Proceedings of the 3rd (2011) CUTSE International Conference
Miri, Sarawak, Malaysia, 8-9 Nov, 2011
Application of Microbial Profile Modification for In
Situ Enhanced Oil Recovery
Seyed Hossein Emadi
Chemical engineering department, Faculty of chemical
engineering
University of Tehran
Tehran, Iran
Hosseinemadi@yahoo.com
Abstract— Permeability modification of porous media using
microorganisms to enhance oil recovery has become a useful and
feasible technology for oil production from watered-out oil fields.
The purpose of these experimental experiences was to determine
capability of microorganisms to reduction of the permeable zone.
A sand packed column and a two dimensional glass micromodel
were used to demonstrate the effectiveness of in situ profile
modification with spores. In both cases, the apparatus conditions
were chosen to isolate the plugging mechanisms just associated
with exopolymer production and eliminate any impact of cell
growth to profile modification. Bacillus Licheniformis (an
exopolymer producing bacterium) was employed in this set of
experiments. Temperature, salinity, and incubation times were
three different variables that were studied in these investigations.
The results show that B. licheniformis is proper to make plugging
to profile modification in salty reservoir with temperatures close
to 40ºC. In Addition it was demonstrated that this bacteria can
reduce the permeability of reservoir as incubation time is
increasing. Likewise, the visual results of glass micromodel
experiments were compatible to sand packed surveys. Hence, B.
licheniformis is suggested for microbial enhanced oil recovery
(MEOR) in highly saline sand stone reservoir with about 40ºC
temperature value.
Keywords-component: MEOR; Bacillus Licheniformis; profile
modification; exopolymer; in situ
I. INTRODUCTION
0B
Utilization of microorganisms is one of the well known
methods in enhanced oil recovery operation. In recent years a
number of studies have demonstrated the potential applicability
of some bacteria for enhanced oil recovery [1-3]. Also,
microbial enhanced oil recovery (MEOR) has been proposed as
an effective low cost method of enhanced oil recovery
approaches. There are two major ways in which microbes may
contribute to EOR: (a) they grow in reservoir rock to produce
gases, biosurfactants, biopolymers and other non-toxic
(205 -- 917)
Tohid Nejad Ghaffar Borhani
Chemical engineering department, Faculty of chemical and
natural resources engineering
University Technology Malaysia
Johor Bahru, Malaysia
Tohid.n.borhani@gmail.com
Alireza Soudmand-asli
School of Chemical and Petroleum Engineering
Shiraz University
Shiraz, Iran
a.soudmand@sazeh.co.ir
biochemical to recover trapped oil; and (b) they can selectively
plug high-permeability channels so that the sweep efficiency of
the recovery process can be increased [4]. Producing gases,
biosurfactants, biopolymers and other non-toxic biochemical
may be carried out in situ (in reservoir rock) or ex situ (in the
controlled environment of a fermenter). Beside the mentioned
classification, microbial enhanced oil recovery can be
classified in aspect of manner of performing the process to well
bore clean up [5], cyclic Microbial Recovery [6], and
Microbial flooding [7]. The mechanisms by which MEOR
processes operate can be quite complex and may involve
multiple biochemical and biophysical process steps such as:
• Reduction of Interfacial tension [8].
• Changes in wettability [9, 10].
• Changes in flow pattern [11].
• Acid production [12-14].
• Biopolymer production [13].
• Solvent production [13]
• Degradation of long chain hydrocarbon and reducing
oil viscosity [13, 15].
One of the microbial technologies under development uses
microbial metabolism to trigger gelation of an in-situ produced
biopolymer.
Bacteria and/or nutrients preferentially enter the reservoir
along high-permeability pathways. Biopolymer produced by
microbe, in those laminas plugs the pore throats, thus
decreasing the permeability in what had once been the high-
permeability zones [4]. This process may improve the sweep
efficiency and thereby the displacement of oil initially
bypassed [16].
The main objective of this study was to evaluate the effect
of environmental conditions, such as temperature and salinity
on the capability of an exopolymer-producing bacterium,
Bacillus licheniformis in blocking the thief zone area. Also, the
 Proceedings of the 3rd (2011) CUTSE International Conference
Miri, Sarawak, Malaysia, 8-9 Nov, 2011
effect of incubation times on permeability reduction of the sand
packed column was investigated. In addition, the pore-scale
development of reservoir pore throats and blocking the porous
media, under the optimal environmental conditions, was
confirmed by conducting a series of 2-d glass micromodel
experiments looking at imbibition of oily phase liquid in water-
saturated models.
The microbial plugging techniques studied within this
paper, encompasses only biopolymer induced permeability
reductions. It means plugging causes by cells which consist of
just biopolymers, not biomass produced by the
microorganisms.
II. MATERIALS
