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Anaerobic degradation of cyclohexane by sulfate-reducing bacteria from hydrocarbon-contaminated marine sediments.

Jaekel U, Zedelius J, Wilkes H, Musat F - Front Microbiol (2015)

Bottom Line: Quantitative growth experiments showed that cyclohexane degradation was coupled with the stoichiometric reduction of sulfate to sulfide.Substrate response tests corroborated with hybridization with a sequence-specific oligonucleotide probe suggested that the dominant phylotype apparently was able to degrade other cyclic and n-alkanes, including the gaseous alkane n-butane.Other metabolites detected were 3-cyclohexylpropionate and cyclohexanecarboxylate providing evidence that the overall degradation pathway of cyclohexane under anoxic conditions is analogous to that of n-alkanes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology Bremen, Germany.

ABSTRACT
The fate of cyclohexane, often used as a model compound for the biodegradation of cyclic alkanes due to its abundance in crude oils, in anoxic marine sediments has been poorly investigated. In the present study, we obtained an enrichment culture of cyclohexane-degrading sulfate-reducing bacteria from hydrocarbon-contaminated intertidal marine sediments. Microscopic analyses showed an apparent dominance by oval cells of 1.5 × 0.8 μm. Analysis of a 16S rRNA gene library, followed by whole-cell hybridization with group- and sequence-specific oligonucleotide probes showed that these cells belonged to a single phylotype, and were accounting for more than 80% of the total cell number. The dominant phylotype, affiliated with the Desulfosarcina-Desulfococcus cluster of the Deltaproteobacteria, is proposed to be responsible for the degradation of cyclohexane. Quantitative growth experiments showed that cyclohexane degradation was coupled with the stoichiometric reduction of sulfate to sulfide. Substrate response tests corroborated with hybridization with a sequence-specific oligonucleotide probe suggested that the dominant phylotype apparently was able to degrade other cyclic and n-alkanes, including the gaseous alkane n-butane. Based on GC-MS analyses of culture extracts cyclohexylsuccinate was identified as a metabolite, indicating an activation of cyclohexane by addition to fumarate. Other metabolites detected were 3-cyclohexylpropionate and cyclohexanecarboxylate providing evidence that the overall degradation pathway of cyclohexane under anoxic conditions is analogous to that of n-alkanes.

No MeSH data available.


Response of cyclohexane-grown, dense-cell suspensions of the enrichment culture Cyhx28-EdB to additions of cycloalkanes (A), and n-alkanes (B), determined as hydrocarbon-dependent sulfate reduction. Cyclohexane-dependent sulfate reduction is shown in both panels as reference (•). The enrichment culture was apparently able to use all cyclic alkanes tested (A), as well as n-pentane and n-hexane (B). A response to addition of n-butane was recorded after a lag phase of 4 days (B, ×). No sulfide production could be detected in incubations with ethane, propane (B), and in substrate-free controls (◯, in both panels). The experiments were performed in anoxic tubes with 10 ml of a 15 × concentrated cell suspension, and confirmed by two independent incubations (here only one data set is shown). CARD-FISH with the sequence-specific probe Cyhx28-EdB_152 showed that the phylotype Cyhx28-EdB-clone63 was dominant in all positive test cultures at the end of the incubation time (Figure SI2).
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Figure 5: Response of cyclohexane-grown, dense-cell suspensions of the enrichment culture Cyhx28-EdB to additions of cycloalkanes (A), and n-alkanes (B), determined as hydrocarbon-dependent sulfate reduction. Cyclohexane-dependent sulfate reduction is shown in both panels as reference (•). The enrichment culture was apparently able to use all cyclic alkanes tested (A), as well as n-pentane and n-hexane (B). A response to addition of n-butane was recorded after a lag phase of 4 days (B, ×). No sulfide production could be detected in incubations with ethane, propane (B), and in substrate-free controls (◯, in both panels). The experiments were performed in anoxic tubes with 10 ml of a 15 × concentrated cell suspension, and confirmed by two independent incubations (here only one data set is shown). CARD-FISH with the sequence-specific probe Cyhx28-EdB_152 showed that the phylotype Cyhx28-EdB-clone63 was dominant in all positive test cultures at the end of the incubation time (Figure SI2).

