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Researc
h o
n M
icro
bia
l R
esto
ration
of
Researc
h o
n M
icro
bia
l R
esto
ration
of
Researc
h o
n M
icro
bia
l R
esto
ration
of
Researc
h o
n M
icro
bia
l R
esto
ration
of
Meth
an
e D
ep
osit w
ith
Su
bsu
rface
Meth
an
e D
ep
osit w
ith
Su
bsu
rface
Meth
an
e D
ep
osit w
ith
Su
bsu
rface
Meth
an
e D
ep
osit w
ith
Su
bsu
rface
CO
CO
CO
CO
2 222S
equ
estr
ation
in
to t
he D
ep
lete
d
Sequ
estr
ation
in
to t
he D
ep
lete
d
Sequ
estr
ation
in
to t
he D
ep
lete
d
Sequ
estr
ation
in
to t
he D
ep
lete
d
Oil
Fie
lds
Oil
Fie
lds
Oil
Fie
lds
Oil
Fie
lds
Researc
h o
n M
icro
bia
l R
esto
ration
of
Researc
h o
n M
icro
bia
l R
esto
ration
of
Researc
h o
n M
icro
bia
l R
esto
ration
of
Researc
h o
n M
icro
bia
l R
esto
ration
of
Meth
an
e D
ep
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ith
Su
bsu
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Meth
an
e D
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ith
Su
bsu
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Meth
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ep
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ith
Su
bsu
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Meth
an
e D
ep
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ith
Su
bsu
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CO
CO
CO
CO
2 222S
equ
estr
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in
to t
he D
ep
lete
d
Sequ
estr
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in
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d
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Oil
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CH
UG
AI TE
CH
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K. Fuj
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atto
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atto
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K. Fuj
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atto
ri,
K. Fuj
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a, Y
. H
atto
ri, H
.Ohta
gaki
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(Jap
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Meta
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apan
Oil,
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and
Meta
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atl.
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apan
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Meta
ls N
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apan
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ls N
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apan
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and
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apan
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and
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and
Meta
ls N
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IEA EOR 31st Annual Symposium and W
orkshop
19th October 2010
Outlin
eO
utlin
eO
utlin
eO
utlin
e
•Background of this research
•Microbial restoration process
•Technical significance of this process
•Approaches of our research
•Our previous research and major
results
•Future challenges
•Conclusion
Background of this research
Background of this research
CO
CO
22DDisposal
isposal
TTechnologies
echnologies
Ocean (abyssal) CO
Ocean (abyssal) CO
22sequestration
sequestration
Aquifer storage of CO
Aquifer storage of CO
22
CO
CO
22Adsorption in coal seams
Adsorption in coal seams
Subsurface (
Subsurface (depleted
depletedoil field
oil field))
CO
CO
22disposal and storage
disposal and storage
Environmentally friendly form
of energy.
Environmentally friendly form
of energy.
One of the m
ost desirable sources of energy.
One of the m
ost desirable sources of energy.
CO2
COCO22
(A m
ajor factor of global warm
ing)
(A m
ajor factor of global warm
ing)
Focusing…
Natural gas
Natural gas
Natural gas
Development of perpetual sources of natural gas
Development of perpetual sources of natural gas
will become increasingly valuable in the future
will become increasingly valuable in the future
Evaluation of the technological possibility
Microbial restoration of natural gas, using subsurface sequestered CO
2
and indigenous anaerobes in oil field
Microbial restoration of methane
deposits with CO
2sequestration
Microbial restoration of methane
deposits with CO
2sequestration
Additives
MicrobesCH
