Bacteria 1

ngineering 58 (2007) 161–172www.elsevier.com/locate/petrol

Journal of Petroleum Science and E

The in situ microbial enhanced oil recovery in fracturedporous media

Alireza Soudmand-asli a, S. Shahab Ayatollahi a,⁎, Hassan Mohabatkar c,Maryam Zareie a, S. Farzad Shariatpanahi b

a School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iranb Department of Chemical Engineering, Faculty of Engineering, University of Tehran, Tehran, Iran

c Department of Biology, School of Sciences, Shiraz University, Shiraz, Iran

Received 29 March 2006; received in revised form 9 November 2006; accepted 24 December 2006

Abstract

These experiments aim to investigate the microbial enhanced oil recovery (MEOR) technique in fractured porous media usingetched-glass micromodels. Three identically patterned micromodels with different fracture angle orientation of inclined, verticaland horizontal with respect to the flow direction were utilized. A non-fractured model was also used to compare the efficiency ofMEOR in fractured and non-fractured porous media. Two types of bacteria were employed: Bacillus subtilis (a biosurfactant-producing bacterium) and Leuconostoc mesenteroides (an exopolymer-producing bacterium). The results show that higher oilrecovery efficiency can be achieved by using biosurfactant-producing bacterium in fractured porous media. Further investigationon the effect of the mentioned bacteria on oil viscosity, porous media permeability and wettability suggests that the plugging ofmatrix-fracture interfaces by an exopolymer is the main reason for the low performance of the exopolymer-producing bacterium.Oil viscosity reduction as well as the reduction of IFT was also found to be the reason for better microbial recovery efficiencies ofbiosurfactant-producing bacterium in the fractured models.© 2007 Elsevier B.V. All rights reserved.

Keywords: Fractured porous media; Microbial Enhanced Oil Recovery (MEOR); Glass micromodel; Biopolymer; Biosurfactant

1. Introduction

Naturally fractured oil reservoirs represent over 20%of the world's oil reserves (Saidi, 1983). However,relatively little success has been achieved in increasingoil production from these complex reservoirs (Delshadet al., 2002). Although in recent years a number of

⁎ Corresponding author. P.O. Box: 71345-1719, Shiraz, Iran. Tel./fax:+98 711 6287294.

E-mail address: [email protected] (S.S. Ayatollahi).

0920-4105/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.petrol.2006.12.004

results from fields have shown microbial enhanced oilrecovery (MEOR) as an applicable and efficient method(Portwood, 1995; Dietrich et al., 1996; Karim et al.,2001; Nagase et al., 2002), the use of MEOR processesin enhanced oil recovery from the fractured reservoirshas been particularly neglected.

No core flooding or micromodel experimentation hasbeen carried out to investigate the MEOR in fracturedporous media. Only Zekri and El-Mehaideb (2002)presented an experimental work on bacterial flooding(secondary recovery) through different fractured

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http://dx.doi.org/10.1016/j.petrol.2006.12.004

Table 2The properties of the used fluids

Property Dyed oil Water

Density (g/cm3) at 28 °C 0.86 1.0Viscosity (mPa⁎s) at 35 °C 29.0 1Flash point (°C) 210 –Pour point (°C) 11

162 A. Soudmand-asli et al. / Journal of Petroleum Science and Engineering 58 (2007) 161–172

systems. The results indicated that fracture orientationstrongly affects the performance of bacterial floodinginto the fractured system.

In this study, the tertiary microbial oil recovery wasinvestigated using etched-glass micromodels havingdifferent fracture angle orientation. B. subtilis andL. mesenteroides were chosen as the treating bacteria.B. subtilis is well known to produce surfactin as abiosurfactant, the chemical structure of which is welldocumented (Donaldson et al., 1989; Banat, 1995).L. mesenteroides is able to produce substantial amountsof dextran, an insoluble exopolymer, in the presence ofsucrose (Xiu-Yuan andWang, 1991; Stewart and Fogler,2001). In addition, to analyze the oil recovery efficiencyof the MEOR in fractured models, the effect of bacteriaon wettability, oil viscosity and permeability of themedia was also studied. The final goal of our study wasto identify the best bacterium for microbial enhanced oilrecovery in fractured reservoirs with regard to thebioproducts of the bacterium.

