Progress in Earth andPlanetary Science

Sugiyama et al.
Progress in Earth and Planetary Science(2024) 11:1
https://doi.org/10.1186/s40645-023-00604-9Open Access

RESEARCH ARTICLE

A unique prokaryotic vertical distribution
in the groundwaters of deep sedimentarygeological settings in Hokkaido, Japan
Ayumi Sugiyama1* , Tetsuo Ibara1, Kazuyo Nagaosa2, Atsunao Marui3 and Kenji Kato2

Abstract
The purpose of this study is to clarify the vertical prokaryotic distribution in groundwater in a terrestrial subsurface sedimentary environment with a geological complex. Six groundwater samples were collected from a coastal
1200-m-deep borehole in which digging strata deposited between 2.3 and 1.5 Ma in Horonobe, Hokkaido, Japan. The
studied succession was divided into three vertical zones that were geochemically differentiated according to theirchloride contents and water-stable isotopes. The upper zone (UZ; shallower than 500 m) primarily contained fresh
water supplied by penetrating meteoric water, the connate water zone (CWZ; deeper than 790 m) contained paleoseawater, and the diffusion zone (DZ; 500–790 m depth) located between UZ and CWZ. Fluctuations in the prokaryotic density and constituents were observed across these three zones. The prokaryotic density decreased from UZtoward DZ, and the density of DZ was two orders of magnitude lower than that of UZ and CWZ. High prokaryotic
activity was observed in CWZ below DZ. The upward expansion of prokaryotic distribution from CWZ, where highprokaryotic potential expressed by biomass can be maintained almost equivalent to that in the marine environment,
probably occurred on a geological timescale from 80 ka to 1.3 Ma, as shown by the groundwater age of DZ. The
DZ is a zone where the geochemistry has changed drastically owing to the mixing of penetrating meteoric waterand the diffusion of deep paleo-seawater, preserving a unique subsurface environment. This chemically mixed zone
might be considered as a buffering zone for prokaryotes to prevent the expansion of prokaryotic density and activity
provided by diffusion and their in situ growth from both above and below the zones, which is expected to be maintained over a geological timescale. This zone is considered important for using subsurface space in the deep subsurface environment of the island arc.
Keywords Prokaryotic distribution, Diffusion zone, Meteoric water, Paleo-seawater, Sedimentary environment, Islandarc, Prokaryotic buffering zone

*Correspondence:
Ayumi Sugiyama
sugiyama-ay@atk-eng.jp
1
Asano Taiseikiso Engineering Co., Ltd., 2‑8‑7 Kitaueno, Taitou‑Ku,
Tokyo 110‑0014, Japan2
Department of Geosciences, Graduate School of Science, Shizuoka
University, 836 Oya, Suruga‑Ku, Shizuoka 422‑8529, Japan
3
Geological Survey of Japan, National Institute of Advanced Industrial
Science and Technology (AIST), 1‑1‑1 Higashi, Tsukuba 305‑8567, Japan

1 Introduction
The deep subsurface represents the last territory in the
search for life on Earth, and the findings of these studies have rewritten our understanding of the biosphere inthis planet. Intensive research on microbial abundance
and the possible activity of microbes in subsurface environments has only begun in the last few decades. Deep,dark, and oxygen-limited large subsurface environments
have been explored in subseafloor habitats since thepioneering work of Parkes et al. (1989; 1994). Whitman
et al. (1998) and Kallmeyer et al. (2012) reported a large© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/.

Sugiyama et al. Progress in Earth and Planetary Science(2024) 11:1

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abundance of microbes in oceanic sedimentary environments, whereas Inagaki et al. (2015) showed that the vertical distribution of microbes extended to a depth of over
2.5 km. Microbial metabolic activity supported by penetrating seawater has attracted the interest of researchers
working in the field of biogeochemistry (e.g., D’Hondt
et al. 2004). Unveiled large subsurface biosphere is crucial for understanding life in extreme environments (Inagaki et al. 2006; 2015; Lipp et al. 2008; Kawagucci et al.
2018).
In contrast to marine subsurface microbial studies, terrestrial studies were launched from the hard-rock geological environment in Scandinavia with the aim of revealing
biogeochemical changes mainly caused by microbial
function (Pedersen 1997; 1999). Studies have revealed
that the total number of microbes in subsurface granitic
environments ranges from ­103 to 1
­ 07 cells/mL in groundwater, in the presence of a hydrogen-driven deep biosphere in crystalline bedrock. Sulfate reducing microbes
are also active in the subsurface environment. Subsequently, intensive deep subsurface microbial studies wereconducted in Olkiluoto, Finland, with similar objectives(e.g., Itävaara et al. 2016; Miettinen et al. 2018). Thesestudies revealed that the density of microbes decreases
with depth and that sulfate reduction and methane oxidation are the main energy sources under steady-state
natural conditions.
In a continental sedimentary environment, Chandler
et al. (1997) found phylogenetically diverse microbialcommunities at the Hanford Site of North America. The
environmental complexity resulting from water–rockinteractions in the terrestrial subsurface environment

affects the abundance of organic compounds and microbial distribution. The path of water through fracturesand pores in rocks supports microbial activity (Fredrickson et al. 1997). Kato et al. (2009) found high microbial
density and associated metabolic activity at the geological boundary between sedimentary units at a depth ofapproximately 300 m in Horonobe. The vertical microbial
distribution in this sedimentary environment was notably
different from the conventional thought that microbial
density decreases linearly with log-depth (e.g., Kallmeyer
et al. 2012; McMahon and Parnell 2014; Magnabosco
et al. 2018).
To elucidate the vertical distribution of microbes ingroundwater, the chemistry of which is affected by thegeological complexity in a terrestrial sedimentary environment in the island arc, we collected groundwater
samples from a borehole with a depth exceeding 1200 m,
constructed in a coastal area of Hokkaido Island in Japan(Fig. 1). The study area is covered by the thick Yuchi
Formation, which was deposited in a shallow marine
environment and overlies the Sarabetsu Formation andalluvium (Ikawa et al. 2014). The examined groundwater was further characterized by an interesting mixing
zone that extended from a depth of 500 to 790 m andwas formed by the penetration and diffusion of meteoric
water and paleo-seawater with an age of millions of years(Ikawa et al. 2014). The upper zone (UZ), 500 m below
the ground surface (bgs), primarily contains fresh watersupplied by penetrated meteoric water. In this zone, thechloride concentration was primarily below 3000 mg/L
(Fig. 2c). In contrast, the lower zone at a depth > 790 m
bgs consisted of a connate water zone (CWZ) of

