Screening of Microorganisms for Microbial Enhanced Oil Recovery Processes

Transcription

1 [Regular Paper] Screening of Microorganisms for Microbial Enhanced Oil Recovery Processes (Received June 9, 1999) The objective of this study is to screen effective microorganisms for the Microbial Enhanced Oil Recovery process (or simply as MEOR). Samples of drilling cuttings, formation water, and soil were collected from domestic drilling sites and oil fields. Moreover, samples of activated-sludge and compost were collected from domestic sewage treatment facility and food treatment facility. At first, microorganisms in samples were investigated by incubation with different media; then they were isolated. By two stage-screening based on metabolizing ability, 4 strains (Bacillus licheniformis TRC-18-2-a, Enterobacter cloacae TRC-322, Bacillus subtilis TRC- 4118, and Bacillus subtilis TRC-4126) were isolated as effective microorganisms for oil recovery. B. licheniformis TRC-18-2-a is a multifunctional microorganism possessing excellent surfactant productivity, and in addition it has gas, acid and polymer productivities. E. cloacae TRC-322 has gas and acid producing abilities. B. subtilis TRC-4118 and TRC-4126 are effective biosurfactant producers, and they reduce the interfacial tension to 0.04 and 0.12dyne/cm, respectively. 1. Introduction The development of Enhanced Oil Recovery (EOR) techniques (redevelopment and stimulation technologies) to produce additional oil must be accelerated to fulfill future oil-requirements, since large oil field discovery is becoming difficult in the recent years. We recognize the microbial FOR process to be one of the effective options among the various FOR methods to redevelop mature reservoirs which have been flooded out by water injection. The history of MEOR process dates back to 1926 when Beckman suggested it1), and after his initial suggestion, additional pioneering studies were reported by ZoBell2)-4), Updegraff et al.5)-7), and Beck8). After their laboratory work, many field trials have been carried out. A review of MEOR field application was presented by Lazar9). Outstanding advantages of MEOR are its lower investment costs in comparison with those of other FOR processes. These advantages make the application of this process to flooded reservoirs to recover additional oil that is difficult even by use of more expensive techniques. The MEOR process involves the following oil recovery mechanisms: pressurization of the driving energy and swelling of oil by in-place gas production, * To whom correspondence should be addressed. improvement of porosity and permeability by acid production in carbonates, control of mobility by biopolymers or biomass, decrease in interfacial tension between oil and water by biosurfactants, and/or degradation of hydrocarbons. Here, the effect of microbial production on oil-displacement is emphasized. In MEOR research, much still remain to be studied because this process depends strongly on the functions and abilities of microorganisms. Hence, our research aims at searching new microorganisms, which possess effective productive abilities for gases, acids, surfactants and/or polymers, and which could be candidates for MEOR field tests. In order to anticipate the effects of recovery mechanisms stated above, it is necessary to select suitable microorganisms for in situ reservoir conditions. Hence, many samples including effective microorganisms for MEOR were collected from domestic drilling sites and oil fields because it was anticipated that they would be suitable for reservoir environment such as anaerobic, high salinity, high temperature and pressurized conditions. Moreover, there are possibilities that both effective and suitable microorganisms exist in other environments than those in oil fields. Thus, activated-sludge and compost were collected as samples from sewage treatment facility and from food treatment facility. After investigation of microflora and isolation of all