2. Materials and methods

2.1. Microorganisms

Two types of bacteria were employed: Leuconostocmesenteroides (PTCC 1059) and Bacillus subtilis(PTCC 1365). They were both obtained from thePersian Type Culture Collection (PTCC), Tehran, Iran.Kamal et al. (2001) reported the ability of PTCC 1059 toproduce considerable amounts of dextran under anaer-obic conditions and in the presence of sucrose. Abtahiet al. (2003) showed that under anaerobic conditions andat low salinity PTCC 1365 is able to reduce remarkablythe oil–water interfacial tension.

2.2. Growth conditions

B. subtilis and L. mesenteroides were cultured onliquid growth medium A and B, respectively. The

Table 1The compositions of growth media

Compositions Growth medium A Growth medium B(g/lit distilled water) (g/lit distilled water)

Sucrose 1.0 0NH4Cl 1.0 2.0Glucose 0 5.0Peptone from meat 10 10Meat infusion 5.0 5.0Na2HPO4 2.0 2.0Sodium chloride 0.3 0.3

composition of each growth medium is presented inTable 1. The bacterial culture was centrifuged at 2500 rev/min for 30 min and collected at the stationary state; it wasthen suspended in autoclaved water. The bacterialsuspension was placed on a magnetic stirrer and allowedto mix at room temperature for 5 min. The solution wascentrifuged and washed once again with water aspreviously described. The bacteria were then suspendedand placed on the magnetic stirrer to make a bacterialsolution. The cell density of the bacterial solution wasadjusted to 109 cells/ml (±107) by spectrophotometricanalysis at 600 nm.

2.3. Fluids

The physical properties of oil and water are given inTable 2. Densities were determined by using a pycnom-eter and viscosity was measured by a Canon-Fenskviscometer, according to the procedures proposed byASTM D 445.

2.3.1. OilSynthetic oil was used as the oleic phase. It was a pure

hydrocarbon and free from other crude oil componentssuch as asphaltene, salts, metals, sulfur, resin acids, andash contents; therefore the effect of these components onmicrobial activities was eliminated in this study. In orderto find the saturation in the transparent models, the oil wasdyed with Sudan Red (C24H21N5); the dyed oil wasfiltered using fine filter papers to remove any solidifieddye particles.

2.3.2. Flooding waterAutoclaved distilled water was used as the flooding

agent. Water was not dyed due to the concerns about the

Table 3The compositions of the nutrient solution

Compositions Concentration (w/v)

Sucrose 4%Di ammonium hydrogen 0.02%Phosphate ((NH4)2HPO4)Ammonium chloride (NH4Cl) 0.04%

Fig. 1. Fracture orientations of the micromodels.

Fig. 2. Porous pattern of the micromodels.

163A. Soudmand-asli et al. / Journal of Petroleum Science and Engineering 58 (2007) 161–172

interference of chemical components on the bacterialgrowth.

2.3.3. Nutrient solutionThe nutrient solution was prepared based on the

investigation of Brown (1982) and Jenneman et al.(1984), who found that the nitrate and phosphatecontaining nutrients are very effective for in situMEOR processes. Table 3 shows the compositions ofthe used nutrient.

2.4. The glass micromodels

Glass micromodels are two dimensional flow-channel networks etched in glass to simulate fluidflow in porous media. These models are usuallyconsidered strongly water-wet because their surfacechemistry is similar to that of clean sandstone (Grattoniet al., 2002). Here, four identically patterned micro-models were constructed. Three of them had a singlefracture with different fracture angle orientation of 45°(inclined-fractured model), 90° (vertical-fracturedmodel) and 180° (horizontal-fractured model) withrespect to the axis of the flow. The schematics of thefracture orientations are shown in Fig. 1.

Corel Draw ® software was used to draw irregularand dead-pore network patterns. The matrix networkconsists of a stochastic distribution of straight pores withlengths distributed in the range of 1.5 and 4 mm andwidths between 0.10 and 0.20 mm. The network patternof the models is shown in Fig. 2.

