Biodegradation of 2-ethylhexyl nitrate (2-EHN) by Mycobacterium austroafricanum IFP 2173

19 2-Ethyhexyl nitrate (2-EHN) is a major additive of fuel which is used to comply with the 20 cetane number of diesel. Because of its wide use and possible accidental release, 2-EHN is a 21 potential pollutant of the environment. In this study, Mycobacterium austroafricanum IFP 22 2173 was selected among several strains as the best 2-EHN degrader. The 2-EHN 23 biodegradation rate was increased in biphasic cultures where the hydrocarbon was dissolved 24 in an inert non-aqueous phase liquid (NAPL), suggesting that the transfer of the hydrophobic 25 substrate to the cells was a growth-limiting factor. Carbon balance calculation as well as 26 organic carbon measurement indicated a release of metabolites in the culture medium. Further 27 analysis by gas chromatography revealed that a single metabolite accumulated during growth. 28 This metabolite had a molecular mass of 114 Da as determined by GC/MS and was 29 provisionally identified as 4-ethyldihydrofuran-2(3H)-one by LC-MS/MS analysis. 30 Identification was confirmed by analysis of the chemically synthesized lactone. Based on 31 these results, a plausible catabolic pathway is proposed whereby 2-EHN is converted to 4-32 ethyldihydrofuran-2(3H)-one, which cannot be metabolised further by strain IFP 2173. This 33 putative pathway provides an explanation for the low energetic efficiency of 2-EHN 34 degradation and its poor biodegradability. 35 3 INTRODUCTION 36