Person: Michael S. Rappé

Abstract

The advanced instrumented GeoMICROBE sleds (Cowen et al., 2012) facilitate the collection of hydrothermal fluids and suspended particles in the subseafloor (basaltic) basement through Circulation Obviation Retrofit Kits (CORKs) installed within boreholes of the Integrated Ocean Drilling Program. The main components of the GeoMICROBE can be converted into a mobile pumping system (MPS) that is installed on the front basket of a submersible or remotely-operated-vehicle (ROV). Here, we provide details of a hydrothermal fluid-trap used on the MPS, through which a gastight sampler can withdraw fluids. We also applied the MPS to demonstrate the value of fixing samples at the seafloor in order to determine redox-sensitive dissolved iron concentrations and speciation measurements. To make the best use of the GeoMICROBE sleds, we describe a miniature and mobile version of the GeoMICROBE sled, which permits rapid turn-over and is relatively easy for preparation and operation. Similar to GeoMICROBE sleds, the Mobile GeoMICROBE (MGM) is capable of collecting fluid samples, filtration of suspended particles, and extraction of organics. We validate this approach by demonstrating the seafloor extraction of hydrophobic organics from a large volume (247L) of hydrothermal fluids.

• We describe the design of a hydrothermal fluid-trap for use with a gastight sampler, as well as the use of seafloor fixation, through ROV- or submersible assisted mobile pumping systems.
• We describe the design of a Mobile GeoMICROBE (MGM) that enhances large volume hydrothermal fluid sampling, suspended particle filtration, and organic matter extraction on the seafloor.
• We provide an example of organic matter extracted and characterized from hydrothermal fluids via a MGM.

Abstract

The degassing of primordial gases from Earth’s interior is evidenced by the high ³He/⁴He ratios in submarine hydrothermal plumes, vent fluids, and rock samples with mantle origin from active hydrothermal systems in mid-ocean ridges (MOR) and subduction zone volcanism. As the largest aquifer on Earth, the uppermost 40-500 m of permeable submarine ridge flank basement (1-65 million-years-old, Myr) holds ∼2% of the ocean volume and accounts for 70% of the seafloor hydrothermal heat flux. However, the degassing of primordial gases through the oceanic ridge flank crust has not yet been directly quantified. Here, we show that high integrity hydrothermal (65 °C) fluids from the sediment-buried 3.5 Myr basaltic crust from the eastern flank of the Juan de Fuca Ridge (JdFR) contain elevated ³He. The ³He/⁴He for the basaltic fluid is 4.5 ± 0.1 R_a (relative to the air ratio), which is greatly elevated when compared to deep seawater (1.05 R_a), but is half of that observed for high-temperature vent fluids (∼8 R_a) emitting from MOR. Only a small fraction of the ³He in ridge flank fluids is derived from the entrainment of high-temperature ridge-axis fluids and is better explained by degassing of the mantle through the mantle-crust boundary. The lower than MOR ³He/⁴He ratios indicate that radiogenic ⁴He originates from aged uranium and thorium decay within the mantle as well as from the ridge-flank basalts. The ³He outgassing through warm ridge flanks (4.9 to 36 mol/yr) accounts for 0.7-6% of the global ³He outgassing, exceeded only by degassing through mid-ocean ridges and subduction volcanism. The presence of mantle ³He suggests that the abiogenic methane present in the ridge flank fluids might be mantle-derived. Based on the ³He outgassing flux, a possibly mantle-derived abiotic methane production rate at the ridge flank is estimated to be 0.3-35 x 10⁸ mol/yr.

Abstract

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin‐dependent transport‐coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO₂ reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H₂ consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.

