SPE 38070 Microbial Enhanced Oil Recovery Field Pilot in a Waterflooded Reservoir H.Yonebayashi* and K.Ono*, Japan National Oil Corporation; H.Enomoto* and T.Chidat, Tohoku University, C-X Hong, China Jilin Province Oifield Administrative Bureau and K Fujiwara, Kansai Research Institute * SPE Members ony 1987, acy ot erie Enger e ‘ina bo tmp Mayu, 16 oe 097 * ener {oe ae waned’ fx peor 2m FE Peg Soman oa in Additional oil recovery amounted to approximately 15% of waterfbod resitual oil using TRC-322, TRC-4118 and TRC- 18-2-a on the average. ‘The field trial will startin middle of 1997. Theobjectivesin this testare two fold. One isto validate the laboratory results, ‘hich have been accumulated. The other isto investigate most predominant microorganisms (i.e. most effective ones on oil recovery), most effective metabolites andior cause of Abstract Japan National Oil Corporation (NOC) / Technology Research Center (TRC) has boen studying the Microbial Enhanced Oil Recovery (MEOR) process which makes effective use of microorganisms metabolism since 1987. Now. SNOC/TRC is planning a field trial to advance our MEOR study to the next stage. This paper describes the feasibility study of the field test. AS a target of MEOR application, a shallow oil fie in China which has been under waterflood for over 20 years has bbeen selected. At frst, the target reservoir was investigated and adaptabilites of TRC's microorganisms tothe field were tested to select candidate microorganisms for injection. The tested ‘TRC's microorganisms were Eloacae TRC-322, B.subilis TRC.4118, B subsilis TRC-4126, B.icheniformis TRC-18-2-2,, Bichenifomis JE-2, B.ickeniformis SP-OI8 and E.cloacae TU-7A which have been especially screened as effective gas, acid, surfactant and/or polymer producers through basic laboratory tests. Inthe former investigation of the reservoir, @ new technique, Polymerase Chain Reaction (PCR) established in the sphere of gene engineering was applied to the study to make clear distinction among reservoir-originated microorganisms. Effects of reservoir conditions, namely, reservoir water, temperature, nutrient, additives and indigenous ‘microorganisms in the reservoir, on the microbial growth and their metabolism were tested to select candidate ‘microorganisms forthe field applicetion, Finally, four microorganisms (i.e. TRC-322, TRC-4118, TRC-18-2-a and TU-7A) were selected for the field trial through these compatibility tests followed by flooding tests 451 ‘obstruction of microbial activity Introduction The oil production from applications of various Improved Oil Recovery (IOREnhanced Oil Recovery (EOR) processeshas been increasing steadily with the exception of a short recess during 1992-1994". Itappears that this upward trend of EOR oil production will continue in the future, because the current ‘worldwide oil-equirements are increasing. Morcover, due to the mpid economé growth in Asia, itis anticipated that the oil-demands will rise substantially in this region. Hence, itis necessary to increase the oil production. The IOW/EOR processes are recognized as one of solutions to cope with such, situation, because the discovery of new big oilfield is becoming rare in recent years, Therefore, the development of the IOREOR techniques (redevelopment and stimulation technologies) to produce additional oil must be boosted to supply the future oil-requirements ‘We recognize microbial EOR process to be one of the cffective options among various IOR/EOR methods to redevelop the waterfboded reservoir. Therefore, research on MEOR has been conducted at INOC/TRC. TRCs research is especially focused on the in-situ process, in which gas production increases pressure and swells oil, polymer production controls mobility and/or surfactant production decreases. interfacial tension. TRC has been searching microorganisms which have useful abilities (i.e. surfactant, polymer, gas and/or acid productivity) for oil recovery. Up to now, about 500 microorganisms have been gathered and many talented ones have been selected through screening, In addition, flooding experiments have been carried out to evaluate their ffects on oil recovery using strains which yielded excellent, results in laboratory tests. It was necessary for TRC’s MEOR study to move the phase of research toa field application. Inthefield test, the ability of TRC’ microorganisms in oil recovery will be tested In addition, there is another motivation in TRC’s research. ‘The potential and the advantege of this process ar significant ‘Many ficki trials" have been reported since Beckman"? suggested this process in 1926. However, many other aspects still remain to be studied. One of the biggest concerns is the ficulty of microbial monitoring in reservoir. In other words difficult to observe in-situ growth and metabolic activity of the injected microorganisms individually. This problem makes it difficult to understand improvement in the oil recovery mechanisms or causes of faire, Therefor, research at TRC aims at making it clear the cause of improving oil recovery ina real reservoir, using TRC’s microorganisms The now bio- technique to detect each microorganism indiviiualy is used to solve the above problem. At frst, the selection of a target oilfield are described Secondly, the screening of microorganisms which have sadaptabiltes to target reservoir environment is explained. In addition, the