2nd International Symposium on Applied Microbiology and MolecularBiology in Oil Systems, June 17-19, 2009, Aarhus, Denmark

MICROBIAL ECOLOGY OF THE HIGH-TEMPERATURE PETROLEUM RESERVOIR

AND RESULTS OF MEOR TECHNOLOGY APPLICATIONC ON

T.N. Nazina, N.M. Shestakova, N.K. Pavlova, E.M. Mikhailova, D.Sh. Sokolova, T.L. Babich, Q. Feng, F. Ni, A.B. Poltaraus, S.S. Belyaev, M.V. Ivanov

•Winogradsky Institute of Microbiology, Russian Academy of Sciences, Russia•Dagang Oil Field Co PetroChina Co Ltd China•Dagang Oil Field Co., PetroChina Co. Ltd., China•Institute of Molecular Biology, RAS, Russia

2nd International Symposium on Applied Microbiology and MolecularBiology in Oil Systems, June 17-19, 2009, Aarhus, Denmark

• 1. Characterization of a microbial community from the Dagang high-temperature oil field (P.R. China) by culture based radioisotope and molecular methodsculture-based, radioisotope and molecular methods.

• 2 The biogeochemical changes in the Dagang oil field in• 2. The biogeochemical changes in the Dagang oil field in the course of application of a biotechnology forenhancement of oil recovery. e a ce e o o eco e y

Microbial Activities and Products Useful for Oil Recovery

• Gas generationA id d ti• Acid production

• Solvent production• Biopolymer production• Surfactant productionSurfactant production• Physical oil displacement

H d b difi ti• Hydrocarbon modification• Viscosity modification

The resident communities include a variety of anaerobic microorganisms:Fermentative, sulfate-reducers, manganese and iron-reducers, acetogens and methanogens.

A bi b t i b d i il fi ld b li d t bAerobic bacteria observed in oil fields are believed to be exogenous contaminants.

Scheme of oil field exploited with waterScheme of oil field exploited with water--floodingflooding

11 1122 22

1 – near-bottom zone of injection well 2 – production well

Mi bi l

Petroleum

Aerobic +O2 H d b Microbial

transformation of petroleum

accompanied

Hydrocarbon-Surfactants, CO2, oxidizing polysaccharides Biomass, Fatty acids, alcohols,

accompanied with production of oil-releasing

agents in a water

Exometabolites aromatic compounds _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Anaerobic agents in a water-

flooded oil field Fermentative bacteria Solvents, gases, lower fatty acidslower fatty acids Propionate, butyrate,

pyruvate, ethanol, propanol

Synthrophic Synthrophic bacteria

Fe3+-

H CO formate reducingThe field tests of

H2, CO2, formate, reducingacetate, methanol

Sulfate- Methanogens reducing

Fe3+ +SO42-

the biotechnologygave positive results; over750000 tons of oil

CH4, CO2 Fe2+, CO2 H2S, CO2

was additionally recovered.

Dagang oil field

The main objective of the project was to

港西三区一断块

试验区 Gangxi bed

carry out the pilot test of the biotechnology for oil recovery enhancement based on the regulation of the stratum microflora activity.

Kongdian bed (North block and block 1)

This biotechnology involved the injection of water air mixture and of mineral nitrogen

Ⅱ

北

water-air mixture and of mineral nitrogen and phosphorous salts for the stimulation of growth of indigenous microorganisms.

ⅠⅠ

ⅢⅣ

南

Methods of investigation1. Analytical (chemistry of water, oil, gases)2. Microbiological methodsUse of selective media for:

A bi t h• Aerobic organotrophs • Hydrocarbon-oxidizing bacteria• Fermentative bacteria• Sulfate-reducing bacteria• Sulfate-reducing bacteria• Methanogens

3. Methods of Polyphase Taxonomy yp yMorphology, physiology and phylogenetic position ofmicroorganisms by 16S rRNA gene sequencing

4. Radioisotope methods• Sulfate reduction – Na2

35SO4• Lithotrophic methanogenesis – NaH14CO3• Aceticlastic methanogenesis 14CH COONa• Aceticlastic methanogenesis - 14CH3-COONa

5. Molecular biology techniques • DNA and RNA extractionDNA and RNA extraction• Amplification and cloning of 16S rRNA genes• Sequencing and phylogenetic identification

Sampling

STAGES OF MEOR APPLICATION ON THE DAGANG OIL FIELDDAGANG OIL FIELD

• Determination of the background level for the physical, chemical and bacteriological parameters of the fluids produced or injected;parameters of the fluids produced or injected;

• Determination of the filtration properties of the collector of the Kongdian bed;g ;

• Development of a technological scheme for the activation of formation microorganisms, development of equipment and carrying out thedevelopment of equipment and carrying out the injection of the water-air mixture and nutrients into the stratum;

• Monitoring the ecological and production characteristics of the oil field.