Abstract

Marine dissolved organic carbon (DOC) is highly depleted in radiocarbon and thus inferred to be largely refractory to removal processes that operate on less than millennial timescales. However, a growing number of reports have shown that a large fraction of marine DOC can be effectively removed during circulation through submarine hydrothermal systems. What is not clear, however, is whether the DOC that remains in hydrothermal fluids is remnant non-reactive DOC from recharged seawater, or DOC that has been largely produced in the subsurface. We collected and characterized warm (∼65 °C) hydrothermal fluids from deep (18, 40, 73, 200 m) within the basalt-hosted basement of the Juan de Fuca Ridge flank in the Northeast Pacific Ocean. DOC concentrations in hydrothermal fluids were 9 to 18 μM, much lower than measured in local deep seawater (37.5 μM). DOCΔ¹⁴C values of −683‰ to −856‰ were much lower than the Δ¹⁴C-dissolved inorganic carbon (DIC) values of −880‰ to −918‰, while DOC δ¹³C values of −23.6‰ to −27.0‰ were much heavier than that of the particulate organic carbon (POC) pool (∼−34‰), suggesting that biological production in the subsurface is not a primary source of DOC. Rather, our data suggest that isotopically enriched DOC are selectively removed from recharged seawater, leaving DOC that is very isotopically depleted in the basaltic basement fluids. Despite the removal of 50–75% of DOC in the subsurface, nuclear magnetic resonance (NMR) functional group analyses indicate that aromatic compounds were added to basaltic basement fluids during passage through the deep subseafloor and may partly contribute to the depleted ¹⁴C DOC in the ridge-flank basement fluids.

Abstract

The exploration of Earth’s terrestrial subsurface biosphere has led to the discovery of several new archaeal lineages of evolutionary significance. Similarly, the deep subseafloor crustal biosphere also harbors many unique, uncultured archaeal taxa, including those belonging to Candidatus Hydrothermarchaeota, formerly known as Marine Benthic Group-E. Recently, Hydrothermarchaeota was identified as an abundant lineage of Juan de Fuca Ridge flank crustal fluids, suggesting its adaptation to this extreme environment. Through the investigation of single-cell and metagenome-assembled genomes, we provide insight into the lineage’s evolutionary history and metabolic potential. Phylogenomic analysis reveals the Hydrothermarchaeota to be an early-branching archaeal phylum, branching between the superphylum DPANN, Euryarchaeota, and Asgard lineages. Hydrothermarchaeota genomes suggest a potential for dissimilative and assimilative carbon monoxide oxidation (carboxydotrophy), as well as sulfate and nitrate reduction. There is also a prevalence of chemotaxis and motility genes, indicating adaptive strategies for this nutrient-limited fluid-rock environment. These findings provide the first genomic interpretations of the Hydrothermarchaeota phylum and highlight the anoxic, hot, deep marine crustal biosphere as an important habitat for understanding the evolution of early life.

Abstract

The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for β-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.

Abstract

Along the eastern flank of the Juan de Fuca Ridge (JdFR), an array of boreholes drilled through sediments and into subseafloor basalt are fitted with CORKs (Circulation Obviation Retrofit Kits) that hydrologically seal off boreholes from sediment pore water and enable direct sampling of pristine basalt-hosted fluids. In this system, the members of the bacterial phylum Nitrospirae, which are closely related to the thermophilic anaerobic sulfate-reducing Thermodesulfovibrio, are among the most abundant bacteria. However, the metabolic and evolutionary profile of this microbe in this ridge system has yet to be fully elucidated. Here, we analyzed previously extracted and sequenced crustal fluid metagenomes from 2 CORKS (U1362A and U1362B) with total lengths of ~269Mbp and ~140Mbp, respectively, post quality control. The 2 metagenomes were assembled into scaffolds in metaSPades and assessed via read recruitment. Read recruitment results were further assessed, and visualized using a suite of programs in Anvi’o. Binning of scaffolds and subsequent manual refinement of bins were done based on the hierarchical clustering of tetra-nucleotide frequencies and GC content of scaffolds via the Anvi’o interactive and refine interface. Bins taxonomically identified as Nitrospirae using the GTDB-Tk classify workflow were placed in a separate Anvi’o collection, manually refined and assessed for completion and contamination using ‘–anvi-summarize’. Based on the preliminary analyses, we have identified seven bins in U1362A and 2 bins in U1362B belonging to the Nitrospirae phylum. All JdFR Nitrospirae bins have >50% completion and <5% redundancy. Also, the presence of two distinct GC contents (~40% vs 60%) of Nitrospirae may suggest that different populations exist within and between samples.