replenishment of nutrients for microorganisms ‘often poses technical and economical challenges. Hence, effect ‘of phosphate additive to accelerate microbial activity is mentioned Finally, candidate micworganisms screened through quantitative evaluation of microbial oil recovering abilities are described, Pilot Test Field and Target Block Pilot test field was selected based on the consideration that the reservoir conditions were aot severe for microorganisms which have been preserved in TRC. As per the general screening criteria of MEOR field application (9220%, K. 2150md, reservoir temp. <70°C, y1s-5-50 cp, pH=5-8, salt content $150,000mg/iter)", Fuyu oilfield in Jilin province, China ‘was chosen as atest field (see Fig.l). Another criterion limited target fieks to the oilfields in which waterflood had been carried out, in order to limit additional investment for facilities This field was discovered in 1959 and developed by China Jilin Province Oilfield Administrative Bureau, ‘The reservoir is shallow at approximately 300-500 m (984- 1,640 ft). The reservoir has a thin alternation of sandstone and shale, andis finely separated into 13 layers, but roughly divided into 4 zones. Total reservoir area is 120 km? (29,650 acre) Other relevant properties of the reservoir are ; permeability = 100-300md (Avg123md, § = 2226 % (Avg25 %), temperature ~ 28-32 °C (Avg30.8 °C) and pressure ~ 20-30 atm. Waterflood has been conducted in whole field since 1973. There were 2,500 producers and 710 injectors, and inverted nine spot flooding pattern was adapted in the earty period. But because of early break through, well pattern was changed to line drive and this pattern has been continued to date. Fuyuoilfield is divided into three arcas : West, Middle and East. The development of this field started fom west to east area. Therefore, two target blocks, “East 24-26 Block” and “East 24-23 Block" (see Fig.2 and Table 1) were selected from the East which can be regarded as the relatively newer area. ‘These blocks are neighbours, but are made independent ofeach other by faults, Properties of crude oil are shown in Table 2. Currently, waterflood with line drive pattern is being 452 H-YONEBAYASI, KONO, H.ENOMOTO, T.CHIDA, C-X.HONG, K FUJIWARA SPE 38070 conducted in both blocks. Investigation of Test Block Samples of reservoir water were collected from four producers and a injector in both blocks to investigate the reservoir condition before injection of microorganisms Sampling wells Were 24-24, 24.23, 22-264, 22-26 and 22-27, The former four wells are producers and the latter one is an injector. The sterilized bottles were carried to well site without contamination of saprophyte, and then were filled with produced (or injected) fluid as quickly as possible, Another noteworthy point was to completely fll the bottle to prevent mixing of any unnecessary microorganisms from air phase Investigation of molasses-utilizing microorgenisms in samples were carried out as soon as possible after sampling Investigation of microorganisms which can assimilate molasses in the reservoir. Existence of many indigenous microorganisms in the reservoir is forecast in general ‘Therefore, influence of indigenous microorganisms on injected foncs must be investigated. However, when MEOR process is applied, it is adequate to take microorganisms which utitze injected nutrients into consideration, Molasses will beused asa ‘main nutrient in this field test, because of its low price and its, high nutritive value for microorganisms. This nutrient is 2 waste product in sugar-manufacturing industry and used in ‘many MEOR projects in all over the worl!’, Therefore, ‘molasses-utilizing microorganisms were investigated and ‘obéained from samples with medium which were consisted of molasses and distilled water (cone. 4% w/v). The competition test between these collected microorganisms and TRC’s ones is discussed later. The isolation of obtained microorganisms was, conducted using plating method and detailed analysis. The former isa basic method according to differences among their colonies, the latter is an_isolation with comparison of their individual 16S FRNA (16S ribosomal ribonucleic acid) gene. Isolation by Plating Method. For the first isolation, agar plates containing 4%(w/v) molasses solution were prepared. Microorganisms inoculated on the plate formed their own colonies and then different kinds of colonies were separated and, inoculated on the new plate again. This cycle was continued until no different kinds of colonies appeared on the plate. As a result, 33 kinds of microorganisms were isolated with this, method according to their own feature of colonics (for example, color, luster, form and so on). However, it is impossible with this method to make a complete isolation, ‘Therefore, the following isolation was conducted asa final step. Isolation with PCR Method. This method was suggested by ‘Saiki et al. in 1985". After that, this tochnigue was improved with the application of w thermostable DNA polymerase by Saiki etal.'