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Energy Sources, Part A: Recovery,

Utilization, and Environmental Effects
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A Laboratory Study for Assessing

Microbial Enhanced Oil Recovery
M. Bao a , T. Liu b , Z. Chen c , L. Guo b , G. Jiang d , Y. Li a & X. Li b
Key Laboratory of Marine Chemistry Theory and Technology ,
Ministry of Education, Ocean University of China , Qingdao , China
Research Institute of Oil Production Technology, Shengli Oil Field
Corporation , Sinopec , Dongying , China
Department of Building, Civil and Environmental Engineering ,
Concordia University , Montreal , Quebec , Canada
College of Petroleum Engineering, China University of Petroleum ,
Beijing , China
Published online: 30 Sep 2013.

To cite this article: M. Bao , T. Liu , Z. Chen , L. Guo , G. Jiang , Y. Li & X. Li (2013) A Laboratory
Study for Assessing Microbial Enhanced Oil Recovery, Energy Sources, Part A: Recovery, Utilization,
and Environmental Effects, 35:22, 2141-2148, DOI: 10.1080/15567036.2010.492380

To link to this article: http://dx.doi.org/10.1080/15567036.2010.492380


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Energy Sources, Part A, 35:2141–2148, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1556-7036 print/1556-7230 online
DOI: 10.1080/15567036.2010.492380

A Laboratory Study for Assessing

Microbial Enhanced Oil Recovery

M. Bao,1 T. Liu,2 Z. Chen,3 L. Guo,2 G. Jiang,4 Y. Li,1 and X. Li2

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Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education,
Ocean University of China, Qingdao, China
Research Institute of Oil Production Technology, Shengli Oil Field Corporation,
Sinopec, Dongying, China
3Department of Building, Civil and Environmental Engineering, Concordia University,

Montreal, Quebec, Canada

4College of Petroleum Engineering, China University of Petroleum, Beijing, China

Microbial enhanced oil recovery utilizes microorganisms and their metabolic products to improve the
oil recovery. A pilot scale study was conducted to investigate the effectiveness of two microorganisms
(D-2 and M-1), presenting as an individual and a mixture, by recovering residual oil from a sandstone
core oil reservoir at Shengli Oil Reservoir, China. Five microbial flooding tests were conducted
sequentially by injecting microbial cultures into the experimental reservoir. The results showed that a
polymer surfactant produced by D-2, M-1, and their mixture, enhanced oil recovery by 5.4, 5.6, and
7.9%, respectively, which are within the reported ranges of increased tertiary oil recovery.

Keywords: microbe, microbial enhanced oil recovery, oil reservoir, polymer, surfactant


Microbial enhanced oil recovery (MEOR) is a process in which microorganisms are used in
situ to alter the oil reservoir microbial environment in order to improve the recovery of oil
trapped in porous media and to increase economic profits (Ollivier and Magot, 2005; Lazar
et al., 2007; Bao et al., 2008). While conventional oil recovery processes (including primary
and secondary recovery) can extract up to 35–45% of the original oil in place, by using tertiary
recovery techniques that are typically performed on nearly depleted oil fields, production may be
increased by 5–15% (Tzimas et al., 2005).
MEOR is a relatively inexpensive and environmentally compatible method for tertiary oil
recovery and is being optimized through the development of more effective microorganisms that
subsist on inexpensive and abundant nutrients present in oil reservoirs (Delshad et al., 2002; Hao
et al., 2004; McInerney et al., 2005). However, most MEOR techniques are application-specific,
and the choice of microorganisms is critical and field sensitive. In addition, even a few percent
of additional oil recovery can contribute an enormous amount, cumulatively and across the whole

Address correspondence to Prof. Zhi Chen, Department of Building, Civil and Environmental Engineering, Concordia
University, Montreal, Quebec H3G 1M8, Canada. E-mail: zhichen@alcor.concordia.ca