45.5oN

Rebun Island

50oN

Study site

Latitude

45oN
45.0oN
Hokkaido Prefecture

40oN

Rishiri Island

Japan Sea
35oN
44.5oN
30oN

Hokkaido Prefecture
130oE

135oE

140oE

Longitude

Fig. 1 Location of the study site

145oE

141.0oE

141.5oE

142.0oESugiyama et al. Progress in Earth and Planetary Science(2024) 11:1

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Fig. 2 Vertical geological, hydrological, and microbiological zonation of the studied borehole. a Geology with estimated aquifer systems basedon Ikawa et al. (2014), b water temperature and geology, c chloride concentration and δD (based on Ikawa et al. 2014), d density of prokaryotes
and FDC, relative density of bacteria and archaea estimated by FISH, and active cells detected by the LIVE/DEAD method

paleo-seawater with a high chloride concentration (maximum concentration of approximately 17,000 mg/L),
which is almost equivalent to that of modern-day seawater (19,000 mg/L). The zone between these two zones at
500–790 m bgs, was defined as the diffusion zone (DZ)
with an intermediate chloride concentration that differedfrom those of the other two zones. The DZ in the sedimentary geological setting formed over the geologicaltimescale showed a drastic change in geochemistry.
Microbial life in this unique environment needs to berevealed not only from a physical aspect but also fromthe historical aspect of geology. To extend our understanding of microbial life in deep and dark subsurface
habitats, it is necessary to demonstrate the microbial
diversity and possible microbial activity in this complex
environment. Compared to microbial studies of marine
subsurface and terrestrial hard-rock environments, there
are limited studies on microbial ecology in the sedimentary geological settings of the island arcs. Further

understanding of microbial ecology in such diverse deep
subsurface environments could provide basic information that not only enables the assessment of life under
extreme environments but also enables the use of subsurface environments.

2 Results
2.1 Groundwater geochemical conditions

The groundwater temperature increased with depth from
9.3 to 43.2 °C at 1152 m bgs (Fig. 2b), with an averagethermal gradient of 29.8 °C/km, ranging from 29.0 °C/
km for the depth range between 100 and 580 m bgs to
33.0 °C/km for the 620–1152 m bgs depth range. All theexamined groundwater samples exhibited a dissolved
oxygen concentration of 0 mg/L.
At 214 m in UZ, the values of pH, electric conductivity(EC), and Eh were 7.94, 0.08 S/m, and − 228.3 mV, respectively (Additional file 3: Table S1). At 476 m in UZ, theEC and Eh values were markedly different from those at

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214 m (2.68 S/m and − 55.0 mV, respectively); however,the pH value was not notably different between the two
depths.
In DZ, at 613 m, the values of pH, EC, and Eh were
7.21, 2.91 S/m, and − 261.3 mV, respectively; at 715 m, thevalues were 7.45, 4.66 S/m, and − 341.1 mV, respectively.
Thus, the groundwater at 715 m was more saline and lesssaline than that at 613 m.
At 943 and 1143 m in CWZ, the values of pH were 6.86
and 6.65, respectively, which were slightly lower thanthose of the other samples because of the dissolution
equilibrium of hydrogen carbonate. EC was 5.79 S/m andEh was − 289.1 mV at 943 m, and at 1143 m EC was 4.58
S/m and Eh was − 293.0 mV (Additional file 3: Table S1).

belonged to 23–42 orders from the examined samples. Clostridiales (Nos. 31–43, Fig. 3, Additional file 3:
Table S3), Desulfuromonadales (represented by No. 18),
and Pseudomonadales (Nos. 7–11) were retrieved fromevery depth and comprised 52–84% of the total retrieved
sequences. Clostridiales, Desulfuromonadales, and Pseudomonadales accounted for 11% (613 m) to 45% (943 m),
1% (1143 m) to 47% (715 m), and 2% (715 m) to 42%(613 m) of sequences, respectively, at the order level.
However, the constituents of Clostridiales differed at thegenus level at different depths.
The vertical distribution of taxa at the order level
exhibited some variation; in DZ, where the density waslow, Pseudomonadales was dominant at 613 m, whereas
the dominant bacterial group at 715 m was Desulfuromonadales. These depths are clearly differentiated by thechloride concentration, from approximately 500 mg/L
in the shallowest part (approximately 500 m) of DZ toapproximately 16,800 mg/L at approximately 790 m(Ikawa et al. 2014).

