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Versatile transformations of hydrocarbons in anaerobic bacteria: substrate ranges and regio- and stereo-chemistry of activation reactions.

Jarling R, Kühner S, Basílio Janke E, Gruner A, Drozdowska M, Golding BT, Rabus R, Wilkes H - Front Microbiol (2015)

Bottom Line: In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures.Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers.The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments.

View Article: PubMed Central - PubMed

Affiliation: Organic Geochemistry, Chemistry of the Earth, Helmholtz Centre Potsdam GFZ German Research Centre for Geosciences Potsdam, Germany.

ABSTRACT
Anaerobic metabolism of hydrocarbons proceeds either via addition to fumarate or by hydroxylation in various microorganisms, e.g., sulfate-reducing or denitrifying bacteria, which are specialized in utilizing n-alkanes or alkylbenzenes as growth substrates. General pathways for carbon assimilation and energy gain have been elucidated for a limited number of possible substrates. In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures. We show that the range of co-metabolically formed alkyl- and arylalkyl-succinates is much broader in n-alkane than in alkylbenzene utilizers. The structures and stereochemistry of these products are resolved. Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers. We propose that these processes play a role in detoxification under conditions of solvent stress. The thermophilic sulfate-reducing strain TD3 is shown to produce n-alkylsuccinates, which are suggested not to derive from terminal activation of n-alkanes, but rather to represent intermediates of a metabolic pathway short-cutting fumarate regeneration by reverse action of succinate synthase. The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments.

No MeSH data available.


Related in: MedlinePlus

Formation of short chain n-alkylsuccinates from n-alkanes with chain length > C6 in strain TD3. (A) Relative amounts of n-alkylsuccinates (as dimethyl esters) found in cultures of strain TD3 grown with the mentioned n-alkane as sole source of carbon and energy. Relative amounts were determined by integration of respective summed mass traces from GC-MS analyses and normalization to 100% (circle diameter represents the relative amount). (B) Potential pathway for formation and further utilization of short chain n-alkylsuccinates.
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Figure 2: Formation of short chain n-alkylsuccinates from n-alkanes with chain length > C6 in strain TD3. (A) Relative amounts of n-alkylsuccinates (as dimethyl esters) found in cultures of strain TD3 grown with the mentioned n-alkane as sole source of carbon and energy. Relative amounts were determined by integration of respective summed mass traces from GC-MS analyses and normalization to 100% (circle diameter represents the relative amount). (B) Potential pathway for formation and further utilization of short chain n-alkylsuccinates.

Mentions: In case of n-alkanes it is generally accepted that these molecules are activated at carbon atom 2, leading to a prominent homologous series of (1-methylalkyl)succinate diastereoisomers, when the respective bacteria anaerobically grow with crude oil (Figure 1A, dark blue; see also Wilkes et al., 2003). Only in a few cases activations at the carbon atom 3 (n-hexane, strain HxN1) or the terminal methyl group (propane, strain BuS5) have been documented to occur as side reactions (Rabus et al., 2001; Kniemeyer et al., 2007). As confirmed by experiments with single substrates or binary substrate mixtures, all tested n-alkane-utilizing strains form succinate derivatives eluting in front of the (1-methylalkyl)succinates (Figure 1A, light blue). These by-products have identical mass spectrometric fragmentation patterns and originate from less favorable transformations of the n-alkanes. The respective product from n-hexane was identified by comparison with a reference standard as (1-ethylbutyl)succinate in strains OcN1 and TD3 (this study) and HxN1 (Rabus et al., 2001), while tentative assignment of its homologs is based on relative retention times and mass spectrometric fragmentation patterns. Moreover, strains OcN1 and TD3 form isopropyl- as well as n-propylsuccinate in cultures with a mixture of their growth substrates (n-octane and n-decane, respectively) and propane, which were identified by comparisons with reference standards. However, these two strains significantly differ with respect to the ratio of isopropyl- to n-propylsuccinate (231.6 in OcN1 vs. 6.5 in TD3). In addition, strain TD3 unexpectedly also formed C1–C7n-alkylsuccinates during anaerobic growth with pure n-alkanes (> C6), binary substrate mixtures containing at least one such n-alkane or crude oil (Figure 1A, red peaks). Methyl-, n-propyl-, and n-butylsuccinate were identified by comparison with reference standards, while assignment of the other n-alkylsuccinates is based on relative GC retention times and mass spectra. Notably, C-even n-alkylsuccinates were predominant upon growth with C-odd n-alkanes and vice versa (Figure 2A).


