Tohoku Forum for Creativity (TFC) International Symposium“Frontier of Understanding Earthʼs Interior and Dynamics”
Tohoku University
13:50-14:20, August 08, 2022

JAMSTEC 海洋研究開発機構
国立研究開発法人

JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY

Exploring the Limits ofthe Deep Subseaﬂoor Biosphere
Fumio Inagaki
Director, Principal Senior Scientist
Mantle Drilling Promotion Oﬃce,
Institute for Marine-Earth Exploration and Engineering (MarE3),
Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
andProfessor, Department of Earth Sciences
Graduate School of Science, Tohoku University1

The ﬁrst look of the subseaﬂoor biosphere“These ﬁndings
signiﬁcantly extend thedepth of the marine
biosphere and indicatethat sedimentaryorganic ma8er,
including molecularfossils, will undergo
con<nued bacterial
modiﬁca<on long a=er
burial.”John Parkes

Barry Cragg
Parkes et al., Nature, 1994; Hydrogeol. J., 2000.

2

Distribu(on of subseaﬂoor sedimentary biomass
Org-rich sediment athigh sedimenta0on
rate areas

Org-poor sediment at
low sedimenta2onrate areas

Cell numbers（Log10 cells/km2）
18192021

A total microbial cell abundance in marine sediment is 2.9 x 1029 cells,corresponding to 4Gt of biomass carbon on Earth.
Kallmeyer et al., PNAS, 2012. 3

Global distribution of aerobic and anaerobic sedimentary habitats
anaerobic
sediment

aerobic
sediment“Oxygen and aerobic communi<es may occur throughout the en<re sediment
sequence in 15-44% of the Paciﬁc and 9-37% of the global sea ﬂoor.”
D’Hondt, Inagaki, et al., Nature Geoscience, 2015.

4

The Deep Biosphere

Particles emitted fromdecaying elements (U, Th, K)

H2O2

H2O
RADIOLYSIS
RADICALS

H2Energy

4.5 Ga to present
long-term survival
over millions of years
DEEP LIFE

Mineralsin the rocks

Nutrients
Organic compounds
5Global distribution of radiolytic H2 production rates in marine sediment“…water radiolysis is the principal source of biologically accessible energy for
microbial communi<es in marine sediment older than a few million years.”
Sauvage, et al., Nature CommunicaDons, 2021.

6

Global survey of sedimentary microbiomes beneath the Ocean~300 deep-frozen sediment core samples collected from 40 diﬀerent sites
were analyzed by using the same sub-sampling and analy=cal approach7

Distribution and abundance of Archaea in the marine sedimentary biosphere“We es&mated thatarchaeal cells
cons&tute 37.3% of
the total microbialcells, corresponding
to 1.1 x 1029 cells in
the global subseaﬂoorsedimentarybiosphere.”Boxplot of archaeal propor2on in microbial 16S rRNA gene (%)
at each drilling site determined by microﬂuidic digital PCR.

Hoshino & Inagaki, ISME J., 2019.

8

Comparison of the microbial community compositions ofmarine sediment, seawater, and topsoil samples
Oxic sediment
Anoxic sediment

Topsoil

Seawater

The Earth’s microbiome contains a dis=nctly diﬀerent community.
Hoshino et al., PNAS, 2020.

9Global microbial amplicon sequence variants (ASVs)
predicted using diﬀerent models• The asympto=c rela=on model best
describe our data, predic=ng total
species diversity in marine sediment
to be 7.8 x 103 for Archaea and 3.2 x
104 for Bacteria.
• Compara=ve analysis of marine
sediment, topsoil, and seawater
microbiomes showed roughly similarlevels of global microbial richness.
• This result indicates that Bacteria are
more diverse than Archaea in Earth’s
global biosphere.
Hoshino et al., PNAS, 2020.

10

What are the ecological roles and limitsof the deep subseaﬂoor sedimentary biosphere?

