LITERATURE OVERVIEW
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 7 th and 8 th 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 19 th 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 19 th 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.
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 physio- chemistry, the Vietnamese Petroleum Company estimated the potential of accumulated underground resources to be 5-6 billion m 3 (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 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 km 2 [201,204,205,207,321,322]:
Petroleum field potential (1000 m 3 of gas equivalent to 1 m 3 of oil)
Exploratory drilling Oil-well Potential (billion m 3 of equivalent oil)
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]
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 sulfate- reducing bacteria, gangrene bacteria,…; anaerobic bacteria: dissimilatory sulfate- reducing 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 sulfate- reducing 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, sulfate- reducing 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: hydrocarbon- oxidizing bacteria, sulfur bacteria, gangrene bacteria, sulfate-reducing bacteria, nitrate-reducing bacteria, cellulose-demolishing bacteria, methane-producing bacteria, and violet sulfur bacteria
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 that microorganisms, especially yeasts (Candida, Torulopsis, Pichia) [298,315] and bacteria, absorb petroleum products well as they metabolize them [58]
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
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 km 2 , 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 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, BH-
5, 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]
The sulfur-converting cycle and dissimilatory - assimilatory sulfate-
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 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 H 2 S 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 SO 4 ) to its most reduced state (sulfide or H 2 S) [156,173]
Hydrogen sulfide and the Biogeochemistry of sulfur [178]
Animals Amino acids, other simple compounds
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]
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 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]
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],
Prokaryote classification and identification 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]
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.
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 undesirable economic consequences [75,143,227]
Annually, almost 16,000 petroleum fields are corroded, resulting in the loss of nearly 16 million USD/year For the explored fields which come into contact with acid, there were no efforts made to reduce corrosion, causing the loss of up to 32 million USD/year In the case of the US’s petroleum field exploration, it was estimated that without using antibacterial substances against corrosion, the US would lose 12.1 million USD/year; the US, however, would probably save 10 million USD/year if it applied antibacterial substances in petroleum exploration [50,59,64,65,103,275,315]
The use of antibacterial substances in explored fields and containers of petroleum is of concern to many countries [46,93,104,315] It was suggested by Meihot that dietylen glycol, trietelen glycol or metoxyetnol should be used for anti- corrosion in aircraft [184,315] In addition, France and Russia used 8-oxyquinolyn effectively in aircraft [181,315] Germany has just discovered a new potential antibacterial substance containing sulfur In his tests, Novikova (Russia) claimed that using 2, 4-dinitroanizol, nitrobrombenzol, or nitropaxetanilide is likely to eliminate 94–100% of sulfate-reducing bacteria [315] Americans used resorsinol, cresol or compounds containing nickel as antibacterial substances to prevent corrosion [64,83,86,112], whereas the British used pentachlorphenclat natri hypochlorid, or formaldehyde [50,107,315]
Other companies in the USA use OILAID-B-10 which is effective over a broad pH range and against many types of microorganisms, including sulfate- reducing bacteria and slime-forming bacteria and algae; it can be used in drilling mud, completion/work over fluids, water floods and packer fluids with different concentrations depending on the contamination [186,187] These companies also use various biocides, including OILAID-B-45, which is a highly effective biocide designed for use in water-based fracturing fluids OILAID-B-45 is a thiocarbamate blend that is effective in systems with high organic contamination [187 OILAID-B-45 has been found to be as effective as conventional biocides in preventing bacterial degradation of fracturing fluid components OILAID-B-45 can control
Glutaraldehyde aerobic bacteria such as slime-formers and anaerobic bacteria such as sulfate- reducers By controlling sulfate-reducers, the chances of immediate H 2 S corrosion are lessened OILAID-B-45 can also control facultative bacteria and is an effective replacement for formaldehyde release agents [7]
In Asia, especially in Vietnam, Myanmar, Indonesia, Malaysia, Bangladesh, Japan, Taiwan, Hong Kong, and Singapore, biocide is used to prevent rust and corrosion, and other specialty chemical products that AP Oil manufactures for the oil industry are also used AP Oil also operates a specialty chemical plant in Singapore to produce products branded as Hexaclean, Hexafuel and Hexatreat
According to WallStraits.com [297], in 1998, the AP Oil company and APP(S) were awarded ISO 9002 certification for Quality Management Systems in the manufacturing of lubricants by SGS Yarsley International Certification Services; the Company and APP(S) were also awarded ISO 9001 certification by SGS Yarsley for manufacturing, research and development of lubricants and specialty products in September 2000; in addition, the Company is an American Petroleum Institute ("API") license holder Their products are licensed under the API's Engine Oil Licensing and Certification System ("EOLCS") This means that the certified products manufactured at their plants meet the standards and specifications set by API They began to export their lubricant products to Vietnam in 1991 They have been in discussion with a potential local partner to build the plant in Dong Nai Province, southern Vietnam Today, about 90% of their products are exported worldwide.[297]
Hexaclean includes products for cleaning and maintenance: tank cleaner, waterless hand cleaner, liquid detergent, filter cleaner, electric motor cleaner, coil cleaner, etc Hexafuel includes products for fuel treatment: emulsion breaker, fuel and sludge conditioner, and diesel care, soot remover, etc
Hexatreat includes products for water treatment: alkaline pH controller, film-forming amine, liquid coagulant, hardness controller, combined boiler water treatment, cooling system corrosion and scale inhibitor, evaporator treatment, corrosion inhibitor, chromate diesel engine cooling water treatment, algae remover, biokill, antifreeze, etc Hexatreat can control SRB and is an effective replacement for glutaraldehyde and nitrate release agents
Today, Vietsovpetro-Vietnam is using the latest antibacterial substance (known as Hexatreat) which belongs to the series of Hexaclean, Hexafuel and
Hexatreat with different functions for each Among which, Hexatreat 1512 is considered to be an antibacterial substance (biocide) against sulfur-reducing bacteria Hexatreat 1512 is used with the aim of reducing financial losses in petroleum exploration In the research for this paper Hexatreat 1512 was used for all experiments.
