Stable Isotopic Studies of n -Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

Abstract

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n -alkanes (C ₆ to C ₁₂ ) and 1- ¹³ C-labeled n -hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d ₁₄ -hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n -hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1- ¹³ C-labeled n -hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that ¹³ C nuclei from 1- ¹³ C-labeled n -hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that ¹³ C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177: 235-243, 2002) for metabolism of deuterated n -hexane by a denitrifying bacterium.