The effect of antimicrobial agent to the sulfate reducing bacteria from the white tiger petroleum oil field in Vietnam :The Dissertation - Major Biotechnology

ACADEMY OF SCIENCES REPUBLIC OF UZBEKISTAN

THE INSTITUTE OF GENETICS AND PLANT EXPERIMENTAL

BIOLOGY

In manuscript rights

UDC: 579.253.43 + 577.213.3

DAM SAO MAI

THE EFFECT OF ANTIMICROBIAL AGENT

TO THE SULFATE REDUCING BACTERIA FROM

THE WHITE TIGER PETROLEUM OIL FIELD IN VIETNAM

03.00.15 – Genetics

THE DISSERTATION

Seeking for degree of candidate of biological sciences

Supervisor: academician

Abdusattor Abdukarimov

Tashkent - 2008

ACADEMY OF SCIENCES REPUBLIC OF UZBEKISTAN

THE INSTITUTE OF GENETICS AND PLANT EXPERIMENTAL

BIOLOGY

In manuscript rights D: 579.253.43 + 577.213.3

Specialized Council D.015.80.01

DAM SAO MAI (ДАМ CAO МАЙ)

THE EFFECT OF ANTIMICROBIAL AGENT

TO THE SULFATE REDUCING BACTERIA FROM

THE WHITE TIGER PETROLEUM OIL FIELD IN VIETNAM

ЭФФЕКТ АНТИМИКРОБНОГО СРЕДСТВА НА

СУЛЬФАТ ВОССТАНАВЛИВАЮЩИЕ БАКТЕРИИ ИЗ

МЕСТОРОЖДЕНИЯ НЕФТИ «WHITE TIGER»

ВО ВЬЕТНАМЕ

03.00.15 – Genetics (генетика)

THE DOCUMENTS

Seeking for degree of candidate of biological sciences

Supervisor: academician Abdusattor Abdukarimov

Official

opponents:

academician D.A. Musaev

senior scientist, PhD. Sh.U.Turdikulova

Tashkent - 2008

ACKNOWLEDGMENTS

Looking back, it is hard to simply turn your back to the Institute of Genetics and Plant

Experimental Biology, Uzbek Academy of Sciences, Tashkent, Uzbekistan and the Faculty of

Food Technology and Biotechnology, Ho Chi Minh University of Industry (HUI), Ho Chi Minh

city, Vietnam where I spent three very fine years. Therefore, at this position, I would like to

thank all people that made these years what they were.

At the first place I have to thank academician Abdullaev Abdumavlyan, to give me the

opportunity to come to Tashkent to perform this work. I have to thank my supervisor

academician Abdukarimov Abdusattor, my laboratory supervisor Dr.Abdurakhmonov Ibrokhim,

and my opinions academician Musaev D.A., Dr. Turdikulova Sh.U, Dr. Tashpulatov D.D. for

their careful critique of this thesis; without their help it would not have gone together quite as

smoothly as it has.

I would also like to thank the Vietsovpetro Company for making this research possible.

Special thanks to Dr. Nghia, who help me receiving the petroleum samples.

The person I owe a lot to is Dr.Chernikova Tatiana, Dr.Zabardast Buriev, Dr.Shermatov

Shukhrat, Dr.Abdullaev Alisher. They taught me research at the best. I have to thank Nguyen

Khanh Hoang who is my co-worker at all of my work in Vietnam. My thanks also go to Tohir

Bozorov, Abdushalom Makamov, Trinh Ngoc Nam, Kieu Phuong Nam for their help with

cloning and sequencing, their help has to be greatly acknowledged.

A very special thanks goes to Dr.Adilova Azoda, Dr.Gafur Makamov, Kushanov

Fakhriddin and Tang Tu Mai, who help me with a substantial amount of the revision all spelling

and grammar of my dissertation; they also help me to complete all documentation for defending.

A big “thank you” to all present and former HUI and the Institute of Genetics and Plant

Experimental Biology, Uzbek Academy of Sciences, Tashkent, Uzbekistan and members, for

their support, discussions, encouragement and other fun activities.

Special thanks to Dr.Ta Xuan Te, the director of HUI, for the technical and

administrative aspects and his encouraging role in organizing lots of social events for the HUI

and the lab.

A Ph.D. student has, against all public opinion, still a private life. At this position I would

like to acknowledge my family in Vietnam- Mom, Dad, my husband, my son, and everyone else

that believed I could reach this goal. I also want to thank Long’s family, who became a second

family for me, especially when I needed one.

