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Applied and Environmental Microbiology, December 2005, p. 8174-8182, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8174-8182.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Irene A. Davidova,^1,2 Lisa M. Gieg,^1,2 Mark Nanny,^2,3 Kevin G. Kropp,^1,2, and Joseph M. Suflita^1,2*

Department of Botany & Microbiology,¹ Institute for Energy and the Environment,² Department of Civil Engineering and Environmental Science, The University of Oklahoma, Norman, Oklahoma 73019³

Received 13 June 2005/ Accepted 4 August 2005

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C₆ to C₁₂) and 1-¹³C-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 d₁₄-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 1-¹³C-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 ¹³C nuclei from 1-¹³C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that ¹³C-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.

Present address: Guardian Chemicals Inc., Ft. Saskatchewan, AB, Canada.

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