B53C-0499
The Extent of the Oceanic Biosphere Throughout Geologic Time
The extent of microbial habitability in the ocean crust is a subject of considerable interest. Current data suggests that microbial activity ceases above approximately 120° C, and we use this isotherm as a proxy for determining the depth of the biosphere in the ocean crust. We first estimate the depth of this isotherm as a function of lithospheric age between 1 to 180 Ma by using theoretical conductive heat loss estimates. This model predicts that the maximum depth of the biosphere ranges from approximately 0.5 km at 1 Ma to nearly 5 km at 180 Ma. We then take into account the effect of hydrothermal activity in lithosphere less than 65 Ma. We first approximate this effect by using a close fit to observed conductive heat flow versus age to construct a geotherm. This approach suggests that the 120° C isotherm may be more than 1 km deeper in younger lithosphere than estimated from the theoretical conductive cooling curve. We then attempt to account for hydrothermal cooling more realistically by constructing temperature profiles that would result from either fluid upflow or downflow at constant velocity and that fit observed conductive heat flow data versus age between 1 and 65 Ma. These profiles suggest that estimates based on conductive geotherms in young crust provide over estimates of the depth of the biosphere as a function of lithospheric age. Consequently, the models provide good end-member constraints on the depth of the biosphere as a function of lithospheric age even in the presence of hydrothermal heat loss. By multiplying the estimate for the depth of the biosphere by the rate of plate production and integrating over time, we obtain estimates of the volume of the biosphere in the present oceanic crust. Finally, we extend our results backward through geologic time by considering the effects of greater rates of plate production, younger lithospheric subduction ages, and models of continental growth.
B53C-0500
Using HSDP Samples to Evaluate the Staudigel et al. Model of Euendolithic Microbial Alteration of Marine Basalt Glass: A Peek at the Unseen Majority
Staudigel et al. (2008, Earth Science Reviews 89 156-176.) recognize separate alteration processes of basalt glass in oceanic crust and elsewhere: Formation of palagonite and pore-filling cements is abiotic, while microbes form granular and tubular textures. Granular alteration begins shortly after glass cools to a habitable state and is a product of organisms that etch the surface of basalt glass by adjusting pH. These organisms ultimately dissolve the glass surface to form an agglomeration of micrometer-scale sub-spherical cavities. Smectite, zeolites, Fe-oxide, and Ti-rich structures occupy the cavities. Together with microbes, they form a bio- and mineral film that ultimately shuts down the system. Tubular structures reflect the activity of hypha-bearing organisms that mine the glass to get nutrients. Hyphae allow the organisms to remain in contact with circulating water while penetrating any biofilm and mining nutrients from unaltered glass. Tubules are about 1 micrometer in diameter and 10s to 100s of micrometers long. Both suites of organisms use reduced elements from the glass as sources of electrons for their metabolism. Activity ceases when secondary minerals reduce permeability to circulating seawater and availability of some nutrients. HSDP results, from an ocean island, show some areas of agreement. Petrographic studies indicate that initiation of tubules post-dates crushing of grains during early burial and formation of isopachous smectite grain coatings. Growth of tubules may be very slow; they grow during a time window of about 30000 to 60000 years, although duration of growth of individual tubules may be much shorter. Tubules and other textures of alteration are commonly coated with ferric hydroxide, perhaps a product of microbial oxidation. However, some features of biotic alteration in HSDP hyaloclastites are inconsistent with the proposed model. Smectitic grain replacement, which resembles granular alteration, forms in HSDP samples after and in association with tubular structures. Smectite replacement of glass is relatively less abundant than tubular alteration; many areas of tubular alteration lack any granular associates. Biotic alteration of glass does not itself develop pore-blocking minerals, but early isopachous smectite and later zeolites formed during palagonitic alteration reduce porosity to a few percent, effectively terminating boring. Tubules undergo modification from cylindrical to steep cones and develop minute branches. Tubule configuration indicates that boring organisms were attracted to olivine crystals and avoided plagioclase.
