Screening of Microorganisms for Microbial Enhanced Oil

石油学会誌 Sekiyu Gakkaishi, 43, (1), 59-69 (2000) 59

[Regular Paper]

Screening of Microorganisms for Microbial Enhanced OilRecovery Processes

Hideharu YONEBAYASHI†1)*, Shinichiro YOSHIDA†2), Kenji ONO†3), and Heiji ENOMOTO†4)

†1) Japan National Oil Corp., Fukoku Seimei Building, 2-2 Uchisaiwaicho 2-chome, Chiyoda-ku, Tokyo 100-8511, JAPAN

†2) Div. of Microbiology, Japan Food Research Laboratories, 52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo 151-0062, JAPAN

†3) Technology Research Center, Japan National Oil Corp., 2-2 Hanriada 1-chome, Mihamalku, Chiba 261-0025, JAPAN

†4) Dept. of Geoscience and Technology, Graduate School of Engineering, Tohoku University, 01 Aramaki-aza-Aoba, Aoba-ku,

Sendai 980-8579, JAPAN

(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 fromdomestic drilling sites and oil fields. Moreover, samples of activated-sludge and compost were collected fromdomestic sewage treatment facility and food treatment facility. At first, microorganisms in samples were investi-

gated by incubation with different media; then they were isolated. By two stage-screening based on metaboliz-ing 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. licheni-formis TRC-18-2-a is a multifunctional microorganism possessing excellent surfactant productivity, and in addi-tion 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 to0.04 and 0.12dyne/cm, respectively.

1. Introduction

The development of Enhanced Oil Recovery (EOR)techniques (redevelopment and stimulation technolo-

gies) to produce additional oil must be accelerated tofulfill future oil-requirements, since large oil field dis-covery is becoming difficult in the recent years. Werecognize the microbial FOR process to be one of the

effective options among the various FOR methods toredevelop 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 sug-

gestion, additional pioneering studies were reported byZoBell2)-4), Updegraff et al.5)-7), and Beck8). After

their laboratory work, many field trials have been car-ried out. A review of MEOR field application was

presented by Lazar9). Outstanding advantages ofMEOR are its lower investment costs in comparisonwith those of other FOR processes. These advantagesmake the application of this process to flooded reser-voirs to recover additional oil that is difficult even byuse of more expensive techniques.

The MEOR process involves the following oil recov-ery mechanisms: pressurization of the driving energyand swelling of oil by in-place gas production,

improvement of porosity and permeability by acid pro-

duction in carbonates, control of mobility by biopoly-

mers or biomass, decrease in interfacial tension be-

tween oil and water by biosurfactants, and/or degrada-

tion 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, surfac-

tants and/or polymers, and which could be candidates

for MEOR field tests.

In order to anticipate the effects of recovery mecha-

nisms stated above, it is necessary to select suitable

microorganisms for in situ reservoir conditions.

Hence, many samples including effective microorgan-

isms 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 pressur-ized 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 sam-

ples from sewage treatment facility and from foodtreatment facility.

After investigation of microflora and isolation of all* To whom correspondence should be addressed.

石油学会誌 Sekiyu Gakkaishi, Vol. 43, No. 1, 2000

60

microorganisms, two stage-screening of isolates was

carried out based on their metabolizing abilities, andthose finally screened were identified.

2. Experimental Methods and Procedures

2.1. Sampling of Microorganisms

Microorganisms were collected from two explorato-ry drilling sites (Sagara and Niigata-Heiya), three oilfields (Amarume in the Yamagata prefecture,Toyokawa and Kurokawa in the Akita prefecture), andtwo facilities (sewage treatment facility in the Chiba

prefecture and food treatment facility in the Miyagiprefecture).

The Sagara drilling site consists of eight formations

(C', A-C, D1-D3, E) and the total depth of the well is3230m (Fig. 1a). Drilling cuttings were gatheredfrom two points in the well. One sampling point

(sample #1) was at 425m MD (Measured Depth) in D3formation and another (#2) was at 1112m MD in D1formation. Both formations consist mainly of sandy-siltstone and siltstone. Porosity and air permeabilityof sandstones, which sedimented at shallower forma-tions than formation C are 20-30% and 5-50 and (milli-darcy), respectively.