2142 M. BAO ET AL.

region. Thus, MEOR studies are continuously being performed on specific oil fields worldwide
(Lazar et al., 2007).
Shengli oil fields and most of China’s other oil fields started production in the early 1960s
and most of the reservoirs are reaching the end of their life. These reservoirs now require new
enhanced oil recovery technologies (such as MEOR) to increase the oil production and at the
same time to reduce the environmental impact caused by the injection of chemicals into the oil
The purpose of the present study was to conduct a pilot experiment using the MEOR technique
on Shengli oil fields. The success of MEOR depends on the selection of suitable microorganisms
for the specific reservoirs studied. Two species of bacteria (D-2 and M-1), previously isolated from
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oil produced at the Shengli oil fields, were chosen for this study. The products of their bacterial
metabolism (surfactant and polymer) have previously been characterized by Bao et al. (2008).


2.1. An Overview of Shengli Oil Fields Reservoir Conditions

The pilot test was conducted on the Ng3 cell, Central Region I, Gudao reservoir, Shengli oil
fields, China. The study area was 0.84 km2 with a depth ranging from 1,173 to 1,230 m. The oil-
bearing layers of the Ng3 cell consist of a sand polymer oil reservoir deposited by a winding river.
The sand is characterized by a high permeability, high viscosity, and high water content. The
reservoir was formed by sandstone at a temperature of 69 ıC and an average pressure of 10.79 MPa.
The porosity of the reservoir was 33%, the average effective thickness was 10.4 m, and the air
permeability was 1.5–2.5 µm2. This data were used to design and build the experimental
sandstone core/oil reservoir used in the MEOR pilot experiment.

2.2. Analysis of Shengli Oil Fields Crude Oil Samples (Oil and Brine)
Crude oil samples were obtained from oil wells 8X815, 7N11, 3C125, and 6-13. The crude oil
samples contained oil plus produced water (brine) and sand. The oil and brine were isolated
separately from the crude oil samples by letting the samples settle overnight at 45ıC. Second,
the lower layer (brine) was carefully removed and the upper layer was taken (oil) into separate
test tubes using a sterile pipette. The percentage of wax content and the solidification point of the
crude oil samples were relatively low (<3.1%, and 15ıC, respectively).
The analysis of the brine was performed to help understand the characteristics of formation
water (water present naturally within the pores of the oil reservoir rock) and the possible effects
of the brine on the growth and metabolism of the injected MEOR microorganism(s). Based
on the analysis, the total mineral content of the natural oil reservoir influent flooding water
(brine) was about 7,900 mg L—1, which was close to the mineral content in the produced water
(brine) obtained from the crude oil samples (6,734–7,738 mg L—1). The produced water contained
NaHCO3 at a weak alkaline pH ranging from 7.94 to 8.30. Ions in the formation water may provide
inorganic nutrients required by microorganisms. Thus, the metal and sulfate were measured in this
experimental study using the oil reservoir water analysis standard method SY/T 5523-2006. Both
the oil reservoir flooding water and the oil sample produced water did not contain heavy metal ions
(such as Hg, Cr), and the amount of SO24— was also low, which would be good for inhibiting the
growth and metabolism of sulfate-reducing bacteria; therefore, the low ion concentrations would
not be expected to prohibit the growth of the injected D-2 and M-1 test microorganisms.

2.3. Pilot Scale MEOR Experiment

For the pilot scale laboratory MEOR study, D-2 and M-1 microorganisms were isolated from
the water contaminated by 200 mL of the L1N1 crude oil sample. The crude oil had 27.6 ıAPI
(American Petroleum Institute gravity) and a viscosity of 46.3 mPas at 69ıC. LIN1 oil was chosen
based on the preliminary experiments, being representative of the crude oil in Shengli oil fields.
The bacterial cultures (and zero culture control) described below were then incubated for 15 days
in culture media to allow time to produce and accumulate products of bacterial metabolism. The
cultures were then mixed with LIN1 oil (oil C brine) as shown in Figure 1 and injected into a
pilot scale experimental sandstone core/oil reservoir in order to achieve experimental reservoir
“flooding” with the microorganisms, and the flooded core oil reservoirs were incubated for a
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further 15 days to allow the microorganisms and the products of their metabolism to interact
with the oil inside the core. The core reservoir effluent was then sampled and the samples were
analyzed for the amount of oil present as previously described (Bao et al., 2008).