B53C-0501
Experimental Study of Hydrogen Generation During Low Temperature Basalt-Water Interaction
Hydrogen generated from the reaction of water with basalt has been proposed as a source of metabolic energy to support subsurface autotrophic microbial communities in both terrestrial and seafloor settings. However, few experimental studies have examined the production of hydrogen from basalt-water interactions at low temperature (e.g., Stevens and McKinley, Science, 1995; ES & T, 2000), and the reactions that might be responsible for hydrogen production remain poorly known. In order to address this issue, we have initiated an experimental study to examine hydrogen production in basaltic systems. Initial experiments have been conducted by reacting basalt powder with water in glass vials, and monitoring hydrogen in the headspace. The experiments encompass a range of temperature (25-100 ° C), pH (5-9) and grain size. Results indicate that within the first few days hydrogen is produced rapidly, in agreement with previous studies. However, the production rates taper off rapidly with continued reaction, indicating that initial rates are not indicative of long-term hydrogen generation in natural systems. Significantly, although previous studies (Stevens and McKinley, 1996, 2000) had concluded that reaction of the mineral olivine is the primary source of hydrogen production in basalt, we find this mineral to be unreactive at temperatures of 100 ° C and lower.
B53C-0502
Kinetic and energetic limitations on the distribution of microbes in the deep subsurface oceanic crust
Marine subsurface environments contain a significant fraction of the total biomass on Earth, which raises fundamental new questions about the limits of life and its evolution. It is so far largely unknown which metabolic pathways the microorganisms in this 'deep biosphere' use for energy generation and how the energy dependence influences the distribution of microbial life in these energy poor systems. To identify thermodynamic and kinetic restrictions on the activity of deep subsurface microbial communities and determine which metabolic pathways might be preferentially used is important to better understand energetic constraints on organisms and the implications they have for the evolution and distribution of life. In the oceanic crust at the flankes of the Midocean Ridge the relatively oxidized seawater is flushed through the relatively reduced basaltic basement rock creating redox gradients that could potentially deliver energy for microbial metabolisms based on reduced minerals containing Fe2+ and reduced sulfur as sources of electrons for metabolic growth. At low temperatures abiotic dissolution of the iron minerals is very slow and the oxidation of Fe2+ could therefore serve as an energy source for microorganisms who can overcome the kinetic limitation of this process. We are applyying both thermodynamic and kinetic calculations to estimate the available energy sources and their kinetic contraints in potential metabolic reactions in the oceanic crust. For each possible reaction it was determined how sensitive the energetic yield is towards temperature changes and changes in the concentrations of each substrate and product involved in the reaction, in order to predict which pathways might be most likely to occur. First results reveal that there is abundant potential energy present that could be harvested in a variety of metabolic reactions known to be used by microorganisms in other habitats. As some of these reactions are severely limited by kinetic considerations the distribution of microbial activity is probably to a large extend regulated by the kinetic drive of different reactions under the ambient concentrations. These theoretical estimations will be tested in an upcoming research cruise where we will analyze the diversity of the microbial community, trying to identify organisms using the metabolic pathways that seem most feasible in our prediction and determine by their distribution how they might have colonized the isolated newly formed oceanic rock.
B53C-0503
Chemical and Physical Characteristics of Basaltic Formation Fluids on a Ridge Flank: Using Drilling Perturbations to Elucidate Water-Rock-Microbial Reactions
Holes 1301A and 1301B were drilled, cased, and instrumented with long-term, subseafloor observatories (CORKs) on the eastern flank of the Juan de Fuca Ridge in Summer 2004. These holes penetrate 265 m of sediment and the uppermost 108 to 318 m of 3.5 Ma basaltic basement, in an area of vigorous, warm (64C) hydrothermal circulation. The new boreholes were located 1 km south and 2.4 km southwest of instrumented Holes 1026B and 1027C, respectively, that were emplaced eight years earlier. This network of four instrumented boreholes was established as part of a long-term, cross-hole experiment that will elucidate hydrologic properties and the nature and dynamics of microbial ecosystems within the upper oceanic crust, in a well defined geochemical and physical context. Downhole instrumented OsmoSampler packages in Holes 1301A and 1026B were replaced by submersible in summer 2008, as part of a program of observatory servicing in preparation for the next drilling expedition and the initiation of cross-hole experiments in this area. The borehole instrument package from Hole 1301A sampled borehole fluids within the upper 107.5 m of basaltic crust during a four-year period of drilling disturbance, self-sustaining flow of cold bottom water into basement, and subsequent recovery to near-predrilling chemical and thermal conditions. Because the borehole was incompletely sealed at the time of initial installation, bottom seawater flowed down into the borehole during the first three years following emplacement, driven by the higher density of cold bottom water relative to warm formation fluid. Borehole thermal records during the first 1.5 years show that temperatures in basement were below 10 C, and fluid samples from the borehole have a chemical composition similar to bottom seawater. Temperatures fluctuated for the next 1.5 years between 10 and 30 C, and the fluid composition began to shift towards that seen in regional basement fluids sampled at nearby Baby Bare outcrop and from Hole 1026B. In early September 2007 the natural formation overpressure overcame the excess pressure of cold bottom water and began to vent a mixture of recently-recharged bottom water and warm formation fluid. The present day composition of fluid venting from Hole 1301A is very similar to that sampled from Baby Bare outcrop. The progression from bottom seawater to formation fluid chemistry is not conservative relative to temperature, most likely because of water-rock and microbial reactions within basaltic basement.