Mentions: We tested the ability of the enrichment culture Cyhx28-EdB to degrade other hydrocarbons than cyclohexane. To prevent false positive results by the enrichment of microorganisms other than the dominant phylotype upon addition of new substrates, the experiments were done with concentrated cell suspensions and incubated for a relatively short time. The enrichment culture responded without a lag phase to additions of other cyclic alkanes, e.g., cyclopentane, methylcyclopentane and methylcyclohexane (Figure 5A). Of the n-alkanes tested, the enrichment culture was apparently able to grow with n-pentane and n-hexane (Figure 5B). The enrichment culture showed n-butane-dependent sulfate-reduction after a lag phase of 4 days (Figure 5B). No sulfate reduction could be detected in incubations with ethane or propane (Figure 5B), or with the aromatic hydrocarbons benzene and toluene (not shown). Hybridizations with the specific probe Cyhx28-EdB_152 showed that the phylotype Cyhx28-EdB-clone63 was highly abundant in all positive substrate test incubations (Figure SI2). These results suggest that the phylotype Cyhx28-EdB-clone63 was most likely responsible for the degradation of the tested hydrocarbons. To date, all reports about microorgansims capable of degrading cycloalkanes under anaerobic conditions showed degradation of single substrates, such as cyclohexane (Musat et al., 2010), ethylcyclopentane (Rios-Hernandez et al., 2003), or cyclopentene, methylcyclopentene, methylcyclopentane, cyclohexane and methylcyclohexane by distinct sulfate-reducing enrichment cultures (Townsend et al., 2004). In addition, it has been reported that the nitrate reducing strain HxN1 is able to co-activate (but not grow with) cyclopentane and methylcyclopentane during growth on n-hexane or crude oil (Wilkes et al., 2003). Given these results and the dominance of the phylotype Cyhx28-EdB-clone63 (>80%), we hypothesize that Cyhx28-EdB-clone63 is relatively versatile with respect to the range of hydrocarbons utilized, including C4-C6n-alkanes as well as methyl substituted and unsubstituted five- and six-ring cycloalkanes. Future studies should establish whether other cycloalkane degraders also display a broad substrate range as proposed for the phylotype Cyhx28-EdB-clone63.


Anaerobic degradation of cyclohexane by sulfate-reducing bacteria from hydrocarbon-contaminated marine sediments.

Jaekel U, Zedelius J, Wilkes H, Musat F - Front Microbiol (2015)

