Microbial DNA; A brand-new tracer of groundwater flow
1,2

Ayumi Sugiyama, 3Kenji Kato, 3Kazuyo Nagaosa, 1Tetsuo Ibara, 1Kazuyoshi Takenobu,
2
Maki Tsujimura and 4Atsunao Marui
1

Asano Taiseikiso Engineering Co., Ltd.,
2-8-7, Kitaueno, Taitou-ku, 110-0014, Tokyo, Japan,
2
Faculty of Life and Environmental Sciences, University of Tsukuba
3
Department of Geoscience, Faculty of Sciences, Shizuoka University
4
Geological Survey of Japan, National Institute of Advanced Industrial Science and Technology (AIST)

Key words; Microbial DNA, groundwater flow path, Brand-new tracer

Abstract
To understanding better how the groundwater flow passes and its influence on the water chemistry we employ abrand-new method to analyze microbes with abundance and community constituents transported by groundwater.
In the previous studies, estimation of transit time and groundwater flow path by groundwater flow analysis,
estimation of averaged residence time of groundwater by chemical analysis such as various radioactive elements
and stable isotopes have been widely used in hydrological studies. These methods show an averaged value ofchemistry of the investigated water which was blended by various water with different sources and flow passes
in subsurface environment. In order to improve the reliability of the estimation of the transport of groundwater,
we focus on microbes in particular Microbial DNA. Particles represent the place from which they weretransported. Transported microbes express the environment being appropriate for their growth. Interaction
among rocks - groundwater - microorganisms is studied to evaluate biogeochemical influences on natural barrierof geological disposal facility for high-level-radioactive wastes. This study shows the possibility to reveal thegroundwater history such as groundwater flow and the environment from which groundwater flow throughanalyzing the density of prokaryotes and microbial community structure analysis.

1. Introduction
In order to evaluate the long-term performance of
natural barrier, it is also necessary to evaluate thestability of it on the order of a thousand years or more.
Long-term assessment of geological environment has
been studied such as long-term stability of geologicalenvironment (earthquake/fault, volcano/magmatism
activity, uplift/erosion and topography variation) andgeological environmental characteristics (geology,
rocks and groundwater). In this study, we try tounderstand geological condition, groundwatercondition and physicochemical properties throughgroundwater. We focus on groundwater influence on
geological environment.
In order to estimate the groundwater flow paths,
groundwater flow simulation and chemical tracer
approach have been carried out (e.g., Kazemi et al.,2006; Tosaka et al., 2010; Hasegawa et al., 2013;
Klaus and McDonnell, 2013). Although groundwaterflow simulation estimates the travel time ofgroundwater and groundwater flow paths, some areas
are difficult to adapt it. For example, the place, whichheterogeneous groundwater flow is dominated or

obtaining the physical information such as geology is
not easy. The mean residence time of groundwater
was estimated by analysis of chemical components ingroundwater such as various radioactive isotopes andstable isotopes (Hasegawa et al., 2013). In particular,
the ancient groundwater age (more than million
years) is accurately measured by 4He, 14C and 36Cl
(e.g., Kasemi et al., 2006; Munnich, 1957; Bentley etal., 1986). However, these methods lead just an
averaged value of investigated groundwater. And, theabovementioned results are different from each otherbecause they are analyzing different substance.
Althoughstudiesofrelationshipamongrocks-groundwater-microbes are under way as an
assessment of geochemical and microbial influencesin natural barriers (e.g., Nagaoka et al., 2009), themethod of microbial DNA for the estimation ofgroundwater flow paths is novel. We propose themicrobial DNA as a new tracer for grasping the
groundwater flow paths. Table 1 summarizes thecharacteristics of different methods including the
microbial method proposed in this paper for clarify
the groundwater flow system.

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan

Table 1. Comparison of three different approaches to clarify groundwater flow system.
Methods/Approachs

Tracers/Softwares

Obtained informations

Trait

Refferences

Groundwater flow
simulation

GETFLOWS,
MODFLOW, FEFLOW
etc.

Travel time
Groundwater flow path・It's inappropriate in areas where
heterogeneous groundwater flow isdominant
・It's not easy to adapt it in areas where
physical information such as geology
is difficult to obtain

Chemical tracer
approach

Stable/radioactive
isotopes, inorganic ions,
CFCs, SF6 etc.