B53C-0504
Real-Time Detection of Redox Species in Basement Fluids Accessed Through IODP CORK Observatories
Growing evidence suggests that a substantial subseafloor biosphere extends throughout the immense volume of sediment-buried basement that underlies the global system of mid-ocean ridge (MOR) flanks and ocean basins. CORK (Circulation Obviation Retrofit Kit) observatories affixed to Integrated Ocean Drilling Program (IODP) boreholes offer an unprecedented opportunity to study intriguing questions regarding biogeochemical properties and microbial diversity in circulating fluids from buried ocean basement. Here, we describe voltammetric measurements collected from DSV Alvin using an in situ electrochemical analyzer (ISEA) coupled to CORK Observatory Fluid Delivery Lines in Cascadia Basin on the Juan de Fuca Ridge Flanks. The ISEA allows for deployment of up to four solid-state gold amalgam working electrodes, capable of providing simultaneous detection of oxygen, iron, sulfur, and other species in real time or continuous data logging modes. We also present traditional and electrochemical on-deck measurements taken on discrete samples collected during voltammetric seafloor scanning to illustrate changes in speciation and oxidation rates that occur between sample collection and on-deck analyses.
B53C-0505
Microbial Observatories Designed to Assess the Basaltic Ocean Crust Deep Biosphere
Although the current census of life indicates a large biomass of life in deep marine subsurface sediments, there is a relative dearth of information regarding microbial life in the hard rock environment of the ocean crust. Considering that the ocean crust comprises the largest hydrologically active environment on Earth, that crustal rocks can be out of redox equilibrium with percolating crustal fluids, and that recent evidence shows that seafloor exposed basalts harbor abundant and highly diverse microbial communities, it is likely that ocean crust basalt supports a significant microbial habitat. Due to current obstacles faced by the scientific community in sampling the oceanic hard rock seafloor, innovative microbial observatories have been deployed at the seafloor and deep in ocean crust to address whether sub-seafloor ocean crust basalts harbors a deep biosphere. These observatories are designed to encourage growth of in situ organisms on specific mineral surfaces for spatially resolved characterization of the microbial processes and populations involved in deep basalt weathering.
B53C-0506
Sub-seafloor Microbial Colonization of Igneous Minerals and Glasses
Understanding how subsurface microorganisms (MOs) contribute to mineral weathering and global element cycling requires an initial investigation into the differential colonization of minerals by distinct physiological types of MOs. We initiated a sub-seafloor experiment utilizing a variety of igneous minerals and glasses at IODP borehole site 1301A on the eastern flank of the Juan de Fuca Ridge. We selected twelve different igneous minerals and glasses and placed them in flow cell chambers at 278 meters below the seafloor in borehole 1301A. This horizon is approximately 15 meters into 3.5 million year old basalt basement, which underlies 263 meters of sediment. The samples were incubated in the borehole for four years and recovered in summer 2008. We report total colonization of the igneous minerals and glasses measured by cell density after DAPI staining and microscopic counting. To understand the relationship between MOs and mineral surfaces, we analyzed thin sections made from DAPI-treated minerals and glasses included in low- fluorescence resin.