CH
44
Methane
Methane
Restoration
Restoration
Oil
Oil
Gas
GasOil/Gas
Oil/Gas
Production
Production
Oil
Reservoir
CO
CO
22
CO
CO
22
Sequestration
Sequestration
３ ３３３
Field operation of in situ
microbial conversion
Field operation of in situ
microbial conversion
Injection well
Nutrient
Nutrient
Cap Rock
Efficient utilization of
indigenous anaerobes
Efficient utilization of
injection anaerobes
Nutrient
+
Anaerobes
Nutrient
+
Anaerobes
The m
echanism of microbial conversion
The m
echanism of microbial conversion
Injection
* Inorganic reaction of therm
al water and
reducing agent of rock (i.e. Fe)
*Hydrogen production with rock form
ation
(i.e. serpentine rock)
DDepleted
epleted
oil
oil/gas field
/gas field
Organic
matter
(i.e.carbohydrate,
hydrocarbone)
H２ ２２２
Hydrogen-producing
therm
ophiliceubacteria
Methane-producing
therm
ophilicarchaea
CO
２ ２２２CH4
2H
２ ２２２O
4H
２ ２２２+
+
４ ４４４Economically viable velocity :0.4Nml/L-m
ed/h
Economically viable velocity:1.6Nml/L-m
ed/h
HPTB
MPTA
CH4
CH4
CO
2
CO
2③ ③③③③ ③③③CCS (
CCS (CO
CO
22ccapture
apture
and Storage
and Storage))
① ①①①① ①①①Production
Production
resources (CH
resources (CH
44))
② ②②②② ②②②Combustion
Combustion
・ ・・・・ ・・・Emission
Emission
④ ④④④④ ④④④Microbial
Microbial
conversion
conversion
(CO
(CO
22to CH
to CH
44))
Sustainable carbon recycling system
by subsurface anaerobes
Sustainable carbon recycling system
by subsurface anaerobes
Task of enveloping the
petroleum and natural
gas resoueces
・ ・・・reducing CO2 emissions
・ ・・・Effective utilization of
Depleted Oil Fields
・ ・・・Recovery of residual crude
oil
・ ・・・Development of sources of
perpetual natural gas
Remedies
CCS
(Carbon dioxide Capture and Storage )
・ ・・・・ ・・・M
icro
bia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
Mic
robia
l R
est
ora
tion
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
of M
eth
ane D
eposi
ts
(MR
MD
) sy
stem
(MR
MD
) sy
stem
(MR
MD
) sy
stem
(MR
MD
) sy
stem
(MR
MD
) sy
stem
(MR
MD
) sy
stem
(MR
MD
) sy
stem
(MR
MD
) sy
stem
Technical significance of the Microbial
restoration process
Technical significance of the Microbial
restoration process
Approaches of our research
(already conducted)
(Step 1) Analysis of microbial diversity in the oil reservoir
(Step 1) Analysis of microbial diversity in the oil reservoir
(reservoir brine
(reservoir brineand crude oil).
and crude oil).
(Step 2) Ascertainment of hydrogen and m
ethane production by
(Step 2) Ascertainment of hydrogen and m
ethane production by
indigenous anaerobes.
indigenous anaerobes.
(Step 3) Feasibility study of MRMD system by primitive
(Step 3) Feasibility study of MRMD system by primitive economic
economic
assessment.
assessment.
(Step 4) Evaluation of hydrogen and m
ethane production potential
(Step 4) Evaluation of hydrogen and m
ethane production potentialunder
under
real reservoir condition and estimation of microbi
real reservoir condition and estimation of microbi al methane
al methane
producing pathway.
producing pathway.
(Step 5) Direct verification of microbial conversions
(Step 5) Direct verification of microbial conversions
(crude oil to hydrogen and
(crude oil to hydrogen and CO2 to CH4
CO2 to CH4).).
(Step 6) Detailed studies of accelerating conditions for the vel
(Step 6) Detailed studies of accelerating conditions for the vel ocity of
ocity of
hydrogen production by HPTB.
hydrogen production by HPTB.
(Step 7) Detailed studies of accelerating conditions for the vel
(Step 7) Detailed studies of accelerating conditions for the vel ocity of
ocity of
methane production and conversion efficiency to m
methane production and conversion efficiency to m
ethane
ethane
by MPTA
by MPTA
(Step 8)
(Step 8)Construction of
Construction of suitable numerical sim
ulation m
odel
suitable numerical sim
ulation m
odelfor
for MRMD
MRMD
system in order to evaluate experimental results
system in order to evaluate experimental results step1 to 7..
step1 to 7..
Sampling of
Sampling of
Reservoir brine
Reservoir brine
3-5
Res.Pressure (MPa)
50-75
Res. Temp
（ （（（℃ ℃℃℃
） ）））
2600-12000
Salinity conc. (ppm)
Depth
（ （（（kｍ
）ｍ
）ｍ
）ｍ
）1.2-1.6
Yabase
（ （（（Akita） ）））
200 Km
Candidate
Candidate
Fields
Fields
Sea of Japan
Sea of Japan
Yabase
Nibroi
Nibroi
Pacific
Pacific
Ocean
Ocean
Matsuzaki
Minami Aga
Minami Nagaoka
Higashi
Kashiwazaki
Kubiki
Meiji
Offshore Iwaki
Tokyo Line
Tokyo
Naruto
Oil Fields
Oil Fields
Gas Fields
Gas Fields
Our previous research and m
ajor results
Our previous research and m
ajor results
1.