The micromodels were made by etching the mirrorimage pattern of pore networks onto a glass plate usinghydrofluoric acid. The etched plat has an inlet and anoutlet port drilled at either end. A second glass plate wasthen placed over the etched side and fused together in aprogrammable furnace. To manufacture uniform-depthglass micromodels, the quality of the etching processeswas controlled by adjusting the sequence of the

processing operations, time of etching and the concen-tration of the acid. The physical and hydraulic propertiesof the micromodels are given in Table 4. The highcontrast in both the width of the fractures and width ofthe pores ensured that very different capillary propertieswere associated with the fractures and the matrixblocks. The pore volume (PV) was determined byweighting the etched plates before and after the etchingprocess. The pore void area was measured by an imageanalyzer software. The thicknesses and porosities of the

Table 4The physical and hydraulic properties of the micromodels

Property Non-fractured

Inclined-fractured

Vertical-fractured

Horizontal-fractured

Model Model Model Model

Fracture width (mm) – 1.0 ≈1.1 1.5Porosity (%) 36.3 37.5 37.1 28.4Etched thickness (μm) 112 117 119 109Pore volume (ml) 0.142 0.153 0.154 0.212Permeability (μm2) 6.1 7.9 7.1 Not

measured

Table 5The experimental results of MEOR in the micromodels

Exp.no.

Fracture angleorientation

Soia Sorwf Ew

b Sorm Er

(%PV) (%PV) (%) (%PV) (%)

Bacterium: B. subtilis1 Non-fractured 92.3 50.7 45.0 39.0 23.12 Inclined-fractured 93.0 57.6 38.0 40.4 29.93 Vertical-fractured 94.3 53.8 42.9 38.3 28.84 Horizontal-fractured ≈98.6 87.5 11.2 84.7 3.2

Bacterium: L. mesenteroides5 Non-fractured 92.3 50.5 45.2 39.8 21.26 Inclined-fractured 93.0 57.7 37.9 50.7 12.17 Vertical-fractured 94.3 53.6 43.1 44.7 16.68 Horizontal-fractured ≈98.6 87.8 11.0 87.8 0a Initial oil saturation.b Water flooding efficiency.


micromodels were determined by the ratio of the mea-sured pore void area to the total area. The absolutepermeability of the models was determined using fallinghead method (Dullien, 1992).

2.5. Image analysis

While glass micromodels have been used mostly astools for qualitative study, some researchers haveemployed image-analyzing techniques for the satura-tion measurement in micromodels (Soll et al., 1993;Corapcioglu et al., 1997; Jeong et al., 2000; Sohrabiet al., 2004). In this study, a computerized imageprocessing system was used to measure the oil satu-ration before and after microbial treatment. The basicsystem consists of a Pentium PC, camera, opticalmicroscope and a professional scanning machine,which allowed us to capture high quality images atcertain times. An image analysis software wasdeveloped to measure the total area occupied by dyedoil. The relatively uniform depth of the models allowedus to determine oil saturation by measuring the areaoccupied by the dyed oil. The experimental setup isshown in Fig. 3.

Fig. 3. Experime

2.6. Experimental procedure

The experimental procedure used in this investiga-tion is as follows:

1- The glass micromodel was washed with chromicacid, acetone and sterilized with 70% ethanol.

2- It was then evacuated and saturated with autoclavedwater.

3- The model was placed horizontally in an incubator ata constant temperature of 37°C.

4- Oil was injected at the rate of 4 ml/h until thecondition of connate water saturation was reached.

5- Then water was injected by a syringe pump at the rateof 0.5 ml/h until no more oil was produced in theeffluent.

6- One pore volume of the mixture of bacterial solutionand nutrient solution (50:50) was injected in to themodel.

ntal setup.


7- The model was incubated anaerobically for a periodof 3 days (shut-in period) at 37°C.

8- After the shut-in period, the model was again floodedwith water until oil production was complete.

The microbial oil recovery efficiency, Er, is re-ported as the percentage of original oil in place and iscalculated as:

Er ¼ ðSorwf−Sormf=Sorwf Þ � 100

where Sorwf and Sormf are the matrix residual oilsaturation after water flooding and after microbialtreatment, respectively. Both are in percentage of porevolume (%PV).

3. Results

In order to study the effect of fracture angleorientation on the performance of the MEOR, eightexperiments were conducted using micromodels withdifferent fracture angles. A non-fractured micromodelwas also used to compare MEOR in fractured and non-

Fig. 4. Non-fractured micromodel after microbial treatment by usingB. subtilis (Experiment 1).

