The Integrated Ocean Drilling Program (IODP) Expedition
337 was the first expedition dedicated to subseafloor microbiology that used
riser-drilling technology with the drilling vessel
Subseafloor sediments harbor a remarkable number of microbial cells, with
its concentrations decreasing logarithmically with increasing burial depth
(Parkes et al., 1994; D'Hondt et al., 2004; Lipp et al., 2008; Kallmeyer et
al., 2012). Active microbial populations exist at great depths below the
ocean floor, which can only be explored by scientific ocean drilling. For
example, the Integrated Ocean Drilling Program (IODP) Expedition 317 found microbial cells in sediments of the
Canterbury Basin, off the coast of New Zealand, down to 1922 meters below
the seafloor (m b.s.f.) (Site U1352, 344 m water depth; Ciobanu et al., 2014).
However, the extent of the deep biosphere and factors limiting life at its
lower boundaries remain largely unknown. For similar reasons, microbial
ecosystems in marine subsurface hydrocarbon reservoirs on the continental
margin are among the least characterized systems on Earth. In fact, our
knowledge base of the interactions of the deep biosphere with offshore
hydrocarbon reservoirs has long suffered from the highly limited
opportunities to target natural gas and oil fields with scientific ocean
drilling initiatives. Despite the biogeochemical significance of hydrocarbon
reservoirs for the global carbon cycle (Head et al., 2003; Jones et al.,
2008), there have been no studies of coal layers that are deeply buried in
the subseafloor, mainly because of the safety regulations related to
hydrocarbon gas-related hazards during non-riser-type drilling. In
continental sediments, large quantities of gaseous hydrocarbons and their
derivatives (e.g., H
The drilling Site C0020 is located in a forearc basin formed by the
subduction of the Pacific Plate (
Bathymetric map of IODP Site C0020 (JAMSTEC Site C9001) and existing drill holes off the Shimokita Peninsula, Japan. Inset map shows plate configuration around Japanese islands and the location of the index map (gray square). This figure is slightly modified from Fig. S1 in Inagaki et al. (2015), and reprinted with permission from American Association for the Advancement of Science (AAAS).
The Hidaka Trough, a sedimentary basin formed by subsidence in the drilling
area, originates just offshore southwest of Hokkaido and extends to the
Japan Trench. Along the coastal area of the Shimokita Peninsula, both
sedimentary and volcanic rocks younger than Late Cretaceous lie scattered on
Triassic to Early Cretaceous sedimentary rocks or Cretaceous granites.
Several scientific drilling expeditions have been carried out off the
Shimokita Peninsula: Deep Sea Drilling Program (DSDP) Legs 56 and 57 in
1977, Leg 87 in 1982, and Ocean Drilling Program (ODP) Leg 186 in 1999. In
addition, well data are available from hydrocarbon drilling exploration
carried out between 1977 and 1999 (Japan Natural Gas Association and Japan
Offshore Petroleum Development Association, 1992; Osawa et al., 2002).