11

IODP Expedition 337: The deep coalbed biosphere oﬀ Shimokita
The ﬁrst deep biosphere-dedicated riser drilling expedition
targeting deeply buried coalbeds in the Ocean

Water depth: 1200 m
Drilling depth: 2,466 mbsf
Inagaki, Hinrichs, Kubo, et al., IODP Sci. Prosp., 337, 201012

Cell abundance and geochemical proﬁles at Site C0020 oﬀ Shimokita“Mul&ple lines ofgeochemical,sedimentological, andmicrobiologicalevidences indicate thatmicrobial life is present
even down to 2.5 km
below the ocean ﬂoor
and may play animportant role in carbon
cycling.”Inagaki, et al., Science, 2015.

13

Taxonomic composition of indigenous bacterial communities
AB
050

Actinobacteria
Chloroflexi

Proteobacteria

Firmicutes

Bacterioidetes

Atribacteria (JS1)

Cyanobacteria

Spirochaetes

Gemmatimonadetes
Tenericutes

OD1

BHI80-139

Deinococcus-Thermus

Planctomycetes
Nitrospirae

Deferribacteres“deep”

deep-sea shallow marineor lacustrine

wetland, peat or tidal flat

4
5
2
3
1
16
21
10
12
13
11
14
29
17
26
19
928
25
24
31
32
30
23
22
8
6
7
15
20
27
18

“deep”

9.5
35.4
84.0
177.4
364.0
1279.11290.01372.61378.71492.31496.41608.3
1628.41740.31754.51762.5
1823.5
1922.7
1930.01940.5
1956.5
1964.81972.01989.5
1994.3
1998.42115.62202.22305.92403.32449.82458.8

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

“shallow”

100 Depth(mbsf)

4523
116
21
101213
1114
29
1726
19
928
25
24
3132
30
23
22867152027
18“shallow”

Bacterial 16S rRNA genes (%)

Fusobacteria
Synergistetes

Armatimonadetes

OP11

diatom ooze
siltstonesandstone
sand-silt

shale
coal

1

“…communi&es diﬀeredmarkedly from shallower
subseaﬂoor communi&es
and instead resembledorganotrophic
communi&es in forest
soils.”

0Bray-Curtis dissimilarity

Inagaki, et al., Science, 2015.

14

Taxonomic composition of indigenous bacterial communities
AB
050

ActinobacteriaChloroflexi

Proteobacteria

Firmicutes

Bacterioidetes

Atribacteria (JS1)

Cyanobacteria

Spirochaetes

Gemmatimonadetes
Tenericutes

OD1

BHI80-139

Deinococcus-Thermus

Planctomycetes
Nitrospirae

Deferribacteres“deep”

deep-sea shallow marineor lacustrine

wetland, peat or tidal flat

Hemipelagic
Lacustrine to coastal“deep”

9.5
35.4
84.0
177.4
364.0
1279.11290.01372.61378.71492.31496.41608.3
1628.41740.31754.51762.5
1823.5
1922.7
1930.01940.5
1956.5
1964.81972.01989.5
1994.3
1998.42115.62202.22305.92403.32449.82458.8

12345678910
1112131415161718
1920
21
22
23
2425
26
27
28
2930
31
32

“shallow”

100 Depth(mbsf)

4
52
3116
21
1012
13
11
14
29
1726
19
928
25
24
31
32
30
23
22
8
6
7
15
20
27
18

“shallow”

Bacterial 16S rRNA genes (%)

Terrestrial

Fusobacteria
Synergistetes

Armatimonadetes

OP11

diatom ooze
siltstonesandstone
sand-silt

shale
coal

1

4
52
3
1
16
21
101213
11
14
29
1726
19
928
25
24
31
32
30
23
22
8
6
7
15
20
27
18

This suggests that
terrigenous sediments
retain indigenous
microbial communi&es
over 20 millions of years
aKer burial in theseabed.

0Bray-Curtis dissimilarity

Inagaki, et al., Science, 2015.

15

Down-hanging sponge (DHS) ﬂow-through bioreactor system

Imachi et al., ISME J., 2011: Inagaki et al., Science, 2015; Imachi et al., ScienDﬁc Reports, 2019.

16

Inagaki et al., Science, 2015; Imachi et al., Sci. Repts., 2019.

Methanobacterium sp.
Imachi et al., ISME J., 2011.

