Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58
https://doi.org/10.1186/s40645-018-0217-2

Progress in Earth andPlanetary Science

RESEARCH ARTICLE

Open Access

Geophysical constraints on microbial biomassin subseafloor sediments and coal seams
down to 2.5 km off Shimokita Peninsula, Japan
Wataru Tanikawa1,2* , Osamu Tadai3, Yuki Morono1,2, Kai-Uwe Hinrichs4 and Fumio Inagaki1,2,5Abstract
To understand the ability of microbial life to inhabit a deep subseafloor coalbed sedimentary basin, the correlation
between fluid transport properties and the abundance of microbial cells was investigated based on core samples
collected down to about 2.5 km below the seafloor during the Integrated Ocean Drilling Program Expedition 337
off the Shimokita Peninsula, Japan. The overall depth profiles for porosity and permeability exhibited a decreasing
trend with increasing depth. However, at depths greater than 1.2 km beneath the seafloor, the transportcharacteristics of the sediments were highly variable, with the permeability ranging from 10−16 to 10−22 m2 and thepore size ranging from < 0.01 to 100 μm. This is mainly attributed to the diversity of the lithology, which exhibits arange of pore sizes and pore geometries. Fracture channels in coal seams had the highest permeability, while shale
deposits had the smallest pore size and lowest permeability. A positive correlation between permeability and pore size
was confirmed by the Kozeny-Carman equation. Cell abundance at shallower depths was positively correlated withporosity and permeability, and was less strongly correlated with pore size. These findings suggest that one of thefactors affecting the decrease in microbial cell abundance with increasing depth was a reduction in nutrient andwater supply to indigenous microbial communities as a result of a decrease in porosity and permeability due tosediment compaction. Anomalous regions with relatively high cell concentrations in coal-bearing units could be
explained by the higher permeability and larger pore size for these units compared to the surrounding
sediments. Nutrient transport through permeable cleats in coal layers might occur upwards toward the upper
permeable sandstone layers, which are well suited for sustaining sizable microbial populations. Conversely,
impermeable shale and siltstone with small pores (< 0.2 μm, which is smaller than microbial cell size) may act asbarriers to water and energy-yielding substrates for deep microbial life. We propose that the pore size andpermeability govern the threshold for microbial habitability in the deep subseafloor sedimentary biosphere.
Keywords: Deep biosphere, Lignite coal, Limits of life, Microbial community, Pore size, Porosity, Permeability,
Habitability

Introduction
Previous scientific ocean drilling investigations have demonstrated that microbial communities are widespread inshallow and deep subseafloor sediments down to depths
of 2.5 km below the ocean floor (Parkes et al. 1994; Lippet al. 2008; Kallmeyer et al. 2012; Inagaki et al. 2015).
These studies of sediment core samples generally showed* Correspondence: tanikawa@jamstec.go.jp
1
Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Scienceand Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan
2
Research and Development (R&D) Center for Submarine Resources, JAMSTEC,
2-15 Natsushima-cho, Yokosuka, Kanagawa 273-0061, Japan
Full list of author information is available at the end of the article

that microbial cell concentrations decrease logarithmicallywith increasing depth (Parkes et al. 1994; Lipp et al. 2008;
Morono et al. 2009; Kallmeyer et al. 2012). The abundance
and activity of microbial communities in deep subseafloor sediments depend on a variety of interrelated factors including the availability of water and nutrients(Abu-Ashour et al. 1994). The spatial variation in geological and geophysical conditions, such as temperature,pressure, pore water chemistry, pH, water activity (Aw),grain and pore geometries, and permeability, is manifested
as spatiotemporal changes in microbial cell abundance,with in situ microbial population size and activity© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

potentially affected by these conditions (Sutherland etal. 1994; Abu-Ashour et al. 1994; Rebata-Landa andSantamarina 2006).
Fluid permeability in sediments, which denotes the efficiency of advective transport of water and nutrients
through porous media along a hydraulic gradient, is also
markedly affected by a variety of interrelated environmental factors, which in turn affect the nature of microbial communities in situ. In addition to water andnutrients, small microbial cells and particulate organic
matter are transported through the more permeable
sandstones (Raiders et al. 1986; Huettel and Gust 1992).
Impermeable layers act as barriers or cap rocks that
limit the transport of fluids and solid materials, including microbial cells (Jørgensen 2000).
Porosity, pore size distribution, and grain size determine
the permeability, and potentially constrain the physical extent of the deep microbial biosphere (Rebata-Landa andSantamarina 2006). Sandy sediments, which are coarser
than fine clay particles, are considered to harbor denser
microbial populations than clay sediments (Phelps et al.
1994; Inagaki et al. 2004). Thus, in the deep sedimentary
biosphere, microbial metabolic activity has been detected in terrestrial sandstone sediments with pore sizesof > 0.2 μm, while no activity was observed in shales
with pore sizes of < 0.2 μm (Fredrickson et al. 1997).
The critical size limit of about 0.2 μm is quite similar
to the lower size limit for typical living cells, whichranges from 0.3 to 0.5 μm in diameter for sphericalcells and < 0.2 μm for rod-shaped cells. Interestingly,
the majority of subseafloor cells are considered to havediameters of 0.25 to 0.7 μm (Kallmeyer et al. 2012).
Microbial cells cannot be found in micropore spaces
smaller than the cells themselves; indeed, a previous study
suggested that the pores in sediments need to be approximately twice the size of a cell in order to facilitate thetransport of microbial cells in subsurface environments(Updegraff 1983). Smaller cells are consequently better
adapted to life in narrow pore spaces in the deep sedimentary biosphere (0.1 to 0.2 μm; Schulz and Jørgensen
2001). Thus, pore size is one of the major factors affecting
microbial cell abundance and microbial activity in deep
shale-sandstone sedimentary environments.
Expedition 337 of the Integrated Ocean Drilling
Program (IODP) was conducted by the riser drilling vessel Chikyu at Site C0020 near the Western Pacificmargin off Shimokita Peninsula, Japan. The primaryscientific objective of Expedition 337 was to investigate
deeply buried microbial communities in lignite coalbearing sediments down to a depth of 2466 m below theseafloor (mbsf ), and to characterize their biogeochemicalrole in carbon cycling (Inagaki et al. 2012, 2015). The
microbial cell abundance decreased sharply in deep sediment layers below 1200 mbsf, with most sediment