®. Now, the PCR procedure is applied to diverse fickls which include fundamental genetic research, clinical medicine and legal medicine. At first, each 16S rRNA gene was extracted from preceding 33 kinds of microorganisms and amplifiad by the PCR. The double-stranded 16S RNA gene were digested by three kinds ‘of restriction enzymes (ie. Hpa ll, Hha I and Alul) to get specific cutting patterns of 16S rRNA gene. The RNA gene‘SPE 38070 MICROBIAL ENHANCED OIL RECOVERY FIELD PILOTIN A WATERFLOODED RESERVOIR consists of 4 kinds of nucleotide (ie. Adenine, Guanine, Cytosine and Thymine). Each RNA gene has its own nucleotide sequence and each restriction enzyme cuts specific positions in the chain. If microbial species are diferent, there are difference among their nucleotide sequence in 16S 1RNA gene, Hence, different size of fragments of RNA gene cut by enzymes are’ obtained and species of microorganisms can be identified by comparison of cutting pattern. For example, Hall cents a C-C connection in the chain of S'—CCGG—3", Hha 7 cuts a G-C connection which is put near the side of 3° in the chain of $—GCGC—3" and Alu [cuts aG-C connectionin the chain of 5AGCT—3", After cutting the chain of nucleotide, frogments of RNA gene were penetrated through gel by clectrophoresis. Because small fragments can penetrate distantly but big ones can not, differences occur in moving distance according to their size. Figure 3 are photographs of the results of electrophoresis. Light bands show fragmentsof RNA gene. The number from 1 to 33 mean discriminating name of ‘microorganisms obtained withthe plating method for the sake of convenience. The “M” is marker as ¢ comparison. As ¢ final resut, 28 pure microorganisms were obtained from collected samples. These 28 kinds of microorganisms were renamed from 101 to 128 in sequence. Table 3 shows species of reservoir-originated microorganisms, which can utilize molasses, in cach sample. Most of their viable cell ‘counts were 10°10" cells/ml and highest counts amounted to 10° cells Analysis of the reservoir water. Two samples of reservoir ‘water collected from each block wereanalyzed (see Table) to determine the effects of their composition on microorganisms ‘Synthetic brine was made with the components similar to those obtained in chemical analysis (see Table), and effects of ‘major components in reservoir Water on microorganisms are being evaluated in the next section, All components in synthetic brine were specified according to the average of both results Adaptabilities of Microorganisms to Target Reservoir ‘The important point to consider is whether the TRC's microorganisms can adapt to the target reservoir conditions or not, Therefore, effects of reservoir water, temperature, ‘molasses and indigenous molasses-utilizing microorganisms in Fuyu oilfield on candidate were investigated, TRC’s microorganisms. Samples of drilling cuttings, formation water and soil near wels were collected in the frst stage to gather microorganisms which can live and make effective production in the reservoir, because it was thought that microorganisms from such origin have high adaptabilties to reservoir environment : anacrobic, high salinity, high temperature and high pressure, Table 6 liss TRC’s main ‘microorganisms which have been selected among all gathered ‘ones to date. TRC-18-2-a and TRC-322 were selected through screening among rescrvoir-originated microorgenisms Her, concentrating on the oil recovery due to microbial metabolism, the producers of gas, acid, surfactant and polymer are emphasized. The former microorganism has all productivties 453 (eas, acid, surfactant and polymer) and the latter is the gas producer. Furthermore, other samples were also collected from. enriched mud and compost of activated-sludge, TRC-41 18nd TRC-4126 were discovered from these environment where ‘were not conventional origin at MEOR research, but these ‘microorganisms are best surfactant producers. In addition, B. licheniformis JF-2 and SP-O18 were provided by Oklahoma University for a baseline comparison. Stain JF-2 is a well evaluated microorganism which produces effective surfactant at typical reservoir conditions, and has been investigated by many authors". From the above mentioned microorganisms, candidate for the field pilot test were selected through the following experiments Effects of reservoir water and molasses. Reservoir water usually includes many inorganie salts and minerals. It is necessary to investigate their influence on microorganisms, because some components often affect microbial multiplication and their metabolism, Molasses isthe only nutrient for injection to the reservoir, so that injected microorganisms are required to have abilities to assimilate this nutrient. Chinese molasses will bbe used inthe field, which is locally available and smade from beet, In case of field trial, nutrient must be preparedat the field from a economical point of view. Therefore, following fundamental incubation was carried out to investigate effects ‘of major components of reservoir water and Chinese molasses fon these 8 useful microorganisms with medium “MSB (Molasses based on Synthetic Brine)” shown in Table 5. All ‘microorganisms were cultured using the Hungate tube filled with 150 ml of sterilized medium. The medium included ‘Chinese molasses and synthetic brine whose components were. specified according to the reservoir water analysis. Headspace of the tube was displaced with nitrogen to keep anaerobic condition, At the constant temperature of 30°C, changes of bacterial counts, interfacial tension (IFT) between n-Octane and the nutricat solution and viscosity were measured during cultivation. As a result, 7 microorganisms withthe exceptionof ‘TRC-4126 grew up over 