2 60 and the formation from middle to lower consists of silty-mudstone, sandstone and a thin layer of tuff. Porosity is estimated to be good for the reservoir rock at shallower depths than 3000m MD. Temperatures tively. The Amarume field is one of the large oil fields in Japan discovered in The relevant properties of the reservoir are: reservoir depth= m VD (Vertical Depth), rock type is sandstone, permeability= 2-800md, effective reservoir thickness=32m, porosi- salinity (Cl-)=16,500mg/l. Other two oil fields, Toyokawa and Kurokawa, are small oil fields. The relevant properties of the reservoir in the Toyokawa field are: reservoir depth=29-340m VD, rock type is Fig. 1 Cross Section of Exploratory Drilling (a. Sagara, b. Niigata-Heiya) microorganisms, two stage-screening of isolates was carried out based on their metabolizing abilities, and those finally screened were identified. 2. Experimental Methods and Procedures 2.1. Sampling of Microorganisms Microorganisms were collected from two exploratory drilling sites (Sagara and Niigata-Heiya), three oil fields (Amarume in the Yamagata prefecture, Toyokawa and Kurokawa in the Akita prefecture), and two facilities (sewage treatment facility in the Chiba prefecture and food treatment facility in the Miyagi prefecture). The Sagara drilling site consists of eight formations (C', A-C, D1-D3, E) and the total depth of the well is 3230m (Fig. 1a). Drilling cuttings were gathered from two points in the well. One sampling point (sample #1) was at 425m MD (Measured Depth) in D3 formation and another (#2) was at 1112m MD in D1 formation. Both formations consist mainly of sandysiltstone and siltstone. Porosity and air permeability of sandstones, which sedimented at shallower formations than formation C are 20-30% and 5-50 and (millidarcy), respectively. The Niigata-Heiya drilling site consists of four formations, and all drilling cuttings were collected from the Uonuma group-haizume formation (Fig. 1b). Sampling points were 519m MD (#3), 1120m MD (#4) and 1860m MD (#5) in depth. The upper formation consists of unconsolidated medium-coarse sand, salinity (Cl-)=9500mg/l, and those of the reservoir in the Kurokawa field are: reservoir depth= m VD, rock type is rhyolite or tuff, permeability=80md, effective reservoir thickness=10m, porosity=40%, 14,500mg/l. In the Amarume oil field, two formation water samples were collected from well SK-27D (#6) and SK-71D (#8), and soil sample which was blurred with oil was collected near well SK-27D (#7). In the Toyokawa oil field, formation waters from well R-101 (#9) and soil (#10) were collected. In the Kurokawa oil field, only a sample (#11) of formation water was collected. Activated-sludge (#12) and compost (#13) were collected from the sewage treatment facility, and activated-sludge (#14) was collected from the food treatment facility. ph and Temperature of each sample were: 2.2. Investigation of Indigenous Microorganisms Media The following media were prepared to investigate microflora in the samples: Soil-extract Agar (SEA), Soil-extract Agar with Yeast-extract and Peptone (SEAYEP), Kerosine Agar (KA), VL Medium (VLM), Sulfur Reducing Bacteria Medium (SRBM), Waksman- Starkey Medium (WSM), Ammonia Oxidizing Bacteria Medium (AOBM), Nitrate Oxidizing Bacteria Medium (NOBM), Nitrate Bouillon (NB) and Molasses Bouillon (MB). For the samples from the activatedsludge and compost, PGY Agar (PGYA), VL Medium (VLM) and Molasses Bouillon (MB) were prepared. The reason for preparing so many kinds of media was to detect all microorganisms and to investigate them individually according to the following classification: aerobes, anaerobes, SRB, Sulfur Oxidizing Bacteria (SOB) and Denitrifying Bacteria (DB). SEA, SEAYEP, KA, VLM and PGYA were used for detection of aerobes and/or anaerobes. WSM and NB were

3 61 used for detecting SOB and DB, respectively. In addition, MB was prepared for Molasses Utilizing Bacteria (MUB). The composition of SEA is in g/l: K2HPO4, 0.2: and agar, 15; and it was made up with a soil-extract solution. The soil extraction was conducted by autoclav- ing, the bottle containing the sample was vibrated in the Two kinds of soil were extracted and the soil-extract solution was prepared by mixing equivalent amount of the soils, one of which was easily available near the laboratory and another from the collected sample. SEAYEP has the same composition as that of SEA with addition of 1.0g/l each of yeast-extract and peptone (both from Difco Laboratories, Detroit, MI, USA). The composition of KA is, in g/l: kerosine, 10; NH4Cl, diluent on SEA, SEAYEP, KA and PGYA. For NaCl, 2.0; and agar, 15. VLM contains, in g/l: tryptone (Difco), 10; NaCl, 5.0; lab-lemco powder (Oxoid, UK), 2.0; yeast extract, 5.0; cysteine hydrochrolide, 0.4; and agar, 0.6. The composition of SRBM is in g/l: K2HPO4, 0.5; yeast extract, 1.0; NH4Cl, 1.0; glycolate, 0.1; sodium ascorbate, 0.1; and agar, 3.0. The composition of WSM is: (NH4)2SO4, 0.3g; yeast