2.3.1. MEOR Experiment Set Up

The experimental equipment consisted of three units: a fluid injection system, a stainless steel
tube containing the test sandstone core, and a production unit (Figure 1). The fluid injection system
consisted of microorganisms, oil, and brine injection sections. The sandstone core was compacted
in a stainless steel tube with a pressure sensor, which recorded the pressure differences between
the inlet and outlet of the cylinder. Samples were obtained from the effluent of the sandstone core
flooding tests and were analyzed in the lab.

2.3.2. MEOR Experiment Procedures

Based on the zone horizontal permeability (1.5–2.5 µm2) in the case field, appropriate quartz
sands were chosen, including 13% at size 0.30–0.40 mm, 16% at size 0.40–0.60 mm, 29% at 0.60–
0.80 mm, 25.8% at 0.80–1.00 mm, 11.5% at 1.00–1.50 mm, and 4.7% at 2.00–3.00 mm of quartz
sands; to form five experimental sandstone cores. These sizes of quartz sands were mixed until
they were homogenous and packed into the cylinder in order to form the experimental sandstone
core/oil reservoir, using a pressure of 30 MPa for 30 sec. The core was fit tightly between two
end plates of the cylinder. A vacuum was applied for 30 min until the negative pressure of the
sandstone core cylinder reached 0.1 MPa. After that, brine produced from the study region was
injected into the cylinder at a low rate initially to prevent the equipment from being damaged.

FIGURE 1 Pilot experiment set up and procedures.

2144 M. BAO ET AL.

Experimental Conditions for the Five MEOR Test Runs

Dimension of Core
Cultured Experimental Total Volume of
Test Microorganisms Incubation Core/Reservoir, Pore Core Core Crude Oil
Run Applied in the Media Diameter — Volume, Porosity, Permeability, Oil in the Saturation,
Number Flooding Test Name Length mL % µm2 Core, mL %

Blank — — 38 — 600 mm 213 31.3 1.67 190 89.2

1 D-2 C 1a 214 31.5 1.72 194 90.7
2 M-1 C 2b 212 31.2 1.75 188 88.7
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3 D-2 C M-1 C 1a 225 33.1 1.85 204 90.7

4 D-2 C M-1 C 2b 218 32.3 1.78 198 90.8
a Incubation medium C for bio-surfactant producing by D-2 bacteria: KH PO 0.3 g, Na HPO 0.6 g, FeSO 0.2 g,
1 2 4 2 4 4
yeast 0.5 g, 2% liquid paraffin added as carbon source.
b Incubation medium C for bio-polymer producing by M-1 bacteria: KH PO 0.5 g, NaNO 1 g, sucrose 10 g, citrate
2 2 4 3
acid 1.3 g, yeast 0.5 g.

The core should be 100% saturated by brine. The experimental sandstone cores were placed in
the experimental set up shown in Figure 1 and were used for the MEOR pilot experiments.
The inlet tubing of the core was connected to the experimental LIN1 oil accumulator. The LIN1
crude oil sample was injected continuously until the core was saturated, and the core was placed
for 3 days at 69ıC in order to allow time for equilibration prior to the microbiology flooding
The inlet tubing of the core was connected to the accumulator, which contained the test
microbiological culture. Pore volume (PV) of the sample core was measured ranging from 212
to 225 with an average of 216.4 mL (i.e., 1 PV D 216.4 mL). The culture was added two times,
the first was added with 0.25 PV culture and 0.25 PV log phase bacteria; the second was added
with 0.5 PV culture. The time interval to add culture was 15 days. Analysis showed that the
surfactant and polymer produced during the period of culture contained lipopolysaccharide at a
concentration of 2:2 ~ 10—2 gL—1 and extracellular polymer at a concentration of 1.1 gL—1, as
well as protein. The core was continuously injected/flooded with the test culture. The procedure
stopped when the outlet of the core contained only the test culture/surfactant/polymer.
In addition to these tests, a control run (“Blank” in Table 1) was included where the core was
injected and flooded with pure nutrient medium (zero microorganisms/surfactant/polymer). Five
experimental runs were performed sequentially using the same experimental pilot scale sandstone
core/oil reservoir. The core was refilled between runs. Table 1 shows the details of each run,
which included a blank culture injection and four microbial injections.