10’cells/ml (sce Table 7) Effects of phosphate, The incubation with medium “MSB' ‘mentioned that 7 candidates had enough adaptabilties against the reservoir temperature, major components in the reservoir ‘water and Chinese molasses. However, the value of metabolite could not often reach earlier data (IFT , pH and viscosity) Which were measured during cultivation using Japanese molasses (cane molasses) at the screening, except for total produced gas amount. Major components of both molasses ; Chinese beet molasses and Japanese cane one are shown in Table 8 Remarkable difference between them is the phosphorus concentration, Amount of phosphorus in Japanese molasses is approximately 7 times as much as inthe Chinese ‘one. This component generally has significant inuence on microbial growth and metabolism. Therefore, following ‘experiment was carried out to investigate effects of additional phosphate on microbial metabolism in cases where Chinese molasses was used. For example, in order to compare total produced gas amount, TU-7A was incubated with two kinds of media, one was 4%(w/v) molasses medium and the other was consisted of the basic medium and phosphats additive (0.1%4 H.YONEBAYAS|, KONO, H.ENOMOTO, T-CHIDA, C:X HONG, K.FUSIVWARA SPE 36070 wiv K:HPO,), Amount of supplemented additive was specified tomake concentration of phosphorus in Chinese molasses the same level as was in case of Japanese one. The experimental conditions were 30°C of cultivation temperature, a week of cultivation period and 100 mi of medium amount. This ‘experiment showed definite influence. Amount of total produced gas increased from 4Sml to 138ml by supplement. This result has a useful implication ; it will be effective to supply additives such as cheap fertiizers which have an abundance of phosphate in the case that Chinese molasses are used as @ main nutrient Competition for indigenous microorganisms in the reservoir. The preceding discussion suggests that 28 ‘microorganisms exist originally in the reservoir. This means that competition between candidate and competitor in the reservoir must be investigated to select predominant candidate ‘ones. At first, target microorganisms for competition were selected from 28 kinds of rescrvoir-originated ones which can assimilate molasses. As a result of mixedincubation, two kinds of predominant strains, No.1!3 and No.118, were screened as competitors, because strain 113 showed highest cell ‘concentration and strain 118 was the second among thetn. Two kinds of competition test were carried outagainst these ‘two microorganisms One was Hala-Test in which clear-zone (Halo) around target microorganism on the plate was observed to-evehuate antimicrobial action ofthe target. Theexperimental procedure involves, (1) preparing en agar plate based on 42%(wiv) molasses medium, (2) spreading competitor on the whole of the plate, 3) inoculating candidate on the middle of, plate immeditely, (4) incubating and (5) observing. Figure 4 are typical examples of the test. In the case of TRC-4126,vs. strain 113, there wereno colonies ofthe former (see Fig.4a). All colonies were made by the latter competitor. This result means that TRC-4126 could not grow and metabolize under existence ‘of No.113. On the other hand, the plate of TU-7A.vs. strain 113 included both colonies (see Figb). The feature of this case ‘was that a big colony of TU-7A extended on many small colonies of No.113 without the formation of a clear-zone in ‘between the both, This fact suggests that each microorganism ‘was not under the influence of an opponent, in other words, there was no antimicrobial action. The last case showed antimicrobial actin of TRC-18-2-2 against strin 118 (see Figdc). The clear-2one existed around colonies of candidate ‘The reason why no colony zone appeared was that lysis ofstrain 118 happened. This lytic response was caused by surfactant which were produced by TRC-18-2-aas antimicrobial agent. The other examination was carried out using liquid ‘medium. The experimental procedure was basically the same fas was in the case of the Halo-Test. In other words, (1) preparing medium “MSB", (2) inoculating competitor in the ‘medium, (G3) inoculating candidate in the medium without interruption, (4) incubating and (5) measuring each bacterial count were continuous steps. Typical resuits are shown in Figures 5-12. The same feature was observed on growth and metabolism curves of TU-7A and TRC-322 against strain 113 (ee FigS-8). Each candidate grew up with competitor and produced gas withoutthe influence of itsrival Produced gas ‘amount in the case of competitive culture was slightly more ‘than that in the case of pure culture, because strain 113 had a small gas productivity, too. It is expected that these two candidates would affect additional oil with their gas production, when they are applied. the reservoir. On the other hand, TRC-4118 and TRC-18-2-a showed another feature on their growth curve (see Fig9 and 11). These two kinds of candidates suppressed competitors completely. It was anatural result tat the difference was not inmetabolismcurves between pure culture and competitive one (See Fig.10 and 12). These resus concluded that the microorganisms, TRC-4118 and ‘TRC-18-2-a are applicable for the tet. Finally, edaptabilities of following candidate microorganisms ; TRC-322, TRC-4118, TRC-18-2-a and TU: 7A were estimated to be high