1. Analysis of microbial diversity
Analysis of microbial diversity(( sampling from
sampling from
well head
well head))
Sampling of
Sampling of
crude oil
crude oil
Descent of
Descent of
sampler
sampler
Direct recovery of
bottom hole sample
Direct recovery of
Direct recovery of
bottom hole sample
bottom hole sample
Analysis of microbial diversity
Analysis of microbial diversity(( sampling from
sampling from
bottom hole
bottom hole))
Yabaseoil field in Japan
Prefilter
（ （（（φ φφφ=2.7
μ μμμm)
Cells collecting
filetr
（ （（（φ φφφ=0.2
μ μμμm)
DNA
DNA
Extraction
Extraction
Sample concentration
Sample concentration
PCR
PCR
(for
(for Eubacteria
Eubacteria))
(for
(for Archaea
Archaea))
DN
A S
equ
ence
DN
A S
equ
ence
DN
A S
equ
ence
DN
A S
equ
ence
DN
A S
equ
ence
DN
A S
equ
ence
DN
A S
equ
ence
DN
A S
equ
ence
CGTGGAGGAT
GACTTATTTC
GGTGCGTAAC
TA…
ＤＧ
ＧＥ
ＤＧ
ＧＥ
ＤＧ
ＧＥ
ＤＧ
ＧＥ
ＤＧ
ＧＥ
ＤＧ
ＧＥ
ＤＧ
ＧＥ
ＤＧ
ＧＥ
Anal
ysis
A
nal
ysis
A
nal
ysis
A
nal
ysis
A
nal
ysis
A
nal
ysis
A
nal
ysis
A
nal
ysis
（ （（（（ （（（D
enat
uring
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Den
aturing
Gra
dient
Gel Ele
ctro
phore
sis)
Investigation of
Investigation of mmicrobial
icrobial ddiversity
iversity
①①Thermotoga
Thermotogasp.
sp.
②②Petrotoga
Petrotogasp.
sp.
③③Thermoanaerobacter
Thermoanaerobactersp.
sp.
④④Anaerobaculum
Anaerobaculumsp.
sp.
⑤⑤Thermaacetogenium
Thermaacetogenium
phaeum
phaeum
⑥⑥Clostridium
Clostridium sp.
sp.
⑦⑦Geobacter
Geobactersp.
sp.
⑧⑧Desulfotomaculum
Desulfotomaculumsp.
sp.
⑨⑨Desulfitobacter
Desulfitobactersp.
sp.
Eubacteria
Eubacteria
①①Methanobacter
Methanobacter
thermoautotrophicus
thermoautotrophicus
②②Methanoculleus
Methanoculleussp.
sp.
③③Methanocalculus
Methanocalculus
halotorerance
halotorerance
④④Methanosarcina
Methanosarcinamazeii
mazeii
⑤⑤Methanosaeta
Methanosaetasp.
sp.
Archaea
Archaea
Some strains of HPTB and MPHTA which have been discovered
Some strains of HPTB and MPHTA which have been discovered
in oil reservoirs around the world, were successfully isolat
in oil reservoirs around the world, were successfully isolat ed.
ed.
Anaerobes
Anaerobes ddiscovered
iscovered from
from
ooililand
and ggas
as rreservoirs
eservoirs
・ ・・・N NNN
2 222/C
O/C
O/C
O/C
O2 222
・ ・・・D
eoxid
izer
Deoxid
izer
Deoxid
izer
Deoxid
izer
・ ・・・S
terile
brin
eS
terile
brin
eS
terile
brin
eS
terile
brin
eor
Sim
ula
ted
or
Sim
ula
ted
or
Sim
ula
ted
or
Sim
ula
ted
brin
eb
rin
eb
rin
eb
rin
e・ ・・・
Isola
tes
Isola
tes
Isola
tes
Isola
tes
・ ・・・C
rud
e o
ilC
rud
e o
ilC
rud
e o
ilC
rud
e o
il
・ ・・・In
cu
bation
In
cu
bation
In
cu
bation
In
cu
bation
at
75
at
75
at
75
at
75
℃ ℃℃℃
2.
2. Evaluation of hydrogen and m
ethane production potential
Evaluation of hydrogen and m
ethane production potential
under reservoir condition and estimation of microbial
under reservoir condition and estimation of microbial
methane producing pathway.
methane producing pathway.
