The effect of antimicrobial agent to the sulfate reducing bacteria from the white tiger petroleum oil field in vietnam

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.

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 sulfatereducing 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

2


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

3


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
4


PGA ........ medium Potato Glucose Agar
Post A .... medium Postgate A
Post B...... medium Postgate B
RNA ........ Ribonucleic Acid
SDS ......... Sodium Dodecyl Sulphate
SEM ........ Scanning Electron Microscopy
SNP ......... Single Nucleotide Polymorphism.
SRB......... Dissimilatory or Assimilatory Sulfate-/Sulfur-Reducing Bacteria

T .............. Thymine
TE ........... solution of Tris HCl and EDTA
TSR ......... Template Suppression Reagent
TT ........... facultative aerobic
UPGMA .. Unweighted Pair Group Method with Arithmetic mean

5


INTRODUCTION
Actuality of the work. It has been realized that crude petroleum oil and gas
are the important resources giving economic potential in Vietnam. The oil industry,
with its complex and demanding production techniques, must pay the cost for
problems caused by corrosion. The main factors causing the corrosion are: oxygen
corrosion, water and carbon dioxide corrosion, acid corrosion and hydrogen sulfide
corrosion.
There have been many studies on the elimination of sulfate-reducing bacteria.
Biocides have been found to be one of the best methods of protecting equipment.
This method has applied effectively in many places in our world. At present
Vietsovpetro, the biggest oil and gas company in Vietnam, is using the biocide,
which is imported with prices of 3.04 – 3.17 USD/liter [280]. About 80,000 –
100,000 liters of this agent are required for the space around one oil well [91]. This
underlies the necessity to investigate the biocidal effect of chemicals to the sulfatereducing bacteria (SRB)
The status of the problem. Many kinds of bacteria are found from crude oil.
Some of them corrode metals, the others biodegrade the petroleum. However,
despite decades of study it is still not known with certainty how many species of
microorganisms contribute to the metal corrosion; how to reliably detect their
presence prior to corrosion events; or how to rapidly assess the efficacy of biocides
and mitigation procedures [34,43,114]. Most researchers have directed their
attention to the activities of sulfate-reducing bacteria (SRB) and have invested in

biocide treatment programs for seawater injection, with the principal aim of killing
or controlling this group of microorganisms [97,116]. In Vietnam, there are many
projects investigating the biocides which could inhibit the metal corrosion to a
transparent petroleum oil production [315,322]. But there is no project to
comprehensively investigate the impact of the biocides on the SRB-s existed
around the Vietnamese petroleum oil, field and the competence of these bacteria in
a biocide resistance [315, 325].
6


Objective. The objective of this work is to study the SRB from the White
Tiger petroleum field at the microbial and molecular level as well as explore the
biocidal and mutational effects of chemical agents to SRB
Tasks of the investigations. Following were the specific task of the work:
-

Sampling of microorganisms from the White Tiger petroleum oil field at
Vungtau province and colonizing of sulfate reducing bacteria (SRB) strain.

-

Studying of the metal-corrosion ability of SRB to estimate its impact to the
White Tiger petroleum oil production rate.

-

Determination of the resistance of SRB to the biocide (Hexatreat 1512) used
in Vietnam.

-

Investigation of SRB growth habits and its adaptation ability to different
environments in Vietnamese petroleum oil field.

-

Identification of microbiological taxonomy of SRB-s at molecular level.

-

Exploration of the mutational effects of biocides to the SRB at the molecular
level.
Novelty of the work. In Vietnam, there are many research projects dealing

with the biocides that could inhibit the metal corrosion. But no project that
comprehensively studied the impact of biocides on the sulfate-reducing bacteria
existing around the Vietnamese oil field. This research studied the corrosion ability
of the sulfate- reducing bacteria and the effects of antimicrobial agents on the
White Tiger’s petroleum sulfate-reducing bacteria. Furthermore, for the first time,
we taxonomically identified the SRB and determined possible genomic mutations
of SRB after biocide treatment in a sample taken from the White Tiger petroleum
oil field. The results showed that Klebsiella pneumoniae caused corrosion and its
corrosion abilities were not reduced under the biocide treatment. These bacteria
with the biocide treatment are able to grow worse than wild ones.
The main findings to be defended.
1.

