Intellectual merit: This proposal describes a series of laboratory studies designed to assess the genomic and molecular patterns of petroleum biodegradation under a range of conditions relevant to the Earth?s surface and subsurface. The concerted application of comprehensive, two-dimensional gas chromatography, Fourier transform ion cyclotron resonance mass spectrometry, and pyrosequencing-based metagenomics will provide unparalleled insight into petroleum biodegradation and the responsible microbes, and distinguishes this work from any previous studies. The primary hypotheses to be tested are the following: 1) Hydrocarbon biodegradation by microbes acts simultaneously on thousands of distinct compounds, not in a strictly stepwise fashion. 2) Hydrocarbon preference pattern and associated genomic potential for a microbial community are controlled primarily by the redox potential of the terminal electron-accepting process. 3) High-molecular-weight and polar compounds are bio-transformed through incomplete oxidation and structural alteration, not through complete remineralization. These hypotheses will be tested by conducting time-series laboratory experiments in which petroleum is biodegraded with different electron acceptors and at different temperatures. The loss and gain patterns for molecules within each treatment and differences across treatments in these patterns and in genomic content will provide the data to test these hypotheses. Results are further anticipated to reveal novel metabolic actions and genomic capacity, and yield molecular degradation patterns that can relate environmental genomic and petroleum content to the relevant biological processes. New data analysis tools will also be developed and validated.
Broader Impact: Results from this research will contribute broadly to an understanding of petroleum biodegradation and carbon cycling in the Earth system, and will be broadly disseminated through popular outlets with assistance from a professional artist. Knowledge gained from this research will also be translated directly to federal agencies including the NOAA?s Assessment and Restoration Division, as well as to private industry through existing corporate ties. Direct educational impacts of this research include the training and education of high school, undergraduate, and graduate students, as well as the advanced training of postdoctoral researchers. High school students will be incorporated through existing summer research programs targeting students from underserved regions. Undergraduate students will be incorporated into all aspects of the proposed research through integration into coursework and REU support. Mentoring of a graduate student and postdoctoral researchers will be provided by the PIs.
This study provided numerous key insights on how oil is microbially degraded in surface reservoirs as well as following releases into the surface from natural seeps and oil spills. The initial goal was to focus mainly on the natural seeps off the coast of Santa Barbara, CA, but we took advantage to study the Deepwater Horizon (DWH) disaster. The latter provided an opportunity to investigate degradation under a wide range of conditions exhibited in the Gulf of Mexico following the explosion of the rig. And we argue that the DWH disaster could be considered an analogue for a very large oil seep. The most noteworthy breakthrough at Santa Barbara was using comprehensive two-dimensional gas chromatography (GC×GC) to study the biodegradation of seep oils in laboratory cultures. In particular, we were interested in the concurrent degradation of different classes of petroleum hydrocarbons. Approximately 90 selected components of petroleum were quantified and oxidized during this study. We found that many compounds were consumed concurrently, which in turn, supports the concept that hydrocarbon-degrading communities have the ability to consume simultaneously many compounds, perhaps numbering in the thousands. These results further demonstrate a capacity for aerobic microbial communities to consume diverse hydrocarbons beyond those typically investigated with conventional analytical techniques. The most unique outcome from studying the Deepwater Horizon site was that we used GC×GC to refine current biodegradation indices, which are used to gauge the extent of petroleum-hydrocarbon degradation, in particular the classes of saturated hydrocarbons. Despite that oils are predominantly composed of saturated hydrocarbons, surprisingly little is known about biodegradation of different saturates classes in surface environments, owing to the limited ability of conventional gas chromatography (GC) to resolve this compound group. We studied moderately weathered oil field samples collected from Gulf of Mexico beaches 12 to 19 months after the DWH disaster. With GC×GC analysis, we could effectively separate and quantify several distinct saturates classes from these field samples. We found that biodegradation proceeds simultaneously for different saturate classes, but to different relative extents, with ease of biodegradation decreasing in the order n-alkanes > methyl alkanes and alkylcycloalkanes > cyclic and acyclic isoprenoids, for compounds in the n-C22-n-C29 range. These results led us to develop a new saturates biodegradation index designed to characterize the extent of weathering in field samples. Unlike previously developed biodegradation indices, the new index incorporates broad information about saturates, the quantitatively dominant GC-amenable component of moderately weathered oils. For example, the index correctly identified accelerated biodegradation in wet environments compared to dry environments, illustrating the importance of local environmental conditions for facilitating natural attenuation. Both of these studies, laboratory and field-based, reveal that the standard dogma that degradation is linear and is much more nuanced. Instead degradation occurs concurrently although different classes are degraded easier than others and at faster rates. With such knowledge on the progression of degradation, we have a greater understanding of how highly reduced carbon is remineralized and also how to assess and restore oiled areas after spills and releases. These are major breakthroughs and are revolutionizing how oil seeps and oil spills are studied. That is, by using GC×GC we can expand our analytical windows to characterize with greater detail petroleum hydrocarbons in the environment.My knowledge about natural oil seeps and the behavior of oil in the ocean have been incredibly useful during and following the Deepwater Horizon disaster. I have discussed natural oil seeps during briefings to Capitol Hill staffers, Congressman, and members of the executive branch. I have given numerous talks about oil spills with a goal of being an honest broker of information to the public and policymakers. This may be the most important outcome of this project. Providing novel and useful science that can be used to make informed decisions about pressing issues.
