Molecular microbial ecology and biogeochemistry of gassy and oily cold seeps

We have several projects studying cold seeps in the Gulf of Mexico. We collaborate with Dr. Ian MacDonald (Florida State Univeristy), Dr. Peter Girguis (Harvard Univeristy), Dr. Harry Roberts (Louisiana State University), Dr. Chuck Fisher (Penn State University) and Drs. Jim Brooks and Bernie Bernard (TDI-Brooks International LLC) on the Deep Slope Chemosynthetic Ecosystems Study. The Gulf of Mexico is a prolific hydrocarbon basin; dynamic patters of sediment topography result from salt tectonics, generating basin and ridge type structures on the seafloors. Researchers in the Joye group

Adapted from figure from Dr. Harry Roberts at LSU

quantify rates of microbial processes, like anaerobic oxidation of methane, methanogenesis, alkane oxidation, and sulfate reduction, study the controls on cold seep microbial metabolism, and use molecular ecological techniques to determine which microbes are involved in key biogeochemical processes. We have examined a suite of ancillary geochemical parameters in water, sediment and vent gas samples (gases only), including, nutrient concentrations, redox species (e.g., H2S, Fe2+, dissolved inorganic carbon, etc.), organic matter (e.g., DOC, volatile fatty acids) and dissolved gas (e.g., CH4, C2H6, C3H8, H2) concentrations.

Gulf of Mexico Study Sites

Most of our work on cold seeps is conducted in the Gulf of Mexico, along the Louisiana and Texas continental shelf and slope (500-3500m water depth; see GOM map above). We have worked at sites in Green Canyon (GC234, GC185, GC233, GC852, GC600, and GC415), in Mississippi Canyon (MC118, MC853, MC640), Atwater Valley (AT340), Garden Banks (GB 425), Walker Ridge (WR269), and Alaminos Canyon (AC601, AC818, AC645). Gas hydrate mounds (left) are abundant and this portion of the Gulf of Mexico is a rich petroluem basin and oil and gas harvesting platforms (right) are common.

Sampling the Seafloor

To access cold seep habitats, we conduct research cruises using a mother ship, usually the R/V Seward Johnson II or the R/V Atlantis, and manned submersibles, the Johnson Sea Link (JSL) or ALVIN, which are operated by the Harbor Branch Oceanographic Institute and Woods Hole Oceanographic Institution (WHOI), respectively. We have also used the ROV JASON to sample Gulf of Mexico cold seeps, an unmanned remotely operated vehicle, which is also operated out of WHOI.

The ALVIN is launched from the R/V Atlantis. The ALVIN can dive to depths up to 4000m while the JSL can dive to only 1000m. We often work with the JSL on the upper slope and either the ALVIN or JASON on the deep slope. The image on left shows Dr. Joye talking with the ALVIN pilots prior to a dive. One of the traditions with the ALVIN is that you get an ice water bath after your first dive of the cruise (here Dr. Joye gets an ice water bath from Guy and Marshall).

The JSL is launched from the Seward Johnson II, as seen here from inside the submersible, twice per day. Two scientists accompany a pilot and engineer on each dive. Dr. Joye enjoys being in the "sphere", i.e., the front compartment, of the submersible because the view on the bottom is amazing.

Gas Hydrates

Methane hydratesrepresent one of the most important reservoirs of organic carbon on Earth. Methane hydrates are found along continental margins around the world. Hydrates represent a unique extreme environment that could serve as a novel niche for microbial life. The picture on the left shows a hydrate breaching the surface of the sediment. The picture at the right shows a close up view of the hydrate surface. Hydrates are either white or orange in color. The surface is very uneven because the hydrate is somewhat unstable and dissolution may cause pitting on the surface. The orange coloration results from the incorporation of oil in the ice lattice. These structure II gas hydrates are rich C2-C6 alkanes and hydrogen sulfide and carbon dioxide.

Microbial Mats

Beggiatoa is a chemoautotrophic (=uses inorganic CO₂ as structural carbon source) bacteria that makes a living by coupling sulfur oxidation with nitrate reduction to ammonium (or possibly N₂). Beggiatoa comes in white and orange varieties. Beggiatoa mats are a common feature observed around hydrate and brine sites. Beggiatoa is present on the sediment surface but is also observed deep in the sedimnet. Orange Beggiatoa is often found adjacent to white Beggiatoa (left). Note that the bottom water temperature on the figure is incorrect. The actual temperature is around 6-7 ºC on the bottom. Other bacteria, including sulfur-oxidizing bacteria related to Thiomargarita namibiensis (right) are also common (note white "spheres"). The cores are taken back up to the surface and in the shipboard lab, they are used to examine rates of processes and to obtain pore water profiles of chemical species.

Additional information about the new Thiomargarita-like microorganism is provided here.

