AbstractAlthough little is known regarding microbial life within our planet’s rock-hosted deep subseafloor biosphere, boreholes drilled through deep ocean sediment and into the underlying basaltic crust provide invaluable windows of access that have been used previously to document the presence of microorganisms within fluids percolating through the deep ocean crust. In this study, the analysis of 1.7 million small subunit ribosomal RNA genes amplified and sequenced from marine sediment, bottom seawater and basalt-hosted deep subseafloor fluids that span multiple years and locations on the Juan de Fuca Ridge flank was used to quantitatively delineate a subseafloor microbiome comprised of distinct bacteria and archaea. Hot, anoxic crustal fluids tapped by newly installed seafloor sampling observatories at boreholes U1362A and U1362B contained abundant bacterial lineages of phylogenetically unique Nitrospirae, Aminicenantes, Calescamantes and Chloroflexi. Although less abundant, the domain Archaea was dominated by unique, uncultivated lineages of marine benthic group E, the Terrestrial Hot Spring Crenarchaeotic Group, the Bathyarchaeota and relatives of cultivated, sulfate-reducing Archaeoglobi. Consistent with recent geochemical measurements and bioenergetic predictions, the potential importance of methane cycling and sulfate reduction were imprinted within the basalt-hosted deep subseafloor crustal fluid microbial community. This unique window of access to the deep ocean subsurface basement reveals a microbial landscape that exhibits previously undetected spatial heterogeneity.
AbstractThe oceanic basaltic basement contains the largest aquifer on Earth and potentially plays an important role in the global carbon cycle as a net sink for dissolved organic carbon (DOC). However, few details of the organic matter cycling in the subsurface are known because great water depths and thick sediments typically hinder direct access to this environment. In an effort to examine the role of water–rock–microorganism interaction on organic matter cycling in the oceanic basaltic crust, basement fluid samples collected from three borehole observatories installed on the eastern flank of the Juan de Fuca Ridge were analyzed for dissolved amino acids. Our data show that dissolved free amino acids (1–13 nM) and dissolved hydrolyzable amino acids (43–89 nM) are present in the basement. The amino acid concentrations in the ridge-flank basement fluids are at the low end of all submarine hydrothermal fluids reported in the literature and are similar to those in deep seawater. Amino acids in recharging deep seawater, in situ amino acid production, and diffusional input from overlying sediments are potential sources of amino acids in the basement fluids. Thermodynamic modeling shows that amino acid synthesis in the basement can be sustained by energy supplied from inorganic substrates via chemolithotrophic metabolisms. Furthermore, an analysis of amino acid concentrations and compositions in basement fluids support the notion that heterotrophic activity is ongoing. Similarly, the enrichment of acidic amino acids and depletion of hydrophobic ones relative to sedimentary particulate organic matter suggests that surface sorption and desorption also alters amino acids in the basaltic basement. In summary, although the oceanic basement aquifer is a net sink for deep seawater DOC, similar amino acid concentrations in basement aquifer and deep seawater suggest that DOC is preferentially removed in the basement over dissolved amino acids. Our data also suggest that organic carbon cycling occurs in the oceanic basaltic basement, where an active subsurface biosphere is likely responsible for amino acid synthesis and degradation.
AbstractThe basaltic ocean crust is the largest aquifer system on Earth, yet the rates of biological activity in this environment are unknown. Low-temperature (<100°C) fluid samples were investigated from two borehole observatories in the Juan de Fuca Ridge (JFR) flank, representing a range of upper oceanic basement thermal and geochemical properties. Microbial sulfate reduction rates (SRR) were measured in laboratory incubations with 35S-sulfate over a range of temperatures and the identity of the corresponding sulfate-reducing microorganisms (SRM) was studied by analyzing the sequence diversity of the functional marker dissimilatory (bi)sulfite reductase (dsrAB) gene. We found that microbial sulfate reduction was limited by the decreasing availability of organic electron donors in higher temperature, more altered fluids. Thermodynamic calculations indicate energetic constraints for metabolism, which together with relatively higher cell-specific SRR reveal increased maintenance requirements, consistent with novel species-level dsrAB phylotypes of thermophilic SRM. Our estimates suggest that microbially-mediated sulfate reduction may account for the removal of organic matter in fluids within the upper oceanic crust and underscore the potential quantitative impact of microbial processes in deep subsurface marine crustal fluids on marine and global biogeochemical carbon cycling.
AbstractTo expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.