The White Tiger oil field in Vietnam contains high densities of SRB. Most of
the samples had SRB quantities at the very aggressive level.
7

2.

Hexatreat 1512 – the biocide which is used by Vietsovpetro Company –
showed different effects to each SRB. With concentration of 100ppm of
biocide, after 6 hrs treatment, SRB-5KK produced 878.33 mg H2S/L, and
SRB-8KK produced 699.30 mg H2S/L. And after 16 hrs all selected SRB-s
was killed.

3.

The metal corrosion ability of SRB of White Tiger petroleum oil field was at
from 3a (an aggressive level). The corrosion ability of the biocide treated
strain was not reduce. With the higher concentrations of bacteria cells (e.g.,
100,000 cells/ml), those bacteria became more dangerous than the wild strain.

4.

All cells of the wild and biocide treated SRB were ovoid or oval- to rod-shape
and the sizes were from 0.7-1.5 x 1.2-2.2 µm. The optimum pH range was 6 –
9. The optimum temperature range was 20 – 500C. The optimum glucose
concentration range was 10%. The optimum NaCl concentration range was
5%. Bacteria with the biocide treatment were able to grow worse than wild
types.

5.

16S rDNA analysis identified SRB of White Tiger petroleum oil field as
Klebsiella pneumoniae, suggesting the microbiological taxonomy of SRB.

Several SNP mutations were identified in wild and biocide treated strain, from
the SRB samples of the White Tiger petroleum oil field.
Scientific and practical significance of the results.
The 16S rDNA analysis of identified SRB and its mutation under biocide

treatment can be used to the further research to develop effective means/tools to
monitor microbial corrosion.
The results of the corrosion ability of the SRB and the biocide effect to the
SRB can be applied in the researches to inhibit these bacteria and to raise the rate
of the petroleum oil production in the other places. These results also can be used
to compare with the corrosion ability of the SRB from different places and to
determine the microbial corrosion system of the petroleum oil field.

8


Application of the results. Results of this work are very useful to the
laboratory of the petroleum companies, the laboratory of the Universities and
Institutes. The results can be further use to the other researches which have related
with Vietnamese petroleum microorganisms.
Presentation of the work. The results of the work were presented in the 2nd
Scientist conference of the Ho Chi Minh University of Industry, 2007 under the
titles of: “Observation of microbes in oils originating in the White Tiger mine” and
“Observation of metal corrosion ability of sulfate reducing bacteria isolated from
the mine White Tiger with ASTM D130 method”.

9


CHAPTER 1. LITERATURE OVERVIEW

1.1. Overview of the oil and gas origin and the oil exploitation industry
There are many theories of the gas-and-oil origin [321]. The 1958 discussion
emphasized that, “Today the organic origin of petroleum is the theory which
commonly used to explain the definite principle of petroleum-fields distribution,
and which is considered as the scientific background for petroleum exploration”.
There were the 7th and 8th world meetings of petroleum in Mexico and Moscow in
1968 and 1971, respectively. In the meetings, scientific reports confirmed the
above theory and developed some modern theoretical principles of organic origin.
Such principles have been applied by geochemists when seeking land regions of
natural petroleum accumulation [321].
Crude oil was explored many BC years ago [321]. In the early 19th century,
petroleum was explored in inconsiderable amounts by manual methods in some
regions located over petroleum fields [321]. With the development of other
industries in the second half of the century, the exploration of deep underground
petroleum developed. In the 19th century, petroleum began to be explored on an
industrial scale and petroleum was explored commercially [315]. Many petroleum
fields started to be found. The first petroleum field explorations occurred during
the years 1857-1859 [314]. The internationally well-known exploration of a 21.2
meter-deep petroleum field on August 27, 1859 in Oil Creek, Pennsylvania was
performed by Edwin L. Drake at the suggestion of the American industrialist,
George H. Bissel [319].
In 1955 petroleum was explored in 45 countries. Currently, there are 75
countries exploring petroleum. In particular, Vietnam is considered to be one of the
countries that have great potential for raw petroleum exploration.

