AbstractHere, we report genome sequences of two Penicillium isolates from below the seafloor of the oligotrophic South Pacific Gyre. These genomes are the first reported for fungi from deeply buried marine sediment. Both genomes will provide valuable information regarding the role of fungi and carbon cycling in the energy-limited subsurface biosphere.
AbstractNearly 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.
AbstractInternational Ocean Discovery Program (IODP) Expedition 370 aimed to explore the limits of life in the deep subseafloor biosphere at a location where temperature increases with depth at an intermediate rate and exceeds the known temperature maximum of microbial life (~120°C) at the sediment/basement interface ~1.2 km below the seafloor. Drilling Site C0023 is located in the vicinity of Ocean Drilling Program (ODP) Sites 808 and 1174 at the protothrust zone in the Nankai Trough off Cape Muroto at a water depth of 4776 m. ODP Leg 190 in 2000, revealed the presence of microbial cells at Site 1174 to a depth of ~600 meters below seafloor (mbsf), which corresponds to an estimated temperature of ~70°C, and reliably identified a single zone of higher cell concentrations just above the décollement at around 800 mbsf, where temperature presumably reached 90°C; no cell count data was reported for other sediment layers in the 70°–120°C range, because the limit of manual cell count for low-biomass samples was not high enough. With the establishment of Site C0023, we aimed to detect and investigate the presence or absence of life and biological processes at the biotic–abiotic transition with unprecedented analytical sensitivity and precision. Expedition 370 was the first expedition dedicated to subseafloor microbiology that achieved time-critical processing and analyses of deep biosphere samples by simultaneous shipboard and shore-based investigations. Our primary objectives during Expedition 370 were to study the relationship between the deep subseafloor biosphere and temperature. We aimed to comprehensively study the factors that control biomass, activity, and diversity of microbial communities in a subseafloor environment where temperatures increase from ~2°C at the seafloor to ~120°C at the sediment/basement interface and thus likely encompasses the biotic–abiotic transition zone. We also aimed to determine geochemical, geophysical, and hydrogeological characteristics in sediment and the underlying basaltic basement and elucidate if the supply of fluids containing thermogenic and/or geogenic nutrient and energy substrates may support subseafloor microbial communities in the Nankai accretionary complex. To address these primary scientific objectives and questions, we penetrated 1180 m and recovered 112 cores across the sediment/basalt interface. More than 13,000 samples were collected, and selected samples were transferred to the Kochi Core Center by helicopter for simultaneous microbiological sampling and analysis in laboratories with a super-clean environment. Following the coring operations, a temperature observatory with 13 thermistor sensors was installed in the borehole to 863 mbsf.
AbstractThe past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be “hot spots” for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of 13C- or 15N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50–2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell–targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.
AbstractIncreasing anthropogenic CO2 in the atmosphere causes global warming and subsequent environmental changes, which may lead to an increase in natural disasters jeopardizing human society. Prompt technological development for CO2 capture and sequestration is required in the international community. In this study, we performed CO2 emission and shallow-type methane hydrate decomposition experiments at the Joetsu Knoll, offshore Joetsu, Niigata, Japan, as pilot studies to test feasibility of CO2 sequestration and methane recovery using methane-CO2 replacement in shallow-type methane hydrates. An isobaric cylinder pump and probe with a built-in heater (“Heat sonde”) were developed to inject CO2 in deep-sea, high-pressure conditions. Before injecting CO2 into a methane hydrate located in deep-sea sediments, we attempted CO2 emission directly into deep-seafloor. In the experiment, liquid CO2 was emitted at the head of Heat sonde, however, the isobaric cylinder pump became clogged during operation. The result reveals that precipitates of CO2 hydrate, which are generated during mixing of inflow seawater and outflow liquid CO2, blocked flow lines of the isobaric cylinder pump and Heat sonde. This suggests that our developed instruments must be improved for future work. We also observed the collapse of an exposed methane hydrate layer at the seafloor upon contact with the Heat sonde in our experiment.