B53C-0507
Prokaryotic diversity, distribution, and insights into their role in biogeochemical cycling in marine basalts and gabbros
Oceanic crust covers nearly 70% of the Earth's surface, of which, the upper, sediment layer is estimated to harbor substantial microbial biomass. Marine crust, however, extends several kilometers beyond this surficial layer, and includes the basalt and gabbro layers. The microbial diversity in basalts is well characterized, yet metabolic diversity is unknown. To date, the microflora associated with gabbros, including microbial and metabolic diversity has not been reported. In our analyses basaltic and gabbroic endoliths were analyzed using terminal restriction fragment length polymorphism, cloning and sequencing, and microarray analysis of functional genes. Our results suggest that despite nearly identical chemical compositions of basalt and gabbro the associated microflora did not overlap. Basalt samples harbor a surprising diversity of seemingly cosmopolitan microorganisms, some of which appear to be basalt specialists. Conversely, gabbros have a low diversity of endoliths, none of which appear to be specifically adapted to the gabbroic environment. Microarray analysis (GeoChip) was used to assay for functional gene diversity in basalts and gabbros. In basalt genes coding for previously unreported processes such as carbon fixation, methane-oxidation, methanogenesis, and nitrogen fixation were present, suggesting that basalts harbor previously unrecognized metabolic diversity. Similar processes were observed in gabbroic samples, yet metabolic inference from phylogenetic relationships of gabbroic endoliths with other microorganisms, suggests that hydrocarbon oxidation is the prevailing metabolism in this environment. Our analyses revealed that the basalt and gabbro layers harbor microorganisms with the genetic potential to significantly impact biogeochemical cycling in the lithosphere and overlying hydrosphere.
B53C-0508
Linking microbial ultrastructure and physiology to iron depositional processes in deep sea hydrothermal environments
Clara S. Chan, Emily Fleming, David Emerson, Katrina J. Edwards Iron microbial mats have been discovered in a variety of deep-sea hydrothermal environments and are increasingly being recognized as more seafloor is explored. The predominant structures found in many of these mats are iron oxyhydroxide-rich filaments. One of the most common structures is a helical stalk bearing a resemblance to the twisted stalk of the terrestrial iron-oxidizing microbe, Gallionella ferruginea. While Gallionella has not been detected in, or isolated from, these mats microaerophilic iron-oxidizing, a stalk- forming bacterium, Mariprofundus ferrooxydans (PV-1 and related strains) has been isolated from mats at the Loihi seamount in Hawaii (Emerson et al. 2007, PLoS One 2(8): e667). Fossilized aggregates of iron filaments have been observed in the rock record (e.g. Little et al. 2004, Geomicrobiol. J. 21:415), and may represent ancient versions of these microbial mats. If this is shown to be true, such filaments would represent one of the few microfossil morphologies that can be linked to a specific microbial metabolism. We have used a combination of test tube culturing, microslide culturing, time lapse microscopy, and electron microscopy to study Mariprofundus stalk morphology and genesis and link these details to physiological responses to environmental chemistry. The goals include determining specific attributes of stalk morphology that can be used to determine the biogenicity of putative iron microfossils, and interpret the conditions of the depositional environment. Light microscopic observation of microslide cultures over the course of several days allowed for determination of bacterial response to developing oxygen and Fe(II) gradients. Once gradients have been established, given an abundant supply of oxygen, cells congregate in a band perpendicular to the gradient and stalks are formed, growing in the direction of increasing oxygen (and decreasing Fe) concentration. This directional growth may be useful in detecting microscale environmental gradients. Furthermore, this behavior may be preserved in the fossil record, which would allow provide insight into the chemistry of past microenvironments. Experiments are being performed to determine the rate of stalk Fe deposition per cell, in order to relate microbial scale processes to geologic-scale deposits. We are currently investigating the details of taxis and motility. Given available data, we posit a life cycle that involves the following: (1) a "free" stalk-less swimming cell, (2) the cell attaches to a solid surface and starts forming a stalk, (3) depending upon the microenvironment, stalks may grow toward higher oxygen (4) stalk growth stops when conditions no longer sustain cell metabolism and the cell detaches to become a free- swimming cell once again. This study gives yields a better understanding of how these microbes colonize and control chemical gradients in deep-sea microbial mats, and provides criteria to evaluate the biogenicity and environments of fossilized mats. We are beginning cryoelectron tomography studies of M. ferrooxydans, in which we will observe internal and external ultrastructure, allowing us to localize the cellular machinery involved in stalk formation, iron oxidation, and biomineralization.
