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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.


Phylogenetic affiliation of the most abundant phylotype in the enrichment culture, Cyhx28-EdB-clone63 (marked in bold-face). The phylogenetic tree was calculated in ARB by neighbor-joining, using only nearly full-length sequences (>1300 nt), with application of different sets of filters. The numbers next to nodes indicate bootstrap values higher than 50%. The scale bar represents 10% estimated sequence divergence.
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Figure 2: Phylogenetic affiliation of the most abundant phylotype in the enrichment culture, Cyhx28-EdB-clone63 (marked in bold-face). The phylogenetic tree was calculated in ARB by neighbor-joining, using only nearly full-length sequences (>1300 nt), with application of different sets of filters. The numbers next to nodes indicate bootstrap values higher than 50%. The scale bar represents 10% estimated sequence divergence.

Mentions: Microscopic analyses showed an apparent dominance by oval to rod-shaped cells of 1.5 μm length × 0.8 μm diameter, on average (Figure 1). Upon depletion of cyclohexane and formation of high concentrations of sulfide some cells showed an elongated morphology (Figure 1). Since attempts to isolate the cyclohexane-degrading microorganism in pure culture were not successful, the enrichment culture was further analyzed by molecular biology methods. Construction and analysis of a 16S rRNA gene library (n = 96) showed that most of the sequences (n = 53) were affiliated with the Deltaproteobacteria. Of the Deltaproteobacterial sequences, the majority (n = 42) were affiliated with the Desulfosarcina-Desulfococcus clade (>94% sequence identity). Other Deltaproteobacteria-affiliated sequences were closely related to Desulfobacterium anilini (n = 4) and Desulfotignum balticum (n = 6) (Figure SI1). A large number of clones (n = 37) were closely related to sequences belonging to the OP3 cluster, a group within the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, from which no isolated strains have been reported so far (e.g., Rotaru et al., 2012). In an attempt to identify the putative most abundant microorganisms, the 16S rRNA gene was amplified using increasingly diluted DNA template. The PCR products from the highest template dilution yielding a result (10−2) were sequenced without cloning. The sequences obtained were identical with one of the clones affiliated with the Desulfosarcina-Desulfococcus cluster, Cyhx28-EdB-clone63 (Figure 2). Cyhx28-EdB-clone63 was closely related to sequences from the Amsterdam mud volcano (94.7% identity; (Pachiadaki et al., 2011), Gulf of Cadiz mud volcano (>93% identity), clone sequences from a naphthalene-degrading enrichment culture (Selesi et al., 2010), Guerrero Negro hypersaline mat clones (Harris et al., 2013) and Zodletone spring sediment clones (Youssef et al., 2012). The closest cultivated relatives were strain BuS5 (92.7% identity; Kniemeyer et al., 2007), and dominant phylotypes in propane- and n-butane-degrading enrichment cultures from the Gulf of Mexico and Hydrate Ridge sediments (Kniemeyer et al., 2007; Jaekel et al., 2013). Substrate tests with strain BuS5 showed that this microorganism degraded only the gaseous alkanes propane and n-butane (Kniemeyer et al., 2007). Shorter (ethane and methane) or longer (n-pentane and higher) alkanes did not serve as growth subtstrates. A similarly restricted substrate range was found for the enrichment cultures Prop12-GMe, But12-GMe, and But12-HyR, which also degraded only propane and n-butane (Jaekel et al., 2013).


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

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

Phylogenetic affiliation of the most abundant phylotype in the enrichment culture, Cyhx28-EdB-clone63 (marked in bold-face). The phylogenetic tree was calculated in ARB by neighbor-joining, using only nearly full-length sequences (>1300 nt), with application of different sets of filters. The numbers next to nodes indicate bootstrap values higher than 50%. The scale bar represents 10% estimated sequence divergence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352924&req=5

Figure 2: Phylogenetic affiliation of the most abundant phylotype in the enrichment culture, Cyhx28-EdB-clone63 (marked in bold-face). The phylogenetic tree was calculated in ARB by neighbor-joining, using only nearly full-length sequences (>1300 nt), with application of different sets of filters. The numbers next to nodes indicate bootstrap values higher than 50%. The scale bar represents 10% estimated sequence divergence.
Mentions: Microscopic analyses showed an apparent dominance by oval to rod-shaped cells of 1.5 μm length × 0.8 μm diameter, on average (Figure 1). Upon depletion of cyclohexane and formation of high concentrations of sulfide some cells showed an elongated morphology (Figure 1). Since attempts to isolate the cyclohexane-degrading microorganism in pure culture were not successful, the enrichment culture was further analyzed by molecular biology methods. Construction and analysis of a 16S rRNA gene library (n = 96) showed that most of the sequences (n = 53) were affiliated with the Deltaproteobacteria. Of the Deltaproteobacterial sequences, the majority (n = 42) were affiliated with the Desulfosarcina-Desulfococcus clade (>94% sequence identity). Other Deltaproteobacteria-affiliated sequences were closely related to Desulfobacterium anilini (n = 4) and Desulfotignum balticum (n = 6) (Figure SI1). A large number of clones (n = 37) were closely related to sequences belonging to the OP3 cluster, a group within the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, from which no isolated strains have been reported so far (e.g., Rotaru et al., 2012). In an attempt to identify the putative most abundant microorganisms, the 16S rRNA gene was amplified using increasingly diluted DNA template. The PCR products from the highest template dilution yielding a result (10−2) were sequenced without cloning. The sequences obtained were identical with one of the clones affiliated with the Desulfosarcina-Desulfococcus cluster, Cyhx28-EdB-clone63 (Figure 2). Cyhx28-EdB-clone63 was closely related to sequences from the Amsterdam mud volcano (94.7% identity; (Pachiadaki et al., 2011), Gulf of Cadiz mud volcano (>93% identity), clone sequences from a naphthalene-degrading enrichment culture (Selesi et al., 2010), Guerrero Negro hypersaline mat clones (Harris et al., 2013) and Zodletone spring sediment clones (Youssef et al., 2012). The closest cultivated relatives were strain BuS5 (92.7% identity; Kniemeyer et al., 2007), and dominant phylotypes in propane- and n-butane-degrading enrichment cultures from the Gulf of Mexico and Hydrate Ridge sediments (Kniemeyer et al., 2007; Jaekel et al., 2013). Substrate tests with strain BuS5 showed that this microorganism degraded only the gaseous alkanes propane and n-butane (Kniemeyer et al., 2007). Shorter (ethane and methane) or longer (n-pentane and higher) alkanes did not serve as growth subtstrates. A similarly restricted substrate range was found for the enrichment cultures Prop12-GMe, But12-GMe, and But12-HyR, which also degraded only propane and n-butane (Jaekel et al., 2013).

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.