This work was supported by the Institute of Genetics and Plant experimental biology,

Uzbek Academy of Sciences, Tashkent, Uzbekistan.

2

CONTENTS

Page

LIST OF ABBREVIATIONS..............................................................................4

INTRODUCTION ............................................................................................... 6

CHAPTER 1. LITERATURE OVERVIEW ....................................................... 10

1.1. Overview of the oil and gas origin and the oil exploitation industry.........10

1.2. Development of the Vietnamese petroleum industry.................................11

1.3. Overview of the corrosion of the oil industry ............................................13

1.4. Overview of the microorganism system in petroleum...............................14

1.5. Overview of the geographic description of the White Tiger oil field ........17

1.6. The sulfur-converting cycle and dissimilatory - assimilatory sulfate￾reducing bacteria .....................................................................................18

1.7. Prokaryote classification and identification methods ................................21

1.8. Antibacterial substances (biocides)............................................................22

CHAPTER 2. MATERIALS AND METHODS .................................................26

2.1. Experiment plan .........................................................................................26

2.2. Materials and equipments: .........................................................................27

2.3. Methods:.....................................................................................................29

CHAPTER 3. OBSERVATION OF MICROBES IN OILS

ORIGINATING IN THE WHITE TIGER OIL FIELD.................45

3.1. Typical properties of the petroleum samples.............................................45

3.2. Study of the aggressive level of sulfate reducing bacteria.........................46

3.3. Study of the general characteristics of microorganism system..................49

3.4. The corrosion ability of the microorganisms.............................................56

CHAPTER 4. EFFECTS OF BIOCIDES ON MICROBES ISOLATED

FROM THE WHITE TIGER OIL FIELD .....................................59

4.1. Effects of biocides on selected bacteria .....................................................59

4.2. Ability to produce H2S...............................................................................64

3

4.3. Comparison of the effects of biocides on SRB-5KK and SRB-8KK

species .....................................................................................................68

4.4. Observation of the adaptability of selected bacteria to biocides................69

CHAPTER 5. OBSERVATION OF THE METAL CORROSION

ABILITY OF MICROBES ISOLATED FROM THE

WHITE TIGER OIL FIELD .......................................................... 70

5.1. Study of the corrosion ability of selected wild bacteria.............................70

5.2. Study of the corrosion ability of the biocide treated bacteria ....................77

CHAPTER 6. THE PROPERTIES OF THE WILD SRB AND THE

BIOCIDE ACCLIMATED SRB....................................................86

6.1. Adaptability to the environment ................................................................86

6.2. Observation of characteristic .....................................................................93

6.3. The log phase .............................................................................................97

CHAPTER 7. THE MOLECULAR-GENETIC TAXONOMICAL

ANALYSIS OF THE WILD SRB AND THE BIOCIDE

ACCLIMATED SRB STRAINS....................................................99

7.1. PCR-amplification of 16S rDNA genes from samples..............................99

7.2. Clonning results: ......................................................................................100

7.3. Sequencing analysing:..............................................................................103

SUMMARY ........................................................................................................110

CONCLUSION....................................................................................................116

RECOMENDATION .......................................................................................... 118

REFFERENCES..................................................................................................119

APPENDIX ........................................................................................................151

4

LIST OF ABBREVIATIONS

A ............. Adenine

AAS ........ Atomic absorption spectrophotometer

API 20A.. anaerobe analyzer test kit

ASTM..... American Standards Test Methods

C ............. Cytosine

CFU ........ Colony Forming Unit

CTAB ..... Cetyl Trimethyl Ammonium Bromide

DEPC...... diethylpyrocarbonate

DNA ....... deoxyribonucleic acid

dNTP....... Deoxyribonucleotide triphosphate

EDTA ..... Ethylene diamine tetra acetic acid

EtBr......... Ethidium Bromide

G ............. Guanine

GTE ........ Glucose Tris EDTA

HK .......... aerobes

hrs ........... hours

ID 32E .... aerobe analyzer test kit

ISO.......... International Organization for Standardization

Kb ........... Kilo base

KK .......... anaerobes

LB........... Luria Bertani – plats medium

MRB ....... Metal-Reducing Bacteria

OD .......... Optical Density

PAUP...... Phylogenetic Analysis Using Parsimony

PCA ........ medium Plate Count Agar

PCR......... Polymerase Chain Reaction

PEG juice Polyethylene glucose juice