MATERIALS AND METHODS
Experiment plan
Crude oil samples Determinning total SRB
Studying the corrosion’s ability of SRB
Studying the effect of anti- microbial agent on SRB Identifying SRB by 16S rDNA analyze
Studying the effect of anti-microbial agent on 16S rDNA of SRB by DNA sequencing
Selecting SRB Testing the corrosion’s ability of SRB
Studying the adaptation’s ability of SRB in different environment
Experiments were conducted according to the following steps:
1 Determine the principal phio-chemical properties of the samples and of the samples’ region Preserve correctly the samples that will be used in microbial tests
2 Determine the aggressive total number of SRB in the samples by the SRB- BART TM test
3 Identify the microbes in the samples: isolate the groups of aerobic and anaerobic microbes in the samples; enrich the microbes with a peptone solution; identify the microbes by Gram dyeing, Catalase test, ID32E test (REF 32 400 – bioMérieux) and API20A test (REF 20 300 – bioMérieux)
4 Determine the corrosion ability of the SRB: select the SRB from the testing microbes; study the corrosion ability of all SRB by the ASTM 130 D test and
5 Study the effects of the antimicrobial agent on selected SRB by the concentration and by the storage time Test the corrosive ability of living bacteria
6 Study the adaptation ability of SRB in different environments: enrich the selected SRB; study the adaptation ability of SRB in different environments
7 Study the effects of the antimicrobial agent on the 16S rDNA of selected SRB by DNA analyzing: select the SRB that corrode deeply and remain alive after treatment with the antimicrobial agent; enrich the selected SRB Extract DNA from the selected SRB; identify the selected SRB by DNA analyzing.
Materials and equipments
2.2.1 Places and procedures to take samples:
Samples were taken from the petroleum fields on the weathered foundation of the south arch of the White Tiger oil field (BK-2) in Ba Ria-Vung Tau province They were taken at depths of 3,000-5,000 m The explored layers’ water temperature and pressure were ≈80 ° C and 220 – 260 atm respectively The sample- taking locations were in the mouth of the petroleum field
The samples used to determine the SRB populations with the SRB-BART TM test, to identify the microorganisms and to do other experiments were from 11 oil wells Table 2.2.1.1.1 shows the dates of sample-taking and sample-testing
2.2.1.2 Procedure to take and prepare samples
Petroleum samples taken out of the field were immediately put in antiseptic plastic bottles The filled bottles were kept cold and closed, and brought to the lab after a while Lab tests on the samples were conducted within 24 hours after taking the samples To test the samples for anaerobic microbes, the samples’ containers were injected with non-reactive gases The samples were kept at cold temperatures of 3 – 5 ° C The samples were brought back to normal room temperature before being used
− Crude oil samples from the oil well (White Tiger) of Ba Ria, Vung Tau, Vietnam
− SRB – BART TM test (Droy Bioconcepts InC., Canada) to determine the number of SRB
− Medium (Difco, Detroit, Mich.): Postgate A, Postgate B, PCA, PGA, Peptone, Colistine (Appendix 2); Gram dyeing (Difco, Detroit, Mich.); Microbes identified test kit: API20A, ID32E (bioMérieux) (Appendix 1)
− Hexatreat 1512: antimicrobial agent that was received from Vietsovpetro Co
− Chemical for DNA extraction: CTAB, phenol/chloroform, ethanol, TE buffer, NaCl, isopropanol, RNase
− Chemical for PCR reaction: PCR buffer, dNTP, Mg 2 solution, Primer pair 27F/1525R, Sigmal Taq DNA polymerase
− Chemical for cloning and purifying plasmid DNA: salt solution (1.2M NaCl, 0,06M MgCl2), PCR4-TOPO vector 20rnx, S.O.C solution, LB medium, GTE buffer, NaOH/SDS, CH3COOK 3M, isopropanol, TE, RNase, CH3COONa
− Chemical for restriction reaction: Tango Buffer, EcoRI restriction enzyme
− Chemical for Electrophoresis running: agarose , Maker 1kb, Hind III 1kb
− Chemical for sequencing analyzing: PEG juice, ethanol, BigDye, primer pair: M13F, M13R, 27F, 1525R, 520F, and 920R, EDTA 125mM, TSR, HI-DI formadine (Integrated DNA Technologies, IA and Sigma)
Microorganism cabinet (Esco, Singapore), autoclave (Hitachi, Japan), Refrigerated incubator shaker (NBS, USA), Scanning Electron microscope microscope (Nikon, Japan), UV-VIS Scanning Spectrophotometer (Thermo Electron Cooperation, USA), Electrophoresis(A&B Applied Biosystem), PCR system 9700- Gene Amp(A&B Applied Biosystem); Alpha Imager 3400 (Innotech Inc.) 310 Genetic Analyzer(A&B Applied Biosystem), 3130x Genetic Analyzer(A&B Applied Biosystem), GEN-box anaer (REF 96 124 – bioMérieux), Copper Corrosion testing system - ASTM D130, IP 154, ISO 2160 (PETROTEST, Germany) Glass apparatus and other related equipment.
Methods
2.3.1 Check the aggressive level of SRB by the SRB – BART TM test
For the detection of SRB, the SRB-BART TM tester of Droycon Bioconcepts Inc., Canada was used This system is able to detect the growth of SRB through the generation of an aspect ratio within the culturing fluids in the tester The system is based on the formation of a vertical reduction – oxidation gradient while a diffusion gradient is generated from the bottom up of a selective medium specific for the SRB bacteria species, based upon the formulations developed by Postgate [71,94,224,225,226,315] The SRB activity is easily recognized in the BART TM tester because the sulfate reduces to H 2 S This tester received environmental technology verification by ETV Canada Inc in 2002 and is able to detect two distinct groups of SRB: aerobes and anaerobes [95]
SRB-BART TM tester system [94,95]
Aerobic growth of the bacteria will occur at the surface of the medium between the
BART-FID and the wall of the tube.
15 ml-20ml of sample are used to bring the
BART-FID up to the correct level Nutrients will gradually diffuse up the sample column to support the aerobic growth.
Nutrient media for growth is provided as a sterile dried matrix on the floor of the tube.
Floating Intercedent Device (FID) used to create a barrier for oxygen diffusion
Once the oxygen has been used by the aerobes, this zone becomes free of oxygen and anaerobic growth will dominate.
