Person: Steven L. D'Hondt

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Chloroflexi are widespread in subsurface environments, and recent studies indicate that they represent a major fraction of the communities in subseafloor sediment. Here, we compare the abundance, diversity, metabolic potential and gene expression of Chloroflexi from three abyssal sediment cores from the western North Atlantic Gyre (water depth >5400 m) covering up to 15 million years of sediment deposition, where Chloroflexi were found to represent major components of the community at all sites. Chloroflexi communities die off in oxic red clay over 10–15 million years, and gene expression was below detection. In contrast, Chloroflexi abundance and gene expression at the anoxic abyssal clay site increase below the seafloor and peak in 2–3 million-year-old sediment, indicating a comparably higher activity. Metatranscriptomes from the anoxic site reveal increased expression of Chloroflexi genes involved in cell wall biogenesis, protein turnover, inorganic ion transport, defense mechanisms and prophages. Phylogenetic analysis shows that these Chloroflexi are closely related to homoacetogenic subseafloor clades and actively transcribe genes involved in sugar fermentations, gluconeogenesis and Wood–Ljungdahl pathway in the subseafloor. Concomitant expression of cell division genes indicates that these putative homoacetogenic Chloroflexi are actively growing in these million-year-old anoxic abyssal sediments.

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We studied microbially mediated diagenetic processes driven by carbon mineralization in subseafloor sediment of the northeastern Bering Sea Slope to a depth of 745 meters below seafloor (mbsf). Sites U1343, U1344 and U1345 were drilled during Integrated Ocean Drilling Program (IODP) Expedition 323 at water depths of 1008 to 3172 m. They are situated in the high productivity “Green Belt” region, with organic carbon burial rates typical of the high-productivity upwelling domains on western continental margins. The three sites show strong geochemical similarities. The downward sequence of microbially mediated processes in the sediment encompasses (1) organoclastic sulfate reduction, (2) anaerobic oxidation of methane (AOM) coupled to sulfate reduction, and (3) methanogenesis. The sediment contains two distinct zones of diagenetic carbonate formation, located at the sulfate–methane transition zone (SMTZ) and between 300 and 400 mbsf. The SMTZ at the three sites is located between 6 and 9 mbsf. The upward methane fluxes into the SMTZ are similar to fluxes in SMTZs underlying high-productivity surface waters off Chile and Namibia. Our Bering Sea results show that intense organic carbon mineralization drives high ammonium and dissolved inorganic carbon (DIC) production rates (> 4.2 mmol m^− 3 y^− 1) in the uppermost 10 mbsf and strongly imprints on the stable carbon isotope composition of DIC, driving it to a minimum value of − 27‰ (VPDB) at the SMTZ. Pore-water calcium and magnesium profiles demonstrate formation of diagenetic Mg-rich calcite in the SMTZ. Below the SMTZ, methanogenesis results in ¹³C-enrichment of pore-water DIC, with a maximum value of + 11.9‰. The imprint of methanogenesis on the DIC carbon isotope composition is evident down to a depth of 150 mbsf. Below this depth, slow or absent microbially mediated carbon mineralization leaves DIC isotope composition unaffected. Ongoing carbonate formation between 300 and 400 mbsf strongly influences pore-water DIC and magnesium concentration profiles. The linked succession of organic carbon mineralization and carbonate dissolution and precipitation patterns that we observe in the Bering Sea Slope sediment may be representative of passive continental margin settings in high-productivity areas of the world’s ocean.

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How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum “Candidatus Atribacteria,” which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. “Ca. Atribacteria” dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing “Ca. Atribacteria” also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD⁺. Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor “Ca. Atribacteria,” as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the “Ca. Atribacteria” can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments.

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Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species–area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 10³ to 6.10 × 10⁵ and 3.28 × 10⁴ to 2.46 × 10⁶ amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth’s global biosphere.

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Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.

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The upper oceanic crust is mainly composed of basaltic lava that constitutes one of the largest habitable zones on Earth. However, the nature of deep microbial life in oceanic crust remains poorly understood, especially where old cold basaltic rock interacts with seawater beneath sediment. Here we show that microbial cells are densely concentrated in Fe-rich smectite on fracture surfaces and veins in 33.5- and 104-million-year-old (Ma) subseafloor basaltic rock. The Fe-rich smectite is locally enriched in organic carbon. Nanoscale solid characterizations reveal the organic carbon to be microbial cells within the Fe-rich smectite, with cell densities locally exceeding 10¹⁰ cells/cm³. Dominance of heterotrophic bacteria indicated by analyses of DNA sequences and lipids supports the importance of organic matter as carbon and energy sources in subseafloor basalt. Given the prominence of basaltic lava on Earth and Mars, microbial life could be habitable where subsurface basaltic rocks interact with liquid water.

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Subseafloor microbial activities are central to Earth’s biogeochemical cycles. They control Earth’s surface oxidation and major aspects of ocean chemistry. They affect climate on long timescales and play major roles in forming and destroying economic resources. In this review, we evaluate present understanding of subseafloor microbes and their activities, identify research gaps, and recommend approaches to filling those gaps. Our synthesis suggests that chemical diffusion rates and reaction affinities play a primary role in controlling rates of subseafloor activities. Fundamental aspects of subseafloor communities, including features that enable their persistence at low catabolic rates for millions of years, remain unknown.