extract, 0.3g; metal solution, 10ml; in 1 liter of distilled water. The metal solution contains 0.3g each The composition of AOBM is in g/l: (NH4)2SO4, 0.5; 7H2O, 0.03; CaCO3, 7.5. The composition of NOBM 7H2O, 0.1. NB contains: Nutrient Broth (Difco), 0.5 g; peptone, 1.0g; and KNO3, 1.0g; in 1 liter of distilled water. MB contains: Nutrient Broth, 8.0g; and molasses, 40g; in 1 liter of distilled water. Molasses is a commercial waste product, and it is used in many MEOR projects all over the world9) because of its low price and its high nutritive value for microorganisms. The composition of PGYA is in g/l: peptone, 2.0; glucose, 0.5; yeast extract, 1.0; and agar, 15. Sterilization of all these media, except SRBMs, was carried words, SRBMs, that is solutions containing sodium thioglycolate and sodium ascorbate, were sterilized by tion containing other components was autoclaved. The ph of SEAYEP was adjusted to 6.8 and 9.0, and there was no adjustment of ph in other media. The initial ph of these media was SEA=6.8, KA=7.2, VLM= , SRBM= , WSM=4.5, NB= and PGYA=7.1, respectively Procedure of Enrichment Culture and Isolation A 30g sample (drilling cutting, soil, activatedsludge, or compost) was mixed in a sterilized glass bottle filled with 270ml of phosphate buffered saline fil- supersonic waving machine for 1min to free microorganisms from the solid surface. This solution was identified as undiluted original solution. Also samples of untreated formation water from oil wells were used as undiluted original solutions. Diluted solutions were prepared for evaluation using the dilution end-point method. The incubation of aerobes and anaerobes was prepared by plating the original solution and its VLM, these solutions were inoculated in 10ml of the medium, and then the surface of the medium was coy- min. The preparation of SRB incubation involves, (1) inoculating the original solution and its diluent in 20 ml-tubes, (2) pouring 15ml of SRBM into the tubes, and (3) putting 3-4ml of autoclaved paraffin on the surface. For the incubation of SOB, AOB, NOB, and DB, the original solution and the diluent were inoculated into 10ml of WSM, AOBM, NOBM and NB in 20 ml-tubes, respectively. For NB, 3-4ml of autoclaved paraffin was put on the surface of the medium after inoculation. All microorganisms were incubated at 30 were carried out under anaerobic condition, and SOB was incubated by shaking the culture. After days of incubation (60 days for SOB, 30 days for AOB and NOB), the viable cell count and most probable number were measured by plate count and by MPN method, respectively. In addition, enrichment incubation with MB was conducted to collect MUB from the #6-#14 samples directly. Because most of MEOR field applications use the molasses only as the main nutrient, microorganisms for this process are required to assimilate with this nutrient. The procedure of the enrichment culture involves, (1) for inoculating putting 1 or 10ml of origi- into 150ml of MB in Hungate tubes (sealed with rub- days, (3) estimating gas, ph, emulsification, and slime forming, (4) isolating MUB from all tubes which were estimated to be positive in the testing. After investigation, all types of microorganisms were isolated from all colonies on the plates First Screening of Isolates for MEOR Process The first screening of microorganisms isolated by the investigation mentioned above was conducted according to their gas, acid, surfactant and/or polymer producing abilities of the MEOR process. The following

4 62 four media were prepared for the tests of each ability: MB for test of gas and/or acid, Defibrinated Sheep Blood Agar (DSBA) for test of surfactant, and Sucrose Agar (SA) for test of polymer. Composition of DSBA and SA were: 50ml of defibrinated sheep blood and 23 g of nutrient agar; and 20g of sucrose and 23g of nutrient agar in 1 liter of distilled water, respectively. The preparation of each incubation was conducted by inoculating isolates into 15ml of MB in a Hungate tube, which was then sealed (the head space was filled with purified nitrogen gas) and plating strains on DSBA and SA plates, respectively. After incubation conditions (except gas and/or acid tests for which only anaerobic condition was used), the amount of gas produced was measured by piercing the rubber cap of the tube with a syringe, ph of the medium was measured the existence of slime on the plates were visually observed. Moreover, all strains, which were estimated inoculated