throughout the above investigations. In addition, effective productivities were ‘maintained under the reservoir conditions. Flooding Experiments Following flooding experiments wore carried out to evaluate effects of microorganisms ; TRC-322, TRC-4118 and TRC. 18-2-2 on oil recovery quantitatively under the reservoir conditions; 30°C of temperature, existence of reservoir water ‘components and in porous media Equipment. Experimental spparatus is shown in Figure 13. ‘The size of core is 1.5 inch in diameter and 1 ft in length. ‘Changes of both injection pressure and differential pressure are ‘monitored with two pressure gauges Which are connected to inlet and outlet lines. ‘These changes in pressures are registered by recorder. Fluids, (except nutrients and microorganisms), ciland brine ae injected through 0.45 jim membrane filter to prevent contamination Material. Crude oil produced from target block was used as, residual cil. This is stocktank oil with the following propertics, its specific gravity is 0.87 at 20/4°C and viscosity is 30 mPa-s. In addition, synthetic brine is used. Molasses is used as the only nutrient for injection. Table $ lists medium composition. Procedure. The experimental procedure is outlined in Figure 14, A Berea sandstone is mounted in the core holder, and then the core holder is sterilized in an autoclave. The sterilization of, injection lines is carried out for each injection. After its absolute permeability to brine is measured, the core is saturated with the crude oil and waterflooded with the synthetic brine to ‘obtain residual oil saturation. Then, microorganisms and their nutrients which demonstrate best growth in the preparatory culture, are injected into the core. After 10-11 days cultivation at 30°C, driving water is injected torecoverthe residual oil. All produced fluids are collected and bacterial counts are measured {o evaluate the growth of microorganisms, Furthermore, oil end. ‘water production rate and pH, IFT, viscosity of produced fluids are measured. The effects of microorganisms on oil recovery ae investigated by these data. These measurement will be conducted at the field test to investigate the oil recovery ‘mechanisms, in addition, the measured value will be compared, ‘with estimated ones in this study. Especially, the measurement of bacterial counts is recognized to be important. TheSPE 28070 MICROBIAL ENHANCED OlL RECOVERY FIELD PILOTIN A WATERFLOODED RESERVOIR abovementioned PCR method will be applied to microbial counting at the field test. Result. The results presented in Table 9 and Figures 15-18 show that incremental oil were obtained in all eases. The oil recovery was 15.5% of the residual oil after microbial injection inthe case of TRC-322 (see Fig.16), The presence of gas could contribute towards improving oil recovery, because the increase of pressure was observed during cultivation (see Fig1S). The main gas component was identified as carbon dioxide from a resuit of chemical analysis, so that it was presumed that oil swelled with produced CO:, Figure 16 shows that additional oil were recovered continuously. This suggested that removal oil in the core was increased by swelling, In the case of TRC-4118 which is a surfactant producer, the oil covery was 14.0% of Sy, (see Fig-17). The reason for improved oil recovery was considered to be a decrease of interfacial tension between oil and water. Figure 17 shows that incremental oil were produced intermitenthy. This sporadic behaviour of oil recovery suggested that the following mechanism was at work: some kind of surfactants and/or solvents removed a small amount of oil continuously, and then the oil bank was formed gradually. Finally, the additional oil was produced as soon as oil bank was large enough to be mobile and displaced by the driving water. Inthe last case of TRC-18-2-a, the oil ecovery reached 13.7% of Su (See Figit8). This microorganism has four kinds of productivities : ie. gas, acid, surfactant and polymer. ‘Therefor, this improved oil recovery was attributed probably tothe combination of above mechanisms and mobility control by polymer production Current Study Furthermore, various culture conditions and some kinds of, additives have been examined to study the optimum conditions for the microorganisms that exhibit best abilities in the reservoir. In addition, conditions for microbial injection have been investigated through flooding test In this pilot test, the important point is to trace activities of injected microorganisms and to ascertain the bacteria which exhibit maximum effect on improving oil recovery. Therefore, anew technology with the PCR procedure was introduced into our MEOR rescarch to make a accurate diagnosis of target microorganisms However, a problem still remains in this technology. This diagnosis technique takes a few days, because collected microorganisms must be incubated before the analysis. Hence, application of the fluorescent in-situ hybridization (FISH) method” has been prepared to save time. If specific probes for candidate microorganisms arc ‘established, it will be possible to detect and measure the injected microorganisms quickly and easily. With regard to field operations, surface facilities are now being constructed at the field. The first huff and pu test will bbe conducted in this year. Based on the results of the huff and puff tes, subsequent flooding test will be planned. Conclusions To dete, many microorganisms were gathered and tested for their abilities for MEOR, All available microorganisms were selected. Based on the feasibility test using TRC's ‘microorganisms forthe field trial, the following conclusions are drawn 1, Fuyu oilfield in China was chosen as a target field fora pilot test, because of its suitable reservoir conditions (i.e, high @ and K, low temp, waterflooded reservoir and so on) for MEOR process. 