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To assess the influence of 16S ribosomal RNA (rRNA) tag choice on estimates of microbial diversity and/or community composition in seawater and marine sediment, we examined bacterial diversity and community composition from a site in the Central North Atlantic and a site in the Equatorial Pacific. For each site, we analyzed samples from four zones in the water column, a seafloor sediment sample, and two subseafloor sediment horizons (with stratigraphic ages of 1.5 and 5.5 million years old). We amplified both the V4 and V6 hypervariable regions of the 16S rRNA gene and clustered the sequences into operational taxonomic units (OTUs) of 97% similarity to analyze for diversity and community composition. OTU richness is much higher with the V6 tag than with the V4 tag, and subsequently OTU-level community composition is quite different between the two tags. Vertical patterns of relative diversity are broadly the same for both tags, with maximum taxonomic richness in seafloor sediment and lowest richness in subseafloor sediment at both geographic locations. Genetic dissimilarity between sample locations is also broadly the same for both tags. Community composition is very similar for both tags at the class level, but very different at the level of 97% similar OTUs. Class-level diversity and community composition of water-column samples are very similar at each water depth between the Atlantic and Pacific. However, sediment communities differ greatly from the Atlantic site to the Pacific site. Finally, for relative patterns of diversity and class-level community composition, deep sequencing and shallow sequencing provide similar results.

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Ammonia-oxidizing archaea (AOA) dominate microbial communities throughout oxic subseafloor sediment deposited over millions of years in the North Atlantic Ocean. Rates of nitrification correlated with the abundance of these dominant AOA populations, whose metabolism is characterized by ammonia oxidation, mixotrophic utilization of organic nitrogen, deamination, and the energetically efficient chemolithoautotrophic hydroxypropionate/hydroxybutyrate carbon fixation cycle. These AOA thus have the potential to couple mixotrophic and chemolithoautotrophic metabolism via mixotrophic deamination of organic nitrogen, followed by oxidation of the regenerated ammonia for additional energy to fuel carbon fixation. This metabolic feature likely reduces energy loss and improves AOA fitness under energy-starved, oxic conditions, thereby allowing them to outcompete other taxa for millions of years.

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Many studies have examined relationships of microorganisms to geochemical zones in subseafloor sediment. However, responses to selective pressure and patterns of community succession with sediment depth have rarely been examined. Here we use 16S rDNA sequencing to examine the succession of microbial communities at sites in the Indian Ocean and the Bering Sea. The sediment ranges in depth from 0.16 to 332 m below seafloor and in age from 660 to 1,300,000 years. The majority of subseafloor taxonomic diversity is present in the shallowest depth sampled. The best predictor of sequence presence or absence in the oldest sediment is relative abundance in the near-seafloor sediment. This relationship suggests that perseverance of specific taxa into deep, old sediment is primarily controlled by the taxonomic abundance that existed when the sediment was near the seafloor. The operational taxonomic units that dominate at depth comprise a subset of the local seafloor community at each site, rather than a grown-in group of geographically widespread subseafloor specialists. At both sites, most taxa classified as abundant decrease in relative frequency with increasing sediment depth and age. Comparison of community composition to cell counts at the Bering Sea site indicates that the rise of the few dominant taxa in the deep subseafloor community does not require net replication, but might simply result from lower mortality relative to competing taxa on the long timescale of community burial.

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The most recent decadal phase of scientific ocean drilling through the International Ocean Discovery Program (IODP) has resulted in paradigm-​shifting understanding of life below the seafloor. Enabled by new drilling and coring approaches, cutting-edge methodologies, and novel observatory science, IODP expeditions have significantly advanced understanding of the amount and diversity of subseafloor life, the metabolic strategies that this life uses to survive under extreme energy limitation, and consequences of this life for the Earth system. Here, we summarize highlights from recent IODP expeditions focused on life beneath the seafloor and emphasize remaining major science challenges in investigating the form and function of life in this environment.

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Nearly half of the global seafloor is overlain by sediment oxygenated to the basement. Yet, despite the availability of oxygen to fuel aerobic respiration, organic carbon persists over million-year timescales. Identifying the controls on organic carbon preservation requires an improved understanding of the composition and distribution of organic carbon within deep oligotrophic marine sediments. Here we show that organic carbon in sediment from the oligotrophic North Atlantic and South Pacific is low (<0.1%), yet stable to depths of 25 m and ages of 24 million years. This organic carbon is not bound in biomass and has a low carbon/nitrogen ratio. X-ray imaging and spectroscopic analyses reveal that the chemical composition of this old, deep organic carbon is dominated (40–60%) by amide and carboxylic carbon with a proteinaceous nature. We posit that organic carbon persists in oxic oligotrophic sediment through a combination of protective processes that involve adsorption to mineral surfaces and physical inaccessibility to the heterotrophic community. We estimate that up to 1.6 × 10²² g of organic carbon are sequestered on million-year timescales in oxic pelagic sediment, which exceeds current estimates of the total global sediment organic carbon and constitutes an important, previously overlooked carbon reservoir.

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Extracellular DNA has been reported to comprise a large fraction of total DNA in near-seafloor sediment. However, the potential effect of extracellular DNA, arising from dead or moribund cells, on sequencing surveys is a critical concern that has largely not been addressed for marine sedimentary habitats. To address this concern, we interrogated freshly collected Arctic and Pacific sediment for extracellular 16S rRNA genes using the photoactive DNA-binding dye Propidium Monoazide. Significant differences between relative abundances of total (intracellular + extracellular) Bacterial 16S rRNA genes and relative abundances of intracellular Bacterial 16S rRNA genes are only detected in three of twelve shallow [10 cm below seafloor (cmbsf)] samples. Relative abundances of total Bacterial 16S rRNA genes are statistically indistinguishable from relative abundances of intracellular Bacterial 16S rRNA genes in all interrogated samples from depths greater than 10 cmbsf. 16S rRNA gene sequencing shows that even where significantly higher abundances of extracellular genes are detected, they have little or no influence on prokaryote community composition. Taxon-level analyses suggest that extracellular DNA, arising from in situdeath, may be sourced from different organisms in sediment of different ages. However, the overall effect of extracellular genes on sequencing surveys of marine sedimentary prokaryotes is minimal.