Two types of experimental setups were considered: sand
pack and micromodel. In the sand pack experimental setup, the
effects of temperature, salinity and incubation time were
investigated. To visualize the bacteria plugging in porous
media, micromodel setup was used. The Bacterial strain and
growth conditions were identical in both experimental setups.
A. Bacterial strain
In this study, the microorganism Bacillus Licheniformis
(PTCC 1595), was employed. This bacterium which is
commonly being used in microbial enhanced oil recovery
obtained from the Persian Type Culture Collection (PTCC),
Tehran, Iran. B. licheniformis produces lipopeptides as
biosurfactants which could release more trapped oil in the
small pores and necks in the porous oil media. Beside it can
produce polyglumatic acid (PGA) as an exopolymer in the
presence of sucrose [17]. We applied this bacteria strain
because of its potential to produce exopolymer to use for
porous medium profile modification.
B. Growth Conditions
The bacterial cells were prepared from frozen stock
cultures, maintained at -25°C, which were subcultured on a
weekly basis. The bacteria were cultured anaerobically on five
20ml-liquid growth mediums (Merck ® nutrient broth) for 24
hrs. B. licheniformis was cultured at 34°C in growth medium
(see Table I). The pH of medium was approximately 7.1.
Bacterial culture was centrifuged at 2500 RPM for 30 min and
then suspended in autoclaved water. The bacterial suspension
was placed on a magnetic stirrer and allowed to mix at room
temperature for 5 min. Again, the solution was centrifuged and
rinsed. The bacterial solution was then suspended in water and
placed on the magnetic stirrer to make a fresh bacterial
solution. The optical density of the bacterial solution was
measured by spectrophotometric analysis at 600 nm. The cell
density was determined using the direct count method and
adjusted at 5×108 (±107 ) cells/ ml. The salinity of the bacteria
solution was maintained at the same amount of the salinity of
the injected brine.
C. Fluids
1) Nutrient Solution
The nutrient solution was prepared according to Brown and
Jenneman et al. [18, 19]. Since feeding of microbes with nitrate
and phosphate containing nutrients has been found to be
effective in plugging, diammonium hydrogen phosphate and
(206 -- 917)
ammonium chloride were added to the nutrient solution [18,
19]. Table II shows the composition of the used nutrient. The
salinity of the nutrient solution was also adjusted to the same
salinity of the injected brine. All the media were purchased
from Merck ® Company.
2) Brine
Distilled water and four different brines with the salinity of
2, 5, 7, 10% w/v of NaCl are used in this study.
3) The n-decane
The n-decane (C10 H22 ) was used as the oily phase for
visual investigations in glass micromodel experiments. To trace
the n-decane movement into the etched glass micromodel, it
was dyed with Sudan red ( C24 H21 N5 ). Then, the dyed
nonaqueous phase was filtered using fine filter paper to remove
any solidified dye particles.
D. Porous Media
1) Sand pack column:
Sand-packs with grain distribution of 300-500 µm and
absolute permeability between 60-90 Darcy and porosity
between 44% − 48% were used in this investigation. The
sand-packed column was 30 cm in length and 2.8 cm in
diameter and was made of stainless steel. Sand pack column
with median grain size of 0.3-0.50 mm is used as a porous
media because sand is the major part of the reservoir. These
grain sizes were chosen to limit the plugging mechanisms to
exopolymer production and eliminate any impact of cell
growth [20] .To avoid the existence of other bacteria, column,
valves and sand grains were sterilized in autoclave for an hour.
Then they were re-sterilized with 70% ethanol.
2) 2D Glass Micromodel
Micromodel is a two-dimensional flow-channel network
etched in glass to simulate fluid flow in porous media. This
model has two distributors at the top and the bottom, the
bottom distributor was used for cleaning the model while the
top one was used for injection of oleic phase. The occurrence
of any physical phenomena in the simulated pore throats and
bodies during fluid flow can be observed through a stereo-
microscope recorded with a video camera and recorder.
Micromodels were made by etching mirror image pattern of
pore networks onto a glass plate using hydrofluoric acid (Fig.
1). Holes drilled on the lower plate serve either as inlet or
outlet ports. The etched plates are then sintered in a
programmable furnace. The pattern of matrix model is
completely random with the throats width varying randomly
between 0.1 to 0.2 mm and with random lengths. The fracture
width is 1.3 mm [21].
E. Permeability measurement:
Absolute permeability of the sand-packed column was
determined prior to the injection of the bacteria and after the
incubation time using the falling head method. Absolute
permeability, which is a measure of the ability of a porous
media to transmit fluids, is a characteristic property of porous
media and it is expressed by Darcy Law:
𝑄𝜇𝐿
𝑘= (1)
𝐴∆𝑃
 Proceedings of the 3rd (2011) CUTSE International Conference
Miri, Sarawak, Malaysia, 8-9 Nov, 2011 (207 -- 917)