The Niigata-Heiya drilling site consists of four for-mations, 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 forma-tion consists of unconsolidated medium-coarse sand,

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

at #3-#5 sampling depth were 32, 43 and 62℃, respec-

tively.The Amarume field is one of the large oil fields in

Japan discovered in 1960. The relevant properties ofthe reservoir are: reservoir depth=530-840m VD

(Vertical Depth), rock type is sandstone, permeability=2-800md, effective reservoir thickness=32m, porosi-ty=14-32%, reservoir temperature=40-58℃ and

salinity (Cl-)=16,500mg/l. Other two oil fields,Toyokawa and Kurokawa, are small oil fields. Therelevant properties of the reservoir in the Toyokawafield are: reservoir depth=29-340m VD, rock type istaffaceous sandstone, reservoir temperature=33℃ and

salinity (Cl-)=9500mg/l, and those of the reservoir inthe Kurokawa field are: reservoir depth=225-335mVD, rock type is rhyolite or tuff, permeability=80md,effective reservoir thickness=10m, porosity=40%,reservoir temperature=38℃ and salinity (Cl-)=

14,500mg/l. In the Amarume oil field, two formationwater samples were collected from well SK-27D (#6)and SK-71D (#8), and soil sample which was blurredwith oil was collected near well SK-27D (#7). In theToyokawa oil field, formation waters from well R-101

(#9) and soil (#10) were collected. In the Kurokawaoil field, only a sample (#11) of formation water wascollected.

Activated-sludge (#12) and compost (#13) were col-lected from the sewage treatment facility, and activat-ed-sludge (#14) was collected from the food treatmentfacility. pH and Temperature of each sample were:#12, pH 7.1, 20℃; #13, pH 8.3, 60℃; and #14, pH 6.6,

27℃, respectively.

2.2. Investigation of Indigenous Microorganisms2.2.1. Media

The following media were prepared to investigatemicroflora 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 BacteriaMedium (AOBM), Nitrate Oxidizing Bacteria Medium

(NOBM), Nitrate Bouillon (NB) and MolassesBouillon (MB). For the samples from the activated-sludge and compost, PGY Agar (PGYA), VL Medium

(VLM) and Molasses Bouillon (MB) were prepared.The reason for preparing so many kinds of media wasto detect all microorganisms and to investigate themindividually 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 detec-tion of aerobes and/or anaerobes. WSM and NB were

Fig. 1 Cross Section of Exploratory Drilling (a. Sagara, b.

Niigata-Heiya)


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used for detecting SOB and DB, respectively. Inaddition, MB was prepared for Molasses UtilizingBacteria (MUB).

The composition of SEA is in g/l: K2HPO4, 0.2: andagar, 15; and it was made up with a soil-extract solu-tion. The soil extraction was conducted by autoclav-ing 500g soil in 500ml water at 120℃ for 20min.

Two kinds of soil were extracted and the soil-extractsolution was prepared by mixing equivalent amount ofthe soils, one of which was easily available near thelaboratory and another from the collected sample.SEAYEP has the same composition as that of SEAwith addition of 1.0g/l each of yeast-extract and pep-tone (both from Difco Laboratories, Detroit, MI, USA).The composition of KA is, in g/l: kerosine, 10; NH4Cl,2.0; Na2HPO4, 1.0; K2HPO4, 0.5; MgSO4・7H2O, 0.5;

NaCl, 2.0; and agar, 15. VLM contains, in g/l: tryp-tone (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;Na2SO4, 1.0; CaCl2・2H2O, 0.1; FeSO4・7H2O, 0.2;

MgSO4・7H2O, 0.1; sodium lactate, 3.5; sodium thio-

glycolate, 0.1; sodium ascorbate, 0.1; and agar, 3.0.The composition of WSM is: (NH4)2SO4, 0.3g;K2HPO4, 4.0g; KH2PO4, 1.5g; MgSO4・7H2O, 0.5g;

yeast extract, 0.3g; metal solution, 10ml; in 1 liter of

distilled water. The metal solution contains 0.3g each

of Na2EDTA, ZnSO4・7H2O, CaCl2・2H2O, MnCl2・

4H2O, CoCl2・6H2O, (NH4)6Mo7O2・4H2O, FeSO4・

7H2O, and CuSO4・5H2O in 1 liter of distilled water.