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Hydrogen, produced by water radiolysis, has been suggested to support microbial communities on Mars. We quantitatively assess the potential magnitude of radiolytic H₂ production in wet martian environments (the ancient surface and the present subsurface) based on the radionuclide compositions of (1) eight proposed Mars 2020 landing sites, and (2) three sites that individually yield the highest or lowest calculated radiolytic H₂ production rates on Mars. For the proposed landing sites, calculated H₂ production rates vary by a factor of ∼1.6, while the three comparison sites differ by a factor of ∼6. Rates in wet martian sediment and microfractured rock are comparable with rates in terrestrial environments that harbor low concentrations of microbial life (e.g., subseafloor basalt). Calculated H₂ production rates for low-porosity (<35%), fine-grained martian sediment (0.12–1.2 nM/year) are mostly higher than rates for South Pacific subseafloor basalt (∼0.02–0.6 nM/year). Production rates in martian high-porosity sediment (>35%) and microfractured (1 μm) hard rock (0.03 to <0.71 nM/year) are generally similar to rates in South Pacific basalt, while yields for larger martian fractures (1 and 10 cm) are one to two orders of magnitude lower (<0.01 nM/year). If minerals or brine that amplify radiolytic H₂ production rates are present, H₂ yields exceed the calculated rates.

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BCO-DMO Project Info

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To quantify the potential for biological contamination associated with the coring process, we conducted perfluorocarbon tracer (PFT) analysis on 556 sediment samples from Integrated Ocean Drilling Program (IODP) Expedition 329. The expedition cored deep-sea sediment at seven sites in the South Pacific Gyre (Sites U1365–U1371). We analyzed two types of sediment samples: (1) samples taken in the central part of the core (i.e., interior samples) and (2) samples taken near the core edge (i.e., exterior samples). We calculated the amount of potential drilling fluid intrusion from the mass of PFT that we measured in each sample. For the seven Expedition 329 sites (15 holes analyzed), PFT content ranges from below detection to levels where contamination is extremely apparent. The centers of the sediment cores (interior samples) contained generally less PFT than the core margins (exterior samples) and thus have lower potential drilling fluid (DF) contamination. The majority of sediment samples (interior) at Sites U1370 and U1371 have a contamination potential close to or below detection levels (i.e., 1.19 × 10^–4 ng_PFT/g_sediment or 1.78 × 10^–3 µL_DF/g_sediment on average). We observed higher contamination values (i.e., 7.28 × 10^–3 ng_PFT/g_sediment or 6.98 × 10^–2 µL_DF/g_sediment on average) at Sites U1365, U1366, and U1367. Finally, we measured much higher PFT concentrations throughout the sediment of Sites U1368 and U1369 (i.e., 5.48 × 10^–2 ng_PFT/g_sediment or 6.60 × 10^–1 µL_DF/g_sediment on average). We observe no apparent correlation of sample PFT content to sediment lithology, degree of sediment disturbance, core section number, or porosity.

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Extracellular DNA, attributed to dead or dying organisms, is known to be significant component of shallow marine sediment. The prevalence of extracellular DNA in sediment deeper than 10cm below the seafloor and the degree to which extracellular DNA signatures may skew surveys of microbial life in this habitat had remained untested. To address these pertinent questions, I embarked on two oceanographic expeditions to the Mid-Arctic and Eastern Pacific ocean and collected fresh subseafloor sediment samples amenable to intra- vs. extra-cellular DNA interrogation using the DNA-binding photoactive dye propidium monoazide. Photoactive dye-treated samples allow the amplification and subsequent high-throughput sequencing of intracellular DNA, exclusively. Thus, comparisons to untreated controls, where both extra- and intra-cellular signals are amplified and sequenced, show the influence of the environmental extracellular DNA component in gene quantification and community sequencing surveys. Significantly higher 16S rRNA gene counts from total versus intracellular DNA pools in shallow sediment confirm the presence of extracellular DNA pools in shallow marine sediment. All samples collected from depths greater than 10 cm did not show differences in 16S rRNA genes quantified from total vs. intracellular DNA pools. 16S rRNA gene sequencing shows that even where there are significant differences in intracellular vs. total (intra- and extra-cellular) gene counts, the reported microbial community composition remains the same. Overall, we show that despite its intermittent detection in shallow sediment, extracellular DNA is not detected and does not affect 16S rRNA gene-targeted community composition surveys in sediment as deep as 10 meters below the seafloor.

