Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

Abstract

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly (13)C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.

Figure 1

Phylogenetic tree of SIP clones and isolated strains from surface and plume waters. SIP clones and isolates are shown in bold along with the highest similarity sequences and type strains (Yarza et al., 2010) from the SILVA SSU NR 111 NR database (Quast et al., 2013). A neighbor-joining tree was constructed with Jukes–Cantor correction and bootstrap replication (n=1000). Nodes with bootstrap support of at least 65% (○) and 90% (•) are marked. Accession numbers of all sequences and the hydrocarbon(s) utilised (NAP, naphthalene; PHE, phenanthrene; HEX, n-hexadecane) by the isolated strains are given in parentheses. Akkermansia muciniphila (CP001071) and Thalassospira xiamenensis (AY189753) were used as the outgroup. The scale bar indicates the difference of number of substitutions per site.

Figure 2

Cumulative ¹⁴CO₂ recovered from incubations with [¹⁴C] phenanthrene (PHE), anthracene (ANT), pyrene (PYR), fluoranthene (FLU), naphthalene (NAP), benz[a]anthracene (BaA), or n-hexadecane (HEX) by surface (solid bar) and plume (open bar) water during incubation at 21 °C. Bars are the averages and standard deviation from triplicate incubations. *, no data (surface water was not incubated with n-hexadecane).

Figure 3

Abundance of 16S rRNA genes of (a) Cycloclasticus (OTU-1) and Colwellia (OTU-2) during incubation with unlabelled phenanthrene, (b) Alteromonas (OTU-12) and Cycloclasticus (OTU-13) during incubation with unlabelled naphthalene, and (c) Alcanivorax (OTU-15) and Marinobacter (OTU-16) during incubation with unlabelled n-hexadecane. Bars are the averages and standard deviations of results from triplicate qPCRs measuring the abundance of group-specific 16S rRNA genes. Circles are the means and standard deviations of triplicate measurements of the total mass of DNA per sample.