10


1.2. Development of the Vietnamese petroleum industry
1.2.1. Development process of the Vietnamese petroleum industry

In Vietnam the petroleum industry has been developing. Before 1975 much
exploratory drilling was done pleasantly [321,325]. In September 1975 the
government decided to organize the General gas-and-petroleum Managing
Authority. In August 1977 the Vietnamese Petroleum Company (Petro Vietnam)
was set up [66,321]. In July 1980 the Vietnamese and Russian governments
officially signed an agreement to geologize and explore petroleum in Vietnam [66]
and to establish the petroleum corporation named Vietsovpetro in June 1981. Its
meaningful activities included geologizing in most Vietnamese regions, creating
technological facilities, and training Vietnamese staff for the young petroleum
industry. In 1986 Vietnamese petroleum exporting was a signal of the development
of the petroleum industry [202]. The more considerable event, however, was the
exploration of the commercial petroleum source in the central granite region,
Dome, of the White Tiger petroleum field, resulting in some changes in the
scientific principles of petroleum in Vietnam specifically and in the world
generally. From 1988, with the Vietnamese opening policies and the laws of
“foreign investment” and of “Vietnamese petroleum,” petroleum exploration
activities in Vietnam have increased, due to the increasing number of other
countries’ petroleum companies run in Vietnam [66,321]. Since Vietnamese
petroleum exportation was up to 3 million tons per year, Vietnam was considered
one of the petroleum exporting countries in 1991. So far, the amount of petroleum
in exploration and exporting accounts for 20 million tons per year.
Based on data from petroleum field exploration and lab parameters of physiochemistry, the Vietnamese Petroleum Company estimated the potential of
accumulated underground resources to be 5-6 billion m3 (including equivalent
gases) and petroleum volume by 2010 to be more than 35-40 million tons per year
[66]. These potentials are the basis for long-term plans to develop the petroleum

11


industry. The more petroleum exploration research there is the more exact can be

the information of the petroleum industry development.
1.2.2. Petroleum field distribution and potential
Based on the research of geological organizations and Vietnamese and foreign
petroleum companies, eight accumulated underground petroleum resources were
found from 1960, with a total area of one million km2 [201,204,205,207,321,322]:
The Vietnamese oil field [203]

Figure 1.2.2.1.
Table 1.2.2.1.
3

3

Petroleum field potential (1000 m of gas equivalent to 1 m of oil)
No
1
2
3
4
5
6

Petroleum field
Red River
Phu Khanh
Cuu Long
Nam Con Son
Malay-Tho Chu
Vung May – Tu
Chinh

Area
(km2)
160
40,000
60,000
100,000
40,000

Exploratory drilling Oil-well
Total drilling
Containing
fields
petroleum (%)
155
55
17
54
42
31
32
76

60,000

-

12

-

Potential (billion
m3 of equivalent
oil)
0.6
0.3 – 0.7
0.7 – 0.8
0.65 – 0.85
0.25 – 0.35
No exploratory
drilling


1.3. Overview of the corrosion of the oil industry
Corrosion in every aspect of the oil industry. From generalized corrosion
caused by oxygen rich environments on marine structures to sulfide stress
corrosion in hostile wells, the corrosion engineer is faced with a whole gamut
of problems.[85]

Figure 1.3.1.

13


1.4. Overview of the microorganism system in petroleum
1.4.1. Microorganism system in some petroleum fields
In 1901 the engineer Seiko V. (Шейко В.) was the first who found bacteria in
petroleum

fields

in

Baku

[23,315].