Fig. 5. Non-fractured micromodel after microbial treatment by usingL. mesenteroides (Experiment 5).

fractured porous media. The potential of mentionedbacteria for microbial enhanced oil recovery wasinvestigated in the core flooding system at 40°C anddifferent salinities of 0, 5 and 10% (Soudmand-asliet al., 2005). The results showed that for both bacteria,the microbial oil recovery efficiency in absence ofsalinity was more than those observed at 5 and 10%salinity. Thus, current experiments were performed at37°C and 0% NaCl. The results are given in Table 5. Itmust be mentioned that the image analysis procedurewas used only for matrix parts of the models thus, therecoveries efficiencies indicate the matrix recoveryefficiencies. In order to analyze the results of theexperiments, three different sets of experiments werecarried out to examine the effects of the bacteria on oilviscosity, porous media wettability and permeabilitywhich are explained in the Appendices.

3.1. The non-fractured model

Experiments 1 and 5 were conducted on the non-fractured model and resulted in 45% water flooding


efficiency. After treating the water flooded modelwith B. subtilis and L. mesenteroides, 23.1% and21.2% of water flood residual oil were recoveredrespectively. Figs. 4 and 5 show the model aftermicrobial treatments. Soudmand-asli et al. (2005)reported that the microbial recovery efficiencies in thecore flooding experiments were 27.1% for B. subtilisand 22.0% for L. mesenteroides. These show theresults of micromodel experiments and core floodingexperiments are comparable, as was also reported byBryant and Douglas (1988).

3.2. The vertical-fractured model

Experiments 3 and 7 were performed in the vertical-fractured model. The water flooding efficiencies were42.9% and 43.1%. After treating the model withB. subtilis, oil recovery increased by 25% from 23.1%in the non-fractured model to 28.8% in the vertical-fractured model, while by employing L. mesenteroidesoil recovery decreased by 22% from 21.2% in the non-

Fig. 6. Vertical-fractured micromodel after microbial treatment byusing B. subtilis (Experiments 3).

Fig. 7. Vertical-fractured micromodel after microbial treatment byusing L. mesenteroides (Experiments 7).

fractured model to 16.6% in the vertical-fracturedmodel. Figs. 6 and 7 show the models after microbialtreatment.

3.3. The inclined-fractured model

The water flooding efficiencies in this model were38.0% and 37.9%. In this case, the tertiary microbialrecovery efficiencies were 29.9% for B. subtilis and12.1% for L. mesenteroides. Figs. 8 and 9 show themodel after microbial treatments. The microbial recov-ery efficiency for B. subtilis shows considerableincrease by 29% for this model compared to the non-fractured one. However, microbial recovery efficiencyfor L. mesenteroides in this model was less than theother models. It was dropped dramatically to 12.1%;43% and 27% reduction when it was compared withmicrobial efficiencies in the non-fractured and thevertical-fractured models respectively. The resultsclearly indicate that, B. subtilis is more efficient forMEOR in fractured porous media.

Fig. 8. Inclined-fractured micromodel after microbial treatment byusing B. subtilis (Experiments 2).

Fig. 9. Inclined-fractured micromodel after microbial treatment byusing L. mesenteroides (Experiments 6).


3.4. The horizontal-fractured model

This model was used to better clarify the effect ofexisting bacteria and their bioproducts in the fractureon matrix oil recovery. This model had two fractures.The second fracture was used just for cleaning andsaturation steps. During the water flooding step, theinlet and outlet of the second fracture were closed,and water was injected from the inlet of the firstfracture and drained from its outlet. The oil recoveryefficiencies by water flooding were 11.0% and 11.2%.After water flooding, one pore volume of bacterialsolution was injected and the model was thenincubated. Thus during the shut-in period the fracturewas saturated with bacterial solution. After the shut-inperiod, several pore volumes of water were injectedagain into the fracture. No oil was recovered afterincubating the model with L. mesenteroides, and themicrobial recovery using B. subtilis was only 3.2%. Itshould be mentioned that in this model water as wellas bacterial solution was imbibed through the matrix-

fracture interface, however no water or bacterialsolution was injected to the matrix directly.