On site inoculation
00
50
50
100
100
150
150
200
200
250
250
300
300
00100
100
200
200
300
300
400
400
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod （ （（（（ （（（
day
dayday
day
day
dayday
day
） ）））） ）））
Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH44444444generation generation generation generation generation generation generation generation （（（（（（（（Nml Nml Nml Nml Nml Nml Nml Nml/L/L/L/L/L/L/L/L））））））））
ARAR-- 3939
ARAR-- 3939
ARAR-- 3939
OR
OR-- 99
７７
ARAR-- 3939
Electron
acceptor
Temp.
Na2S
L-cystein
- +
50℃
55℃
Na2S
-50℃
Na2S
Na2S
-
55℃
+55℃
Ascertainment of hydrogen and m
ethane production
Ascertainment of hydrogen and m
ethane production
potential by indigenous anaerobes .
potential by indigenous anaerobes .
Velocity of CH4generation ： ：：：0.13Nml/L/h
Hydrogen production
Hydrogen productionfrom
from
crude o
crude oililand
and m
ethane generation
methane generation
(indigenous anaerobes in brine, reservoir temp., 0.2MPa)
(indigenous anaerobes in brine, reservoir temp., 0.2MPa)
Deoxidizer
For isolates ; 105, For reservoir brine ; 108
Initial conc. of microbes (cells/m
l)
5 (at YabaseOil Field)
Initial pressure (M
Pa)
50, 75 (at YabaseOil Field)
Initial temperature (degC)
Water phase 200 , Gas phase 800
Culture scale (m
l)
Values
Specifications
N2
ダ ブ ル エ ン ド ボ ン ベダ ブ ル エ ン ド ボ ン ベダ ブ ル エ ン ド ボ ン ベダ ブ ル エ ン ド ボ ン ベ
１ １１１L
容 容容容1000ml
<Setting>
<Setting>
・ ・・・・ ・・・Reservoir brine
Reservoir brine
(Sterile or non
(Sterile or non-- Sterile)
Sterile)
・ ・・・・ ・・・Nutrient with deducing
Nutrient with deducing
agent (Glucose, Yeast extract)
agent (Glucose, Yeast extract)
For Hydrogen producing
therm
ophiliceubacteria
ダ ブ ル エ ン ド ボ ン ベダ ブ ル エ ン ド ボ ン ベダ ブ ル エ ン ド ボ ン ベダ ブ ル エ ン ド ボ ン ベ
１ １１１L
容 容容容1000ml
For Methane producing
therm
ophilicarchaea
H2/
CO
2
<Monitoring>
<Monitoring>
・ ・・・・ ・・・HH22, CH
, CH
4
4 : GC
: GC
・ ・・・・ ・・・Organic Acids : HPLC
Organic Acids : HPLC
・ ・・・・ ・・・Anaerobes:
Anaerobes: RTm
RTm
--PCR
PCR
Culture experiments using cylinder bottles
Culture experiments using cylinder bottles
(indigenous anaerobes, reservoir temp. and pressure)
(indigenous anaerobes, reservoir temp. and pressure)
Core holder
N2
Accumulator
Berea
sandstone
70
10
Culture scale
Water phase (ml)
Gas phase (m
l)
300
Perm
eability (m
d)
80
Pore volume (ml)
105
108
Initial conc. of
microbes (cells/m
l)
For Isolates
For Reservoir brine
5
Initial pressure (MPa)*
50, 75
Initial temp. (degC)*
11.3
Surface area (cm
2)
23.4
Porosity (%)
30
Length (cm)
Values
Specifications
Medium
+
Microbes
Vacuum pump
H2/
CO2
<Setting>
<Setting>
・ ・・・・ ・・・Reservoir
Reservoir
brine
brine
(Sterile or
(Sterile or
non
non-- Sterile)
Sterile)
・ ・・・・ ・・・Nutrient
Nutrient
with deducing
with deducing
agent
agent
(Glucose,
(Glucose,
Yeast extract)
Yeast extract)
<Monitoring>
<Monitoring>
* * Values of
Values of Yabase
YabaseOil Field
Oil Field
・ ・・・・ ・・・HH22, CH
, CH
44: GC
: GC
・ ・・・・ ・・・Organic Acids : HPLC
Organic Acids : HPLC
・ ・・・・ ・・・Anaerobes:
Anaerobes: RTm
RTm
--PCR
PCR
Culture experiments using
Culture experiments using berea
bereasandstone cores
sandstone cores
(indigenous anaerobes, reservoir temp., pressure,
(indigenous anaerobes, reservoir temp., pressure, microspace
microspace))
0
50
100
150
200
250
050
100
150
200
250
培養
日数
（日
）
メタン生成量（Nml/L）
ＣＯ
ＣＯ
ＣＯ
ＣＯ
２ ２２２添
加添
加添
加添
加、 、、、
無添
加無
添加
無添
加無
添加
ケー
スケ
ース
ケー
スケ
ース
培養
後培
養後
培養
後培
養後
の のののメ
タン
メタ
ンメ
タン
メタ
ン生
成菌
相比
較生
成菌
相比
較生
成菌
相比
較生
成菌
相比
較2M
3M
4M
5M
6M
7M
初期
初期
初期
初期
Clostridiaceae
Clostridiaceaestr
str..