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DNA in marine sediment contains both fossil sequences and sequences from organisms that live in the sediment. The demarcation between these two pools and their respective rates of turnover are generally unknown. We address these issues by comparing the total extractable DNA pool to the fraction of sequenced chloroplast DNA (cpDNA) in sediment from two sites in the Bering Sea. We assume that cpDNA is a tracer of non-reproducing fossil DNA. Given >150,000 sequence reads per sample, cpDNA is easily detectable in the shallowest samples but decays with depth, suggesting that sequencing-based richness assessments of communities in deep subseafloor sediment are relatively unaffected by fossil DNA. The initial decrease in cpDNA reads suggests that most cpDNA decays within 100–200 k.y. of deposition. However, cpDNA from a few phylotypes, including some that match fossil diatoms, are present throughout the cored sediment, ranging in age to 1.4 Ma. The relative fraction of sequences composed by cpDNA decreases non-linearly with increasing sediment age, suggesting that detectable cpDNA becomes more recalcitrant with age. This can be explained by biological activity decreasing with sediment age and/or by preferential long-term survival of only the most thoroughly protected DNA. The association of cpDNA reads with published records of siliceous microfossils, including diatom spores, at the same sites suggests that microfossils may help to preserve DNA. This DNA may be useful for studies of paleoenvironmental conditions and biological evolution on time scales that approach or exceed 1 m.y.

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Subseafloor sediment hosts a large, taxonomically rich and metabolically diverse microbial ecosystem. However, the factors that control microbial diversity in subseafloor sediment have rarely been explored. Here we show that bacterial richness varies with organic degradation rate and sediment age. At three open-ocean sites (in the Bering Sea and equatorial Pacific) and one continental margin site (Indian Ocean), richness decreases exponentially with increasing sediment depth. The rate of decrease in richness with depth varies from site to site. The vertical succession of predominant terminal electron acceptors correlates to abundance-weighted community composition, but does not drive the vertical decrease in richness. Vertical patterns of richness at the open-ocean sites closely match organic degradation rates; both properties are highest near the seafloor and decline together as sediment depth increases. This relationship suggests that (i) total catabolic activity and/or electron donor diversity exerts a primary influence on bacterial richness in marine sediment, and (ii) many bacterial taxa that are poorly adapted for subseafloor sedimentary conditions are degraded in the geologically young sediment where respiration rates are high. Richness consistently takes a few hundred thousand years to decline from near-seafloor values to much lower values in deep anoxic subseafloor sediment, regardless of sedimentation rate, predominant terminal electron acceptor, or oceanographic context.

Importance Subseafloor sediment provides a wonderful opportunity to investigate drivers of microbial diversity in communities that may be isolated for millions of years. Our manuscript shows the impact of in situ conditions on bacterial community structure in subseafloor sediment. Specifically, it shows that bacterial richness in subseafloor sediment declines exponentially with sediment age, and in parallel with organic-fueled oxidation rate. This result suggests that subseafloor diversity ultimately depends on electron donor diversity and/or total community respiration. Despite the extraordinary nature of this ecosystem, we believe this is the first study of how and why biological richness changes over time in subseafloor sediment.

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Subsurface microbial communities undertake many terminal electron-accepting processes, often simultaneously. Using a tritium-based assay, we measured the potential hydrogen oxidation catalyzed by hydrogenase enzymes in several subsurface sedimentary environments (Lake Van, Barents Sea, Equatorial Pacific, and Gulf of Mexico) with different predominant electron-acceptors. Hydrogenases constitute a diverse family of enzymes expressed by microorganisms that utilize molecular hydrogen as a metabolic substrate, product, or intermediate. The assay reveals the potential for utilizing molecular hydrogen and allows qualitative detection of microbial activity irrespective of the predominant electron-accepting process. Because the method only requires samples frozen immediately after recovery, the assay can be used for identifying microbial activity in subsurface ecosystems without the need to preserve live material. We measured potential hydrogen oxidation rates in all samples from multiple depths at several sites that collectively span a wide range of environmental conditions and biogeochemical zones. Potential activity normalized to total cell abundance ranges over five orders of magnitude and varies, dependent upon the predominant terminal electron acceptor. Lowest per-cell potential rates characterize the zone of nitrate reduction and highest per-cell potential rates occur in the methanogenic zone. Possible reasons for this relationship to predominant electron acceptor include (i) increasing importance of fermentation in successively deeper biogeochemical zones and (ii) adaptation of H₂ases to successively higher concentrations of H₂ in successively deeper zones.

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Hydrogen (H₂) is produced in geological settings by dissociation of water due to radiation from radioactive decay of naturally occurring uranium (²³⁸U, ²³⁵U), thorium (²³²Th) and potassium (⁴⁰K). To quantify the potential significance of radiolytic H₂ as an electron donor for microbes within the South Pacific subseafloor basaltic aquifer, we use radionuclide concentrations of 43 basalt samples from IODP Expedition 329 to calculate radiolytic H₂ production rates in basement fractures. The samples are from three sites with very different basement ages and a wide range of alteration types. U, Th, and K concentrations vary by up to an order of magnitude from sample to sample at each site. Comparison of our samples to each other and to the results of previous studies of unaltered East Pacific Rise basalt suggests that significant variations in radionuclide concentrations are due to differences in initial (unaltered basalt) concentrations (which can vary between eruptive events) and post-emplacement alteration. However, there is no clear relationship between alteration type and calculated radiolytic yields. Local maxima in U, Th, and K produce hotspots of H₂ production, causing calculated radiolytic rates to differ by up to a factor of 80 from sample to sample. Fracture width also greatly influences H₂ production, where microfractures are hotspots for radiolytic H₂ production. For example, H₂ production rates normalized to water volume are 190 times higher in 1 μm wide fractures than in fractures that are 10 cm wide. To assess the importance of water radiolysis for microbial communities in subseafloor basaltic aquifers, we compare electron transfer rates from radiolysis to rates from iron oxidation in subseafloor basalt. Radiolysis appears likely to be a more important electron donor source than iron oxidation in old (>10 Ma) basement basalt. Radiolytic H₂ production in the volume of water adjacent to a square cm of the most radioactive SPG basalt may support as many as 1500 cells.

