Summary:
Recent studies suggest that microbial communities played important roles in the degradation of oil discharged from the Deepwater Horizon wellhead. Specifically, DNA sequences collected from the oil plume showed a predominance of one phylogenetic Order of Bacteria: the Oceanospirillales, and an increase in hydrocarbon degrading genes compared to control sites (Hazen et al. 2010. Deep-sea oil plume enriches indigenous oil-degrading bacteria. Science 330:204-208).
Our proposed pilot study builds upon the Hazen et al. study through microcosm experiments that are aimed at determining if similar microbial responses can be elicited under laboratory conditions. The second aim of our study was to demonstrate the utility of interrogating microbial cells in environmental samples using a metagenomic approach. Our proposed study differs from Hazen et al. in three ways: (i) rather than amplifying DNA sequences, this study used non-amplified DNA sequences - eliminating the biases associated with PCR amplification; (ii) rather than directly collecting DNA from in situ water samples, DNA will be collected from oil-amended and non-amended seawater microcosms that simulate in situ conditions; (iii) rather than using DNA microarrays and cloning/sequencing approaches to interpret the microbial response, we will interpret the response using high throughput Roche/454 DNA sequencing followed by metagenomic analysis.
Our hypotheses (H) are the following: H1, a substantially lower diversity of DNA sequences will be obtained from the oil-amended microcosms than non-amended microcosms because metabolic networks of hydrocarbon-utilizing genes are established, which decreases the relative number of DNA sequences involved in other metabolic activities; H2, the oil and dispersant microcosms will yield a substantially lower sequence diversity than those obtained from the other microcosms because an increased surface to volume ratio of the oil droplets will increase hydrocarbon availability leading to the rapid establishment of a dominant hydrocarbon-degrading community; and H3, hydrocarbon-utilizing genes will be more abundant in the oil-amended microcosms than the non-oil amended microcosms.
Metagenomic analysis depends upon post-processing approaches for interpreting the sequence data. For this study, custom-designed software programs will be used to prune the 454 sequence data, and identify and remove sequences containing ambiguous nucleotides (e.g., Ns). RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq/) was used to determine sequence identity. Sequences not identified by RefSeq will be considered ambiguous. Taxonomic information of ribosomal DNA sequences will be determined using the Ribosomal Database Project (http://rdp.cme.msu.edu/).
The diversity of DNA sequences will be determined using the Shannon Weaver Diversity index. This study deals with "biological degradation of the contaminants" and the "technology developments for improved detection, characterization, and mitigation of offshore oil spills" (21). Successful demonstration and validation of the metagenomic approach will go a long way towards understanding the response of hydrocarbon-utilizing microbes in marine environments and developing strategies to minimize the impact of oil discharges in the field.