into 15ml of MB in test tubes (18mm under the same conditions used in the test with DSBA. After incubation, the surface tension of the medium was measured with a Du Nouy tensiometer. Finally, all strains, which produced gas, showed ph lower than 5.5, reduced the surface tension below 40.0dyne/cm, and/or formed slime, were considered to be positive strains, and those which showed outstanding ability in each function were selected for second screening Second Screening of the Strains Selected in First Screening The second screening of strains, which were selected in first screening and which produced gas, acid, surfactant, and/or polymer, was carried out by inoculating the strains in 15ml of one of the following media: Molasses with Inorganic Salts (MIS) for gas and/or acid producers in a Hungate tube, sealing it whose head space was filled with purified nitrogen gas, MB (for surfactant producers) and Sucrose Bouillon (SB) for polymer producers in test tubes. Regarding the compositions of MIS and SB, the former is in g/l: molasses, 40; NaCl, 12.37; Na2B4O7, 0.34; NH4Cl, 0.1; CaCl2, 0.25; and MgCl2, 0.215, and the latter is also in g/l: nutrient broth, 8; and sucrose, 20. The formation water generally includes many inorganic salts and minerals. Hence, it is necessary to take account of their influence on strains because some components often affect the microbial multiplication and their metabolism. Then the components of these inorganic salts were specified similar to those in representative domestic oil fields. gas produced and ph of medium resulted were measured by the same methods used in first screening. In order to evaluate surfactant and polymer producing in abilities, interfacial tension between n-octane and media was measured with a spinning drop interfacial tensiometer, and the viscosity of media was measured with a B-type viscometer. Surfactant and polymer producers, Bacillus licheniformis JF-2 and Bacillus licheniformis SP-018, which were provided by Oklahoma University, were incubated as positive controls, respectively. The strain JF-2 is a well evaluated microorganism, which produces effective surfactant under typical reservoir environment, and it has been investigated by many authors10)-12). Strain SP-018 is recognized as an excellent polymer producer. Finally, the screened strains were identified and their characteristics investigated. 3. Results and Discussion 3.1. Indigenous Microorganisms Indigenous Microorganisms in Exploratory Drilling Sites and Oil Fields Cell concentrations of indigenous microorganisms in all samples are presented in Table 1. The incubation of microorganisms from Segawa drilling cuttings showed cells/g mesophilic aerobes and anaerobes existed in the shallow formation (#1). However, the presence of thermophilic aerobes was less than that of mesophilic, and no thermophilic anaerobe was detected. In addition, there were less aerobes or no anaerobes in the deeper formation (#2). SRB and DB were detected in high cell concentrations tion was low in #2 sample. Similar trend was observed with the two Niigata-Heiya samples, #3, 519 m MD and #4, 1120m MD, which included more aerobes and anaerobes than those of #5 (1860m MD). The cell counts of most predominant aerobes and anaerobes attained 107 and 106cells/g, respectively, at attained cells/g. Mesophilic DB existed in high population in levels down to 1120m MD in this region. It was expected at first that many thermophilic microorganisms could be detected from #5 the results fell short of expectation. Their highest concentration was in the range of cells/g. The formation water of the Amarume and Kurokawa oil fields (#6, #8 and #11) included indigenous microorganisms in low concentrations, and that of the Toyokawa oil field (#9) contained more mesophilic microorganisms than others; however, its highest concentration was cells per 100ml. Chemical analysis of the formation waters of Amarume and Kurokawa showed a small difference in most of the components; however, some components in Toyokawa differed clearly from those in other two fields. For