Page 2 of 17

samples containing a maximum of several hundred microbial cells per cubic centimeter (Inagaki et al. 2015).
However, a larger microbial biomass was observed in lignite coal layers. Physical and chemical characteristics ofthe cored sediment samples were determined at onboard
and offshore laboratories (Gross et al. 2015; Glombitza
et al. 2016; Tanikawa et al. 2016; Ijiri et al. 2017;
Trembath-Reichert et al. 2017), but the key factors responsible for constraining the vertical distribution of the
microbial populations in the deep sedimentary biospherehave not yet been well clarified (Hinrichs and Inagaki2012). While permeability and pore characteristics potentially govern not only microbial biomass but also thereservoir capacity of microbially produced coalbed methane (Gamson et al. 1993; Clarkson and Bustin 1996;Strąpoć et al. 2011), the transport dynamics associatedwith these characteristics have not yet been reported.
In this study, to determine the key physical properties
that influence the habitability for microbial life of deep
subseafloor environments, we present the permeability
and pore size distribution of core samples obtained fromthe coal-bearing sedimentary basin off Shimokita
Peninsula, Japan (Sanriku-oki subbasin), obtained during
IODP Expedition 337. The variations in the permeability
and pore characteristics of sediments were investigatedto clarify whether the decreasing trend in microbial cellabundance with increasing sediment depth could be
explained, at least in part, by depth-dependent physicalproperties (Rebata-Landa and Santamarina 2006). The
relationship among permeability, lithology, porosity, andpore size distribution in the sediment core samples was
examined in order to clarify the factors that affect fluid
transport and microbial cell abundance in the approximately 2.5-km-deep subseafloor sedimentary biosphere.

Methods/Experimental
Core materials

Riser drilling site C0020 off the Shimokita Peninsula,
Japan, is located in a sedimentary forearc basin off theSanriku Province that developed along the Ishikari-Sanriku-oki forearc zone, which extends along the N-S trending
narrow zone from southern Hokkaido to off Kuji in Iwate
Prefecture (Fig. 1, Takano et al. 2013). Gas exploration
drilling by the Ministry of International Trade and Industry of Japan (MITI) confirmed the presence of an exploitable amount of natural gas in Cretaceous to Eocene coalformations in the Sanriku-oki subbasin (Fig. 1b, Osawa et
al. 2002). Scientific ocean drilling was conducted near the
MITI sites in the Sanriku-oki basin approximately 80 km
offshore from Hachinohe City during Chikyu shakedown
cruise CK06-06 (Expedition 902) in 2006 (Aoike 2007)
and IODP Expedition 337 in 2012 (Inagaki et al. 2012).
Using a non-riser hydraulic piston coring system, the initial shakedown cruise CK06-06 recovered shallow

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

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Fig. 1 a Location of IODP Expedition 337 Site C0020A (the same site as Chikyu shakedown cruise CK06-06) (Modified after Inagaki et al. 2012). b A
close-up map showing the distribution of the Ishikari-Sanriku-oki forearc basins, the Sanriku-oki subbasin, and the location of the Ministry of International
Trade and Industry of Japan (MITI) Sanriku-oki well (Modified after Takano et al. 2012)sediment core samples from the seafloor down to 365
mbsf in Hole C9001C at Site C9001 (41° 10.5983′ N, 142°
12.0328′ E, water depth 1180 m), and then established ariser pilot hole (Hole C9001D) down to 647 mbsf (Aoike
2007; Masui et al. 2008). IODP Expedition 337 drilled
deeper in the same riser hole (the C9001D riser hole
was designated IODP Hole C0020A, and the site namewas changed from Site C9001 to Site C0020). During Expedition 337, deep-sediment core samples were retrieved
from 1276.5 to 2466 mbsf with riser drilling, and well logging data were acquired at the same depth interval(Fig. 2a).
Shallow sediment core samples from 0 to 340 mbsf
(units A and B in Hole C9001C) obtained during cruise
CK06-06 were composed mainly of unconsolidated
olive-black or dark olive-gray diatomaceous silty clays,
containing quartz, plagioclase, and clay minerals (Aoike

2007). Gas hydrates were observed patchily in volcanic
ash and sandy sediments from ~ 190 to 360 mbsf
(Tomaru et al. 2009). During Expedition 337, a variety oflithologies was observed in core samples collected fromthe deeper horizons (e.g., coal, shale, siltstone, sandstone, beach sand, carbonate cemented sandstone, andconglomerates) (Fig. 2a, Inagaki et al. 2012). Unit I, a
Pliocene unit at 647 to 1256.5 mbsf, primarily comprised
diatom-bearing silty clay. Unit II, a Miocene unit at
1256.5 to 1826.5 mbsf, mainly comprised silty shales intercalated with sandstone and siltstone layers. Unit III,an early to middle Miocene unit at 1826.5 to 2046 mbsf,
comprised coarse- to fine-grained clastic deposits with
13 lignite coal layers (Inagaki et al. 2012; Gross et al.
2015). The coal seams in unit III were typically approximately 1 m thick, with the exception of two layers thatare 3.5 and 7.3 m thick. Unit IV, an early Miocene to lateTanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

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Fig. 2 a Core lithology, biostratigraphy, and lithological units at Site C0020 identified on Chikyu shakedown cruise CK06-06 and IODP Expedition
337, b cell concentration, c thermal conductivity, d predicted temperature profile, and e water activity at Site C0020. Figures are modified after Aoike(2007), Inagaki et al. (2012, 2015), and Tanikawa et al. (2018). Raw cell concentrations plotted in (b) were obtained from the microscopic counts reported by
Inagaki et al. (2015)

Oligocene unit at 2046.5 to 2466 mbsf, was composed ofsilty shale, and sandstone intercalated with siltstone andshale. A single 0.9 m thick coal seam was observed at2449 mbsf near the base of the unit (Inagaki et al. 2012).
The total organic carbon (TOC) in coal and coaly
shale (40–76 wt.%) was significantly higher than that inshale (0.43 ± 0.29 wt.%), sandstone (0.26 ± 0.18 wt.%),and shallow clay-rich silty sediments (1.2 ± 044 wt.%, 0
to 363 mbsf ) (Aoike 2007; Inagaki et al. 2012; Gross et al.
2015). Concentrations of CO and total nitrogen dissolvedin a sediment core and measured by the extraction
method were higher in the coal-bearing units (1900 to2000 mbsf, and ~ 2450 mbsf) than those in the adjacentsediments (Expedition 337 Scientists 2013). The depth
profile for the C1/C2 hydrocarbon gas ratio estimated by
mud-gas monitoring suggested the enhancement of microbial methane in the coal-bearing units (Inagaki et al. 2012).
Microbial cell concentrations at Site C0020 measured
during CK06-06 (Morono et al. 2009) and Expedition 337(Inagaki et al. 2015) showed that, at depths shallower than
365 mbsf, cell concentrations were considerably high(Morono et al. 2009), whereas cell concentrations in deeper layers decreased sharply below the expected global regression line (Fig. 2b; cf. Parkes et al. 2000; Inagaki et al.
2015). Nevertheless, lignite coal samples from two lithological units (unit III and the bottom of unit IV) showed