B53C-0509
Bacterial Diversity in Deep Sea Hydrothermal Environments as Determined by Massively Parallel Pyrosequencing
Seafloor alteration of rocks and minerals exerts influence on many biogeochemical elemental cycles, but the controls, rates, and influence on and by microbiological processing are not well understood. Furthermore, because mineral-microbe interactions and biologically mediated alteration processes may propagate well into the subseafloor, it is important to ground our understanding of these processes where samples can be accessed and hypotheses tested. Here, the bacterial diversity in deep sea hydrothermal sulfides from the EPR 9°N region and basalts from the Loihi Seamount near Hawaii were investigated using massively parallel pyrosequencing (454 sequencing) of the V6 hypervariable region of the 16S rRNA gene. Our goal was to determine the relative diversity of these samples, which are all influenced by hydrothermal fluids, versus other seawater habitats unaffected by hydrothermal processes. Using the 454 sequencing method, we obtained ~60 base pair reads for thousands of sequences per sample, allowing for vastly more detailed fingerprinting of the microbial community than previously possible. We retrieved seven samples from hydrothermal vent chimneys high in sulfide content from the EPR 9°N region and one sample from basalts at the Loihi Seamount off Hawaii. Rarefaction curves of total species number per sample were constructed and diversity between samples was compared. In addition, for the basalt sample a comparison of 454 data to full length sequences of the 16S rRNA gene sequenced via traditional capillary sequencing was made to try to understand the different results garnered by using these two different methods.
B53C-0510
Linking Geochemical Models and Microbial Populations Within Hydrothermal Chimneys on the East Pacific Rise
We present here a statistical comparison of the reported phylogenetic diversity from two studies: a 'proto-chimney' at Q vent, sampled after 92 hours (McCliment et al., 2006), and a mature beehive chimney at Bio9 vent (Kormas et al., 2006), both on the East Pacific Rise. The mature chimney displayed a significantly lower diversity than expected using the delta test, a measure of functional diversity, relative to a master database of phylogenetic data reported from vents along the East Pacific Rise. This result may indicate that the beehive environment present at the time of sampling at Bio9 vent (2000) harbored a more unique or endemic population, despite the chimney being well-established. In comparison, the proto-chimney harbored a similar abundance of Archaeal clones but had a functional diversity score closer to the expected average. To determine linkages between these phylogenetic surveys, observed chemistry of endmember fluids and mineralogy of these structures, we use geochemical modeling to simulate the micro-environments within the chimney wall consistent with observed mineral paragenesis. The proto-chimney was modeled as new growth strictly caused by mixing of the endmember vent fluid and seawater, while the mature chimney was modeled as mixing within an established chimney wall, similar to models of Tivey (1995). Results indicate that despite differences in chimney age/maturity and predicted fluid chemistry across the chimney wall, the predicted free energy for various autotrophic metabolic reactions is very similar between the two vents. Iron oxidation is predicted at temperatures higher than microorganisms are known to tolerate (>160 degrees C), but sulfate reduction and methanogenesis are predicted at all temperatures. Oxidation of elemental sulfur is predicted only at temperatures below about 15 degrees C. Noticeably absent are elemental sulfur reduction, sulfide oxidation, and the knallgas reaction, all of which are inferred to be present based on closest relatives of observed clones. Future modeling efforts may need to incorporate the energetic feedbacks among consortia of microorganisms in order to explain the complexity of natural systems.
B53C-0511
Geochemical Characterization of Subsurface Microbial Habitat Produced by Serpentinization: Preliminary Results from Coast Range Ophiolite Formation Fluids
We examined the mineralogy and aqueous geochemistry associated with five wells sunk (up to 45 m deep) in actively serpentinizing bodies of the Coast Range Ophiolite to assess their potential as subsurface habitats for life. Through production of hydrogen, serpentinization has the potential to support a variety of subsurface microbial metabolisms (e.g., methanogenesis), and is thus of interest both in charting the deep biosphere on Earth and in assessing the habitability of other planetary bodies with ultramafic components. This site, at which repeated monitoring and ready access to the subsurface are possible, offers great potential for characterizing the long-term life-hosting potential of serpentinizing systems. Inspection of archived core cuttings from the same locality, sampling serpentinite up to 110 meters below the surface, reveals nearly completely serpentinized parent rock, with polished and massive serpentine, and prominent bastite (serpentinized pyroxene) grains. Historical records of fluid geochemistry from the wells, dating to their installation in the 1980's, show consistently elevated pH (9.12-10.15) and Ca2+ concentrations (12-84 ppm), consistent with formation under serpentinizing conditions. In general, these formation fluids are depleted in major ions and enriched in Fe and Mn with respect to seawater. New data, including fluid concentrations of methane and hydrogen, are similarly consistent with active serpentinization. Geochemical modeling of these data constrains the free energy yields that could be accessed by H2 - consuming microbial metabolisms, including methanogenesis. These models constitute the first in a series of quantitative assessments of the habitability of a subsurface serpentinizing system.