（ （（（（ （（（Thermaacetogenium
Thermaacetogeniumphaeum
phaeum
） ）））） ）））
Desulfotomaculum
Desulfotomaculumthermobenzoicum
thermobenzoicum
Thermotoga
Thermotogasp.
sp.
Coprothermobacter
Coprothermobactersp.
sp.
Methane generation
Methane generationunder real reservoir condition
under real reservoir condition
(indigenous anaerobes in brine, reservoir temp., 5MPa)
(indigenous anaerobes in brine, reservoir temp., 5MPa)
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod （ （（（（ （（（
day
dayday
day
day
dayday
day
） ）））） ）））
Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH44444444generation generation generation generation generation generation generation generation
（（（（（（（（Nml Nml Nml Nml Nml Nml Nml Nml/L/L/L/L/L/L/L/L））））））））
Hydrogenotroph
Hydrogenotrophmethane
methane
producing
producing archaea
archaea
・ ・・・・ ・・・M
ethanobacte
rium
Met
hanobacte
rium
therm
oauto
trophic
um
therm
oauto
trophic
um
・ ・・・・ ・・・M
ethanoculleu
sM
ethanoculleu
ssp
.sp
.
・ ・・・・ ・・・Arc
haeoglo
bus
Arc
haeoglo
bussp.
sp.
Methane
Crude oil
(Alkane,aromatic
hydrocarbone）
Hydorogen
Acetic acid
Acetoclast
Acetoclasthydrogen
hydrogen
producing bacteria
producing bacteria
・ ・・・・ ・・・Clo
stridia
ceae
Clo
stridia
ceaestr
str..
（ （（（（ （（（Therm
aaceto
geniu
mTherm
aaceto
geniu
mphaeum
phaeum
） ）））） ）））
・ ・・・・ ・・・Therm
oanaero
bacte
rTherm
oanaero
bacte
rspsp
..
・ ・・・・ ・・・Syntrophom
onas
Syntrophom
onassp.
sp.
Hydrocarbon degrading
Hydrocarbon degrading
hydrogen producing bacteria
hydrogen producing bacteria
・ ・・・・ ・・・Petroto
ga
Petroto
gaspsp
. (
. ( m
ioth
erm
am
ioth
erm
a))
・ ・・・・ ・・・Therm
oto
ga.
Therm
oto
ga. spsp..
（ （（（（ （（（hypogea
hypogea） ）））） ）））
・ ・・・・ ・・・Therm
oanaero
bacte
rTherm
oanaero
bacte
rspsp
..
・ ・・・・ ・・・Therm
odesu
lfobacte
rium
Therm
odesu
lfobacte
rium
spsp..
・ ・・・・ ・・・D
esu
lfoto
macullum
Desu
lfoto
macullum
therm
obenzo
icum
therm
obenzo
icum
Microbial methane producing pathway in real reservoir
175
Met
hano
ther
mob
acte
rM
etha
noth
erm
obac
ter
Met
hano
ther
mob
acte
rM
etha
noth
erm
obac
ter
ther
moa
utot
roph
icus
ther
moa
utot
roph
icus
ther
moa
utot
roph
icus
ther
moa
utot
roph
icus
11
1111
11
Num
ber
of hy
droge
n p
rodu
cin
g an
aero
bes
Num
ber
of hy
droge
n p
rodu
cin
g an
aero
bes
Num
ber
of hy
droge
n p
rodu
cin
g an
aero
bes
Num
ber
of hy
droge
n p
rodu
cin
g an
aero
bes
75
50
50, 7
5
75
50
50
75
50
Tem
p. o
f Tem
p. o
f Tem
p. o
f Tem
p. o
f is
ola
tion
isola
tion
isola
tion
isola
tion
（ （（（℃ ℃℃℃
） ）））
2 2221222221
Num
ber
Num
ber
Num
ber
Num
ber
of
ofof
of
isola
tes
isola
tes
isola
tes
isola
tes
Met
hano
culle
usM
etha
nocu
lleus
Met
hano
culle
usM
etha
nocu
lleus
palm
olei
palm
olei
palm
olei
palm
olei
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
sp.
sp.
sp.
sp.