Seismic profiles around Site C0020 show pull-up blanking reflections below
bottom-simulating reflectors at around 360 m b.s.f., suggesting the
occurrence of methane hydrates in shallow sedimentary realms and a strong
upward flux of free hydrocarbon gases from deep reservoirs. A thick and
prominent Quaternary sedimentary unit onlaps to a Pliocene unit and is
thought to be composed mainly of alternating beds of mud and sand with
intercalations of thin volcanic tephras and locally developed gravel/sand
layers. The Pliocene unit consists primarily of alternating beds of mudstone
and sandstone. Below these relatively recent formations, there are
sedimentary deposits ranging from Cretaceous to Miocene in age that are cut
by many landward-dipping normal faults. The presence of coal formations
underneath the seafloor has been confirmed by the natural gas drilling
exploration at nearby site MITI Sanriku-Oki, approximately 50 km southward
from Site C0020 (Fig. 1; see Osawa et al., 2002). Sonic logging data in the
MITI Sanriku-Oki well showed that three major tuff layers involving coal
layers with 30, 45, and 80 m thickness (40–60 % total organic carbon (TOC) in lignite coal
layer and 0.5–2 % TOC in tuffs) are present in Eocene and Pliocene–Upper
Cretaceous horizons, in which vitrinite reflection values (Ro) were in the
range between 0.5 and 0.7 %, indicating relatively immature coals. The
in situ temperatures are well within the range of the habitable zone of microbes,
based on the reported thermal gradient of 22.5
During the
Microbial cell numbers in 365 m sediment from Site C9001 were evaluated by
the fluorescent image-based automated cell count system, showing that the
sediment contained abundant microbial cells with counts over 10
Cultivation of aerobic and anaerobic microorganisms has been conducted at
Site C9001 and a variety of microbes and their enzymatic activities were
observed (Kobayashi et al., 2008). Using a continuous-flow bioreactor system
called down-flow hanging sponge reactor, phylogenetically diverse, strictly
anaerobic microbes were also successfully cultivated, including methanogens
such as the genera
During Expedition 337, we tackled a number of fundamental questions
regarding deep subseafloor hydrocarbon systems. For example:
What role does subsurface microbial activity play in the formation of
hydrocarbon reservoirs? Do deeply buried hydrocarbon reservoirs, such as natural gas formations and
coalbeds, act as geobiological reactors that sustain subsurface life by
releasing nutrients and carbon substrates? Do the conversion and transport of hydrocarbons and other reduced compounds
influence biomass, diversity, activity, and functionality of deep subseafloor
microbial populations? What are the fluxes of both thermogenically and biologically produced
organic compounds and how important are these for the carbon budgets in the
shallower subsurface and the ocean? What paleoenvironmental information and sedimentary regimes are recorded at
Site C0020? What is the extent of the subseafloor biosphere? What environmental factors
define the lower boundaries of the biosphere?
The
While the surface pressure test of the blow out preventer (BOP) was being
carried out, the science party was shuttled on board by helicopter flights on
31 July. Technical problems were found during the function test of the BOP
and troubleshooting continued until 8 August. A successful BOP landing was
confirmed on 11 August. Extensive BOP tests were carried out until
completion of the pressure test on 14 August. Subsequently, riser drilling
started. Due to the delay in operations, planned spot-coring in the interval
647–1220 m b.s.f. was canceled and instead a 17-1/2
Coring operation started on 25 August. While riser drilling continued, spot cores were taken by rotary core barrel (RCB) until core 7R hit a low rate of penetration interval at 1604.0 m b.s.f. The core included gravels of volcanic origin and showed different lithology from previous cores. To retrieve core samples at this depth, an industry-type large diameter core (LDC) was tested. The LDC operation was, however, stopped before reaching the targeted 27 m of drilling advance, because an increase in pump pressure and no further penetration indicated core jamming. The LDC was recovered on deck on 30 August and the recovered core was 10.0 m from 21.5 m of advance. The core was cut into 1.0 m long sections at the middle pipe rack and transferred to the laboratory. Spot coring resumed with RCB and cores 9R to 14R were taken before continuous coring through the coal-bearing horizons started at 1919 m b.s.f. After four consecutive RCB cores were recovered, the drill bit appeared to be worn out. Consequently, it was decided to change the drill bit.
After coring of core 18R, it was also decided that we continue RCB coring and cancel a planned operation of the LDC. The new drill bit was installed and coring operations resumed on 4 September. Another seven consecutive RCB cores were taken from 1950 to 2003.5 m b.s.f. Core recovery was high, and coal-bearing sequences were obtained. With the successful sampling of various lithologies within and around the coal-bearing formation, our operational mission in this interval was fulfilled, and we then advanced the borehole by drilling and spot coring in 100 m intervals to more completely examine the hydrocarbon system and explore the limits of life at greater depths. We reached the terminal depth of Hole C0020A at 2466 m b.s.f. on 9 September, exceeding the previous deepest hole of scientific ocean drilling (i.e., 2111 m b.s.f., Hole 504B off Costa Rica, ODP Leg 148). The core recovery through riser drilling was remarkably high, often close to 100 %, even at great burial depths of 2000 m b.s.f. and deeper.