Black only around Ball/Top (BT)- Aerobic SRB consortium
Black in Base and around Ball (BA)-
Combination of anaerobic (BB) and aerobic (BT) SRB
Solution Cloudy (CG) – Anaerobe bacteria present
2.3.1.2 Method of using SRB-BART TM test [95] :
1 Remove the inner tube from the outer tube Using the outer tube from the BART collect at least 20ml of sample Fill the inner tube with the sample until the level reaches the fill line
2 Tightly screw the cap back on the inner tube Return the inner tube to the outer tube and screw the outer cap on tightly Allow the medium to dissolve slowly, and the ball to rise at its own speed Do not shake or swirl the tube Label the outer tube with the date and sample origin
3 Place the BART tube away from direct sunlight and allow it to incubate at room temperature Check the BART visually for reaction daily
2.3.1.3 Time Lag to SRB-BART™ Populations
The populations of SRB given in Table 2.3.1.3.1 reflect the higher recovery rates and comparisons with other tests may show the SRB-BART™ to be the more sensitive
Relationship between time lag and correlative population for SRB [94]
Time Lag (days) Population (CFU/ml) Time Lag (days) Population (CFU/ml)
The risk potential for the severity of a detected SRB event can be expressed through the shortness of the time lag (in days) as follows:
1 Very aggressive (treatment should be started as early as convenient)
2 Aggressive (treatment should be considered before the condition degenerates further)
3 Moderately Aggressive (treatment may not be required but vigilance through ongoing testing should be practiced)
4 Normal Background Levels (routine testing is recommended)
Relationship between the time lag to the first reaction in an SRB-BART™ and the aggressively of the SRB [94]
Very aggressive Aggressive Moderate Not
PCA media was used to determine the aerobic and anaerobe microbes; PGA for the molds; Post B for SRB Use the micropipette to take up 0.1ml of the petroleum sample into each Petri dish Use a different micropipette-tube for each petroleum sample Pour about 15 – 20ml the available culture Move the Petri dish in a gentle circle so that the culture and petroleum sample are mixed properly Close the Petri dish Let the materials in the Petri dish solidify naturally
Incubate microbes: For the testing of aerobic microbes : incubate at 37 ° C For the testing of anaerobic microbes : Put the Petri dish containing the samples into a closed incubation container Tear the package of GEN-box anaer (REF 96 124 – bioMérieux), take out the pack inside and put it quickly into the same incubation container Close and incubate at 37 ° C
To make sure that the microbes were dispersed correctly in the cultures and improve their microbial population so they will be available for later experiments
- Transfer the 0.1ml petroleum samples directly into the prepared cultures without diluting the samples Each sample is isolated into 4 test tubes with Post A and 4 Petri dishes with Post B Half in aerobic conditions and half in anaerobic conditions
- Prepare the test tubes containing 10ml sterilized Post A; a bottle containing 10ml Post B Sterilized solution of FeSO 4 H 2 O 5%
- Isolate SRB in liquid cultures: place 0.1ml of the solution of FeSO4 into each test tube (Post A) Shake the test tubes Place 0.1ml of the petroleum sample into each test tube
- Isolate SRB in solid cultures: Use the micropipette to take up 0.1ml solution FeSO 4 in each Petri dish Transfer 0.1ml of the petroleum sample into each Petri dish Pour about 15 – 20ml (Post B) of the available culture Let the materials in the Petri dish solidify naturally
2.3.2.3 Method of isolating and purifying the selected microbes
Isolate selectively and purify microbes Transfer and preserve microbes Create a single microbial colony on a series of Petri dishes Each type of microbe is isolated on at least one Petri dish (the number of Petri dishes depends on the experimental requirement) Prepare the Petri dish containing 20ml culture Post B (for SRB), and PCA (for other microorganisms) Enrich microbial population for liquid culture and for solid culture Transfer microbes attached on the microbial loop to a surface of the Petri dish Some microbe-transferring techniques are T-shape, corner, spray, and continuous technique Incubate microbes (See 2.3.2.1.)
2.3.2.4 Method of isolating facultative anaerobic SRB
Look for the presence of non-obligated anaerobic microbes in raw petroleum Isolate non-obligated anaerobic microbes from the liquid mixture or colonies of anaerobic microbes by using the microbe-transferring Petri dish Incubate in aerobic conditions If these microbes grow, they are able to live in both aerobic and anaerobic conditions Isolate SRB in the Petri dish containing 20ml culture Post B Isolate the microbes: (See 2.3.2.3.)
2.3.3 Method of enriching microbial population [57,131,225,308, 315]
Transfer microbes to new nutritive cultures so that they grow and reproduce, resulting in the increase of the microbial population Prepare the test tubes (filled with liquid or solid cultures) and Petri dishes containing microbes involved Petri dishes contain 20ml Post B and PCA Sloping test tube of culture contains 10ml Post B and PCA Test tube contains 10ml Peptone or Post A
Increase the microbial population: Enrich the microbial population for the liquid culture and for the solid culture Transfer microbes to cultures for the Petri dish, for the sloping-culture test tube and for the liquid-culture test tube Incubate microbes (See 2.3.2.1.)