However,

research

on

petroleum

microorganisms has been actually considered since Bastin (American) and
Karagiteva (Карагитева-Russian) discovered sulfate-reducing petroleum bacteria
in 1926 [6,42,142,315].
As far as Russian scientists are concerned, the research on regions of
petroleum microorganisms is essential [1,2,3,8,11,1314,17,18,19,20,25,315].
According to Kuznexova (Кузнецова)

and Svex (Швец) [10,100,315], the

regional microorganism systems in Russia’s petroleum fields include: aerobic
bacteria: hydrocarbon-oxidizing bacteria, sulfur bacteria, assimilatory sulfatereducing bacteria, gangrene bacteria,…; anaerobic bacteria: dissimilatory sulfatereducing bacteria, nitrate-reducing bacteria, cellulose-demolishing bacteria,
methane-producing bacteria, violet sulfur bacteria,….
Research on the petroleum microbes system has been conducted in China.
Wang pointed out [298,315] that the field Laojunmiao contained gangrene
bacteria, nitrate-reducing bacteria, hydrocarbon-metabolizing bacteria, and sulfatereducing bacteria.

Some Japanese scientists [315] stated that the petroleum microorganisms
system in the country is rather diverse, including Micrococcus, Brevibacterium,
and Achromobacter [163-168].
In Germany, Heyer and Schwartz [19,315] isolated bacteria in some natural
underground petroleum fields, showing the presence of Pseudomonas, sulfatereducing bacteria, Mycobacterium and Nocardia.
In Vietnam, Lai Thuy Hien, Dang Cam Ha and Ly Kim Bang [151] have
studied the microorganisms in the Thai Binh and Vung Tau oil fields. They found
most of the microbes which the other researchers had isolated: hydrocarbonoxidizing bacteria, sulfur bacteria, gangrene bacteria, sulfate-reducing bacteria,
14


nitrate-reducing bacteria, cellulose-demolishing bacteria, methane-producing
bacteria, and violet sulfur bacteria.
1.4.2. Application of microorganisms
All microorganisms in petroleum and gas fuel use hydrocarbon as the carbon
source for their metabolization and energy [53,62,312,315]. In 1961, Fush grouped
26 microbial species, including 75 types of demolishing linear-chain hydrocarbons,
and 75 types of demolishing ring-chain hydrocarbons [51]. They are present in the
following fields:
Index microorganisms: In 1939 and 1941, Mogichevxki G.A. found the
method of using index microorganisms to look for petroleum [4,15,315]. Some
other nations applied this method [5,12,13,16,21,22,315].
Microorganisms in the second-stage of petroleum exploration: In practice, the
one-stage petroleum exploration process probably recovers 30% of the total
petroleum product. For this reason, most nations use two-stage petroleum
exploration. The second stage in such exploration is usually conducted by physical,
chemical, or microbiological methods [39,52,315]. Today, the microbiological
method is used commonly [83,306]. This method makes use of a mixture of
microorganisms

pumped

into

petroleum

fields

[15,37,54,55,56,83,

145,163,164,306,315].
Microorganisms in environmental treatment: The microbial ability to degrade
hydrocarbon has been applied successfully in refining petroleum (e.g., in purifying
paraffin or separating sulfur), in protecting the environment from petroleum-based
pollution, and in cleaning water waste from petroleum-filtering factories
[70,123,151,215,276,311,315,316].
Protein-producing microorganisms: The fact that microorganisms are
cultivated on a culture containing petroleum or natural gases is of great interest to
worldwide scientists of chemistry, microbiology, and nutrition [5,7,9,17,24,36,
38,92,147,298,312,315]. It can be seen that petroleum products are used for the
protein production because of the numerous and cheap materials. Another reason is
15


that microorganisms, especially yeasts (Candida, Torulopsis, Pichia) [298,315]
and bacteria, absorb petroleum products well as they metabolize them [58].
1.4.3. Metal-corroding microorganisms
Metal corrosion by bacteria is a great trouble in the petroleum industry. The
fact that electrical systems, ships, and transport-tube systems off coasts are
corroded by bacteria is the main difficulty in operating the petroleum exploration