Versatile transformations of hydrocarbons in anaerobic bacteria: substrate ranges and regio- and stereo-chemistry of activation reactions.

Jarling R, Kühner S, Basílio Janke E, Gruner A, Drozdowska M, Golding BT, Rabus R, Wilkes H - Front Microbiol (2015)

Formation of short chain n-alkylsuccinates from n-alkanes with chain length > C6 in strain TD3. (A) Relative amounts of n-alkylsuccinates (as dimethyl esters) found in cultures of strain TD3 grown with the mentioned n-alkane as sole source of carbon and energy. Relative amounts were determined by integration of respective summed mass traces from GC-MS analyses and normalization to 100% (circle diameter represents the relative amount). (B) Potential pathway for formation and further utilization of short chain n-alkylsuccinates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561516&req=5

Figure 2: Formation of short chain n-alkylsuccinates from n-alkanes with chain length > C6 in strain TD3. (A) Relative amounts of n-alkylsuccinates (as dimethyl esters) found in cultures of strain TD3 grown with the mentioned n-alkane as sole source of carbon and energy. Relative amounts were determined by integration of respective summed mass traces from GC-MS analyses and normalization to 100% (circle diameter represents the relative amount). (B) Potential pathway for formation and further utilization of short chain n-alkylsuccinates.
Mentions: In case of n-alkanes it is generally accepted that these molecules are activated at carbon atom 2, leading to a prominent homologous series of (1-methylalkyl)succinate diastereoisomers, when the respective bacteria anaerobically grow with crude oil (Figure 1A, dark blue; see also Wilkes et al., 2003). Only in a few cases activations at the carbon atom 3 (n-hexane, strain HxN1) or the terminal methyl group (propane, strain BuS5) have been documented to occur as side reactions (Rabus et al., 2001; Kniemeyer et al., 2007). As confirmed by experiments with single substrates or binary substrate mixtures, all tested n-alkane-utilizing strains form succinate derivatives eluting in front of the (1-methylalkyl)succinates (Figure 1A, light blue). These by-products have identical mass spectrometric fragmentation patterns and originate from less favorable transformations of the n-alkanes. The respective product from n-hexane was identified by comparison with a reference standard as (1-ethylbutyl)succinate in strains OcN1 and TD3 (this study) and HxN1 (Rabus et al., 2001), while tentative assignment of its homologs is based on relative retention times and mass spectrometric fragmentation patterns. Moreover, strains OcN1 and TD3 form isopropyl- as well as n-propylsuccinate in cultures with a mixture of their growth substrates (n-octane and n-decane, respectively) and propane, which were identified by comparisons with reference standards. However, these two strains significantly differ with respect to the ratio of isopropyl- to n-propylsuccinate (231.6 in OcN1 vs. 6.5 in TD3). In addition, strain TD3 unexpectedly also formed C1–C7n-alkylsuccinates during anaerobic growth with pure n-alkanes (> C6), binary substrate mixtures containing at least one such n-alkane or crude oil (Figure 1A, red peaks). Methyl-, n-propyl-, and n-butylsuccinate were identified by comparison with reference standards, while assignment of the other n-alkylsuccinates is based on relative GC retention times and mass spectra. Notably, C-even n-alkylsuccinates were predominant upon growth with C-odd n-alkanes and vice versa (Figure 2A).

Bottom Line: In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures.Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers.The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments.

View Article: PubMed Central - PubMed

Affiliation: Organic Geochemistry, Chemistry of the Earth, Helmholtz Centre Potsdam GFZ German Research Centre for Geosciences Potsdam, Germany.

ABSTRACT
Anaerobic metabolism of hydrocarbons proceeds either via addition to fumarate or by hydroxylation in various microorganisms, e.g., sulfate-reducing or denitrifying bacteria, which are specialized in utilizing n-alkanes or alkylbenzenes as growth substrates. General pathways for carbon assimilation and energy gain have been elucidated for a limited number of possible substrates. In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures. We show that the range of co-metabolically formed alkyl- and arylalkyl-succinates is much broader in n-alkane than in alkylbenzene utilizers. The structures and stereochemistry of these products are resolved. Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers. We propose that these processes play a role in detoxification under conditions of solvent stress. The thermophilic sulfate-reducing strain TD3 is shown to produce n-alkylsuccinates, which are suggested not to derive from terminal activation of n-alkanes, but rather to represent intermediates of a metabolic pathway short-cutting fumarate regeneration by reverse action of succinate synthase. The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments.

No MeSH data available.


Related in: MedlinePlus