2.2 Vertical distribution of prokaryotes

The density and frequency of dividing cells (FDC) inprokaryotes were determined in the groundwater samples collected from six different depths (Fig. 2d; Additional file 3: Table S2). The density of prokaryotes in
UZ was 6.71 × ­104 ± 0.23 × ­104 cells/mL at 214 m and1.98 × ­105 ± 0.16 × ­105 cells/mL at 476 m. The sample collected from 214 m showed the highest FDC value (2.44%)
among the examined samples. FDC, an indicator of thegrowth potential of a given population, was low in DZ
and at 943 m. LIVE/DEAD staining, which was used
to estimate the contribution of living cells to the entire
prokaryotic density, was greater than 60% in all samples,with the highest value of 80–82% in UZ. This suggeststhat these prokaryotes have the potential to exert theirbeneficial effect.
The prokaryotic density sharply decreased in DZ byapproximately one or two orders of magnitude relative tothe values in UZ, being 4.75 × ­103 ± 0.20 × ­103 cells/mL at
613 m and 2.68 × ­103 ± 0.03 × ­103 cells/mL at 715 m.
The CWZ exhibited a high density of prokaryotes,
with 1.87 × ­105 ± 0.09 × ­105 cells/mL at 943 m and1.15 × ­106 ± 0.06 × ­106 cells/mL at 1143 m. FDC increased
at 1143 m to 1.79%, from 0.56% at 943 m. The densityof bacteria was as high as 9.1 × ­104 cells/mL at 943 m
and 3.8 × ­104 cells/mL at 1143 m, and that of archaea
increased from 2.9 × ­103 cells/mL at 943 m to 2.9 × ­104
cells/mL at 1143 m (Fig. 3). Although the estimates ofMethanomicrobiales and Methanobacteriales by fluorescence in situ hybridization (FISH) contributed to the total
prokaryotic cell counts of FISH small (0.79% and 0.63%,
respectively, at 1143 m, Additional file 3: Table S2), theseresults suggest that these groups might be active because
they were detectable by RNA staining.
2.3 Community constituents

Next-generation sequencing (NGS) retrieved between
535 and 1,553 operational taxonomic units (OTUs)

2.3.1 Upper zone

The NGS data analyzed at the genus level are plotted inFig. 3, and detailed data are provided in Additional file 3:
Table S3. The order Clostridiales, comprising 22% of
the total retrieved sequences at 214 m, was represented
by Acetobacterium, some genera of Eubacteriaceae,
and some genera of Clostridium (Nos. 38, 39, and 43 in
Additional file 3: Table S3). Acetobacterium are strictly
anaerobic bacteria with an optimal temperature range of
27–30 °C for mesophilic species, and 20–30 °C for psychrotolerant species. They oxidize H­ 2 by reducing C­ O2
or fermentation (Simankova and Kotsyurbenko 2009). In
addition, some constituents of Desulfuromonadales wereretrieved from UZ, specifically, some genera of Pelobacteraceae (No. 18), which comprised 10% of the community. Pseudomonas (No. 8) comprised 8% of the retrieved
sequences.
Some genera of Lachnospiraceae (No. 36; order
Clostridiales) dominated the prokaryotic community at 476 m, accounting for 32% of the total retrieved
sequences. After Clostridiales, some genera of Pelobacteraceae (No. 18) from the order Desulfuromonadales
contributed 14% of the total sequences. In the order
Pseudomonadales, Pseudomonas, Acinetobacter, andsome genera of Pseudomonadales (Nos. 8, 10, and 11)
were retrieved from 476 m and together accounted for
16% of the total. In addition to these major constituents,
Oleibacter (order Oceanospirillales, No. 12) was retrieved
from a depth of 476 m in the lower part of UZ.
For archaea, some genera of Methanobacteriaceae (No.
57) were retrieved from 214 m, accounting for 8% ofSugiyama et al. Progress in Earth and Planetary Science

100%(2024) 11:1

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Pseudoalteromonas
1

1

Others >1%

8

2
5

2
5

2

11

2

3
8

2

80%

3
7
8

10
11
12

Pseudomonas
8

some Genus of
Pelobacteraceae

12

Relative ratio (%)

18
18
27

40%

10

some Genus of
Lachnospiraceae
Acetobacterium

0%

57

214 m
UZ

Oleibacter

31

37

12

Anoxynatronum
Alkalibacter
37

36

18

37

36
37

476 m
UZ

29

34
18

37

56
57

23

32

39
43

36

11

38

20%

18

21
24

10
11

Acinetobacter
60%

2

613 m
DZ

38
41

Anaerovirgula
41

Methanoculleus
54

57

57

715 m
DZ

943 m
CWZ

40
43
47
52

Methanobacterium56

some Genus of
Methanobacteriaceae
1,143 mCWZ

Fig. 3 Relative contribution of prokaryotic constituents revealed by NGS analysis at genus level. The abundances of each fraction and captions areprovided in Additional file 3: Table S3

the total. At the depth of 476 m, the contribution to theentire community was 1.7%.
2.3.2 Diffusion zone

The community constituents of DZ, where the prokaryotic density was very low, markedly differed between
613 and 715 m, reflecting environmental changes alonga sharp physicochemical gradient. The major constituents at 613 m were some genera of Pelobacteraceae (No.
18) of the order Desulfuromonadales, which contributed
22% of the total retrieved sequences. Pseudomonas, Acinetobacter, and some genera of Pseudomonadales (Nos.
8, 10, and 11) accounted for 39% of the total sequences.
These bacteria also occurred at the depth of 476 m with arelative abundance of 16%. Oleibacter (Oceanospirillales,
No. 12) contributed 10% of the total retrieved sequences
at 613 m and 5% at 476 m. The lowest detected proportion of Clostridiales, comprising up to 11% of the total

community, was found at 613 m, whereas this group was
the major member of the community at 476 m.
Members of Pelobacteraceae (No. 18) comprised 47% of
the total retrieved sequences at the depth of 715 m in the
deeper part of DZ. These bacteria are strictly anaerobic,
mesophilic, chemolithoheterotrophs or chemoorganotrophs (Kuever et al. 2005). Alkalibacter, Acetobacterium, and Anaerovirgula (Nos. 37, 38, and 41) were themajor constituents of Clostridiales at 715 m, comprising
34% of the total retrieved sequences. Alkalibacter are
strictly anaerobic, obligately alkaliphilic, and halotolerant bacteria with an optimal growth temperature of 35 °C(Garnova and Zhilina 2009a). Pseudoalteromonas (order
Vibrionales, No. 5) was retrieved from depths of 715 and943 m, but was not a major member of the assemblages atother depths (maximum 0.5% at 613 m). Another featureof the community at 715 m was the presence of archaea
and members of the family Methanobacteriaceae (Nos.

