Simulation of Deepwater Horizon oil plume reveals substrate specialization within a complex community of hydrocarbon degraders Ping Hua, Eric A. Dubinskya,b, Alexander J. Probstc, Jian Wangd, Christian M. K. Sieberc,e, Lauren M. Toma, Piero R. Gardinalid, Jillian F. Banfieldc, Ronald M. Atlasf, and Gary L. Andersena,b,1 aEcology Department, Climate and Ecosystem Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; bDepartment of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720; cDepartment of Earth and Planetary Science, University of California, Berkeley, CA 94720; dDepartment of Chemistry and Biochemistry, Florida International University, Miami, FL 33199; eDepartment of Energy, Joint Genome Institute, Walnut Creek, CA 94598; and fDepartment of Biology, University of Louisville, Louisville, KY 40292 Edited by Rita R. Colwell, University of Maryland, College Park, MD, and approved May 30, 2017 (received for review March 1, 2017) The Deepwater Horizon (DWH) accident released an estimated Many studies of the plume samples reported that the structure 4.1 million barrels of oil and 1010 mol of natural gas into the Gulf of the microbial communities shifted as time progressed (3–6, 11– of Mexico, forming deep-sea plumes of dispersed oil droplets and 16). Member(s) of the order Oceanospirillales dominated from dissolved gases that were largely degraded by bacteria. During the May to mid-June, after which their numbers rapidly declined and course of this 3-mo disaster a series of different bacterial taxa were species of Cycloclasticus and Colwellia dominated for the next enriched in succession within deep plumes, but the metabolic capa- several weeks (4, 5, 14). Following containment of the DWH bilities of the different populations that controlled degradation rates wellhead, all of these dominant members declined in abundance. of crude oil components are poorly understood. We experimentally Multiple hypotheses have been put forth regarding the drivers of reproduced dispersed plumes of fine oil droplets in Gulf of Mexico microbial community succession during the period of hydrocarbon seawater and successfully replicated the enrichment and succession contamination. Redmond and Valentine (14) proposed that the of the principal oil-degrading bacteria observed during the DWH input of natural gas structured the microbial community response. event. We recovered near-complete genomes, whose phylogeny A modeling study suggested it was a consequence of different MICROBIOLOGY matched those of the principal biodegrading taxa observed in the metabolic growth rates combined with ocean fluid dynamics (6). field, including the DWH Oceanospirillales (now identified as a Dubinsky et al. (3) found succession was primarily controlled by Bermanella species), multiple species of Colwellia, Cycloclasticus, hydrocarbon rather than nutrient availability. A 16S rRNA gene- and other members of Gammaproteobacteria, Flavobacteria, and Rho- based study using surrogate oil suggested that dispersant might dobacteria. Metabolic pathway analysis, combined with hydrocarbon compositional analysis and species abundance data, revealed sub- Significance strate specialization that explained the successional pattern of oil- degrading bacteria. The fastest-growing bacteria used short-chain The Deepwater Horizon drilling accident was the first major alkanes. The analyses also uncovered potential cooperative and com- release of oil and natural gases in the deep ocean, and con- petitive relationships, even among close relatives. We conclude that siderable uncertainty remains about the fate of vast amounts patterns of microbial succession following deep ocean hydrocarbon of hydrocarbons that never reached the surface. We simu- blowouts are predictable and primarily driven by the availability of lated the deep-sea plumes of dispersed oil microdroplets and liquid petroleum hydrocarbons rather than natural gases. measured biodegradation of crude oil components. We suc- cessfully reproduced the successive blooms of diverse bacte- hydrocarbon biodegradation | Gulf of Mexico | microbial communities | ria observed in the field and obtained near-complete Macondo oil | genome succession genomes of all major hydrocarbon-degrading species, pro- viding an assessment of the metabolic capabilities of the uring April to July 2010, 4.1 million barrels of crude oil were microbial community responsible for biodegradation. Our Dreleased into the Gulf of Mexico due to the Deepwater results show that rapidly degraded components of oil were Horizon (DWH) accident. The DWH accident was the first consumed by bacteria with highly specialized degradation major spill to occur in the deep ocean (1,500-m depth), and the capabilities