AbstractMarine sediments are a primary reservoir for the long-term storage of organic matter, and the rate of burial and oxidation of this sedimentary organic material help to regulate both atmospheric oxygen and carbon dioxide concentrations. To evaluate the impact of circulating basement fluid on the preservation of deeply buried organic carbon, sedimentary profiles of dissolved and particulate organic carbon (DOC and POC) near the sediment/basement interface were obtained from sediment coring at Site U1363 during Integrated Ocean Drilling Program Expedition 327. Sedimentary DOC increased from 0.25 mM at 1 m below the seawater/sediment interface to a maximum of 0.86 mM at mid-depth (8–11 meters below seafloor [mbsf]), before subsequently decreasing to a minimum of 0.10 mM at the sediment/basement interface (222.7 mbsf). Thus, the oceanic basement appears to be a net sink for sedimentary DOC. Sedimentary DOC and alkalinity profiles were similar and inversely mirror those of sulfate, suggesting that the buildup of DOC in sediment pore water is related to remineralization of sedimentary POC. The sedimentary POC content at Site U1363 ranged from 47 to 391 µmol-C/g, with δ13C values from –25.3‰ to –22.4‰. The total particulate nitrogen (PN) content ranged from 4.1 to 32.9 µmol-N/g, with δ15N values from 1.8‰ to 7.2‰ and a POC:PN ratio of 12 ± 2 (n = 54). No depth-specific systematic variations in POC, PN, POC:PN ratio, δ13C-POC or δ15N-PN were detected, and no significant correlations between sedimentary DOC and POC concentrations were observed.
AbstractThe oceanic basaltic crust is the largest aquifer on Earth and has the potential to harbor substantial subsurface microbial ecosystems, which hitherto remains largely uncharacterized and is analogous to extraterrestrial subsurface habitats. Within the sediment-buried 3.5 Myr old basaltic crust of the eastern Juan de Fuca Ridge flank, the circulating basement fluids have moderate temperature (∼65 °C) and low to undetectable dissolved oxygen and nitrate concentrations. Sulfate, present in high concentrations, is therefore expected to serve as the major electron acceptor in this subsurface environment. This study focused on the availability and potential sources of two important electron donors, methane (CH4) and hydrogen (H2), for the subseafloor biosphere. High integrity basement fluids were collected via fluid delivery lines associated with Integrated Ocean Drilling Program (IODP) Circulation Obviation Retrofit Kits (CORKs) that extend from basement depths to outlet ports at the seafloor. Two new CORKs installed during IODP 327 in 2010, 1362A and 1362B, were sampled in 2011 and 2013. The two CORKs are superior than earlier style CORKs in that they are equipped with coated casing and polytetrafluoroethylene fluid delivery lines, reducing the interaction between casing materials with the environment. Additional samples were collected from an earlier style CORK at Borehole 1301A. The basement fluids are enriched in H2 (0.05–1.8 μmol/kg), suggesting that the ocean basaltic aquifer can support H2-driven metabolism. The basement fluids also contain significant amount of CH4 (5–32 μmol/kg), revealing CH4 as an available substrate for subseafloor basaltic habitats. The δ13C values of CH4 from the three boreholes ranged from −22.5 to −58‰, while the δ2H values ranged from −316 to 57‰. The isotopic compositions of CH4 and the molecular compositions of hydrocarbons suggest that CH4 in the basement fluids is of both biogenic and abiotic origins, varying among sites and sampling times. The δ2H values of CH4 in CORK 1301A fluid samples are much more positive than found in all other marine environments investigated to date and are best explained by the partial microbial oxidation of biogenic CH4. In conclusion, our study shows that CH4 and H2 are persistently available to fuel the deep biosphere and that CH4 is both produced and potentially consumed by microorganisms in the oceanic basement.