Mean residence time・Different tracer has different information
・It shows averaged value of chemistry of
the sampled water

Hasegawa et al. (2013)
Klaus and McDonnell (2013)

Microbes

Biogeochemical influences
on natural barrier

・We focus on interaction among rocksgroundwater-microorganisms

Nagaoka et al. (2009)

Viruses

Short mean residence time
Preferential flow paths

・We focus on anthropogenic pollution

Hunt et al. (2014)

Microbial DNA

Groundwater flow history

・It expresses the environment
appropriate for their growthThis study

Microbial tracer
approach

Advantages of employing microbial DNA for the
groundwater flow system studies are shown in thefollowing,1) It is possible to estimate the ‘State of the place’such as physicochemical condition by microbialcommunity structure analysis.
2) It is possible to suggest the existence of a specificgroundwater flow path.
3) It is possible to estimate with high reproducibility
to use the different kind of tracers.
Therefore, in this paper, we introduce a case study of
estimating the groundwater flow paths by applying
the microbial DNA analysis in groundwater.

Kasemi et al. (2006)
Tosaka et al. (2010)

2. Methods
Analytical methods and the process are shown in
Figure 1, and the cautions of the analytical techniques
are also shown. Details of the method of microbial
analyses are given in Sugiyama et al. (submitted to
Biogeosciences).
Water samples were collected from boreholes andspring water. After counting total direct counts(TDC), microbial community was identified by using
fluorescence in situ hybridization (FISH) and
Next-generation sequencing.

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan

Figure 2. Geological map of Mt. Fuji (Segawa et al., 2015 and Sugiyama et al., 2017).
We collected the samples, which are rainwater (R1 to R5) and groundwater (SP-0m and GW-550m).
SP-0m is spring water and GW-550m is well water with deep groundwater (the depth is 550 m).
3. Results and Discussion
3.1. Case of Mt. Fuji
3.1.1. BackgroundA huge amount of groundwater is stored insubsurface environment of Mt. Fuji (3,776 m a.s.l.),which is the largest volcanic mountain in Japan(Figure 2). The groundwater characteristics aredescribed as follows (Sugiyama et al., 2017).
1. Temperature of groundwater ranges from 10 to 15o
C in the foot of Mt. Fuji.
2. Almost saturated dissolved oxygen was included ingroundwater.
3. Major reservoir is in the lava where the height isabout 100~200m.
4. The density of prokaryotes was very low (103cells/mL), although the density of prokaryotes ofrainwater and soil were 104 to 105 and 108 to 109
cells/mL, respectively.
5. Microbial components of groundwater weredifferent from that of rainwater.
6. Assuming piston flow transport an apparent
residence time was estimated to about 30 years by
36
Cl/Cl ratio (Tosaki et al., 2011).
3.1.2. Suggestion of the depth information ofgroundwater
Finding thermophilic bacterial DNA (e.g.,
Allobaculum stercoricanis) in spring water, whose
temperature was as low as 10 to 15oC throughout theyear, suggested at least some of the groundwater
source was expected at a depth of 600 m or greater,
based on a temperature gradient of 4oC/100 m,
because thermophilic prokaryotes are optimally
adapted to temperatures > 40oC. This depth is far
below the lava layer that was taken to be a substantial
pool of groundwater. Thus, analysis of microbial
DNA can suggested the depth information ofgroundwater (Segawa et al., 2015).

3.1.3. Possibility to reveal the groundwater flow
paths
Multiple analyses (stable isotope analysis,
chemical analysis and microbial analysis) performed
to elucidate the groundwater flow paths under theheavy rainfall for groundwater (SP-0m and
GW-550m; details were shown in Figure 2).
The oxygen isotopic ratio of spring water(SP-0m) was increased and silica concentration was
decreased during just a couple of weeks after theheavy rainfall exceeding 300 mm/event. The densityof Bacteria also sharply increased. This suggests thatheavy rainfall promotes shallow subsurface flow
contributing to the discharge in addition to thegroundwater in the deep aquifer.
On the other hand, although deep groundwater at
GW-550m did not show the signatures, which are
oxygen isotopic ratio and silica concentration, densityof Archaea was increased. Due to the density ofArchaea increased with depth in both terrestrial
subsurface environments (Kato et al., 2009) andmarine (Karner et al., 2001), this suggests thatstrengthened piston flow resulting from the heavy
rain transported archaeal particles from the deep
geologic layer along the groundwater flow.
Intensive observation suggests the presence ofshort time-scale flow pattern (contribution of shallow
groundwater flow) and long time-scale flow pattern(contribution of deep groundwater flow) rather than
mean residence time, which can help us to reveal thegroundwater flow paths. Though chemical analysis ofgroundwater shows an averaged value of theinvestigated water which was blended by various
water with different sources and routes in subsurface
environment, microbial DNA analysis may suggest
the place where they are originated, which may giveinformation of the source and flow paths of theinvestigated water.