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6 64 instance, the representative different components (in mg/l) between Toyokawa and the other two fields were: K+, 382.4, 504.5, 69.9, 227.1; Ca2+, 957.4, 512.6, 52.9, 227.0; Cl-, 15932, 14299, 8116, 13632; HCO3-, 302.9, 506.6, , 670.1; CO32-, 0.0, 0.0, 271.5, 35.1: in the order of #6, #8, #9, and #11. The former two cations and chloride ion of Tooyokawa were less than those of others, and the latter two anions were clearly more than those in Amarume and Kurokawa. It was presumed that these differences affected the distribution of indigenous microorganisms. In addition, positive results in the surfactant forming test were observed only in the 10ml original solutions from acti- be mild for microbial habitation. As expected, both soil samples included a high concentration of microorganisms, approximately up to 108 cells per 100g. Because these soils were collected at well sites, and because they always contained spilled oil, mesophilic hydrocarbon-utilizing microorganisms were detected in high cell concentrations with KA under aerobic condition. In addition, the enrichment culture with MB showed (#7 and #10) included active gas and acid producing microorganisms because all tubes ranged up to thousand-fold dilution were observed to be positive. However, most of them were mesophilic gas and acid producers because the diluents showed negative results in the gas productivity test and low activity in the acid microorganisms existed in only two soil samples, #7 and #10. Isolates were obtained from all colonies on the agar plates that were used for the investigation of microflora. In addition, MUB were isolated from the enrichment culture. Finally, 123 kinds of microorganisms were isolated and their features investigated (cell shape, Gram stain, spore, motility, oxidase, catalase, OF, and colony). Representative isolates are listed in Table Indigenous Microorganisms in Activatedsludge and Compost Table 3 lists cell concentrations of indigenous microorganisms in each sample. The incubation of microorganisms in all samples showed mesophilic aerobes in high concentration ( cells/ml) and mesophilic anaerobes in low concentration (less than 150cells/ml). Thermophilic aerobes were detected in low concentration (#14, 90cells/ml) or at least not in high concentration (#12, 104cells/ml) in activated-sludge. Sample #13 included as much thermophilic aerobes as mesophilic (107cells/g). As to thermophilic anaerobes, they were not detected in samples of activated-sludge; only the compost included them in low concentrations. According to the investigation of spores, most of the microorganisms in the compost were spore-forming. Because the growth range temperature for such microorganisms is generally broad, both mesophilic and thermophilic aerobes were detected from the compost. The enrichment culture with MB showed the follow- and acid tests were positive in all range of dilution, but positive results in the polymer forming test were observed in the 10ml undiluted solution from #12 and in the original solutions (1 and 10ml) from #13, and compost in all dilution ranges were positive in gas and acid forming tests, but the activated-sludge solution showed low activity for gas forming microorganisms (#14; only 10ml original solution) and acid forming microorganisms (1 and 10ml original solutions). There was no result estimated to be positive in the surfactant and polymer forming tests. These results suggested that (1) mesophilic gas and/or acid producing MUB were predominant in all samples, (2) thermophilic ones were only predominant in the compost, and (3) surfactant and/or polymer producing microor- temperatures. Isolates were obtained from all colonies on PGYA and VLMA plates. In addition, MUBs were isolated from enrichment culture. As a final result, 44, 41, and 42 types of microorganisms were isolated from #12, #13 and #14 samples, respectively. The total amount of isolates was 127. They were numbered , , and in the order of #12, #14, and #13 samples. There were: five mesophilic aerobes ( ), three thermophilic aerobes ( ), six mesophilic anaerobes ( ), 14 mesophilic aerobic MUB ( ), ten mesophilic anaerobic MUB ( ), five thermophilic aerobic MUB ( ), and only one thermophilic anaerobic MUB (4144) from #12 activated-sludge; eight mesophilic aerobes ( ), three thermophilic aerobes ( ), eleven mesophilic anaerobes ( ), ten mesophilic aerobic MUB ( ), six mesophilic anaerobic MUB ( ), two thermophilic aerobic MUB ( ) and one thermophilic anaerobic MUB (4241) from #14 compost. In addition there were five mesophilic aerobes ( ), six thermophilic aerobes ( ), six mesophilic anaerobes ( ), four thermophilic anaerobes ( ), seven mesophilic aerobic MUB ( ), six mesophilic anaerobic MUB ( ), four thermophilic aerobic MUB ( ), and four thermophilic anaerobic MUB ( ) from #13 activated-sludge.