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
subt
erra
neus
subt
erra
neus
subt
erra
neus
subt
erra
neus
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
The
rmoa
naer
obac
ter
teng
cong
ensi
ste
ngco
ngen
sis
teng
cong
ensi
ste
ngco
ngen
sis
The
rmot
oga
The
rmot
oga
The
rmot
oga
The
rmot
oga
naph
toph
ilana
phto
phila
naph
toph
ilana
phto
phila
Num
ber
of m
eth
ane p
rodu
cin
g an
aero
bes
Num
ber
of m
eth
ane p
rodu
cin
g an
aero
bes
Num
ber
of m
eth
ane p
rodu
cin
g an
aero
bes
Num
ber
of m
eth
ane p
rodu
cin
g an
aero
bes
The
rmot
oga
The
rmot
oga
The
rmot
oga
The
rmot
oga
sp.
sp.
sp.
sp.
Ana
erob
acul
umA
naer
obac
ulum
Ana
erob
acul
umA
naer
obac
ulum
ther
mot
errn
umth
erm
oter
rnum
ther
mot
errn
umth
erm
oter
rnum
Str
ain
Str
ain
Str
ain
Str
ain
Isolates participated in MRMD system
Isolates
Isolates participated in MRMD system
participated in MRMD system
※ ※※※Alm
ost all anaerobes have isolated
from bottom hole sample
3. Isolation of hydrogen and m
ethane producing m
icrobes
3. Isolation of hydrogen and m
ethane producing m
icrobes
participated in MRMD system and Direct verification of
participated in MRMD system and Direct verification of
microbial reactions (crude oil to hydrogen and CO
microbial reactions (crude oil to hydrogen and CO
22to CH
to CH
44).).
Ingredients of crude oil (
Ingredients of crude oil (hexadecane
hexadecane, ,
toluene
toluene） ）））） ）））labeled by stable isotope (
labeled by stable isotope (13
13C)
C)
・ ・・・・ ・・・Extraction of
Extraction of
DNA/RNA
DNA/RNA
・ ・・・・ ・・・Density gradient
Density gradient
centrifugation
centrifugation
13C
13C
DNA/RNA from
DNA/RNA from
anaerobes which can
anaerobes which can
use the Ingredients of
use the Ingredients of
crude oil
crude oil
13C
13C
13
13C
C fraction
fraction
13C
13C
HPYB (Therm
oanaero
bacte
rsp
. )
13C
13C
DNA
DNA
analysis
analysis
12
12C fraction
C fraction
13C
13C
Substrate
Substrate
＊ ＊＊＊＊ ＊＊＊13
13CC-- DNA Including
DNA Including Thermoanaerobacter
Thermoanaerobactersp.
sp.
have
have detected
detected
＊ ＊＊＊＊ ＊＊＊Conversion ratio of hydrogen from hexadecane
Conversion ratio of hydrogen from hexadecane
based on the hydrogen balance
based on the hydrogen balance ： ：：：： ：：：approx.
approx. 60
60％ ％％％％ ％％％
＊ ＊＊＊＊ ＊＊＊Conversion ratio of hydrogen from toluene
Conversion ratio of hydrogen from toluene
based on the hydrogen balance
based on the hydrogen balance ： ：：：： ：：：approx. 5
approx. 5％ ％％％％ ％％％
M ① ①①①
SIP
SIP-- DGGE
DGGE
analysis
analysis
Incubation (
Incubation (80
80℃ ℃℃℃℃ ℃℃℃XX100
100days)
days)
Direct verification of hydrogen production by HPTB Id
entification
Identification
22
13C
DNA from m
etabolic
DNA from m
etabolic
microbes
microbes
13C
13C
13C
13C
13C
13C
13C
DNA analysis
DNA analysis
13CH4
13C
13C
Metabolite
Metabolite
13CCalculate the m
icrobial
Calculate the m
icrobial
conversion rate
conversion rate
CO
CO
22labeled by
labeled by
stable isotope (
stable isotope (13
13C)
C)
13CH4
13CH4
・ ・・・・ ・・・Extraction of
Extraction of
DNA/RNA
DNA/RNA
・ ・・・・ ・・・Density gradient
Density gradient
centrifugation
centrifugation
MPTA
MPTA
（ （（（（ （（（M
ethanoth
erm
obacte
rM
ethanoth
erm
obacte
r
therm
oauto
trophic
us
therm
oauto
trophic
us
） ）））） ）））
Identification
Identification
13
13C
C fraction
fraction
Incubation (
Incubation (80
80℃ ℃℃℃℃ ℃℃℃X30days)
X30days)
＊ ＊＊＊＊ ＊＊＊13
13CH
CH
44have
have detected.
detected.