In each experiment the sand-packed column's permeability TABLE II. THE COMPOSITION OF THE NUTRIENT SOLUTION
was determined before the injection of the bacteria and after Composition Concentration, w/v
shut-in period using the falling head method [22]. In the falling Sucrose 2%
head method (Fig. 2), the following equations apply: Diammonium hydrogen phosphate,
0.01%
𝑄 = 𝑑𝑉/𝑑𝑡 (2) ((NH 4 ) 2 HPO 4 )
Ammonium chloride, (NH 4 Cl) 0.02%

𝑑𝑉 = −𝑎 × 𝑑ℎ (3)

∆𝑃 = 1.0133 × 10−5 𝑔ℎ𝜌 (4)

Substituting equations 2, 3, and 4 into Darcy's Law gives

the following equation:
𝑎𝜇𝐿 𝑑ℎ
𝑘 = −1.0133 × 105 × (5)
𝐴𝜌𝑔ℎ 𝑑𝑡

The equation for calculation of permeability is easily

integrated to:
𝑎𝜇𝐿 ℎ
𝑘 = 1.0133 × 105 × ln � 0� (6)
𝐴𝑡𝑔𝜌 ℎ1

By monitoring changes of h during time t, permeability will

be determined from equation 6.
The permeability reduction can be calculated as follow:
𝑘𝑖 −𝑘𝑜 (7)
Permeability Reduction % (P.R.) = × 100
𝑘𝑖 Figure 1. Micromodel pattern.

F. Porosity measurement
The dried sand packed column/micromodel was vacuumed
and saturated with degassed distilled water. Pore volume and
porosity were determined from the change in weight as follow:
Pore Volume ( saturated weight − dried weight ) / 0.998
φ= = × 100
Bulk Volume bulk volume
G. Sand-packed column experimental procedure:
To avoid the existence of other bacteria, column, valves
and sand grains were sterilized in autoclave for an hour. Then
they were re-sterilized with 70% ethanol. Thereafter, the sand
packed was dried at 90°C to constant weight and subsequently
III. METHODS
In each experiment the sand–packed column is flushed out
with CO2 for 15 minutes to simulate a zone for an anaerobic
bacterium. Next, five pore volumes (PV) of brine (with salinity
of 0% – 7% NaCl, pH ≈ 7 and made from sterile water) were
(8) passed through each column at a constant flow rate of 80 ml/s
and 0.2 PV of bacterial solution ( 5 × 108 cells/ml) was
injected to the column using syringe pump. After injecting of
0.2 PV of nutrient (2% sucrose, 0.01% (NH4 )2 HPO4 ) at 10
ml/s, the column was pressurized with N2 (nitrogen) to 5 bar to
simulate the reservoir condition. Then it mounted horizontally
in a constant temperature- controlled room (25℃ up to 55℃ ) P P