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan

Figure 3. Location of Horonobe area in Hokkaido (Iwatsuki et al., 2009 a); Niizato et al., 2007 b) and c)).
Table 2. Density of prokaryotes in the three geological formations (*Kato et al., 2009).
Site(borehole)
DD-1

TDC
(cells/mL)

Yuchi

Sampling depth(m)714.5-715.5

DD-1

Yuchi

H17*

Koetoi

Formation

2.68x103

FDC
(%)
0.46

Bacteria(%)
14.9

Archaea(%)
1.1

942.5-943.5

1.87x105

0.56

48.8

1.5

35.5-37.5

6

2.57x10

0.55

7.51

2.6

5

HDB-6*

Koetoi-Wakkanai

281-3122.20x10

5.63

61.9

3.5

HDB-6*

Wakkanai364-4094

4.61x10

1.08

-

-

224-23358.36x10

0.83

14.0

10.3

6

HDB-4*

Minor fault

HDB-4*

Minor fault

281-291

5.06x10

1.82

6.41

10.9

HDB-4*

fault

475-482

6

0.45

12.8

19.1

1.13x10

Total Direct Count: TDC, Frequency of Dividing Cells: FDC.

3.2. Case of Horonobe
3.2.1. Background
The Horonobe coastal area, which is locatedalong northwestern coast of Hokkaido, is an alluvial
plain. It is a sedimentary layer that formed from thelatter half of the Paleogeneity to the present.
Sedimentary rocks and Quaternary strata are
distributed. Sedimentary rocks are formed including
Soya coal-bearing Formation, Onishibetsu Formation,
Masuporo Formation, Wakkanai Formation and
Koetoi Formation. Quaternary strata are formed
including Yuchi Formation, Sarabetsu Formation,
terrace deposits and alluvium deposits.
Kato et al. (2009) performed analysis of microbes
in groundwater collected by boreholes drilled into
sedimentary rock within two formations (Wakkanai
Formation and Koetoi Formation). The density ofprokaryotes was between 4.61 x 104 and 5.06 x 106
cells/mL, which is similar to the numbers observed at
the marine subsurface.

3.2.2. Distribution of prokaryotes in deep
groundwater
In this study, we performed microbial analysis ofdeep groundwater taken from different depth (Yuchi
Formation at 943m and 715m) in order to understandthe investigated groundwater environment andperform the scientific discussions from different
viewpoint based on previous studies.
Table 2 shows density of prokaryotes in threegeological formations and fault. Although density ofprokaryotes at 943m depth (1.87x105 cells/mL) was
not high as a sedimentary environment, which issimilar to the numbers published in Kato et al. (2009),
density at 715m depth was low (2.68x103 cells/mL)
as a sedimentary environment. Sample of 943m depth
in comparison to that of 715m depth shows highnumber of prokaryotes and high ratio of Bacteria(48.8%) next to Koetoi-Wakkanai bounder (61.9%).
The vertical distribution of the prokaryotes did notshow a simple decrease in abundance with increasing
depth.

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan

Characteristics of detected major genus levelprokaryotes are absolutely anaerobic or anaerobic
bacteria accounted for the majority. The
characteristics are mesophilic or thermophilic,
halophilic or halotolerant and alkalophilic bacteria.
We interpreted these prokaryotes live in the seawater
environment which is adaptive and inhabitable in thatenvironment. Methanoculleus and some of
Methanobacteriaceae, which produce methane under
the absolutely anaerobic environment is detected.
This suggests the metabolism of deep subsurface
environment.
It is important to clarify the presence of
microorganisms in the given subsurface environment
because cells with significant activity are observed in
the sample, which promotes a better understanding of
the current environment.
4. Conclusions
Summary of this study are shown in Table 3.
Though chemical analysis of groundwater shows anaveraged value of the investigated water which was
blended by various water with different sources androutes in subsurface environment, microbial DNA
analysis may suggest the place where they are
originated, which may give information of the source
and flow paths of the investigated water. It is
important to clarify the presence of microorganisms
in the given subsurface environment because cellswith significant activity are observed in the sample,
which promotes a better understanding of the current
environment. However, it is necessary to consider
some analytical techniques according to eachenvironment, and further accumulation of data isnecessary in the future.
The distribution of prokaryotes and their possiblefunctions are clearly constrained by the geologicalproperties of deep terrestrial subsurface environments.
Consequently, given the potential influence of
microbial activity on the geochemistry of a given
geological setting, particularly within the area ofinfluence of radioactive waste disposal candidate site,