Abstract

It is generally accepted that diverse, poorly characterized microorganisms reside deep within Earth’s crust. One such lineage of deep subsurface-dwelling bacteria is an uncultivated member of the Firmicutes phylum that can dominate molecular surveys from both marine and continental rock fracture fluids, sometimes forming the sole member of a single-species microbiome. Here, we reconstructed a genome from basalt-hosted fluids of the deep subseafloor along the eastern Juan de Fuca Ridge flank and used a phylogenomic analysis to show that, despite vast differences in geographic origin and habitat, it forms a monophyletic clade with the terrestrial deep subsurface genome of “Candidatus Desulforudis audaxviator” MP104C. While a limited number of differences were observed between the marine genome of “Candidatus Desulfopertinax cowenii” modA32 and its terrestrial relative that may be of potential adaptive importance, here it is revealed that the two are remarkably similar thermophiles possessing the genetic capacity for motility, sporulation, hydrogenotrophy, chemoorganotrophy, dissimilatory sulfate reduction, and the ability to fix inorganic carbon via the Wood-Ljungdahl pathway for chemoautotrophic growth. Our results provide insights into the genetic repertoire within marine and terrestrial members of a bacterial lineage that is widespread in the global deep subsurface biosphere, and provides a natural means to investigate adaptations specific to these two environments.

Abstract

The global deep subsurface biosphere is one of the largest reservoirs for microbial life on our planet. This study takes advantage of new sampling technologies and couples them with improvements to DNA sequencing and associated informatics tools to reconstruct the genomes of uncultivated Bacteria and Archaea from fluids collected deep within the Juan de Fuca Ridge subseafloor. Here, we generated two metagenomes from borehole observatories located 311 meters apart and, using binning tools, retrieved 98 genomes from metagenomes (GFMs). Of the GFMs, 31 were estimated to be >90% complete, while an additional 17 were >70% complete. Phylogenomic analysis revealed 53 bacterial and 45 archaeal GFMs, of which nearly all were distantly related to known cultivated isolates. In the GFMs, abundant Bacteria included Chloroflexi, Nitrospirae, Acetothermia (OP1), EM3, Aminicenantes (OP8), Gammaproteobacteria, and Deltaproteobacteria, while abundant Archaea included Archaeoglobi, Bathyarchaeota (MCG), and Marine Benthic Group E (MBG-E). These data are the first GFMs reconstructed from the deep basaltic subseafloor biosphere, and provide a dataset available for further interrogation.

Abstract

This paper is dedicated to the late James P. Cowen, whose collegiality, infectious enthusiasm for science, and pioneering studies of the ocean basement microbiome inspired the work.

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Abstract

Although fluids within the upper oceanic basaltic crust harbor a substantial fraction of the total prokaryotic cells on Earth, the energy needs of this microbial population are unknown. In this study, a nanocalorimeter (sensitivity down to 1.2 nW ml^-1) was used to measure the enthalpy of microbially catalyzed reactions as a function of temperature in samples from two distinct crustal fluid aquifers. Microorganisms in unamended, warm (63°C) and geochemically altered anoxic fluids taken from 292 meters sub-basement (msb) near the Juan de Fuca Ridge produced 267.3 mJ of heat over the course of 97 h during a step-wise isothermal scan from 35.5 to 85.0°C. Most of this heat signal likely stems from the germination of thermophilic endospores (6.66 × 10⁴ cells ml^-1_FLUID) and their subsequent metabolic activity at temperatures greater than 50°C. The average cellular energy consumption (5.68 pW cell^-1) reveals the high metabolic potential of a dormant community transported by fluids circulating through the ocean crust. By contrast, samples taken from 293 msb from cooler (3.8°C), relatively unaltered oxic fluids, produced 12.8 mJ of heat over the course of 14 h as temperature ramped from 34.8 to 43.0°C. Corresponding cell-specific energy turnover rates (0.18 pW cell^-1) were converted to oxygen uptake rates of 24.5 nmol O₂ ml^-1_FLUID d^-1, validating previous model predictions of microbial activity in this environment. Given that the investigated fluids are characteristic of expansive areas of the upper oceanic crust, the measured metabolic heat rates can be used to constrain boundaries of habitability and microbial activity in the oceanic crust.