The sedimentary fungi have experienced an evolutionary stasis
Paleo-deposi2onalenvironment

Depthbelow
seaﬂoor(m)I24






V538106
V538106C





C

Schizophyllum commune 20R-7-F01


isolated from 20 million-year-old lignite coalbeds
at 2 km-deep beneath the ocean

Genomes of subseaﬂoor fungal
isolates have 3 orders ofmagnitude lower nucleoDdediversity, subsDtuDon andhomologous recombinaDon rates
than other terrestrial strains.

Liu et al., Environ. Microbiol., 2012; Liu et al., iScience, 2022.

19The limits to the deep biosphere

DepthOrg

Cell concentra2ons in marine sediment.

•••

Temperature
PressurepH•••

Age
Porosity
Permeability•
••

Water
Nutrients
Energy substrates“Although the new study constrains some of theenvironmental factors that control the popula&on
density of subseaﬂoor microbes, cri&cal gaps remain
before we can conﬁdently model the size and extensionof deep subseaﬂoor life. For example, the limits of lifethat deﬁne the habitable zones remain poorlyconstrained.”
Hinrichs & Inagaki, Science, 2012:
D’Hondt, Inagaki, OrcuW, and Hinrichs, Oceanography, 2019.

20

Geophysical limits to the deep coalbed biosphere:
porosity and permeability“…one of the factors aﬀecDng thedecrease in microbial cellabundance with increasing depth
was a reducDon in nutrient andwater supply to indigenous
microbial communiDes as a resultof a decrease in porosity andpermeability due to sedimentcompacDon.”“…impermeable shale andsiltstone with small pores(<0.2µm) may act as barriers towater and energy-yielding
substrates for deep microbial life.”

Tanikawa et al., PEPS, 201821

Temperature is a critical factor for microbial persistence
Rate of biomolecule damage (k yr-1) and cell concentration (cells cm-3)
10-4

10-2

110104

106

108

1010

1002DNAhydrolysis

2.2

4.4

1000

26.0
2,466 mbsf
IODP Site C0020

60.0

Temperature (ºC)

Sediment depth (m)102global reg
reon the Pa sion line of cell cocific marg
ncentratio
insn

10-6
1

amino acidracemizaGon

The energy requirement
for biomolecule damage
ﬁxa=on increases withtemperature in situ.
The cell abundance
and the energy supply
should be balanced inthe deep biosphere.

242

104
Biomolecule damage rate
DNA depurination
glutamic acid and glutamine
alanineCell concentration
raw cell concentration (CK06-06)
most concervative cell concentration
based on taxonomic filtration

Inagaki et al., Science, 2015; Lever et al., FEMS Microbiol. Rev., 2015.

22

IODP Expedition 370:
Temperature limits to the deep biosphere oﬀ Muroto (T-Limit)

CapeMuroto

Nankai Trough~125 km

Site C0023: Water depth: 4776 m, Drilling depth: 1177 mbsf
Hinrichs, Inagaki, Heuer, et al., IODP Sci. Prosp. 370, 2016; Heuer et al., IODP Prel. Rept., 2017.

23

Substantial impact of temperature on microbial abundance and activity
Depth: 652.0 mbsf, 76℃（43R-2）“Multiple lines of geochemical and43R-3̲02.tif
microbiological evidence show thattemperature is a critical environmental
factor for deﬁning the abundance,activity, and distribution of microbial
cells in the deep subseaﬂoor
biosphere.”
Heuer, Inagaki, Morono, et al., Science, 2020. 24

Substantial impact of temperature on microbial abundance and activity
Depth: 1176.8 mbsf, 120℃（112R-2）

43R-3̲02.tif〜400 cells/cm3“..it moreovershows that life in
112R-1̲cell̲02.tif
the deep subseaﬂoor is NOT
constrained by an upper
Bars120ºC.”: 20 µm
temperature limit below

Heuer, Inagaki, Morono, et al., Science, 2020. 25

Geochemical signals of microbial metabolisms at Site C0023

mesophic methanogenesis
biogenic CH4

biogenic CH4

acetateincrease

thermophilic
methanogenesis

anaerobic
oxidation of
methane (AOM)thermogenic
CH4
hyperthermophilic
methanogenesis

acetateconsumption
hyperthermophilic
methanogenesis

sulfatefrom basaltic
aquiferGeochemical proﬁles of
microbial substrates
and products provide
evidence for microbialactivity down to the~16-million-year-old
oceanic crust.
The transient heating
events may have locallysterilized sediment, but
microbial cells, acetate
consumption, andmethanogenic activity
prevail again in >100ºC
sediments toward thesediment-basement
interface.