4. Discussion

To compare oil recovery efficiencies in the differentmodels, it was decided to keep the experimental variablesuch as the pattern of the micromodels, flow rate andconcentration of bacterial and nutrient solution un-changed in all the experiments. The results show thatfor biosurfactant-producing bacterium, B. subtilis, micro-bial recovery efficiencies in fractured porous media aremuch higher than the non-fractured one. UsingL. mesenteroides, the biopolymer-producing bacteriumshow completely different results. Microbial recoveryefficiency for this bacterium diminishes considerably inthe fractured models.

Matrix oil recovery in fractured porous media isinfluenced by interaction between fluids in the fractureand matrix. In general, when the miscibility conditions

Fig. 10. The pore-level photo that was taken after aging the non-fractured micromodels with bacterial solution of B. subtilis.

Table 6The results of the oil viscosity reduction in flask type experiments(μbm=29.0 cP at 35 °C)

Bacterium μam (mPa⁎s) at 35 °C μr (%)

B. subtilis 19.1 34.1L. mesenteroides 28.3 ≈2.4


are not present the following parameters affect theultimate oil recovery in fractured porous media:

• Matrix permeability size and shape (Babadagli andErshaghi, 1992; Cuiec et al., 1994; Ma et al., 1997),

• Wettability (Ma et al., 1994; Babadagli, 1997),matrix boundary conditions (Cuiec et al., 1994;Zhang et al., 1996; Ma et al., 1997),

• Viscosities of the phases (Ghedan and Poetmann,1990; Babadagli, 2001),

• Interfacial tension (IFT) (Keijzer and De Vries, 1990;Al-Lawati and Saleh, 1996).

Regarding the recovery mechanism involved in theMEOR processes, wettability alteration, porous mediapermeability reduction, changes in the interfacialtension and oil viscosity reduction by microorganismsand their bioproducts were the factors which can

Fig. 11. The pore-level photo that was taken after aging the non-fractured micromodels with bacterial solution of L. mesenteroides.

influence the microbial efficiencies in our experiments.The effect of the above parameters on matrix oilrecovery will be discussed as follows:

4.1. Wettability alteration

The wettability alteration tests are described inAppendix A. Comparing microscopic images of thefresh models with microbial treated ones (see Figs. 10and 11) shows no change in fluids distributions. In allcases, water films cover the surfaces of the pores,indicating that original wettability as well as wettabilityof treated model is strongly water wet and no wettabilityalteration occurs in the experiments.

4.2. Viscosity and interfacial tension reduction

Evaluation of bacteria effect on the oil viscosity isdescribed in Appendix B. As can be seen in Table 6,B. subtilis reduces the oil viscosity by 34.1% while nosignificant change in oil viscosity occurs whenL. mesenteroides is used. In general, bacteria affect theoil viscosity on two possible mechanisms: 1) reductionof the average molecular weight of heavy components(Bailey et al., 2001) and, 2) production of specific bio-logical products that would alter the physical propertiesof the oil (Ulmer et al., 1983; Streeb and Brown, 1992;Li et al., 2002). The flask experiments were carried outat atmospheric pressure; therefore there was nopossibility of biogas production effect on the oilviscosity. In addition, no clue was found in the literatureabout the production of gas by the bacteria. Thealternate mechanism of microbial depolymerizationand/or degradation of the high molecular weight consti-tuent such as long-chain hydrocarbon asphaltenes andresins to the smaller molecular products is also not

Table 7The sand packed physical parameters and results of effect of thebacteria on sand packed permeability

Bacterium Porosity ki ko Permeability reduction(%) (μm2) (μm2) (%)

