The primary study site, Mississippi Canyon Lease Block 118 (MC118), is an intensively studied site that is the focus of the Gulf of Mexico Gas Hydrates Research Consortium (GOM-HRC, which is managed through the University of Mississippi). We collaborate with a variety of individuals associated with the GOM-HRC, including Drs. Ian MacDonald and Jeff Chanton (Florida State University), Drs. Andreas Teske and Chris Martens (University of North Carolina), and Dr. Laura Lapham (University of Maryland).
Our work focuses on gas hydrate mounds and the associated sediments and water bodies overlying these features. We are conducting an integrative study that utilizes both standard and state-of-the-art biogeochemical, microbiological, and molecular ecological techniques to unravel the interactions between carbon and sulfur cycling in the unique deep sea habitat of gas hydrates. We will characterize the poorly understood microbial populations mediating important processes in the carbon and sulfur cycles and further elucidate survival strategies of microbes living in these ecosystems.
The main objectives of this work are
- to estimate microbial abundance and the diversity of microbial communities in gas hydrate and associated sediments;
- to quantify rates of methane oxidation and sulfate reduction in gas hydrate and sediment samples in ex situ and slurry incubations;
- to determine whether methane oxidation and sulfate reduction occur within “intact” (i.e., solid) gas hydrates, and;
- to document the stability and persistence of gas hydrate mounds using temperature sensors and time-lapse digital photographic monitoring.
This project is an interdisciplinary effort to quantify microbial processes in gas hydrates, a poorly understood microbial niche, while broadly clarifying the microbial processes driving carbon and sulfur cycling in cold seep environments. Building on the success of previous research, the project will continue and expand our study of microbial distributions and activity in gas hydrate ecosystems. In addition to providing fundamental information on the distribution of microorganisms and microbial processes in gas hydrates, a novel environment on Earth that may be present elsewhere in the Universe, our work is pertinent to a range of studies of microbial biogeochemistry and biodiversity in gas hydrate environments and has relevance to efforts to understand the conditions that may have supported life early in Earth history.
Why study methane seeps?
Methane seeps are ubiquitous features of active and passive continental margins. In the Gulf of Mexico, sediments overlie enormous reservoirs of liquid and gaseous hydrocarbons that rest upon Jurassic-age salt deposits. Methane seepage and gas hydrates are abundant at MC118 (Figure 1).
Salt-driven tectonics generate fault networks that act as conduits for the rapid transfer of oil, gas, and brines from deep reservoirs through the overlying sediments and into the water column. On the seafloor, these conduits give rise to gas vents and seeps, subsurface and surficial methane hydrates, brine pools, and mud volcanoes. Sediments around areas of active seepage are characterized by elevated concentrations of simple (C1-C5) and complex hydrocarbons (oil) and hydrogen sulfide (H2S). The sediments at MC118 are rich in alkanes and oil, as seen in this picture, showing oil-saturated pore water being collected from a sediment squeezer
Gas hydrates are solid ice-like structures comprised of water and entrapped gases, predominately methane, but also ethane, propane, iso-butane, butane, pentane, carbon dioxide, hydrogen sulfide, and nitrogen. Gas hydrates form spontaneously where a steady source of gas occurs within an appropriate pressure-temperature window (in continental margin sediments, T < 10 ºC and/or P 1-5 MPa;) and are a dynamic component of the global carbon cycle (Figure 1). The amount of methane present in gas hydrates likely exceeds conventional fossil fuel reserves (oil and gas). The largest fraction of the hydrate reservoir lies beneath ~200m of sediment at the base of continental margins. In these diffusion-dominated settings, rates of microbial activity are low. However, gas hydrate deposits also occur in the upper few meters of seafloor sediments in shallow advection-dominated regions. In advection-dominated systems, particularly if a hydrate deposit is covered by a drape of sediment, methane oxidation and sulfate reduction rates may be much higher. Because of their occurrence along continental margins, the stability of shallow gas hydrate reservoirs is sensitive to changes in global climate. Increased water temperature alters the hydrate stability field and may lead to dissociation of gas hydrates and release of methane to the associated sediments and water column. In fact, hydrate dissociation has been correlated with significant variations in global climate, and periodic pulses of hydrate-derived methane to the atmosphere may have driven past increases in global temperatures and changes in global carbon fluxes.
Though the global distribution of gas hydrates is fairly well documented, relatively little is known of the microbial diversity of gas hydrate habitats or the magnitude of microbially-mediated transformations of carbon and sulfur within these areas. Potential feedbacks between global climate and hydrate dissociation provide justification for studying the molecular biogeochemistry of hydrate systems, as microbial activity, such as the anaerobic oxidation of methane, may reduce methane exchange between the hydrate reservoir and the hydrosphere, and ultimately to the atmosphere. At the microbial scale, gas hydrates simultaneously serve as a unique support surface and an anhydrous (low water content) obstacle for life Knowing whether gas hydrates support diverse and active microbial assemblages will also help in evaluating the possibility for similar geomicrobiological interactions on other worlds (e.g., Europa or Titan) where gas hydrates may also occur.
