Enhanced Oil Recovery Through Microbial Treatment

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Enhanced Oil Recovery Through Microbial TreatmentM. A. Rauf a , M. Ikram b & N. Tabassum ba Chemistry Department , UAE University , Al-Ain, UAEb Chemistry Department , Quaid-I-Azam University , Islamabad, PakistanPublished online: 24 Jun 2008.

To cite this article: M. A. Rauf , M. Ikram & N. Tabassum (2003) Enhanced Oil Recovery Through Microbial Treatment, Journalof Trace and Microprobe Techniques, 21:3, 533-541, DOI: 10.1081/TMA-120023069

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JOURNAL OF TRACE AND MICROPROBE TECHNIQUES

Vol. 21, No. 3, pp. 533–541, 2003

SOIL AND ENVIRONMENTAL SCIENCES

Enhanced Oil Recovery Through Microbial Treatment

M. A. Rauf,1,* M. Ikram,2 and N. Tabassum2

1Chemistry Department, UAE University, Al-Ain, UAE2Chemistry Department, Quaid-I-Azam University,

Islamabad, Pakistan

ABSTRACT

Two strains of Bacillus species, namely BS-I and BS-II were used to degrade the

crude oil samples. The ability of these strains and their mixed culture to produce

gases, organic acids and other solvents were monitored. Under anaerobic condi-

tions, both the strains produced considerable gases. BS-I produced 287 cc of gas

per week accompanied by lowering the pH of the system and an oil consumption

up to 56%. On the other hand, BS-II produced 353 cc of gas per week with

lowering of pH and oil consumption up to 16%. In mixed strains, 240 cc of gas

was produced with lowering of pH and an oil consumption of 16%. Four gases,

namely CO2, N2, O2 and C3H8 were identified by gas chromatography. Carbon

dioxide was produced in major amounts in all the cases. Nine organic acids were

also identified in molasses based medium. These were quantified by GLC

technique using standards as reference sources. In sand packed columns, the oil

recovery efficiency was calculated to be 83% in the case of BS-II.

Key Words: Microbial treatment; Enhanced oil recovery.

*Correspondence: M. A. Rauf, Chemistry Department, UAE University, P.O. Box 17551,

Al-Ain, UAE; E-mail: [email protected].

533

DOI: 10.1081/TMA-120023069 0733-4680 (Print); 1532-2270 (Online)

Copyright & 2003 by Marcel Dekker, Inc. www.dekker.com

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INTRODUCTION

Oil is used as a major source of energy in many cases. In its usable form, it needsto be extracted and refined from the crude form. All oil recovery methods arecomplicated and expensive at the end. This translates in the high price of theusable commodity for the consumer. It is generally recognized that only about30% of the oil can be recovered using the current technology, while 70% remainsin the reservoir.[1] A scope for developing such refining methods exists, and variousprocesses are suggested in this regard. The recovery of this remaining oil is possiblethrough microbial treatment of the crude oil in the oil well.[2] This unique processrequires in situ injection of some suitable microorganisms, which can produce thedesired enhanced oil recovery compounds, such as gases, acids, solvents, polymersetc. These can then be pumped out through suitable techniques. This method savesthe cost of oil recovery in secondary and tertiary phases employing methods such asmiscible flooding, surfactant flooding, polymer flooding and steam flooding etc.[3]

All these mentioned process are costly and are selective in use. The microbialenhanced oil recovery (MEOR) is one such method, which has been suggested,tested and used in many developed countries.[4,5] The results were found to bepromising and further investigations in this direction are under progress. In thispaper we report the use of two microbial strains, namely BS-I and BS-II to theireffect on the crude oil samples. The products produced by such treatments wereanalyzed by different instrumental techniques, and results are compared in termsof the efficiency of the microbes.

EXPERIMENTAL

All the chemicals used in this work were of analytical grade and were procuredfrom either E. Merck or Sigma chemicals and used as such.

The crude oil samples were collected from the Balkasar oil field. This oil wasvery viscous, thick and black in color, and suitable for microbial treatment. Thebacterial strains, namely BS-I and BS-II were obtained from the MicrobiologyLaboratory of the Biology Department of Quaid-I-Azam University (QAU),Islamabad.

