SyntrophicSyntrophic Acetate Degradation to Acetate ...
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ISMOS-2, Aarhus, June 17-19, 2009Winogradsky Institute of Microbiology, Russian Academy of SciencesINMI
SyntrophicSyntrophic Acetate Degradation to Acetate Degradation to Methane in a HighMethane in a High--Temperature Temperature
Petroleum ReservoirPetroleum ReservoirPetroleum ReservoirPetroleum ReservoirNatalya M. Shestakova1, Tamara N. Nazina1, Qingxian Feng2, Fangtian Ni2, Tatiana
P T 1 A d i B P lt 3 S S B l 1 Mikh il V I 1P. Tourova1, Andrei B. Poltaraus3, Sergey S. Belyaev1, Mikhail V. Ivanov1
1Winogradsky Institute of Microbiology, Russian Academy of Sciences, Russia
2Dagang Oilfield Company, China
3Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russia
*E-mail: genomnat@mail.ru
Microorganisms Isolated from High-Microorganisms Isolated from HighTemperature Oil Fields
• Hydrocarbon oxidizing bacteria • Fermentative bacteria• Fermentative bacteria• Sulfate-reducing bacteria
Acetogens• Acetogens• Methanogens Methanothermobacter sp.
Only H2-utilizing methanogens were isolated from high-temperatureoil fields:
Methanobacterium thermoaggregansMethanothermobacter thermautotrophicusMethanococcus thermolithotrophicus
No Acetate-utilizing methanogens were isolated
Aim and objects:
Our aim was to examine the distribution,biodiversity and geochemical activity of themicroorganisms in a high-temperaturemicroorganisms in a high temperaturepetroleum reservoir1. Characterization of a microbial community
combining culture-based, radioisotope andl l th dmolecular methods
2. Investigation of microbial interactions duringacetate degradation to methane under thermophiliccondition
Dagang Oilfield
Horizon 1200 –Horizon depth
1200 1400 m
T t 59oCTemperature 59oC
pH 7.1-7.6
Salinity 5.8-6.6 g/l
The biotech trialThe biotech trial
G lGoal:Enhancement of the oil recovery
Proposal:Production of oil-displacing compounds by microbes in situp g p y
Method:I j t t + i i t t ti l t th itInject water+air mixture to stimulate the community
We will study the water from production wellsWe will study the water from production wells
Materials and MethodsMaterials and Methods
1. Analytical (chemistry of water, oil, gases)
2. Microbiological methods
3. Radioisotope methods
4 Molecular biology techniques4. Molecular biology techniques
Sampling
Culture-based
7
5
6
3
4
ells
/ml)
2
3
Log(
ce
000 0 SRB
Met-H2
Met-A
Ferment
0
1
VI, 2
0
XII,
200
VII,
2001
I, 20
02
VI, 2
002
I, 20
03
IX, 2
003
HOB
Time
Rates of Methanogenesis
Near-bottom zone of the injection wellProduction wells
35
40
45
1200
1400
ion
20
25
30
800
1000
e pr
oduc
t
10
15
20
400
600
Met
hane
0
5
1
1002
-1
1008
-1
1012
-1
1017
1017
-3
1017
-5
1032
1050
-1
1050
-3
1094
0
200
8 m3 25 m3 1094-1
Methanogenesis was registered from both labeled bicarbonate and acetate
Increase of Acetate in Formation Waters
160
180
120
140
160
80
100
20
40
60
1
1002
-1
008-
1
12-1
17 -3
Jan. 2003
May 20050
20
10 101
10
1017
-
1017
-5
1032
1050
-1
1050
-3
1094
July 2001
Production wells
Methanogenesis and Acetate contentMethanogenesis and Acetate contentcorrelate
101750 200
1008-10 25 60
40
50
160
200
0,2
0,25
48
60
y
30 1200,15 36
, μg/
l day
ate,
mg/
l
10
20
40
80
0,05
0,1
12
24
Met
hane
,
Acet
a
0
10
VII, 2001 I, 2002 VI, 2002 I, 2003 IX, 2003 XII, 20040
40
0
0,05
VII, 2001 I, 2002 VI, 2002 I, 2003 IX, 2003 XII, 20040
M
VII, 2001 I, 2002 VI, 2002 I, 2003 IX, 2003 XII, 2004
- Acetate content - From NaH14CO3 - From 14CH3COONa
16S rRNA analysis
methanogenic enrichmentsmethanogenic enrichmentsnatural formation water formation water from the zoneformation water from the zone of the pilot trialmethanogenic enrichmentmethanogenic enrichment culture on acetate
Clone Libraries
Phylotypes Combined Natural Formation Methano- Totalmethano-genicEnrichments
formationwater
water in the zone of the pilot trial
genicenrichmentculture
ota
on acetate
Archaeal clones 102 403 181 96 782Ph l t 101 403 164 96 64Phylotypesbelonged toMethanothermo-
101 403 164 96 764
bacterBacterial clones 20 314 457 46 837Number of clones 122 717 638 142 1619
Archaeal 16S rDNAArchaeal 16S rDNA from the Formation Water
Clones from Methanogenic CulturesClones from Methanogenic Cultures
Domain Closest relatives Clons b
% d inumber Identity
ArchaeaMethanobacteriales Methanothermobacter thermautotrophicus 56 97,3 - 100Methanobacteriales p
Uncultured archaea clon(Methanobacteriaceae sp.)