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We present a mathematical model that quantifies the rate of water radiolysis near radionuclide-containing solids. Our model incorporates the radioactivity of the solid along with the energies and attenuation properties for alpha (α), beta (β), and gamma (γ) radiation to calculate volume normalized dose rate profiles. In the model, these dose rate profiles are then used to calculate radiolytic hydrogen (H₂) and hydrogen peroxide (H₂O₂) production rates as a function of distance from the solid–water interface. It expands on previous water radiolysis models by incorporating planar or cylindrical solid–water interfaces and by explicitly including γ radiation in dose rate calculations. To illustrate our model’s utility, we quantify radiolytic H₂ and H₂O₂ production rates surrounding spent nuclear fuel under different conditions (at 20 years and 1000 years of storage, as well as before and after barrier failure). These examples demonstrate the extent to which α, β and γ radiation contributes to total absorbed dose rate and radiolytic production rates. The different cases also illustrate how H₂ and H₂O₂ yields depend on initial composition, shielding and age of the solid. In this way, the examples demonstrate the importance of including all three types of radiation in a general model of total radiolytic production rates.

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We investigated compositional relationships between bacterial communities in the water column and those in deep-sea sediment at three environmentally distinct Pacific sites (two in the Equatorial Pacific and one in the North Pacific Gyre). Through pyrosequencing of the v4–v6 hypervariable regions of the 16S ribosomal RNA gene, we characterized 450 814 operational taxonomic units (OTUs, 97% similarity). Hierarchical clustering and non-metric multidimensional scaling partition the samples into four broad groups, regardless of geographic location: a photic-zone community, a subphotic community, a shallow sedimentary community and a subseafloor sedimentary community (≥1.5 meters below seafloor). Abundance-weighted community compositions of water-column samples exhibit a similar trend with depth at all sites, with successive epipelagic, mesopelagic, bathypelagic and abyssopelagic communities. Taxonomic richness is generally highest in the water-column O₂ minimum zone and lowest in the subseafloor sediment. OTUs represented by abundant tags in the subseafloor sediment are often present but represented by few tags in the water column, and represented by moderately abundant tags in the shallow sediment. In contrast, OTUs represented by abundant tags in the water are generally absent from the subseafloor sediment. These results are consistent with (i) dispersal of marine sedimentary bacteria via the ocean, and (ii) selection of the subseafloor sedimentary community from within the community present in shallow sediment.

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The depth of oxygen penetration into marine sediments differs considerably from one region to another. In areas with high rates of microbial respiration, O₂ penetrates only millimetres to centimetres into the sediments, but active anaerobic microbial communities are present in sediments hundreds of metres or more below the sea floor. In areas with low sedimentary respiration, O₂ penetrates much deeper but the depth to which microbial communities persist was previously unknown. The sediments underlying the South Pacific Gyre exhibit extremely low areal rates of respiration. Here we show that, in this region, microbial cells and aerobic respiration persist through the entire sediment sequence to depths of at least 75 metres below sea floor. Based on the Redfield stoichiometry of dissolved O₂ and nitrate, we suggest that net aerobic respiration in these sediments is coupled to oxidation of marine organic matter. We identify a relationship of O₂ penetration depth to sedimentation rate and sediment thickness. Extrapolating this relationship, we suggest that oxygen and aerobic communities may occur throughout the entire sediment sequence in 15–44% of the Pacific and 9–37% of the global sea floor. Subduction of the sediment and basalt from these regions is a source of oxidized material to the mantle.

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Knowledge of salinity in the deep ocean is important for understanding past ocean circulation and climate. Based on sedimentary pore fluid chloride measurements of a single Pacific site, Adkins et al. (2002) suggested that, during the Last Glacial Maximum (LGM), the Pacific deep bottom water was saltier than expected based on lower sea level alone. Here we present high‐resolution salinity profiles from five sites in the South, Equatorial, and North Pacific Ocean. Our study greatly constrains understanding of LGM salinity in the Pacific Ocean. Our results show that LGM chloride concentrations of deep Pacific bottom water were 4.09 ± 0.4% greater than today’s values. Pacific Ocean bottom water salinity was also indistinguishable from being homogeneous across the wide range of latitudes studied here. These LGM salinity reconstructions are on average slightly higher (~1.4 to 1% higher) than expected from sea level of the time, which is generally inferred to have been ~120 to ~135 m lower than today.

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Dissolved inorganic carbon (DIC) concentration and total alkalinity in marine sediment vary with biological activity, mineral diagenesis and past bottom ocean water composition. Reliable interpretation of this data is often compromised due to precipitation of calcium carbonate (CaCO₃) during sediment recovery, processing and sample storage.

Here we present and test a method that corrects for this precipitation and consequently allows quantification of in situ carbonate system chemistry. Our method relies on the over-determination of the dissolved carbonate system by (i) measuring DIC, alkalinity and calcium, and (ii) explicitly assuming CaCO₃ saturation in the sediment. We experimentally tested this method using data from Integrated Ocean Drilling Program (IODP) Site U1368 in the South Pacific Gyre. Our results show that we can accurately reproduce in situ aqueous carbonate system chemistry if DIC, alkalinity and calcium concentration are measured simultaneously. At Site U1368, the correction for sampling associated precipitation is equivalent to 4.5 and 8.9% of the measured DIC and alkalinity, respectively. The method is well suited for any sediment porewater that is saturated with respect to calcium carbonate; consequently, it is applicable for approximately 50% of the global oceanic seafloor.

