46685-AC2
Characterizing Anaerobic Microbial Oxidation of Hydrocarbons: Tracing the Fate of C3 and C4-derived Carbon in Marine Seeps
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 hypotheses and objectives in this project are as follows:
Hypothesis A) C1-C4 oxidation and sulfate reduction are interrelated in hydrocarbon seep communities.
Analyses: Quantify rates of anaerobic microbial oxidation of C1-C4 alkanes and sulfate reduction during standard radiotracer incubations and during "artificial seep" incubations.
Hypothesis B) Specific assemblages of uncultivated phylotypes are responsible for mediating the anaerobic oxidation of C2-C4 hydrocarbons.
Analyses: Constrain the identity of microbial phylotype(s) that mediate anaerobic C2-C4 oxidation.
Hypothesis C) Growth rates --and therefore the population dynamics-- of microbes mediating C2-C4 oxidation is unknown and likely affects the geochemical environment and the ratios of C1-C4 oxidation in situ.
Analyses: 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.
Hypothesis D) C2-C4 derived carbon may end up as methane, dissolved inorganic carbon (DIC) or other organic compound. This is turn can affect the isotopic ratios of other microbial phylotypes such as AMO archaea and SRB.
Analyses: Determine the fate of C2-C4-derived carbon using 13C-labeled hydrocarbons during time-course experiments.
This project began in September 2007. During these months, we have made significant progress in fabricating the artificial seep system to address hypothesis A, and have set up a series of enrichments to address hypothesis B. It is our objective to have completed these experiments (i.e. those which address hypotheses A and B) by the end of this calendar year.
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. The system is designed to incubate four separate continuous flow sediment reactors under geochemical conditions that mimic those found at thermogenic gas seeps in the Gulf of Mexico. The Anaerobic Hydrocarbon Incubation System (AHIS) consists of a water-conditioning column that is used to produce fluids of similar chemical composition to the porewaters in the Gulf of Mexico (a detailed description is found below), a high-pressure flow through sediment incubation reactor, and a packed bead bed flow through reactor.
Incubation experiments in progress
Sediment core samples were recovered from a thermogenic gas hydrate seep site WR269 in 2007. The sediments were homogenized and packed into the steel reactors under N2 stream. Reactors were 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. Incubations have been running continuously for approximately 3 months, and will continue until December 2008, at which point we will sample for DNA, RNA, and protein, as well as fix biomass for imaging, and live biomass samples for AOM and sulfate reduction (SR) rate measurements. Dissolved gas concentrations will be determined by a Seawater-inlet-GC and Quadropole-MS. Subsequently, DNA will be extracted from discrete horizons and amplified with general Bacterial and Archaeal primers for community analyses. Samples for proteomic analysis will be extracted by a recently established sediment extraction method and sequenced at the Harvard Microchemistry and Protoemics Facility.
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
Few studies aim to employ a full range of (appropriate) analytical and molecular biological approaches to study these phenomena. Most deep-sea microbiological research depends upon isolating and incubating cultures at one atmosphere pressure, in conditions that bear little resemblance to those found in situ. We are studying these communities at environmentally-relevant conditions, as a means of coupling microbiological dynamics (e.g. population dynamics) to biogeochemical rates (alkane oxidation, sulfate reduction) and subsequently assess the potential impact on local and global geochemical cycles. The proposed research will thus fill a critical data gap by developing our understanding of the linkages between hydrocarbon oxidation and sulfate reduction in these environments; a question with clear implications for both carbon cycling and climate change.