45 97,4 – 99,5
Thermococcales Thermococcus sibiricus 1 99,1e ococca es
BacteriaThermoanaerobacteriales Thermanaeromonas toyohensis ToBET 1 97
Thermoanaerobacter ethanolicus 1 99,5
Coprothermobacter sp. P1 2 97
Thermovenabulum ferriphilus 3 92
Thermacetogenium phaeum 3 94 - 95,3
Clostridiales Desulfotomaculum thermobenzoicum 1 93,1
Thermotogales Unidentified Thermotogales 4 91,9 - 93,6Thermotogales Unidentified Thermotogales 9 ,9 93,6
Nitrospirales Thermodesulfovibrio sp. 3 97,4 - 97,8
Planctomycetales Unidentified Planctomycetales 1 98,1
Bacterial 16S rDNA f th F ti W t
Ecological group Number Ecological group Number
from the Formation Waterg g p
of clones
Aerobic organotrophic bacteria
Sphingomonas (α) 33
g g pof clones
Fermentative bacteria
Thermoanaerobacter 1Sphingomonas (α) 33
Afipia 2
Labrys 2
Thermoanaerobacter 1
Thermovenabulum 1
Thermoterrabacterium 1
Variovorax (β) 2
Curvibacter 5
Polaromonas 3
Caldotoga 5
Thermotoga 1
Fervidobacterium 2
Pseudomonas (γ) 28
Hydrocarbon-oxidizing bacteria
Dictyoglomus 5
Pedobacter 2
D 1Rhodococcus 1
Thauera (β) 1
Sulfate-reducing bacteria
Dysgonomonas 1
Syntrophic bacteriaSyntrophaceae (delta-) 1g
Desulfotomaculum 8
Thermodesulfovibrio 1
y p ( )
Thermacetogenium 1
Syntrophomonadaceae 1
Thermophilic Syntrophic Acetate-p y pOxidizing Bacteria
Strain AOR Thermacetogenium phaeum
Negative stained electron micrograph of the AOR in pure culture. Bar, 0.2 μm (Lee, Zinder, 1988). Transmission electron micrographs of pure culture
strain PBT cell. Bars, 1 μm (Hattori et al., 2000).
СН3СОО- + Н+ + 2H2O 4Н2 + 2СО2
Strain AOR
СН4
Methanobacterium
CO2
2H2O
4H2
Isolation of Anaerobic Organotrophs g p
Property Strain 1017-7d
Temperature range, 0С 40-75
рН range 5.8-8.516S rRNA genes of isolates were highly similar (99 6%) to genes of
Thermoanaerobacter ethanolicus
р g
NaCl range 0-2
Substrates used:
similar (99.6%) to genes of
Н2 + СО2 +
Fructose +
Maltose +
Starch +
Pyruvate +
A biArabinose -
Ethanol -
Acetate -
Reduction of S2O32- +
Reduction of SO4- -
Isolation of Thermophilic Methanogensp gM. thermautotrophicus Z-245, X68712
Strain KZ3Strain KZ3M. defluvii DSM 7466, X99046M. thermoflexus DSM 7268, X99047M. thermautotrophicus ΔHT, AE000666p ,M. thermautotrophicus GC-1, AY196661M. thermautotrophicus HN4, X68719M. wolfeii DSM 2970T AB104858M. thermautotrophicus SF-4, X68718Strain KZ24M. thermautotrophicus ZH3, Z37156M. marburgensis, X15364M. subterraneum DSM 11074, X99044Methanobacterium formicicum DSM 1312, M36508M th th f id DSM 2088 M59145
9.2
8 6 4 2 0
Methanotorris formicicus, AB100884Methanothermus fervidus DSM 2088, M59145
8 6 4 2 0
Nucleotides (x100)
Ph i l f I l t d M thPhysiology of Isolated Methanogens
Property KZ3 KZ24aSalinity, % NaCl 0 - 1 0 - 1
Temperature range,0C 40-65-60 40-65- 65
S b t tSubstrates:
H2+CO2 + +
Foramate +/Foramate +/- -
Acetate - -
Methane Production from Acetate by Pure Cultures and Co-cultures of Methanothermobacter -
Thermoanaerobacter
3,5
4
140
160
day
2
2,5
3
100
120
140
or µ
g/l d
1
1,5
2
40
60
80
hane
, %
0
0,5
Pure cultures Co culturesPure cultures Co-cultures0
20
40
Met
h
Pure cultures Co culturesPure cultures Co-cultures Pure cultures Co-cultures
Conclusions
1. The processes of methanogenesis from both bicarbonate and acetatewere registered in the formation waters by culture-based andradioisotope methods.
2 By 16S rRNA gene analysis was revealed that H utilizing2. By 16S rRNA gene analysis was revealed that H2-utilizingmethanogens of the genus Methanothermobacter predominated in themicrobial community. Phylotypes of acetoclastic methanogens were notf d i th i i l f ti tfound in the original formation water.
3.Phylotypes of fermentative bacteria and archaea, sulfate-reducing andsyntrophic (Thermacetogenium) bacteria were revealed in the high-syntrophic (Thermacetogenium) bacteria were revealed in the hightemperature Dagang oil field.
4. It was shown that a syntrophic association of T. ethanolicus and M.thermautotrophicus carried out the reaction of acetate degradation tomethane.
AcknowledgementsAcknowledgements
Dagang Oilfield Company, China
Winogradsky Institute of Microbiology, Russia
Engelhardt Institute of Molecular Biology, Russia
Thank you!
AcknowledgementsAcknowledgements
The work was supported by the Dagang OilfieldCompany (DFT04-122-IM-18-20RU), Russian Ministry ofEducation and Science (4174.2008.4) and the RussianA d f S i (P "M l l d C ll lAcademy of Sciences (Program "Molecular and CellularBiology").
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