Hydrocarbon-degrading bacteria and the bacterial community response in gulf of Mexico beach sands impacted by the deepwater horizon oil spill

Abstract

A significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize the in situ response of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C₈ to C₄₀) concentrations ranging from 3.1 to 4,500 mg kg⁻¹ in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarily Gammaproteobacteria, including representatives of genera with known oil degraders (Alcanivorax, Marinobacter, Pseudomonas, and Acinetobacter). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 10⁷ to 10.2 × 10⁷ copies g⁻¹) versus clean (0.024 × 10⁷ to 1.4 × 10⁷ copies g⁻¹) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genus Alcanivorax showed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of the Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) as key players in oil degradation there.

Fig. 1.

Principal component analysis of bacterial SSU rRNA gene abundance and days since oil arrival. Dark bubbles represent visibly oil-contaminated samples, whereas light ones indicate visibly clean samples. Bubble size is relative to the number of bacterial SSU rRNA gene copies per gram of sand. Vectors represent variables used to generate the euclidean distance. SSU rRNA abundance data were initially fourth root transformed to meet the assumptions of normality.

Fig. 2.

Linear regression analysis of the abundance of overall bacteria in comparison to the abundance of Alcanivorax spp. for each sampling trip, with the abundance determined as SSU rRNA gene copies per gram of sand.

Fig. 3.

Phylogenetic comparison of SSU rRNA gene sequences from oil-degrading bacterial isolates (★) and sequences retrieved from oiled Pensacola Beach sands (boldface). Only Gammaproteobacteria lineages are included in the analysis. The most similar sequences identified by BLAST are indicated by GenBank accession number. The bootstrapped neighbor-joining phylogenetic tree was generated in MEGA using the maximum composite likelihood model with gamma-distributed rates and pairwise deletion (39). SSU rRNA sequences from oil-degrading isolates, similar environmental OTU from the pyrosequencing data set, and top isolated BLAST hits were aligned by using the GreenGenes NAST aligner (16). Nodes with >70% bootstrapping support, out of 500 replications, are shown.

Fig. 4.

Multidimensional scaling plot of SSU rRNA pyrosequence libraries derived from oiled Pensacola Beach sands (black), clean Pensacola sands (gray), and pristine sands (gray) from St. George Island. Samples above the black line were obtained from gDNA extracts, while samples below the line were derived from total RNA extracts reverse transcribed using a bacterial SSU rRNA gene primer. Lowercase letters (a to e) indicate pyrosequence libraries derived from the same sand sample. Averaged Bray-Curtis distance is shown. Bubble size is relative to percent abundance of Alcanivorax-like OTU as assigned by the RDP classifier.

Fig. 5.

Phylum- and class-level phylogenetic analysis of RNA and DNA-based bacterial SSU rRNA pyrosequence libraries for Pensacola Beach sand samples collected on 2 July 2010.