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 INTRODUCTION CHAPTER 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 MATERIALS AND METHODS 26 2.1 Experiment plan 26 2.2 Materials and equipments: 27 2.3 Methods: 29 CHAPTER 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 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 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 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 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 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 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 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 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] 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 The White Tiger oil field in Vietnam contains high densities of SRB Most of the samples had SRB quantities at the very aggressive level 3.4 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 by using the spectrometer Prepared samples with 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 8KK Standar of Absorbance at 580 nm of 5KK Absorbance at 580 nm Absorbance at 580 nm 0.8 y = 9*10-8x R2 = 0.9989 0.6 0.4 0.2 0 200 400 600 800 1000 1200 Bacteria*10 (CFU) 0.8 -8 y = 8*10 x 0.6 R = 0.9997 0.4 0.2 0 200 400 600 800 1000 1200 Bacteria*10 (CFU) Figure 3.4.1.1 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 ~107cells/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 H2S 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 107 cells/ml, adding 15 ml 56 peptone solution and NaCl to each test tube Corrosion ability tests, which were conducted at h, 72 h, and 144 h, gave the following results Table 3.4.2.1 Microbial ability to corrode metal (ASTM method) Population of microbes: 0.1 ml Code Oil well 0h 72 h Blank (no microbes) Freshly polished 1KK CKB-402 Freshly polished 1a 4KK CKB-402 Freshly polished 5KK CKB-2001 8KK Population of microbes: 0.2 ml 144 h Code Oil well 0h 72 h 144 h Blank (no microbes) Freshly polished 1b 1KK CKB-402 Freshly polished 2b 2c 2b 2b 4KK CKB-402 Freshly polished 2b 2b Freshly polished 2b 2c 5KK CKB-2001 Freshly polished 2b 3a 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 57 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) 0h 72h 144h % corrosion mass 0.20 0.15 0.10 0.05 0.00 k an Bl K 1K ) ml (0 K( K m 0.2 l) K( K m 0.1 l) ) ) l) l) l) l) l) l) l) ml ml 2m 1m 2m 1m 2m 1m 2m 0 K( K( K( K( K( K( K( K( K( K K K K K K K K K 5 8 9 12 12 Bacteria sample Figure 3.4.2.1 With their corrosion ability and biochemical characters, the facultative anaerobic bacteria, 5KK and 8KK, were isolated from the CKB-421 and CKB2001 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 58 CHAPTER EFFECTS OF BIOCIDES ON MICROBES ISOLATED FROM THE WHITE TIGER OIL FIELD 4.1 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 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 106 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 hours It is essential to note that the microbe populations increased in the first 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 hours, and its 59 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 The population of SRB-5KK, C.10 (cells/ml) 1400 1200 1000 800 600 400 200 0h 2h 4h 6h 8h 16h 20h 24h 26h Times ppm 60 ppm 10 ppm 70 ppm 20 ppm 80 ppm 30 ppm 90 ppm 40 ppm 100 ppm 50 ppm Figure 4.1.1.1 4.1.2 Effects of biocides on SRB-8KK species Concentrations of Hexatreat 1512 added to each sample were 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 106 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 60 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 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 1400 1306 1200 The population of SRB-8KK, C.10 (cells/ml) Populations of SRB-8KK species after Hexatreat 1512 was added 1000 800 600 400 200 0h 2h 4h 6h 8h 16h 20h 24h 26h Times ppm 10 ppm 20 ppm 30 ppm 40 ppm 60 ppm 70 ppm 80 ppm 90 ppm 100 ppm 50 ppm Figure 4.1.2.1 In the samples with Hexatreat 1512 concentrations of 20 and 30 ppm, the microbe populations decreased for 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 