Microbes were identified according to Bergey’s manual of Determinative Bacteriology and Bergey’s manual of Systematic Bacteriology and the following process: test ID32E (REF 32 400–bioMérieux)(Appendix 1.1) to identify the aerobic bacteria; test API20A (REF 20 300–bioMérieux)(Appendix 1.2) to identify the anaerobic bacteria; Gram dyeing (complex Gram dyeing); antibiotic test: with Colistine (from Nam Khoa Co.); catalase test
2.3.5.1 Detection of Copper Corrosion from Petroleum Products by the Copper
Strip Tarnish Test [63, 95] ỉ Principle: This test determines the corrosiveness of fuel to copper by submerging a copper strip in a heated sample of fuel for a specified time period At the end of the time period, the strip is compared to the ASTM Copper Strip Corrosion Standards to determine the level of the fuel’s corrosiveness ỉ Procedure:
- Prepare the solution of selected microbes with concentrations of 10 7 CFU/ml, the Peptone and Postgate A medium
- Pour 19.9 ml or 19.8 ml of each medium into each tube; fill every tube with 0.1 or 0.2ml of the prepared bacteria solution (equivalent 50,000-100,000 cells/ml/tube)
- Immerse the clean copper strip (which was treated with abrasive-paper, alcohol and acetone and compared with strip corrosion standard) into the tube by times: 0, 72, 96, 120, 144, and 168 hrs
- Clean and measure the corrosion after the elapsed time with the corrosion bomb (at 85 0 C, 1 hr) and the ASTM Copper Strip Corrosion Standards ỉ Result: The final colors of the strips were compared with the ASTM Copper Strip Corrosion Standards
ASTM Copper Strip Corrosion Standards (ASTM D130 – 94)
1 Slight tarnish a Light orange, almost the same as freshly polished strip b Dark orange
2 Moderate tarnish a Claret red b Lavender c Multicolored with lavender blue or silver, or both, overlaid on claret red d Silvery e Brassy or gold
3 Dark tarnish a Magenta overcast on brassy strip b Multicolored with red and green showing (peacock), but no gray
4 Corrosion a Transparent black, dark gray or brown with peacock green barely showing b Graphite or lusterless black c Glossy or jet black
OBSERVATION OF MICROBES IN OILS
Typical properties of the petroleum samples
Petroleum is a liquid having a color ranging from light brown to black It can be pale blue under light It typically has an unpleasant odor Petroleum in the White Tiger oil field contains a mixture of hydrocarbons in which there is much paraffin (30%), little sulfur, much methane (20%), and much ion sulfate (16.46- 617.25 mg/l) Water in the oil-bearing bed contains only small amounts of minerals (4.1-28.76 mg/l) and O 2 [315] (Fig.3.1.1.)
The samples were extracted from fields in the south arch of the White Tiger oil field (Bach Ho), in Ba Ria-Vung Tau province They were picked up at 3,000- 5,000 m depths; the explored layers’ water temperature and pressure were ∼ 80°C and 220 – 260 atm respectively The petroleum samples were taken from the mouths of the fields
The samples used to determine the SRB populations with the SRB-BART TM test and to identify the microorganisms were from 11 oil wells and were taken on
April 24, 2006 On 9/13/2005, 11/15/2005, and 4/24/2006 samples were taken from 6 of the oil wells to do other experiments also (Table 3.1.1.)
Properties of petroleum fields (data from Vietsovpetro Co.)
No Lot Depth of sample (m)
Depth from the lot to the sample (m)
With materials such as sulfur, ion sulfate, and water in the oil well one can see that the crude oil of the White Tiger oil field is rich in SRB that produce H2S The presence of SRB is the reason so much equipment used in the petroleum industry corrodes Therefore, it is understandable that so many antimicrobial agents are bought every year to kill SRB
At this time, Vietsovpetro uses Hexatreat 1512 as an antimicrobial agent Hexatreat 1512 was also used in our experiments.
Study of the aggressive level of sulfate reducing bacteria
All samples were tested with the SRB-BART TM test Most results are aggressive or very aggressive after 2 days (Fig.3.2.1 and Table 3.2.1.) Figure 3.2.1 shows the states of the petroleum samples after the SRB-BART TM test
The petroleum samples with SRB-BART TM test after 48hrs
The correlative SRB from White Tiger oil field with SRB-BART TM test
2 CKB-2001 04//24/2006 BA 700,000 + + included petroleum and water
According to tables 2.3.1.3.2 and 3.2.1 we can see that:
- The petroleum samples from CKB 002 were taken on 11/15/2005 and 4/24/2006 After 2 days one could see the state at the BT level This means that this oil well had aerobic SRB, whose quantity is at the aggressive level Treatment of the bacteria should be considered before the condition degenerates further
But for the samples taken on 9/16/2005, the BT level was reached after 4 days This means that the quantity of the SRB was not at very aggressive levels at this time
- The petroleum samples from CKB 2001, CKB 402, CKB 421, CKB 422 were taken three times (9/16/2005, 11/15/2005, and 4/24/2006) All of the samples had a combination of reactions (BB and BT) 2 days after the SRB-BART TM test Blackening occurred both in the base and around the ball, although the length of the inner test vial was not always blackened According to the table 2.3.1.3.2., the SRB density of these samples was very aggressive Treatment for SRB should be started as soon as possible
- The petroleum samples from CKB 420 were taken on 9/16/2005, 11/15/2005, and 4/24/2006 After 5 or 6 days the signs of SRB appeared These signs were not the same at the three times, but were moderately aggressive Treatment for the SRB may not be required but attention through ongoing testing should be practiced
- The other samples were taken on 4/24/2006 Most of them had SRB quantities at the aggressive level (reactions BT or BA after 2 days), except the samples from CKB 428 (reaction BT after 6 days)
The petroleum samples from White Tiger oil field, which is located in Vung Tau – Vietnam, contained high populations of SRB, resulting in oil-rig corrosion there, as well as the corrosion of containers, oil-conveying ships, and tubes
In the samples there are both aerobes and anaerobes at the aggressive level That is why the explored oil which is exposed to the air can also be corroded by microbes In fact, anti-microbe substances used by Vietsovpetro have effects on the microbes only temporarily at the places of exploration; there are no effects on the SRB during the periods of conveyance and storage of oil and the SRB are thus able to cause corrosion
The microbial corrosion proceeds as follows: SRB reduce sulfate into H 2 S
H 2 S in turn corrodes steel, producing FeS As a result, FeS functions as the cathode of a galvanic pin whereas Fe functions as the anode The ferric anode is corroded and hydrogen gas is formed at the cathode, FeS, through a process in which the SRB take electrons from the surface of the ferric anode to proteins in bacterial cells
[327] It is important to prevent such corrosion; otherwise the corrosion process will speed up.
Study of the general characteristics of microorganism system
3.3.1 Study of the microbial growth, morphology and colony appearance
3.3.1.1 Study of the colony appearance
Microbial growth made the liquid culture cloudy, and in some samples microbes formed black sediment that accumulated at the test tube bottom
Microbes’ growth in liquid cultures
(a)Post A medium, anaerobe condition: CKB 2001_KK (black), CKB 420_KK
(white); (b) Post A medium, aerobe condition: CKB 421_HK (black), CKB
2001_HK (white) (c) Peptone medium, anaerobe condition; (d) Post B medium, anaerobic condition
For the samples tested in Post A, black FeS particles formed, usually by the SRB, i.e., the SRB converted sulfur in the oxidizable state (sulfate compounds, such as FeSO 4 ) into the reducible state (sulfite, or H 2 S) This reaction was used to detect the presence of SRB (Fig.3.3.1.1.1.)