cell concentrations that were two to four orders of magnitude higher than those in the adjacent sediments (Inagaki
et al. 2015).
The thermal conductivity increased gradually withdepth, and this trend was explained by the reduction inporosity associated with sediment compaction during
burial (Fig. 2c, Tanikawa et al. 2016). The scatter in thermal properties observed in the same horizontal section
is most likely due to the high variation in the lithology
at Site C0020. A temperature-depth profile at Site
C0020 indicated that the temperature at the bottom of
the hole was 63.7 °C (Fig. 2d; Tanikawa et al. 2016).
The water activity (Aw), a measure of how efficiently
pore water facilitates biological (i.e., enzymatic) reactions,is defined as the ratio of the vapor pressure of core samples to that of pure water (Grant 2004). The water activity
for the sediment core samples at Site C0020, measured
using a commercial Aw sensor, ranged from 0.954 to 0.983(Tanikawa et al. 2018) and showed no clear correlation
with depth or cell concentration in the sediment sample(Fig. 2e). The Aw values in coal layers (0.977 to 0.979) wereslightly lower than those in shallow sediments, but tended
to be higher than those in sandstone and siltstone layers.
The sediment core samples used in this study were obtained from Hole C0020A during IODP Expedition 337
and from Hole C9001C during cruise CK06-06. We

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

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selected core interiors for analysis to eliminate the likelihood of contamination by drilling mud from the core exteriors (Inagaki et al. 2012; Expedition 337 Scientists 2013).
Analytical methods

All of the physical properties of the core samples were
measured at the Kochi Core Center.
Pore size distribution

The pore size distribution for core samples was evaluatedby mercury injection capillary pressure (MICP) measurements at room temperature (25 °C) using a mercury porosimetry analyzer (PoreMaster 60 GT, Quantachrome
Instruments, FL). To measure the wide range of pore sizes
in sediment core samples with high accuracy, a PoreMaster
60GT with low- and high-pressure stations and dual
high-pressure transducers was used. Briefly, the mercury intrusion pressure ranged from 7 to 350 kPa and from140 kPa to 206 MPa (420 MPa for several core samples) forthe low- and high-pressure stations, respectively. These
pressures were used to measure pore diameters in therange of about 0.004 to 1000 μm. Before measuring thepore size distribution, sediment core samples were dried atroom temperature for 72 h and then vacuum-dried for
1 week. An assemblage of two to five fragments that gave
total grain matrix volumes of 0.12 to 0.68 cm3 (i.e., 0.3 to
1.8 g) was used for each measurement.
The pore size into which mercury intrudes in coresamples is given by the Washburn equation:
Pm  ðd=2Þ ¼ −2γcosθ;

ð1Þ

where Pm is the injected mercury pressure, d is the pore
diameter, γ is the surface tension of mercury, and θ isthe contact angle between the mercury and the sample.
To evaluate the pore size distribution from Eq. (1), we
selected 480 erg/cm2 (1 erg/cm2 = 10−3 N·m/m2) as thesurface tension and a contact angle of 140° (León y León
1998). The motor speed for pressure generation was set
to 5 (the motor speed of the PoreMaster 60 GT system
is variable from 1 [slowest] to 25 [fastest]) in fixed ratemode, which corresponds to a generation speed ofaround 37 MPa/min, for high-pressure analysis. For
low-pressure analysis, we were unable to control the injection pressure speed, which is around 40 kPa/min. By
assuming that the intrusion volume is equivalent to thepore volume in sediments, the mercury intrusion porosity was estimated using mercury injection capillary pressure data (Filomena et al. 2012). The accuracy of thepressure transducer was ± 0.11%, and the volume change
resolution was ± 0.0001 cm3, which corresponds to apore-size error of ± 0.1% and a mercury intrusion porosity error of 0.01 to ± 0.2%.

Permeability measurements

The changes in permeability and porosity under high effective pressure (= confining pressure – pore pressure)
were measured at room temperature (20 to 25 °C) using
a high-pressure apparatus (Tanikawa et al. 2013). High
confining pressure and pore fluid pressure were applied
to sediment core samples to reproduce the in-situ effective pressure conditions and to estimate in-situ physicalproperties. All of the test samples, except for the coalsamples, were cut and shaped into cylindrical forms witha length of 6 to 20 mm for permeability measurements.
The cylindrical samples had faces measuring 20 mm indiameter. Since coal is very fragile, the coal samples were
cut into cuboids using a small diamond cutting saw tomeasure the permeability. The faces of the cuboid samples measured 15 or 20 mm; i.e., the upper (inflow) andlower (outflow) faces were 15 × 15 mm or 20 × 20 mm.
A constant differential pore-pressure method was applied to estimate the permeability (Bernabe 1987). The following equation was used to calculate the permeability, k: Qη ∂P −1
QηL
;
k¼
¼ 
A L
A P up −Pdown

ð2Þ

where Q is the volume of fluid drained from the sample, A
is the cross-sectional area of the sample, η is the fluid viscosity, δP/L is the pore pressure gradient in the core samples, which is determined by the pore pressure at the upper
end, Pup, and the lower end, Pdown, of the specimen, and L
is the length of the specimen. The inlet pore pressure wasvaried from 1 to 2 MPa, and the outlet pore pressure was
kept constant at 1 MPa. The confining pressure and outlet
pore pressure were kept constant by using an ISCO syringe
pump (100DX, Teledyne ISCO Inc., Lincoln, NE), and theinlet pore pressure was controlled using a fluid/gas regulator. The same differential pore pressure (i.e., from 0.05 to
1 MPa) was applied until the flow rate, which was calculated from the change in the storage volume of the pump,
became constant. The upper and lower limits for the flowrate were 10 ml/min and 5 × 10− 5 ml/min, respectively,
which is equivalent to a permeability of 10−13 to 10−20 m2
for the specimen sizes in the present study. The accuracy ofthe pore pressure data was ± 0.001 MPa, which corresponds to ± 2% of the maximum standard error for thepermeability (the standard error for the confining pressure
was ± 0.007 MPa). Pure water (Elix, Merck Millipore
Corporation, Germany) was used for the pore fluid in thepermeability and porosity measurements. The confiningpressure was increased from 2 to 31 MPa in steps ranging
in size from 1 to 10 MPa.
The pore fluid was drained from the samples to the pore
fluid flow line during a stepped increase in confining pressure, and the drainage of pore fluid corresponded to the
reduction in porosity. Therefore, to measure the porosity