The composition of AOBM is in g/l: (NH4)2SO4, 0.5;NaCl, 0.3; K2HPO4, 1.0; MgSO4・7H2O, 0.3; FeSO4・

7H2O, 0.03; CaCO3, 7.5. The composition of NOBM

is in g/l: NaNO3, 0.006; K2HPO4, 1.0; FeSO4・7H2O,

0.03; CaCl2, 0.3; NaCl, 0.3; CaCO3, 1.0; MgSO4・

7H2O, 0.1. NB contains: Nutrient Broth (Difco), 0.5

g; peptone, 1.0g; and KNO3, 1.0g; in 1 liter of distilledwater. MB contains: Nutrient Broth, 8.0g; andmolasses, 40g; in 1 liter of distilled water. Molassesis a commercial waste product, and it is used in manyMEOR 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; glu-cose, 0.5; yeast extract, 1.0; and agar, 15. Sterili-zation of all these media, except SRBMs, was carriedout by autoclaving them at 120℃ for 15min. In other

words, SRBMs, that is solutions containing sodium

thioglycolate and sodium ascorbate, were sterilized by

filteration with a 0.2μm membrane filter, and the solu-

tion containing other components was autoclaved.The pH of SEAYEP was adjusted to 6.8 and 9.0, andthere was no adjustment of pH in other media. Theinitial pH of these media was SEA=6.8, KA=7.2,VLM=7.2-7.4, SRBM=7.1-7.4, WSM=4.5, NB=7.0-7.2 and PGYA=7.1, respectively.

2.2.2. Procedure of Enrichment Culture and Iso-lation

A 30g sample (drilling cutting, soil, activated-sludge, or compost) was mixed in a sterilized glass bot-tle filled with 270ml of phosphate buffered saline fil-tered through a 0.2μm membrane filter. After mix-

ing, the bottle containing the sample was vibrated in the

supersonic waving machine for 1min to free microor-

ganisms 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

diluent on SEA, SEAYEP, KA and PGYA. For

VLM, these solutions were inoculated in 10ml of the

medium, and then the surface of the medium was coy-

ered with 3-4ml of autoclaved paraffin at 120℃ for 15

min. The preparation of SRB incubation involves, (1)inoculating the original solution and its diluent in 20ml-tubes, (2) pouring 15ml of SRBM into the tubes,and (3) putting 3-4ml of autoclaved paraffin on thesurface. For the incubation of SOB, AOB, NOB, andDB, the original solution and the diluent were inoculat-ed into 10ml of WSM, AOBM, NOBM and NB in 20ml-tubes, respectively. For NB, 3-4ml of autoclaved

paraffin was put on the surface of the medium afterinoculation. All microorganisms were incubated at 30and 55℃. The incubation of anaerobes, SRB, and DB

were carried out under anaerobic condition, and SOBwas incubated by shaking the culture. After 10-14days of incubation (60 days for SOB, 30 days for AOBand NOB), the viable cell count and most probablenumber were measured by plate count and by MPNmethod, respectively.

In addition, enrichment incubation with MB wasconducted to collect MUB from the #6-#14 samplesdirectly. Because most of MEOR field applicationsuse the molasses only as the main nutrient, microorgan-isms for this process are required to assimilate with thisnutrient. The procedure of the enrichment cultureinvolves, (1) for inoculating putting 1 or 10ml of origi-nal solution and 1ml of its diluent (×10, 102 or 103)

into 150ml of MB in Hungate tubes (sealed with rub-ber caps), (2) incubating at 30 and 55℃ during 10-14

days, (3) estimating gas, pH, emulsification, and slimeforming, (4) isolating MUB from all tubes which were

estimated to be positive in the testing. After investi-

gation, all types of microorganisms were isolated fromall colonies on the plates.2.3. First Screening of Isolates for MEOR Process

The first screening of microorganisms isolated by theinvestigation mentioned above was conducted accord-ing to their gas, acid, surfactant and/or polymer produc-ing abilities of the MEOR process. The following


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four media were prepared for the tests of each ability:MB for test of gas and/or acid, Defibrinated SheepBlood Agar (DSBA) for test of surfactant, and SucroseAgar (SA) for test of polymer. Composition of DSBAand SA were: 50ml of defibrinated sheep blood and 23

g of nutrient agar; and 20g of sucrose and 23g ofnutrient agar in 1 liter of distilled water, respectively.