It can be seen that there were a lot of black particles This was supported by the above SRB-BART TM test on the number of samples containing SRB and on the populations of SRB in the samples, in terms of both aerobes and anaerobes (Fig 3.3.1.1.1)
Microbes’ growth in liquid cultures - Post A - in the aerobic condition
Figure 3.3.1.1.2 shows that in the aerobic condition, microbes’ still caused corrosion Especially the bacteria of the samples from CKB 402, CKB 2002, CKB
2001, CKB 421, and CKB 422 were very dangerous The contents of the tubes became black quickly and spread out into a large area In the other samples the color did not change completely black, but in the bottom of the tube it became dark This means that these bacteria also produced H 2 S
Microbes’ growth in liquid cultures - Post A - in anaerobic condition
In the anaerobic condition (Fig 3.3.1.1.3), the microbes grew in the Post A (liquid culture) They also produced H2S This chemical reacts with FeSO4 (in
PostA medium) and produces FeS It can be seen that the black to dark brown color that appeared at the bottom of the tubes came from samples from the following oil wells: CKB002, CKB2002, CKB402, CKB428, CKB556, CKB491, CKB421, CKB422, and CKB2001 In the samples from CKB 409 and CKB 420 the colors were light brown This means that in those samples the bacteria produced only small amounts of H2S
The samples that had quantities of SRB at the aggressive level (Table 3.2.1.) were chosen From the test with the Post A medium in both aerobic and anaerobic conditions, the samples that contained bacteria that can produce a lot of H 2 S (Fig 3.3.1.1.2 and 3.3.1.1.3.) were chosen Then the samples were checked again with the Post B medium (Fig 3.3.1.1.1./d) The medium became black This means that these samples had a lot of SRB at the very aggressive level It is necessary to treat these SRB immediately, before they destroy all the equipment used in the oil industry
Observed in a Petri dish used for microbial isolation, the microbes grew from the petroleum stain and spread widely on the agar surface (Fig 3.3.1.1.4) The surrounding microbial layer had a color of turbid white to pale yellow, was unsmooth, and had a curving border When incubated for a longer time, the layer spread completely over the agar surface
Microbes’ growth on the culture PCA and Post B
Culture PCA: (a) CKB 421_HK; (b) CKB 420_HK); culture Post B: (c) CKB
On the agar surface, the microbes grew in different colonies The colony characteristics of the aerobic and anaerobic microbes were pretty similar, such as pale yellow, round, and smooth (Fig 3.3.1.1.4/a-b) Some colonies were black on the Postgate B medium (Fig 3.3.1.1.4/c-d)
The Post B medium containing SO4 2- was used for the SRB tests Samples with the appearance of black colonies were isolated for further observation of their characteristics These bacteria, which can corrode metals by reducing sulfate, were the microbes of interest in this thesis (Fig 3.3.1.1.4/d)
On the slopping agar, the microbes grew in different colors and different shapes Some of them were white (Fig 3.3.1.1.5/1a), but others were black (Fig 3.3.1.1.5/1b)
For the samples observed in the agar-slopping tubes with the Postgate B medium, SRB also appeared with black agar areas (Fig 3.3.1.1.5/1b)
Microbes on the slopping agar culture Post B and PGA
1.a-b Microbes on the slopping agar culture Post B (a.CKB 2001-HK, b.CKB2001- KK);2.a-b Molds’ growth on the agar culture PGA ((a) CKB 402, (b) CKB 422)
SRB-BART TM tests with the Post A and Post B media indicated the actual presence of SRB in the samples from the White Tiger oil field
In the observed samples there were molds in addition to bacteria (Fig 3.3.1.1.5/2a-b) Though the populations of the molds were small, their presence showed the complexity of the microbial system in White Tiger oil field Mold appearances were: round colony with white or yellowish brown color, fine branches (Fig 3.3.1.1.5/2.a-b)
The color and the shape of microbes that was present on the different media shows that the crude oil of the White Tiger oil field is rich in microorganisms They are of various kinds, such as: molds, aerobic bacteria, anaerobic bacteria, SRB, etc These microorganisms would be identified in the following experiments
3.3.1.2 Study of the microbial growth over time
Each microbial species had a different growing time
Comparison of the microbes’ growth time
Before isolation Post A After 3 – 5 days After 7 – 10 days
Post B After 2 – 4 days After 7 – 10 days Pure species Post A, Peptone After 2 – 3 days After 3 – 5 days
Post B After 6 hour- 2 days After 3 – 5 days
Table 3.3.1.2.1 shows that aerobes grew faster than anaerobes: 2-3 days for aerobes whereas 7 days for anaerobes in the samples before isolation Furthermore, both aerobes and anaerobes in the samples after isolation grew faster than those in the samples before isolation Possibly, among the original microbes before isolation, some species could prevent the growth of others; whereas, the objective microbial species, which were pure after isolation, can grow well In particular, aerobes also grew fast, especially in the Postgate B medium; for example, some aerobic species could produce colonies after only 6 hours It was interesting that anaerobes growing in either the Postgate A medium or the Postgate B medium can produce colonies in the same length of time Aerobes showed the same behavior However, pure species grew faster
Practically, there is originally a complex population of microbes in the samples Thus, at the first stage after crude oil exploration, such a complex population of microbes affects the explored oil At the next stages, the more adaptable microbes overgrow the others As a result, the number of microbial species in oil is fewer but their effect on the surroundings is more considerable
3.3.1.3 Study of the microbial morphology:
Each of our bacteria samples was scanned at a magnification of 15,000x by SEM Our bacteria had different shape, such as: spherical, ovoid, rod-shaped or vibrioid-shape (Fig 3.3.1.3.1)