2. Application of the PCR method made it possible to detect resarvoir-origineted microorgenisms completely and individually. In this study, 28 molasses-assimilating igenous microorganisms were detected with this method. Atte field trial, in-situ microbial activities will bemeasured ‘with this technique, in order to make the oil recovery mechanisms clear. 3.£, cloacae TRC-322, B, subtilis TRC-4118, B, licheniformis TRC-18-2 and E cloacae TU-7A are applicable to the target reservoir conditions (i.e. brine component reservoir temp. and competitiveness to indigenous microorganisms), 4. The examination of additive showed a small amount of cheap artificial and easily avaible fertiizer rich in phosphorus was able to enhance microbial abilities, 5. Laboratory flooding experiments showed that microbial effectson oil recovery were high inall cascs. Recovery of the waterflood residual oil was 15.5% with TRC-322. This, nprovel oil recovery was attributed to produced carbon dioxide. Inthe casc of TRC-4118, 14.0% of Se was obtained by its surfactant metabolism. The oil recovery by TRC-18- 2a reached 13.7% of Sq. The mixed mechanisrns worked inthis case inally, TRC-322, TRC-4118, TRC-18-2-2 and TU-7Awere screened as candidates for the field tial through adaptability tests, competition tests and flooding tests, 7. Field trial will be carried out soon Nomenclature (C= bacterial counts, cells/ml porosity, % K, = absolute permeability to water, md e= oil viscosity, ep Ap = differential pressure, psi tial oil saturation, % residual oil saturation, Acknowledgments The authors wish to thank Japan National Oil Corporation and China Jitin Province Oilfield Administrative Burcau for the permission to present this paper. We also thank staffof Tohoku University, Kansai Research Institute and Japan Food Research Laboratories fot their experimental data. We thank HK, Sarma for his assistance during preparation of this paper. 4556 H.YONEBAYAS!, K ONO, H ENOMOTO, T.CHIDA, C-X HONG, K.FUJMWARA SPE 28070 References I. 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K.: “Mixed Culture Miobial Enhanced Waterflood : Tertiary MEOR Case Study.” SPE 24820, 1992, Knapp, RM, Melnemey, M.J., Coates J.D., Chisholm, J.L., Mewie, DE and Bhupahirau, V.K “Design and Implementation of a Mirabially Enhanced Oil Recovery Field Pilot, Payne County, Oklahoma,” SPE 24818, 1992 Bryant, R. S, Stepp, AK., Bers, K.M. and Burchfield, T. E “Migobial Entanced Waterfloodng Field Tests,” SPEDOE 27751, 1994 8. Wang, XY,, Xue, YF, Dai, Gand Zhao, L lication of Bio-Hufiw-Put Techoology at Iiin Oil Field,” The Sth International Conference on Microbial Enhanced Oil Recoveryand Related Biotechnology for Soling Environmental Problens, 115- 127, 1995 Zhang, CY. and Zhang, 1.C. “A Pilot Test of EOR by In-Sita “Miacerganism Fermentation in the Daging Oilfel.” Microbial Enhancement of Oil Recovery. RecentAdvances:proceedingsofihe 1982 international conference on microbial enkanced ll recover E Premuric and A Woodhead (ed), 231-244. 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Thomas, CP., Duvall, ML, Roberton, EP., Barret, KB. and Bala, GA. : “Surfactant - Basal EOR Mediated by Naturally Occurring Microorganisms,” SPE Reservoir Engineering, 285-291, November 1993, 18 Ivaheri, M, Janneman, GE, Melaemey, MI, and Knapp, RM: “Anaerobic Prodidion of Biosufactant’ by Bacillus licheniformis Strain F-2,” Appl Environ. Microbiol, $0, 698-700, 1985 18, Amana RL, Krumborz.L. ant SiahlD.A. : “Fluorescent Oligonudeotite Probing of Whole Cells for Determinative, Phylogenetic, and Environmental Studies in. Micobiology,” J Bacteriol, 172, 762-770, 1990 20, Amann,R I, Binder,B.J, Olson,R J, Chisholm,S. W., Devereux, R. and StahlD.A. “Combination of 168 rRNA-Targeted Oligonudeatide Probes with Flow Cytomary for Analyaing Mixed Migobial Populations” Appl Environ Microbial, $6, 1919-1925, 1990 Appendix — Investigation of Microflora In Target, Reservoir Molasses-uilizing indigenous microorganisms were separated and investigated. At the same time, all reservoir-onginated microorganisms were investigated, too. Samples of reservoir water were collected from four producers (24-24 and 24-23. in Eaxt24-23 block, 22-26. and 22-26 in East 24-26 block) and two injectors. (26-233 in Eas24-23 block and 22-27, in East 24-26 block). Inaddition, a sample of treated water for injection at waterflood plant was also collected to investigate the propagation of microorganisms between surface and reservoir. The sampling was carried out similarly in the case of investigation of molasses-assimilating microorganisms In order to separate all indigenous microorganisms fromcollected samples, specific cultivative conditions (see Table A-1) and specific kinds of medium (see Table A-2) were used for each kinds of microorganisms. The result arc shown in Figure A-I. There were litle ‘Aciduric Suifur Oxidizing Bacteria” and “Ammonia Oxidizing Bacteria” which were autotrophic