Anaerobic Oxidation of n -Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture

Abstract

We identified trace metabolites produced during the anaerobic biodegradation of H 26 -and D 26 -n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by COH or COD addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H 26 -and D 26 -dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated n-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H 26 -dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.The anaerobic biodegradation of n-alkanes has recently been demonstrated under nitrate-reducing (6,11,19), sulfatereducing (1,2,7,9,(20)(21)(22), and methanogenic (3, 25) conditions. Pure bacterial cultures that couple the reduction of the first two electron acceptors to the oxidation of alkanes have been isolated (1,2,11,(20)(21)(22), but little is known about the initial mechanism(s) of anaerobic activation and subsequent metabolism of these hydrocarbons. Previous studies of the total cellular fatty acid composition of pure sulfate-reducing cultures grown on alkanes have suggested that activation does not occur via dehydrogenation to the corresponding 1-alkene (2) but does occur by addition of an unknown organic carbon fragment to the C-2 position of the original molecule (22).Recent research has shown that the anaerobic bacterial metabolism of toluene and xylenes is initiated by addition of the methyl group to the double bond of fumarate, forming optically pure (R)-(ϩ)-benzylsuccinic acid and methylbenzylsuccinic acids, respectively (4,5,(12)(13)(14). These addition reactions occur with retention of the proton abstracted from the methyl group in the succinyl moiety of the metabolite (4, 5). The detection of 3-phenyl-1,2-butanedicarboxylic acid from ethylbenzene (L. M. Gieg and J. M. Sufl...

Microbial nitrate-dependent cyclohexane degradation coupled with anaerobic ammonium oxidation

“…A metabolite with prominent fragment ions at m/z 114, 146, 155 and 197 was identified as cyclohexylsuccinic acid dimethyl ester on the basis of comparison with an authentic standard (Figure 1). This suggests that cyclohexane is anaerobically activated by addition to fumarate, similar as n-alkanes (Kropp et al, 2000;Rabus et al, 2001) and ethylcyclopentane (Rios-Hernandez et al, 2003). Furthermore, two other metabolites were tentatively identified (on the basis of the mass spectra) as 9-cyclohexylnonanoic acid and 11-cyclohexylundecanoic acid (Figure 2).…”

“…Anaerobic growth of pure cultures with cycloalkanes has not been observed (for example, Wilkes et al, 2003). An activation of alicyclic hydrocarbons by a radical-catalyzed addition to fumarate, analogous to n-alkane activation (Kropp et al, 2000;Rabus et al, 2001), was postulated on the basis of metabolite analysis in a sulfate-reducing enrichment culture with ethylcyclopentane (Rios-Hernandez et al, 2003). A denitrifying bacterium growing anaerobically with n-alkanes from petroleum co-metabolized cyclopentane and formed cyclopentylsuccinate (Wilkes et al, 2003); cyclopentane did not allow productive growth.…”

Recent Advances in Petroleum Microbiology

“…Similar operons may be present in other strains, as the novel benzylsuccinate synthase reaction, catalyzing the addition of fumarate to toluene (110,181), may also be involved in the metabolism of xylenes (349,444,445), alkylnaphthalenes (20,23), n-hexadecane (497), and n-dodecane (351). For example, dodecylsuccinic acids were detected from a sulfate-reducing enrichment culture growing on n-dodecane (351), and an nhexane-utilizing denitrifying bacterium with a protein similar to BssC has been isolated from the toluene-degrading denitrifying bacteria (664).…”

“…For example, dodecylsuccinic acids were detected from a sulfate-reducing enrichment culture growing on n-dodecane (351), and an nhexane-utilizing denitrifying bacterium with a protein similar to BssC has been isolated from the toluene-degrading denitrifying bacteria (664). In addition, the metabolites (1-methylpentyl)succinate and (1-ethylbenzyl)succinate from the anaerobic metabolism of n-hexane by a denitrifying strain indicate a C-2 and a C-3 addition of fumarate, analogous to the toluene activation reaction (497).…”

Anaerobic degradation of propane and butane by sulfate-reducing bacteria enriched from marine hydrocarbon cold seeps

“…Strain BuS5 can degrade both propane and butane and is phylogenetically affiliated with the Desulfosarcina-Desulfococcus cluster of the Deltaproteobacteria. Analysis of metabolites showed that propane and butane are activated similar to higher alkanes by addition to fumarate (Kropp et al, 2000;Rabus et al, 2001) primarily at the secondary carbon atom yielding iso-propyl-and (1-methylpropyl)-succinate, respectively (Kniemeyer et al, 2007;Savage et al, 2010).…”