B. subtilis 30.9 15.9 15.6 ≈1.8L. mesenteroides 29.0 13.0 7.8 40.0


applicable here. Because, these components were notpresent in the used oil, therefore the oil viscosity reductionwhich is accomplished by B. subtilis treatment is mostlydue to the biosurfactants, organic acids or solvents that areproduced during the microbial growth in the flask typeexperimentation. To further clarify this matter, the pH ofthe solution was also monitored during the course of thisinvestigation. No considerable changes in the pH of thebacterial solution were observed during the incubationperiod of the oil and bacterial solution in the flasks.Besides, there is no evidence in the literature of B. subtiliscapability to produce solvents. Thus production ofsurface-active agents by B. subtilis, which cause thegeneration of emulsions of water-in-oil, is the mostimportant factor behind oil viscosity reduction. Otherinvestigators also have reported the oil viscosity reductiondue to production of surfactants (Bertrand et al., 1994;Banat et al., 2000; Mulligan, 2005). It is mentioned in theliterature that one of the most important limitations ofmatrix oil recovery is oil viscosity (Babadagli, 1996a,b).Thus oil viscosity reduction by B. subtilis could be thereason for higher microbial oil recovery in the fracturedmodels. Moreover, reduction in interfacial tension due tosurfactant addition reported a positive effect on the matrixultimate recovery. (Babadagli et al., 1999; Babadagli,2003). B. subtilis can reduce the interfacial tensionbetween oil and water by producing surfactin. Asmentioned the remarkable reduction of IFT by usingB. subtilis (PTCC 1365) under anaerobic condition and atlow salinity was reported by Abtahi et al. (2003). Hence,oil viscosity reduction as well as reduction of IFT, wasfound to be the reason for better microbial recoveryefficiencies of B. subtilis in fractured models. Thesebacterial activities also can improve oil recoveryefficiency in the capillary porous media (Brown, 1992;Bryant and Lindsey, 1996). Released oil from thehorizontal-fractured model confirms the B. subtilis(biosurfactant-producing bacterium) capability of drain-ing the oil from the matrix blocks to the fractures.

4.3. Permeability reduction

Evaluation of bacterial activities on the permeabilitiesof porous media are explained in Appendix C. Aconsiderable permeability reduction of 40.0% from 13.0to 7.8 μm occurred when L. mesenteroides was used (seeTable 7), while no reduction in permeability was observedin the sand-packed column in which B. subtilis wasincubated. We have also seen earlier when L. mesenter-oides was used in the fractured models, the microbial oilrecovery efficiencies were drastically reduced. The reasonfor this reduction is due to the fact that this bacterium is

able to produce dextran (an insoluble exopolymer) whichcauses plugging in parts of the matrix-fracture interfacesand reduces the interaction between the matrix andfracture. After water flooding, the fractures were com-pletely saturated with water. Once the bacterial solutionwas injected, the fractures became saturated with thebacterial solution. Thus, considerable amounts of dextranwere produced during the incubation period (shut-inperiod) in the matrix-fracture interface, causing severepermeability reduction in these regions. The photos weretaken after MEOR confirms this conclusion. By compar-ing Figs. 6 with 7 and 8 with 9 shows that in the case ofusing L. mesenteroides, the saturation of trapped oils nearthe matrix-fracture interfaces is much higher than thosemodels treated with B. subtilis. Plugging of matrix-fracture interfaces was also reported during bacterialflooding by Zekri and El-Mehaideb (2002), who utilizedthe scanning electron microscope (SEM) technique tovisualize the plugging of the fractures. The differences inthe L. mesenteroides microbial recovery efficiencies inthe vertical-fractured and inclined-fractured models couldbe explained by the fact that the matrix-fracture interfacein the inclined-fracturedmodel is much longer than that ofvertical-fractured model. Hence plugging by the exopo-lymer was more severe there. While production ofbiopolymers can reduce oil recovery in fractured porousmedia, several investigations have acknowledged thisbacterial activity as an important factor to increase oilrecovery in capillary porous media (Stepp et al., 1996;Yusuf et al., 1999). It must be mentioned that in all theexperiments (micromodels and sand-packed column) theconditions were chosen to isolate the plugging mechan-isms associated with exopolymer production. It is wellknown that the plugging of porous media during themicrobial treatment is caused by bacterial cells and theproduced exopolymer (Donaldson et al., 1989). In thecase of plugging by bacterial cells, the cells adhere to rocksurfaces and reduce the permeability of the highpermeable zone to increase the sweep efficiency duringsecondary and tertiary oil recovery. To evaluate accurateeffects of the biopolymer in the MEOR process infractured porous media, it was decided to isolate theplugging mechanisms associated with exopolymer pro-duction only. The relatively high permeabilities of themicromodels and the sand-packed columns suggest thatthe average throat size of the pores in the models is largeenough to allow the free flow of dispersed bacterial cellsas predicted by Gruesbeck and Collins (1982). Based onthe results reported in the literature, a grain size of 300 to500 μm in the sand-packed experiments and the porewidths between 0.10 and 0.20 mmwere selected in whichthe bacterial cells do not contribute to plugging the porous


media (Mac Leod et al., 1988; Fontes et al., 1991;Lappan and Fogler, 1996; Stewart and Fogler, 2001).Thus, plugging only occurred in the experiments whereL. mesenteroides was used.