B53C-0512
Why heavy oilfields exist? The dynamic interplay of oil charge, basin dynamics, caprock leakage and gas generating biodegradation that produces heavy oilfields
Heavy oil and bitumen resources develop by extensive in-reservoir oil biodegradation resulting in a wide range of oil compositional gradients that reflect the complex interplay of oil charge rate and composition, biodegradation in oil-water transition zones at the base of oil columns and geologically controlled in-reservoir diffusive mixing over geological time. Worldwide, observed compositional gradients are maintained by unaltered oil charge near the top of reservoirs and concomitant degradation at the base of the reservoirs at rates comparable to the charge rates of oil fields. Across the Alberta oil sands, elevated CO2, high CH4 and low C2+ gas contents, steep oil compositional gradients, high aqueous bicarbonate concentrations and isotopic values in equilibrium with enriched d13CCO2 gas signatures are indicative of active persistence of active biodegradation to the present. Numerical models of carbon isotope systematics identify the dominant reaction pathway of subsurface hydrocarbon biodegradation as methanogenic alkane degradation by CO2 reduction, which produces large volumes of isotopically light methane and heavy CO2 in solution gas. Simple charge-degrade numerical models predict generation of 3 to 6 times reservoir volumes of biogenic gas in the genesis of heavy oil over geological time, which would have displaced oils from the traps. Gas caps in shallow reservoirs are small at best, suggesting seal leakage is pervasive and this is confirmed by degraded oil in many heavy oil caprocks. Also much less CO2 is measured in biodegraded oil field gases than is predicted based on reaction stoichiometry. The paucity of large gas caps, evidence of methane-rich and sometimes oil charged cap rocks, anomalously high formation water alkalinity and enriched aqueous d13Ccarbonate values in shallow Alberta biodegraded oil reservoirs point to leaky reservoir top seals and dissolution of biogenic gas into the water and oil phases. Indeed we consider top seal leakage of biogenic gas required to produce heavy oil and super heavy oil fields to maintain active biodegradation at the oil-water contact by transport of nutrients through the water phase. This would otherwise be curtailed by petroleum completely filling the reservoir to the underseal. The abundance of heavy oil and super heavy oil in shallow reservoirs reflect leaky reservoir seals at these depths and systematic removal of large volumes of gas generated by petroleum biodegradation and sometimes spill of oil. In uplifted basins with shallow heavy oil resources, degassing of oil and discharge of biodegradation generated gas may also have episodically contributed substantial carbon loads to the atmospheric carbon budget.
B53C-0513
Activity and Extent of Carbon Dioxide and Acetate Utilizing Methanogens in Deep Organic- rich Aquifers Within the Illinois Basin, USA
Global climate change and rising energy demands are motivating investigations of CO2 sequestration in deep geologic reservoirs within interior sedimentary basins, such as the Illinois Basin. Here in the organic rich Upper Devonian New Albany Shale and Pennsylvanian coal beds, potential CO2 sequestration sites coincide with microbial communities that are actively producing economic volumes of methane. Microbes generate methane via two major metabolic pathways, CO2 reduction and acetate fermentation. Injecting CO2 into organic-rich reservoirs may affect in-situ microbial communities and may result in enhanced methane generation, however predicting these affects is difficult unless accompanied by an understanding of the distribution and activity of life in the deep biosphere in various hydrogeochemical settings. This study investigates the extent, controls on, and metabolic pathways for methane generation in the New Albany Shale and Pennsylvanian coalbeds across the eastern margin of the Illinois Basin. We hypothesize that CO2 reduction is the dominant metabolic pathway, however acetate fermentation is enhanced in areas where there is more labile organic matter, lower salinity concentrations, and more rapid recharge rates, such as in shallow coal zones and near the shale subcrop. To test this hypothesis we coupled compound specific isotopes of CO2, CH4, DIC, and H2O with acetate, cation, and anion concentrations, and microbial studies to determine the extent of acetoclastic versus hydrogenotrophic methanogenesis, groundwater residence times, and major environmental controls on metabolic pathways. These results have implications for CO2 sequestration and stimulation of methanogenesis in deep organic rich formations, which may help to reduce global impacts of climate change and prolong economic reservoirs of methane.