microorganisms. These kinds of microorganisms did not require organic compound, but their activities were very low. “Sulfur Reducing Bacteria (SRB)" were detected at low level of bacterial count, too. On the other hand, aerobes which included “Coryneform Becteria” and “No-fermentable Gram-negative Bacteria” existed at high cell concentration, This was a noteworthy resuk, because reservoir was usually in anaerobic cavironment and it was difficult for aerobes to live in such condition. Hence, the real origins of these aerobes were guessed to be at the surface and they probably came to the underground with injected water, Incase of anaerobes, bacterial counts from producers were 10-10" cells/ml, but there were little anaerobes in samples from injector 22-27, andwater flood plant, Most predominant microorganisms were No-fermentable Gram-negative Bacteria with a maximum bacterial count of 10'-10* cells. These were heterotrophic microorganisms and they demanded organic compound which were supplied from the outside. 456SPE 38070 MICROBIAL ENHANCED OIL RECOVERY FIELD PILOT IN A WATERFLOODED RESERVOIR Table 1 Summary of the Test Block in Fuyu Oilfield Test Block Eaet2e73 East 24-26 Reservoir Area Tom’ 0.562 0219 Depth Im) 300-450 320-450 Temperature re} 28.0 280 Thickness net (rm) 152 148 Permeability Ind] 0 230 Porosity rl 26 26 Producer {well 4 8 Injector we] 4 4 Weil Spacing Im 100 100 Water Cut ra ot ne Table2 Properties of Crude Oil from Fuyu Oilfield Properties of Analysis Sample Source lock East 24 2423 EE 242 ‘pacific Gravity ‘014°C 0.0506 0.0837 201420 0970s 0.8729 Viscosity (Kinematic) [mPa-s] 20.31 423 Soliditying Point re] 18 1” Water Content ral 23s 90 Sultur Content Imgfiter] No* No Salt Content {onghiter) 197334137334 OW Acid Number 0.08 0.06, ‘Asphaltene Content (1) 038 1.93 Got Content ol 273 ara Paraffin Content rT 5.80 720 Wax Melting Point re} 390-410 39.0-41.0 Initia! Bolling Point re} at fat ‘oon sample wor cara and anya 192 Table 3 _Reservoir-originated Microorganisms which can assimilate Molass Block __Well___Original Microbes in Sample East2e26 22-26 103,106,107,108,109,110,111,112 22.27% 101,102,103,106,105,106,108,113,116,195,116,117,118,119,122 22-26, 106,109,120,121,128 East 2429 24-24 109,102,103,105,113,194,115,118,122,123,124,125,126 242% 127 487H.YONEBAYAS!, K.ONO, H.ENOMOTO, T.CHIDA, C-X HONG, K.FUJIWARA, ‘SPE 38070 Table 4 Chemical Analysis of Table 5 Medium Composition Researvoir Water based on Synthetic Brine Major ‘Sample Source ‘Medium Composition Components “Well 22.27, Well 2424 —Wse Molasses —SSCSC~*~ eos” 2100 2000 Nac taeng Total Fe 14 oss Ker aed) Fett <02 <02 NalicOy 2820 mg ret <02 028 Gach, 2H,0 187 mg Mn oat o19 MgC, 6H,0 114mg ea a FeCl, 64,0 amg ne a KH,PO, 10mg ral a NaHs0,-H,0 3mg 1 1“ Distiled Water 1,000 mi 8 “4 a 55 6a 73 049, 096 ar 24 28 r 16 16 soe aa oss nit: (motte ‘ALC Zn Sn, Co, Hy Ca Pound Grwere not data Table6 — TRC’s Main Microorganisms Selected through Screenings No. Genus and Species Main Origin Productivity ‘Sample Source Location of Sampling TRC-322 Enterobactorcioacee = G Formation Water Toyokawa Oitfield” TRC-4118 Bacitus subtilis 8 ‘Compost of Activated-Sludge Sewage Treatment Faciity’*** TRC-6126 Bacllus subtits 8 Compost of Activated-Sludge Sewage Treatment Faciity"*™* TRC-18-2-a Bacilus licheniformis G,A.8,P*** Drilling Cuttings Sagara Drilingt Tura" — Entarobacturcloacso—G,S Soil near Wall Site Minamiaga Oifiots"*** apa" Bacillus cheniformis Formation Water Oi Wellin Carter County, Oke $P.018"* — Bacilus licheniformis “To snd TA a aco Teno Uninet. 75,4, 8 and Pea usa, vrata and pty reece winaendEPOt8 on povdedby Ohsomt Unwry, ‘Texan donate Table 7 _Adaptabilities of TRC’s Microorganisms to the Test Block Microorganisms Bacterial Produced Vis. Microorganisms Bacterial Produced Vis. Count Gas Count Gas ‘Amount ‘Amount Feetsimt] {mij eR) Ceetisir) mij —_eP) B.lcheniformis TRC-AB20 12x10" 1534.7 —B. licheniformis JF-2 40x 50S Eclocae TRC-322 A4x402 2324.8 B.iicheniformis SP-O18 = 2Axt07 1281.5 subtilis TRC-4118 12107 5618 Ecloncao TU-TA 18x10 264-26 B subtilis TRC-4126 42x18 618 458SPE 38070 MICROBIAL ENHANCED OIL RECOVERY FIELD PILOT IN A WATERFLOODED RESERVOIR Table 8 Chemical Analysis of Molasses Major Components Molasses Unit Major Components. Molasses unit made made ‘made made in in china Japan China Japan Total Nitrogen 08% % DirectReducing Sugar 66 17.0% ‘Ash Content 15 * Total Sugar “3 9% Volatile Basic Nitrogen 6.0 mg/100g Amino Nitrogen 150 40.0 mg/t00g ° mg/100g Sulfur Dioxide 0002 ND’ hg Fe mgi100g Nitrate 6B 005 ghkg ca mgi00g Cu 1% 2 ppm Ne mgit00g Zn 803 985 ppm K mgi100g Mn 623 27.8 ppm Ma mg/100g Siz 4070 2310 ppm. cr mg/'00g a 4 37 ppm Furctose % Dextros % Sucrose 283 % Table 9 Conditions and Results of Flooding Experiments Ne. 1 2 3 tm 23 23 2 Permeability Ky Ima] a4 542 sas Initial Ol Saturation Su Ped 85 a n Residual Oil Saturation after Water Flooding Su Cl a 26 40 Microorganisms TRC-322, TRC-4118 TRO-18-28 ‘Bacterial Counts In injected Fula x 108 (cetts/mt) 0.04 33 14 Cultivation Time days) 10 " 10 Cultivation Temperature re} 30 0 30 Olt Recovery after Microbial Flooding 1% 80d) 155 140 437 Table A-1 Conditions of Cultivation to detect Indigenous Microorganisms Target Microorganisms Medium Condition of Cultivation ‘Aerobes: Soll Extract Agar 30°C, 10-14days, aerobic Kerosine Agar 30°C, 10-14days, aerobic ‘Anacrobes we 3076, 