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 sulfatereducing 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. The anaerobic biodegradation of n-alkanes is now a welldocumented process that has been demonstrated to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Several sulfate-reducing and denitrifying bacterial strains and enrichment cultures capable of complete n-alkane mineralization have been described (1,7,8,13,16,17,19,20). Anaerobic metabolism of n-alkanes has been shown to occur by at least two mechanisms, both of which include addition of carbon to the parent substrate. An activation mechanism involves an initial carboxylation reaction. Using the sulfate-reducing isolate strain Hxd3, So et al. (21) showed that 13 Cbicarbonate was added to the C-3 position of the parent alkane, while subsequent reactions involved elimination of the two adjacent terminal carbon atoms, yielding a fatty acid with one less carbon atom than the original hydrocarbon. Alternately, the primary attack on an alkane can be initiated by addition to the double bond of fumarate with the formation of alkylsuccinic acid derivatives (13,18). This mechanism seems to be more widespread as it has now been found in three separate anaerobic cultures (6,7,13,18,24). This reaction resembles the anaerobic biodegradation of toluene and xylene, in which the aryl methyl carbon of the substrate is added across the double bond of fumarate to form benzylsuccinate or methylated derivatives (2, 3). However, alkanes are not activated at the terminal methyl group. The succinyl moiety is attached to the parent alkane at the C-2 or C-3 position (7,13,18). The biochemistry of this conversion is incomplete, but electron paramagnetic resonance spectroscopy has suggested a possible radical mechanism since an organic radical was detected in n-hexane-grown cells of...

Comparison of Mechanisms of Alkane Metabolism under Sulfate-Reducing Conditions among Two Bacterial Isolates and a Bacterial Consortium

“…These observations demonstrate that the succinyl moiety in the metabolites is not attached to the terminal carbon atom in the hexadecyl moiety but is, rather, in a subterminal position yielding chromatographically distinct stereoisomers (diastereomers). These findings are consistent with those previously reported (20,29,40).…”

“…This product could result from direct decarboxylation of MPA or decarboxylation after C-skeleton rearrangement of MPA. This study and others (20,29,40) have shown that all deuterium atoms are retained in the fumarate addition metabolite. For MPA, the retained deuterium atom is attached to the methylene atom (C-2 position) on the succinyl moiety, based on the observation of a fragment ion at m/z 388 (Fig.…”

“…The mass spectra of these compounds were identical, and the fragmentation features suggest these metabolites are diastereomers of methylpentadecylsuccinic acid (MPA), the presumed fumarate addition product. An example is shown in (20,29,40). A McLafferty rearrangement produced an ion at m/z 264, which fragments further to m/z 173 (29).…”

“…This has been shown for sulfate-reducing consortia (20,29), a denitrifying isolate (40,55), and a sulfate-reducing isolate (19). Fumarate addition to cyclic alkanes has also recently been demonstrated (42,54).…”

Reactivity and fate of secondary alkane sulfonates (SAS) in marine sediments

“…During this sequence, a C3 unit is released ( Fig. 6D and E), which may be used for the regeneration of fumarate and later activation of other SAS molecules, according to Wilkes et al (2002) and Davidova et al (2005).…”