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Scientific ocean drilling has greatly advanced the understanding of subseafloor sedimentary life. Studies of Ocean Drilling Program (ODP) and Integrated ODP samples and data show that mean per-cell rates of catabolic activity, energy flux, and biomass turnover are orders of magnitude slower in subseafloor sediment than in the surface world. They have also shown that potentially competing metabolic pathways co-occur for hundreds of meter depth in subseafloor sediment deposited over millions of years.

Our study of an example site (eastern equatorial Pacific ODP Site 1226) indicates that the energy yields of these competing reactions are pinned to a thermodynamic minimum. The simplest explanation of this long-term coexistence is thermodynamic cooperation, where microorganisms utilize different but coexisting pathways that remove each other’s reaction products.

Our Site 1226 results indicate that the energy flux to subseafloor sedimentary microbes is extremely low. Comparison to biomass turnover rates at other sites suggests that most of this flux may be used for building biomolecules from existing components (e.g., amino acids in the surrounding sediment), rather than for de novo biosynthesis from inorganic chemicals.

Given these discoveries, scientific ocean drilling provides a tremendous opportunity to address several mysteries of microbial survival and natural selection under extreme energy limitations. Some of these mysteries are centered on microbial communities: To what extent do counted cells in subseafloor sediment constitute a deep microbial necrosphere? How do different kinds of microbes interact to sustain their mean activity at low average rates for millions of years? Other mysteries relate to individual cells: How slowly can a cell metabolize? How long can a cell survive at such low rates of activity? What properties allow microbes to be sustained by low fluxes of energy? In what ways do subseafloor organisms balance the benefit(s) of maximizing energy recovery with the need to minimize biochemical cost(s) of energy recovery?

A strong scientific ODP will be critical to address these mysteries.

Abstract

The nature of the energy yielding mechanisms in the lowenergy organic-poor sedimentary environment underlying the South Pacific Gyre (SPG) is not fully constrained. We used the approach of Wang et al. (2008) to quantify rates of organic-fuelled metabolic activities at most IODP Expedition 329 Sites (U1365 through U1370). At Site U1366 and U1370 net rates of oxygen-reducing organic oxidation averaged 1.77E^-2 and 1.64E^-3 fmol O₂ cell^-1 yr^-1, respectively, representing a tremendously low cellular metabolism. At Site U1370, we observe net oxygen reduction throughout the entire sediment column. At Site U1366, statistically significant net oxygen reduction is not detected at depths greater than 11 meters below seafloor. Despite these low rates of organic oxidation, most cell counts are above the minimum detection limit throughout the entire sequence at both sites. Hydrogen from natural radioactive splitting of water has been hypothesized to be a significant electron donor in organic-poor sediment of the SPG. Becauses water radiolysis produces H₂ and – O₂ simultaneously, oxidation of this H₂ does not contribute to net O₂ reduction in the sediment. Our calculation of radiolytic H₂ production, based on radioactive element content and sediment physical properties, indicate that on average 5.63E^-1 and 9.79E^-2 fmol H₂ yr^-1 cell^-1 is available throughout the sequence at Sites U1366 and U1370, respectively. Despite these relatively high production rates, dissolved H₂ abundances are below detection at both sites. These results suggest that H₂ from in situ water radiolysis fuels the predominant energy-yielding pathway for microbes in SPG sediment.

Abstract

In recent years, multiple research groups have tremendously advanced understanding of subseafloor sedimentary life. Microbes in subseafloor sediment are now known to be abundant, diverse and characterized by extraordinarily low mean rates of activity. Some discoveries challenge our sense of what is possible. For example, per-cell energy fluxes are far below the rates believed necessary for reproduction. What mechanisms might allow cells to reproduce at such low rates? Or do many of them live for millions of years without reproducing? Bulk population studies show that a very large fraction of these cells is active. However, we know essentially nothing about cell-to-cell variation in respiration, biomass turnover or reproduction. Furthermore, we do not clearly understand how organic-fueled respiration can persist for tens of myrs at very slow rates. Subseafloor community structure is largely unexplored. We have very limited understanding of the ways in which subseafloor microbes compete and almost no understanding of how they cooperate. Roles of viruses, eukaryotes, resting stages and bacterial spores in subseafloor ecosystems are largely unknown. The proximate causes and ultimate consequences of natural selection in subseafloor communities remain unknown. For the most part, we do not yet know the genetic potential of subseafloor microbes, the extent to which their potential is expressed, or the conditions under which they are expressed. The actual limits to subseafloor life are not yet known. Advancing understanding of these issues will yield fundamental insight into the nature of life.

Abstract

High-temperature water–rock reactions produce large quantities of hydrogen, which must be transported to cooler settings to sustain life. Lower-temperature hydrogen generation could potentially support life in situ and free subsurface microbes from photosynthetic constraints.

Abstract

Concentrations of uranium (U), thorium (Th), and potassium (K) in geological materials provide insight into many important lithological characteristics and geologic processes. In marine sediment, they can aid in identifying clay compositions, depositional environments, and diagenetic processes. They can also yield information about the alteration and heat production of rocks (Ketcham 1996; Barr et al., 2002; Revillon et al., 2002; Brady et al., 2006; Bartetzko, 2008). Measurements of the concentrations of these elements in geological materials are relatively straightforward in shore-based laboratories. Rapidly determining their abundance within cores of sedimentary and igneous rock sequences onboard a research vessel is a more challenging but potentially very useful method to non-destructively and quickly provide important geochemical information about the concentrations of U, Th, and K within the sequences being cored.