Response of cyclohexane-grown, dense-cell suspensions of the enrichment culture Cyhx28-EdB to additions of cycloalkanes (A), and n-alkanes (B), determined as hydrocarbon-dependent sulfate reduction. Cyclohexane-dependent sulfate reduction is shown in both panels as reference (•). The enrichment culture was apparently able to use all cyclic alkanes tested (A), as well as n-pentane and n-hexane (B). A response to addition of n-butane was recorded after a lag phase of 4 days (B, ×). No sulfide production could be detected in incubations with ethane, propane (B), and in substrate-free controls (◯, in both panels). The experiments were performed in anoxic tubes with 10 ml of a 15 × concentrated cell suspension, and confirmed by two independent incubations (here only one data set is shown). CARD-FISH with the sequence-specific probe Cyhx28-EdB_152 showed that the phylotype Cyhx28-EdB-clone63 was dominant in all positive test cultures at the end of the incubation time (Figure SI2).
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: Response of cyclohexane-grown, dense-cell suspensions of the enrichment culture Cyhx28-EdB to additions of cycloalkanes (A), and n-alkanes (B), determined as hydrocarbon-dependent sulfate reduction. Cyclohexane-dependent sulfate reduction is shown in both panels as reference (•). The enrichment culture was apparently able to use all cyclic alkanes tested (A), as well as n-pentane and n-hexane (B). A response to addition of n-butane was recorded after a lag phase of 4 days (B, ×). No sulfide production could be detected in incubations with ethane, propane (B), and in substrate-free controls (◯, in both panels). The experiments were performed in anoxic tubes with 10 ml of a 15 × concentrated cell suspension, and confirmed by two independent incubations (here only one data set is shown). CARD-FISH with the sequence-specific probe Cyhx28-EdB_152 showed that the phylotype Cyhx28-EdB-clone63 was dominant in all positive test cultures at the end of the incubation time (Figure SI2).
Mentions: We tested the ability of the enrichment culture Cyhx28-EdB to degrade other hydrocarbons than cyclohexane. To prevent false positive results by the enrichment of microorganisms other than the dominant phylotype upon addition of new substrates, the experiments were done with concentrated cell suspensions and incubated for a relatively short time. The enrichment culture responded without a lag phase to additions of other cyclic alkanes, e.g., cyclopentane, methylcyclopentane and methylcyclohexane (Figure 5A). Of the n-alkanes tested, the enrichment culture was apparently able to grow with n-pentane and n-hexane (Figure 5B). The enrichment culture showed n-butane-dependent sulfate-reduction after a lag phase of 4 days (Figure 5B). No sulfate reduction could be detected in incubations with ethane or propane (Figure 5B), or with the aromatic hydrocarbons benzene and toluene (not shown). Hybridizations with the specific probe Cyhx28-EdB_152 showed that the phylotype Cyhx28-EdB-clone63 was highly abundant in all positive substrate test incubations (Figure SI2). These results suggest that the phylotype Cyhx28-EdB-clone63 was most likely responsible for the degradation of the tested hydrocarbons. To date, all reports about microorgansims capable of degrading cycloalkanes under anaerobic conditions showed degradation of single substrates, such as cyclohexane (Musat et al., 2010), ethylcyclopentane (Rios-Hernandez et al., 2003), or cyclopentene, methylcyclopentene, methylcyclopentane, cyclohexane and methylcyclohexane by distinct sulfate-reducing enrichment cultures (Townsend et al., 2004). In addition, it has been reported that the nitrate reducing strain HxN1 is able to co-activate (but not grow with) cyclopentane and methylcyclopentane during growth on n-hexane or crude oil (Wilkes et al., 2003). Given these results and the dominance of the phylotype Cyhx28-EdB-clone63 (>80%), we hypothesize that Cyhx28-EdB-clone63 is relatively versatile with respect to the range of hydrocarbons utilized, including C4-C6n-alkanes as well as methyl substituted and unsubstituted five- and six-ring cycloalkanes. Future studies should establish whether other cycloalkane degraders also display a broad substrate range as proposed for the phylotype Cyhx28-EdB-clone63.

Bottom Line: Quantitative growth experiments showed that cyclohexane degradation was coupled with the stoichiometric reduction of sulfate to sulfide.Substrate response tests corroborated with hybridization with a sequence-specific oligonucleotide probe suggested that the dominant phylotype apparently was able to degrade other cyclic and n-alkanes, including the gaseous alkane n-butane.Other metabolites detected were 3-cyclohexylpropionate and cyclohexanecarboxylate providing evidence that the overall degradation pathway of cyclohexane under anoxic conditions is analogous to that of n-alkanes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology Bremen, Germany.

ABSTRACT
The fate of cyclohexane, often used as a model compound for the biodegradation of cyclic alkanes due to its abundance in crude oils, in anoxic marine sediments has been poorly investigated. In the present study, we obtained an enrichment culture of cyclohexane-degrading sulfate-reducing bacteria from hydrocarbon-contaminated intertidal marine sediments. Microscopic analyses showed an apparent dominance by oval cells of 1.5 × 0.8 μm. Analysis of a 16S rRNA gene library, followed by whole-cell hybridization with group- and sequence-specific oligonucleotide probes showed that these cells belonged to a single phylotype, and were accounting for more than 80% of the total cell number. The dominant phylotype, affiliated with the Desulfosarcina-Desulfococcus cluster of the Deltaproteobacteria, is proposed to be responsible for the degradation of cyclohexane. Quantitative growth experiments showed that cyclohexane degradation was coupled with the stoichiometric reduction of sulfate to sulfide. Substrate response tests corroborated with hybridization with a sequence-specific oligonucleotide probe suggested that the dominant phylotype apparently was able to degrade other cyclic and n-alkanes, including the gaseous alkane n-butane. Based on GC-MS analyses of culture extracts cyclohexylsuccinate was identified as a metabolite, indicating an activation of cyclohexane by addition to fumarate. Other metabolites detected were 3-cyclohexylpropionate and cyclohexanecarboxylate providing evidence that the overall degradation pathway of cyclohexane under anoxic conditions is analogous to that of n-alkanes.

No MeSH data available.