Brine Pools {Learn More}

Hypersaline brine pools are another feature of the Gulf of Mexico petroleum basin. Brine pools form when warm, salty fluids migrate through the sediments through fissures in the sediment. The brine is more dense than sea water, so it pools on the surface after cooling to ambient temperature. To date, we have examined two brine pools, one stable brine pool with apparently lower rates of fluid flow (GC233 brine). This brine pool has been stable long enough for a dense community of methanotrophic mussels to develop around the pools edge (A). Such chemoautotrophic symbiotic associations are common at sites of fluid and gas seepage, as seen in the Gulf of Mexico, along the Florida Escarpment and along the Cascadia margin. The other brine site is an active mud volcano that is known for high rates of fluid flow (GB425, B). The GB425 brine has frequent eruptions of warmer (10 or more ºC warmer than bottom waters) fluid (B) and macrofaunal communities are not common here. We sample the brines using a novel device called the 'brine trapper' (C, 3-m long gray PVC device in lower part of figure), which is deployed from the side of the submersible. A photo of the brine trapper deployed in the GB425 brine pool is shown in panel (D). The brine is sediment is particle rich (E), as noted by the change in color deeper in the brine (from L to R in the image). A unique feature of the mud volcano site is the abundance of barite (Barium-Sulfate) chimneys (F). Barium originating from the brine precipitates when it comes into contact with sulfate-rich seawater.

A		B
C		D
E		F

MC118 GAS HYDRATE MICROBIAL OBSERVATORY: STUDYING LONG TERM PATTERNS IN THE ABUNDANCE, IDENTITY, AND ACTIVITY OF MICROORGANISMS ASSOCIATED WITH GULF OF MEXICO GAS HYDRATES

Overview

This project is a collaborative effort between the Joye group and Dr. Ian MacDonald (Florida State University). Our 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. Jeff Chanton and Laura Lapham (Florida State University), Drs. Andreas Teske and Chris Martens (University of North Carolina), and Drs. Ray Highsmith and Carol Lutken (University of Mississippi).

Our work focuses on gas hydrate mounds and the associated sediments. 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 a unique deep sea habitat: gas hydrates. We will characterize the poorly understood microbial populations mediating important processes in the carbon and sulfur cycles and to further elucidate survival strategies of microbes living in gas hydrate ecosystems.

The main objectives of this work are to:

1. to estimate microbial abundance and the diversity of microbial communities in gas hydrate and associated sediments;
2. to quantify rates of methane oxidation and sulfate reduction in gas hydrate and sediment samples in ex situ and slurry incubations;
3. to determine whether methane oxidation and sulfate occur within ‘intact’ (i.e., solid) gas hydrate, and;
4. 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 proposed project will continue and expand our study of microbial distributions and activity in gas hydrate ecosystems. As well as 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 MC110.

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 (oils) hydrocarbons 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

Figure 2

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 moderate 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 obstacle for life. Knowing whether gas hydrates support diverse and active microbial assemblages will 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, has 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 we know about microbial activity in gas hydrates?

Due to the chemical composition of Gulf of Mexico gas hydrate, a variety of biogeochemical processes may occur within the gas hydrate niche. We are looking for: (1) aerobic methane oxidation, (2) anaerobic oxidation of methane 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 aerobic methane oxidation (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.

The anaerobic oxidation of methane (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 supports 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, see Joye CV for the reference), 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).

Figure 3: Gulf of Mexico gas hydrates support a visibly diverse biological assemblage. (A) Exposed gas hydrate with an orange Beggiatoa mats beneath it (image is about 75 cm wide). (B) Orange Beggiatoa overlying cm-thick sediment drape on top of a gas hydrate. (C) Hydrate surface showing ice worms (Hesiocaeca methanicola), ice worm burrows and sediment loosely attached to the hydrate surface. The ice worms are about 3 cm long.

Figure 4: Rates of AOM and SR in hydrate material collected from Gulf of Mexico hydrate sites in 2001 and 2002. SR rates (white columns with black error bars) are plotted on the left axes. AOM rates (black columns with white error bars) are plotted on the right axes. Note the different scales. Sample types are shown on the x-axis. Significant rates of AOM and SR have been observed in all the samples we have analyzed to date. Experimental conditions were 8ºC, 1 atm and 125 µM CH4.

Figure 5: Microscope image of ANME-1 archaea from a gas hydrate sample that was fixed with formalin and later analyzed using CARD-FISH to determine the abundance of specific microbes (see methods). Bacterial numbers in interface hydrate-sediment (4x108) exceeded those in the interior (2x106; Orcutt et al. 2004). The scale bar is 5 µm.

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 “intact” gas hydrate that is incubated at in situ pressures using a hydrostatic pressure vessel. Documenting microbial activity within solid gas hydrate using radiotracers would represent a substantial advance towards understanding the importance of gas hydrate as a viable microbial niche. These data may also help elucidate the impact of microbial activity on gas hydrate dynamics in the environment.

Our Hypotheses

We will test the hypotheses presented below by comparing microbial distributions, abundance, and activity in a variety of gas hydrate samples. We will apply 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 will be determined using techniques we developed during previous research projects in combination with high pressure incubation technique that will allow us to simulate in situ conditions. We will use 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 will 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 from in situ bioreactors and from hydrate samples collected at the sea floor.

Hypothesis 2: Rates of AOM and SR within gas hydrates are significant.

We will determine rates of AOM and SR in hydrate samples using radioisotope tracer techniques. Rates will be determined in experiments using slurries and in experiments with ‘intact’ (i.e., solid) gas hydrate 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 will conduct slurry experiments amending gas hydrate and sediment samples with important substrates (e.g., CH4, SO42-, or oil) and monitoring 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 will acquire time-lapse images of the main gas hydrate mound, and the bioreactors we deploy, and will 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.