CHARACTERISTICS OF THE KONGDIAN AREA OF THE DAGANG OIL FIELDDAGANG OIL FIELD

Depth 1206 – 1434 m

T 9 CTemperature 59 oC

pH 7.1-7.6

S li it 0 6 %Salinity 0.6 %

Water cut 95%

22productionwells

1111injectionwells

Layout of injection and production wells at the experimental site of the

Kongdian oil field

Chemical and bacteriological parameters of the Kongdian bed before the trial

• Near-bottom zone*:Characteristic Parameters

HCO3-, mg/l 419-698

Low fatty acids mg/l 0-5

Near bottom zone :• Aerobic bacteria <103 cells/ml• Oil-oxidizing - 10-103 cells/ml• Production wells Low fatty acids, mg/l 0 5

Aerobic organotrophic bacteria, cells/ml 0-102

Aerobic oil-oxidizing bacteria, cells/ml 0-103

Production wells• Aerobic bacteria <10 cells/ml• Oil-oxidizing <10

Fermentative, cells/ml 102-105

Sulfate-reducing, cells/ml 0-103

Methanogens, cells/ml - H2+CO2, 0-103

- on acetate 0-102

Sulfate-reduction rate, μg of S2- L-1day-1 0.002-19.904

5

/ml

Methanogenesis rate, μg of CH4 L-1 day-1

from H2+CO2 0-1.595

from acetate 0-5 611

2

3

lg c

ells

/

Ferment.Sufate-red.

Meth-H2Meth- acet

from acetate 0-5.61

δ 13C/CH4, o/oo -41.2 - -43.1

δ 13C/ΣCO2+ HCO3-+ CO3

2-, o/oo -1.5 - +6.4

0

1

98-27* 1008 1012 1015-1 1032-1 1050-3

AerobicOil-oxid.

δ 13C/of oil, o/oo -26.5

Aerobic bacteria isolated from the Dagang oil fieldAerobic bacteria isolated from the Dagang oil field

•34 aerobic strains Geobacillus jurassicus DS1Т

•45-65 °С:Geobacillus

j

(G. subterraneus, G. pallidus,G. stearothermophilus,

G. thermoglucosidasius) GLC tracing of crude oil satrated hydrocarbons after

exposure to degradation by G. jurassicus DS214

Thermoactinomyces

20 45 °С 02468

101214

Abu

ndan

ce,

%weathered oil, control incubated at 60 0C

•20-45 °С:Bacillus, Oceanobacillus,

0

С10

С12

С14

С16

С18

С20

С22

С24

С26

С28

С30

С32

С34

С36

Numbers of carbons

02468

Abu

ndan

ce, %

,Micrococcus,Cellulomonas,Pseudomonas

С12

С14

С16

С18

С20

С22

С24

С26

С28

С30

С32

Number of carbons

Branched alkanes Unbranched alkanes

PseudomonasAcinetobacter

Diversity of alkB homologs in bacteria of the genus Geobacillus

alkBhomolog

Nearest alkane 1-monooxygenase according to BLAST analysis

Nucleotidesequencehomolog BLAST analysis sequencesimilarity, %

alkB-geo1 alkB4 from R. erythropolis NRRL B-16531 99.2

alkB-geo2 alkB4 from R. erythropolis NRRL B-16531 90.0

alkB-geo3 alkB3 from Nocardia sp. H17-1 87.7

alkB-geo4 alkB3 from R. erythropolis NRRL B-16531 96.7alkB-geo5 alkB2 from R. erythropolis 50-V 95.4alkB-geo6 alkB2 from R erythropolis NRRL B- 99 0alkB-geo6 alkB2 from R. erythropolis NRRL B- 99.0alkB-geo7 alkB, environmental clone alkG4-35k 69.8

alkB-geo8 alkB1 from Rhodococcus sp Q15 70 0alkB-geo8 alkB1 from Rhodococcus sp. Q15 70.0

50 80

A B

3035404550

ones

50

60

70

80

nes

1015202530

Num

ber o

f clo

20

30

40

50

Num

ber o

f clo

05

10

bacil

lusom

onas

diphilu

sacte

rium

icocc

usom

onas

cocc

usob

acte

rord

onia

cteriu

mob

acter

0

10

20a

N

Geoba

Pseud

omTep

idip

Therm

oana

erobac

Lapil

lico

Sphing

omLa

ctoco

Acinetob Gor

Carno

bac

Caulob

Geobacillus

PseudomonasShigellaGordonia

Leuconostoc

Williamsia

Methylobact

erium

16S rDNA 16S crDNA 16S rDNA 16S crDNA

• 16S rDNA and 16S crDNA clone libraries from hydrocarbon-oxidizing enrichment cultures isolated from near-bottom zone of injection well (A) and from production well (B) of the Dagang high-j ( ) p ( ) g g gtemperature oilfield