5. Conclusion

• The performance of the in situ MEOR process infractured porous media can be improved by theselection of suitable bacterium with respect to itsbioproducts.

• The In situ microbial enhanced oil recovery efficien-cy in fractured media is predominantly affected bythe fracture orientations.

• Biopolymer-producing bacteria (i.e. L. mesenter-oides) cannot improve the oil recovery efficiency inthe fractured porous media as much as they can do inthe non-fractured media, because of the matrix-fracture plugging effects.

• Considerable permeability reduction was observedwhen the biopolymer-producing bacteria were incu-bated in sand-packed column.

• The microbial oil recovery efficiency by usingbiosurfactant-producing bacteria (i.e. B. subtilis) inthe fractured porous media is higher than that of thenon-fractured media.

• High oil recovery efficiency was achieved in thefractured porous media when the biosurfactant-producing bacteria were used as the microbialtreating agent mostly due to the interfacial tensionand viscosity reduction.

• No sign of wettability alteration was observed duringthe MEOR process using both biosurfactant andbiopolymer-producing bacteria.

Acknowledgements

Authors would like to thank staffs of Enhanced OilRecovery Lab. of Shiraz University and staffs of Up-stream Petroleum Engineering Lab. of Tehran University.

Appendix A. Investigation on effect of the bacteriaon wettability

The wettability was measured qualitatively using themicroscopic examinationmethod by looking at the oil andwater saturation distribution in the micromodel (Ander-son, 1986). Microscopic observation of the clean freshmodel before microbial treatment shows an absolutelywater wet condition. To determine the degree of water oroil wetness after microbial treatment, the clean non-fractured model was saturated with the mixture of

bacterial and nutrient solutions. The model was thenincubated for 4 days, and after that it was water floodedwith 1 pore volume of dyed water with methylene blue.Connate water condition was achieved by flooding themodels with oil. Pore level photos taken after oil flooding(see Figs. 10 and 11) clearly show nowettability alterationof the microbial treated micromodel.

Appendix B. Evaluation of bacterial effect on oilviscosity

The reduction of oil viscosity during the MEORprocess is contributed to higher oil recovery. Todemonstrate this effect on oil recovery efficiency usingthe mentioned bacteria, two more flask type experimentswere performed. Flasks were placed in a shaker incubatorat 60 rev/min. Each flask contained 50 ml of the dyed oil,30 ml of the bacterial solution and 30 ml of the nutrientsolution. Flasks were incubated at 35°C for 3 days. Afterinoculation period the oil was centrifuged and its viscositywas determined at 35°C by the Canon-Fensk viscometer.The results are given in Table 6. Significant reduction inthe oil viscosity is observed using B. subtilis compared toL. mesenteroides. Moreover the pH of the aqueous phasein the flasks was also measured before and after theincubation period. There is no significant change of pHindicating no acidic byproducts were produced during theincubation period. The percentage of oil viscosityreduction, μr, is calculated as:

lr ¼ ðlbm−lam=lbmÞ � 100

where μbm and μam are the oil viscosity before microbialtreatment and after microbial treatment respectively.

Appendix C. Evaluation of bacterial activities onporous media permeability

The effect of the bacteria on the permeability ofporous media during the MEOR process was alsoinvestigated using sand-packed columns. Sand-packedcolumns were filled and packed with the sterile andfresh quartz sand having the grain size of 300 to 500 μm.The permeability of the sand-packed column wasmeasured using the falling head method. 0.2 porevolume of bacterial solution was then injected into thesaturated column followed by 0.2 pore volume ofnutrient solution at the flow rate of 10 ml/s. The shut-inperiod (3 days) started just after the injection of thenutrient solution by closing the valves at the two ends ofthe column. The column was then incubated for 3 days.After the shut-in period one PV of water was injected


into the column and then the final permeability of thesand-packed was measured. The percentage of perme-ability reduction due to bacterial treatment wascalculated as follows:

Permeability reduction ð%Þ ¼ ki−koki

� 100

where ki and ko are the permeability of the sand-packedbefore the injection of bacteria and after the shut-inperiod respectively. The results are given in Table 7. It isobvious that L. mesenteroides had a considerablecapability to reduce the porous media permeability.

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