Abstract

Integrated Ocean Drilling Program (IODP) Expedition 329 made major strides toward fulfilling its objectives. Shipboard studies documented (1) fundamental aspects of habitability and life in this very low activity subseafloor sedimentary ecosystem and (2) first-order patterns of habitability within the igneous basement. A broad range of postexpedition studies will complete the expedition objectives. Throughout the South Pacific Gyre (SPG; Sites U1365–U1370), dissolved oxygen and nitrate are present throughout the entire sediment sequence, and sedimentary microbial cell counts are lower than at all previously drilled IODP/ Ocean Drilling Program (ODP)/Deep Sea Drilling Program (DSDP) sites. In contrast, at Site U1371 in the upwelling zone just south of the gyre, detectable oxygen and nitrate are limited to the top and bottom of the sediment column, manganese reduction is a prominent electron-accepting process, and cell concentrations are higher than at the same depths in the SPG sites throughout the sediment column. Geographic variation in subseafloor profiles of dissolved and solid-phase chemicals are consistent with the magnitude of organic-fueled subseafloor respiration declining from outside the gyre to the gyre center.

Chemical profiles in the sedimentary pore water and secondary mineral distributions in the basaltic basement indicate that basement alteration continues on the timescale of formation fluid replacement, even at the sites with the oldest basement (84–120 Ma at Sites U1365 and U1366).

Abstract

The Integrated Ocean Drilling Program site U1365 drilled into the basement of the southwest Pacific crust formed from the mid-Cretaceous Osbourn Trough that rifted apart the Manihiki and Hikurangi Plateaus (the Greater Manihiki). The basalt geochemistry at this site is crucial for understanding the magmatic processes and mantle source of the mid-Cretaceous Osbourn Trough. The recovered fresh basalts were low-K tholeiitic normal (N) and depleted (D) mid-ocean ridge basalt (MORB). Their trace element and Sr–Nd isotope compositions indicate a Pacific-type mantle source rather than any significant influences from the nearby Louisville Seamount Chain or from the Greater Manihiki Plateau. Despite the presence of a plume head underneath the Osbourn Trough at its initial stage, the insignificance of a plume head could be explained by the long-distance (> 1000 km) southward migration of the Osbourn Trough. Lavas at site U1365 vary from low-MgO (< 6.9 wt.%) N-MORB at the bottom to high-MgO (8 wt.% to 9.5 wt.%) D-MORB and, then, to medium-MgO (7.3 wt.% to 8.2 wt.%) N-MORB according to their eruption sequences, which was accompanied by magma mixing in the magma reservoir. The D-MORB group lavas have higher melting degrees than those of N-MORB group based on their concentrations of TiO₂, Na₂O and CaO corrected for crystallization relative to MgO = 7.8 wt.%. The major element compositions of the high-MgO D-MORB lavas were consistent with partial melting in the spinel–peridotite zone over a pressure interval from ~ 3.1 GPa to 2 GPa in the mantle. The significant overlap of N-MORB and D-MORB in Sr–Nd isotopes suggests that chemical differences between the two groups were derived from the mantle melting processes. Based on comparison with lavas from the East Pacific Rise where a positive correlation of mantle melting degree vs. spreading rate is shown, we suggest that the Osbourn Trough might have a full spreading rate of ~ 140 mm/yr. Thus, the slow ridge-like axial morphology of the Osbourn Trough should be a character of an extinct fast ridge.

Abstract

In the last three decades we have learned a great deal about microbes in subsurface environments. Once, these habitats were rarely examined, perhaps because so much of the life that we are concerned with exists at the surface and seems to pace its metabolic and evolutionary rhythms with the overt planetary, solar, and lunar cycles that dictate our own lives. And it certainly remains easier to identify with living beings that are in our midst, most obviously struggling with us or against us for survival over time scales that are easiest to track using diurnal, monthly or annual periods. Yet, research efforts are drawn again and again to the subsurface to consider life there. No doubt this has been due to our parochial interests in the resources that exist there (the water, minerals, and energy) that our society continues to require and that in some cases are created or modified by microbes. However, we also continue to be intrigued by the scientific curiosities that might only be solved by going underground and examining life where it does and does not exist.

But really, is life underground just a peculiarity of most life on the planet and only a recently discovered figment of life? Or is it actually a more prominent and fundamental, if unseen, theme for life on our planet? Our primary purpose in this chapter is to provide an incremental assembly of knowledge of subsurface life with the aim of moving us towards a more complete conceptual model of deep life on the planet. We aim to merge the consideration of the subsea-floor and the continental subsurface because it is only through such a unified treatment that we can reach a comprehensive view of this underground life. We also provide some thoughts on a way forward with what we consider to be interesting new research areas, along with the methods by which they might be addressed as we seek new knowledge about life in this Stygian realm.