Diversity of bacterial and archaeal 16S rRNA genes in the DNA clone library from formation water of the Dagang oilfield

30,0

35,0AlphaproteobacteriaBetaproteobacteriaGammaproteobacteria

15,0

20,0

25,0

of c

lone

s

DeltaproteobacteriaClostridiaThermotogaeDictyoglomiBacteroidetes

5,0

10,0

,

% ActinobacteriaNitrospirae

0,0

Phylum Closest identified relative (acc number)

Number of clones(acc. number) of clones

Methanobacteria Methanothermobacter thermautotrophicus

402

Delta H (AE000666)

Uncultured archaeal clone LCA (AB084240)

1

Total 403

• Rheological characteristics of culture media of aerobic eo og ca c a acte st cs o cu tu e ed a o ae ob cthermophilic bacteria from the Kongdian bed

Strain, substrate

Emulsifying activity, %

Surface tension,mN/m

Interfacial tension*, mN/m

Viscosity, mPaxs(at 60 °C)

Microbial community 1098-25 m3

Sucrose 50 53.1 19.3 0.68Acetate 30 51.3 19.0 0.66C10-C20 paraffin

40 51.5 25.1 0.62

Crude oil 30 41.9 11.8 0.63Geobacillus jurassicus DS1Т

Sucrose 30 45.3 15.5 0.64A t t 30 47 3 15 3 0 61Acetate 30 47.3 15.3 0.61C10-C20 paraffin

5 51.3 22.7 0.64

Crude oil 30 41.9 12.5 0.60

Determination of the filtration properties of the collector of the Kongdian bed

10921017-7 1094

6

--

1,76 Бк/л 1015

1094-1

64,2 Бк/л

418,3 Бк/л

Бк/л

1013-11017-5

1,96 Бк/л

1,44

Бк/л

13,78 Бк/л

1,89 Бк/л

10171015-1

115,6 Б

10081008-1 01 1050-3

Appearance of tracers

1017

1050-360,2 1050-1

in formation waters from production wells located around the

1050

36,0 Бк/л58,9 Бк

/л

,2 Бк/л89,0 Бк/л

1050 1

injection wells1032-1

1002-11050-2

•Development of a technological scheme of the activation of formation microflora;formation microflora;•Development of a field engineering process for injecting water-air mixture and mineral salts into the stratum;;•Injection of the water-air mixture and nutrients into the stratum.

Monitoring the microbiological,Monitoring the microbiological, biogeochemical and production

characteristics of the oil field in thecharacteristics of the oil field in the course of the pilot trial

Approximately 40 parameters of the fluids from 22production wells, of backflushed water from the injection wellproduction wells, of backflushed water from the injection welland of injection water were monitored for 5 years.

The numbers of microorganisms in formation The rate of sulfate reduction in

f i f h K diwater from wells 1015-1 in the course of the pilot trial

formation water of the Kongdian bed in the course of the pilot trial

1015-1 300

678

lg

200

250

ug S

2-/l/

day

23456

lls n

umbe

r,

100

150

fate

redu

ctio

n ra

te, u

e,20

00.,2

000

,200

120

0200

200

3

HOBMet-H2

Ferment01C

e

Dec July1092

631017-4

0

50

Sulf

June

Dec

.Ju

ly,

Jan.

,2Ju

ne,2

Jan.

,20

HOB SRB Met H2 Met Acet Ferment

2000July2001

Jan2002

June2002

Jan2003

Dec2004

May2005

Dec2005

May2006

Dec2006

1050-11094-1

1050-1 1094-1 1092 63 1017-4HOB SRB Met-H2 Met-Acet Ferment

Peak values of methanogenesis rate in formationPeak values of methanogenesis rate in formation water in the course of the trial

(μg of CH4 L-1 day-1)(μg 4 y )

40

45 1400

30

35

40

1000

1200

20

25

600

800

10

15

200

400

0

5

1 63

1002

-1

1008

1008

-1

1012

1012

-1

1015

-1

1017

1017

-2

1017

-3

1017

-4

1017

-5

1017

-7

1032

1032

-1

1050

-1

1050

-2

1050

-3

1092

1094

0

8 m3 25 m3 1094-1

From NaH14CO3 From 14CH3-COONa From NaH14CO3 From 14CH3-COONa

Physicochemical parameters of formation water and gas of the Kongdian bed during the biotechnological trial