SEM micrographs of typical bacteria morphology
(a) from oil-well CKB002, (b) from oil-well CKB2001, (c) from oil-well CKB421
3.3.2 Study of the microbes in raw petroleum samples from the White Tiger oil field
Yeast was not found in the test samples The frequencies of molds in samples were different In the samples from oil wells CKB 409, CKB 421, CKB 428, and CKB 2001, there was no appearance of molds In the samples from CKB 556, the frequency of the molds was the highest (40 cells/ml) The genera of the molds were
Penicillium and Aspergillus Both fungal species are known to produce citric acid, which is involved in iron attack [161]
Through microbial isolation in raw petroleum samples, the frequencies of the aerobic bacteria in the samples varied The species of aerobic microbes that were isolated from samples were tested with the biochemical test ID32E From the biochemical tests by ID32E and according to Bergey’s manual [57], bacteria were identified in the test samples by looking up the respective reference codes available in the bacteria-identifying table The common aerobic bacteria were: Enterobacter spp.; Serratia spp; Sphingomonas spp; Vibrio spp, Pseudomonas spp One of the species found in all the oil samples is Pseudomonas spp, considered metal- reducing bacteria (MRB) The bacterium can reduce iron oxide, causing corrosion in aerobic conditions It is important to pay attention to such conditions for test observation in oil storage and oil conveyance
The species of anaerobic microbes that were isolated from the samples were tested with the biochemical test called API 20 A From the biochemical tests by API 20 A and according to Bergey’s manual [57], bacteria were identified in the test samples by looking up the respective reference codes available in the bacteria- identifying table There were 12 common anaerobe microbial species, i.e.,
The corrosion ability of the microorganisms
3.4.1 The standard diagram of microbial populations
Slurry of selected microbes was prepared with a translucence measurement of less than 1 by using the spectrometer Prepared samples with 5 different contents, and then measured the absorption at 580 nm in order to set up a standard diagram
The standard diagrams of microbial populations
Standar of Absorbance at 580 nm of 5KK y = 9*10 -8 x
Ab so rb an ce a t 58 0 n m
Standar of Absorbance at 580 nm of 8KK y = 8*10 -8 x
Ab so rb an ce a t 58 0 n m
The standard diagrams (Fig 3.4.1.1) showed the high accuracy of methods of spectrometry and of microbial population measurement So, these methods were used for the next tests
Slurries with populations of ~10 7 cells/ml were used in order to observe metal corrosion and the effects of antibacterial substances The volumes of the microbial slurry were 0.1 and 0.2 ml, respectively - equivalent to 50000 and 100000 cells/ml
3.4.2 The corrosion ability of the microorganisms
After isolation, the microbial species that corroded metals most and producing
H 2 S in the samples were chosen for further tests ASTM 130D and Copper mass measuring methods were used to test the extent of microbial corrosion In the two testing methods, the above chosen microbial species were tested, using the 0.1 ml and 0.2 ml tested samples with microbial populations of 10 7 cells/ml, adding 15 ml peptone solution and NaCl to each test tube Corrosion ability tests, which were conducted at 0 h, 72 h, and 144 h, gave the following results
Microbial ability to corrode metal (ASTM method)
Population of microbes: 0.1 ml Population of microbes: 0.2 ml
Code Oil well 0 h 72 h 144 h Code Oil well 0 h 72 h 144 h
Blank (no microbes) Freshly polished Blank (no microbes) Freshly polished 1KK CKB-402 Freshly polished 1a 1b 1KK CKB-402 Freshly polished 2b 2c 4KK CKB-402 Freshly polished 2b 2b 4KK CKB-402 Freshly polished 2b 2b 5KK CKB-2001 Freshly polished 2b 2c 5KK CKB-2001 Freshly polished 2b 3a 8KK CKB-421 Freshly polished 2b 3b 8KK CKB-421 Freshly polished 2b 3b 9KK CKB-420 Freshly polished 2a 2b 9KK CKB-420 Freshly polished 2b 2c 12KK CKB-422 Freshly polished 2a 2a 12KK CKB-422 Freshly polished 2b 2c
It can be seen that the microbes corrode the metal in different This might be due to either the changeable amount of produced substances or to those produced substances’ effects on the microbes Furthermore, there were some microbes that corrode metals in the condition of high microbial populations
For the ASTM 130D method, the 4KK species showed an unchanged ability to corrode metal from 72 h to 144 h with two concentration of bacteria The 1KK species with low populations in the samples showed an unstable ability to corrode metal, i.e., without corrosion in the first 72 h but with significantly increasing corrosion afterward It indicates that the 1KK species had low metal-corrosion ability in low microbial populations
Additionally, the 5KK, 8KK, 9KK and 12KK species showed an increase in corrosion according to the populations of the microbes and the working time of the microbes But the 5KK and 8KK had higher corrosion ability than the others
The two tests showed that the corrosion activity 5KK and 8KK species increased with the increase in bacteria populations in the tested samples and with the increase in observed time In other words, these bacteria were highly adaptable to the environment, producing increasing corrosion ability Additionally, the corrosion ability of those bacteria was highest That is why the observation of these bacteria was a significant consideration
Ability of microbes to corrode metal (Copper mass measuring method)
With their corrosion ability and biochemical characters, the facultative anaerobic bacteria, 5KK and 8KK, were isolated from the CKB-421 and CKB-
2001 samples From now, we named them as SRB-5KK and SRB-8KK because of their ability of sulfate/sulfur reducing In addition, they were also chosen in order to observe all the other studies in this research.