Preparation for Microbial Treatment

Inoculum’s Preparation

Inoculums of each bacterial strain and the mixed culture was prepared as fol-lows: Peptone (5 g) and beef extract (3 g) were carefully weighed and dissolved in 1Lof distilled water. The pH of the nutrient was adjusted to 6.8. A 100mL portion ofthis nutrient was poured in 250mL flasks, which were plugged and autoclaved at121�C and 15 lb/in2 for 20min. Thereafter, 1% of the crude oil and a loopful ofbacterial strain were then added to each flask under sterilized conditions. The flaskswere then placed in an incubator shaker at 100 rpm at 37�C for 48 h.

534 Rauf, Ikram, and Tabassum

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Enrichment Culture Technique

The Bennet medium was prepared as reported.[6] The pH was adjusted to 7.1 inthe media and the contents of the flask were autoclaved. Then 10mL of the preparedinoculum’s preparation and 2mL of the crude oil was added to the flask andproperly labeled. The flasks were then plugged and incubated at 37�C and100 rpm for seven days. After the incubation period, 10mL of this medium and2mL of crude oil was added and the flasks were again incubated at 37�C at100 rpm for another seven days.

Gas Production Under Anaerobic Conditions

This was done by a method proposed by Lazer and Constantinescu.[7] Accordingto this method, 100mL of deionized water was poured in each of the 250mL flasksand were autoclaved. To these flasks, then 2% of the crude oil and 4% molasses wasadded. The pH of the solution was adjusted to 7 with 10% NaOH. 10% inoculum’spreparation was then added at this stage under sterilized conditions. The flasks weresealed at this stage with injectible rubber septum, and 50 cc syringe was injected intothe flask via septum for the collection of gases produced during the fermentationprocess.

Analysis of Gas by GC

Gases produced in the above steps were analyzed by gas chromatographic tech-nique. In this case, Hewlett Packard Instrument, (model no. 50880A) with a FlameIonization Detector, was used. Helium was used as a carrier gas, and the stationaryphase was a ‘‘Porapak Q’’ packed column (6� 1/8 OD, 80/100mesh). The flowrate was 30mL/min at 70�C and the gas pressure was maintained at 64 psi.Retention times of the standard gases were also monitored for comparison andidentification purposes. All the peaks were subjected to auto integration processfor quantification.

pH Determination and Oil Extraction

At the end of the fermentation process, the pH of the solution was noted downusing a pH meter. The amount of oil utilized by the microorganism and their mixedculture was calculated using the standard method.[8] The oil was extracted from thebroth by using a mixture of ether and benzene (1:3 ratio). The solvent mixture wasadded to all the flasks and the contents were mixed thoroughly. These were thentransferred to a separatory funnel and allowed to stand for 3–4 h for completeseparation of the phases. The water-soluble fractions were discarded, whereas theorganic solvent fraction was collected out and weighed in petriplates. The petriplateswere placed in an oven at 50�C until the solvent completely evaporated, leavingbehind the oil. These contents were weighed again.

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Qualitative Test for Organic Acid Production

Standard literature method[9] was employed for this purpose. The medium usedfor this test was used as slants. The slants were streaked by two bacterial strains, andany organic production was checked by a change in color after 24 h of incubation at27�C.

Organic Acid Production and Extraction

In this standard procedure,[7] 100mL of 2% yeast extract and 1% peptone wereweighed and dissolved in 1 L of distilled water. It was then autoclaved and 250mL ofthis was added to separate conical flasks and the 4% molasses and 2% oil wereadded and the pH was adjusted to 7 with the help of 10% NaOH under sterileconditions. At this stage, 10% of the bacterial inoculums was added to theseflasks and were plugged. A control flask was also prepared under similar conditionsexcept the presence of bacterial inoculums. Fermentation was allowed at 37�C and100 rpm for seven days. After this process, 20mL of the solution was taken out andcentrifuged at 10,000 rpm for 15min. The supernatant liquid was used for organicacid analysis by GLC.