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

change induced by the change in the confining pressure,the drained fluid volume was measured using the ISCO
syringe pump by keeping the pore pressure constant at
1 MPa under undrained conditions (Wibberley 2002). The
absolute porosity value was calculated from the change inthe pore volume and the initial pore and grain matrix volumes at atmospheric pressure. The initial pore volume
was calculated from the wet and dry weights of the sediment samples (the latter obtained following drying in anoven at 105 °C for 24 h), and the grain matrix volume wasmeasured using a commercial helium gas pycnometer(Penta Pycnometer, Quantachrome Instruments, FL). The
grain matrix volume was measured five times and theaverage value was used to calculate the initial porosity.
The standard deviation of the grain matrix volume was ±0.004 cm3, which is equivalent to ± 0.3% of the standard
errors for the grain matrix volume and porosity.
Microstructural observations

Microstructural observations of four dried samples
after the permeability measurements were performed

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using a micro-focus X-ray computed tomography(μX-CT) system (HMX225-ACTISt3, Tesco Co. Ltd.,Tokyo, Japan). The voltage and current used for X-ray
irradiation were 90 kV and 30 μA, respectively. The
resolution of the images was 1024 pixels × 1024 pixels
in a 10 mm2 area (1 pixel = 10 μm). ImageJ software(https://imagej.nih.gov/ij/) was used to reconstruct thesliced image of the sample from the Digital Imaging andCommunications in Medicine (DICOM) format data.

Results
Representative pore size distribution data

Figure 3 shows six examples of the relationship between
the pore size and pore volume fraction obtained using thelogarithmic pore volume distribution normalized by the
total pore volume. The mean, modal, and median poresize for each treatment are given in the same figure. For
unconsolidated silty clay at a shallow depth (Core 20H4,
177.7 mbsf; Fig. 3a), the unimodal pore size distribution
was characterized by a single peak in the volumetric fraction at a pore size of around 0.8 μm. The results obtained

Fig. 3 Examples of pore size distribution for core samples at Site C0020. Mean (a), modal (b), and median (c) pore size distributions are plotted in
the same figures: a silty clay at 177.7 mbsf, b sandstone at 1490.8 mbsf, c shale at 1610.8 mbsf, d coal at 1920 mbsf, e well-cemented sandstone
at 1975.8 mbsf, and f siltstone at 2201.3 mbsf

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

for the median pore size were similar to those obtained
for the modal size, although the mean pore size wassmaller than the modal size. The pore size for sandstone(Core 5R1, 1490.8 mbsf; Fig. 3b) shows a bimodal distribution characterized by two major peaks at around 2 and20 μm, with the latter being sharper and more intense.
This suggests that the larger mode corresponds to porespaces between coarser sand grains, while the smaller
mode represents those between matrix clays. The peak obtained for shale (Core 8L8, 1610.8 m; Fig. 3c) is very sharp,
indicating that the pore sizes are distributed within a very
narrow range centered at 0.005 μm. Both the highest andsecond highest volumetric fractions for the coal sample(Core 15R3, 1920.9 m; Fig. 3d) were near the highest andlowest measureable pore sizes for the experimental system. This suggests that coal has a bimodal pore size distribution with peaks at < 0.004 μm and > 100 μm. The poresize for silty clay was lower than that for sandstone, butlarger than that for siltstone and shale.

Pressure dependence of transport properties

The permeability of sedimentary rock samples at an initially applied effective pressure of 1 to 3 MPa (defined as
the initial permeability) varied from 10−13 to 10−19 m2,
and decreased with effective pressure in all specimens(Fig. 4a). The initial permeability of coal was the highest
among the tested specimens (> 10−13 m2). At an effective

Page 7 of 17pressure of 20 MPa, the coal permeability decreased by
two to three orders of magnitude from the initial permeability, while the permeability of sandstone and siltstone
at an effective pressure of 20 MPa decreased by one orderof magnitude. At a high effective pressure (> 20 MPa), thepermeability of coal (Cores 15R3 and 30R2) was lower
than that of sandstone. Among the other lithologies
examined, shale showed the lowest permeability at any effective pressure, reaching the lower measurable limit(about 10−20 m2) at an effective pressure of 10 to 20 MPa.
The reduction rate for permeability with increasing effective pressure varied with lithology, and coal exhibited thehighest pressure sensitivity. The reduction curve for coal
permeability (Core 30R2) as a function of effective pressure could be expressed by an exponential decay function,
while the permeability-effective pressure correlation forsandstone, siltstone, and silty clay could be fitted using a
power-law curve with an exponent of around − 1 (Fig. 4a).
The porosity decreased from the initial value at atmospheric pressure (defined as the initial porosity in thispaper) by 5 to 12% at an effective pressure of 20 MPa(Fig. 4b). In all samples, the porosity reduction rate decreased with increasing effective pressure, and the reduction curves could be fitted using logarithmic andexponential curves. Among the specimens tested, the reduction rate for porosity was the highest for the silty
clay samples (Core 20H4, 8.5% reduction at an effective
pressure of 5 MPa), and the lowest for carbonate

Fig. 4 Effective pressure dependence of a permeability and b porosity of core samples at Site C0020. The fits estimated by the least squares method
and errors are also shown. Pe: effective pressure (MPa), k: permeability (m2), Φ: porosity, and R2: coefficient of determination for least squares fit

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

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cemented sandstone (Core 22R2, 1.2% reduction at
5 MPa) and shale (Core 8L8, 1.5% reduction at 5 MPa).
Depth profile of physical properties

The experimental results showed that the permeability
for all specimens decreased with increasing effective
pressure (Fig. 4a). Therefore, the laboratory results forthe permeability-effective pressure profiles were used toestimate the depth profiles for the in-situ permeability(defined as the permeability at an effective pressure thatis equivalent to the depth of the core samples in thisstudy) at Site C0020. According to Wangen (1997), thevertical effective pressure (Pe) at a depth z = h below theseafloor under hydrostatic pressure is defined as
Z hZ hρb gδz−ρ f gδz
0
Z0 hh
Z h
i
¼
ð1−ΦðzÞÞρr þ ΦðzÞρ f gδz−
ρ f gδz;

