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Biosurfactant production in bioreactors by using heavy crude oil fractions as substrate and evaluation of the ability of biosurfacatns to enhance crude oil biodegradation

Mandalenaki Athina

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Summary

Biosurfactants (BS) are amphiphilic biological molecules produced extracellularly or as a part of the cell membrane, from a variety of yeasts, bacteria and fungi. They are used in a range of applications one of which is bioremediation. During bioremediation of crude oil in sea, BS increase the bioavailability of the pollutant and thus, the rate of degradation by the native microorganisms.Biosurfactant production is highly dependent on the carbon source used in the culture. Most commonly, the carbon source is soluble and the biosurfactant product has substrate impurities. This fact makes the production of purified BS more difficult and expensive. During this experiment, heavy oil fractions (HOF) were used as carbon source. The aim of this investigation was to extract purified BS with a simple solvent extraction from the culture.The microorganism used was Alcanivorax borkumensis SK2. This bacterium was isolated from Elefsina bay during previous investigation of our lab. This microorganism is known for its glycolipid production and its oil degradation ability. Furthermore, it is commonly found in oil polluted marine environments.For the biosurfactant production, a Bioengineering bioreactor was used, as to have stable and controlled conditions in the culture but also a bigger scale. The maximum BS production in the bioreactor was 20 mg/L. According to other investigations and the bibliography, this concentration is quite low, but the main asset of the mythology used, is the fact that purified BS were isolated by a simple solvent extraction.Thin layer chromatography was carried out to compare the BS produced in the lab with a pure rhamnolipid sample (R-95 RL95% της Aldrich Chemistry). The BS produced by A. borkumensis SK2 contains mono- and di- rhamnolipids, and two other non-detected biosurfactants.Furthermore, the baffled flask test (BFT) protocol (according to the EPA) was implemented as to detect the dispersion of crude oil in water, by using BS. BS were previously diluted in soybean oil and the different BS concentrations tested were 0%, 5%, 7,5% and 15% w/v. The percent increase of oil dispersion in each case was 0%, 25.85%, 74.85% and 77.76% respectively.Last but not least, a bioremediation test was carried out as to test the effect of BS in the biodegradation of crude oil. BS were diluted in water and crude oil. GC-MS was used as to estimate the degradation of crude oil after specific time frames. Indicatively, the average mass reduction of saturated hydrocarbons, after 28 days of experiment, was 52,18% for BS diluted in water (2 mg active agent / 10 ml seawater) and 29,45% for BS diluted in soybean oil (5 mg active agent / 10 ml seawater).

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