AbstractMicrobial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~104 cells cm−3. Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.
AbstractThe 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.
AbstractGene sequencing of natural microbial communities in the deep subsurface has provided access to a biosphere of Bacteria, Archaea, and Eukaryotes, characterized by unexpected evolutionary depth and diversity. Despite phylogenetic overlaps with surface environments, the predominant groups of Bacteria and Archaea in the subseafloor differ from those found in surface seafloor environments. The extent and diversity of the deep subsurface biosphere has been mapped to a large extent using gene and genome sequence analysis; these approaches have extended ongoing cultivation efforts on subseafloor microbial communities, including new types of Bacteria, Archaea, and Eukaryotes in pure culture. This chapter starts by introducing the most commonly used, highly conserved, and phylogenetically informative marker gene, the 16S ribosomal RNA gene, then provides an overview on sequence analysis of functional genes that code for proteins and enzymes with distinct biological and process-relevant functions, and concludes with recent metagenome and single-cell sequencing surveys that allow novel insights into microbial diversity and function of the deep subseafloor biosphere.
AbstractScientific 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.
AbstractThe hydrogeologic properties of igneous ocean crust have been tested directly in only a few locations during IODP, but more common studies of crustal structure and rock alteration (using core samples and wireline logs) provide insight as to how water–rock interactions modify the crust over time. Collectively these studies reveal strong lithologic and hydrogeologic control on the nature of water–rock interactions, with hydrogeology following crustal architectures and histories. Permeability is generally greatest in the upper crust, but is heterogeneously distributed with depth and (at least in one location) may be azimuthally anisotropic. There appears to be a spreading rate dependence of basic patterns of rock alteration in the upper oceanic crust, with more variable and extensive alteration observed in crust created at slow- and medium-rate spreading centers. There may also be a spreading rate dependence of hydrogeologic properties, but we currently lack direct observations to test this hypothesis. The evolution of crustal properties with age is consistent with sustained ridge-flank water–rock interactions, and a continued dependence on fluid flow rates and reaction temperatures.
AbstractThe origin, evolution, and distribution of life throughout the universe can be better understood by determining the limits to life on Earth. A broad range of many of the physical and chemical constraints that determine the limits to life, such as temperature, pressure, physical space, water content, and the availability of energy and nutrients, are found in subseafloor environments. In fact, several expeditions (Ocean Drilling Program (ODP) and Integrated Ocean Drilling Program (IODP: now International Ocean Discovery Program)) have been at least partially motivated by the desire to explore the boundaries between the habitable and the uninhabitable parts of the subseafloor. In this chapter, the possible subseafloor environments and their physical and chemical characteristics that could signify the limits of the biosphere, particularly the hydrothermally active subseafloor environments, are reviewed. Although the nature and distribution of extreme or fringe biospheres are unknown, previous ODP- and IODP-expedition-based microbiological investigations have shown that the subseafloor hydrothermal systems with relatively abundant energy supplies (sediment-derived organic compounds and serpentinization-derived H2) provide targets for seeking the limits (boundary conditions) in subseafloor environments. Here, we also discuss predicted patterns of the abundance and composition of potential microbial catabolisms in the fringe microbial communities of subseafloor hydrothermal fluids based on the thermodynamic potential of particular catabolic strategies and the computed cost of anabolism in these settings.
AbstractIntegrated 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).
AbstractThe 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.
Sediment-covered basalt on the flanks of mid-ocean ridges constitutes most of Earth's oceanic crust, but the composition and metabolic function of its microbial ecosystem are largely unknown. By drilling into 3.5-million-year-old subseafloor basalt, we demonstrated the presence of methane- and sulfur-cycling microbes on the eastern flank of the Juan de Fuca Ridge. Depth horizons with functional genes indicative of methane-cycling and sulfate-reducing microorganisms are enriched in solid-phase sulfur and total organic carbon, host δ13C- and δ34S-isotopic values with a biological imprint, and show clear signs of microbial activity when incubated in the laboratory. Downcore changes in carbon and sulfur cycling show discrete geochemical intervals with chemoautotrophic δ13C signatures locally attenuated by heterotrophic metabolism.