EFFECTS OF BIOCIDES ON MICROBES ISOLATED
Effects of biocides on selected bacteria
4.1.1 Effects of biocides on SRB-5KK species
Concentrations of Hexatreat 1512 added to each sample were 0 ppm, 10 ppm,
20 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 80 ppm, 90 ppm, and 100 ppm Each sample contained microbes at a concentration of 10 6 CFU/ml The tested samples were tested with the spectrometry method [35,77,121,222,250] In terms of the effects of Hexatreat 1512 on SRB-5KK species, the absorbance values, which depend on the biocide concentration and the survey time, were measured Based on the established standard diagram (Fig.3.4.1.1.), the conversion of absorption to respective populations of SRB-5KK species was done, showing the same resulting decreases in microbe populations
In the blank sample without biocide, the bacteria grew fast in the first period of time, showing the greatest bacterial population after 16 hours (Fig 4.1.1.1.) The population then went down sharply This reduction of population was probably caused by the decrease in nutrients for the microbes
In the samples with a Hexatreat 1512 concentration of 10 ppm, the microbe population decreased and completely died off in 24 hours In the samples with Hexatreat 1512 concentrations of 20-90 ppm, the different decreases in microbe populations depended on the biocide concentration; but the microbes completely died off after only 20 hours For the samples with a Hexatreat 1512 concentration of 100 ppm, the microbe population decreased significantly and completely died off after only 6 hours
It is essential to note that the microbe populations increased in the first 2 hours after Hexatreat 1512 was added, and the increase depended on the biocide concentration in each sample This indicated that Hexatreat 1512 did not have immediate effect on SRB-5KK species Its effect came only after 2 hours, and its effect depended on its concentration In the low concentrations of less than 80 ppm, the effect of Hexatreat 1512 on SRB-5KK species was not completely certain In other words, the needed concentration of Hexatreat 1512 must be greater than 90 ppm
The populations of SRB-5KK species after Hexatreat 1512 was added
Th e po pu la tio n of SR B -5 K K , C 1 0 4 (c el ls /m l)
0 ppm 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm
60 ppm 70 ppm 80 ppm 90 ppm 100 ppm
4.1.2 Effects of biocides on SRB-8KK species
Concentrations of Hexatreat 1512 added to each sample were 0 ppm, 10 ppm,
20 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 80 ppm, 90 ppm, and 100 ppm Each sample contained microbes at a concentration of 10 6 CFU/ml The tested samples were tested with the spectrometry method In terms of the effects of
Hexatreat 1512 on SRB-8KK species, the absorbance values, which depend on the biocide concentration and the survey time, were measured Based on the established standard diagram of absorbance (Fig 3.4.1.1.), the conversion of absorption to respective populations of SRB-8KK species was done, showing the same resulting decreased in microbe populations
In the blank sample without biocide, the bacteria grew fast in the first period of time, showing the greatest bacterial population after 16 hours (Fig 4.1.2.1.) The population then went down sharply This decrease in population was probably caused by the decrease in nutrients for the microbes
In the samples with a Hexatreat 1512 concentration of 10 ppm, the microbe population was unchanged after the first 4 hours, and then gradually increased to a peak population at 16 hours The population then went down sharply This indicated that Hexatreat 1512 with a concentration of 10 ppm did not have a considerable effect on SRB-8KK species
Populations of SRB-8KK species after Hexatreat 1512 was added
Th e po pu la ti on of S R B -8 K K , C 1 0 4 ( ce lls /m l)
0 ppm 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm
60 ppm 70 ppm 80 ppm 90 ppm 100 ppm
In the samples with Hexatreat 1512 concentrations of 20 and 30 ppm, the microbe populations decreased for 2 hours, then increased gradually to their greatest populations at 16 hours Next, the populations went down sharply, corresponding to the biocide concentrations This indicated that Hexatreat 1512 at concentrations of 20 - 30 ppm did not have a considerable effect on SRB-8KK species The bacteria were gradually adaptable to the cultures with the above concentrations of the biocide
In the samples with Hexatreat 1512 concentrations of 40 ppm, 50 ppm, and 60 ppm, the microbe populations increased, with the greatest populations at 8 hours The populations then went down sharply This indicated that Hexatreat 1512 in concentrations of 40 ppm, 50 ppm, and 60 ppm had a noticeable effect on SRB- 8KK species However, the bacteria were gradually adaptable to the cultures with the above concentrations of the biocide
In the samples with Hexatreat 1512 concentrations of 70 - 100 ppm, the microbe populations’ decreased for 2 hours, but then decreased gradually; they were completely killed after 8 hours This indicated that Hexatreat 1512 at these higher concentrations had a gradual effect of completely killing off SRB-8KK species
It is essential to note that the microbe populations increased in the first 2 hours after Hexatreat 1512 was added, and the increase depended on the biocide concentration in each sample This indicated that Hexatreat 1512 did not have immediate effect on SRB-8KK species Its effect came only after 2 hours, and its effect depended on its concentration In the low concentrations of less than 70 ppm, the effect of Hexatreat 1512 on SRB-8KK species was not optimum The higher the concentration of Hexatreat 1512 that was used, the more effective was the biocide In other words, the needed concentration of Hexatreat 1512 must be greater than 70 ppm, and the time needed to kill the bacteria was 16 hours
4.1.3 The dependence of the selected SRB on the biocide concentration
The figure 4.1.3.1 shows that if we increased the biocide concentration, the SRB-5KK killing time would be shorter For example, with 10 ppm of the biocide concentration the SRB-5KK killing time was 24 hrs in the tubes; if we increased the biocide concentration to 100ppm, the needed time to kill SRB-5KK was 6hrs
The dependence of the population of SRB-5KK on the Hexatreat 1512 concentration with different isolation times
0 ppm 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm 60 ppm 70 ppm 80 ppm 90 ppm 100 ppm
Th e p op ul at ion of S R B- 5K K , C 10 4 (c ell s/m l)
The dependence of the population of SRB-8KK on the Hexatreat 1512 concentration with different isolation times
0 ppm 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm 60 ppm 70 ppm 80 ppm 90 ppm 100 ppm
Hexatreat 1512 concentration T he p op ul at ion of S R B -8K K , C 10 4 (c el ls /m l)
In the real time, at Vietsovpetro, the cycle that gives the biocide into the packer fluid around the oil well, is 6 hrs/time So, if we use the Hexatreat 1512 to destroy the SRB-5KK, the concentration of this biocide should be 100ppm
Compare the figure 4.1.3.1 and 4.1.3.2 we can see that the SRB-8KK can stand the same biocide affection better than the SRB-5KK With Hexatreat 1512 concentration ≤ 60ppm, after 26 hrs, the SRB-8KK was not killed According to the experiments, if we use the Hexatreat 1512 to destroy completely the SRB-8KK after 6 hrs, the concentration of this biocide should be more than 100ppm
Ability to produce H 2 S
During the bacteria’s growth and death, H2S is produced It is a fact that H2S produced by bacteria causes metal corrosion The question is how much of the H 2 S is produced by bacteria
4.2.1 H 2 S produced by SRB-5KK species
In the growth of SRB-5KK, the bacteria produced H2S, which corroded metal, causing harm in the petroleum industry The following table and diagram show the amount of H 2 S produced by SRB-5KK in the presence of biocide
The amount of H 2 S produced by SRB-5KK in the presence of biocide
In the blank sample without biocide, the bacteria grew fast in the first period of time, producing H2S in this time When the bacteria population was at its highest level (at 20 hours), the H 2 S was produced 1900 mg H 2 S/l (Table 4.2.1.1.) The bacteria populations then went down sharply This decreasing population was probably caused by a decrease in nutrients for the microbes So that, after a certain time of increase in H2S, the amount of H2S was increased slightly after 20 hours, because of reducing the amount of bacteria population
The amount of H 2 S produced by SRB-5KK in the presence of Hexatreat 1512
H 2 S prod uc ti on by S R B -5 K K ( m g/ l)
0 ppm 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm
60 ppm 70 ppm 80 ppm 90 ppm 100 ppm
In the samples with Hexatreat 1512 concentrations of 10 – 90 ppm, the microbe populations decreased due to the effect of the biocide But, the living bacterial cells produced H 2 S well, increasing this by-product up to its maximum
H 2 S amount at 16 hours It then kept stable In the other words, from this time, all bacteria were killed, so the produced H2S was unchanged (Fig 4.2.1.1.)