it is necessary to consider the microbial community
composition and the geological attributes of the sites
at which they are the most prevalent.
Acknowledgements
This research is part of the outcome of received
order from AIST, sampling and well logging of deep
groundwater in Horonobe-cho, Teshio-gun, Hokkaido
and microbial analysis was performed at Shizuoka
University. This research was supported by Asano
Taiseikiso Engineering Co. Ltd. We thank Dr.
Kawanishi, Mr. Nakano and Ms. Sung for theircomments on an earlier version of the manuscript.
References
1. Kasemi, G. A., Lehr, J. H. and Perrochet, P.,
Groundwater Age. Hoboken, NJ: Jphn Wileyand Sons. 325 pp. 2006.
2. Tosaka, H., Mori, K., Tada, K., Tawara, Y.,
Yamashita, K.: A general-purpose terrestrial
fluids/heat flow simulator for watershed systemmanagement.
In:
IAHR
International
Groundwater Symposium 2010.
3. Hasegawa, T., Nakata, K., Kondo, H., Goto, K.,
Muramoto, S., Tomioka, Y., Goto, K. andKashiwaya, K.: Evaluation of Groundwater

Mobility in a Coastal Area Using
Groundwater Dating: Borehole Investigationin the West Coastal Area of the Miura
Peninsular, Journal of Geography, 122,
116-138, 2013.
4.

5.

6.

Klaus, J. and McDonnell, J. J.: Hydrographseparation using stable isotopes: Review andevaluation, Journal of Hydrology. 505, 47-64,
2013.
Munnich, K. O.: Messungen des C14 Gehaltes
von hartem Grundwasser. Naturwissenshaften.
44, 32-33, 1957.
Bentley, H. W., Phillip, F. M., Davis, S. N.,
Habermehl, M. A., Airey, P. L. Calf, G. E.,
Elmore, D. Gove, H. E. and Torgersen, T.:
Chlorine 36 dating of very old groundwater 1.

Table 3. Summary of interpreted of groundwater flow paths.

Study area

Comment on the methods spesific on the site

Mt. Fuji・(Due to the low biomass,)
A lot of sample are needed for filteration.

Horonobe

・Filtration was hindered by many obstacles.
・A lot of sample and time are needed
for filteration.
・Extracted DNA needs to be purified.

Obtained information spesific on the site
・Analysis of microbial community constituentssuggests the depth information of groundwater.
・Intensive observation suggests the presence of shortand long time-scale flow pattern rather than mean
residence time.
・The vertical distribution of the prokaryotes does notshow a simple decrease in abundance withincreasing depth.
・Analysis of microbial community constituentssuggests seawater mixture and deep subsurface
environmental metabolism.

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan

7.

8.

9.