Could microorganisms live inside methane ice?
The abundance and activity of microbes in solid structures, including ice, salt, and mineral crusts, have received increased attention in recent years. Sea ice microbial communities survive, and may even thrive, inside interconnected brine channels that concentrate nutrients and organic substrates. The development of such organic-rich fluid inclusions containing active microbial clusters was observed in laboratory studies of ice-VI crystals at high pressure (~1250 MPa). Samples from deep within Vostok ice cores revealed that active microbes survive within this icy environment, where liquid water is likely limiting, for thousands of years. Examples of actively growing microbes in icy habitats suggest that ice is a viable microbial niche. In fact, cryotolerant microbial communities may have served as oases for life during certain periods (e.g. snowball phases) of Earth history. Experimental evidence from salt crystals also illustrates that microbes can survive over short (weeks to months) and long (years) periods inside solid structures.
Gas hydrates represent a solid microbial habitat characterized by a variety of environmental extremes, including high pressure, cold temperatures, high salinities inside brine inclusions, major and minor nutrient stress, water stress, and high concentrations of potentially toxic gases such as hydrogen sulfide. Gas hydrates are unique compared to the previously described solid structures where life proliferates, such as ice, salt, and mineral crusts, because hydrates also contain abundant reduced substrates that could fuel microbial metabolism, including methane, other alkanes, hydrogen sulfide, aromatics, and oils. In surface-breaching gas hydrate deposits, dissolved ions, such as SO42- are trapped within hydrate structures as they form and could serve as the electron acceptor for the anaerobic oxidation of methane or respiration of other organic compounds, such as alkanes and oil. Though a variety of microbes have been observed to occur in close physical contact with hydrates, the activity of these microorganisms is largely undocumented.
What do we know about microbial activity in gas hydrates?
Due to the chemical composition of Gulf of Mexico gas hydrates, a variety of biogeochemical processes may occur within the gas hydrate niche. We are looking for: (1) aerobic methane oxidation (MOX), (2) anaerobic oxidation of methane (AOM) and sulfate reduction (SR), and (3) oxidation of oil and other hydrocarbons.
Methane oxidation occurs by both aerobic and anaerobic mechanisms. The physiology of and controls on MOX in sediments are well documented. In gas hydrate ecosystems, MOX is limited to the outer surface of gas hydrates where methane and oxygen co-occur, thus, a great deal of the methane oxidation observed in gas hydrate ecosystems occurs via an anaerobic pathway.
AOM occurs in anoxic sediments and water columns, in both freshwater and marine systems. However, the biochemical mechanism(s) of AOM are debated, the controls on AOM in the environment are unclear, and our understanding of the diversity and associations of microorganisms involved in the process is limited. Most work on AOM has been conducted in marine sediments, where rate measurements of AOM and modeling results suggest that a lot of the upward CH4 flux is oxidized anaerobically near the sulfate-methane interface. Syntrophic coupling between methane oxidizing and sulfate reducing microorganisms supposedly mediates AOM. Over the past few years, organic geochemical biomarker and molecular biological data from marine systems have provided support for this hypothesis. Multiple putative methanotrophic archaea and SO42- reducing bacterial partner organisms have been identified in several environments. Biomarker evidence suggests the involvement of multiple archaeal and bacterial groups in AOM.
Despite the probable involvement of methanogens and sulfate reducers, the biochemical mechanism of AOM remains a subject of intense debate. Standard thermodynamic calculations imply that AOM consortia live under energetically marginal conditions; however, their abundance and proliferation in some environments point to novel metabolic capabilities that provide them with significant competitive advantages. Understanding the distribution and physiology of syntrophic associations is required to elucidate their potentially important metabolic role in gas hydrate environments.
Surface breaching gas hydrates are abundant in the Gulf of Mexico (Figure 3). We observed high rates of both AOM and SR in gas hydrate sub-samples (Figure 4, adapted from Orcutt et al. 2004, including sediment at the surface of gas hydrates, in a mix of frozen sediment and hydrate at the solid surface, and in interior samples that contained small sediment inclusions. Trends in the spatial distribution of AOM and SR were evident in all gas hydrate samples from numerous sites in the Gulf of Mexico (GC232, GC234, and GC185, ~500m; GC415, ~1000m; and a newly described site in the southern Gulf with asphalt volcanism, ~3000m).
Data from 16s rRNA signal-enhanced fluorescence in situ hybridization (CARD-FISH) of hydrate samples shows that two groups of the putatively methane-oxidizing archaea (i.e., ANME-1 and ANME-2 groups) and their supposed sulfate-reducing bacterial partners (Desulfosarcina/Desulfococcus spp.) comprise a significant fraction of the microbial population (>25%; Figure 5). These data suggest that active microbial communities are a general feature of gas hydrates and that microbes living inside gas hydrates are at the very least capable of active metabolism.
Based on the results of our work to date, we know that a variety of microorganisms are present within gas hydrates and that these microorganisms mediate AOM and SR. These rate data were obtained in slurry incubations, which are quite different from in situ conditions. Slurry incubations alter substrate concentrations and availability and may potentially alter the distribution and association of microorganisms. One of the most novel aspects of the work proposed here is the determination of rates of microbial activity within an “intact” gas hydrate that is incubated at in situ pressures using a hydrostatic pressure vessel. Documenting microbial activity within a solid gas hydrate using radiotracers would represent a substantial advance towards understanding the importance of gas hydrates as a viable microbial niche. These data may also help elucidate the impact of microbial activity on gas hydrate dynamics in the environment.
We are testing the hypotheses presented below by comparing microbial distributions, abundance, and activity in a variety of gas hydrate samples. We are applying combined microscopic and culture-independent molecular ecological techniques to determine the distribution of microorganisms and to identify specific microbes involved in key biogeochemical processes, such as AOM and SR. Rates of microbial activity are determined using techniques we developed during previous research projects in combination with high pressure incubation techniques that allow us to simulate in situ conditions. We are using time-lapse digital photography and temperature loggers to evaluate the persistence and stability of gas hydrates over time.
Hypothesis 1: Gas hydrates support a viable microbial community.
We compare the microbial abundance, distribution, and the identity of key functional (e.g., aerobic and anaerobic methanotrophs, sulfate reducers, and methanogens) and phylogenetic (e.g., Bacteria and Archaea) groups in gas hydrate samples collected at the sea floor.
Hypothesis 2: Rates of AOM and SR within gas hydrates are significant.
We determine rates of AOM and SR in hydrate samples using radioisotope tracer techniques. Rates are determined in experiments using slurries and in experiments with “intact” (i.e., solid) gas hydrates to determine whether microbes are active within the gas hydrate solid.
Hypothesis 3: Substrate availability influences microbial distributions and activity within gas hydrates and associated sediments.
We conduct slurry experiments amending gas hydrate and sediment samples with important substrates (e.g., CH4, SO42-, or oil) and monitor changes in rates of microbial activity.
Hypothesis 4: Mobile benthic fauna and dynamic physical processes play key roles in influencing the distribution of microbial mats associated with gas hydrates.
We acquire time-lapse images of the main gas hydrate mound, and the bioreactors we deploy, and use these images to quantify the temporal and spatial characteristics of the gas hydrate mound and associated macro and microbial (e.g., giant sulfur oxidizing bacteria) fauna.
We collect samples from MC118 once or twice per year during research cruises. We use research submersibles, like the Johnson Sea Link, to dive down to the seafloor, 1000m below the surface, and collect samples of gas hydrates and sediments. Sometimes, the views when we return to the sea surface are spectacular, as shown here (Figure 6), when we were treated to see a beautiful sunset from inside the submersible.
Bowles, M.W., and S.B. Joye, 2010. High rates of denitrification and nitrate consumption in cold seep sediments. The ISME Journal, 5: 565-567; doi:10.1038/ismej.2010.134.
Bowles, M.W., V. A. Samarkin, K. L. M. Bowles, and S.B. Joye, 2010. Weak coupling between sulfate reduction and the anaerobic oxidation of methane in methane-rich seafloor sediments in ex situ incubations. Geochimica et Cosmochimica Acta, doi:10.1016/j.gca.2010.09.043.
Bowles, M.W., V.A. Samarkin, and S.B. Joye, 2011. Improved measurement of microbial activity in deep-sea sediments at in situ pressure and methane concentration. Limnology and Oceanography: Methods, 9: 499-506.
Bowles, M.W., L.M. Nigro, A.P. Teske, and S.B. Joye, 2012. Denitrification and the environmental factors influencing nitrate removal in Guaymas Basin hydrothermally-altered sediments. Frontiers of Microbiology, doi:10.3389/fmicb.2012.00377.
Joye, S.B., 2012. A piece of the methane puzzle. Nature, 491: 538-539; doi:10.138/nature11749.
Treude, T., L. Levin, C. Smith, and S.B. Joye, 2013. Marine ecosystems associated with near surface methane gas hydrate. In United Nations Environmental Program GRID program, Methane Dynamics in Ocean Ecosystems: Past and Present, (in press).
Adams, M.M., A.L. Hoarfrost, A. Bose, S.B. Joye, and P.R. Girguis, 2013. Anaerobic short-chain alkanes in hydrothermal sediments: influences on sulfur cycling and microbial diversity. Frontiers in Microbiology, doi: 10.3389/fmicb.2013.00110.
Wankel, S., M. Adams, D. Johnstone, S.B. Joye, and P.R. Girguis, 2012. Anaerobic methane oxidation in metalliferous hydrothermal sediments: influence on carbon flux and decoupling from sulfate reduction. Environmental Microbiology, 14: 2726-2740.
Dekas, A.E., G. Chadwick, S. Connon, M. Bowles, S.B. Joye, and V.J. Orphan, 2013. Investigating methane seep nitrogen fixation at Mound 12, Costa Rica: Spatial distribution of diazotrophy and significance of the ANME archaea. Environmental Microbiology (in press).
Marshall Bowles (received his PhD 2011)
Ryan Sibert (PhD student)
Lindsey Fields (Post Doc)
The National Institute for Undersea Science and Technology (NIUST)