7 65 Table 2 Characteristics of Representative Isolates a) Not tested. b) Characteristic pigment not produced. Table 3 Cell Concentrations in Samples Collected from Sewage Treatment and Food Treatment Facilities The unit of cell concentration in #13 sample is cells/g and those in #12 and #14 are cells/ml.

8 First Screening Strains from Exploratory Drilling Sites and Oil Fields As a result of the first screening of isolates for MEOR process, 69 and 61 strains with gas and acid producing abilities, respectively, were considered to be positive among the 123 strains tested. Among them the following 19 strains 107 (7.1), 108 (8.8), 113 (6.1), 201 (7.0), 204 (6.8), 207 (6.8), 208 (7.0), 322 (6.8), 325 (6.2), 402 (6.5), 412 (7.2), 413 (8.1), 504 (6.1), 515 (7.1), 516 (6.5), 533 (6.8), 611 (7.5), 620 (6.1), and 637 (7.2) exibited high gas producing ability (more than 6.0 ml of gas amount). Values in parentheses show the amounts of gas produced in milliliter. The microorganism with the highest gas producing ability was strain 108, which produced 8.8ml. The main gas component was identified as carbon dioxide. The following 14 strains 18-2-a, 24-1-c, 103, 107, 112, 323, 324, 330, 524, 537, 610, 620, 626, and 636 gave phs less than 5.3. Estimation of surfactant producing abil- ever there were only 15 following strains 18-2-a (36.1), 18-2-b (36.6), 24-1-c (36.3), 206 (35.7), 330 (38.9), 415 (37.9), 418 (32.6), 522 (31.2), 524 (32.5), 536 (35.1), 537 (34.6), 538 (36.8), 610 (33.4), 635 (32.1), and 637 (38.7), that reduced the surface tension less than 40.0dyne/cm. Values in parentheses indicate the surface tension in dyne/cm. The lowest surface tension was 31.2dyne/cm shown by strain 522. There were 16 following strains 18-2-a, 18-2-b, 21-2-a, c, 414, 418, 502, 507, 508, 522, 536, 601, 616, 622, 624, and 635 that were considered to be slime forming. In addition, strains 18-2-a, 522, and 635 had four different abilities (gas, acid, surfactant and polymer production); and strain 418 (acid, surfactant, and polymer production) and strains 524, 537, and 637 (gas, acid, and surfactant production) had three different abilities shown in parentheses. Finally, the following 18 strains 18-2-a, 108, 206, 322, 330, 412, 414, 418, 507, 522, 524, 537, 538, 611, 616, 622, 635, and 637 were selected from isolates by first screening based on their single or pluralistic ability Strains from Activated-sludge and Compost The screening results of representative isolates are listed in Table 4. By the first screening for MEOR process, 20 strains with gas producing ability were considered to be positive among all 127 strains tested. (more than 6.0ml of gas). Thus, these two were selected as gas producers in first screening. Ten isolates (strains 4119, 4122, 4139, 4141, 4142, 4224, 4230, 4239, 4311, and 4327) gave values of ph lower than 5.0, and especially strain 4141 gave the low- the acid producer in first screening. As regards the estimation of surfactant producing sis in which 12 strains were positively reactive only under aerobic condition, 13 strains reactive only under anaerobic condition, and the remaining 23 strains produced surfactants under both conditions. However, there were only 12 strains, namely 4104, 4105, 4116, 4118, 4123, 4126, 4141, 4143, 4204, 4206, 4225, and 4309 that reduced the surface tension to less than 40.0 dyne/cm. These 12 strains were selected as surfactant producers in first screening. The lowest surface tension (32.5dyne/cm) was registered by strain 4116 at The following 10 strains 4104, 4107, 4115, 4116, 4126, 4142, 4210, 4240, 4324, and 4335 were considered to be slime