Heuer, Inagaki, Morono, et al., Science, 2020. 26

Rapid microbial life in the deep and hot subseaﬂoor biosphere“ At this high temperature, a small
community of microorganisms
subsisted with excepDonally highcell-speciﬁc rates of energy
metabolism, similar to rates insurface sediments and laboratory
cultures.”“This discovery is in stark contrast
to the extremely slow microbial
life otherwise observed in thedeep subseaﬂoor, but isconsistent with a high energy
demand needed to withstand andrepair thermal cell damage.”
Beulig et al., Nature CommunicaKons, 2022.

27

Some signiﬁcant questions remain to be addressed:••••

How is the deep biosphere evolu(on associated with plate
tectonics and mantle convec(on?
What are the roles of the deep crustal biosphere in Earth’s
element cycling?
Is plate tectonics required for the emergence of life?
Why the planet Earth is habitable?

28

The oceanic lithosphere has been largely unexplored

2050 Science Framework: Michibayashi et al., Oceanography,29

Alternation of oceanic volcanic glass:
Textural evidence of microbial activity“…autotrophic microbes living on the chemical energy of Earth’s
volcanic rocks could also exist within other planetary bodies
where ﬂuid ﬂow establishes gradients in redox poten&al, as
long as there are sources of required nutrients, water, andcarbon.”Fisk et al., Science, 1998.

IODP Science Plan 2013-2023.

30

Direct evidence for microbial activity in 3.5 Ma-oceanic basalt

Site 1301, IODP Expedi2on 301“Downcore changes incarbon and sulfur cycling
show discreate
geochemical intervals withchemoautotrophic d13C
signatures locallyaUenuated byheterotrophic metabolism.”
Lever et al., Science, 2013.

31

Expansive microbial habitats in ~100 Ma-aged crustal fractures
Microbes

Dense microbial
popula=ons
nontronite-ﬁllingveins (like bioﬁlms)
IODP
Exp. 329 within
South PaciﬁcGyre (104 Ma)

Suzuki et al., CommunicaDons Biology, 2020.

32

Is there life in the deep oceanic crust (and even in the upper mantle)?
Time-integrated ﬂuid ﬂuxes

Primary hypothesis
In oceanic crust cooler than thethermal limit to life, water-rockinteractions sustain microbial life
as deeply as seawater-derived
ﬂuid penetrates.
ü Microbes in deep oceanic crust arestrongly associated with speciﬁc
secondary mineral phases.
ü The composition of microbialcommunities and the processes
that sustain the communities
change with depth in the crust, inresponse to lithologic boundaries
and/or chemical and physicalgradients.

D.A. Teagle, COMPLEX Report, 1999.

33

The Earth

Oceanic Ridge
ChikyuCore Sample

Magma Chamber

Subduction zone

Upper Mantle©JAMSTEC

34

Technologies

SpaceSatellite

Atmosphere

Helicopter

Neutrino, Muon

ChikyuSlim-Riser Pipes
BOPPilot

Stage 1

Stage 2

Main

Stage 3

OceanPost

Stage 4

Sediment
Pillow Lava
Sheeted Dike

Casing Pipes

GabbroObservatory Sensors

MohoCore Sample
Drill Bit

Nature©JAMSTEC

Upper Mantle

nnnn

Geo-hazards
Geo-health
Carbon Neutral
Deep Earth Technology35

JAMSTEC 海洋研究開発機構
国立研究開発法人Acknowledgement•••••

JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGYODP/IODP, JR and Chikyu crews/operators
Globally distributed collaborators and friends (esp. ODP Leg 201, IODP Exp. 329, 337, 370)
Kochi Core Center, MarE3, Mantle Drilling Promotion Oﬃce, JAMSTEC
J-DESC, Deep Carbon Observatory (DCO), CIFAR Earth4D Program
Funding agencies: MEXT/JSPS, NSF36