evacuated and saturated with autoclaved water. After 2 hours, and was incubated anaerobically. After the shut-in period,
the porosity was calculated from the change in weight. Initial varying from 3-21 days, the columns were flooded with 1 PV
permeability, 𝑘𝑖 , was measured as mentioned earlier. of autoclaved water and final permeability, 𝑘𝑜 , of the sand
packed was measured [21]. The experimental apparatus for the
TABLE I. THE COMPOSITION OF THE GROWTH MEDIA packed–bed system is shown in Fig. 3.
Growth medium A a
Composition
gr/lit distilled water
Sucrose 1.0
h
NH4Cl 1.0 Stand Glass
Glucose 0.0 Pipe
Peptone from meat 10.0
A
Meat infusion 5.0 Sand
Na 2 HPO 4 2.0
Solium chloride 0.3
L

Figure 2. Schematic system for Permeability Measurement

 Proceedings of the 3rd (2011) CUTSE International Conference
Miri, Sarawak, Malaysia, 8-9 Nov, 2011 (208 -- 917)

Syringe pump The experiment first was carried out in packed-bed to

determine the effect of temperature and salinity on
permeability reduction. Then, micromodel experiments were
88:8 carried out to directly observation of bacteria effects on profile
Air Bath modification in porous media. Volumes of injected bacteria
and nutrients in each stage of experiments were similar and in
agreement with sand pack column procedure. The maximum
reduction in permeability for incubation time of 3 days was
Sand-Packed Column
48% that observed at salinity of 5% and 40℃ . This is theP

optimum conditions for maximum potential of B. licheniformis

Figure 3. Experimental apparatus for the packed–bed system
A. Micromodel Experimental Procedure
As it is shown in Fig. 4 to perform two set of experiments,
the model was maintained vertically in full-faced in front of a
programmable camera in the incubator by fix temperature at
25℃ . Two sets of procedures were done as follow:
P
to plugging.
A. Sand pack column experimental results:
1) Effect of water salinity:
Fig. 5 is shown the variation of permeability reduction in
different temperatures as a function of salinity in the sand-
packed models. The maximum plugging of B. licheniformis
was occurred at 5% NaCl. The incubation time was 3 days for
each experiment. The figure illustrates that the permeability

For the first experiment, first the dried micromodel was reduction increases for all temperature up to the salinity of 5%
saturated with water, the bottom distributor was closed. After and then decreasing as salinity is increasing. Also, permeability
that the top distributor was filled with n-decane ( C10 H22 ) reduction at 7% and 10% NaCl are higher than the measured
colored with Sudan red as the oily phase. Transparent values at 0% and 2% NaCl. Hence, B. licheniformis is suitable
micromodel was dyed with Sudan Red (C24 H21 N5 ) to trace the for permeability reduction in highly saline soils.
decane movement. Then, after filling the distributor with the
colored decane, enough time was given to decane to move
downward, pushing the water onwards. At the last stage,
photos were taken from the model at different time, clearly
show the oily phase movement velocity.
For the second experiment, in first stage, the glass
micromodel was cleaned by acetone and dried using a stream
of N2 . In the second stage, to sterile the model, 5 PVs of 70%
ethanol was injected to the model. Then the model was died
and several pore volumes of sterile water were passed through.
Afterward, one pore volume of a mixture (50:50) of the
bacteria solution (108 cells/cc of Bacillus Licheniformis) and
the nutrient ( 4% w/v, sucrose + 0.02% w/v, (NH4 )2 HPO4
+ 0.04%, w/v NH4 Cl) was injected from the bottom of the Figure 4. Schematic Diagram of Micromodel Experimental Set-up
model till it was saturated. Subsequently the model was
incubated at 25℃ for 4 days. At the end, after the shut-in
P

period passed the same procedure as described in first

experiment was employed to investigate the n-decane
movement using the photo camera.
IV. RESULTS AND DISCUSSIONS
A combination of sand-packed column and micromodel
experiments were used to evaluate the exopolymer plugging of
porous media. This combination provides direct measurement
as well as direct observations of plugging. In both cases, the
conditions were chosen to isolate the plugging mechanisms
associated with exopolymer production. It is observed that in
absence of exopolymer production, permeability reduction was
small and straining occurred due to the cell growth. Figure 5. Effect of Salinity on Permeability Reduction

