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Journal of Petroleum Science and Engineering 58 (2007) 161 172 www.elsevier.


The in situ microbial enhanced oil recovery in fractured porous media

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

School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iran 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 using etched-glass micromodels. Three identically patterned micromodels with different fracture angle orientation of inclined, vertical and horizontal with respect to the flow direction were utilized. A non-fractured model was also used to compare the efficiency of MEOR in fractured and non-fractured porous media. Two types of bacteria were employed: Bacillus subtilis (a biosurfactantproducing bacterium) and Leuconostoc mesenteroides (an exopolymer-producing bacterium). The results show that higher oil recovery efficiency can be achieved by using biosurfactant-producing bacterium in fractured porous media. Further investigation on the effect of the mentioned bacteria on oil viscosity, porous media permeability and wettability suggests that the plugging of matrix-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 of biosurfactant-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 increasing oil production from these complex reservoirs (Delshad et 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: shahab@shirazu.ac.ir (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 oil recovery (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 processes in enhanced oil recovery from the fractured reservoirs has been particularly neglected. No core flooding or micromodel experimentation has been carried out to investigate the MEOR in fractured porous media. Only Zekri and El-Mehaideb (2002) presented an experimental work on bacterial flooding (secondary recovery) through different fractured


A. Soudmand-asli et al. / Journal of Petroleum Science and Engineering 58 (2007) 161172 Table 2 The properties of the used fluids Property Density (g/cm ) at 28 C Viscosity (mPas) at 35 C Flash point (C) Pour point (C)

systems. The results indicated that fracture orientation strongly affects the performance of bacterial flooding into the fractured system. In this study, the tertiary microbial oil recovery was investigated using etched-glass micromodels having different fracture angle orientation. B. subtilis and L. mesenteroides were chosen as the treating bacteria. B. subtilis is well known to produce surfactin as a biosurfactant, the chemical structure of which is well documented (Donaldson et al., 1989; Banat, 1995). L. mesenteroides is able to produce substantial amounts of dextran, an insoluble exopolymer, in the presence of sucrose (Xiu-Yuan and Wang, 1991; Stewart and Fogler, 2001). In addition, to analyze the oil recovery efficiency of the MEOR in fractured models, the effect of bacteria on wettability, oil viscosity and permeability of the media was also studied. The final goal of our study was to identify the best bacterium for microbial enhanced oil recovery in fractured reservoirs with regard to the bioproducts of the bacterium. 2. Materials and methods

Dyed oil 0.86 29.0 210 11

Water 1.0 1

composition of each growth medium is presented in Table 1. The bacterial culture was centrifuged at 2500 rev/ min for 30 min and collected at the stationary state; it was then suspended in autoclaved water. The bacterial suspension was placed on a magnetic stirrer and allowed to mix at room temperature for 5 min. The solution was centrifuged and washed once again with water as previously described. The bacteria were then suspended and placed on the magnetic stirrer to make a bacterial solution. The cell density of the bacterial solution was adjusted to 109 cells/ml (107) by spectrophotometric analysis at 600 nm. 2.3. Fluids

2.1. Microorganisms Two types of bacteria were employed: Leuconostoc mesenteroides (PTCC 1059) and Bacillus subtilis (PTCC 1365). They were both obtained from the Persian Type Culture Collection (PTCC), Tehran, Iran. Kamal et al. (2001) reported the ability of PTCC 1059 to produce considerable amounts of dextran under anaerobic conditions and in the presence of sucrose. Abtahi et al. (2003) showed that under anaerobic conditions and at low salinity PTCC 1365 is able to reduce remarkably the oilwater interfacial tension. 2.2. Growth conditions B. subtilis and L. mesenteroides were cultured on liquid growth medium A and B, respectively. The
Table 1 The compositions of growth media Compositions Sucrose NH4Cl Glucose Peptone from meat Meat infusion Na2HPO4 Sodium chloride Growth medium A (g/lit distilled water) 1.0 1.0 0 10 5.0 2.0 0.3 Growth medium B (g/lit distilled water) 0 2.0 5.0 10 5.0 2.0 0.3