AbstractA remarkable number of microbial cells have been enumerated within subseafloor sediments, suggesting a biological impact on geochemical processes in the subseafloor habitat. However, the metabolically active fraction of these populations is largely uncharacterized. In this study, an RNA-based molecular approach was used to determine the diversity and community structure of metabolically active bacterial populations in the upper sedimentary formation of the Nankai Trough seismogenic zone. Samples used in this study were collected from the slope apron sediment overlying the accretionary prism at Site C0004 during the Integrated Ocean Drilling Program Expedition 316. The sediments represented microbial habitats above, within, and below the sulfate–methane transition zone (SMTZ), which was observed approximately 20 m below the seafloor (mbsf). Small subunit ribosomal RNA were extracted, quantified, amplified, and sequenced using high-throughput 454 pyrosequencing, indicating the occurrence of metabolically active bacterial populations to a depth of 57 mbsf. Transcript abundance and bacterial diversity decreased with increasing depth. The two communities below the SMTZ were similar at the phylum level, however only a 24% overlap was observed at the genus level. Active bacterial community composition was not confined to geochemically predicted redox stratification despite the deepest sample being more than 50 m below the oxic/anoxic interface. Genus-level classification suggested that the metabolically active subseafloor bacterial populations had similarities to previously cultured organisms. This allowed predictions of physiological potential, expanding understanding of the subseafloor microbial ecosystem. Unique community structures suggest very diverse active populations compared to previous DNA-based diversity estimates, providing more support for enhancing community characterizations using more advanced sequencing techniques.
AbstractIntegrated 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).
I was honored to receive a DEBI RCN Graduate Student Education Exchange grant for research at the IODP Kochi Institute for Core Sample Research in Kochi, Japan. During the month of July 2010, I worked with Dr. Fumio Inagaki and other members of his lab group to learn his techniques of cell enumeration and flow cytometry. The experience of working in his lab allowed me to go beyond the typical collaboration based on brief meetings and email exchanges alone. I was able to step outside of my comfort zone and have a research experience in an unfamiliar culture. I learned much more than research techniques including overcoming communication barriers, building collaborations, and cultural exchanges. I discovered that the basic standards of science are global and although communication was difficult with a few lab members, the language of science transcended that and we continued to learn from each other regardless. The methods I have learned in Japan have given me the ability to expand on my skill set and apply it to various environments. Since working with Dr. Inagaki, the techniques I have learned have allowed me to work on samples collected from research cruises in the Gulf of Mexico and IODP Leg 325 in the Great Barrier Reef. Dr. Inagaki encouraged me to ship and work on my own samples in order to return to my home institution with data that I am able to directly incorporate into my dissertation. We have since discussed other collaboration opportunities and I look forward to what the Research Coordination Network can provide in the future.
Enumeration of microbial cells in subsurface samples is an important baseline approach in our understanding of microbial life and ecosystems. This method has proven a challenge as non-specific fluorescent signals due to sediment particles impede efficient detection and counting of microbial cells. Therefore, the proposed travel exchange took place at Fumio Inagaki’s laboratory (JAMSTEC) in Kochi (Japan) in order to carry out computer-based automatic cell counting for gravity core sediments fixed on board during the DARCSEAS cruise in the Eastern Mediterranean Sea. This method is based on washing sediments with hydrofluoric acid, and staining with SYBR Green I in order to eliminate fluorescence of non-biological background while discriminating at the same time against background fluorescence of unspecifically stained organic material whose emission wavelengths are slightly offset from the peak of the SYBR Green I fluorescence emission window (Morono, 2009). This innovative technique will allow processing the large number of cell count samples generated during the cruise. This will in turn enable robust statistical comparison between samples as it will eliminate the bias of human counting. This information will prove useful in quantifying the microbial population in the sediment samples of the Mediterranean Sea. This travel exchange will also be very beneficial for learning this new method and training with the leading experts on this field of research.