B51F-01 INVITED
Using Crustal Fluids to Peer Into the Subseafloor Microbial Habitat
In hard rock seafloor environments, fluids emanating from the basement are one of the best windows into the subseafloor and its resident microbial community. These low-temperature crustal fluids are ubiquitous at both active hydrothermal systems and ridge flank environments. Over the last 15 years, studies of the microbial communities in crustal fluids from eruptive events, drill holes, ridge flanks, and hydrothermal seamounts have revealed a phylogenetically and physiologically diverse microbial community, representing a wide spectrum of thermal tolerances and metabolic strategies from both the subseafloor and the deep sea. In addition, emerging technologies in seafloor sampling capacity and microbial ecology are rapidly increasing our ability to study this difficult habitat. This presentation will provide an overview of what we have learned about the population structure, genomic repertoire, and physiological function of microbes in crustal fluids and what the future holds for subseafloor biosphere research. Data will be integrated with geochemical measurements in crustal environments to better define the subseafloor habitat and its resident microbial community.
B51F-02
The Biogeochemistry and Ecology of Deep Sediment-Buried Basement Biosphere: Juan de Fuca Ridge Flanks
Our Deep Biosphere project (funded by NSF-Microbial Observatory) is designed to exploit the unprecedented opportunities provided by the new generation of long-term borehole-CORK observatories (advanced CORKs) installed on the flanks of the Juan de Fuca Ridge (JdFR) by the Integrated Ocean Drilling Program (IODP), to study the microbial geochemistry and ecology of the sediment-buried ocean basement. We will present a description of the new CORK's attributes with respect to deep biosphere studies, and the instrumentation sleds and associated equipment that we have built in support of these studies. The instrumentation sleds (e.g., the GeoMICROBE sled) allow for "in situ" (i.e., at the seafloor) geochemical analyses (e.g., electrochemistry) of the 30-65oC fluids that circulate within the sediment-buried basement., as well as the in situ filtration of fluids for ship and shore-based molecular biological, culture, biomass and geochemical procedures. We will also present the challenges, solutions and preliminary results of our recent (August 2008) Atlantis/Alvin cruise to the JdFR flank CORKs.
B51F-03
The BOSS: a novel approach to coupling temporal changes in geochemistry and microbiology in the deep subsurface biosphere.
Though our knowledge of deep subsurface environments is burgeoning, our understanding of the physiological capacity and activity of deep subsurface microbial communities is in its infancy. Specifically, the quantitative relationship between microbial diversity, density, and activity and geochemical cycles is poorly understood, as is true for most marine environments. This is due to the difficulty of concurrently sampling and quantifying both biological and chemical factors over time and space, especially in remote or inhospitable locales. To address this limitation, we have developed a BioOsmoSampling System (or BOSS). Osmosamplers use osmotic pressure to continuously sample seawater, and can be deployed on the order of weeks to years. These osmosamplers have been previously used for geochemical sampling, and we have developed the hardware and reagents to enable their use in microbial sampling. In particular, we have developed the capacity to collect and preserve fluid samples for nucleic acid and protein analyses, as well as lipids and cellular structure (for microscopy). Molecular degradation in a variety of preservatives was <50% (DNA) and <15% (proteins, lipids) after one year, and ongoing efforts suggest that three-year preservation of DNA and proteins is possible. Here we present data from a pilot study, in which we deployed the BOSS at a diffuse flow vent site at the Juan de Fuca Ridge, and an IODP borehole in Middle Valley. We observed co- registered changes in geochemistry (both ions and volatiles), temperature and microbial community composition. We also recovered high densities of previously enigmatic microbes. This proof-of-concept experiment allowed us to correlate changes in microbial population structure with changes in the physicochemical environment in the diffuse hydrothermal flow, and set the stage for future deep subsurface deployments.
B51F-04
Discriminating enumeration of subseafloor life using automated fluorescent image analysis
Enumeration of microbial cells in marine subsurface sediments has provided fundamental information for understanding the extent of life and deep-biosphere on Earth. The microbial population has been manually evaluated by direct cell count under the microscopy because the recognition of cell-derived fluorescent signals has been extremely difficult. Here, we improved the conventional method by removing the non- specific fluorescent backgrounds and enumerated the cell population in sediments using a newly developed automated microscopic imaging system. Although SYBR Green I is known to specifically bind to the double strand DNA (Lunau et al., 2005), we still observed some SYBR-stainable particulate matters (SYBR-SPAMs) in the heat-sterilized control sediments (450°C, 6h), which assumed to be silicates or mineralized organic matters. Newly developed acid-wash treatments with hydrofluoric acid (HF) followed by image analysis successfully removed these background objects and yielded artifact-free microscopic images. To obtain statistically meaningful fluorescent images, we constructed a computer-assisted automated cell counting system. Given the comparative data set of cell abundance in acid-washed marine sediments evaluated by SYBR Green I- and acridine orange (AO)-stain with and without the image analysis, our protocol could provide the statistically meaningful absolute numbers of discriminating cell-derived fluorescent signals.
B51F-05
Methanogenesis in the hot and deep: implication for the deep biosphere
Microbial methanogenesis in the deep-sea and deep subseafloor is a key process in the carbon cycle of Earth. Hyperthermophilic methanogens are important primary producers in the deep, hot ecosystem and may represent the most ancient type of life flourishing in the early Earth. Nevertheless, the biogeochemical function and impact of methanogens in deep sea and deep subseafloor are poorly understood, in part because it is difficult to replicate the high temperatures and hydrostatic pressures in the laboratory. We develop a new technique for cultivation of chemolithoautotrophs under high hydrostatic pressures. Using this technique, growth, survival and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. The results renewed the previous record of upper temperature limit (UTL) for life and the stable carbon isotopic fractionation of the microbiological methane production. These new findings are of great implication for the limits of life and function in the deep biosphere.