Acid Analysis by GLC

For analysis by GLC, a 25 mL sample was injected into a Perkin ElmerInstrument, model no. 3920. The GLC column was packed with 15% SP 1220and 1% H3PO4 and was operated at 130�C. Nitrogen gas at a flow rate of25mL/min was used as a carrier gas. FID detector was employed for signal detec-tion. Retention times of the standard acids were also monitored for comparison andidentification purposes. All the peaks were subjected to auto integration process forquantification.

Recovery of oil from Sand Packed Columns by the Action of Bacteria

Heat resistant glass columns of 20–25 cm length and 4–5 cm width were used tostudy the role of microorganism (bacterial strains) in residual oil recovery fromporous media. The columns were initially filled with quartz sand, (0.1–0.5mm.grain size), and was plugged with glass wool at the bottom end. The upper inletswere sealed with injectible rubber septum, and the columns were autoclaved. Afterautoclaving the columns were placed in a sterile environment and oil sample wasintroduced into the column using a 50 cc syringe, till the column was completelysaturated with oil. Deionized autoclaved water was added from the top to letexcess oil drain down. The amount of remaining oil in the column was recorded.At this stage 4% molasses and 10% bacterial inoculums was added to the columnthrough the septum. The bacterial inoculum was prepared in a nutrient broth at pH 7with 5% salinity. The column after injection was incubated at 45�C for one week.


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After one week, the column was brought in sterile conditions and the remainingmolasses was added to it and incubated again at 45�C for another week. Total oilrecovery (TOR) and Residual oil recovery (ER) were estimated as per literaturemethod.[9]

ER ¼ðSOWF� SOCEÞ � 100%

SOWF

TOR ¼ðSOI� SOCEÞ � 100%

SOI

where, SOI¼ residual oil saturation prior to water flooding, SOWF¼ residual oilsaturation after water flooding, SOCE¼ residual oil saturation after microbial treat-ment, ER¼ residual oil recovery and TOR¼ total oil recovery.

RESULTS AND DISCUSSION

Initial screening of all the bacterial strains available at Microbiological ResearchLab of the Quaid-I-Azam University (QAU) was done. Only two bacterial strainsproduced more than 50 cc of gas on 24 h of incubation. These samples, identified asBS-I and BS-II were selected for the present work. The bacterial strains were revivedby sub-culturing technique and were then processed by Gram straining. These wereidentified as Gram-positive Bacillus species.[10] These bacteria have a well-knownproperty of their tolerance to salt, sugar, high temperature, alkaline, and evenextreme pH conditions.[11] Their spore forming capacity makes it possible forthem to move more easily through the reservoir rocks down to greater depths andto make their way into many habitats, which serve as selective environment fortheir growth.[12] It has been reported that culture Bacillus species are capable ofproducing organic acids, surfactants, solvents like ethanol, acetone, isopropanoland methanol.[12]

Gas production by two identified bacteria by standard procedures as narrated inthe experimental section, was carried out under anaerobic conditions. After only24 h, gas produced by each bacterial strain was estimated quantitatively on dailybasis. On the first day, BS-I produced 75 cc of gas, while BS-II produced 94 cc of gas,whereas 81 cc of gas was produced in a mixed culture. The gas production decreasedin amount by every day passing and became zero for all the cases on the sixth day.The data is shown in Table 1 for comparison purposes in all the three cases.

Thus one can see that during the six-day period, BS-II produced the maximumamount of gas (353 cc) as compared to other strains. This was followed by BS-I andthen mixed strain.

At any given time of gas production, the gas mixture was analyzed by using gaschromatography technique. Mainly four gases were identified. These being CO2, N2,C3H8 and O2. Besides this, BS-1 also produced H2. The major quantity of gasidentified on GC was CO2, while the others were produced in smaller quantities.During the fermentation of carbohydrates, a number of gases are produced whichhelp in repressurization of pressure depleted reservoir. Under anaerobic conditions,production of gases was observed to test the efficiency of individual strains for gas


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production. Maximum amount of gas was produced by BS-II during fermentation ofmolasses and produced CO2, O2 and N2. In the case of mixed culture, although thelevel of gas production was lesser than BS-I and BS-II, the gaseous mixture con-tained all the four gases, namely N2, CO2, O2 and C3H8. BS-II seems to be a betterstrain as it produces more amount of gas as compared to other. This fact is impor-tant, as the gas production by microbes is believed to be an important mechanism foroil recovery from the wells.[13] Table 2 summarizes the data on the production ofvarious gases by various strains.