Pe ¼

0

ð3Þ

0

where Φ(z) is the porosity at depth z, ρb is the wet bulk
density, ρr is the grain density, ρf is the fluid density, andg is the gravitational acceleration. The porosity-depth relationship at this site was reported in a previous study(Tanikawa et al. 2016), and is characterized by a negative
exponential curve:Φ ¼ Φ0 exp ð−αzÞ;ð4Þwhere Φ0 is the porosity at the seafloor (z = 0 mbsf ) and
α is a consolidation factor that is evaluated empirically.
Values of Φ0 = 0.740 and α = 0.000456 m−1 fit theporosity-depth curve at this site well (Fig. 5a). To

simplify the model, pore fluid pressure was assumed ashydrostatic pressure, and the grain density and fluid
density were assumed to be constant at 2650 kg/m3 and1000 kg/m3, respectively. Using the assumptions above,a 2 km depth off Shimokita corresponds to an effective pressure of 16.7 MPa. The in-situ porosity (defined as the porosity at an effective pressure that is equivalent to the depth ofthe core samples under hydrostatic pressure in this study)
was also estimated from the pressure-porosity relationshipdetermined in the laboratory, which is the same methodology as that employed for the permeability estimation.
The in-situ porosity results obtained in this study generally agreed well with measurements performed at atmospheric pressure onboard, as well as the logging data
calculated using the gamma-ray bulk density data fromthe three-detector lithology density (TLD) logging tool(Fig. 5a; Inagaki et al. 2012). At larger depths, the in-situ
porosity is much closer to the logging data than the onboard porosity data.
The in-situ permeability of silty clay at shallow horizons
was approximately 10−16 m2 (Fig. 5b), implying that thein-situ permeability decreases with increasing depth, although the data are highly scattered around coal-bearing
layers (unit III and lower portion of unit IV). In unit III,the in-situ permeability of sandstone and coal was 4 to 5
orders of magnitude larger than that of siltstone and shale.
The lowest in-situ permeability of < 10−21 m2 was observed in shale at around 2100 mbsf. Coal materials ataround 1900 mbsf exhibited the highest in-situ permeability of 9 × 10−15 m2, but relatively low in-situ permeability
values were also obtained for other coal samples(10−19 m2) in the same unit III (Fig. 5b; Ijiri et al. 2017).

Fig. 5 Depth profiles for a in-situ porosity, b in-situ permeability, and c modal pore size distribution. The legend shows the lithology of the coresamples. Logging data and porosity data are modified from Inagaki et al. (2012). In-situ porosity and in-situ permeability values were estimatedbased on the relationship between porosity/permeability and effective pressure, as shown in Fig. 4. The initial permeability plotted in (b)is the measured value at low effective pressure (1 to 3 MPa). The modal pore size was obtained from MICPT measurements under
unconfined conditions

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

Since the pore size distribution was measured under
unconfined conditions, the depth profile of pore sizethat we determined in the present study tended to yieldvalues that were larger than in-situ values. The depth
profile for the pore size shows that the pore size ishighly scattered due to lithological variation; for example, the pore size for sandstone, siltstone, and coallayers ranges from 3 to 30 μm, 0.03 to 0.1 μm, and 100
to 200 μm, respectively (Fig. 5c). No pore size dependence on depth was observed for any of these lithologies.
The smallest pore size of around 0.01 μm was observed
for shale at 1600 mbsf, while the largest pore size wasobserved for coal samples at 1900 mbsf, which is in good
agreement with the horizons at which high in-situ permeabilities were observed.
Relationship between cell abundance and physical properties

The correlation between cell abundance and in-situ physical properties is plotted in Fig. 6, using the cell concentration data obtained from microscopic direct counts(Morono et al. 2009; Inagaki et al. 2015). The depth profile
for cell concentration at Site C0020 was compiled using
data from core samples collected at depths closest to thoseused for our laboratory experiments.
Despite not having sufficient data for in-situ porosities
in the range of 0.45 to 0.6, the cell concentration appeared
to decrease with decreasing in-situ porosity (Fig. 6a). The

Page 9 of 17

correlation between the decrease in in-situ porosity andthe logarithmic decrease in cell concentration with depth
could be well described by an exponential decay function,
which is similar to the function employed for theporosity-depth reduction curve described by Eq. (4).
However, in the low in-situ porosity range (< ~ 0.4), noclear influence of in-situ porosity on cell concentration in
our dataset was observed.
In-situ permeabilities above 10−17 m2 were correlatedwith higher cell concentrations (> 104 cell/cm3), and cellabundance decreased markedly for in-situ permeabilitiesof less than 10−17 m2 (Fig. 6b). This relationship was welldescribed by a power-law function and/or an exponential
decay function. However, no clear dependence of thein-situ permeability on the cell concentration was observed for the low-permeability samples. In these samples,the correlation between cell density and pore size wasmore complicated, and no well-defined relationship wasobserved (Additional file 1: Figure S1).
Relationship among initial porosity, initial permeability,and pore size

No obvious correlation was observed between the initialporosity and the initial permeability (Fig. 7a). However,
the effect of the lithology on the cross-plot data is apparent; e.g., coal has a very high initial permeability, which is
independent of the initial porosity, and the dependence ofFig. 6 Relationship between cell density and in-situ physical properties and their semi-empirical curves: a in-situ porosity and b in-situ permeability.
Cell concentrations at depths closest to the depths where physical properties were measured (0.79 ± 0.68 m and 0.89 ± 0.68 m apart for (a) and (b),respectively). Gray shading indicates cell concentrations below the minimum quantification limit. Fits estimated by the least squares method using
in-situ values and the errors are also shown. The initial porosity data is modified from Inagaki et al. (2012). Φ: porosity, ρcell: cell density (cells/cm3), andk: permeability (m2)Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

Page 10 of 17

Fig. 7 a Initial porosity vs. initial permeability for the core samples used for the permeability tests. b Initial permeability plotted against the modal
pore size distribution. Dashed lines show regression curves. k: permeability (m2), Φ: porosity, and d: pore size (μm)

the initial permeability on the initial porosity is very weak
for sandstone. Silty clay has a high initial porosity and a
relatively low initial permeability, and the initial permeabilities of siltstone and shale show a slight initial porosity
dependence. The fitting curves based on the integrated
initial porosity data for silty clay, siltstone, and shale—allof which are classified as fine-grained materials—are described by a power-law function with an exponent of ~ 3.
The initial permeability was found to decrease with decreasing modal pore size (Fig. 7b). This relationship waswell fit by a power-law function for all lithologies. The
coal group has a large pore size and a high initial permeability, while shale has a small pore size and a moderately
low initial permeability. The relationship between the median pore size and the initial permeability is similar to thatfor the modal pore size, whereas the mean pore size is lessdependent on the initial permeability (Fig. 7b).

The pore size for the sandstone in Fig. 8b is the largest
among the four samples. The siltstone (Core 27R2) andshale (Core 8L8) exhibit smaller pore sizes, and the poresare more homogenously distributed. The μX-CT analysis
revealed that the coal sample (Core 15R3; Fig. 8) has open
parallel cracks, or cleats (see Laubach et al. 1998 for naturally occurring fractures). Most cleats developed in the
direction parallel to the horizontal bedding plane, but asmall number also formed in the vertical direction, and in
so doing, connected fractures oriented parallel to thebedding itself (Fig. 8a). The aperture size of the cleats
ranged from < 10 to 100 μm. Isolated pores in the coalmatrix were very small, and the μX-CT images showedthat the average density of vertical fractures was around 1
fracture/mm.