Analysis results from five flooding tests of the experimental sandstone core/oil reservoir are shown
in Table 2. The indigenous bacteria were found in the formation water; the microbial content was
measured using the blood counting chamber. As the D-2 and M-1 were different at the bacterial
colony, the bacterial count was measured through a maximum probable number. The cultures of
D-2 and M-1 bacteria were analyzed for the number of bacteria present in the experiment effluents.
The number of indigenous bacteria were observed to be 60 cells mL—1, which were reduced from
130 cells mL—1 measured in the influent before injection into the system; the cultures of D-2
and M-1 bacteria became the following: D-2 are 6:4 ~ 107 mL—1 (compared to 8:4 ~ 107 mL—1,

Results of the Five Sequential MEOR Tests Performed on the Experimental Core Oil Reservoir

Oil Recovery Oil Recovery Total Oil Oil Recovery Oil Recovery Total Oil
After First After Second Recovery from After First After Second Recovery from
Test Run Water Water Water Flooding, Microbial Microbial Microbial
Number Flooding, % Flooding, % % Flooding, % Flooding, % Flooding, %

Blank 53.4 10.5 63.9 1.2 0.5 1.7

1 54.1 11.3 65.4 4.2 1.2 5.4
2 54.2 10.6 64.8 4.0 1.6 5.6
3 51.6 8.8 60.4 3.9 1.4 5.3
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4 53.1 10.2 63.3 5.6 2.3 7.9

before injection), M-1 are 1:2 ~ 108 mL—1 (compared to 2:3 ~ 107 mL—1 before injection), and
particularly the D-2/M-1 mixture are 7:1 ~ 107 (compared to 1:6 ~ 108 mL—1 before injection).
As seen in Table 1, the dimensions of the core did not change throughout the sequence of five
flood tests/runs and remained constant at 38 ~ 600 mm. The core total pore volume also remained
constant with a mean of 216 mL. At the time that the flood test/run was performed with the
injected blank culture (zero microorganisms), the core was determined to have a core total pore
volume of 213 mL, porosity of 31.3%, and permeability of 1.67 µm2 (Table 1). The flooding
process using the blank culture contained 190 mL oil in the core, demonstrating 89.2% residual
oil in the core.
As shown in Table 2, for the four microbial test runs, the oil recovery resulting from the water
flooding was in the range of 60–65%. After the injection of test cultures, run #1 produced an
additional 3.7% of oil and run #2 increased oil recovery by an additional 3.9% compared to the
blank run, demonstrating the improved efficiency of oil recovery. For flooding tests using the
mixture of the two cultures (D-2 plus M-1), oil recovery in the nutrient medium C1 (run #3) is
lower than that in the nutrient medium C2 (run #4). However, Table 2 indicates that results from
the two mixture runs (run #3 and run #4) are better than the single culture (run #1 and run #2),
especially the result obtained in the nutrient medium C2 (run #4), which showed a 6.2% additional
oil recovery compared to the blank run.
The relation of the injected culture volume with the experimental sandstone core inlet pressure
for each test run is shown in Figure 2a. The inlet pressure increased sharply when the injected
culture/surfactant/polymer injection reached 1.0–1.5 PV and then remained relatively constant.
The pressure change induced in test run #4 by the injection of the mixture of D-2 and M-1 was
significantly greater than that observed in the other experiments. The pressure changes observed
during the test culture injections indicated that the mobilized oil was released after 1.0–1.5 PV
(Figure 2a).
Recovered oil in the effluent showed a similar trend as shown in Figure 2b. The amount of oil
detected in the effluent at the core outlet in each experiment decreased with increasing injected
culture volume. The core effluent oil concentration observed at 5.0 PV of the injected culture was
between 15–20% of that observed at 0.5 PV (Figure 2b). The oil concentration in the effluent
tended to be highest for the test runs where the mixture of D-2 plus M-1 was injected (test run
Figure 2a shows that the volume of oil recovered from the core increased with the injected
culture volume. The oil recovered after the injection of the culture containing the mixture of D-2
plus M-1 (test run #3, Figure 2c triangles) was greater (1.25–1.9 mL) than the other cultures after
about 3.5 PV culture injection and the largest amount of oil recovered was 11.5 mL obtained at
5.0 PV (Figure 2c).
2146 M. BAO ET AL.
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FIGURE 2 The relation of injected culture volume with (a) the experimental sandstone core inlet pressure,
(b) the amount of oil in the effluent, and (c) the amount of oil recovered.