61 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 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 SRB8KK 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 hours, but then decreased gradually; they were completely killed after 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 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 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 62 The dependence of the population of SRB-5KK on the Hexatreat 1512 concentration with different isolation times 1200 The population of SRB-5KK, C.10 (cells/ml) 1100 1000 900 0h 2h 4h 800 700 6h 8h 16h 600 500 20h 24h 26h 400 300 200 100 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 Figure 4.1.3.1 The dependence of the population of SRB-8KK on the Hexatreat 1512 concentration with different isolation times 1200 The population of SRB-8KK, C.10 (cells/ml) 1100 1000 0h 2h 4h 6h 8h 16h 20h 24h 26h 900 800 700 600 500 400 300 200 100 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 Figure 4.1.3.2 63 In the real time, at Vietsovpetro, the cycle that gives the biocide into the packer fluid around the oil well, is 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 hrs, the concentration of this biocide should be more than 100ppm 4.2 Ability to produce H2S 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 H2S is produced by bacteria 4.2.1 H2S 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 H2S produced by SRB-5KK in the presence of biocide Table 4.2.1.1 The amount of H2S produced by SRB-5KK in the presence of biocide (mg H2S/L) ppm of Biocide Survey Times 0h 4h 6h 8h 16h 20h 24h 26h ppm 821.67 1076.67 1416.67 1700.00 1900.00 1918.33 1928.33 10 ppm 793.33 1020.00 1360.00 1700.00 1710.00 1710.00 1710.00 20 ppm 765.00 991.67 1331.67 1671.67 1671.67 1671.67 1671.67 30 ppm 765.00 991.67 1303.33 1643.33 1643.33 1643.33 1643.33 40 ppm 736.67 991.67 1275.00 1643.33 1643.33 1643.33 1643.33 50 ppm 736.67 963.33 1246.67 1586.67 1586.67 1586.67 1586.67 60 ppm 708.33 963.33 1246.67 1586.67 1586.67 1586.67 1586.67 70 ppm 708.33 963.33 1218.33 1586.67 1586.67 1586.67 1586.67 80 ppm 708.33 935.00 1190.00 1190.00 1190.00 1190.00 1190.00 64 ppm of Biocide Survey Times 0h 4h 6h 8h 16h 20h 24h 26h 90 ppm 708.33 878.33 1161.67 1190.00 1190.00 1190.00 1190.00 100 ppm 708.33 878.33 878.33 878.33 878.33 878.33 878.33 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 H2S was produced 1900 mg H2S/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 H2S produced by SRB-5KK in the presence of Hexatreat 1512 H2S production by SRB-5KK (mg/l) 2250.00 2000.00 1750.00 1500.00 1250.00 1000.00 750.00 500.00 250.00 0.00 0h 4h 6h 8h 16h 20h 24h 26h Times Biocide concentration: ppm 60 ppm 10 ppm 70 ppm 20 ppm 80 ppm 30 ppm 90 ppm 40 ppm 100 ppm 50 ppm Figure 4.2.1.1 65 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 H2S well, increasing this by-product up to its maximum H2S 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 H2S was at hours when the bacteria were completely killed off Then the amount of H2S was unchanged The microbe populations decreased for hours, but then increased gradually; they were completely killed in 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 H2S is not great 4.2.2 H2S 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 H2S produced by SRB-8KK in the presence of biocide Table 4.2.2.1 The amount of H2S produced by SRB-8KK in the presence of biocide (mg H2S/L) ppm of Biocide Survey Times 0h 4h 6h 8h 16h 20h 24h 26h ppm 821.67 1190.00 1530.00 1756.67 1833.33 1933.33 1993.33 10 ppm 793.33 1105.00 1530.00 1700.00 1773.33 1833.33 1893.33 20 ppm 708.33 1076.67 1473.33 1680.00 1743.33 1776.67 1786.67 30 ppm 694.17 1076.67 1473.33 1671.67 1733.33 1772.67 1776.67 40 ppm 694.17 1048.33 1443.33 1548.33 1626.67 1636.67 1636.67 50 ppm 694.17 1046.33 1443.33 1526.67 1563.33 1570.00 1580.00 60 ppm 694.17 1046.33 1443.33 1490.00 1506.67 1520.00 1540.00 70 ppm 561.20 792.10 970.33 970.33 970.33 970.33 970.33 80 ppm 557.50 774.80 923.50 923.50 923.50 923.50 923.50 66 Survey Times ppm of Biocide 0h 4h 6h 8h 16h 20h 24h 26h 