The physical properties of oil and water are given in Table 2. Densities were determined by using a pycnometer and viscosity was measured by a Canon-Fensk viscometer, according to the procedures proposed by ASTM D 445. 2.3.1. Oil Synthetic oil was used as the oleic phase. It was a pure hydrocarbon and free from other crude oil components such as asphaltene, salts, metals, sulfur, resin acids, and ash contents; therefore the effect of these components on microbial activities was eliminated in this study. In order to find the saturation in the transparent models, the oil was dyed with Sudan Red (C24H21N5); the dyed oil was filtered using fine filter papers to remove any solidified dye particles. 2.3.2. Flooding water Autoclaved distilled water was used as the flooding agent. Water was not dyed due to the concerns about the
Table 3 The compositions of the nutrient solution Compositions Sucrose Di ammonium hydrogen Phosphate ((NH4)2HPO4) Ammonium chloride (NH4Cl) Concentration (w/v) 4% 0.02% 0.04%

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Fig. 1. Fracture orientations of the micromodels.

interference of chemical components on the bacterial growth. 2.3.3. Nutrient solution The nutrient solution was prepared based on the investigation of Brown (1982) and Jenneman et al. (1984), who found that the nitrate and phosphate containing nutrients are very effective for in situ MEOR processes. Table 3 shows the compositions of the used nutrient. 2.4. The glass micromodels Glass micromodels are two dimensional flowchannel networks etched in glass to simulate fluid flow in porous media. These models are usually considered strongly water-wet because their surface chemistry is similar to that of clean sandstone (Grattoni et al., 2002). Here, four identically patterned micromodels were constructed. Three of them had a single fracture with different fracture angle orientation of 45 (inclined-fractured model), 90 (vertical-fractured model) and 180 (horizontal-fractured model) with respect to the axis of the flow. The schematics of the fracture orientations are shown in Fig. 1. Corel Draw software was used to draw irregular and dead-pore network patterns. The matrix network consists of a stochastic distribution of straight pores with lengths distributed in the range of 1.5 and 4 mm and widths between 0.10 and 0.20 mm. The network pattern of the models is shown in Fig. 2. The micromodels were made by etching the mirror image pattern of pore networks onto a glass plate using hydrofluoric acid. The etched plat has an inlet and an outlet port drilled at either end. A second glass plate was then placed over the etched side and fused together in a programmable furnace. To manufacture uniform-depth glass micromodels, the quality of the etching processes was controlled by adjusting the sequence of the

processing operations, time of etching and the concentration of the acid. The physical and hydraulic properties of the micromodels are given in Table 4. The high contrast in both the width of the fractures and width of the pores ensured that very different capillary properties were associated with the fractures and the matrix blocks. The pore volume (PV) was determined by weighting the etched plates before and after the etching process. The pore void area was measured by an image analyzer software. The thicknesses and porosities of the

Fig. 2. Porous pattern of the micromodels.


A. Soudmand-asli et al. / Journal of Petroleum Science and Engineering 58 (2007) 161172 Table 5 The experimental results of MEOR in the micromodels Exp. no. Fracture angle orientation Soi a (%PV) 92.3 93.0 94.3 98.6 Sorwf (%PV) 50.7 57.6 53.8 87.5 Ew b (%) 45.0 38.0 42.9 11.2 Sorm (%PV) 39.0 40.4 38.3 84.7 Er (%) 23.1 29.9 28.8 3.2

Table 4 The physical and hydraulic properties of the micromodels Property NonInclined- Vertical- Horizontalfractured fractured fractured fractured Model Model Model Model 36.3 112 0.142 6.1 1.0 37.5 117 0.153 7.9 1.1 37.1 119 0.154 7.1 1.5 28.4 109 0.212 Not measured

Fracture width (mm) Porosity (%) Etched thickness (m) Pore volume (ml) Permeability (m2)

Bacterium: B. subtilis 1 Non-fractured 2 Inclined-fractured 3 Vertical-fractured 4 Horizontal-fractured

micromodels were determined by the ratio of the measured pore void area to the total area. The absolute permeability of the models was determined using falling head method (Dullien, 1992). 2.5. Image analysis While glass micromodels have been used mostly as tools for qualitative study, some researchers have employed image-analyzing techniques for the saturation measurement in micromodels (Soll et al., 1993; Corapcioglu et al., 1997; Jeong et al., 2000; Sohrabi et al., 2004). In this study, a computerized image processing system was used to measure the oil saturation before and after microbial treatment. The basic system consists of a Pentium PC, camera, optical microscope and a professional scanning machine, which allowed us to capture high quality images at certain times. An image analysis software was developed to measure the total area occupied by dyed oil. The relatively uniform depth of the models allowed us to determine oil saturation by measuring the area occupied by the dyed oil. The experimental setup is shown in Fig. 3.

