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Élodie Nicolau

Biodegradation of 2-ethylhexyl nitrate by Mycobacterium austroafricanum IFP 2173

Published on 7 October 2008


Thesis presented October 07, 2008

Abstract:
Compounds such as 2-ethylhexyl nitrate (2-EHN) are added to diesel fuel to improve ignition and boost cetane number. The production of 2-EHN reaches around 100 000 tons per year in France, principally. Risks associated to its utilization are however poorly known because, in case of accidental release in the environment, nothing is known about its biodegradability.
In this study, we aimed to (i) identify bacterial strains able to degrade 2-EHN and compare their capabilities, (ii) elucidate the degradation pathway, and (iii) identify the enzymes involved. Biodegradation of 2-EHN was first tested in biphasic cultures under conditions that reduce the toxicity and increase the availability of the hydrophobic substrate. Using optimized culture conditions, we showed that several strains of Mycobacterium austroafricanum were able to degrade 2-EHN. One of the most efficient strain (IFP 2173) which could grow at 2-EHN concentrations up to 6 g.l-1, was chosen to investigate the degradation pathway. ​On the basis of carbon balance determination and gas chromatographic (GC) analysis on the culture medium, I found that the degradation of 2-EHN was incomplete and gave rise to the accumulation of a metabolite. This metabolite was identified as ß-methyl-
γ-butyrolactone by GC-MS and LC-MS/MS analysis. The structure of the lactone indicated that 2-EHN was degraded through a pathway involving hydroxylation the methyl group of the main carbon chain, its oxidation into aldehyde an acid and a subsequent cycle of ß-oxidation.
Enzymes involved in the 2-EHN biodegradation pathway were looked for by a proteomic approach. Analyses by two-dimensional gel electrophoresis showed that, when exposed to 2-EHN, strain IFP 2173 triggered the synthesis of a bunch of enzymes specialized in fatty acid metabolism such as ß-oxidation enzymes, as well as alcohol and aldehyde dehydrogenases. An exhaustive analysis of the IFP 2173 proteome resulted in the identification of more than 200 proteins induced on 2-EHN, including a cytochrome P450 predicted to function as an alkane monooxygenase (CYP153). Moreover, I also cloned genes encoding two trans-membrane alkane hydroxylases, which passed undetected in the proteomic analysis. Hence, strain IFP 2173 syntheses three alkane hydroxylases potentially able to catalyze the initial attack on 2-EHN. In order to determine which of the three monooxygenases was responsible for this reaction, their structural gene was cloned in plasmids designed for the expression in either E. coli or M. smegmatis mc2. Preliminary results indicated that the recombinant proteins were produced in some cases. These constructions will be used to separately study the activity of each enzyme with respect to alkane and 2-EHN hydroxylation.

Keywords:
2-ethylhexyl nitrate, Mycobacterium austroafricanum, biodegradation, 4-ethyldihydrofuran2-(3H)one, alkane hydroxylase, branched alkane, cyp153 and alkB genes

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