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56 and 57), whereas at 613 m, the contribution of thesemethanogens was very small.

The density of prokaryotes in DZ was of the order of­103 cells/mL, which was two orders of magnitude lower
than that in UZ; however, it increased again to ­105 cells/
mL at 943 m in CWZ composed of paleo-seawater.
This density of prokaryotes in CWZ demonstrates thatthe deep, old groundwater retained a biomass similar
to that of the ocean surface environment (Murakami
et al. 1999; Karner et al. 2001; Salka et al. 2008). The
deeper sample collected at 1143 m exhibited a higher
prokaryotic density and possible growth potential, as
suggested by the FDC, than the sample collected at
943 m in the upper part of CWZ. This finding can be
partly explained by the relatively high archaeal density
detected by NGS and FISH at 1143 m. The groundwatertemperature increased linearly from 9.3 °C at the surface to 42 °C at 1143 m. The prokaryotic distribution
found in this terrestrial sedimentary geological setting
differed from that reported for a hard-rock geologicalsetting. This vertical prokaryotic profile has not beenpreviously described and is unique.
The most interesting finding was the apparent low density of prokaryotes in DZ compared to the high density
observed in CWZ. Thus, a low prokaryotic density wasobserved in DZ between UZ and CWZ. The density andgrowth potential suggested by the FDC of the prokaryotic communities in DZ were very low compared tothose at shallower (476 m) and deeper (943 m) depths.
The unique vertical profiles of prokaryotes may haveresulted from differences in water chemistry with depth.
The chloride concentration and stable isotope signatures indicate that the groundwater of UZ was suppliedby meteoric water, whereas the deeper zone of CWZ,
below 790 m, was filled with paleo-seawater with an ageof approximately 1.3 Ma (Ikawa et al. 2014). The chloride concentration and δD changed markedly with thedepth in DZ (Fig. 2c), thus suggesting a drastic verticalchange in the chemical condition for prokaryotes causedby the mixing of meteoric water with paleo-seawaterthat had gradually diffused vertically over the geologicaltime. The difference in prokaryotic constituents between
the upper and deeper DZ may result from changes inchemical conditions, as discussed below. These major
environmental changes resulted in the observed distribution of prokaryotes in DZ, which was different fromthat reported previously wherein a linear decrease in thedensity of prokaryotes was solely governed by an increasein temperature until more than 45 °C (e.g., Heuer et al.
2020; Dai et al. 2021) or a sharp decrease in the density
occurred due to high salinity (Nisson et al. 2023). The ageof the groundwater in DZ ranges from 80 ka to 1.3 Ma(Ikawa et al. 2014); thus, the mixing of meteoric andpaleo-seawater restricted the distribution and density ofprokaryotes over a long geological timeframe.

2.3.3 Connate water zone

For the order Clostridiales, Alkalibacter and Anaerovirgula (Nos. 37 and 41) were retrieved from 943 m,with relative abundances of 29% and 12% of the total
sequences, respectively. In addition to Clostridiales,
some genera of Pelobacteraceae, Oleibacter, and Pseudoalteromonas (Nos. 18, 12, and 5) were retrieved from
943 m. Some methanogens, such as Methanobacterium,
Methanocorpusculum, and Methanoculleus (Nos. 53–57),
were detected at 943 m, accounting for 10% of the total
retrieved sequences. A relatively high density of archaea
compared to the overlying zones was also detected by
FISH analysis (Fig. 2d).
The deepest sample obtained (1143 m depth) showed aclearly different community structure with a large diversity of prokaryotes. A typical feature of this sample was
the presence of various genera of Clostridiales, such as
Soehngenia, Dethiosulfatibacter, Desulfitobacter, Alkalibacter, Anoxynatronum, Thermotalea, and Clostridium(Nos. 31, 32, 34, 37, 40, 42, and 43), which are anaerobic, alkaliphilic, mesophilic, or thermophilic bacteria(Parshina and Stams 2009; Garnova and Zhilina 2009a;b). Among these genera, Anoxynatronum is capable offermentative metabolism. For the order Pseudomonadales, Pseudomonas, Acinetobacter, and some generaof Pseudomonadales (Nos. 8, 10, and 11) were retrieved
from 1143 m. Some genera of Pelobacteraceae (No. 18),
which were the major constituents of DZ, contributed
minimally to the total reads at 1143 m, relative to samples
from higher and lower depths. The sample from 1143 m
exhibited the largest proportion of methanogens among
the examined groundwater samples, accounting for 15%of the total retrieved sequences.

3 Discussion
3.1 Vertically unique distribution of prokaryotes

Previous studies have reported that the density ofprokaryotes in the subseafloor and terrestrial subsurface environments decreases almost linearly with
log-depth from ­108 to 1
­ 03 cells for the examined sample volume at depths ranging from near the surface toover 3 km (Kallmeyer et al. 2012; McMahon and Parnell 2014; Parkes et al. 2014; Magnabosco et al. 2018).
The vertical distribution of prokaryotes in the deep
groundwater of the sedimentary geological setting wasobserved herein, wherein a low density was found at
the intermediate depth named DZ, which contained
paleo-seawater that had infiltrated upward from 790 to
500 m depth, while the density of prokaryotes increased
again in the deeper zone at depths of 943 and 1143 m.