forming, and strains 4107, 4142, 4210, and 4240 showed the equivalent slime forming ability to that of a positive control, Bacillus licheniformis SP Hence, these four strains were selected. Finally, the following 18 strains, 4104, 4105, 4107, 4116, 4118, 4123, 4126, 4134, 4141, 4142, 4143, 4204, 4206, 4210, 4225, 4240, 4309, and 4324, were selected from isolates in the first screening based on their productive ability. It should be mentioned that many microorganisms were found in activated sludge and compost as expected, and those microorganisms found in activated sludge were potentially effective for MEOR process Second Screening Strains from Exploratory Drilling Sites and Oil Fields The results of second screening are presented in Table 5. High performance of gas and/or acid production was shown by strains 108, 322, 412, and 611, all of which were Enterobacter. Especially, strain 322 produced most gas (7.3ml) and the lowest ph (4.7). On the other hand, lower IFTs were obtained by 18-2-a, 206, and 418, all of which belong to Bacillus. Moreover strain 18-2-a reduced IFT to 1.2dyne/cm, which was lower than IFT (1.7dyne/cm) of the positive control, Bacillus licheniformis JF-2. There was no strain which increased the viscosity of the medium more than 5.6cp of the positive control, Bacillus licheniformis SP-018. As a final result, two strains were selected as candidates for MEOR field testing. One is strain 18-2-a, which is an excellent surfactant producer, and also excellent producer of gases, acids and polymers. The other strain 322 is also an excellent gas and acid producer. These two strains were renamed TRC-18-2-a and TRC-322, respectively. They were identified as Bacillus licheniformis and Enterobacter cloacae, respectively. According to the investigation of their characteris-

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10 68 Table 5 Results of Second Screening of Exploratory Drilling Fields or Oil Fields Originated Strains Table 6 Results of Second Screening of First Screened Strains Isolated from Activated-sludge and Compost a) Interfacial tension between n-octane and medium. b) Not tested. c) Provided by Oklahoma University. d) Measured for 18-2-a, testing. e) Measured for all microorganisms, except 18-2-a, testing. tics, the maximum temperature of growth of TRC conducted with nutrient broth in a temperature gradient incubator. Based on these results, the former was found applicable to the approximately 1000m VD reservoir, and the latter was limited to shallow oil fields. In addition, the optimum ph ranges of growth were for TRC-18-2-a and for TRC Therefore, these microorganisms could be used in most of sandstone reservoirs Strains from Activated-sludge and Compost The results of second screening are presented in Table 6. The high performance of gas production was shown by strains 4134 (5.5ml) and 4324 (5.7ml). However, the total amount of gas produced by them could not reach that attained by TRC-322. Strains 4141 and 4142 reduced ph to its lowest value (4.7) which was equal to that of TRC-322. Lower IFT was obtained with 4104, 4105, 4116, 4118, 4123, and 4126, and particularly strains 4118 and 4126 reduced IFT to 0.04 and 0.20dyne/cm, respectively. They were a) Interfacial tension between n-octane and medium. b) Not tested. c) Provided by Oklahoma University. lower than IFT (1.7dyne/cm) of the positive control, Bacillus licheniformis JF-2, and the result of TRC-18-2-a (1.2dyne/cm). There were no strains which increased the viscosity of medium more than 5.6 cp, the value attained by the positive control, Bacillus licheniformis SP-018. Two strains, 4118 and 4126, were finally selected as candidates for MEOR field testing. These two strains were renamed TRC-4118 and TRC-4126, respectively; they were also identified as Bacillus subtilis. 