Abstract

Although 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.

Abstract

The 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.

Abstract

To 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.

Abstract

Marine 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.

Abstract

The 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.

Abstract

The detection of life in extreme environments and the amazing capacity of microbes to obtain energy from the environment has led to the hypothesis that heat, not water or energy sources, is the final barrier that constrains life on Earth. As such, life is expected to extend far into Earth’s crust, potentially inhabiting an environment as extensive as the world’s oceans [1]. This chapter aims to summarize what is currently known about life in the oceanic crust, which contains all three domains of life and is a vastly underexplored habitat. This is not the first summary of this topic, and additional content can be found in numerous review and primary research papers [2–16], book chapters [17, 18], and books [19–21].

Abstract

The value of cultivating microbial strains that are representative of abundant microorganisms in situ is generally acknowledged amongst marine microbial ecologists, primarily because they provide the means to determine phenotypic properties and detailed physiological characteristics of living cells in a controlled setting. In the shadow of the rapid, ongoing expansion in environmental genomic, transcriptomic, and proteomic surveys of marine systems, a minor resurgence in experiments designed to isolate and grow free-living marine microorganisms has met some success. Interestingly, the most immediate impact that many of the resulting strains have had on our understanding of marine microbial communities has not resulted from experiments aimed to interrogate cellular physiology, but rather from their sequenced genomes. It is predicted, however, that their prolonged impact on marine ecology will result from basic laboratory research that links cellular physiology with its molecular underpinnings.

Abstract

Microorganisms 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.

Abstract

Though generally not perceived as an “extreme” environment, the global open ocean is a desert with respect to the organic and inorganic building blocks required to promote the growth of any potential inhabitants. In illuminated surface waters where light energy available for photosynthesis abounds, thermal stratification of the water column results in the constant starvation of (primarily microscopic) photoautotrophs for inorganic nutrients such as nitrogen and phosphorus. In the dark ocean interior, inorganic nutrients are generally in excess; however, organic carbon compounds are in short supply, and the stocks that are present are generally considered to be inaccessible as substrates to sustain active cell growth and metabolism. In spite of all of this, massive populations of microscopic cells abound in the global open ocean. Assessments based on gene- and genome-based measures have revealed a tremendous amount of diversity within the microbial plankton, but also that a vast majority of this diversity is not currently represented in existing culture collections of marine microorganisms. However, a combination of approaches that permit the interrogation of in situ microbial communities, such as environmental metagenomics, and methodological developments aimed at cultivating microorganisms present in numerically high abundance in the environment, such as high-throughput cultivation, are facilitating an unprecedented understanding of oligotrophic marine microorganisms and the roles they play in global ocean ecology.

Abstract

Despite 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.

Abstract

We are investigating the deep biosphere of the subseafloor basaltic crust on the eastern flank of the Juan de Fuca Ridge by accessing pristine crustal fluids via Circulation Obviation Retrofit Kit (CORK) sampling and instrumentation platforms. Second-generation CORKs installed within Integrated Ocean Drilling Program boreholes provide unprecedented opportunities to study the microbial ecology and biogeochemistry of fluids circulating in the sediment-covered oceanic basement. In the summers of 2008-2011, a unique large-volume sampling system was used to collect some of the only large volume pristine basement fluid samples available for microbiological studies to date, from a 3.5 Myr-old basalt-hosted basement aquifer that is covered by 260m of sediment. On an annual scale, the microbial community appears to be temporally dynamic; however, some common community members are shared between sample years. We will obtain nucleic acid sequence data from the metagenome of two deep subsurface microbial community samples (one each from field seasons 2010 and 2011) and the metatranscriptome of one corresponding sample, as well as amplify the genomes of a subset of single cells. Due to the well-known difficulties in obtaining pristine sub-seafloor ocean crustal fluids, these samples offer an unmatched opportunity to investigate the community genomics of cells inhabiting the deep subseafloor biosphere.