Bacterium: L. mesenteroides 5 Non-fractured 92.3 6 Inclined-fractured 93.0 7 Vertical-fractured 94.3 8 Horizontal-fractured 98.6
a b

50.5 57.7 53.6 87.8

45.2 37.9 43.1 11.0

39.8 50.7 44.7 87.8

21.2 12.1 16.6 0

Initial oil saturation. Water flooding efficiency.

2.6. Experimental procedure The experimental procedure used in this investigation is as follows: 1- The glass micromodel was washed with chromic acid, acetone and sterilized with 70% ethanol. 2- It was then evacuated and saturated with autoclaved water. 3- The model was placed horizontally in an incubator at a constant temperature of 37C. 4- Oil was injected at the rate of 4 ml/h until the condition of connate water saturation was reached. 5- Then water was injected by a syringe pump at the rate of 0.5 ml/h until no more oil was produced in the effluent. 6- One pore volume of the mixture of bacterial solution and nutrient solution (50:50) was injected in to the model.

Fig. 3. Experimental setup.

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7- The model was incubated anaerobically for a period of 3 days (shut-in period) at 37C. 8- After the shut-in period, the model was again flooded with water until oil production was complete. The microbial oil recovery efficiency, Er, is reported as the percentage of original oil in place and is calculated as: Er Sorwf Sormf =Sorwf 100 where Sorwf and Sormf are the matrix residual oil saturation after water flooding and after microbial treatment, respectively. Both are in percentage of pore volume (%PV). 3. Results In order to study the effect of fracture angle orientation on the performance of the MEOR, eight experiments were conducted using micromodels with different fracture angles. A non-fractured micromodel was also used to compare MEOR in fractured and non-

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

fractured porous media. The potential of mentioned bacteria for microbial enhanced oil recovery was investigated in the core flooding system at 40C and different salinities of 0, 5 and 10% (Soudmand-asli et al., 2005). The results showed that for both bacteria, the microbial oil recovery efficiency in absence of salinity was more than those observed at 5 and 10% salinity. Thus, current experiments were performed at 37C and 0% NaCl. The results are given in Table 5. It must be mentioned that the image analysis procedure was used only for matrix parts of the models thus, the recoveries efficiencies indicate the matrix recovery efficiencies. In order to analyze the results of the experiments, three different sets of experiments were carried out to examine the effects of the bacteria on oil viscosity, porous media wettability and permeability which are explained in the Appendices. 3.1. The non-fractured model
Fig. 4. Non-fractured micromodel after microbial treatment by using B. subtilis (Experiment 1).

Experiments 1 and 5 were conducted on the nonfractured model and resulted in 45% water flooding


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efficiency. After treating the water flooded model with B. subtilis and L. mesenteroides, 23.1% and 21.2% of water flood residual oil were recovered respectively. Figs. 4 and 5 show the model after microbial treatments. Soudmand-asli et al. (2005) reported that the microbial recovery efficiencies in the core flooding experiments were 27.1% for B. subtilis and 22.0% for L. mesenteroides. These show the results of micromodel experiments and core flooding experiments are comparable, as was also reported by Bryant and Douglas (1988). 3.2. The vertical-fractured model Experiments 3 and 7 were performed in the verticalfractured model. The water flooding efficiencies were 42.9% and 43.1%. After treating the model with B. subtilis, oil recovery increased by 25% from 23.1% in the non-fractured model to 28.8% in the verticalfractured model, while by employing L. mesenteroides oil recovery decreased by 22% from 21.2% in the non-

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

fractured model to 16.6% in the vertical-fractured model. Figs. 6 and 7 show the models after microbial treatment. 3.3. The inclined-fractured model The water flooding efficiencies in this model were 38.0% and 37.9%. In this case, the tertiary microbial recovery efficiencies were 29.9% for B. subtilis and 12.1% for L. mesenteroides. Figs. 8 and 9 show the model after microbial treatments. The microbial recovery efficiency for B. subtilis shows considerable increase by 29% for this model compared to the nonfractured one. However, microbial recovery efficiency for L. mesenteroides in this model was less than the other models. It was dropped dramatically to 12.1%; 43% and 27% reduction when it was compared with microbial efficiencies in the non-fractured and the vertical-fractured models respectively. The results clearly indicate that, B. subtilis is more efficient for MEOR in fractured porous media.