The preparation of each incubation was conductedby inoculating isolates into 15ml of MB in a Hungatetube, which was then sealed (the head space was filledwith purified nitrogen gas) and plating strains onDSBA and SA plates, respectively. After incubationat 40℃ for 5-7 days under both aerobic and anaerobic

conditions (except gas and/or acid tests for which onlyanaerobic condition was used), the amount of gas pro-duced was measured by piercing the rubber cap of thetube with a syringe, pH of the medium was measuredwith a glass electrode pH meter, and β-hemolysis and

the existence of slime on the plates were visually

observed. Moreover, all strains, which were estimated

to be semipositive according to their β-hemolysis, were

inoculated into 15ml of MB in test tubes (18mm indiameter×180mm in length) and were incubated

under the same conditions used in the test with DSBA.After incubation, the surface tension of the mediumwas measured with a Du Nouy tensiometer. Finally,all strains, which produced gas, showed pH lower than5.5, reduced the surface tension below 40.0dyne/cm,and/or formed slime, were considered to be positivestrains, and those which showed outstanding ability ineach function were selected for second screening.2.4. Second Screening of the Strains Selected in

First ScreeningThe second screening of strains, which were selected

in first screening and which produced gas, acid, surfac-tant, and/or polymer, was carried out by inoculating thestrains in 15ml of one of the following media: Molas-ses with Inorganic Salts (MIS) for gas and/or acid pro-ducers in a Hungate tube, sealing it whose head spacewas filled with purified nitrogen gas, MB (for surfac-tant producers) and Sucrose Bouillon (SB) for polymer

producers in test tubes. Regarding the compositionsof 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: nutrientbroth, 8; and sucrose, 20. The formation water gener-ally includes many inorganic salts and minerals.Hence, it is necessary to take account of their influenceon strains because some components often affect themicrobial multiplication and their metabolism. Thenthe components of these inorganic salts were specifiedsimilar to those in representative domestic oil fields.

After 5-7 days of incubation at 40℃, the amount of

gas produced and pH of medium resulted were mea-sured by the same methods used in first screening. In

order to evaluate surfactant and polymer producing

abilities, interfacial tension between n-octane andmedia was measured with a spinning drop interfacialtensiometer, and the viscosity of media was measuredwith a B-type viscometer. Surfactant and polymer

producers, Bacillus licheniformis JF-2 and Bacilluslicheniformis SP-018, which were provided byOklahoma University, were incubated as positive con-trols, respectively. The strain JF-2 is a well evaluatedmicroorganism, which produces effective surfactantunder typical reservoir environment, and it has beeninvestigated by many authors10)-12). Strain SP-018 isrecognized as an excellent polymer producer. Finally,

the screened strains were identified and their character-istics investigated.

3. Results and Discussion

3.1. Indigenous Microorganisms3.1.1. Indigenous Microorganisms in Exploratory

Drilling Sites and Oil FieldsCell concentrations of indigenous microorganisms in

all samples are presented in Table 1.The incubation of microorganisms from Segawa

drilling cuttings showed 102-104cells/g mesophilic aer-obes and anaerobes existed in the shallow formation

(#1). However, the presence of thermophilic aerobeswas less than that of mesophilic, and no thermophilicanaerobe was detected. In addition, there were lessaerobes or no anaerobes in the deeper formation (#2).SRB and DB were detected in high cell concentrationsfrom #1 sample by 30℃ incubation, but their popula-

tion was low in #2 sample. Similar trend wasobserved with the two Niigata-Heiya samples, #3, 519m MD and #4, 1120m MD, which included more aer-

obes and anaerobes than those of #5 (1860m MD).The cell counts of most predominant aerobes andanaerobes attained 107 and 106cells/g, respectively, at30℃ with SEAYEP. SRB were detected from #4 cut-

tings at 30℃ and from #5 at 55℃, and their counts

attained 103-104cells/g. Mesophilic DB existed in

high population in levels down to 1120m MD in this

region. It was expected at first that many ther-

mophilic microorganisms could be detected from #5

sample because its temperature was 62℃. However,

the results fell short of expectation. Their highestconcentration was in the range of 102-103cells/g.