90 ppm 543.10 734.20 902.30 902.30 902.30 902.30 902.30 100 ppm 520.10 699.30 879.10 879.10 879.10 879.10 879.10 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 16 hours), the H2S was produced 1756.67 mg H2S/L (Table 4.2.2.1.) The bacteria populations then went down sharply After 16 hours the H2S 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 H2S produced by SRB-8KK in the presence of Hexatreat 1512 H2S production by SRB-8KK (mg /l) 2250 2000 1750 1500 1250 1000 750 500 250 0h 4h 6h 8h 20h 24h 26h Times Biocide concentration: ppm 60 ppm 16h 10 ppm 70 ppm 20 ppm 80 ppm 30 ppm 90 ppm 40 ppm 100 ppm 50 ppm Figure 4.2.2.1 67 It is likely that, for samples with 10 – 30 ppm concentrations of Hexatreat 1512, the amounts of H2S that were produced by bacteria, are described by the similar diagram plots However, the changes in H2S as a result of production depended on the concentrations of the biocide used These little changes in H2S 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 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 H2S was at 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 H2S 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 H2S produced by each species, has the same importance Figure 4.2.1.1 and 4.2.2.1 reveals the amounts of H2S produced by SRB-5KK and SRB-8KK species during their growth and death It can be seen that the amount of H2S produced by SRB-5KK increase gradually in different concentrations of biocide Although the population of SRB5KK decreased greatly with the increase in concentration of biocide, the surviving 68 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 H2S with the biocide concentration of from - 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 H2S production by bacteria, to decrease the concentration of H2S to below 1000 mg/L H2S, Hexatreat 1512 should be used with the concentrations of 100 ppm for SRB-5KK, and 70 ppm for SRB-8KK, respectively 4.4 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 69 CHAPTER OBSERVATION OF THE METAL CORROSION ABILITY OF MICROBES ISOLATED FROM THE WHITE TIGER OIL FIELD In the observation of metal-corrosion ability, ASTM D130 and Copper Mass methods were used Used the bacteria solution which was isolated from the White Tiger oil field: SRB-5KK, SRB-5KK(B), SRB-8KK, SRB-8KK(B) (Chapter 4) 5.1 Study of the corrosion ability of selected wild bacteria 5.1.1 Study of the corrosion ability of selected bacteria by the ASTM D130 method Added 0.1 ml and 0.2 ml prepared samples of bacteria, equivalent to 50,000 cells/ml and 100,000 cells/ml in each tube Copper bars were put in a peptone solution with NaCl After the observed time of h, 72 h, 96 h, 120 h, 144 h, and 168 h, the copper bars were corroded The colors of the corroded copper bars depended on the corrosion speed Based on the standard color table, the respective values of corrosion ability are indicated In the observation, to ensure the accuracy of the test, all samples were tested times Cu-corrosion was shown after being tested by the corrosion analyzer Colors of the Cu-bars were compared with the copper strip corrosion standards, showing respective corrosion values It is likely that the corrosion of the Cu-bars was due to the metabolization of corrosion-causing bacteria For these tests, peptone cultures with NaCl were considered to be pretty natural conditions Figure 5.1.1.1 shows the corrosion effects of SRB-5KK species on Cu-bars Copper bars of the tested samples were kept parallel for comparison, showing the change in the colors of the copper bars For the blank samples, there was a little color change from the “freshly polished” point to 1b, as an actual result of dipping the copper bars into the peptone solutions with NaCl (natural sea water containing NaCl) With the effect of the salt solution, the copper bars were corroded slightly, still under the risk point Different populations of bacteria produced different 70 ... 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,... 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. .. 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