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

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fracture interface, however no water or bacterial solution was injected to the matrix directly. 4. Discussion To compare oil recovery efficiencies in the different models, it was decided to keep the experimental variable such as the pattern of the micromodels, flow rate and concentration of bacterial and nutrient solution unchanged in all the experiments. The results show that for biosurfactant-producing bacterium, B. subtilis, microbial recovery efficiencies in fractured porous media are much higher than the non-fractured one. Using L. mesenteroides, the biopolymer-producing bacterium show completely different results. Microbial recovery efficiency for this bacterium diminishes considerably in the fractured models. Matrix oil recovery in fractured porous media is influenced by interaction between fluids in the fracture and matrix. In general, when the miscibility conditions

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

3.4. The horizontal-fractured model This model was used to better clarify the effect of existing bacteria and their bioproducts in the fracture on matrix oil recovery. This model had two fractures. The second fracture was used just for cleaning and saturation steps. During the water flooding step, the inlet and outlet of the second fracture were closed, and water was injected from the inlet of the first fracture and drained from its outlet. The oil recovery efficiencies by water flooding were 11.0% and 11.2%. After water flooding, one pore volume of bacterial solution was injected and the model was then incubated. Thus during the shut-in period the fracture was saturated with bacterial solution. After the shut-in period, several pore volumes of water were injected again into the fracture. No oil was recovered after incubating the model with L. mesenteroides, and the microbial recovery using B. subtilis was only 3.2%. It should be mentioned that in this model water as well as bacterial solution was imbibed through the matrix-

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


A. Soudmand-asli et al. / Journal of Petroleum Science and Engineering 58 (2007) 161172 Table 6 The results of the oil viscosity reduction in flask type experiments (bm = 29.0 cP at 35 C) Bacterium B. subtilis L. mesenteroides am (mPas) at 35 C 19.1 28.3 r (%) 34.1 2.4

influence the microbial efficiencies in our experiments. The effect of the above parameters on matrix oil recovery will be discussed as follows: 4.1. Wettability alteration The wettability alteration tests are described in Appendix A. Comparing microscopic images of the fresh models with microbial treated ones (see Figs. 10 and 11) shows no change in fluids distributions. In all cases, water films cover the surfaces of the pores, indicating that original wettability as well as wettability of treated model is strongly water wet and no wettability alteration occurs in the experiments. 4.2. Viscosity and interfacial tension reduction Evaluation of bacteria effect on the oil viscosity is described in Appendix B. As can be seen in Table 6, B. subtilis reduces the oil viscosity by 34.1% while no significant change in oil viscosity occurs when L. mesenteroides is used. In general, bacteria affect the oil viscosity on two possible mechanisms: 1) reduction of the average molecular weight of heavy components (Bailey et al., 2001) and, 2) production of specific biological products that would alter the physical properties of the oil (Ulmer et al., 1983; Streeb and Brown, 1992; Li et al., 2002). The flask experiments were carried out at atmospheric pressure; therefore there was no possibility of biogas production effect on the oil viscosity. In addition, no clue was found in the literature about the production of gas by the bacteria. The alternate mechanism of microbial depolymerization and/or degradation of the high molecular weight constituent such as long-chain hydrocarbon asphaltenes and resins to the smaller molecular products is also not

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

are not present the following parameters affect the ultimate oil recovery in fractured porous media: Matrix permeability size and shape (Babadagli and Ershaghi, 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 the MEOR processes, wettability alteration, porous media permeability reduction, changes in the interfacial tension and oil viscosity reduction by microorganisms and their bioproducts were the factors which can

Table 7 The sand packed physical parameters and results of effect of the bacteria on sand packed permeability Bacterium Porosity ki ko Permeability reduction (%) (m2) (m2) (%) 15.9 13.0 15.6 7.8 1.8 40.0