AbstractMicroorganisms inhabiting sediment in close proximity to recharging basement outcrops are of interest because of the enhanced advective fluid flow in these locations, which is expected to exert unique selective pressures on the resident microbial communities. Here, Integrated Ocean Drilling Program (IODP) boreholes were used to access sediment microbial communities near Grizzly Bare recharge seamount on the Juan de Fuca Ridge eastern flank. The two locations examined in this study, Holes U1363G and U1363B, are 50 and 177 m away from the center of the outcrop, respectively. In general, small subunit ribosomal RNA gene clones from all three domains of life were detected; these groups were predominantly related to microorganisms known to reside in marine sediment. A large fraction of environmental gene clones recovered from Hole U1363B and U1363G sediment were related to uncultivated, candidate phyla of Bacteria such as BHI80-139, BRC1, JS1, OPB41, and TA06. Hole U1363B and U1363G sediment clone libraries were generally dominated by the domain Bacteria and particularly the phylum Chloroflexi, which comprised approximately one-quarter of the total gene clones identified. However, borehole sediment also contained several archaeal lineages that were phylogenetically affiliated with the Miscellanenous Crenarchaeotal Group. Eukaryotic fungi were only detected within the interstitial water sample from Hole U1363B. Finally, a minor portion of clones recovered from sediment in this study were also recovered from basement fluid samples previously characterized from Baby Bare discharge seamount and Ocean Drilling Program and IODP borehole Circulation Obviation Retrofit Kit Observatories (CORKs) in Holes 1026B and U1301A, which are ~50 km to the north-northeast.
The permeable rocks of the upper oceanic basement contain seawater-sourced fluids estimated to be ~ 2% of the global ocean volume. This represents a very large potential subsurface biosphere supported by chemosynthesis. Recent collection of high integrity samples of basement fluid from the sedimented young basaltic basement on the Juan de Fuca Ridge flanks, off the coasts of Vancouver Island (Canada) and Washington (USA), and subsequent chemical analyses permit numerical modeling of metabolic redox reaction energetics. Here, values of Gibbs free energy for potential chemolithotrophic net reactions were calculated in basement fluid and in zones where basement fluid and entrained seawater may mix; the energy yields are reported both on a per mole electrons transferred and on a per kg of basement fluid basis. In pure basement fluid, energy yields from the anaerobic respiration processes investigated are anemic, releasing < 0.3 J/kg basement fluid for all reactions except methane oxidation by ferric iron, which releases ~ 0.6 J/kg basement fluid. In mixed solutions, aerobic oxidation of hydrogen, methane, and sulfide is the most exergonic on a per mole electron basis. Per kg of basement fluid, the aerobic oxidation of ammonia is by far the most exergonic at low temperature and high seawater:basement fluid ratio, decreasing by more than two orders of magnitude at the highest temperature (63 °C) and lowest seawater:basement fluid ratio investigated. Compared with mixing zones in deep-sea hydrothermal systems, oceanic basement aquifers appear to be very low energy systems, but because of their expanse, may support what has been labeled the ‘starving majority’.
AbstractSubmarine volcanic eruptions and intrusions construct new oceanic crust and build long chains of volcanic islands and vast submarine plateaus. Magmatic events are a primary agent for the transfer of heat, chemicals, and even microbes from the crust to the ocean, but the processes that control these transfers are poorly understood. The 1980s discovery that mid-ocean ridge eruptions are often associated with brief releases of immense volumes of hot fluids (“event plumes”) spurred interest in methods for detecting the onset of eruptions or intrusions and for rapidly organizing seagoing response efforts. Since then, some 35 magmatic events have been recognized and responded to on mid-ocean ridges and at seamounts in both volcanic arc and intraplate settings. Field responses at mid-ocean ridges have found that event plumes occur over a wide range of eruption styles and sizes, and thus may be a common consequence of ridge eruptions. The source(s) of event plume fluids are still debated. Eruptions detected at ridges generally have high effusion rates and short durations (hours to days), whereas field responses at arc volcanic cones have found eruptions with very low effusion rates and durations on the scale of years. New approaches to the study of submarine magmatic events include the development of autonomous vehicles for detection and response, and the establishment of permanent seafloor observatories at likely future eruption sites.
AbstractDespite its immense size, logistical and methodological constraints have largely limited microbiological investigations of the subseafloor basement biosphere. In this study, a unique sampling system was used to collect fluids from the subseafloor basaltic crust via a Circulation Obviation Retrofit Kit (CORK) observatory at Integrated Ocean Drilling Program borehole 1301A, located at a depth of 2667 m of sediment, which serves as a barrier to direct exchange with bottom seawater. At an average of 1.2 × 104 cells ml−1, microorganisms in borehole fluids were nearly an order of magnitude less abundant than in surrounding bottom seawater. Ribosomal RNA genes were characterized from basement fluids, providing the first snapshots of microbial community structure using a high-integrity fluid delivery line. Interestingly, microbial communities retrieved from different CORKs (1026B and 1301A) nearly a decade apart shared major community members, consistent with hydrogeological connectivity. However, over three sampling years, the dominant gene clone lineage changed from relatives of Candidatus Desulforudis audaxviator within the bacterial phylum Firmicutes in 2008 to the Miscellaneous Crenarchaeotic Group in 2009 and a lineage within the JTB35 group of Gammaproteobacteria in 2010, and statistically significant variation in microbial community structure was observed. The enumeration of different phylogenetic groups of cells within borehole 1301A fluids supported our observation that the deep subsurface microbial community was temporally dynamic.