activities, resulting in economic losses [177, 279]. Such bacteria exist commonly
in petroleum fields and in petroleum-product preservation systems [99,315]. So the
adequate detection of metal-corroding microorganisms and the prevention of their
bad influences could contribute economically to international petroleum
exploration [72,74]. Small amounts of antibacterial substances could be useful in
this area [50,214,268,]. Coduen confirmed that 44% of the 8,215 sulfur petroleum
fields were corroded, badly affecting the economy [49]. Baca [315] suggested that
80% of the sulfur petroleum fields should be treated with anticorrosive substances.
Some petroleum companies conducted tests involving metal-corrosion
bacteria’s effects on biofilms, by using chromium as a steel component with
different contents of 0.03%, 1% and 13% [67]. In the experiment, a circulation
looping system made of PVC tubes was designed properly. After 15 days, the total
number of bacteria appearing on the biofilm was counted and their corrosion rate
was determined. The colony of bacteria was found to be greatest in the case of
0.03% crom. It was concluded that the higher the crom content in steel, the greater
the number of bacteria on biofilms and the less the corrosion rate.
[76,160,237,296]
Among corrosive bacteria in petroleum fields, the most significant were those
participating in the sulfur-converting process, such as SRB, Thiobacillus, etc.
[18,26,29,73,119,126]

16


1.5. Overview of the geographic description of the White Tiger oil field
The White Tiger oil field is located at the hollow region Cuu Long in Vung
Tau province.
The basin Cuu Long has an area of 60,000 km2, is situated along the southeast
coast of Vietnam, and includes the delta Cuu Long and the inland of the southeast
provinces [202,321]. The geological structure of the hollow region Cuu Long is as

follows: a) the root layer - Cenozoic - includes mainly granite, grandiosity, and
diorite in unsmooth states because these materials changed much during the
formation process; b) the upper layer includes walls forming Cenozoic, Eocene
sediment-Ta Coi formation, Oligocene sediment (including Ta Cu-lower Oligocene
and Tra Tan-upper Oligocene), Miocene sediment (lower, medium and upper
Miocene), and Pliocene sediment-East Ocean formation. [326,328].
Recently, the basin Cuu Long has been an attractive region for petroleum
exploration. In 1986, Vietnam started to seriously explore petroleum. From 1988,
based on the great amount of petroleum that was found in the cracks on granite
stones in the White Tiger oil field, the hollow region Cuu Long became the main
place of petroleum exploration, producing 5% of the total production in Vietnam
[128,202].
Many petroleum fields were discovered and explored [325]: Rang Dong,
Phuong Đong (lot 15-2); Ruby, Emerald, Pearl and Topaz North (lot 01-02); White
Tiger and Dai Hung (lot 09); Vai Thieu (lot 17); Black Lion, Gold Lion and White
Lion (lot 15-1); Thang Long (lot 02/97) and Gold Tuna (lot 09/2), etc., and
especially the White Tiger oil field with its 2 petroleum pouches, which was found
by Mobil. Ten other petroleum pouches in the Oligocene-accumulated
underground layers also were observed. 1986 was the first year that commercial
petroleum was announced for the granite center of the White Tiger oil field.
The White Tiger oil field is 120 km away from Vung Tau, in lot 9, and has
been explored since 1986 [325], with petroleum production of 7-9 million

17


tons/year. However, if the high technology of the two-stage petroleum exploration
is invested, there will be an increase in production and exploration time.
The petroleum-containing area in the hollow granite region Cuu Long is the
most productive area in the region [202]. As a result of any change in magma,