The formation water of the Amarume and Kurokawaoil fields (#6, #8 and #11) included indigenousmicroorganisms in low concentrations, and that of theToyokawa oil field (#9) contained more mesophilicmicroorganisms than others; however, its highest con-centration was 104-106 cells per 100ml. Chemicalanalysis of the formation waters of Amarume andKurokawa showed a small difference in most of thecomponents; however, some components in Toyokawadiffered clearly from those in other two fields. For


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instance, the representative different components (inmg/l) between Toyokawa and the other two fieldswere: 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, 3652.1, 670.1; CO32-, 0.0, 0.0, 271.5,35.1: in the order of #6, #8, #9, and #11. The formertwo cations and chloride ion of Tooyokawa were lessthan those of others, and the latter two anions wereclearly more than those in Amarume and Kurokawa.It was presumed that these differences affected the dis-tribution of indigenous microorganisms. In addition,reservoir temperature of Toyokawa (#9) was 33℃.

Because this temperature was lower than temperatures

of others (#6, 42-54℃; #8, 52℃; #11, 38℃), it might

be mild for microbial habitation.

As expected, both soil samples included a high con-

centration 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

the following results. At 30℃, two samples of soil

(#7 and #10) included active gas and acid producingmicroorganisms because all tubes ranged up to thou-sand-fold dilution were observed to be positive.However, most of them were mesophilic gas and acid

producers because the diluents showed negative resultsin the gas productivity test and low activity in the acid

productive test at 55℃ incubation. The polymer pro-

ducing ability tests at 30℃ showed that positive

microorganisms existed in only two soil samples, #7and #10.

Isolates were obtained from all colonies on the agar

plates that were used for the investigation of microflo-ra. In addition, MUB were isolated from the enrich-ment culture. Finally, 123 kinds of microorganismswere isolated and their features investigated (cell shape,Gram stain, spore, motility, oxidase, catalase, OF, andcolony). Representative isolates are listed in Table 2.3.1.2. Indigenous Microorganisms in Activated-

sludge and CompostTable 3 lists cell concentrations of indigenous

microorganisms in each sample.The incubation of microorganisms in all samples

showed mesophilic aerobes in high concentration (107-108cells/ml) and mesophilic anaerobes in low concen-

tration (less than 150cells/ml). Thermophilic aerobeswere detected in low concentration (#14, 90cells/ml)or at least not in high concentration (#12, 104cells/ml)in activated-sludge. Sample #13 included as muchthermophilic aerobes as mesophilic (107cells/g). Asto thermophilic anaerobes, they were not detected insamples of activated-sludge; only the compost included

them in low concentrations. According to the investi-

gation 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-

ing results of temperature. At 30℃ incubation, gas

and acid tests were positive in all range of dilution, but

positive results in the polymer forming test wereobserved in the 10ml undiluted solution from #12 and

in the original solutions (1 and 10ml) from #13, and

positive results in the surfactant forming test wereobserved only in the 10ml original solutions from acti-

vated-sludge (#12 and #14). At 55℃ incubation, the

compost in all dilution ranges were positive in gas andacid forming tests, but the activated-sludge solutionshowed low activity for gas forming microorganisms

(#14; only 10ml original solution) and acid formingmicroorganisms (1 and 10ml original solutions).