Sugiyama et al. Progress in Earth and Planetary Science(2024) 11:1

3.2 Characteristics of community constituents

To elucidate the drastic vertical changes in prokaryotic
abundance in DZ, community similarity analysis at thegenus level was conducted using the Jaccard index (Jaccard 1912) and nonmetric multidimensional scaling(NMDS) (Borcard et al. 2011). In the Jaccard analysis, thecommunity similarity of the prokaryotic constituents ofsamples from 613 and 715 m depths in DZ was low. However, the similarities between those from 613 and 476 m
depths and between those from 715 and 943 m depths
were high (Additional file 3: Table S5). The NMDS analysis classified the examined prokaryotic communities into
three different clusters: Cluster A, consisting of prokaryotic components at 214 and 476 m in UZ and at 613 m inthe upper part of DZ; Cluster B, consisting of prokaryotes at 715 m in the lower part of DZ and at 943 m inthe upper part of CWZ; and Cluster C, consisting solelyof prokaryotes at 1143 m in CWZ (Fig. 4). The similarityof the examined prokaryotic community constituents ingroundwater may reflect the responses of prokaryotes toenvironmental changes caused by the mixing of meteoric
water and paleo-seawater. Details are provided as follows:
The major prokaryotic constituents of Cluster A at theorder level were Clostridiales, Desulfuromonadales, andPseudomonadales. These prokaryotes were also retrieved
from seawater-based formations in the Minami-Kanto
gas field (Ishii et al. 2019) and the deep groundwater ofan accretionary prism in Japan (Matsushita et al. 2016).
Genus-level analysis revealed that some genera of Lachnospiraceae (No. 36) dominated the order Clostridiales
at 476 and 613 m. Some genera of Pelobacteraceae (No.
18) appeared at all depths in Cluster A with a similar

Fig. 4 Genus-level NMDS analysis of the prokaryotic community

Page 7 of 13

contribution to the community. Pseudomonas, Acinetobacter, and some genera of Pseudomonadales (Nos.
8, 10, and 11) were the major constituents of the 613 m
community and were also retrieved from 214 and 476 m.
Pseudomonas grows aerobically in general, but in somecases, they can grow anaerobically using nitrate as anelectron acceptor (Palleroni 2009). Acinetobacter is frequently present in soil and water environments (Juni
2005). Oleibacter (No. 12) was retrieved from both 476
and 613 m, as well as from 214 m, although its relative
abundance was low at 214 m. They are mesophilic (Teramoto 2015) and an optimum growth temperature for thetype species O. marinus are 25–30 °C (Teramoto et al.
2011). The small contribution of Oleibacter at 214 m
probably resulted from low temperatures. DZ exhibited
drastic changes in its chemistry, which were reflected by
the differences in prokaryotic constituents between thetwo samples from the upper (613 m) and lower (715 m)
parts of DZ. The prokaryotic constituents in the upper
part of DZ resemble those in UZ.
Cluster B, in which Alkalibacter and some genera of
Pelobacteraceae (Nos. 37 and 18) were the major prokaryotic constituents, was retrieved from depths of 715 and943 m. Some genera of Pelobacteraceae were retrieved
from every examined depth; however, their contributions
were particularly high in the community of prokaryotes
in Cluster B. Anaerovirgula and Pseudoalteromonas (Nos.
41 and 5), which are marine bacteria, were the characteristic constituents of Cluster B. Anaerovirgula is obligately
anaerobic, alkaliphilic, and mesophilic, and an optimum
growth temperature for the type species A. multivorans is
35 °C (Pikuta et al. 2006), which was slightly higher than
the temperature at 715 m. Pseudoalteromonas is anaerobic and halophilic bacteria (Bowman and McMeekin2005); this genus was not retrieved from UZ. The
increasing temperature and salinity through the chemical diffusion zone formed with a geological time length in
the depth interval of 715 to 943 m might provide appropriate conditions for the development of prokaryotic
communities in Cluster B. Another characteristic of thecomponents in Cluster B was reflected by the low valuesof the Chao 1, Shannon, and Simpson diversity indices(Additional file 3: Table S4). All indices were the lowest
at 715 m, followed by those at 943 m. This indicates thatthese depths are in a transitional state from paleo-seawater to dilution by penetrating meteoric water.
Cluster C consisted solely of prokaryotes at 1143 m in
CWZ. The Shannon and Simpson indices of the 1143 m
depth sample were the highest among the examinedsamples (Additional file 3: Table S4). The major prokaryotes in Cluster C belonged to the order Clostridiales(Nos. 31, 32, 34, 37, 40, 42, and 43). In addition, therelative contribution of methanogens, represented by

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Methanobacteriaceae (Nos. 57 and 56), which utilize­H2/CO2 or formic acid (e.g., Maestrojuán et al. 1990;
Skerman et al. 1980), was as high as 15% of the total
sequences. These prokaryotes are characterized by the
deep groundwater of an accretionary prism and various hot springs retrieved from depths greater than 1 km
(Matsushita et al. 2020; Sugiyama et al. 2021a).
Geochemical analysis showed that more than 90% of
the dissolved gases at 1143 m were methane, which wasshown to be biogenic based on its stable carbon isotope
value (Agency for Natural Resources and Energy 2018).
The presence of methanogens has been demonstrated not
only by DNA analysis, but also by RNA detection through
FISH analysis (Amann et al. 1995). These findings suggest that methanogenic activity may have occurred in the
paleo-seawater environment. Substrates for methanogenesis, namely ­H2/CO2 or formic acid, can be produced by
Clostridiales. The number of reads retrieved from NGS
suggested a high correlation between methanogens andClostridiales (t-test; t [10] = 0.833, not significant). Thus,
a syntrophic consortium of ­H2-producing fermentative
bacteria and ­H2-utilizing methanogenic archaea may bepresent for an accretionary prism, as inferred by Matsushita et al. (2020). The abundance of methanogens estimated from the NGS decreased upward from CWZ,
became very low at 613 m depth in DZ, and increaseagain upward. Some genera of Methanobacteriaceae,
such as Methanobacterium (No. 56), which can performhydrogenotrophic methanogenesis, have been detected
in deep marine sediments (Shlimon et al. 2004; Ishii et al.
2019) and terrestrial surface or subsurface environments(Zhu et al. 2011; Nyyssönen et al. 2014; Sugiyama et al.
2021a). The reason for the decrease in the relative abundance of methanogens from 476 to 613 m depth throughthe geologically changing environment from the sedimentary boundary of the Sarabestu and Yuchi formations
at 471.5 m to the chemical diffusion zone of approximately 500 to 790 m is not yet fully understood.