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

B. subtilis 30.9 L. mesenteroides 29.0

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applicable here. Because, these components were not present in the used oil, therefore the oil viscosity reduction which is accomplished by B. subtilis treatment is mostly due to the biosurfactants, organic acids or solvents that are produced during the microbial growth in the flask type experimentation. To further clarify this matter, the pH of the solution was also monitored during the course of this investigation. No considerable changes in the pH of the bacterial solution were observed during the incubation period of the oil and bacterial solution in the flasks. Besides, there is no evidence in the literature of B. subtilis capability to produce solvents. Thus production of surface-active agents by B. subtilis, which cause the generation of emulsions of water-in-oil, is the most important factor behind oil viscosity reduction. Other investigators also have reported the oil viscosity reduction due to production of surfactants (Bertrand et al., 1994; Banat et al., 2000; Mulligan, 2005). It is mentioned in the literature that one of the most important limitations of matrix oil recovery is oil viscosity (Babadagli, 1996a,b). Thus oil viscosity reduction by B. subtilis could be the reason for higher microbial oil recovery in the fractured models. Moreover, reduction in interfacial tension due to surfactant addition reported a positive effect on the matrix ultimate recovery. (Babadagli et al., 1999; Babadagli, 2003). B. subtilis can reduce the interfacial tension between oil and water by producing surfactin. As mentioned the remarkable reduction of IFT by using B. subtilis (PTCC 1365) under anaerobic condition and at low salinity was reported by Abtahi et al. (2003). Hence, oil viscosity reduction as well as reduction of IFT, was found to be the reason for better microbial recovery efficiencies of B. subtilis in fractured models. These bacterial activities also can improve oil recovery efficiency in the capillary porous media (Brown, 1992; Bryant and Lindsey, 1996). Released oil from the horizontal-fractured model confirms the B. subtilis (biosurfactant-producing bacterium) capability of draining the oil from the matrix blocks to the fractures. 4.3. Permeability reduction Evaluation of bacterial activities on the permeabilities of porous media are explained in Appendix C. A considerable permeability reduction of 40.0% from 13.0 to 7.8 m occurred when L. mesenteroides was used (see Table 7), while no reduction in permeability was observed in the sand-packed column in which B. subtilis was incubated. We have also seen earlier when L. mesenteroides was used in the fractured models, the microbial oil recovery efficiencies were drastically reduced. The reason for this reduction is due to the fact that this bacterium is

able to produce dextran (an insoluble exopolymer) which causes plugging in parts of the matrix-fracture interfaces and reduces the interaction between the matrix and fracture. After water flooding, the fractures were completely saturated with water. Once the bacterial solution was injected, the fractures became saturated with the bacterial solution. Thus, considerable amounts of dextran were produced during the incubation period (shut-in period) in the matrix-fracture interface, causing severe permeability reduction in these regions. The photos were taken after MEOR confirms this conclusion. By comparing Figs. 6 with 7 and 8 with 9 shows that in the case of using L. mesenteroides, the saturation of trapped oils near the matrix-fracture interfaces is much higher than those models treated with B. subtilis. Plugging of matrixfracture interfaces was also reported during bacterial flooding by Zekri and El-Mehaideb (2002), who utilized the scanning electron microscope (SEM) technique to visualize the plugging of the fractures. The differences in the L. mesenteroides microbial recovery efficiencies in the vertical-fractured and inclined-fractured models could be explained by the fact that the matrix-fracture interface in the inclined-fractured model is much longer than that of vertical-fractured model. Hence plugging by the exopolymer was more severe there. While production of biopolymers can reduce oil recovery in fractured porous media, several investigations have acknowledged this bacterial activity as an important factor to increase oil recovery in capillary porous media (Stepp et al., 1996; Yusuf et al., 1999). It must be mentioned that in all the experiments (micromodels and sand-packed column) the conditions were chosen to isolate the plugging mechanisms associated with exopolymer production. It is well known that the plugging of porous media during the microbial treatment is caused by bacterial cells and the produced exopolymer (Donaldson et al., 1989). In the case of plugging by bacterial cells, the cells adhere to rock surfaces and reduce the permeability of the high permeable zone to increase the sweep efficiency during secondary and tertiary oil recovery. To evaluate accurate effects of the biopolymer in the MEOR process in fractured porous media, it was decided to isolate the plugging mechanisms associated with exopolymer production only. The relatively high permeabilities of the micromodels and the sand-packed columns suggest that the average throat size of the pores in the models is large enough to allow the free flow of dispersed bacterial cells as predicted by Gruesbeck and Collins (1982). Based on the results reported in the literature, a grain size of 300 to 500 m in the sand-packed experiments and the pore widths between 0.10 and 0.20 mm were selected in which the bacterial cells do not contribute to plugging the porous