AbstractThe permeable upper oceanic basement serves as a plausible habitat for a variety of microbial communities. There is growing evidence suggesting a substantial subseafloor biosphere. Here new time series data are presented on key inorganic species, methane, hydrogen and dissolved organic carbon (DOC) in ridge flank fluids obtained from subseafloor observatory CORKs (Circulation Obviation Retrofit Kits) at Integrated Ocean Drilling Program (IODP) boreholes 1301A and 1026B. These data show that the new sampling methods (Cowen et al., 2012) employed at 1301A result in lower contamination than earlier studies. Furthermore, sample collection methods permitted most chemical analyses to be performed from aliquots of single large volume samples, thereby allowing more direct comparison of the data. The low phosphate concentrations (0.06–0.2 μM) suggest that relative to carbon and nitrogen, phosphorus could be a limiting nutrient in the basement biosphere. Coexisting sulfate (17–18 mM), hydrogen sulfide (∼0.1 μM), hydrogen (0.3–0.7 μM) and methane (1.5–2 μM) indicates that the basement aquifer at 1301A either draws fluids from multiple flow paths with different redox histories or is a complex environment that is not thermodynamically controlled and may allow co-occurring metabolic pathways including sulfate reduction and methanogenesis. The low DOC concentrations (11–18 μM) confirm that ridge flank basement is a net DOC sink and ultimately a net carbon sink. Based on the net amounts of DOC, oxygen, nitrate and sulfate removed (∼30 μM, ∼80 μM, ∼40 μM and ∼10 mM, respectively) from entrained bottom seawater, organic carbon may be aerobically or anaerobically oxidized in biotic and/or abiotic processes.
AbstractIntegrated Ocean Drilling Program borehole CORK (Circulation Obviation Retrofit Kit) observatories provide long-term access to hydrothermal fluids circulating within the basaltic crust (basement), providing invaluable opportunities to study the deep biosphere. We describe the design and application parameters of the GeoMICROBE instrumented sled, an autonomous sensor and fluid sampling system. The GeoMICROBE system couples with CORK fluid delivery lines to draw large volumes of fluids from crustal aquifers to the seafloor. These fluids pass a series of in-line sensors and an in situ filtration and collection system. GeoMICROBE's major components include a primary valve manifold system, a positive displacement primary pump, sensors (e.g., fluid flow rate, temperature, dissolved O2, electrochemistry-voltammetry analyzer), a 48-port in situ filtration and fluid collection system, computerized controller, seven 24 V–40 A batteries and wet-mateable (ODI) communications with submersibles. This constantly evolving system has been successfully connected to IODP Hole 1301A on the eastern flank of the Juan de Fuca Ridge. Also described here is a mobile pumping system (MPS), which possesses many of the same components as the GeoMICROBE (e.g., pump, sensors, controller), but is directly powered and controlled in real time via submersible operations; the MPS has been employed repeatedly to collect pristine basement fluids for a variety of geochemical and microbial studies.
AbstractIntegrated Ocean Drilling Program (IODP) Expedition 327 (summer 2010) was designed to resolve the nature of fluid-rock interactions in young, upper volcanic crust on the eastern flank of the Juan de Fuca Ridge. Expedition 327 drilled, cased and cored two new basement holes, conducted hydrogeologic experiments, and installed subseafloor borehole observatories (Circulation Obviation Retrofit Kits, CORKs). These CORKs were intended to allow borehole conditions to recover to a more natural state after the dissipation of disturbances caused by drilling, casing, and other operations; provide a long-term monitoring and sampling presence for determining fluid pressure, temperature, composition, and microbiology; and facilitate the completion of active experiments to resolve crustal hydrogeologic conditions and processes. Expedition 327 was followed (summer 2011) by R/V Atlantis Expedition AT18-07, with the remotely-operated vehicle (ROV) Jason, to service these CORKs, collect subseafloor pressure data, recover and deploy autonomous fluid and microbial samplers, collect large volumes of borehole fluids, and initiate a cross-hole hydrogeologic experiment using an electromagnetic flow meter. In addition, Atlantis Expedition AT18-07 refurbished an old CORK that could not be replaced during IODP Expedition 327, completing a critical part of the three-dimensional observation network that is currently being used to monitor a large-scale, directional formation response to long-term fluid flow from the crust.