Discussion

Microstructure

Which physical factors affect depth-dependence of microbial
cell abundance?

Microstructural observations by μX-CT showed that sandstone (Core 5R1; Fig. 8) consists of a mixture of coarse
particles (0.1 to 0.2 mm in diameter) and very fine particles that fill some of the pores between the larger grains.
This leads to the bimodal pore-size distribution seen in
Fig. 3b, with a broad peak at about 2 μm and a sharperand more intense peak at about 20 μm. This suggests thatthe larger mode corresponds to pore spaces between
coarser sand grains, while the smaller mode represents
those between matrix clays.

Both the porosity and the permeability decrease with increasing effective pressure (Fig. 4), and the correlation between the initial porosity and the initial permeability forfine-grained sediments is well defined (Fig. 7a). The increasing compaction of sediments with depth results in
reduced porosity and permeability, which may correspondto lower tortuosity and lower pore connectivity. Increasing
compaction may thus constrain the habitability of deep
sediments for microbial communities (Prasad 2003;
Rebata-Landa and Santamarina 2006). Compaction is also

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

Page 11 of 17

Fig. 8 μX-CT image of four core samples used for permeability tests. a Original images sliced in the vertical direction relative to the flow direction. The
sliced images for Cores 5R1, 27R2, and 8L8 are parallel to the bedding, and the image for Core 15R3 is perpendicular to the bedding. b Modified sliced
images of the cut section, shown as a broken line in (a), parallel to the flow direction. c Binary images of (b), adjusted based on initial porosity data

likely to influence the transport of water, nutrients, andsecondary products of microbiological activity (Yakimov
et al. 1997). Therefore, the positive correlation between
cell concentration and in-situ porosity observed at Site
C0020 suggests that mechanical sediment compaction
was responsible for the low density of microbes at this site
(Figs. 2a and 6a). Indeed, the positive correlation between
cell concentration and in-situ permeability also supports
this notion (Fig. 6b). The influence of sediment compaction on cell abundance would be more apparent in shallower environments than in deeper environments, as thevariation in cell concentration is less constrained in the
low in-situ porosity and in-situ permeability regimes.
Why do coal-bearing units have anomalously high microbial
biomass?

At Site C0020, the coal-bearing sections in unit III andthe lower portion of unit IV had relatively high microbial
cell concentrations. These units/sections were characterized by having relatively permeable coal and sandstonelayers with a relatively large pore size (Fig. 5b, c). Thechanges that occur with increasing depth, which aremainly due to variations in the lithology, are larger for thepermeability and the pore size than for the porosity.
Consequently, because of the large variation in lithology
at Site C0020, there was no clear correlation between theporosity and the permeability below 1000 mbsf (Fig. 7a).

The large pore size determined by MICP for coal likely
reflects the existence of voids in the fractures (cleats).
Consequently, the high permeability of coal at low effective pressure likely has a marked influence on both verticaland bedding parallel flow through cleats. The permeabilityof coal is very sensitive to the effective pressure, and thein-situ coal permeability can be very different to the initial
permeability. Therefore, the in-situ pore size (aperturesize) for coal is expected to be much smaller than themeasured pore size (Fig. 7b) Nevertheless, voids in fractures may form an important pathway for fluid flow
through coalbeds, because the in-situ permeability of fractured coal is much higher than that of the intact coalmatrix or neighboring siltstone/shale formations (Fig. 5b,
Ijiri et al. 2017). Therefore, it is considered that nutrientsand energy sources were mainly released from the coallayers through the cleats, where large amounts of energy
sources are stored, and that these compounds seeped into
the overlying permeable sandy sediments. However, theimpermeable shale and siltstone layers, which overlie thepermeable sandstone layers, may act as barriers to verticalfluid flow. As shown in Fig. 5b, the permeability change
between permeable (sandstone, coal) and impermeable(siltstone, shale) layers was two to four orders of magnitude in coal-bearing units. Since almost no nutrient flow
occurs through the impermeable layer, the very low celldensities at 1500 to 1800 mbsf and from 2000 to 2400

Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

mbsf (< 100 cells/cm3) can be explained by the effectiveness of the siltstone barrier for preventing the verticaltransport and dispersal of nutrients and cells. Permeable
layers may sustain more abundant active microbial populations than low-permeability layers, and may be one of
the reasons for the higher cell numbers in coalbed layers(Inagaki et al. 2015). Therefore, in deeper sections, poresize and permeability, rather than porosity, are considered
to have a greater influence on cell abundance (Phadnis
and Santamarina 2011).
The in-situ permeability was estimated from the effective pressure dependence of the permeability, assumingthat the pore pressure is hydrostatic in deep regions.
However, overpressure (pore pressures higher than thehydrostatic pressure) occurs in deeper regions of mostthick sedimentary/petroleum basins (Bredehoeft andHanshaw 1968; Swarbrick and Osborne 1998) and incoal sediments (Law 1984; Su et al. 2003). Since siltstoneand shale at Site C0020 form an impermeable barrier to
fluid migration, overpressure in and below impermeable
formations can easily occur due to inhibition of verticalfluid/gas transfer and hydrocarbon generation in coal
units (Hunt et al. 1994, 1998; Osborne and Swarbrick
1997; Zhao et al. 2018). The permeability of coal is much
more sensitive to the effective pressure than that ofother rock types in the Sanriku-oki subbasin. Therefore,
overpressure would be expected to increase the in-situ
permeability and pore size for coal, so allowing an abundant microbial population to be sustained. Overpressure
can also increase the methane sorption and storage capacity of coal (Scott 2002).
In the past, the shale layer may have supplied organic
matter that was consumed by microbes in the adjacentsandstone layers (Fredrickson et al. 1997). However, because the shale deposits at Site C0020 currently have amuch lower TOC content and a lower diffusion rate thanthose for coal, the nutrients that support indigenous microbial communities in sandstone and coal are considered
to be derived from lignite coal. Cleats and fractures in coalincrease the substrate surface area available for microbial
action (Strąpoć et al. 2011). Since young lignite coal is susceptible to microbial attack, microbial coalbed methane
would have been readily produced in the coalbeds of theSanriku-oki basin.
Influence of pore size on sedimentary microbial biomass

Pore size can restrict both the size of microbial populations and the cellular volume in porous sediments(Fredrickson et al. 1997). At Site C0020, the modal
pore size for shale (0.028 ± 0.035 μm) and siltstone(0.044 ± 0.009 μm) adjacent to coal units is less than
0.2 μm (Fig. 5c), whereas the pore size for sandstone andcoal is > 2 μm (fracture aperture size of > 1 μm). A poresize of 0.2 μm for lithologies with low in-situ permeability