B53C-0514
Meter-long microbial ropes from euxinic cave lakes
Cave divers exploring a remote conduit in the Frasassi cave system (Italy) discovered unusual 1 to 2 meter-
long, rope-like microbial biofilms in the anoxic layer of a permanently stratified cave lake. Organic carbon in
the rope-like biofilm has a delta 13C value of -33 per mil, indicating in situ lithautotrophic primary production
and little or no input from surface-derived plant carbon. SEM/EDS of critical point dried samples showed
interlocking strands of microbial-sized filaments with trapped mineral particles including S, CaCO3, silicate
clays, and sulfate and phosphate minerals. Staining with the nucleic acid dye DAPI further showed that the
ropes are composed of closely packed, intact microbial cells. Geochemical profiles of the stratified lake
(conductivity, pH, ORP, T, oxygen, sulfide, sulfate, ammonium) delineate a sharp chemocline at ~2.3 m water
depth, several meters above the ropes. Geochemical data for the water surrounding the microbial ropes
suggest that little redox energy is available, and that sulfate reduction and methanogenesis should be the
most favorable reactions. Radiocarbon ages for the ropes are associated with a large uncertainty due to
potentially changing contributions of radiocarbon-dead limestone carbonate over time. However, the data
suggest that the ropes are thousands of years older than animals in the cave system, consistent with extreme
energy limitation and slow growth.
Based on phylogenetic analyses of archaeal, bacterial and universal 16S rDNA clone libraries from the
microbial ropes, approximately 50 percent of bacterial clones affiliate with sulfur-reducing
deltaproteobacteria. Approximately 61 percent of archaeal clones (20 percent of all clones) are associated
with an environmental clade of euryarchaeota commonly retrieved from deep sea sediments (MBG-D). Most
other clones in the libraries grouped in clades without cultivated representatives. No clones associated with
known methanogens or anaerobic methane oxidizers were retrieved.The diversity of genes associated with
methane metabolism (mcrA) and dissimilatory sulfate reduction (dsrAB) are currently under investigation
using PCR. Future work will employ a full cycle rRNA approach in addition to stable and radioisotope labeling
to identify specific phylotypes in the ropes and link them with geochemical processes.
http://www.geosc.psu.edu/~jmacalad/cavemicro.html
B53C-0515
Bacteria-Driven Carbon Metabolisms in Methane Hydrate-Bearing Deep Marine Sediments from the Bay of Bengal
The methane hydrates in deep marine subsurface sediments play an important role in the global biogeochemical cycling of carbon. However, we have a limited understanding of the microbial communities and their metabolic functioning with respect to carbon assimilation and respiration pathways in those habitats. Our objective was to determine the microbial diversity and the primary functional genes relevant to potential carbon metabolism in sediments that contain the deepest methane hydrates yet discovered. The samples were obtained from offshore India near the Andaman Islands in the Bay of Bengal, representing the deepest methane hydrate sediments found to date, likely due to the low geothermal gradient. The hydrate was found in sediment layers that contain coarse-grained volcanic ash in the depth range from 300 to 650 meters below the seafloor. DNA was extracted from 13 depth horizons, eight of which were found onboard to contain methane hydrates. Microscopic cell enumeration and domain-specific quantitative polymerase chain reaction (qPCR) revealed that those sediments harbor relatively small microbial populations that are composed mainly of Bacteria. For each sample, multiple displacement amplification (MDA) followed by PCR amplification was attempted using primers specific for archaeal and bacterial 16S rRNA, acetyl-CoA carboxylase (accC), citrate lyase (aclB), pyruvate oxidoreductase (porA), oxoglutarate oxidoreductase (oorA), 1,5-bisphosphate carboxylase (RubisCO: cbbL), particulate methane monooxygenase (pmoA), methanol dehydrogenase (mxaF), and methyl co-enzyme M reductase (mcrA) genes. Consistent with the low Archaea abundance predicted by qPCR, archaeal 16S rRNA, mcrA, and aclB genes were never detected, while the bacterial 16S rRNA genes and the functional genes of oorA, accC, cbbL, pmoA, mxaF, and porA were successfully amplified. Clone libraries of 16S rRNA and functional genes indicated that members of Firmicutes and other Gram-positive Bacteria, such as Bacillus spp., dominate both hydrate and non-hydrate sediment layers. These results of functional gene amplification suggest that bacterial populations related to one-carbon (CO2 and CH4/CH3OH) biogeochemical cycling occur in the sediments examined. Compared to other seafloor settings, this Bacteria-dominated deep subseafloor habitat has unique phylogenetic and functional diversity.