There was no result estimated to be positive in the sur-factant and polymer forming tests. These results sug-

gested that (1) mesophilic gas and/or acid producingMUB were predominant in all samples, (2) ther-mophilic ones were only predominant in the compost,and (3) surfactant and/or polymer producing microor-

ganisms were low at 30℃ and never deteted at high

temperatures.Isolates were obtained from all colonies on PGYA

and VLMA plates. In addition, MUBs were isolatedfrom enrichment culture. As a final result, 44, 41, and42 types of microorganisms were isolated from #12,#13 and #14 samples, respectively. The total amountof isolates was 127. They were numbered 4101-4144,4201-4242, and 4301-4341 in the order of #12, #14,and #13 samples. There were: five mesophilic aer-obes (4101-4105), three thermophilic aerobes (4106-4108), six mesophilic anaerobes (4109-4114), 14mesophilic aerobic MUB (4115-4128), ten mesophilicanaerobic MUB (4129-4138), five thermophilic aerobicMUB (4139-4143), and only one thermophilic anaero-bic MUB (4144) from #12 activated-sludge; eightmesophilic aerobes (4201-4208), three thermophilicaerobes (4209-4211), eleven mesophilic anaerobes

(4212-4222), ten mesophilic aerobic MUB (4223-4232), six mesophilic anaerobic MUB (4233-4238),two thermophilic aerobic MUB (4239-4240) and onethermophilic anaerobic MUB (4241) from #14 com-

post. In addition there were five mesophilic aerobes(4301-4305), six thermophilic aerobes (4306-4311), sixmesophilic anaerobes (4312-4317), four thermophilicanaerobes (4318-4321), seven mesophilic aerobic MUB

(4322-4328), six mesophilic anaerobic MUB (4329-4334), four thermophilic aerobic MUB (4335-4338),and four thermophilic anaerobic MUB (4339-4342)from #13 activated-sludge.


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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.


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3.2. First Screening3.2.1. Strains from Exploratory Drilling Sites and

Oil FieldsAs 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 bepositive among the 123 strains tested. Among themthe 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 6.0ml of gas amount). Values in parentheses show theamounts of gas produced in milliliter. The microor-

ganism with the highest gas producing ability wasstrain 108, which produced 8.8ml. The main gascomponent was identified as carbon dioxide. The fol-lowing 14 strains 18-2-a, 24-1-c, 103, 107, 112, 323,324, 330, 524, 537, 610, 620, 626, and 636 gave pHsless than 5.3. Estimation of surfactant producing abil-ity indicated that 35 strains showed β-hemolysis; how-

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 lessthan 40.0dyne/cm. Values in parentheses indicate thesurface tension in dyne/cm. The lowest surface ten-sion was 31.2dyne/cm shown by strain 522. Therewere 16 following strains 18-2-a, 18-2-b, 21-2-a, 24-1-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 dif-ferent abilities (gas, acid, surfactant and polymer pro-duction); and strain 418 (acid, surfactant, and polymer

production) and strains 524, 537, and 637 (gas, acid,and surfactant production) had three different abilitiesshown 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 byfirst screening based on their single or pluralistic abili-ty.3.2.2. Strains from Activated-sludge and Com-

postThe screening results of representative isolates are

listed in Table 4. By the first screening for MEOR

process, 20 strains with gas producing ability were con-sidered to be positive among all 127 strains tested.Two strains 4134 (6.0ml at 40℃) and 4324 (6.4ml at

40℃) especially showed high gas producing ability

(more than 6.0ml of gas). Thus, these two wereselected as gas producers in first screening.

Ten isolates (strains 4119, 4122, 4139, 4141, 4142,4224, 4230, 4239, 4311, and 4327) gave values of pHlower than 5.0, and especially strain 4141 gave the low-

est(45 at 55℃). Hence, strain 4141 was selected as

the acid producer in first screening.

As regards the estimation of surfactant producing

ability, 48 strains showed positive results in β-hemoly-

sis in which 12 strains were positively reactive onlyunder aerobic condition, 13 strains reactive only underanaerobic condition, and the remaining 23 strains pro-duced surfactants under both conditions. However,there were only 12 strains, namely 4104, 4105, 4116,4118, 4123, 4126, 4141, 4143, 4204, 4206, 4225, and4309 that reduced the surface tension to less than 40.0dyne/cm. These 12 strains were selected as surfactant

producers in first screening. The lowest surface ten-sion (32.5dyne/cm) was registered by strain 4116 at40℃.