In the samples with a Hexatreat 1512 concentration of 100 ppm, the greatest amount of H 2 S was at 6 hours when the bacteria were completely killed off Then the amount of H 2 S was unchanged The microbe populations decreased for 2 hours, but then increased gradually; they were completely killed in 6 hours This indicated that Hexatreat 1512 with the higher concentration had a gradual effect of completely killing SRB-5KK species The changed in the amount of H 2 S is not great
4.2.2 H 2 S produced by SRB-8KK species
In the growth of SRB-8KK, the bacteria produced H2S which corroded metal, causing harm in the petroleum industry The following table and diagram show the amount of H 2 S produced by SRB-8KK in the presence of biocide
The amount of H 2 S produced by SRB-8KK in the presence of biocide
In the blank sample without biocide, the bacteria grew fast in the first period of time, producing H 2 S in this time When the bacteria population was at its highest level (at 16 hours), the H 2 S was produced 1756.67 mg H 2 S/L (Table 4.2.2.1.) The bacteria populations then went down sharply After 16 hours the H 2 S amount increased slightly, indicating that the bacterial cells were largely killed off, so the amount of H2S produced by the surviving cells not very high as the first period of time
The amount of H 2 S produced by SRB-8KK in the presence of Hexatreat 1512
0 ppm 10 ppm 20 ppm 30 ppm 40 ppm 50 ppm
60 ppm 70 ppm 80 ppm 90 ppm 100 ppm
It is likely that, for samples with 10 – 30 ppm concentrations of Hexatreat
1512, the amounts of H 2 S that were produced by bacteria, are described by the similar diagram plots However, the changes in H 2 S as a result of production depended on the concentrations of the biocide used These little changes in H 2 S indicated that the biocide concentrations of 10 – 30 ppm were insignificant In the samples with Hexatreat 1512 concentrations of 40 – 60 ppm, the amount of H2S increased From 8 hours it then increased slightly, depending on the biocide concentration In the samples with Hexatreat 1512 concentrations of 70 - 100 ppm, the greatest amount of H 2 S was at 8 hours when the largest population of bacteria decreased Then it kept stable The change in the H2S amount was not much, probably due to the quick death of many bacterial cells (Fig 4.2.2.1.)
In other words, SRB-8KK species were able to produce great amounts of H 2 S even in the presence of biocide In general, SRB-8KK species produced more H2S and were more adaptable to biocide than SRB-5KK species
4.3 Comparison of the effects of biocides on SRB-5KK and SRB-8KK species
Figure 4.1.1.1 and 4.1.2.1 show that Hexatreat 1512 greatly effected on SRB-5KK species, with only a concentration of 10 ppm However, the biocide gradually affected SRB-8KK species, shown by the fact that the population went down insignificantly with different concentrations of biocide, whereas the greatest decrease in the population with the biocide concentration of 70 ppm In the others word, Hexatreat 1512 effected on the population of SRB-5KK more than on the population of SRB-8KK
In fact, the population of bacteria is not the only factor that affects corrosion Another factor, the amount of H 2 S produced by each species, has the same importance Figure 4.2.1.1 and 4.2.2.1 reveals the amounts of H 2 S produced by SRB-5KK and SRB-8KK species during their growth and death
It can be seen that the amount of H 2 S produced by SRB-5KK increase gradually in different concentrations of biocide Although the population of SRB-5KK decreased greatly with the increase in concentration of biocide, the surviving bacteria cells still produced a lot of H2S In addition, the amount of H2S was not considerable in the case that the bacteria were eliminated with the Hexatreat 1512 concentration of 100 ppm
For SRB-8KK species, there was a significant increase in H 2 S with the biocide concentration of from 0 - 60 ppm With concentration of biocide from 70 – 100ppm, there was a little increase in H2S, where the SRB-8KK species were eliminated pretty
In terms of H 2 S production by bacteria, to decrease the concentration of H 2 S to below 1000 mg/L H 2 S, Hexatreat 1512 should be used with the concentrations of 100 ppm for SRB-5KK, and 70 ppm for SRB-8KK, respectively.
Observation of the adaptability of selected bacteria to biocides
Observation reveals that there were some bacteria that remain alive in the presence of biocide The method of incubation in liquid cultures containing biocide was used to assess the existence of bacteria, and the method used incubation on Petri agar to determine the living bacteria
It can be seen that some bacteria survived after treatment with Hexatreat
1512, i.e., up to 60ppm after 48 hrs treatment We called them SRB-5KK(B) and
SRB-8KK(B) Those bacteria were able to adapt to the new cultures Obviously, the real existence of bacteria in the presence of high concentrations of Hexatreat
1512 revealed to some extent the changes in the character and corrosion ability of the bacteria This is possibly an ability caused by the change in gene codes of the bacteria These were issues for further research.