disposal is one of the most urgent requirements for utilizing geological space.
Microbes present in reduced subsurface environments
pose potential risks associated with geochemical instability (Parkes et al. 2014; Miettinen et al. 2018). Regarding
the function of microbes in the subsurface environment
in relation to the subsurface space, the following possible
microbial effects should be considered: (1) methanogenesis and ­H2-producing metabolic processes resulting in
the release of explosive gases; (2) metabolic activity thatchanges the environmental redox potential, leading toenhanced metal corrosion (Yoshikawa et al. 2009; Katoet al. 2020); and (3) geochemical reactions and migrationof embedded chemical species (Utsunomiya et al. 2020).
Elucidating the potential activities of methanogenesisand ­H2-producing processes is important subjects among
them, because explosive gases are released into the environment. Possibility remains that the geological structure
plays a role as a cap rock to trap the methane and hydrogen gases produced below (Caesar et al. 2019; Drake et al.
2019). Thus, the biogeochemical effect of the subsurface
environment is governed by the strength of microbialactivity (Whitman et al. 1998; Parkes et al. 2014). However, it is necessary to clarify the in situ concentrationof related chemical compounds, such as hydrogen, ­CO2,
and methane to estimate the activity of methanogenesis,as the analysis of microbial DNA and RNA does not show
this activity.
Previous studies have confirmed that sedimentary geological settings promote the abundance and growth of
a diverse range of microbial species compared to hardrock systems (Parkes et al. 2014; Inagaki et al. 2015).
In the present study, we discovered a zone of reduced
microbial activity, i.e., DZ, where environmental conditions changed drastically; this zone was located above
a deeper zone with higher microbial activity provided
by paleo-seawater. The DZ exhibited a marked fluctuation in the vertical distribution of microbes, which was
associated with differences in community constituents.
The upward expansion in the distribution of microbes
from CWZ, where the high microbial potential can be
maintained equivalent to that in the marine environment in the examined paleo-seawater, probably occurred
through a geological timescale shown by the groundwater age of DZ from 80 ka to 1.3 Ma (Ikawa et al. 2014).
The DZ is characterized by drastic changes in geochemistry because of the mixing of penetrating meteoric waterand the diffusion of deep paleo-seawater, preserving aunique subsurface environment over a long period oftime. This zone mitigates the high microbial activity inthe deeper zone with high temperatures in the presenceof anoxic paleo-seawater, which reduces the possible
risk of microbial hydrogen and methane gas production.

3.3 Microbiological studies in extension of utility
for subsurface environment

Environmental and social needs have developed thefocus of studies on deep subsurface environments, fromsearching for natural resources to the utilization of spacefor carbon dioxide capture and storage and radioactive
waste (Pedersen 1999; Morozova et al. 2011; Momper
et al. 2017). Global warming and climate change concerns have triggered global efforts to reduce atmosphericcarbon dioxide, and carbon dioxide capture and storage.
One of the major strategies for reducing atmospheric carbon dioxide is to embed it into subsurface space (Leunget al. 2014; Kumar et al. 2018). In addition, constructing
a high-level radioactive waste repository for geologicalSugiyama et al. Progress in Earth and Planetary Science(2024) 11:1

Page 9 of 13

Therefore, this zone could be regarded as a bufferingzone for prokaryotes, preventing the expansion of theirdensity and activity in deep subsurface sedimentary environments. The findings from this zone indicate a unique
vertical prokaryotic profile in a terrestrial sedimentarygeological setting. We suggest that this zone is importantfor planning the future use of subsurface environments in
the island arcs.

to 1205 m bgs in a sedimentary geological setting inHoronobe, Hokkaido, Japan. The well is located 300 m
from the coastline. The details of DD-1 are describedby Ikawa et al. (2014) based on HRISE (2005). The location of the study site and its geology are shown in Figs. 1
and 2b, respectively. The geological formations encountered in this study are the Yuchi, the Sarabetsu, andAlluvium formations. The Yuchi Formation is estimatedto be approximately 1100-m thick, and consists mainlyof fine sand, clay, and silt, indicating that the unit was
deposited in a shallow marine environment. The formation was deposited at 2.3 Ma, and is overlain by the Sarabetsu Formation (1.5 Ma and younger). In well DD-1, theboundary between the Yuchi and the Sarabetsu formations is located at 471.5 m bgs. The geological succession
is topped by alluvium from 86.4 m bgs upward. Ikawa
et al. (2014) divided the studied aquifer down to 1004 m
into several systems-Alluvium Aquifer, Upper Sarabetsu
Aquifer, Lower Sarabetsu Aquifer, Yuchi Aquifer, DZ,
and CWZ-based on differences in the geological setting
inferred from measurements of chloride concentrationand δD of pore water. The age of each aquifer was estimated from δD and δ18O values of groundwater to be
12–42 ka for the Upper Sarabetsu Aquifer, > 80–120 ka
for the Lower Sarabetsu Aquifer and Yuchi Aquifer, 80 ka
to 1.3 Ma for DZ, and > 1.3 Ma for CWZ (Fig. 2a).