Abstract

The global geographic distribution of subseafloor sedimentary microbes and the cause(s) of that distribution are largely unexplored. Here, we show that total microbial cell abundance in subseafloor sediment varies between sites by ca. five orders of magnitude. This variation is strongly correlated with mean sedimentation rate and distance from land. Based on these correlations, we estimate global subseafloor sedimentary microbial abundance to be 2.9⋅10²⁹ cells [corresponding to 4.1 petagram (Pg) C and ∼0.6% of Earth’s total living biomass]. This estimate of subseafloor sedimentary microbial abundance is roughly equal to previous estimates of total microbial abundance in seawater and total microbial abundance in soil. It is much lower than previous estimates of subseafloor sedimentary microbial abundance. In consequence, we estimate Earth’s total number of microbes and total living biomass to be, respectively, 50–78% and 10–45% lower than previous estimates.

Abstract

Microbial communities can subsist at depth in marine sediments without fresh supply of organic matter for millions of years. At threshold sedimentation rates of 1 millimeter per 1000 years, the low rates of microbial community metabolism in the North Pacific Gyre allow sediments to remain oxygenated tens of meters below the sea floor. We found that the oxygen respiration rates dropped from 10 micromoles of O₂ liter⁻¹ year⁻¹ near the sediment-water interface to 0.001 micromoles of O₂ liter⁻¹ year⁻¹ at 30-meter depth within 86 million-year-old sediment. The cell-specific respiration rate decreased with depth but stabilized at around 10⁻³ femtomoles of O₂ cell⁻¹ day⁻¹ 10 meters below the seafloor. This result indicated that the community size is controlled by the rate of carbon oxidation and thereby by the low available energy flux.

Abstract

Two decades of scientific ocean drilling have demonstrated widespread microbial life in deep sub-seafloor sediment, and surprisingly high microbial–cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in this vast buried ecosystem are not yet understood. It is not known whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a dormant, spore-like state. Here we apply a new approach—the D:L-amino–acid model—to quantify the distributions and turnover times of living microbial biomass, endospores and microbial necromass, as well as to determine their role in the sub-seafloor carbon budget. The approach combines sensitive analyses of unique bacterial markers (muramic acid and D-amino acids) and the bacterial endospore marker, dipicolinic acid, with racemization dynamics of stereo-isomeric amino acids. Endospores are as abundant as vegetative cells and microbial activity is extremely low, leading to microbial biomass turnover times of hundreds to thousands of years. We infer from model calculations that biomass production is sustained by organic carbon deposited from the surface photosynthetic world millions of years ago and that microbial necromass is recycled over timescales of hundreds of thousands of years.

Abstract

Integrated Ocean Drilling Program Expedition 329 made major strides toward fulfilling its objectives. Shipboard studies documented (1) fundamental aspects of habitability and life in this very low activity subseafloor sedimentary ecosystem and (2) first-order patterns of basement habitability. A broad range of postexpedition studies will complete the expedition objectives.

Throughout the South Pacific Gyre (Sites U1365–U1370), dissolved oxygen and nitrate are present throughout the entire sediment sequence. Concentration profiles of oxygen and nitrate indicate that subseafloor heterotrophic respiration is oxic and proceeds very slowly. In contrast, at Site U1371 in the upwelling zone just south of the gyre, detectable oxygen and nitrate are limited to the top and bottom of the sediment column and manganese reduction is a prominent electron-accepting process.

Geographic variation in subseafloor profiles of dissolved and solid-phase chemicals are consistent with the magnitude of organic-fueled subseafloor respiration declining from outside the gyre to the gyre center.

Microbial cell counts are lower than at all sites previously drilled. Countable cells disappear with increasing depth in the sediment at every site in the South Pacific Gyre (Sites U1365–U1370). Concentrations of dissolved oxygen and nitrate, total organic carbon, and total nitrogen stabilize as countable cells fall below the minimum detection limit. The downhole disappearance of cells and measurable organic oxidation appears to result from the disappearance of organic electron donors.

At the South Pacific Gyre sites, dissolved hydrogen concentration is low but often above detection in deep sediment. At Site U1371, where most of the sediment is anoxic, dissolved hydrogen concentration is above detection through much of the column.

High-resolution chemical and physical measurements provide the opportunity for reconstructing glacial seawater characteristics through the South Pacific Gyre. Such reconstruction will greatly contribute to understanding the global ocean-climate system.

Dissolved chemical profiles and igneous petrology indicate that basement alteration continues on the timescale of formation fluid replacement, even at the sites with oldest basement (84–120 Ma at Sites U1365 and U1366). Profiles of dissolved chemicals indicate that microbial habitability of the entire sediment sequence and the uppermost basalt is not limited by access to electron acceptors (oxygen and nitrate) or major nutrients (carbon, nitrogen, and phosphorus).

Abstract

This project aims to address one of potentially many novel metabolisms present in the deep biosphere of which little or nothing is known. Considering energy-yielding pathways, several studies focusing on Gibbs energy calculations and known geochemical parameters in other extreme environments have shown hundreds of potentially life supporting redox couples; while these reactions may not be favorable under standard conditions, the deep biosphere is anything but standard. As microbial life can eke out a living using metabolisms right at the edge of possibility, the potential for not only novel organisms, but geochemically relevant ones, is tremendous. Specifically, we will be looking at a zone present in a few deep sediment cores, first noted by Schrum et al. (2009) which exhibit ammonium loss in the absence of typical oxidants (i.e. no oxygen, no nitrite). Uptake for biomass production doesn’t appear feasible, either – hence the mystery. Using genetic and isotopic tools, we hope to figure out what organisms may be unique to these zones and investigate the feasibility, previously proposed, of ammonium oxidation coupled to sulfate reduction. Our main sample set includes cores from the Bay of Bengal, though similar features have also been noted off the east coast of the U.S.; potentially, processes such as this could be of global importance.