Page 12 of 17

is consistent with the proposed threshold size for microbial metabolic activity (Fredrickson et al. 1997). The poresize for shale and siltstone is probably smaller than the
majority of in situ microbial cells at C0020, since microbial cells in deep marine sediments are approximately
0.5 μm in size (Kallmeyer et al. 2012; Braun et al. 2016),
and the microbial cell size estimated after incubating coaland shale samples from C0020 ranged from 0.3 to 1.2 μm(Trembath-Reichert et al. 2017). If the pore size is smaller
than the cell size, then cell dispersal will not occur despite
the presence of intense hydrological fluid flows, and microbial activity in such sediments will be low.
At Site C0020, it is conceivable that microbial cells wereburied and trapped in shale and siltstone, promoting the
agglutination of cells, and limiting the physical transport
and/or migration of cells and eukaryotic spores fromsandstone and coal layers to shale and siltstone layers(Inagaki et al. 2015; Gross et al. 2015; Liu et al. 2017).
Other potential geophysical constraints on deep subseafloor
microbial biomass

In addition to the physical properties of sedimentaryrock, other environmental factors also affect the microbial habitability of the deep subseafloor biosphere, e.g.,temperature, pressure, water content, Aw, pore fluid
chemistry, and pH (Beuchat 1974; Fernández Pinto et al.
1991; Zhang et al. 1998; Jenkins et al. 2002; Musslewhite
et al. 2003; Grant 2004; Williams and Hallsworth 2009;
Stevenson et al. 2015). These different geophysical parameters control the availability of nutrients and energy
substrates required for the maintenance of essential cellular functions (e.g., biomolecule repair) over geologicaltime (Hoehler and Jørgensen 2013; Lever et al. 2015).
In general, soil bacteria exhibit optimal growth at highAw, but several bacteria have a high tolerance to desiccation (Aw < 0.8, Griffin 1972; Lavelle and Spain 2001) andhighly saline water (Aw ≈ 0.61, Grant 2004). The Aw
values at Site C0020A range from 0.954 to 0.983 and arealmost unaffected by depth or lithology (Fig. 2e, Tanikawa et al. 2018). Since the Aw values are considerably
higher than the Aw limit for the biosphere, water activity
is not considered to affect the microbial population sizeor activity in the Sanriku-oki subbasin.
Although the temperature at Site C0020 increases almost linearly with depth (24.4 °C/km, Tanikawa et al.
2016), it is not clear whether such temperature increases
limit the population size, activity, or microbial community structure in the deep subseafloor biosphere(Hinrichs and Inagaki 2012).
Permeability estimates based on pore characteristics

Our results indicate that differences in pore size aremainly responsible for the observed variation in initial
permeability (Fig. 7b). To date, numerous attempts haveTanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

Page 13 of 17

been made to relate the pore size distribution and theporosity to the fluid permeability (Dullien 1992). The relationship between the initial porosity and initial permeability also indicates that the permeability characteristics offine-grained sediments are governed by the traditional
Kozeny-Carman equation (Fig. 7a). This equation is widely
applied to mathematical models of microbiological processes (Taylor et al. 1990; Vandevivere et al. 1995) or prediction of permeability in well logging tools (Prasad 2003;
Osterman et al. 2016). The formula describes the relationship between the permeability and the pore size, porosity,and geometry of porous sediments based on the assumption that the pore structure consists of a bundle of cylindrical capillaries (Dullien 1992). The Kozeny-Carman
equation is expressed as follows:
k¼

Φd 2
;
16 f τ 2

ð5Þ

where k is the permeability, ϕ is the porosity, d is thediameter of a volume-equivalent spherical pore, f is afactor that accounts for pores with different crosssectional shapes, τ is the tortuosity factor, and fτ2 is the
Kozeny-Carman coefficient. In this study, the mercury
intrusion porosity and representative pore size evaluatedfrom the MICP measurements were used to calculate
the permeability based on Eq. (5), where f was assumed
to have a value of 2.
Poremaster software (Poremaster for Windows® 8.01,
Quantachrome Instruments, FL) was used to calculate
the tortuosity values from the MICP measurement results. The empirical formula introduced by Carniglia(1986) is based on Fick’s first law and a cylindrical pore
structure model, and is expressed as follows:

 


4 X vi 1þετ ¼ 2:23−1:13V ρb 0:92 
;
ð6Þ
di
S
where V is the total specific pore volume, ρb is the bulk
density of the sediment, S is the total surface area of
sediment particles, vi is the change in pore volume
within a pore size interval i, di is the average pore size
within a pore size interval i, and ε is the pore shape factor exponent (0 < ε < 1). A value of ε = 1, which corresponds to cylindrical pores, was used in this study. To
estimate the permeability using Eqs. (5) and (6), themean, modal, and median pore sizes were used (seeAdditional file 1: Figure S2 and Table S1).
In the low-permeability region (< 10−17 m2), the permeabilities estimated using the mean and modal poresizes were in reasonable agreement with the measured
permeabilities under a low effective pressure of 3 MPa(Fig. 9). In the higher permeability region, use of themean pore size underestimated the permeability, while
use of the modal or median pore size overestimated it

Fig. 9 Comparison of measured permeability at an effective pressureof 3 MPa with predicted permeability. The permeability was calculated
from the modal pore size distribution using Eqs. (5) and (6)(Fig. 9). The modal pore size was therefore considered
to be representative of the fluid transport properties overa wide permeability range.
Fracture permeability model

Using the average pore size, the Kozeny-Carman equation (Eq. 5) did not accurately reproduce the measured
permeability when the permeability was high (Fig. 9).
Microstructural images of the coal material (Fig. 8, Core
15R3) showed that fluids could selectively flow throughfractures; consequently, a fracture permeability modelseems more appropriate than the capillary-tube-based
Kozeny-Carman model. The well-known empirical equation for single fracture flow, known as the cubic law(Witherspoon et al. 1980; Raven and Gale 1985), isexpressed as follows:
Qfr ¼

b3 ΔP
ð1:06 < R < 1:65Þ;12R μLð7Þ

where Qfr is the flow rate per unit width normal to theflow direction, b is the mean aperture size of the fracture, R is a fracture surface characteristic factor, μ is thefluid viscosity, and ΔP/L is the pressure gradient in the
direction of the fluid flow. Here, we consider the cuboid
coal samples that were used in the permeability measurements in this study. Fracture planes develop parallelto the sample length, L, and sample width, W, and normal to the H axis. If multiple parallel fractures developed
parallel to the L and W directions, then the flow rate perunit area, q, measured in the permeability tests can be
expressed as follows:Tanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

q¼

Qfr  W ρfr H
W H

b3 ΔP
ρ ;
¼
12R μL fr

Page 14 of 17

ð8Þ

where ρfr is the fracture density (number of fractures perunit length normal to the fracture surface). Based on Eq.
(2), the fracture permeability is then given by:
kf ¼

b3 ρfr12R:

ð9Þ

The μX-CT data for the coal sample (Fig. 8c) showedthat b ranged from 1 to 10 μm, and ρfr was 1000 m−1.
Therefore, assuming that R = 1, the coal permeability approaches 10−13 to 10−14 m2, which is more consistent
with the experimentally determined permeability thanthat estimated using the Kozeny-Carman model. This result suggests that fluid transport within coal is mainly affected by the fracture (or cleat) geometry, rather than by
the coal matrix, for which the permeability model consists of an assembly of circular pipes.

