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MAJOR RESEARCH AND EDUCATION ACTIVITIES

The overarching goal of this project was to examine the relation between anaerobic alkane oxidation and sulfate reduction. The precise nature and extent of anaerobic alkane oxidation (other than anaerobic methane oxidation, or AMO) is largely unconstrained.  To date, the anaerobic oxidation of C2-C4 hydrocarbons has largely been inferred from stable carbon isotope data, and from the enrichment of two anaerobic alkane oxidizing sulfate reducers. 

As such, our objectives in this project were to: A) Quantify rates of anaerobic microbial oxidation of C1-C4 alkanes and sulfate reduction during standard radiotracer incubations and during "artificial seep" incubations, B) Constrain the identity of microbial phylotype(s) that mediate anaerobic C2-C4 oxidation, C) Examine the population dynamics of microorganisms associated with anaerobic C2-C4 alkane oxidation, i.e. cell density in relation to measured oxidation rates via qPCR and isotopic tracer studies, and D) Determine the fate of C2-C4-derived carbon using 13C-labeled hydrocarbons during time-course experiments.

During these last twenty-four months, we have made significant progress in accomplishing these objectives (described in detail below).  In addition, our collaborator Dr. Samantha Joye at the University of Georgia and her group have been working with us on measuring the differential influence of methane and other alkanes on sulfate reduction.  We have completed the first set of flow-through experiments, and have presented these results at a gas hydrate workshop in Bremen, Germany.  In addition, we have completed the batch reactor experiments to examine the differential influence of alkanes on sulfate reduction.  We are submitting our paper on the effects of alkanes on sulfate reduction by January 2010. 

The following is a brief description of our efforts to date:

The Anaerobic Hydrocarbon Incubation System (AHIS)

We have completed the fabrication of a high-pressure continuous flow artificial seep system as well as the first set of incubations (described in detail in the previous report and the "nugget".  Anaerobic methane oxidation rates of ca. 100 nmol per cubic centimeter of sediment per day were measured for these sediments (Joye et al., unpublished data). The sediments were homogenized under an N2:CO2 atmosphere, and 200 cc of sediment were mixed with 800-cc quartz sand (pre-soaked in sulfidic seawater to remove trace oxygen) and packed into the steel reactors under N2-stream.  Reactors were then incubated under continuous flow of sulfidic hydrocarbon rich seawater (containing only one of the four short-chain alkanes: methane, ethane, propane, or n-butane) at 7C.

After running for four months, we examined the reactors for changes in alkane and sulfate reduction rates.  We observed that sulfate reduction rates varied among the reactors being fed different alkanes.  There were substantial differences in rate within each reactor at the different sediment horizons.  Most interestingly, we observed dramatic shifts in the bacterial population among all three reactors (Table 1, below).  There was a dramatic increase in the number of epsilon proteobacteria that were recovered from ethane and propane reactors.  While we initially believed this was attributable to the high sulfide, is it important to note that sulfide was elevated among all three reactors.  Given that the sediment inoculum was consistent among all three reactors, and that the only difference was the introduction of alkanes, the results below are intriguing in that they suggest that alkanes play a role in influencing epsilon proteobacterial communities.  To date, the role of the epsilon proteobacteria in hydrocarbon oxidation has not been examined, and these results provide intriguing evidence that these alkanes play a role -directly or indirectly- in stimulating their growth.

Table 1. Bacterial and Archaeal 16S rDNA clone libraries from each bioreactor after 30-days incubation at 7°C under constant flow.

Currently we are also working on batch enrichments using different combinations of electron donors (methane or alkanes) and acceptors (sulfate, nitrate, iron and manganese).  We are now preparing for isolation and will continue with our current enrichment efforts.  It is apparent that we have stimulated the activity of anaerobic alkane oxidizers, as there is considerable increase in headspace pressure and sulfide concentration.

Summary

           The continual discoveries of new hydrates and seep sites suggest that the frequency, distribution and environmental significance of gas hydrates and "chemosynthetic communities", is greater than previously assumed. Furthermore, correlations between the flux of methane from sediments and significant variations in global climate have been noted throughout Earth's history.  It is well-known that sediment-hosted microbial communities represent an important sink for these low molecular weight hydrocarbons, effectively acting as a biological "filter" reducing the flux to the atmosphere.  As such it is of global importance that we understand these communities and the geochemical cycles that they mediate.  Our research studied these communities at environmentally-relevant conditions, as a means of coupling microbiological dynamics (e.g. population dynmamics) to biogeochemical rates (alkane oxidation, sulfate reduction) and subsequently assess the potential impact on local and global geochemical cycles.  We have developed novel technologies to reproduce in situ conditions in the lab, where we can more easily govern much of the variability.  This research has elucidated the interactions between the phylotypes responsible for alkane oxidation, the factors that control alkane oxidation rates, their relation to AMO, and the fate of alkane-derived carbon, and thus fills a critical data gap by furthering, for the first time, our understanding of the linkages between hydrocarbon oxidation (other than methane) and sulfate reduction in these environments; a question with clear implications for both carbon cycling and climate change.