B51F-06 INVITED
Patterns of Respiration and Sources of Electron Donors in Subseafloor Sediment
Patterns of co-variance between subseafloor respiration and oceanographic properties show promise for estimating the global extent of microbial activity in subseafloor sediment. Organic-fueled respiration by the subseafloor sedimentary community varies by almost four orders of magnitude from one site to another. It is highly correlated with several oceanographic properties, including distance from shore, annual seasurface chlorophyll concentration, and depth-integrated sedimentary cell abundance. The predominant electron acceptor also varies with the rate of organic-fueled respiration, shifting from sulfate to metals to oxygen as respiration rate declines. The energetic limits to life are extremely low in subseafloor sedimentary communities, particularly in anoxic sediment. Per-cell energy fluxes are orders of magnitude lower in subseafloor sediment than in microbial cultures and surface environments. Per-cell rates of organic-fueled respiration are about an order of magnitude higher (in electron equivalents) in oxic subseafloor sediment of the South Pacific gyre (SPG) than in anoxic subseafloor sediment. Buried organic matter is the principal source of electron donors for subseafloor sedimentary activity. However, mass-balance calculations indicate that hydrogen from in situ radiolysis of water may be the predominant electron-donor source in SPG sediment, where the organic-fueled respiration rate is extremely low. The rate of microbial respiration and the significant reliance on radiolytic hydrogen in SPG sediment may be characteristic of subseafloor sedimentary communities throughout almost half of the world ocean.
B51F-07
Quantifying Global Subseafloor Microbial Abundance: Method and Implications
The marine subseafloor biosphere is estimated to be up to 1/3rd of all life on Earth or 50-80% of the Earth's microbial biomass. These estimates are based on theoretical calculations and cell counts from ODP sites located in areas of high primary productivity. The cell counts range from 108 to 1010 cells- cm-3 in surface sediments and rapidly decrease to 1029 cells-cm-3 within 10 to 100 m of the seafloor. In contrast, recent cell counts from the low primary productivity region of the South Pacific Gyre are four orders of magnitude lower with an even more rapid decrease in cell count with depth. Based on these new observations, the previous estimates of subseafloor biosphere are likely exaggerated upper limits. To obtain a more accurate measure of subseafloor biosphere, we combined well-correlated cell count and depth relationships with the global distribution parameters of sea-surface chlorophyll, organic carbon burial rates, and distance to land. Specifically, we performed linear regressions for a compilation of published subseafloor cell counts plotted as a function of depth in log-log space. The y-intercept (i.e., cell count at 1 m depth) and slope (i.e., log-rate of cell count decrease with depth) for all correlations with R-square values greater than 0.5 were then compared to each of the three global distribution parameters at corresponding geographic locations. The relationship between each global distribution parameter and the remaining y- intercepts and slopes were then used to create a global grid of predicted y-intercept and slope. These grids were integrated as a function of depth to the corresponding sediment thickness (maximum sediment thickness of 4 km) and then integrated spatially to obtain an estimate of global cell counts. The resulting total cell counts are surprisingly similar for each of the global parameters (e.g., 4.2 x 1029 for sea- surface chlorophyll, 5.9 x 1029 for organic carbon burial rates, and 3.8 x 1029 for distance from land) and suggest cell counts that are 10-20% of the previous estimate of 3.5 x 1030. These results imply a significantly smaller marine subseafloor biosphere that only comprises 1/20th of all life on Earth or 5-15% of the Earth's microbial biomass.
B51F-08
Milankovitch-scale correlations between deeply-buried microbial populations and biogenic ooze lithology
Active populations of buried microbes are unevenly distributed in the sub-seafloor of the world's ocean. Globally, the rates of microbial activity in the sub-seafloor of open-ocean, oligotrophic basins are much lower than in ocean-margin eutrophic basins. Variations of cell abundances and metabolic activity are often independent from sediment depths with increased prokaryotic activity at geochemical and/or sedimentary interfaces. At the scale of lithologic units, higher microbial activity has been detected in units with abundant diatom ooze. Given these broad-scale relationships between paleoceanography and sub-seafloor microbial life it is plausible that variations in microbial populations at scales finer than lithologic units may also occur, if properties, such as organic carbon (OC), porosity, or solid-phase electron acceptors, vary within individual beds. In this study we demonstrate that microbial populations vary at the scale of individual beds in the biogenic oozes of a drill site in the eastern equatorial Pacific (Ocean Drilling Program Leg 201, Site 1226). We relate bedding-scale changes in biogenic ooze sediment composition to OC and microbial cell concentrations using high-resolution color reflectance data as proxy for lithology. Our analyses demonstrate that microbial concentrations are larger by an order of magnitude in the more organic-rich diatom oozes than in the nannofossil oozes. The variations mimic small-scale variations in diatom abundance and OC indicating that the modern distribution of microbial biomass is ultimately controlled by Milankovitch-frequency variations in past oceanographic conditions. Because OC becomes more refractory with depth, bedding-scale differences in OC and microbial concentrations are no longer apparent below 200 meters below seafloor (mbsf). The evidence presented in this study suggests that future microbiology sampling schemes that account for small- scale lithologic variations should be part of the study design. Moreover, the correlations between microbial numbers and color reflectance; and between color reflectance and OC, suggest that OC content can be used as proxy for microbial biomass in the sub-seafloor of the eastern equatorial Pacific, and potentially in other sedimentary basins characterized by similar biogenic pelagic sediments.