After seven days of fermentation, the pH of the fermentation broth was checked.There was a considerable decrease in pH as compared to the initial pH value of 7.BS-II showed maximum decrease in pH value from 7 to 4.7, while in the mixedculture, the pH value decreased to 5.1, whereas in the case of BS-I the decreasewas from pH 7 to 5. At the end of the fermentation process, the oil consumedwas also calculated for each strain. BS-II showed maximum oil consumption ofnearly 56%. BS-I consumed 22% of oil, whereas the mixed strain consumednearly 16% of oil. Similar studies using different strains are cited in the literature.[14]

Production of gases and a decrease in pH, both help in oil release process from theoil shale. This also helps to lower the viscosity of crude oil and thus facilitates itsrecovery.[15]

Organic acid production was observed on molasses based medium, which wassupplemented with peptone and yeast extract to enhance acid production. Sampleswere taken from fermentation broth after 48 h of incubation because by that timeproduction of organic acids initiated. The production of organic acid in the brothdropped its pH value. A maximum number of nine organic acids were identified in

Table 1. Gas production (in cc) by various bacterial strains.

Day BS-I BS-II Mixed strain

1 75 94 81

2 68 83 70

3 56 68 55

4 50 57 25

5 38 36 10

6 0 0 0

Total 287 353 241

Table 2. Gas chromatographic analyses of gases produced by various

strains (results reported in mole%/mole).

Component BS-I BS-II Mixed strain

Carbon dioxide 71 81 68

Oxygen 25 8.5 21

Nitrogen 4 3 8.5

Propane — 7.5 2.5


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the case of BS-II. All these acids were analyzed by GLC using the standards as areference source. The results are shown in Table 3.

It has been suggested that organic acids produced by microbes attack anddissolve the limestone in the oil well and thus promote bacterial movement inthe porous rocks, which helps in the release of trapped oil.[16] The importance ofthis mechanism is supported by lab and field trials, wherein the pH of water loweredby a value of 1 to 2 units during MEOR, thus indicating that the acid can bemicrobially produced in quantities sufficient to affect an entire reservoir.[17]

The ability of organic acids and solvents produced as a result of bacterial actionon oil was also subjected to sand packed glass columns. The oil trapped in sandparticles after the water flooding was actually released by the action of bacteria. Theresidual oil recovery efficiency (ER) and the total oil recovery (TOR) were calculatedfor all the strains, and the results are shown in Table 4.

From this table one can see that maximum ER and TOR were achieved in thecase of BS-II. Some similar studies on sulfate reducing bacteria are reported inthe literature.[18]

CONCLUSION

Microbiological strains BS-I, BS-II and their mixed culture were introduced incrude oil samples. Various analyses with respect to the crude oil degradation weredone using the GC and GLC technique. It was observed that BS-II is a better strain

Table 3. GLC analysis of organic acids (in %) produced by various strains.

Component BS-I BS-II Mixed strain

Formic acid 8.2 31 10.5

Acetic acid 56.5 28.5 53.5

Propionic acid 3.5 3.5 3

Isobutyric acid 0.4 0.5 0.6

Butyric acid 10 28 11

Isovaleric acid 0.75 0.3 0.8

Heptanoic acid 1 2.2 —

Valeric acid — 0.2 —

Isocapranoic acid — 0.2 —

Table 4. Residual oil recovery (ER) and total oil recovery (TOR)

from sand packed columns using various strains.

Strains ER (%) TOR (%)

BS-I 13 73

BS-II 39 83

Mixed stain 4.5 66


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for MEOR process, as it produced the maximum amount of gases and solvents.These can substantially help in oil recovery from the oil wells.