The condition of the riser borehole was excellent, allowing for close-to-perfect acquisition of downhole wireline logging data. After pulling out of the hole, wireline-logging operation started. Logging run 1 (PEX: platform express) started on 10 September, followed by runs 2 (FMI: formation microimager) and 3 (CMR: combinable magnetic resonance). High-permeability layers were selected for the Modular Formation Dynamics Tester (MDT) based on the results from the first three runs. After a wiper trip, run 4 (MDT-GR: MDT-gamma ray) started on 12 September. Pretests for fluid mobility and formation pressure measurements were carried out at 31 horizons. Formation fluid samples were taken by the Schlumberger's QuickSilver MDT-Probe at six horizons of high mobility, and the six bottles were recovered on deck on 14 September. The sample bottles were delivered to the laboratory during the following run 5 (VSP: vertical seismic profile). The last run was completed on 15 September, ending scientific operation on the rig floor.
Lithologic profile of Hole C0020A based on macroscopic observation of cuttings (middle and right column) and cores (right column) recovered during Expedition 337. Gamma-ray log and positions of spot-cores are also plotted. This figure is slightly modified from Fig. 2 in Gross et al. (2015), and reprinted with permission from Elsevier.
Four distinct lithologic units were identified at Site C0020 on the basis of
combined analysis of cuttings and cores, and assisted by inspection of X-ray
computed tomography (CT) scan images and wireline-logging data; 14 coal layers were confirmed
between 1825 and 2466 m b.s.f. On-board micropaleontology included diatoms,
calcareous nannofossils, organic-walled dinoflagellate cysts (dinocysts),
pollen, and spores, indicating a probable age of late Oligocene to early
Miocene at the base at 2466 m b.s.f. A stratigraphic column of the borehole is
provided in Fig. 2. From top to base, the following units were described
(Inagaki et al., 2012; Gross et al., 2015).
Unit I (647–1256.5 m b.s.f.) consists primarily of diatom-bearing silty
clay. This unit resulted from sedimentation in an offshore marine
environment. Diatoms were best and most abundantly preserved in Unit I among
monitored microfossils together with predominantly heterotrophic dinocysts.
Diatom floras in Unit I are consistent with a Pliocene cool-water
continental shelf succession. Heterotrophic dinocyst communities feeding off
diatom blooms are suggestive of elevated marine productivity. Unit II (1256.5–826.5 m b.s.f.) consists mostly of silty shale with
some interspersed intervals of sandstone and siltstone. Cuttings samples
show a lower amount of sand and an increase of silt at the Unit I/II
boundary. The abundance of biogenic siliceous material, glauconite, and plant
remains additionally differentiate Unit II from the overlying unit. Unit II
was subdivided into two different subunits, sandstone and siltstone
associated with marine fossiliferous material (Subunit IIa; 1256.5–1500 m b.s.f.)
and organic-rich shale and sandstone associated with plant remains
(Subunit IIb; 1500–1826.5 m b.s.f.). The upper part of Unit II represents an
offshore paleoenvironment, possibly close to the shelf margin; with
increasing depth the paleoenvironment gradually changes into a shallow
marine setting. The bottom part of Unit II is situated in the intertidal
zone. This shift is consistent with microfossil assemblages that exhibit few
identifiable diatoms and poor dinocysts; reworked dinocyst in Unit II, as in
deeper units, have Paleogene ages that broadly fall in the range of
early–middle Eocene through to late Oligocene. Pollen and spores are
moderately well represented but are abundant near the base of Unit II,
consistent with an increasing terrestrial influence in shallow marine
sediments. Unit III (1826.5–2046.5 m b.s.f.) is dominated by several coal
horizons, which we subdivided into coaly shales, siltstones, and sandstones.