The following 10 strains 4104, 4107, 4115, 4116,4126, 4142, 4210, 4240, 4324, and 4335 were consid-ered to be slime forming, and strains 4107, 4142, 4210,and 4240 showed the equivalent slime forming abilityto that of a positive control, Bacillus licheniformis SP-018. 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 selectedfrom isolates in the first screening based on their pro-ductive ability.

It should be mentioned that many microorganismswere found in activated sludge and compost as expect-ed, and those microorganisms found in activated sludgewere potentially effective for MEOR process.3.3. Second Screening3.3.1. Strains from Exploratory Drilling Sites and

Oil FieldsThe results of second screening are presented in

Table 5. High performance of gas and/or acid pro-duction 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 positivecontrol, Bacillus licheniformis JF-2. There was nostrain which increased the viscosity of the mediummore than 5.6cp of the positive control, Bacilluslicheniformis SP-018.

As a final result, two strains were selected as candi-dates for MEOR field testing. One is strain 18-2-a,which is an excellent surfactant producer, and alsoexcellent producer of gases, acids and polymers. Theother strain 322 is also an excellent gas and acid pro-ducer. These two strains were renamed TRC-18-2-aand TRC-322, respectively. They were identified asBacillus licheniformis and Enterobacter cloacae, re-spectively.

According to the investigation of their characteris-


67

石油学会誌 Sekiyu Gakkaishi, Vol, 43, No. 1, 2000

68

tics, the maximum temperature of growth of TRC-18-2-

aand that of TRC-322 were from 55.1 to 57.4℃ and

from 40.6 to 43.2℃, respectively. However, their

optimum ranges of temperature were 25.4-47.8 and

22.1-39.4℃, respectively, observed in the experiments

conducted with nutrient broth in a temperature gradientincubator. Based on these results, the former wasfound applicable to the approximately 1000m VDreservoir, and the latter was limited to shallow oilfields. In addition, the optimum pH ranges of growthwere 6.0-10.5 for TRC-18-2-a and 6.0-9.0 for TRC-322. Therefore, these microorganisms could be usedin most of sandstone reservoirs.3.3.2. Strains from Activated-sludge and Com-

post The results of second screening are presented in

Table 6. The high performance of gas productionwas 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. Strains4141 and 4142 reduced pH to its lowest value (4.7)

which was equal to that of TRC-322. Lower IFT wasobtained 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

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 in-creased the viscosity of medium more than 5.6 cp, thevalue attained by the positive control, Bacillus licheni-

formis SP-018.Two strains, 4118 and 4126, were finally selected as

candidates for MEOR field testing. These two strainswere renamed TRC-4118 and TRC-4126, respectively;they were also identified as Bacillus subtilis.

4. Summary of Results

(1) Indigenous microorganisms from exploratory drill-ing sites and oil fields included both aerobic and anaer-obic ones, in spite of anaerobic sampling environments.On the other hand, most of the microorganisms fromactivated-sludge and compost were aerobic becausethese samples existed under aerobic conditions. Inaddition, MUB were isolated from formation water,soil (oil fields), activated-sludge and compost (sewagetreatment facility and/or food treatment facility).

(2) By the first screening of isolates from exploratorydrilling sites and oil fields, 19, 14, 15, and 16 strainswere estimated to be of high gas, acid, surfactant, and

polymer producers, respectively. In addition, strainswith pluralistic ability for gas, acid, surfactant, and

polymer production were obtained. Finally, 18 strainswere selected, based on their single ability or on plural-

Table 5 Results of Second Screening of Exploratory DrillingFields or Oil Fields Originated Strains

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.

Table 6 Results of Second Screening of First Screened StrainsIsolated from Activated-sludge and Compost

a) Interfacial tension between n-octane and medium.

b) Not tested.

c) Provided by Oklahoma University.


69

istic ability.

(3) By the first screening of isolates from activated-sludge and compost, 2, 11, 12, and 4 strains possessing

gas, acid, surfactant, and polymer producing abilitieswere considered to be effective for MEOR process.