AbstractSubseafloor borehole observatories (“CORKs”) are currently the best mechanism by which fluids from subsurface hydrologic zones can be collected to evaluate the composition, evolution, and consequence of fluid circulation in oceanic crust. The fluid-sampling capabilities of CORKs have evolved over two decades, spanning the Ocean Drilling Program and Integrated Ocean Drilling Program. The fluid-sampling system for the original CORK design consisted of a single polytetrafluoroethylene (PTFE) tube that connected to a valve at the seafloor and ended at depth in the formation. Through successes and disappointments coupled with community desires and efforts, significant iterations of CORK design and capabilities have led to the development of a range of crustal fluid-sampling systems. These iterations continue today with the development of new borehole capabilities, sensors, and samplers. This paper discusses these developments and transitions, highlighting the pros and cons of various designs, materials, and decisions. Although the evolution of CORK design has taken years because of the infrequency of CORK deployments and sample recovery operations, we as a community are now in a position to report on groundbreaking results that will enhance our understanding of subseafloor hydrogeology, crustal evolution, geochemical fluxes, microbial ecology, and biogeochemical processes, as indicated by the wealth of work referenced herein and by the complexity and flexibility of present and future designs.
AbstractMultiple tracers were pumped into upper basement around Hole U1362B during Integrated Ocean Drilling Program (IODP) Expedition 327 as part of a single- and cross-hole tracer experiment on the eastern flank of Juan de Fuca Ridge. Tracers injected were sulfur hexafluoride (dissolved gas), cesium chloride hexahydrate, erbium chloride, and holmium chloride hexahydrate (solutes), and several sizes of fluorescent microspheres and fluorescent-stained microbes filtered from surface seawater (particles). Tracers were injected as part of a 24 h pumping experiment intended to test a large volume of basement rock around Hole U1362B. We report on the design of the tracer experiment, methods used to prepare and inject tracers using shipboard mud and cement pump systems, and tools developed to permit shipboard and downhole sampling of injectate. Shore-based analysis of injectate samples will be essential for interpretation of long-term samples collected from subseafloor borehole observatories (“CORKs”). Borehole samples are being collected continuously within a long-term CORK installed in Hole U1362B after tracer injection was complete and within similar CORK systems installed in nearby boreholes before and during Expedition 327. CORK servicing expeditions are currently planned for summer 2011 and 2012. These expeditions and additional work in subsequent years will provide data and samples that will permit a quantitative assessment of tracer transport behavior in the upper ocean crust.
AbstractIntegrated Ocean Drilling Program (IODP) Expedition 327 installed two new subseafloor borehole observatory systems (“CORKs”) in 3.5 m.y. old upper ocean crust on the eastern flank of Juan de Fuca Ridge in Holes U1362A and U1362B. Expedition 327 participants also recovered part of an instrument string previously deployed in a CORK in Hole U1301B and deployed a short replacement string. These observatories are part of a network of six CORKs that was designed to monitor, sample, and complete multidisciplinary cross-hole experiments. We present an overview of project goals and describe the design, construction, and deployment of new CORK systems. We also provide an update on the status of preexisting CORK systems as of the start of Expedition 327. Additional CORK servicing and sampling are scheduled for summer 2011 and 2012, including a long-term free-flow perturbation experiment that will test the large-scale directional properties of the upper ocean crust around the observatories.
Sulfate reducing microorganisms (SRM) may play a significant role altering upper oceanic crustal fluids when suitable electron donors, such as hydrogen or organic matter, are available. The habitability of such an environment with respect to sulfate reduction depends on the competition of microbial communities for substrates, which is largely dictated by the energetics of catabolic and anabolic processes. Although sulfate reduction has been observed in fluids taken from the upper ocean crust in Juan de Fuca Ridge flanks, the electron donors (EDs) used by SRM have not been identified, nor has the energy required for organic synthesis been determined. As a result, a collaboration is underway to characterize the EDs that are plausible candidates for the SRM in the Juan de Fuca system and to quantify the amount of energy these microorganisms require to synthesize biomolecules. This is accomplished by carrying out thermodynamic calculations that take into account the physicochemical properties of the resident fluids. Specifically, the Gibbs energy of reactions describing the reduction of sulfate by various EDs and the synthesis of amino acids from inorganic precursors is being calculated at the temperature, pressure and compositional conditions prevailing in particular Juan de Fuca sample site locations.