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").