and that crude oil alone could explain the micro- processes that determined the fate of oil released at this depth bial dynamics observed in the field. were largely unknown. An important phenomenon observed in the deep ocean was the unexpected suspension of small oil Author contributions: P.R.G., R.M.A., and G.L.A. designed research; J.W. and L.M.T. per- droplets (<100 μm) that remained trapped at depth in the water formed research; A.J.P. contributed new reagents/analytic tools; P.H., E.A.D., A.J.P., C.M.S., J.F.B., and G.L.A. analyzed data; and P.H., E.A.D., A.J.P., P.R.G., J.F.B., R.M.A., column because they lacked enough buoyancy to rise to the and G.L.A. wrote the paper. surface. Consequently, vast plumes of oil microdroplets con- The authors declare no conflict of interest. taining not only soluble but also insoluble fractions of oil were This article is a PNAS Direct Submission. retained at depth, largely between 900 and 1,300 m deep (1, 2), and subject to biodegradation by the deep ocean microbial Freely available online through the PNAS open access option. – Data deposition: The whole genome shotgun project and the associated draft genomes community (3 6). Furthermore, the application of dispersants at have been deposited at the DNA Data Bank of Japan (DDBJ), European Nucleotide the wellhead may have enhanced oil droplet formation, oil re- Archive, and GenBank (accession nos. MAAA00000000– MAAU00000000 and tention, and biodegradation at depth, although potential in- MAAW00000000–MABF00000000). The version described in this paper is version hibitory effects of dispersants on biodegradation have been MAAA00000000–MAAU00000000 and MAAW00000000–MABF00000000. The raw reads have been deposited at DDBJ, the European Molecular Biology Laboratory, and GenBank (acces- reported (7). There continues to be considerable uncertainty and sion no. SRP075617). The project description and related metadata are accessible through disagreement about the rates of microbial biodegradation under BioProject PRJNA320927. these conditions and the factors controlling the fate of the 1To whom correspondence should be addressed. Email: [email protected]. complex mixture of crude oil compounds that were trapped deep This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the water column (8–10). 1073/pnas.1703424114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1703424114 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 promote microbes that are able to use dispersant for growth. Therefore, it was thought that the relative abundance of the nat- ural hydrocarbon degraders, such as Marinobacter, was kept low (7). No study, to date, has been able to specify the relationship between hydrocarbon substrate availability and the metabolic ca- pacities of the diverse group of organisms responsible for hydro- carbon degradation in the DWH plume. Our goals in this study were to mimic the conditions present in the deep-sea oil plume at the time of the DWH oil release by producing highly dispersed (∼10-μm) oil droplets in natural seawater, correlate the progression of oil degradation with shifts in the endemic microbial community by analyzing changes in hy- drocarbon chemistry and bacterial populations over a 64-d time course, and recover high-quality draft genomes to determine the metabolic factors that drove the microbial community shifts throughout the oil biodegradation process. Results Microbial Community Structural Changes Correlated with Hydrocarbon Groups. To observe the interaction between the microbial com- munity and the consumption of hydrocarbons we simulated the deep-sea plume observed during the DWH incident in the labo- ratory. Natural seawater collected from Mississippi Canyon block Fig. 2. Response of deep-ocean bacteria to MC252 oil. Heat maps of Phy- loChip hybridization intensities for the incubation experiment (A) and field MC-294 at 1,100- to 1,200-m depth was mixed to a final concen- samples collected in 2010 during the DWH event (B). Hybridization in- tration of 2 ppm oil and 0.02 ppm Corexit EC9500A dispersant. tensities were standardized to the maximum value for each operational Microdroplets were produced using a pressurized flow injection taxonomic unit (OTU). OTUs are shown that represent the 10 most enriched (PFI) droplet generator (15) to produce median-size oil droplets genera in incubation samples compared with starting abundances (t0). OTUs of 10-μm diameter using Macondo (MC252) oil (SI Methods). Oil enriched in 2010 field samples that matched enriched genera in the in- droplets remained in suspension in replicate 2-L bottles for the cubation experiment are shown in B. OTUs that dominated early, in- 64 d of the experiment. termediate, and late