B53C-0516
Microbial Biodiversity in the Subsurface of Carbonate Mounds from the Gulf of Cadiz off Morocco
The study area, El Arraiche mud volcano field, is situated 35 km offshore the north-western Moroccan margin, on top of the accretionary wedge of the Gulf of Cadiz. An exploratory cruise of R/V Belgica in 2002 off Larache (Morocco) has led to the discovery of small mounds topping ridges and structural heights, respectively on Pen Duick Escarpment, Renard Ridge, Vernadsky Ridge and Al Idrisi Ridge. These mounds are found amidst 9 giant mud volcanoes and occur in a setting where focused fluid seepage is observed. Subsequent cruises have confirmed the colonization by dominantly lifeless cold-water corals and have unveiled extensive fields of seep-related carbonates in off-reef regions. We present the microbial biodiversity of the subsurface of two different carbonate mounds (alpha- and beta-mound) flanked by a giant double- peaked mud volcano in the Pen Duick Mound Province in a water depths of 500-600 m. Most of the sediment comprises pelagic calcite (coccoliths), detrital quartz and authigenic dolomite, often observed encasing coccoliths. Stable carbon isotope values of the bulk carbonate range from -7 to -15 permil indicating the involvement of microbes in the production of bicarbonate ions. Pore water analysis evidences a sharp sulphate-methane-transition (SMT) zone at 3.5 m below the mound top, whereas the depth of no sulfate is much deeper in the surrounding sediments. The horizon characterized by a strong corrosion of the coral fragments is just lying above and at the front of the recent location of the zone of anaerobic methane oxidation (AOM). In order to define the primary microbial community involved in carbonate precipitation, we did direct culturing, DNA isolation and PCR analysis of functional genes, including archaeal and bacterial 16S rRNA gene analysis. In combination with a DGGE approach, we developed a microbial biodiversity profile along the two carbonate mounds.
B53C-0517
Geomicrobiology and hopanoid content of sulfidic subsurface vent biofilms, Little Salt Spring, Florida
Sulfide-rich, oxygen-poor environments are widespread in the subsurface and were prevalent at the earth's surface during critical intervals in the geologic past. Modern microbial communities in sulfidic niches have the potential to shed light on the biogeochemistry and biosignatures of anoxia and euxinia in earth history. Caves and sinkholes provide rare windows into microbially-dominated, sulfidic subsurface environments that are otherwise difficult and expensive to access. Little Salt Spring (Sarasota County, Florida) is a cover-collapse sinkhole lake with oxic surface water and anoxic, sulfidic bottom water (Alvarez Zarikian 2005). The site is famous for excellent preservation of human and animal archaeological remains (Clausen 1979), and its microbiology has never been investigated. Abundant white biofilms develop seasonally at a warm vent that feeds into the anoxic bottom water at 73 m depth below the water surface. The biofilms are of interest both as potential sources of biomarker compounds and because of their likely role in sulfuric acid production and limestone dissolution (speleogenesis). Biofilm samples were collected by expert science divers and investigated using microscopy, nucleic acid, and lipid analytical methods. Microscopy of the live biofilm revealed clusters of microbial filaments with holdfasts and dendritic, sulfur-rich colonial structures similar to those described in the 1960s for Thiobacterium, a sulfur-oxidizing genus with undetermined phylogeny. A 16S rDNA library constructed from the biofilm was split into three main phylotypes, with multiple clones representing (1) a Betaproteobacterial clade with no cultivated representatives, (2) filamentous Epsilonproteobacteria, and (3) a major bacterial lineage without named isolates (OP11/OD2). A full cycle rRNA approach is currently underway to link 16S rDNA phylotypes with specific populations in the biofilm. We confirmed using fluorescence in situ hybridization (FISH) that abundant filamentous cells with holdfasts are Epsilonproteobacteria. Additional FISH experiments will target the Betaproteobacterial and OP11/OD2 phylotypes retrieved by cloning. Based on HPLC-MS analyses, the biofilm contains at least 5 membrane hopanoid structures distinct from the suite of hopanoids present in sinking organic particles from the photic zone of the sinkhole. Future efforts will be aimed at linking hopanoid structures to specific sulfur-oxidizing populations and to geochemical parameters such as sulfide and oxygen concentrations. References Alvarez Zarikian,C. A., P. K. Swart, J. A. Gifford, P. L. Blackwelder, Palaeogeography, Palaeoclimatology, Palaeoecology 225, 134 (2005). Clausen, C. J., A. D. Cohen, C. Emiliani, J. A. Holman, J. J. Stipp, Science 203, 609 (1979).