180

140

160

180

l

1200

1400

80

100

120

ceta

te, m

g/l

600

800

1000

НСО

3, m

g/l

20

40

60Ac

0

200

400

163

1002

-110

0810

08-1

1012

1012

-110

15-1

1017

1017

-210

17-3

1017

-410

17-5

1017

-710

3203

2-1

050-

150

-250

-309

294 -1

July 2001June, 2002

Sept. 2003May 2005

0163

1002

-110

0810

08-1

1012

1012

-110

15-1

1017

1017

-210

17-3

1017

-410

17-5

1017

-710

3210

32-1

1050

-110

50-2

1050

-310

9210

9410

94-1

June, 2000

Jan., 2002

Sep., 20030

1 10 10 10 105 10 10

1094

July 2001 Jan. 2002 June, 2002 Jan. 2003 Sept. 2003 Nov 2004 May 20050-200 200-400 400-600 600-800 800-1000 1000-1200 1200-1400

Content of HCO3- and acetate in formation waters of the Kongdian

bed in the course of the pilot trial (mg/l)

The rheological characteristics of formation water from well • Emulsifying activity of formationof formation water from well

1008-1 in the course of the pilot trial

• Emulsifying activity of formation fluids of the Kongdian bed

45

50

25

30

35

40

/m 10 10 11015 1

1017

63

10

15

20

25

mN 02

-108-1

008

15-1 0

12

17-4

3

Dec 2000July 2001

Jan 2002 Interfacial tension

Surface tension0

5

June, 2002Jan 2003

Sept 2003

Interfacial tension

Interfacial tension Surface tension • Viscosity of formation water i d f 0 70 t 0 86increased from 0.70 to 0.86 mPa x s.

Gas composition and Biogeochemical Characteristics of the K di B d

• δ 13C/CH4, o/oo (in the gas)Kongdian Bed

In the course of the trial: CO2 increased on 2-4 1 %;

-15

1 63

1002

-110

0810

08-1

1012

1012

-110

15-1

1017

1017

-210

17-3

1017

-410

17-5

1017

-7

1032

1032

-110

50-1

1050

-210

50-3

1092

1094

1094

-1

CO2 increased on 2-4.1 %;CH4 increased on 2-5.7 % in the gas of several production wells.

•δ13C∑CO2 + HCO3- +CO3

2- -25

1 1 1 1 1 1 1 1 1 1 1 1 1 1

(in the formation water)

Before the trial: -35

+6.4 o/oo ÷-1.5 o/oo

In the course of the trial: +6 4 o/ ÷ 12 9 o/ (14 ll )

-55

-45

+6.4 o/oo ÷ -12.9 o/oo (14 wells)+6.4 o/oo ÷ +10.6 o/oo (3 wells) -65

Dec 2000 July 2001 Jan 2002 June 2002 Jan 2003 Sept 2003

Additional oil production on the North block of the Kongdian bed as a result of application of MEOR technology in V 2001bed as a result of application of MEOR technology in V.2001-

V.2007

Well D li f l ti Additional eNo. Decline formulation dd t o a

oil, t

1002-1 Q0=16.75(1+0.5*0.039t)-2 94951008-1 Q0=9.8（1+0.5*0.02t)-2 38921015-1 Q0=11(1+0.5*0.021t)-2 52781017 2 Q 7 15(1 0 5*0 028t) 2 30521017-2 Q0=7.15(1+0.5*0.028t)-2 30521017-3 Q0=8.31(1+0.5*0.072t)-2 4461017-7 Q0=24.11(1+0.5*0.027t)-2 19321017 7 Q0 24.11(1 0.5 0.027t) 19321032-1 Q0=31.71(1+0.5*0.02t)-2 58681050-3 Q0=5.48(1+0.5*0.027t)-2 50361094-1 Q0=5.99(1+0.5*0.031t)-2 1003

Total 36002Decline curve of oil production Decline curve of oil production on the North block in 1997on the North block in 1997--20072007

CONCLUSIONS

• It was experimentally proved that the high-temperature oil field is an ecosystem in which a biotic community interacts with an abiotic medium so that energy fluxes create a certain trophic structure Thesemedium so that energy fluxes create a certain trophic structure. These energy fluxes are based on the biotransformation of oil in particular trophic chains and can be regulated in a goal-directed manner.

• The possibility of the application of the MEOR technology based on the activation of metabolism of the formation microorganisms was demonstrated in high-temperature oil field. g p

• As the result of the injection of water-air mixture and mineral nutrientsmicrobial transformation of residual oil was accompanied by

l ti f bi b t i l til f tt id bi f t taccumulation of bicarbonate ions, volatile fatty acids, biosurfactants, and microbial biomass in the stratal water, and of CH4 and CO2 in the gas phase.

• Microbial metabolites promoted additional oil replacement from the layer. About 36 thousand tons of oil was recovered additionally on the Dagang oil field as a result of MEOR application.Dagang oil field as a result of MEOR application.