4. Summary of Results (1) Indigenous microorganisms from exploratory drilling sites and oil fields included both aerobic and anaerobic ones, in spite of anaerobic sampling environments. On the other hand, most of the microorganisms from activated-sludge and compost were aerobic because these samples existed under aerobic conditions. In addition, MUB were isolated from formation water, soil (oil fields), activated-sludge and compost (sewage treatment facility and/or food treatment facility). (2) By the first screening of isolates from exploratory drilling sites and oil fields, 19, 14, 15, and 16 strains were estimated to be of high gas, acid, surfactant, and polymer producers, respectively. In addition, strains with pluralistic ability for gas, acid, surfactant, and polymer production were obtained. Finally, 18 strains were selected, based on their single ability or on plural-

11 69 istic ability. (3) By the first screening of isolates from activatedsludge and compost, 2, 11, 12, and 4 strains possessing gas, acid, surfactant, and polymer producing abilities were considered to be effective for MEOR process. (4) TRC-18-2-a, TRC-322, TRC-4118 and TRC-4126 were selected as candidate microorganisms for MEOR field testing. TRC-18-2-a possessing gas and acid productive abilities reduced IFT to 1.2dyne/cm, which was lower than IFT (1.7dyne/cm) of the positive control, B. licheniformis JF-2. TRC-322 produced an optimum amount of gas (7.3ml) and the lowest ph (4.7). TRC-4118 and TRC-4126 reduced IFT to 0.04 and 0.20dyne/cm, respectively, both of which were lower than that of IFT of JF-2. Acknowledgments The authors wish to thank Japan National Oil Corporation (JNOC) for the permission to present this paper. In addition, we gratefully acknowledge the cooperation of Japan Petroleum Exploration Co., Ltd., Tohoku Oil Co., Ltd. and Chuokogyo Co., Ltd. to collect the samples. We also thank the staff of Japan Food Research Laboratories for their experimental data. In addition, we thank K. Suzuki, H. Matsubayashi, Y. Sugihara, T. Yamamoto, T. Ogatsu, S. Murata, Y. Mitsuishi, Y. Yoichi, and K. Oseto, who were in charge of MEOR process research at JNOC during We thank H. K. Sarma for his assistance in preparation of this paper. References 1) Beckman, J. W., Ind. Eng. Chem., 10, 3 (1926). 2) ZoBell, C. E., U. S. Pat (1946). 3) ZoBell, C. E., World Oil, 126, (13), 36 (1947). 4) ZoBell, C. E., World Oil, 127, (1), 35 (1947). 5) Updegraff, D. M., Wren, G. B., U. S. Pat (1953). 6) Updegraff, D. M., Wren, G. B., Appl. Microbiol., 2, 309 (1954). 7) Updegraff, D. M., U. S. Pat (1957). 8) Bech, J. V., Producers Monthly, 11, 13 (1947). 9) Lazar, I. I., "Microbial Enhancement of Oil Recovery-Recent Advances" Proceedings of the 1990 International Conference on Microbial Enhanced Oil Recovery, ed. by Donaldson, E. C., Elsevier, Amsterdam (1991), p ) Lin, S. C., Goursaud, J. C., Kramer, P. J., Georgiou, G., Sharma, M. N., "Microbial Enhancement of Oil Recovery-Recent Advances" Proceedings of the 1990 International Conference on Microbial Enhanced Oil Recovery, ed. by Donaldson, E. C., Elsevier, Amsterdam (1991), p ) Thomas, C. P., Duvall, M. L., Robertson, E. P., Barrett, K. B., Bala, G. A., SPE Reservoir Engineering, 285 (1991). 12) Javaheri, M., Jenneman, G. E., McInerney, M. J., Knapp, R. M., Appl. Environ. Microbiol., 50, 698 (1985). Keywords Improved oil recovery, MEOR, Screening, Surfactant, Bacillus, Enterobacter