abstract
Oil Spill bioremediation often involves using the enzymatic capabilities of indigenous hydrocarbon - degrading microbial populations and modifying environmental factors to achieve enhanced rates of hydrocarbon biodegradation. Seeding involves the introduction of exogenous microorganisms into the natural environment to increase the rate and extent of biodegradation of pollutants. The rational for this approach is that the indigenous microbial population may not be capable of degrading the wide range of potential substrates present in complex mixture as petroleum. This paper presents the results of an extensive laboratory research programe focused on sea water simulated, bench - scale laboratory, studies to evaluate the hydrocarbon biodegradation ability of several induced mutants of Pseudomonas fluorescens compared with the wild strain. UV - irradiation was used to induce genetically improved strains capable of rapid degradation of crude oil.
Monitoring of the biodegradation profile using gas chromatography - flame ionization detector (GC/FID) illustrated that several mutant strains showed increased rates of biodegradation compared with wild organism. Additional tests were conducted to evaluated the biodegradation profile of several mixed cultures of the most effective mutants, the ecological safety (toxicity) of the biodegradation end products and the competition profile between seeded and indigenous microorganisms. Gas chromatography and high performance liquid chromatographic analysis showed that, there were a great improvement in degradation profile of n-alkanes, aromatic and polar compound of crude oil by using mixed cultures of most effective mutants. Additionally, the toxicity of the degradation products were reduced in this case. Moreover, there was various competition profiles between seeded mutant formulations and indigenous microorganisms which are already exist in sea water.

Introduction
Bioremediation is the process of stimulating the growth and activity of microorganisms that naturally feed on spilled hydrocarbons to hasten their decomposition and removal from the environment. In addition to the application of nutrients, in the field, bioremediation is enhanced by inoculation or seeding with allochthonous micooraganisms. This effect is due to the fact that the indigenous microbial populations may not be capable of degrading the wide range of potential substrates present in such complex mixture as crude oil. In general seeding has been considered as a possible bioremediation option in aquatic and shoreline environments.
Through genetic manipulation, efforts are being continuously devoted to creating microbial strains with a wide range of biodegradation activity that can be applied to control oil spill pollution. Using specialized, preselected mutants was a cost - effective environmentally sound method for bioremdiation of crude oil spill. Ultraviolet radiation is one of the earliest known and most commonly used mutagen. The mechanism of mutagensis of UV depends on its direct observation by the nitrogenous bases on DNA. The main effect of which is the covalent linking of adjacent pyrimidine residues. Generally, the criteria to be met by effective seed organisms have been reviewed by Atlas. They include the ability to degrade most petroleum components, grow in the environment, and to complete with indigenous microorganisms in addition to inability to produce toxic metabolites.

Experimental Work

Materials
Abu Sannan crude oil Sea Water from the Mediterranean open sea (El Max beach) Pseudomonas fluorescens was selected as a test organism for biodegradation experiments. The larvae of brine shrimp Artemia salina (El Max Strain) were selected as the test organism for toxicity.

Methods
Induction of Genetic Variabilities. A 25 ml nutrient broth culture (bacto beef extract, 3g/l; bacto peptone, 5g/l; yeast extract, 3g/l and NaCl 3g/l) was inoculated with Pseudomonas fluorescens and incubated at 37 C for 24 hours. P. 371^