The Great Artesian Basin, Australia. Water
Resour. Res. 22, 1991-2001, 1986.
Nagaoka, T., Hirano, S. and Nakamura, T.:
Microbial impacts on geochemistry evolution in
nuclear waste repository - Laboratory
experiment of microbially mediated redox
changes with lake sediment -, Environmental
Science Research Laboratory Rep., V08034,
2009.
Hunt, R. J., Borchardt, M. A. and Bradbury, K.
R.: Viruses as groundwater tracers: using
ecohydrology to characterize short travel times
in aquifers, Ground Water, 52, 187-193, 2014.
Sugiyama, A., Masuda, S., Nagaosa, K.,
Tsujimura, M. and Kato, K.: Tracking thedirect impact of rainfall on groundwater at Mt.
Fuji by multiple analyses including microbial
DNA, Biogeosciences, submitted.
10. Porter, K. G. and Feig, Y. S.: The use of DAPI
for identifying and counting aquatic
microflora, Limnol. Oceanogr., 25, 943-948,
1980.
11. DeLong, E. F., Archaea in coastal marine
environments. Proceedings of the National
Academy of Sciences of the United States ofAmerica, 89, 5685-5689, 1992.
12. Amann, R. I., Binder, B. J., Olson, R. J.,
Chisholm, S. W., Devereux, R. and Stahl, D. A.:
Combination of 16S ribosomal-RNA-targeted
oligonucleotide probes with flow-cytometry for
analyzing mixed microbial-populations, Appl.
Environ. Microb., 56, 1919-1925, 1990.
13. Pernthaler, A., Pernthaler, J. and Amann, R.:
Fluorescence in situ hybridization and catalyzed
reporter deposition for the identification ofmarine bacteria, Appl. Environ. Microbiol., 68,
3094-3101, 2002.
14. Teira, E., Reinthaler, T., Pernthaler, A.,
Pernthaler, J. and Herndl, G. J.: Combining
catalyzed reporter deposition-fluorescence insitu hybridization and microautoradiography todetect substrate utilization by bacteria andarchaea in the deep ocean, Appl. Environ.
Microbiol., 70, 4411-4414, 2004.
15. Stahl, D. A. and Amann, R. I.: Development andapplication of nucleic acid probes, In:
Stackebrandt, E. and Goodfellow, M. (eds.)
Nucleic acid techniques in bacterial systematics,
Wiley, J. and Chichester, S., 205-248, 1991.
16. Kimura, H., Ishibashi, J. I., Masuda, H., Kato,
K., and Hanada, S.: Selective phylogenetic
analysis targeting 16S rRNA genes ofhyperthermophilicarchaeainthedeep-subsurface hot biosphere, Appl. Environ.
Microbiol., 73, 2110-2117, 2007.
17. Somerville, C. C., Knight, I. T., Straube, W. L.
and Colwell, R. R.: Simple, rapid method for
direct isolation of nucleic-acids from aquatic

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

environments, Appl. Environ. Microbiol., 55,
548-554, 1989.
Claesson, M. J., Wang, Q., O’Sullivian, O.,
Greene-Diniz, R., Cole, J. M., Ross, R. P. andO’Toole, P. W.: Comparison of two
next-generation sequencing technologies forresolvinghighlycomplexmicrobiota
composition using tandem variable 16S rRNA
gene regions, Nucleic Acids Res., 38, 1-13,
2010.
Klindworth, A., Pruesse, E., Schweer, T.,
Peplies, J., Christian, Q., Horn, M. andGlöckner, F. O.: Evaluation of general 16S
ribosomal RNA gene PCR primers for classicaland net-generation sequencing-based diversity
studies, Nucleic Acids Res., 41, 1-11, 2012.
Takahashi, S., Tomita, J., Nishioka, K., Hisada,T. and Nishijima, M.: Development of aprokaryotic universal primer for simultaneous
analysis of Bacteria and Archaea using
next-generation sequencing, PLoS One, 9,e105592, 2014.
Sugiyama, A., Segawa, T., Furuta, T., Nagaosa,K., Tsujimura, M. and Kato, K.: Microbial
DNA; a possible tracer of groundwater, EGU
General Assembly 2017, 5883, 2017.
Tosaki, Y., Tase, N., Sasa, K., Takahashi, T. andNagashima, Y.: Estimation of groundwater
residence time using the 36Cl bomb pulse,
Ground Water, 49, 891-902, 2011.
Segawa, T., Sugiyama, A., Kinoshita, T., Sohrin,R., Nakano, T., Nagaosa, K., Greenidge, D. andKato, K.: Microbes in groundwater of a volcanic
mountain, Mt. Fuji; 16S rDNA phylogenetic
analysis as a possible indicator for the transportroutes of groundwater, Geomicrobiol. J., 31,
677-688, 2015.
Kato, K., Nagaosa, K., Kimura, H., Katsuyama,C., Hama, K., Kunimaru, T., Tsunogai, U. andAoki, K.: Unique distribution of deep
groundwater bacteria constrained by geologicalsetting, Environ. Microbiol. Rep., 1, 569-574,
2009.
Karner, B. M., DeLong, F. E. and Karl, M. D.:
Archaeal dominance in the mesopelagic zone of
the Pacific Ocean, Nature, 409, 507-510, 2001.
Iwastuki, T., Ishii, E. and Niizato, T.: Scenario
Development of Long-term Evolution for deep
Hydrochemical Conditions in Horonobe Area,
Hokkaido, Japan, Journal of Geography, 118,
700-716, 2009.
Niizato, T., Funaki, H. and Yasue, K.:
Paleogeography and geological evolution since
the Late Pliocene in and around the Horonobe
area, northern Hokkaido, The Journal of theGeological Society of Japan, 113, 119-135,
2007.

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan