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...

Recent studies have demonstrated that fumarate addition and carboxylation are two possible mechanisms of anaerobic alkane degradation. In the present study, we surveyed metabolites formed during growth on hexadecane by the sulfate-reducing isolates AK-01 and Hxd3 and by a mixed sulfate-reducing consortium. The cultures were incubated with either protonated or fully deuterated hexadecane; the sulfate-reducing consortium was also incubated with [1,2-13 C 2 ]hexadecane. All cultures were extracted, silylated, and analyzed by gas chromatography-mass spectrometry. We detected a suite of metabolites that support a fumarate addition mechanism for hexadecane degradation by AK-01, including methylpentadecylsuccinic acid, 4-methyloctadecanoic acid, 4-methyloctadec-2,3-enoic acid, 2-methylhexadecanoic acid, and tetradecanoic acid. By using d 34 -hexadecane, mass spectral evidence strongly supporting a carbon skeleton rearrangement of the first intermediate, methylpentadecylsuccinic acid, was demonstrated for AK-01. Evidence indicating hexadecane carboxylation was not found in AK-01 extracts but was observed in Hxd3 extracts. In the mixed sulfatereducing culture, however, metabolites consistent with both fumarate addition and carboxylation mechanisms of hexadecane degradation were detected, which demonstrates that multiple alkane degradation pathways can occur simultaneously within distinct anaerobic communities. Collectively, these findings underscore that fumarate addition and carboxylation are important alkane degradation mechanisms that may be widespread among phylogenetically and/or physiologically distinct microorganisms.

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...

Condensed thiophenes comprise a significant portion of the organosulfur compounds in petroleum and in other products from fossil fuels. Dibenzothiophene (DBT) has served as a model compound in biodegradation studies for over two decades. However, until quite recently, few other organosulfur compounds were studied, and their fates in petroleum-contaminated environments are largely unknown. This paper presents a review of the types of organosulfur compounds found in petroleum and summarizes the scant literature on toxicity studies with condensed thiophenes. Reports on the biodegradation of benzothiophene, alkylbenzothiophenes, DBT, alkylDBTs, and naphthothiophenes are reviewed with a focus on the identification of metabolites detected in laboratory cultures. In addition, recent reports on quantitative studies with DBT and naphtho[2,1-b]thiophene indicate the existence of polar sulfur-containing metabolites that have escaped detection and identification.

No abstract