It is observed that oil recovery from test run #3 (the culture containing the mixture of D-2
plus M-1 cultured in C1 medium) was about 1.5 times that in the other three test runs (Figure
2c); especially, it was better than the test run #4, which also contained the mixture of D-2 plus M-
1, but in a different culture medium C2. The main difference between the mediums culture C1 (test
run #3) vs. C2 (test run #4) was the 2% liquid paraffin, added as a carbon source in C 1 vs. sucrose
added in C2. The results indicate that the particular incorporation of 2% liquid paraffin in the C 1
culture medium with the two bacterial cultures D-2 and M-1 synergistically enhanced oil recovery
from the experimental sandstone core/oil reservoir significantly.
Although the core pressure was found to increase with the increased volume of the culture
injection, little gas generation was observed in these experiments. This phenomenon needs to be
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further studied as a number of different gases were reported during a flooding test by Kong et al.
Finally, the metabolites produced by the bacteria in the culture medium were analyzed as

● The D-2 utilized hydrocarbon as a carbon source and produced bioemulsifier containing
polysaccharide-lipid complexes to emulsify the oil. Polysaccharides, including glucose, man-
nose, and rhamnose, were indentified and found stable at a wide range of pH, temperature,
and salinity. Also, D-2 mainly metabolized bioemulsifier at 2:2 ~ 10—2 gL—1 together with
the lipopolysaccharide.
● The M-1 produced extra-cellular polymer, which included 50.8% amylase. The result of
gas chromatography analysis showed that the amylose contained mannose, glucose, and
galactose at 51.8, 25.4, and 22.8%, respectively. ˇ-D-Pyran polysaccharide and ˛-D-furan
polysaccharide were also found in amylase through an IR analysis.


The MEOR technology was examined and applied to Shengli oil field, the second largest oil field
in China, to enhance in-depth oil recovery in this research. The composition of the crude oil
samples obtained was first analyzed, and a pilot scale MEOR laboratory study was conducted.
Cultures of D-2 and M-1 bacteria separated from Shengli oil field produced water were tested
alone or in a mixture, by flooding an experimental sandstone core oil reservoir that had been
saturated with an oil (LIN1) representative of the Shengli oil field. The microorganisms were
grown in two different media for 15 days prior to flooding the core (media C 1 and C2). Five
experimental runs were performed sequentially using the same experimental core, including one
blank cultured injection and four microbial runs. The experimental results showed that M-1 and
the D-2/M-1 mixture both produced polymer surfactant, which enhanced oil recovery by 5.4,
5.6, and 7.9%, respectively. The improved recovery efficiencies were within the reported ranges
of increased tertiary oil recovery. One particular combination (a culture containing a mixture of
both bacteria) grown in the C1 media (containing 2% parafilm) proved to be the most effective
approach for recovering oil from the experimental core compared to other combinations tested,
i.e., D-2 or M-1 alone, D-2 plus M-1 in C2 media containing sucrose instead of 2% paraffin. As
a result, this study contributed with a novel pilot investigation of field application of MEOR.


This research was funded by the National Science Foundation of China (Grant No. 50604013).
2148 M. BAO ET AL.


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