10-t4days, anaerobic Kerosine Agar 30°C, 0-14days, anaerobic Sulfate Reducing Bacteria for SRB 30°C, 10-44days, ‘Ammonia Oxidizing Bacteria for AoB 30°C, 4weeks ‘Acidurle Sulfur Oxidizing Bacteria Silverman $K 30°C, Bweeks, shaking culture Sulfur Oxidizing Bacteria Starkey 30°C, Bweeks, shaking culture Dentritying Bacteria Nitrate Buitlon 30°, 10-14days for NOB 30°C, Bweeks 45910 H.YONEBAYASI, K ONO, HENOMOTO, T.CHIDA, C-XHONG, KFUJIWARA SPE 26070 Table A-2 Medium Composition eam competion adm Conponion SoU Enact AGH KPO dag SlvermanK iS, or ent Cat 109 xe og rerone ie tegpo, 3a Agar 1509 ‘MgS0,+7H,0 sg Gol exrat 000m faa Kerosine 10.09 10.09 wet noe Disied ater 1200 Nash, 195 stay ag04 20 HPO, ase eee fe aSou-7H:0 asa repo, 136 " ‘ feel se mas0,-740 ose Died Water 00m tom 3g vw Tryptone 10.09 Distilled Water 1,000 mi wet 308 : io arenes Meta olton Yeast Extract 50g NegEDTA $30 CynteneHydroctrlie 04g 2804-70 oss wet ose exo) 2440 % Agar 06s Mac, 44,0 039 Distilled Water 1,000 mi CoC, * 6H: 0.39 (migiorOy-4H,0 035 for SRB K,HPO, Osg FeSO,°7H,0 03g ‘Yeast Extract 1.09 CuSO,*5H,0 0.39 NHAC! 10g Distilled Water 1,000 mi pee ae Nitrate Buillon Nutrient Broth 059 Cac,-2H,0 arg Peptone fog Fes, 740 029 rio, 138 was0u-T40 ote Dinted Water 1.900 Sen Lace 355 ferns ——_Nahog 0.0085 Sedum Thogycolate 8 we ea feu Ancorote oie ca Rea oer 09 cach 03a water 1.900 ec ae for AOB (NH) 80, Osa CaCO, 10g act 039 Mg804-7H,0 Or retro, tea Dine Water 1 M980.-7850 038 e800 oes xe0, 133 Diced Water 1 mt 460‘SPE 36070, MICROBIAL ENHANCED OIL RECOVERY FIELD PILOT IN A WATERFLOODED RESERVOIR 100" 110" SO. [woman Beijing * Shanghai Pacific Ocean aor Fig. Location Map of Fuyu Oilfield East 2026 Block 1 Prox [East 2073 Block se x on y fio enas st mf mel oe ean “ps pun, 2% Ser, a ern eon, ot, rem, M262 M6, 26, prem, mI, panff : od “f Fig.2 Map of "East 24-23 Block” and “East 24-26 Block” 461SPE 38070 M19 1097 Ws 4912 11 109 8 2M M33 32 34 2028 2020 M M 90 27 26 2529 22 21167 6 54 3 Priv rr an Setar tape ebetabet-tet etd | f speed fi-tag 32 9027 25 OM 4 1S BSS 21M HO TM m7 8M usa eset 2 iM ae NT 6M Fig.3_ Three kinds of photographs of electrophoresis shows each microbial 16S rRNA gene cut by three kinds of restriction enzymes, respectively. (a) Hpa Il, (b) Hha J and (c) Alu Twere used as an restriction enzyme. 462SPE 38070 MICROBIAL ENHANCED OIL RECOVERY FIELD PILOT !N A WATERFLOODED RESERVOIR 3 Fig Three views of compatition tests between Indigenous Microorganisms and Candidate ones, Gase a is TRC-4126 vs, 113, case b Is TU-TA vs, 113 and case c is TRC-18-2-a vs. 118. 463 E = 2 2 & 3 a os 0 % 0 2 Time {days} Fig.5 Competition between TU-7A and 113 = 1000 £ © Pure Culture zoo * Competitive Culture i 600 ¥ «0 i 3 2 200 = ° os 7 % 2 2s Time (¢ays] Fig.6 _ Effect of 113 on Metabolized Gas Amount of TU-7A ‘o? 10% Bacterial Counts (cells/ml) wt f 0 108 o 5 © 1% 2 Time {days} Fig.7 Competition between TRC-322 and 113“ H.YONEBAYAS|, K.ONO, HENOMOTO, T.CHIDA, CX.HONG, K.FLUINVARA SPE 38070 1000 wo = = 00 wot E 6 fotreana 7 Tr 4 10 © TRO-4118 | & ans } 108 3 10 3 m 10 os © 1% 2 2 os wm 1 2 2 ‘Time [days) ‘Time [days] Fig.8 Effect of 113 on Metabolized Gas Fig.9 Competition between TRC-4118 and 113 ‘Amount of TRC-322 so 10 § a © Pure Culture = i Competitive Culture § (© TRC-AB28 a) 3 oi 5 j 10° Bn bw 3 3 3 3 | 3 ow 10 10° o 5 1% 2 2s . 5 @ i 2 2 Time [days] Time [days] Fig0 Effect of 113 on IFT Reduction by Fig.11 Competition betwoen TRC-18-2-a and 118 TRC-4118 a CTs od = 500 £ © Pure Culture E 400 4 Competitive Culture = 00 é E 200 3 gm ° os 7 1 me Tie [days] Fig.12 Effect of 118 on Metabolized Gas 464‘SPE 38070, MICROBIAL ENHANCED OlL RECOVERY FIELD PILOT IN A WATERFLOODED RESERVOIR, ‘Ale Bath 20°C Water Bath 30°C ea Ce Fraction Collector Fig3_ Flooding Apparatus Preparation 11512" Borea Sandstone — 7 — 1 Microbial Flooding | ona% K_=520md Gear ‘Microorganiems with Nutrients ) | Initial Bacterial Count Injection= 1.03.0 PV Tempra 36 || eas 10-14 dye | Water Flooding Injection > 3.0 PV 8,536% Injection> 6.0 PV Fig.14 A General Flow Diagram of Flooding Experiments6 H.YONEBAYASI, KONO, HENOMOTO, TCHIDA C-XHONG, K FUJIWARA sre seoro (to) 200 20 100 510 3 Tea ts 2 meatie 36 wo Ele 5 @ mc-te28 3 ; & —_ z $1.2 Be, ogise © so» Foo anasassussananos|® £1 E i evr eonnemey | ug jad i ik a onwcrca am | |" 2 4*2 0 an gtitittesescesssess 8 eran we la ORR A Sooo ee ee a ° ° Moll oto eae aaa et eee eos 1 as 2s me [eay] Pore Volume Produced Fig.15 Pressure Response during Cultivation ‘Fig 16 _Oil Recovery, Differential Pressure and Oil Rate vs. Pore Volume Produced a Flooding Experiment using TRC-322 (ao) (107%) 2» 0 oa 200 0 Ws 84°_ Fis baw iso = 1° 2 g i g Hd et j oe] E Fk 100 & < Bb Ey, = aad a tnecatsone|| | sti3 5 2 H.8 35 Oiirate "3 6 ot, yA {fh oo ° os 1 us 2 as 8 os 1 132 a8 Pore Volume Produced Pore Vokime Produc Fig.7_Oll Recovery, Diferntal Pressure and Oil Fig18__Oil Recovery, Differential Pressure and Oi Rate vs. Pore Volume Produced at Flooding Experiment using TRC-6118 Viable Count {cetisi) Fig. At Indicated at the baseline of 0.1 cells/ml. Viable counts of original microorganisms in samples. Microorganis! Rate vs. Pore Volume Produced at Flooding Experiment using TRC-18-2-a a 8 c D E: F G: No-fermentable Gram-negative Bacteria 1: Well 2226, 4 : Well 24.24 2: Woll 22:26 © : Woll 24.23, 3: Well 22:27, 6 : Water Flood Plant 18 with zero-viable count are 466