4 Conclusions
We found a unique prokaryotic vertical distribution in
groundwater at intermediate depths of meteoric waterand paleo-seawater from 500 to 790 m, named the diffusion zone, DZ, in a sedimentary geological settingin Hokkaido, Japan. The examined groundwater was
obtained from a borehole with a depth exceeding 1200 m,
which was constructed as part of a pre-experimental
study of the sedimentary terrestrial subsurface environment for radioactive waste treatment.
The density of DZ prokaryotes was of the order of­103 cells/mL, which was two orders of magnitude lower
than that of the upper zone, UZ. The density increased
again to ­105 cells/mL at 943 m in the connate waterzone, CWZ, composed of paleo-seawater > 1.3 Ma. The
observed unique vertical profile of prokaryotes is suggested to result from the differences in water chemistry
and physics with depth.
The prokaryotic community constituents in the upperand lower DZ differed, indicating a significant change in
the groundwater environment. However, the estimatedcommunity constituent of the upper part of DZ was similar to that of UZ and that of the lower part of DZ was
not different from that of CWZ. This suggests that community constituents remained in the major profiles of
the upper and lower zones. Although prokaryotic density was suppressed, the major community constituents
maintained their abundance within a given condition.
Unlike the understanding of prokaryotes in hard-rockenvironments, the vertical distribution of prokaryotes
in the groundwater constrained by both meteoric waterand paleo-seawater is not simple. These findings led us tohypothesize that the DZ could act as a buffering zone forprokaryotic density and activity over a geological timescale. Sedimentary microbiology is expected to be more
complex than that of hard-rock systems. Thus, we needto expand our understanding of the microbial ecology insedimentary geological environments for the future useof subsurface environments.
5 Methods/experimental
5.1 Study site

The examined groundwater was sampled from an observation well (DD-1) drilled from 5.2 m above sea level5.2 Sample collection and field measurements

We examined groundwater samples obtained from thethree zones of borehole DD-1 between February 2017and December 2018: 214 m and 476 m, in UZ, 613 m
and 715 m, in DZ, and 943 m and 1143 m, in CWZ. Eachgroundwater sample was collected by using a packer
system (Asano Taiseikiso Engineering Co., Ltd., Tokyo,
Japan) and sterilized bottles. The sampling procedures
and microbial analytical processes are summarized in theflow chart of Sugiyama et al. (2021b). The microbial analysis methods are described in the following section. The
individual screen depths and environmental parameters
measured in situ are listed in Additional file 3: Table S1.
The examined water sample at a depth of 214 m was
obtained from the Sarabetsu Formation and at depths of
those at 613, 715, 943, and 1143 m from the Yuchi Formation. A water sample at 476 m was taken from theboundary between the Sarabetsu and Yuchi formations.
Temperature logging was performed to identify aquifers between 30 and 1152 m (Fig. 2b). Preliminary drainage was performed prior to groundwater sampling toconfirm the pH and EC stability and attenuation of
Amino G acid (7-amino-1,3-naphthalene disulfonicacid mono potassium salt; ­C10H8KNO6S2) added during
drilling to ensure the in situ water quality of the studied
depth.

Sugiyama et al. Progress in Earth and Planetary Science(2024) 11:1

Page 10 of 13

Environmental parameters, such as water temperature,
pH, EC, and oxidation–reduction potential (ORP), were
measured in situ by using Ocean Seven 303 (IDRONAUT
S.r.l., Italy). The ORP values were transformed into Eh(STD) values (H conversion) by using Eq. (1):more than 20 microscopic fields, and totally more than
300 prokaryotic cells were counted under an epifluorescence microscope (BX51-FLA; Olympus Corp., Tokyo,
Japan) (Kato et al. 2009). Dividing cells were counted
to determine the FDC, which indicates the growthpotential of a given population (Newell and Christian1981). Counting by microscopy was performed in triplicate, and standard deviations were obtained. Hybridization was performed by using the bacteria-specific
EUB338 probe or the archaea-specific ARC915 probe
and non-ARC915 probe (listed in Additional file 3:
Table S6). Methanomicrobiales and Methanobacteriales
were analyzed in the sample at 1143 m by using probes
MG1200b and MB311, respectively. The ratio of bacteria to archaea was counted and calculated based onimages captured using an epifluorescence microscope(BX51-FLA) equipped with a digital camera (DP71,
Olympus) (Additional file 2: Fig. S2).
To estimate the living cell ratio, LIVE/DEAD staining was performed using a LIVE/DEAD Bac Light
Viability Kit consisting of two fluorescent nucleic acidstains: SYTO9 (green) and propidium iodide (PI; red)(Auty et al. 2001). SYTO9 permeates both viable andnon-viable cell membranes, whereas PI only permeates damaged cell membranes, which in turn negates
the SYTO9 fluorescence. Viable prokaryotic cells fluoresce in green, and those with a damaged or non-viable
membranes fluoresce in red. A subsample of groundwater amount was collected on a nuclepore filter (0.2 μm
pore size). The cells was stained with SYTO9 (final
concentration 0.5 μM) and PI (3 μM), and the numbersof cells were counted under an epifluorescence microscope (BX51-FLA). In this study, the ratio of cells with
viable prokaryotic cell membranes (defined here as living cells) among all cells was calculated.

Eh (STD)[mV] =(Eh[mV] + 206)
− 0.7 × Temperature[◦ C] − 25

...

(1)
Dissolved oxygen was measured by using Ocean Seven
303 for ground-pumped water. Amino G acid concentration was measured using a spectrophotometer (FP-8000,
JASCO Corp., Japan).
5.3 Microbial analysis

The microbial communities were elucidated though cellcounts, LIVE/DEAD staining, FISH to evaluate specific population dynamics. In addition, DNA analysis
was performed to elucidate the community constituentsusing NGS of the 16S rRNA gene. Microbial DNA has
been shown to suggest groundwater flow paths basedon differences in the physiological characteristics of theretrieved DNA, which resulted from the environmental
distribution of microbes (Segawa et al. 2015; Sugiyama
et al. 2018; Schilling et al. 2023).
5.3.1 Microscopic analysis