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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 where L. mesenteroides was used. 5. Conclusion The performance of the in situ MEOR process in fractured porous media can be improved by the selection of suitable bacterium with respect to its bioproducts. The In situ microbial enhanced oil recovery efficiency in fractured media is predominantly affected by the fracture orientations. Biopolymer-producing bacteria (i.e. L. mesenteroides) cannot improve the oil recovery efficiency in the fractured porous media as much as they can do in the non-fractured media, because of the matrixfracture plugging effects. Considerable permeability reduction was observed when the biopolymer-producing bacteria were incubated in sand-packed column. The microbial oil recovery efficiency by using biosurfactant-producing bacteria (i.e. B. subtilis) in the fractured porous media is higher than that of the non-fractured media. High oil recovery efficiency was achieved in the fractured porous media when the biosurfactantproducing bacteria were used as the microbial treating agent mostly due to the interfacial tension and viscosity reduction. No sign of wettability alteration was observed during the MEOR process using both biosurfactant and biopolymer-producing bacteria. Acknowledgements Authors would like to thank staffs of Enhanced Oil Recovery Lab. of Shiraz University and staffs of Upstream Petroleum Engineering Lab. of Tehran University. Appendix A. Investigation on effect of the bacteria on wettability The wettability was measured qualitatively using the microscopic examination method by looking at the oil and water saturation distribution in the micromodel (Anderson, 1986). Microscopic observation of the clean fresh model before microbial treatment shows an absolutely water wet condition. To determine the degree of water or oil wetness after microbial treatment, the clean nonfractured model was saturated with the mixture of

bacterial and nutrient solutions. The model was then incubated for 4 days, and after that it was water flooded with 1 pore volume of dyed water with methylene blue. Connate water condition was achieved by flooding the models with oil. Pore level photos taken after oil flooding (see Figs. 10 and 11) clearly show no wettability alteration of the microbial treated micromodel. Appendix B. Evaluation of bacterial effect on oil viscosity The reduction of oil viscosity during the MEOR process is contributed to higher oil recovery. To demonstrate this effect on oil recovery efficiency using the mentioned bacteria, two more flask type experiments were performed. Flasks were placed in a shaker incubator at 60 rev/min. Each flask contained 50 ml of the dyed oil, 30 ml of the bacterial solution and 30 ml of the nutrient solution. Flasks were incubated at 35C for 3 days. After inoculation period the oil was centrifuged and its viscosity was determined at 35C by the Canon-Fensk viscometer. The results are given in Table 6. Significant reduction in the oil viscosity is observed using B. subtilis compared to L. mesenteroides. Moreover the pH of the aqueous phase in the flasks was also measured before and after the incubation period. There is no significant change of pH indicating no acidic byproducts were produced during the incubation period. The percentage of oil viscosity reduction, r, is calculated as: lr lbm lam =lbm 100 where bm and am are the oil viscosity before microbial treatment and after microbial treatment respectively. Appendix C. Evaluation of bacterial activities on porous media permeability The effect of the bacteria on the permeability of porous media during the MEOR process was also investigated using sand-packed columns. Sand-packed columns were filled and packed with the sterile and fresh quartz sand having the grain size of 300 to 500 m. The permeability of the sand-packed column was measured using the falling head method. 0.2 pore volume of bacterial solution was then injected into the saturated column followed by 0.2 pore volume of nutrient solution at the flow rate of 10 ml/s. The shut-in period (3 days) started just after the injection of the nutrient solution by closing the valves at the two ends of the column. The column was then incubated for 3 days. After the shut-in period one PV of water was injected

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into the column and then the final permeability of the sand-packed was measured. The percentage of permeability reduction due to bacterial treatment was calculated as follows: ki ko Permeability reduction % 100 ki where ki and ko are the permeability of the sand-packed before the injection of bacteria and after the shut-in period respectively. The results are given in Table 7. It is obvious that L. mesenteroides had a considerable capability to reduce the porous media permeability. References
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