Almost all coal horizons consist of fine-detritic to xylodetritic coal with
some layers of xylitic coal. Water content, color, and vitrinite reflectance
measurements of the coal suggest that the coal has low maturity.
Bioturbation and sedimentary features like flaser bedding, lenticular
bedding or cross-bedding suggest a near-shore depositional environment with
tidal flats and tidal channels. The presence of siderite bands at the bottom
of this unit suggests a back barrier marine environment in combination with
wetlands (e.g., salt marsh or swamp). Small terrestrial influence (e.g.,
siderite grains) might occur within sand bodies that overlie coal horizons.
This could be due to channels from deltaic environments. Unit III contains
excellently preserved pollen and spore assemblages in the coals and
associated terrestrial to coastal shallow marine sediments. However,
dinocysts are scarce and contain few useful biostratigraphic markers. The
pollen floras tentatively suggest a likely age of early–middle Miocene for
Unit III. Unit IV (2046.5–2466 m b.s.f.) is dominated by silty shales in the
upper part, sandstone, intercalated with siltstone, and shale associated with
sand in the middle part and with silt and a thin coal layer in the lower
part. Wireline logs and cuttings samples suggest a thick homogeneous shale
layer between the Unit III boundary and core 27R at 2200 m b.s.f. The
depositional environment of Unit IV resembles that of Unit III, except that
the former contains only one thin coalbed layer. Like Unit III, Unit IV
experienced high-frequency fluctuations of the depositional environment.
Within a few meters, there are sediments related to tidal flats and tidal
channels, which are overlain by organic-rich material of a marsh that
resulted in formation of peat. The pollen floras place a maximum age of late
Oligocene for the base of Unit IV.
A series of physical property measurements were performed on core and
cuttings samples from Site C0020. Gamma-ray attenuation density, magnetic
susceptibility, natural gamma radiation,
Distribution and lithological variation of porosity in discrete core samples with comparison of cuttings at Site C0020 (Inagaki et al., 2012; Tanikawa et al., 2016). Note that discrete core samples can be more representative for in situ porosity than porosity of cuttings.
Due to the very good borehole condition, logging data were excellent
quality, resulting in straightforward interpretation of these logging data
with respect to lithology. The logging data generally compensated for the
lack of cores, and ultimately led to the establishment of a database that
fully reconstructs the sedimentation history at Site C0020 (Inagaki et al.,
2012). Borehole temperature was measured with two types of logging tools.
The maximum temperature at the bottom of Hole C0020A was estimated by
examining the temperature build-up pattern during the logging operation. The
estimated temperature gradient was 24.0
Depth profiles of
Expedition 337 was the first riser-drilling expedition to incorporate
extensive on-board microbiological and biogeochemical analyses. All
microbiological samples for the cell count, cultivation, and molecular
studies were obtained from the center of non-disturbed whole-round core
samples after inspection by X-ray CT scan and perfluorocarbon
tracer assays for contamination from drill fluids (see Lever et al., 2006;
Inagaki et al., 2012) immediately after core recovery. The samples were then
processed with special aseptic care in either the microbiology laboratory on
the
Within the lithological setting of the Shimokita coalbed biosphere, where
environmental conditions changed drastically after shallow coastal sediments
subsided below sea-level since the Miocene, we have detected the most deeply
buried microbial communities studied to date (Inagaki et al., 2015).
