Anaerobic degradation of propane and butane by sulfate-reducing bacteria enriched from marine hydrocarbon cold seeps

Abstract

The short-chain, non-methane hydrocarbons propane and butane can contribute significantly to the carbon and sulfur cycles in marine environments affected by oil or natural gas seepage. In the present study, we enriched and identified novel propane and butane-degrading sulfate reducers from marine oil and gas cold seeps in the Gulf of Mexico and Hydrate Ridge. The enrichment cultures obtained were able to degrade simultaneously propane and butane, but not other gaseous alkanes. They were cold-adapted, showing highest sulfate-reduction rates between 16 and 20 °C. Analysis of 16S rRNA gene libraries, followed by whole-cell hybridizations with sequence-specific oligonucleotide probes showed that each enrichment culture was dominated by a unique phylotype affiliated with the Desulfosarcina-Desulfococcus cluster within the Deltaproteobacteria. These phylotypes formed a distinct phylogenetic cluster of propane and butane degraders, including sequences from environments associated with hydrocarbon seeps. Incubations with (13)C-labeled substrates, hybridizations with sequence-specific probes and nanoSIMS analyses showed that cells of the dominant phylotypes were the first to become enriched in (13)C, demonstrating that they were directly involved in hydrocarbon degradation. Furthermore, using the nanoSIMS data, carbon assimilation rates were calculated for the dominant cells in each enrichment culture.

Figure 1

Temperature-dependent sulfate reduction rates (SRR) of the propane- and butane-degrading enrichment cultures from marine hydrocarbon seeps. Incubations were done for up to 11 days. The SRR are expressed as percent of the highest rate determined. The maximum sulfate reduction rates (mM H₂S day^-1) were 0.62 (Prop12-GMe), 0.82 (But12-GMe), 0.9 (But12-HyR) and 1.12 (strain BuS5).

Figure 2

Consumption of propane () and butane () coupled to the production of sulfide () by the enrichment culture Prop12-GMe. Methane (), ethane (), pentane () and isobutane () were not degraded if supplied as single substrates or in mixtures with propane or butane. No sulfide was produced in controls without substrate (). Cultures were set up in 120 ml serum bottles supplied with 79 ml artificial seawater medium and 1 ml inoculum from a dense cell suspension (10 × concentrated).

Figure 3

Microscopic images of the enrichment cultures Prop12-GMe (a, c, e), and But12-HyR (b, d, f). Phase contrast images show densely packed, mostly oval-shaped cells in both enrichment cultures (a, b). These cells affiliated with the Desulfosarcina-Desulfococcus cluster of the Deltaproteobacteria, as demonstrated by hybridization of aggregate cross sections with the probe DSS658 (c, d). Hybridizations of partly homogenized aggregates with sequence-specific oligonucleotide probes further indicated that each of the enrichment cultures was dominated by a unique phylotype (e, f). The hybridization images (c – f) show overlays of probe (orange) and DAPI (blue) signals. Scale bars = 5 μm.

Figure 4

Phylogenetic affiliation of the dominant phylotypes in the enrichment cultures Prop12-GMe and But12-HyR (marked in boldface). Sequences affiliated with the Desulfosarcina-Desulfococcus cluster of the Deltaproteobacteria and those targeted by the DSS658 oligonucleotide probe are marked. The phylogenetic tree was calculated by Maximum Likelihood using only nearly full length sequences (>1400 bp). The numbers next to the nodes indicate bootstrap values (100 replications); only values larger than 50% are shown.

Figure 5

Abundance of ¹³C as determined by bulk analysis of biomass (□) and by nanoSIMS in single cells of the enrichment cultures Prop12–GMe, But12–GMe, and But12–HyR incubated with ¹³C-propane (Prop12-GMe) or ¹³C-butane (But12-GMe and But12-HyR). In order to distinguish cells of the dominant phylotype (•) from accompanying cells (), the values were plotted with an off-scale on the time axis of ±0.2. The dominant cells were identified based on the presence of ¹⁹F signals. Shaded area indicates the range of ¹³C-abundance values obtained for cells at 0 incubation days (natural abundance of ¹³C).

Figure 6

NanoSIMS images of the enrichment cultures Prop12-GMe (a – c), But12-GMe (d – f), and But12-HyR (g – i) after incubation with ¹³C-labeled propane or butane for 3, 5 and 3 days, respectively. Individual cells were identified in the total biomass (¹²C¹⁴N⁻) images (a, d, g), and highlighted by drawings around cell edges, for easier tracking in the ratio images. Cells of the dominant phylotypes were identified based on detection of ¹⁹F (b, e, h) introduced into the cells by deposition of F-containing tyramides after hybridization with HRP-labelled oligonucleotide probes. The ratio images of ¹³C/¹²C show incorporation of label by individual cells (c, f, i). Scale bars = 1 μm.