The 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.
We examined the carbon isotopic and molecular compositions of residual gases within Miocene methane-derived carbonates collected in Japan. Methane, ethane, and propane were extracted by acid ...digestion of powdered carbonates. The isotopic and molecular compositions of the extracted hydrocarbons are inconsistent with conventional thermogenic and microbial gases. Despite a range of δ13C values from −67‰ to −38‰ (relative to Vienna Pee Dee Belemnite (VPDB)), the liberated hydrocarbons yielded consistently low methane to ethane + propane ratios (2–30). The extracted ethane and propane yielded anomalous δ13C values as low as −84‰, lower than those of the coexisting methane. The ethane and propane were most likely produced through thermal cracking of organic compounds preserved within the seep carbonates during burial. The observed unusual isotopic trends may be explained by the mixing of two thermogenic gas components with different carbon isotopic and molecular compositions. Nevertheless, a positive correlation between δ13C values of methane and relatively immature carbonates at one study site (Nakanomata) indicates that while methane was oxidized to bicarbonate from which carbonates precipitated, it was preserved within the host carbonate cements. Such a scenario indicates that the residual methane at least partly originates from the Miocene seep fluid. Smaller amounts of methane were also released during heating and crushing of chipped samples. The results suggest that methane was entrapped mainly within intracrystal inclusions, which is supported by the observation of abundant nanometer-scale voids in an individual crystal.
•Hydrocarbon gases were extracted from Miocene methane-seep carbonates in Japan.•Most hydrocarbons are thermogenic gases produced from organic matter in carbonates.•The original seep methane could partly be preserved in immature carbonate crystals.
Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by ...microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.
The fossil record of tubeworms is scarce compared to that of chemosynthetic bivalves although tubeworms are one of the dominant inhabitants among seep and vent faunas at present. This study reports ...tubeworm fossils observed on a float of muddy dolomite concretion from the Middle Miocene Bessho Formation, central Japan. Vesicomyid bivalve fossils with seep carbonates have been reported from many localities of the Bessho Formation. The concretion float contains abundant tubes, 1 to 4 mm in diameter, with no surface ornamentation and their inner space filled by chalcedony. In microscopic view, the tubes consist of flexible multi-layered organic sheets, over 100 µm thick, which were deformed and delaminated. The tubes gently curve and are unbranched, elongating in an almost same direction; they form aggregation patches, separated from each other in matrix-supported condition. The tube aggregate matrices are more calcareous than the muddy surroundings. The tube size, wall structure and aggregation in calcareous patches are similar to posterior ‘root’ tubes of vestimentiferans in present seep sites such as the Gulf of Mexico and Congo deep-sea fan. Our study represents the first fossil record of tubeworms from a dolomite concretion, suggesting that silicification could be an important factor for fossil preservation of flexible tubes against sub-bottom fluidization, deformation, and dissolution of initial calcium carbonates.
We have discovered deep-sea mud that is extremely enriched in rare-earth elements and yttrium (together called REY) in the Japanese Exclusive Economic Zone around Minamitorishima Island, in the ...western North Pacific Ocean. The maximum total REY concentration reaches approximately 7000 ppm, which is much higher than that reported for conventional REY deposits on land and other known potential REY resources in the ocean. The extremely REY-rich mud is characterized by abundant phillipsite and biogenic calcium phosphate. In addition, the stratigraphic layer with the highest REY concentration occurs just ~3 m beneath the seafloor. The shallow burial of these strata together with the high REY content, especially those of heavy rare-earth elements, suggest that the newly discovered extremely REY-rich mud may be a promising REY resource.
Recent explorations of scientific ocean drilling have revealed the presence of microbial communities persisting in sediments down to ~2.5 km below the ocean floor. However, our knowledge of these ...microbial populations in the deep subseafloor sedimentary biosphere remains limited. Here, we present a cultivation experiment of 2-km-deep subseafloor microbial communities in 20-million-year-old lignite coalbeds using a continuous-flow bioreactor operating at 40 °C for 1029 days with lignite particles as the major energy source. Chemical monitoring of effluent samples via fluorescence emission-excitation matrices spectroscopy and stable isotope analyses traced the transformation of coalbed-derived organic matter in the dissolved phase. Hereby, the production of acetate and
C-depleted methane together with the increase and transformation of high molecular weight humics point to an active lignite-degrading methanogenic community present within the bioreactor. Electron microscopy revealed abundant microbial cells growing on the surface of lignite particles. Small subunit rRNA gene sequence analysis revealed that diverse microorganisms grew in the bioreactor (e.g., phyla Proteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, Spirochaetes, Tenericutes, Ignavibacteriae, and SBR1093). These results indicate that activation and adaptive growth of 2-km-deep microbes was successfully accomplished using a continuous-flow bioreactor, which lays the groundwork to explore networks of microbial communities of the deep biosphere and their physiologies.
To investigate fluid regimes in the Nankai accretionary wedge, we estimated the temperatures of lithium (Li) reservoirs from the Li isotope ratio (7Li/6Li) in fluids recovered from Kumano mud volcano ...No. 5 (KMV#5) in the Nankai forearc basin. The 7Li/6Li ratios in the KMV#5 fluids are among the lightest reported from submarine mud volcanoes. The estimated Li reservoir temperatures (310 °C at maximum) were significantly higher than the smectite-to-illite conversion temperature (60–150 °C), whereas the temperature of fluid from the deeper Nankai décollement was estimated to be 150 °C at maximum. Data obtained from previous conductivity surveys across the trench suggest that there are two fluid reservoirs associated with distinct dehydration processes in the Nankai subduction zone. From these results, we inferred that the discontinuous mud diapirism in the Kumano forearc basin results from the periodic injection of deep-seated fluid accumulated in the corner of the serpentinized forearc mantle wedge that travels upward via a thrust fault. The intermittent upwelling of deep-seated fluid from the Nankai accretionary wedge due to periodic breaches of a low-permeability barrier above the fluid reservoir may be associated with seismogenic fault activity.
