Formation of n-alkane- and cycloalkane-derived organic acids during anaerobic growth of a denitrifying bacterium with crude oil

The formation of metabolites during anaerobic biodegradation of saturated hydrocarbons directly from crude oil in the absence of oxygen was investigated using a denitrifying bacterium, the Azoarcus-like strain HxN1, which can utilise C6–C8 n-alkanes anaerobically as growth substrates. Various alkylsuccinates (apparently diastereomers) with alkyl chains (probably linked at C-2) ranging from C4 to C8 were detected by gas chromatography–mass spectrometry. These metabolites apparently result from the activation reaction of C4–C8 alkanes with cellular fumarate, analogous to the recently established reaction of pure n-hexane with fumarate in strain HxN1 to yield (1-methylpentyl)succinate. Other succinates carried substituents derived from cyclopentane and methylcyclopentane and hence indicated an activation of such cycloalkanes. Since n-butane, n-pentane or cycloalkanes as single compounds did not support growth of strain HxN1, their apparent products point to co-metabolic reactions during utilisation of the C6–C8 n-alkanes. Furthermore, methyl-branched and cyclopentyl-substituted fatty acids were detected. This finding is explained by a further metabolism of the substituted succinates via carbon skeleton rearrangement and decarboxylation. All metabolites detected in the oil-grown cultures were also identified in cultures grown with defined mixtures of saturated hydrocarbons. Results are of potential value for an understanding of metabolite formation in hydrocarbon-rich anoxic environments from the viewpoint of bacterial physiology.