Conclusions
To determine the key physical properties that dominate
the habitability for microbial life of deep subseafloor
coal-bearing environments, the porosity, permeability, andpore size distribution for sedimentary rocks off the
Shimokita Peninsula, Japan, were measured using coresamples obtained during the Chikyu shakedown cruise
CK06-06 and IODP Expedition 337. The porosity and permeability were measured at an effective pressure of up to
30 MPa to correct for the influence of the effective pressure. The in-situ porosity and permeability gradually decreased with increasing depth down to about 2.5 km
below the ocean floor (water depth of 1180 m). However,
the in-situ porosity and permeability, and the pore size forsediments at depths greater than 1200 mbsf depended
strongly of the lithology. For example, the in-situ permeability and pore size for coal and sandstone are 10−16 to10−18 m2 and 100 to 10 μm, respectively, while those forsiltstone and shale are 10−19 to 10−22 m2 and < 0.1 μm, respectively. The higher permeability of coal at low effective
pressure is due to the dominance of fracture (cleat) flow,whereas that of sandstone is due to the higher volume
fraction of large pores. At low effective pressures, themeasured permeability for coal conforms to the fracture
permeability model, whereas that for the other lithologies
is better predicted using the Kozeny-Carman equationand pore size data.
The decrease in microbial cell concentration with increasing depth in shallow sediments at Site C0020 isconsistent with a reduction in in-situ porosity and permeability. This suggests that the gradual decrease in microbial biomass at shallow depths can be explained by agradual decrease in pore connectivity and flow pathways

due to mechanical compaction. On the other hand, thecellular biomass is less dependent on the in-situ porosityand permeability at depths below 1200 mbsf. Anomalously high microbial cell concentrations (~104–5 cells/cm3) were observed in coal-bearing layers comprisinghighly permeable sandstone and coal formations withrelatively large pore sizes, whereas very low cell concentrations were observed in very low-permeability siltstoneand shale formations with small pore sizes. Therefore,
lithological variation of hydrological properties may control the vertical distribution of microbial biomass atgreater depths. The nutrients and energy sources in lignite coals can move through the fracture (cleat) systems
in the coalbeds and can reach adjacent permeable layers,
resulting in locally dense microbial populations.
Conversely, impermeable siltstone and shale layers thatact as cap rocks for fluid transport may limit the habitability for microbial communities by suppressing the
supply of water and energy-rich substrates from the permeable layers. The pore size for siltstone and shale at
Site C0020 is generally < 0.2 μm, potentially limiting microbial life. Fractures in the lignite coal are likely to havepromoted the production of biological coalbed methane,
and in the presence of impermeable formations, thiswould have generated overpressure in the coalbeds,
which would keep fractures open and produce large porespaces. Based on these observations, we suggest thatfluid permeability and pore size are important environmental factors that limit life in deep subseafloor environments and the physical extent of the biosphere in
sedimentary basins.

Additional file
Additional file 1: Figure S1. Relationship between cell concentrationand modal pore size. No meaningful correlation is obtained. Figure S2.
Depth-tortuosity profile at Site C0020. Tortuosity was calculated from Eq.
(6) using mercury injection capillary pressure (MICP) data. The evaluatedtortuosity was used for permeability calculations. The tortuosity estimatedfrom Eq. (6) ranged from 1.48 to 2.16, and decreased with increasing
depth, probably due to the tortuosity having a strong dependence onporosity. Table S1. General information and physical properties for coresamples collected at Site C0020A. Table S2. Permeability calculated from
Eqs. (5) and (6) using pore size distribution data. Data are plotted inFig. 9. Table S3. In-situ porosity and permeability plotted in Fig. 5a andb. (DOC 2321 kb)

Abbreviations
Aw: Water activity; DICOM: Digital Imaging and Communications in Medicine;
IODP: Integrated Ocean Drilling Program; mbsf: Meters below seafloor;
MICP: Mercury injection capillary pressure; TLD: Three-detector lithology
density logging tool; TOC: Total organic carbon; μX-CT: Micro-focus X-ray
computed tomography
Acknowledgements
The present research used core samples and data provided by the Integrated
Ocean Drilling Program (IODP) Expedition 337. We thank the crews, scientists,and laboratory technicians aboard the D/V Chikyu for drilling, coring, andTanikawa et al. Progress in Earth and Planetary Science (2018) 5:58

sample processing during IODP Expedition 337. The manuscript had benefited
from reviews by Ryuji Tada, Yasufumi Iryu, and anonymous reviewers.
Funding
This study was supported in part by the Japan Society for the Promotion of
Science (JSPS) Strategic Fund for Strengthening Leading-Edge Research andDevelopment (to JAMSTEC), the JSPS Funding Program for Next Generation
World-Leading Researchers (NEXT Program, to FI), and the JSPS Grant-in-Aid
for Scientific Research (No. 25800284).
Availability of data and materials
The data supporting this paper are available in the supplementary information
files (Additional file 1: Figure S1 and S2, and Tables S1–S3), and at SIO7 Data
Center (http://sio7.jamstec.go.jp/).
Authors’ contributions
WT, KUH, and FI designed the study. WT and OT conducted physical property
measurements and data analysis. YM performed cell counts. FI led cruise CK0606. FI and KUH led IODP Expedition 337 and contributed to data interpretation
by WT. WT and FI wrote the paper. All of the authors approved the final versionof the manuscript.
Competing interests
The authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.
Author details
1
Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Scienceand Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan.
2
Research and Development (R&D) Center for Submarine Resources, JAMSTEC,
2-15 Natsushima-cho, Yokosuka, Kanagawa 273-0061, Japan. 3Marine Works
Japan Ltd., Monobe B200, Nankoku, Kochi 783-8502, Japan. 4MARUM Center for
Marine Environmental Sciences and Department of Geosciences, University ofBremen, 28359 Bremen, Germany. 5R&D Center for Ocean Drilling Science(ODS), JAMSTEC, Yokohama, Kanagawa 236-0001, Japan.
Received: 23 August 2017 Accepted: 12 September 2018

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