(4) TRC-18-2-a, TRC-322, TRC-4118 and TRC-4126were selected as candidate microorganisms for MEORfield testing. TRC-18-2-a possessing gas and acid

productive abilities reduced IFT to 1.2dyne/cm, whichwas lower than IFT (1.7dyne/cm) of the positive con-

trol, B. licheniformis JF-2. TRC-322 produced anoptimum amount of gas (7.3ml) and the lowest pH

(4.7). TRC-4118 and TRC-4126 reduced IFT to 0.04and 0.20dyne/cm, respectively, both of which werelower than that of IFT of JF-2.

AcknowledgmentsThe authors wish to thank Japan National Oil

Corporation (JNOC) for the permission to present this

paper. In addition, we gratefully acknowledge thecooperation of Japan Petroleum Exploration Co., Ltd.,Tohoku Oil Co., Ltd. and Chuokogyo Co., Ltd. to col-lect the samples. We also thank the staff of JapanFood Research Laboratories for their experimental

data. In addition, we thank K. Suzuki, H. Matsu-bayashi, 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 1987-1993. 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. 2 413 278 (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. 2 660 550 (1953).

6) Updegraff, D. M., Wren, G. B., Appl. Microbiol., 2, 309 (1954).

7) Updegraff, D. M., U. S. Pat. 2 807 570 (1957).

8) Bech, J. V., Producers Monthly, 11, 13 (1947).

9) Lazar, I. I., "Microbial Enhancement of Oil Recovery-RecentAdvances" Proceedings of the 1990 International Conference on

Microbial Enhanced Oil Recovery, ed. by Donaldson, E. C.,Elsevier, Amsterdam (1991), p. 485-530.

10) Lin, S. C., Goursaud, J. C., Kramer, P. J., Georgiou, G., Sharma,

M. N., "Microbial Enhancement of Oil Recovery-Recent Ad-

vances" Proceedings of the 1990 International Conference on

Microbial Enhanced Oil Recovery, ed. by Donaldson, E. C.,

Elsevier, Amsterdam (1991), p. 219-226.

11) 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).

要旨

微生物攻法 (MEOR) のための有用微生物のスクリーニング

米林英治†1), 吉田信一郎 †2), 大野健二†3), 榎本兵治 †4)

†1) 石油公団, 100-8511東京都千代田区内幸町2-2-2

†2) (財)日本食品分析センター, 151-0062東京都渋谷区元代々木町52-1

†3) 石油公団石油開発技術センター, 261-0025千葉市美浜区浜田1-2-2

†4) 東北大学大学院工学研究科地球工学専攻, 980-8579仙台市青葉区荒巻字青葉01

微生物を用いた石油の増進回収 (MEOR) に有用な微生物を

採集することを目的とした研究を行った。サンプリング試料と

しては, 基礎試錐 (しすい) のカッティング, 油層水, 油田土

壌のほか, 下水処理場および食品工場の活性汚泥およびコンポ

ストの合計14試料を取り上げた。まず, これらの試料中の全

てのコロニー形成微生物を採取することを念頭に, 11種類の

異なる培地を用いて培養し, 全ての異なるコロニーを対象に微

生物を分離した。さらに, 微生物の増殖性と代謝産物のMEOR

への機能による2段階のスクリーニングを行った。その結果,

MEORに有用な微生物として Bacillus lichentformis TRC-18-2-a,

Enterobacter cloacae TRC-322, Bacillus subtilis TRC-4118, Ba-

cillus subtilis TRC-4126の4種類の微生物を得た。B. licheni-

formis TRC-18-2-aは, 界面活性物質のほか, ガス, 酸, ポリマ

ーも生産する多機能微生物である。E. cloacae TRC-322は, 多

量のガスと酸を生産する。B. subtilis TRC-4118と B. subtilis

TRC-4126は界面活性物質生産菌であり, 界面張力 (対n-オク

タン) をそれぞれ0.04, 0.12dyne/cmまで低下させた。

Keywords

Improved oil recovery, MEOR, Screening, Surfactant, Bacillus, Enterobacter


Documents

Screening of Microorganisms for Microbial Enhanced Oil