there are natural changes in temperature, heat energy, and weather. However, the
hollow region Cuu Long is different. Test parameters from the regions
underground mine and the geological underground-layer analysis proved that the
geological history of this region, especially the proof of the depression process,
accounts for the petroleum in the granite region.[47]
On June 19, 1981, Vietnam and CCCP signed the agreement for the
geological surveying of and the petroleum exploitation in North Vietnam. At the
same time, the Vietsovpetro Company was established.[203]
At the end of 1981, Vietsovpetro had drilled the first surveying oil well, BH5, in White Tiger. And in May, 1984, industrial petroleum flow was discovered.
On June 29, 1986, the first ton of crude oil was exploited from White
Tiger.[201,203]
1.6. The sulfur-converting cycle and dissimilatory - assimilatory sulfatereducing bacteria
1.6.1. Sulphur-converting process
Sulfur is one of the abundant elements on the Earth and its various elemental,
oxidized and reduced forms are driven by the sulfur cycle involving bacteria and
other microbes. According to Mark D.S and Frederic K.P [178] hydrogen sulfide is
a key compound in the sulfur cycle and the one of the most abundant forms of
sulfur in the environment. Four fundamental types of reactions are involved in the
sulfur cycle (Fig 1.6.1.1.): (a) mineralization or decomposition of organic sulfur
(from living cells or of synthetic origin), (b) microbial assimilation of simple sulfur
compounds into biomass, (c) oxidation of elemental sulfur and inorganic
compounds such as sulfides and thiosulfate and (d) reduction of sulfate and other
18


anions to sulfide. H2S is direct intermediate in three of these reactions:
mineralization, sulfur oxidation and sulfate reduction, all of which can be mediated
by various microbes. The sulfur cycle and the role of H2S and bacteria are in this
biogeochemistry of sulfur [178].
In spite of the fact that it exists in small quantities in cells, sulfur is an

absolutely essential element for living systems. The SRB can transform sulfur from
its most oxidized form (sulfate or SO4) to its most reduced state (sulfide or H2S)
[156,173].
Hydrogen sulfide and the Biogeochemistry of sulfur [178].
Human
(a)
(a)

Animals

Amino acids, other
simple compounds

(a)
Phytoplankton,
terrestrial plants
(b)

(c)

(a)

(a)
Heterotrophic
microorganisms

(c)
So

(d)

(c)

(c)

H2S
(b)
(d)

Sulfate

Figure 1.6.1.1.
Many bacteria are capable of producing hydrogen sulfide from organic
materials. Sulfate reducing bacteria (SRB) are the key players in the global sulfur
cycle. They represent a heterogeneous group of bacteria and Archaea
physiologically unified by their ability to perform dissimilatory sulfate reduction
for energy – generating processes. In contrast to assimilatory sulfate reduction the
use of sulfate as electron acceptor and its reduction to hydrogen sulfide is restricted
to this group.[68,178]
1.6.2. Dissimilatory sulfate-reducing bacteria
Dissimilatory reduction of sulfate to hydrogen sulfide is used by a diverse
group of heterotrophic strict anaerobes as a sink for electrons generated during
oxidation of a carbon source [26,41,60,61,69]. Industrially, this source of sulfide
19


has been used to precipitate metals in metal equipments and has been proposed for
stabilization of metals and for formation of metal sulfide “quantum” particles for
microelectronics applications [40,69,130,136]. However, sulfate-reducing bacteria
are obligate anaerobes and their application is limited to anaerobic environments
[41,69,118].

Dissimilatory sulfate-reducing bacteria comprise several groups of bacteria
that use sulfate as an oxidizing agent, reducing it to sulfide [122,125,140,141].
Most of these bacteria can also use oxidized sulfur compounds such as sulfite and
thiosulfate, or elemental sulfur [89,108,148,182].
The sulfate-reducing bacteria have been treated as phenotypic group, together
with the other sulfur-reducing bacteria, for identification purposes [108,110]. They
are found in several different phylogenetic lines. Three lines are included in
Proteobacteria (Desulfobacterales, Desulfovibrionales, and Syntrophobacterales),
all of which are included in the delta subgroup [87,90,153].
1.6.3. Assimilatory sulfate-reducing bacteria
Sulfide is also produced from sulfate during assimilatory sulfate reduction for
the synthesis of cysteine and methionine [69,102,138,149]. Unlike dissimilatory
sulfate reduction, assimilatory sulfate reduction is tightly regulated so that little or
no excess sulfide is produced and secreted from the cell. Furthermore, assimilatory
sulfate reduction operates under many growth conditions, such that the strict
anaerobic conditions necessary for dissimilatory sulfate reduction are not required.
An aerobic sulfide production pathway could be useful for precipitation and
removal or stabilization of heavy metal contaminants, for the formation of metal
sulfide quantum particles, or for any other use of sulfide under conditions that are
not strictly anaerobic.[69,78,82,84,113,115,117,135].
Assimilatory sulfate-reducing bacteria comprise many groups of bacteria that
use sulfate as an oxidizing agent, reducing it to sulfide, such as: Staphylococcus
aureus [133], Pseudomonas stutzeri [253,254], Clostridium thermoaceticum [79],
Klebsiella pneumoniae [27,28,178,137],..
20