•The 7Li/6Li signature in mud volcano fluid in the Nankai forearc basin is light.•Nankai mud volcano fluid contains Li that experienced a high T of >150 °C.•Nankai mud diapirism involves fluids from the serpentinized forearc mantle.•Deep-seated fluids upwell intermittently in the Nankai accretionary wedge.
Submarine mud volcanoes (SMVs) are formed by muddy sediments and breccias extruded to the seafloor from a source in the deep subseafloor and are characterized by the discharge of methane and other ...hydrocarbon gasses and deep-sourced fluids into the overlying seawater. Although SMVs act as a natural pipeline connecting the Earth's surface and subsurface biospheres, the dispersal of deep-biosphere microorganisms and their ecological roles remain largely unknown. In this study, we investigated the microbial communities in sediment and overlying seawater at two SMVs located on the Ryukyu Trench off Tanegashima Island, southern Japan. The microbial communities in mud volcano sediments were generally distinct from those in the overlying seawaters and in the well-stratified Pacific margin sediments collected at the Peru Margin, the Juan de Fuca Ridge flank off Oregon, and offshore of Shimokita Peninsula, northeastern Japan. Nevertheless, in-depth analysis of different taxonomic groups at the sub-species level revealed that the taxon affiliated with
, heterotrophic anaerobic bacteria that typically occur in organic-rich anoxic subseafloor sediments, were commonly found not only in SMV sediments but also in the overlying seawater. We designed a new oligonucleotide probe for detecting
using the catalyzed reporter deposition-fluorescence
hybridization (CARD-FISH). CARD-FISH, digital PCR and sequencing analysis of 16S rRNA genes consistently showed that
are abundant in the methane plumes of the two SMVs (0.58 and 1.5 × 10
cells/mL, respectively) but not in surrounding waters, suggesting that microbial cells in subseafloor sediments are dispersed as "deep-biosphere seeds" into the ocean. These findings may have important implications for the microbial transmigration between the deep subseafloor biosphere and the hydrosphere.
It is necessary to purify diatom frustules by taxon to perform accurate geochemical analyses of diatom fossils preserved in sediments. However, the small size of diatoms has hitherto prevented ...taxon-specific purification; therefore, previous geochemical analyses of diatom frustules have been performed with mixtures of various taxa. In this study, we developed a taxon-selective collection method of diatom fossils that uses a cell sorter. The experimental material comprised six samples from a sediment core of Hole U1538A in the Scotia Sea, Atlantic sector of the Southern Ocean drilled during the International Ocean Discovery Program Expedition 382. Following conventional pretreatments (removal of organic matter and carbonates as well as clay minerals), we conducted cell sorter experiments focusing on the optical and fluorescence characteristics of the diatom fossils. We succeeded in selectively isolating with high purity five diatom taxa representing the Southern Ocean diatom flora: (1) discoid diatoms (mainly
Thalassiosira
) with a moderate degree of fluorescence; (2)
Fragilariopsis
(mainly
Fragilariopsis kerguelensis
), which exhibits the highest fluorescence values; (3)
Rhizosolenia
; (4)
Eucampia antarctica
; and (5) needle-shaped diatoms (
Thalassiothrix
). This taxon-specific diatom purification method will enable more accurate geochemical analyses, such as the oxygen isotope ratio (δ
18
O) of diatom frustules, which is likely to lead to significant advances in paleoceanography, especially at high latitudes or in upwelling zones where diatoms are abundant. This method will also be useful in paleolimnology, ocean biology, and phycology.
The Earth’s microbial biosphere extends from ambient to extreme environments, including deep-sea hydrothermal vents and subseafloor habitats. Despite efforts to understand the physiological ...adaptations of these microbes, our knowledge is limited due to the technological challenges associated with reproducing in situ high temperature (HT)-high hydrostatic pressure (HHP) conditions and sampling HT-HHP cultures. In the present study, we developed a new high temperature and pressure (HTP) incubation system that enabled the maintenance of HT-HHP conditions while sampling incubation medium and mostly eliminated non-biological reactions, including hydrogen generation or the leakage of small gaseous molecules. The main characteristics of our system are (1) a chamber made of gold with gold-etched lid parts that suppress the majority of non-biological reactions, (2) the exceptional containment of dissolved gas, even small molecules, such as hydrogen, and (3) the sampling capacity of intra-chamber liquid without depressurization and the isobaric transfer of a culture to inoculate new medium. We initially confirmed the retention of dissolved hydrogen in the incubation container at 82°C and 20 MPa for 9 days. Cultivation tests with an obligate hyperthermophilic piezophile (Pyrococcus yayanosii), hydrogenotrophic hyperthermophile (Archaeoglobus profundus), and heterotrophic hyperthermophile (Pyrococcus horikoshii) were successful based on growth monitoring and chemical analyses. During HTP cultivation, we observed a difference in the duration of the lag phase of P. horikoshii, which indicated the potential effect of a pressure change on the physiology of piezophiles. The present results suggest the importance of a cultivation system designed and developed explicitly for HTP conditions with the capacity for sampling without depressurization of the entire system.
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