Microbial cells were detached from sediments by a multi-layer density
gradient technique (Morono et al., 2013), and then their concentration was
determined by both manual and computer-based microscopic image analyses
(Morono et al., 2009, 2013; Morono and Inagaki, 2010). The cell count
analysis revealed that cell concentrations in deep sediments below
A scanning electron micrograph of an anaerobic,
methanogenic microbial community enriched in
By comparison of 16S rRNA gene sequences (i.e., V1–V3 region) from control
samples to those derived from experimental samples, we determined the
taxonomic composition of indigenous bacterial communities. The data
indicated that deep bacterial communities (1278–2458 m b.s.f.) differ profoundly
from communities at shallower depths (0–364 m b.s.f.) (Fig. 6). For example, no
or very low proportions of sequence reads affiliated with the phyla
Chloroflexi or “Atribacteria”, both globally abundant groups in subseafloor
sediments on ocean margins (Inagaki et al., 2003, 2006), were detected in
the deep layers. Instead, the sequence assemblage in deep layers is
represented, in decreasing order of abundance, by the genera
Taxonomic composition of indigenous bacterial
communities based on 16S rRNA gene sequences from
Depth profiles of cell concentrations (black plots), biomolecule damage rates, and in situ temperature at Site C0020. This figure is slightly modified from Fig. S14 in Inagaki et al. (2015), and reprinted with permission from AAAS.
The samples and data from Site C0020 offer the unique opportunity to explore
the lower margins of the habitable zone, i.e., the bottom of the deep
biosphere, and to search for clues regarding physical, chemical, and
bioenergetic factors that limit microbial life below
Taken together, the major goals of the on-board geology, microbiology, and biogeochemistry programs were successfully accomplished, and extensive shore-based studies using samples and data collected during Expedition 337 will significantly expand our knowledge of the deep, dark, and old subseafloor biosphere and contribute to the better understanding of the biogeochemical carbon cycle.
During Expedition 337, our major operational objectives to meet the
scientific goals have been successfully accomplished through use of the
riser-drilling system of the
During Expedition 337, we performed spot coring, instead of conventional
sequential coring strategy, using standard 8 1/2
The use of riser-drilling mud is essential for future deep scientific
explorations. However, we will need to address both microbial and chemical
contamination issues in order to optimize conditions for the examination of
very deeply buried microbial communities and the chemical conditions in
their habitat. For example, the mud used during Expedition 337 contained
about 10
A positive aspect of the deep-riser drilling is the superior borehole stability supported by the use of high-viscosity mud that prevents possible collapse and flow-down of rubbly horizons such as coal and fault layers. This is not only useful for coring materials with high recovery rate, but also essential for successful completion of multiple deployments of logging tools, including downhole in situ fluid sampling and analysis by the QuickSilver MDT-Probe. With the combined use of borehole observatory sensors and subseafloor laboratory equipment, the maintenance of stable deep-riser boreholes will be highly useful for advanced subseafloor research in short- to long-term projects.
Last but not least, our expedition also provided a test ground for the use
of riser-drilling technology to address geobiological and biogeochemical
objectives and was therefore a crucial step toward the next phase of deep
scientific ocean drilling. Since the riser system was originally developed
by the petroleum industry, the
Monika Bihan, Stephen A. Bowden, Marshall Bowles, Marcus Elvert, Clemens Glombitza, Doris Gross, Guy J. Harrington, Verena Heuer, Wei-Li Hong, Tomoyuki Hori, Tatsuhiko Hoshino, Akira Ijiri, Hiroyuki Imachi, Motoo Ito, Masanori Kaneko, Mark A. Lever, Kevin Li, David Limmer, Yu-Shih Lin, Chang-Hong Liu, Barbara A. Methé, Sumito Morita, Yuki Morono, Masafumi Murayama, Naohiko Ohkouchi, Shuhei Ono, Young-Soo Park, Stephen C. Phillips, Xavier Prieto-Mollar, Marcella Purkey, Natascha Riedinger, Yoshinori Sanada, Justine Sauvage, Glen Snyder, Rita Susilawati, Yoshinori Takano, Wataru Tanikawa, Eiji Tasumi, Takeshi Terada, Hitoshi Tomaru, Elizabeth Trembath-Reichert, David T. Wang, and Yasuhiro Yamada.
The authors are grateful to the Integrated Ocean
Drilling Program (IODP) and the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (MEXT) for providing an opportunity to
explore the deep coalbed biosphere off Shimokita during Expedition 337. We
thank all crews, drilling team members, and lab technicians on the drilling
vessel