1.7. Prokaryote classification and identification methods
1.7.1. Traditional methods
Traditional methods of classification and identification are based on:

[134,309]
-

Microbial morphology characteristics, including shape and size of microbial
cells, color of colony, movement of microbes, presence of organisms like
flagella, pill, capsule, spore, storage granule,..

-

Microbial characteristics of physiology and of energy conversion, including
ability of using C- and N-sources, path of energy conversion, products formed
by metabolization, relationship with oxygen, adaptability to endosmosis
pressure, ranges of temperature and pH suitable for microbial growth, ...

-

To identify microbes based on these traditional methods, it is common to use
classification codes, especially Bergey’s manual of systematic bacteriology.

1.7.2. Methods based on genetic factors
Identification methods based on genetic factors show accurate results in
relatively short time, by using techniques such as: nucleic acid probes; PCR;
decoding genetic order of RNA 16S of ribosome
Ribosome 70S of prokaryote, consisting of protein and three kinds of rRNA
(5S, 16S, 23S), mainly contributes to protein generation. rRNA plays an important
role, undertaking a unique function in all microbes, being present in the form of
many copies in cell, being conservative well for genetic code despite some
ribonucleotide-order difference between microbial species. That is the reason why
rRNA is considered as a tool to assess evolutionary processes, esp. to be used for
classification and identification of microbes. [106]

The technique of decoding genetic order is based not only on rRNA but also
on rDNA (rRNA-coding process), because DNA is easier to be taken and more
stable than RNA [124,127].

21


Among kinds of rRNA, rRNA 16S is the most suitable for classification and
identification of microbes. It is due to its proper length of about 1,500
ribonucleotides, whereas rRNA 5S of 120 ribonucleotides (too little genetic
information) and 23S of ~ 3000 ribonucleotides (too long, resulting in difficulty in
decoding). Some areas of rRNA 16S of all prokaryotes are conservative, some
stand for typical characteristics of each microbial species.
As the order of rDNA 16S of a microbe is decoded completely, the
identification of the microbe is well done by comparing its order with those of
rDNA of other microbial species which are conserved in the genetic bank in the
condition of using Blast software or others. Based on the genetic data, the
phylogenic tree is probably drawn, showing evolution relationship between the
microbial species involved and others.
The above method is used commonly, but makes sure that:
-

The order of rDNA 16S must be long. (> 1300 ribonucleotides)

-

The difference between orders of any two rDNA 16S of a microbial species
must not more than 0.5%.
Commonly, the order of rDNA 16S is used for comparison at the level of

microbial species, whereas rDNA 16S – rDNA 23S (internally transcribed spacer,
ITS or intergenic spacer, IGS) for the level of microbial genera.
1.8. Antibacterial substances (biocides)
Hydrogen sulfide is associated with countless problems in the oil industry,
including: the contamination of fuel gas and oil, the corrosion of metal surfaces,
and the plugging of reservoirs and consequent reduced oil recovery due to the
precipitation of metal sulfides. A major source of sulfide is the metabolic end
product of sulfate reducing bacteria (SRB). These organisms reduce sulfate to
sulfide at the expense of the oxidation of a wide range of organic substrates and
hydrogen. If sulfate is available, significant amounts of sulfide can be generated,
posing

an

environmental

risk

and

[75,143,227]
22

undesirable

economic

consequences