ABBREVIATIONS

ER Tesidual oil recoveryFID Flame ionization detectorGC Gas chromatographyGLC Gas liquid chromatographyMEOR Microbiologically enhanced oil recoveryTOR Total oil recoveryQAU Quaid-I-Azam University

ACKNOWLEDGMENT

The authors gratefully thank Dr. A. Hammed from the Microbiology Labs,Quaid-I-Azam University, Islamabad, for providing us the bacterial strains forthis work.

REFERENCES

1. Lake, L.W. Enhanced Oil Recovery; Prentice Hall: New York, USA, 1996.2. Donaldson, E.C.; Grilling, N.G.; Yen, F.T. Microbial Enhanced Oil Recovery.

Development in Petroleum Science; Elsevier Publ. Co.: N.Y. USA, 1989.3. Donaldson, E.C.; Chilingarian, G.V.; Yen, T.F. Enhanced Oil Recovery

Processess and Operation; Elsevier Science: New York, USA, 1989.4. Kemal, B.; Mehmentoglu, T.; Donmez, S. Application of microbial enhanced

oil recovery techniques to Turkish heavy oil. App. Microbiol. Biotechnol. 1992,36 (6), 833–835.

5. Johnson, A.C. Microbial Oil Release Technique for Enhanced Oil Recovery,Conference on Microbiological Process Inject in Enhanced Oil Recovery,San Diego, CA, USA, 1979.

6. Bennett, B.R. Microbial degradation of diesel fuel. Trans. Kansas Acad. Sci.1982, 85 (2), 72–77.

7. Lazar, I.; Constantinescu, P. Field trial results of microbial enhanced oilrecovery. Int. Bioresources Journal 1985, 122–144.

8. Buchaman, R.E.; Gibbons, N.E. Bergey’s Manual of DeterminativeBacteriology, 8th Ed.; Williams and Wilkins Co.: Baltimore, USA, 1974.

9. Lazar, I.; Dorbota, M.; Stefanescu, L.; Sandolescu, P. Constantinescu, C.;Morosanu, N.; Boeta, N.; Illiescu, O. Preliminary results of some recentMEOR trials in Romania. Dev. Petrol. Sci. 1991, 31, 365–385.

10. Choudri, S.F.; Hameed, A. Occurrence of oil mobilizing bacteria in differentecological sources in Pakistan. Rev. Roum. Biol. 1992, 37 (1), 51–55.


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11. Zajic, J.E.; Howard, M. Properties of bacillus from conroe oilfield and otherreservoir sources. Int. Bioresources J. 1985, 295–309.

12. Bryant, R.S.; Burcfield, T.E.; Dennis, D.M.; Hitman, D.O. Microbial EnhancedWater Flooding: Mink Unit Project, Enhanced Oil Recovery Symp. Tulsa, OK,USA, 1988.

13. Zehender, A.J.B. Biology of Anaerobic Microorganisms; John Wiley & Sons:New York, USA, 1988.

14. Ijah, V.J.J.; Ukpe, L.I. Biodegradation of crude oil by Bacillus strain 28A and61B isolated from oil spilled soil. Waste Management 1992, 12 (1), 55–60.

15. Rowe, H.G.; York, S.D.; Ader, J.C. Slaughter estate unit tertiary pilot perfor-mance. J. Pet. Technol. 1982, 34, 613–620.

16. Rhoel, P.O.; Choquelte, P.W. (Eds.). Carbonate Petroleum Reservoirs;Springer-Verlag: New York, USA, 1985.

17. Tanner, R.S.; Udeegbunan, E.; McInerneg, M.H.; Knapp, R.M. Microbiallyenhanced oil recovery from carbonate reservoirs. Geomicrobiol. 1991, 9,169–195.

18. Standards on Assessment and Remediation of Petroleum Sites. ASTM.Committee E-50 on Environmental Assessment. ASTM. Washington D.C,USA, 1999.

Submitted May 16, 2002Accepted November 15, 2002


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Enhanced Oil Recovery Through Microbial Treatment