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

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.     

Comparison of mechanisms of alkane metabolism under sulfate-reducing conditions among two bacterial isolates and a bacterial consortium

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 [1,2-13C2]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 d34-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 sulfate-reducing 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.     

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

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 [1,2-¹³C₂]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 d₃₄-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 sulfate-reducing 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.     

Initial reactions in anaerobic alkane degradation by a sulfate reducer, strain AK-01

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-(13)C(2)]hexadecane or perdeuterated pentadecane was used as the growth substrate, (13)C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the (13)C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two (13)C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1, 2-(13)C(2)]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.     

Initial Reactions in Anaerobic Alkane Degradation by a Sulfate Reducer, Strain AK-01

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-¹³C₂]hexadecane or perdeuterated pentadecane was used as the growth substrate, ¹³C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the ¹³C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two ¹³C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1,2-¹³C₂]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.     

Anaerobic n-alkane metabolism by a sulfate-reducing bacterium, Desulfatibacillum aliphaticivorans strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Anaerobic transformation of alkanes to fatty acids by a sulfate-reducing bacterium,strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and (13)C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and (13)C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-(13)C(2)]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [(13)C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a (13)C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-(13)C(2)]hexadecane-derived fatty acids contained either two (13)C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Anaerobic n-Alkane Metabolism by a Sulfate-Reducing Bacterium, Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803^T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Anaerobic 1-Alkene Metabolism by the Alkane- and Alkene-Degrading Sulfate Reducer Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane- and alkene-degrading, marine sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, known to oxidize n-alkanes anaerobically by fumarate addition at C-2, was investigated for its 1-alkene metabolism. The total cellular fatty acids of this strain were predominantly C-(even number) (C-even) when it was grown on C-even 1-alkenes and predominantly C-(odd number) (C-odd) when it was grown on C-odd 1-alkenes. Detailed analyses of those fatty acids by gas chromatography-mass spectrometry after 6- to 10-week incubations allowed the identification of saturated 2- and 4-ethyl-, 2- and 4-methyl-, and monounsaturated 4-methyl-branched fatty acids with chain lengths that correlated with those of the 1-alkene. The growth of D. aliphaticivorans on (per)deuterated 1-alkenes provided direct evidence of the anaerobic transformation of these alkenes into the corresponding 1-alcohols and into linear as well as 10- and 4-methyl-branched fatty acids. Experiments performed with [¹³C]bicarbonate indicated that the initial activation of 1-alkene by the addition of inorganic carbon does not occur. These results demonstrate that D. aliphaticivorans metabolizes 1-alkene by the oxidation of the double bond at C-1 and by the subterminal addition of organic carbon at both ends of the molecule [C-2 and C-(ω-1)]. The detection of ethyl-branched fatty acids from unlabeled 1-alkenes further suggests that carbon addition also occurs at C-3. Alkylsuccinates were not observed as potential initial intermediates in alkene metabolism. Based on our observations, the first pathways for anaerobic 1-alkene metabolism in an anaerobic bacterium are proposed. Those pathways indicate that diverse initial reactions of 1-alkene activation can occur simultaneously in the same strain of sulfate-reducing bacterium.     

Anaerobic Transformation of Alkanes to Fatty Acids by a Sulfate-Reducing Bacterium, Strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and ¹³C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and ¹³C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-¹³C₂]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [¹³C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a ¹³C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-¹³C₂]hexadecane-derived fatty acids contained either two ¹³C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Production of highly 13C-labeled polyphenols in Vitis vinifera cell bioreactor cultures

The use of Vitis vinifera cells grown in a 2 l-stirred tank bioreactor for producing isotopically 13C-labeled phenolic substances is presented. Several culture parameters were optimized to achieve characteristics of growth and polyphenol metabolism similar to that recorded in shake flasks. Administration of [1-13C]L-phenylalanine (3 mM) to grape cell suspension cultures led to the production of 13C-labeled stilbenes (trans- and cis-piceids), catechins (catechin and epicatechin) and anthocyanins (delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-3-O-beta-glucosides). Incorporation of [1-13C]L-phenylalanine into polyphenols was measured by means of 13C satellites in the proton NMR spectrum and EA-IRMS. The enrichment of labeling obtained for all the compounds (between 40 and 65%) is sufficient to investigate their absorption and metabolism in humans.     