Hence, grain size of 300 to 500 µm was selected for the

packed-bed experiment to ensure that straining did not occur.
Likewise, the porous media in the micromodel was large
enough to avoid straining. Therefore, permeability reduction
due to straining was not considered here.
 Proceedings of the 3rd (2011) CUTSE International Conference
Miri, Sarawak, Malaysia, 8-9 Nov, 2011 (209 -- 917)

unplugged one. These results can be compared by the

microscopic photos from the pores of micromodels before and
after microbial injection as they are shown in Figs. 10 and 11.
The pore size is reduced as a result of biopolymer production.
V. CONCLUSION:
4B

Sand packed column and 2-D glass micromodel were used

Figure 6. Effect of temperature on Permeability Reduction
2) Effect of Temperature:
The observed permeability reduction at different
to evaluate permeability reduction caused by exopolymer-
producing bacteria. A number of experiments at different
salinities (0, 2, 5, 7 and 10 % w/v of NaCl) and temperatures
(25, 40 and 55°C) in both setups have been made. The cell
density, type and concentration of nutrients in all experiments
were also kept constant. By implementation of different sets of
experiments with these two experimental setups, it was realized
that B. licheniformis may be used for the batches or the soils
with high concentration of salt and this type of bacteria can be
applied to temperature ranges of 25℃ to 55℃ and reduced the
P P

temperatures is illustrated in Fig. 6 The maximum potential of permeability of the soil dramatically. The maximum reduction
B. licheniformis to produce bio-polymer has occurred at 40℃ . P

in permeability was 48% at 5% salinity and 40℃.

It is evident that at different salinities the permeability
reduction increases by increasing the temperature from 25℃ to P
In addition in the visual evaluation of profile modification
40℃ and then decreasing as temperature is increasing. The
P
via 2-D glass micromodel injection profiles taken before and
incubation time for each experiment was 3 days. after the microbial treatment demonstrated that the thief zones
were plugged by the biopolymer resulting in fluid diversion.
The results indicate that the proposed method using this The results indicate that microbial plugging may be a useful
type of bacteria may be certainly used for a range of and effective tool for reducing flow in high permeable thief
temperatures between 25℃ to 55℃ . P P
zones.
3) Effect of incubation time:
Three experiments were performed to investigate the effect
of time on permeability reduction (Fig. 7). In each run, the
concentration of bacteria and the concentration of nutrients
were the same. The permeability was measured after 3, 10, and
21 days of bacteria injection. As it is shown, the permeability is
reduced by 45%, 58%, and 63% as the time of incubation
increased in 3, 7 and 21 days, respectively. The results indicate
that bacteria can reduce the permeability of the soil
dramatically as incubation time is increasing.

Figure 9. Oil Migration into Figure 8. Oil Migration into

Micromodel, without permeability Micromodel, with permeability
reduction, after 5 days reduction, after 5 days

Figure 7. Effect of incubation time on pereability reduction (Salinity=1%,

T=40℃ ) P

B. Micromodel experimental results:

14B

Two sets of experiments were performed with one of the

micromodels with bacterial solution and the other without it.
Figs. 8 and 9 clearly comprise the very low movement of oil
from top to bottom in plugged micromodel due to permeability
reduction in porous media and deep movement of oil in Figure 10. Micromodel Pore Size without Bacteria Injection
 Proceedings of the 3rd (2011) CUTSE International Conference
Miri, Sarawak, Malaysia, 8-9 Nov, 2011
Figure 11. Micromodel Pore Size with Bacteria Injection
NOMENCLATURE
𝑘 Absolute permeability (Darcy)
𝑄 Volumetric flow rate (cm3 /s)
𝐴 Cross-sectional area of flow (cm2 )
𝐿 Length of flow (cm)
∆𝑃 Pressure difference (atm)
𝑉 Volume of liquid (cm3 )
𝑡 Time (second)
𝑎 Cross-section area (cm2 )
ℎ Height of liquid (m)
𝜌 Density of the liquid (kg/m3 )
𝜇 Liquid viscosity (cp)
𝑔 9.8 m2 /s
𝐾𝑖 Initial permeability before injection of bacteria (Darcy)
𝐾𝑜 Final permeability after shut-in period (Darcy)
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