The density of prokaryotes and the community structure
were analyzed by total direct count (TDC) and FISH,
following Porter and Feig (1980) and DeLong (1992),
respectively, with some modifications (Kato et al. 2009;
Segawa et al. 2015; Sugiyama et al. 2018). If the sampled
water contained ­Fe2+, ethylenediaminetetraacetic acidwas immediately added to the sample at a final concentration of ~ 6 mM to minimize the formation of F
­ e2O3
aggregates, which interfered with the microscopic observations (Nagaosa et al. 2008). The concentration of ­Fe2+
was determined using the ferrozine method (Viollier
et al. 2000). The sampled water was immediately subsampled and fixed with neutralized formaldehyde (final concentration 2%) for TDC. The subsamples for FISH were
fixed with paraformaldehyde (final concentration 3%) andcollected by the filtration on a nuclepore filter (0.2 μm
pore size; GE Healthcare UK Ltd.). The water samples
for TDC were stored at 4 °C and the filtered samples for
FISH were stored at − 20 °C until analysis.
The subsample of groundwater for TDC was collected by the filtration of 1–100 mL on a nuclepore filter (0.2 μm pore size). Cells were stained with
4′,6-diamidino-2-phenylindole (DAPI, Nacalai TesqueInc., Kyoto, Japan; final concentration, 0.01 μg/mL) and5.3.2 DNA extraction

The sampled groundwater was filtered with a 0.22-μm
Sterivex-GV filter (EMD Millipore) to collect more than­108 cells. Bulk DNA was extracted as described previously (Somerville et al. 1989). Prokaryotic cells were lysedusing a solution of lysozyme and proteinase K in SterivexGV. Bulk DNA was extracted using phenol. As some
substances originating in the geological environment
interfere with the PCR reaction, bulk DNA was purified
using a PowerClean Pro DNA Clean-Up Kit (MO BIO)
and subsequently extracted using a one-step PCR inhibitor removal kit (Zymo Research). The concentrations of
the extracted DNA were determined using a Qubit anddsDNA HS Assay Kit (Thermo Fisher Scientific).

Sugiyama et al. Progress in Earth and Planetary Science(2024) 11:1

5.3.3 16S rRNA gene sequencing

The 16S rRNA genes were amplified using 2-step tailed
PCR by a primer coded V3-V4 regions 341f/805r primer
set. A negative control that did not contain the template
DNA was used to confirm that there was no contaminationin the PCR. The PCR conditions are shown in Additional
file 1: Fig. S1. Sample libraries for sequencing were preparedaccording to the Synergy H1 and QuantiFluor dsDNA System, and checked using a Fragment Analyzer and dsDNA
915 Reagent Kit (Advanced Analytical Technologies). NGS
was run by MiSeq. OTUs were clustered using QIIME at
a 97% similarity level. Chimeric sequences were detectedusing the Qiime. Individual OTUs were assigned based on
representative sequences from the GreenGenes database.
The accession number is DRA012929.
5.3.4 Statistical analysis

The Chao 1, Shannon, and Simpson indices were calculated
according to Chao (1984), Shannon (1948), and Simpson(1949), respectively. Community similarity between thetwo samples was estimated using the Jaccard index (Jaccard 1912), which converts the assemblage data to presence/
absence. The NMDS analysis was used to evaluate the relative similarity among the prokaryotic communities (Borcard
et al. 2011). NMDS analysis was applied to use the similarity
index directly because the process of normality and linearity obtained by principal components analysis is incongruous for analyzing biological communities. Cluster analysis
was performed using k-medoids (Gülagiz and Sahin 2017),
which are more robust than k-means (Borcard et al. 2011).
Abbreviations
TDC	Total direct count
FDC	Frequency of dividing cells
FISH	Fluorescence in situ hybridization
NGS	Next-generation sequencing
OTUs	Operational taxonomic units
NMDS	Nonmetric multidimensional scaling

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40645-​023-​00604-9.
Additional file 1: Fig. S1. Details of the reaction mixture and PCR
conditions.
Additional file 2: Fig. S2. FISH image of groundwater sample at 476 m.
Yellow arrows indicate hybridized cells (living cells) and white arrows
indicate not hybridized cells (not living cells).
Additional file 3: Table S1. Observed in situ environmental parametersat the six sample depths. Table S2. Results of microbial analysis for the six
studied depths. Table S3. Genus-level taxa and their relative ratios (%) in
the prokaryotic community obtained by NGS analysis. Table S4. Results of
NGS and calculated diversity indices. Table S5. Results of the community
similarity analysis using the Jaccard index. Table S6. Oligonucleotide
probes used in this study for target groups.

Page 11 of 13

Acknowledgements
This study was carried out as a part of R&D supporting program titled "Development of enhancing the disposal system in the coastal region (2018
FY)” under the contract with Ministry of Economy, Trade and Industry (METI)(Grant No.: JPJ007597). This study is part of the collaborative research between
the National Institute of Advanced Industrial Science and Technology (AIST)
and Asano Taiseikiso Engineering Co., Ltd. (ATK). This study was also partially
supported by the River Works Technology Research and Development Program of the Ministry of Land, Infrastructure, Transport and Tourism, Japan. We
are grateful to various people for their support and helpful comments on the
manuscript and Mr. Shiga from ATK for his comments on an earlier version of
the manuscript.
Author contributions
AS proposed the topic, conceived, and designed the study. AS, IT, KN, and KK
joined sample collection. IT performed groundwater sampling. KN conducted
the experimental study and analyzed the data. AM and KK collaborated withthe corresponding author (AS) in the interpretation of the data and construction of manuscript. All the authors have read and approved the final version of
the manuscript.
Funding
This study was carried out as a part of R&D supporting program titled "Development of enhancing the disposal system in the coastal region (2018 FY)”
under the contract with Ministry of Economy, Trade and Industry (METI) (Grant
No.: JPJ007597).
Availability of data and material
The datasets supporting the conclusions of this article are included within thearticle and its additional files.

Declarations
Competing interests
The authors declare that they have no competing interest.
Received: 27 April 2023 Accepted: 26 December 2023

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