Anaerobic degradation of n-hexane in a denitrifying bacterium: Further degradation of the initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement

The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3- d1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional beta-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.     

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.     

Anaerobic initial reaction of n-alkanes in a denitrifying bacterium: evidence for (1-methylpentyl)succinate as initial product and for involvement of an organic radical in n-hexane metabolism

A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize n-alkanes anaerobically with nitrate as the electron acceptor to CO(2) formed (1-methylpentyl)succinate (MPS) during growth on n-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on n-hexanoate. Anaerobic growth with [1-(13)C]n-hexane or d(14)-n-hexane led to a 1-methylpentyl side chain in MPS with one (13)C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from n-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in n-hexane-grown cells but not in n-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of n-hexane and that of toluene, even though n-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that n-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-d(2)-fumarate was added to cultures growing on unlabeled n-hexane, 3-d(1)-MPS rather than 2,3-d(2)-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism.     

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3, 4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate

We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.     

Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate.

We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.     

Anaerobic Biodegradation of n-Hexadecane by a Nitrate-Reducing Consortium

Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H₃₄-hexadecane, fully deuterated hexadecane (d₃₄-hexadecane), or H₃₄-hexadecane and NaH¹³CO₃. Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H₂₉]pentadecanoic acid, [H₂₅]tridecanoic acid, [1-¹³C]pentadecanoic acid, [3-¹³C]heptadecanoic acid, [3-¹³C]10-methylheptadecanoic acid, and d₂₇-pentadecanoic, d₂₅-, and d₂₄-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-¹⁴C]hexadecane was demonstrated based on the recovery of ¹⁴CO₂ in active cultures.Linear alkanes account for a large component of crude and refined petroleum products and, therefore, are of environmental significance with respect to their fate and transport (38). The aerobic activation of alkanes is well documented and involves monooxygenase and dioxygenase enzymes in which not only is oxygen required as an electron acceptor but it also serves as a reactant in hydroxylation (2, 16, 17, 32, 34). Alkanes are also degraded under anoxic conditions via novel degradation strategies (34). To date, there are two known pathways of anaerobic n-alkane degradation: (i) alkane addition to fumarate, commonly referred to as fumarate addition, and (ii) a putative pathway, proposed by So et al. (25), involving carboxylation of the alkane. Fumarate addition proceeds via terminal or subterminal addition (C-2 position) of the alkane to the double bond of fumarate, resulting in the formation of an alkylsuccinate. The alkylsuccinate is further degraded via carbon skeleton rearrangement and β-oxidation (4, 6, 8, 12, 13, 21, 37). Alkane addition to fumarate has been documented for a denitrifying isolate (21, 37), sulfate-reducing consortia (4, 8, 12, 13), and five sulfate-reducing isolates (4, 6-8, 12). In addition to being demonstrated in these studies, fumarate addition in a sulfate-reducing enrichment growing on the alicyclic alkane 2-ethylcyclopentane has also been demonstrated (23). In contrast to fumarate addition, which has been shown for both sulfate-reducers and denitrifiers, the putative carboxylation of n-alkanes has been proposed only for the sulfate-reducing isolate strain Hxd3 (25) and for a sulfate-reducing consortium (4). Experiments using NaH¹³CO₃ demonstrated that bicarbonate serves as the source of inorganic carbon for the putative carboxylation reaction (25). Subterminal carboxylation of the alkane at the C-3 position is followed by elimination of the two terminal carbons, to yield a fatty acid that is one carbon shorter than the parent alkane (4, 25). The fatty acids are subject to β-oxidation, chain elongation, and/or C-10 methylation (25).In this study, we characterized an alkane-degrading, nitrate-reducing consortium and surveyed the metabolites of the consortium incubated with either unlabeled or labeled hexadecane in order to elucidate the pathway of n-alkane degradation. We present evidence of a pathway analogous to the proposed carboxylation pathway under nitrate-reducing conditions.     

Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 μM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 ± 0.003 nmol min⁻¹ mg of protein⁻¹ only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.