＊ ＊＊＊＊ ＊＊＊Conversion ratio of methane
Conversion ratio of methane
from CO
from CO
22injecting head space
injecting head space
based on the carbon balance
based on the carbon balance
： ：：：： ：：：approx. 55
approx. 55％ ％％％％ ％％％
Direct verification of methane production by MPTA
4.
4.
4.
4.
4.
4.
4.
4. 水
素水
素水
素水
素・ ・・・
メタ
ンメ
タン
メタ
ンメ
タン
生成
促進
条件
生成
促進
条件
生成
促進
条件
生成
促進
条件
の ののの検
討検
討検
討検
討水
素水
素水
素水
素・ ・・・
メタ
ンメ
タン
メタ
ンメ
タン
生成
促進
条件
生成
促進
条件
生成
促進
条件
生成
促進
条件
の ののの検
討検
討検
討検
討Injection of
Injection of
NN22Gas
Gas
or
or
NN22:CO
:CO
22=90:10
=90:10 vol
vol %%
・ ・・・・ ・・・Incubation at 55
Incubation at 55℃ ℃℃℃℃ ℃℃℃
or 75
or 75℃ ℃℃℃℃ ℃℃℃,,
・ ・・・・ ・・・Analysis of head space gas
Analysis of head space gas
(H(H22and CH
and CH
44) by GC
) by GC
Sterilized
Sterilized
reservoir
reservoir
brine
brine
・ ・・・・ ・・・Crude oil
Crude oil
・ ・・・・ ・・・Isolates (HPTB)
Isolates (HPTB)
・ ・・・・ ・・・Additives
Additives
１２
１２
１２
１２
4. Development of accelerating technique by additives
4. Development of accelerating technique by additives
＊ ＊＊＊Economically viable velocity
Hydorgen:1.6Nml/L-m
ed/h
Methane : 0.4Nml/L-m
ed/h
電子
受容
体の
検討
（Ｈ
ＹＨ
-8+H
YH
-10）
0.00
5.00
10.
00
15.
00
20.
00
25.
00
05
10
15
20
2530
培養
日数
（日
）
Ｈ２ (Nml/L)
24 H
YH
-8
26 H
YH
-8+H
YH
-10 F
e 1
mM
Adding Fe, Se
Unde
r coexi
stence o
f U
nde
r coexi
stence o
f U
nde
r coexi
stence o
f U
nde
r coexi
stence o
f SO
42-at 75℃ ℃℃℃
Non additives
培養
日数
培養
日数
培養
日数
培養
日数
（ （（（日 日日日
） ）））
メタ
ン生
成に
対す
るC
O2濃
度の
影響
0
500
1000
1500
2000
2500
05
10
15
20
2530
培養
期間
（da
ys)
メタン濃度（累計）（Nml/L-med)
No.
1
No.
2
No.
3
No.
4
No.
5
No.
No. 55
No.
No. 44
No.
No. 33
No.
No. 22
No.1
No.1
Series
Series
HH22/C
O/C
O22==550/
0/ 5500
HH22/C
O/C
O22==660/
0/ 4400
HH22/C
O/C
O22==770/
0/ 3300
HH22/C
O/C
O22==880/
0/ 2200
HH22/C
O/C
O22==990/
0/ 1100
Providing gaseous CO
Providing gaseous CO
22・ ・・・・ ・・・Sterile brine
Sterile brine
・ ・・・・ ・・・0.2MPa
0.2MPa
・ ・・・・ ・・・75
75℃ ℃℃℃℃ ℃℃℃
・ ・・・・ ・・・pH=8
pH=8
Thermotoga.sp
Control
Intensity
Tim
e(m
in)
040
20
10
30
Non additives
Adding Fe, Se
Hydrocarbon
degradation
Evaluation of accelerating additives
Evaluation of accelerating additives
for hydrogen production
for hydrogen production
Evaluation of accelerating CO
Evaluation of accelerating CO
22Conc.
Conc.
for methane production
for methane production
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod （ （（（（ （（（
day
dayday
day
day
dayday
day
） ）））） ）））
Amount of H Amount of H Amount of H Amount of H Amount of H Amount of H Amount of H Amount of H22222222generation generation generation generation generation generation generation generation
（（（（（（（（Nml Nml Nml Nml Nml Nml Nml Nml/L/L/L/L/L/L/L/L））））））））
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod
Incubat
ion p
eriod （ （（（（ （（（
day
dayday
day
day
dayday
day
） ）））） ）））
Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH Amount of CH44444444generation generation generation generation generation generation generation generation （（（（（（（（Nml Nml Nml Nml Nml Nml Nml Nml/L/L/L/L/L/L/L/L））））））））
Observation of microbial cells adsorption on the surface of
Observation of microbial cells adsorption on the surface of
reservoir rock by high temperature and pressure resistanc
reservoir rock by high temperature and pressure resistance cell.
e cell.
5. Development of accelerating technique by cells adsorption
5. Development of accelerating technique by cells adsorption
10µm
10µm
10µm
10µm
(1)
(1) Alm
ost all m
icrobial cells have adsorbed into the reservoir rock
Alm
ost all m
icrobial cells have adsorbed into the reservoir rock. .
(2)Adsorption ratio of
(2)Adsorption ratio of microbial cells
microbial cellshas able to change by the decrease
has able to change by the decrease
in pH value and the increase in salinity
in pH value and the increase in salinity
(3)
(3) hydrogen production and m
ethane production by adsorbed
hydrogen production and m
ethane production by adsorbed cells
cells has
has
become higher than that of free bacteria in porous m
edia
become higher than that of free bacteria in porous m
edia
Results
Futu
re c
hal
lenge
sFutu
re c
hal
lenge
sFutu
re c
hal
lenge
sFutu
re c
hal
lenge
sFutu
re c
hal
lenge
sFutu
re c
hal
lenge
sFutu
re c
hal
lenge
sFutu
re c
hal
lenge
s
Crude oil
Hydrocarbon degrading
Hydrocarbon degrading
Hydrogen producing
Hydrogen producing
bacteria
bacteria
Hydrogenotroph
Hydrogenotroph
methane producing
methane producing
archaea
archaea
Gaseous
Gaseous
CO
CO
22
Soluble
Soluble
CO
CO
22
(Step 9) Design a field operation for MRMD system
(Step 9) Design a field operation for MRMD system
(including the
(including the injectivity
injectivityof HPTB and MPTA cells into porous
of HPTB and MPTA cells into porous
media, and the design of state of CO2 that can
media, and the design of state of CO2 that can become the
become the
substrate of the m
ethane production).
substrate of the m
ethane production).
(Step 10) The grasping of microbial diversity
(Step 10) The grasping of microbial diversity
related to MRMD system in the domestic
related to MRMD system in the domestic
and overseas oil reservoir.
and overseas oil reservoir.
(Step 11) Ascertainment of conditions for
(Step 11) Ascertainment of conditions for
methane production by indigenous
methane production by indigenous
anaerobes in reservoir based on the
anaerobes in reservoir based on the
field operation tests.
field operation tests.
(Step 12)
(Step 12) Economic assessment of
Economic assessment of MRMD
MRMD
system
system with high accuracy
with high accuracy
based on the field
based on the field operationtest
operationtest ..
MRMD Setting
MRMD Setting
Conclusion
Conclusion
(1)
(1)The MRMD system m
ay lead to give incentive to CO
The MRMD system m
ay lead to give incentive to CO
22
sequestration technologies such as CCUS
sequestration technologies such as CCUS-- EOR (Carbon dioxide
EOR (Carbon dioxide
Capture, Utilization and Storage with enhanced oil recovery).
Capture, Utilization and Storage with enhanced oil recovery).
(2) In the past findings, indigenous anaerobes in oil reservoir
(2) In the past findings, indigenous anaerobes in oil reservoir which
which
participate in MRMD system have been isolated.
participate in MRMD system have been isolated.
(3)The pathway and the accelerating conditions of microbial
(3)The pathway and the accelerating conditions of microbial
methane generation have also been elucidated.
methane generation have also been elucidated.
(4) But then, there are m
uch obstacles to be resolved for the fi
(4) But then, there are m
uch obstacles to be resolved for the fi eld
eld
operation and the practical use of MRMD system.
operation and the practical use of MRMD system.
Thank you !!
Thank you !!
The end
The end
