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Application of Biosurfactants to
Wettability Alteration and IFT Reduction
in Enhanced Oil Recovery From Oil–Wet
Carbonates
a b c
D. Biria , E. Maghsoudi & R. Roostaazad
a
Department of Biotechnology, Faculty of Advanced Sciences and
Technologies , University of Isfahan , Isfahan , Iran
b
Department of Civil Engineering , École Polytechnique University ,
Montreal , Canada
c
Chemical and Petroleum Engineering Department , Sharif
University of Technology , Tehran , Iran
Published online: 24 May 2013.

To cite this article: D. Biria , E. Maghsoudi & R. Roostaazad (2013) Application of Biosurfactants
to Wettability Alteration and IFT Reduction in Enhanced Oil Recovery From Oil–Wet Carbonates,
Petroleum Science and Technology, 31:12, 1259-1267, DOI: 10.1080/10916466.2011.606554

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 Petroleum Science and Technology, 31:1259–1267, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1091-6466 print/1532-2459 online
DOI: 10.1080/10916466.2011.606554

Application of Biosurfactants to Wettability Alteration

and IFT Reduction in Enhanced Oil Recovery From
Oil–Wet Carbonates
Downloaded by [University of Connecticut] at 01:30 10 October 2014

D. Biria,1 E. Maghsoudi,2 and R. Roostaazad3

1
Department of Biotechnology, Faculty of Advanced Sciences and Technologies,
University of Isfahan, Isfahan, Iran
2
Department of Civil Engineering, École Polytechnique University, Montreal, Canada
3
Chemical and Petroleum Engineering Department, Sharif University of Technology,
Tehran, Iran

To obtain potentially applicable microorganisms to an effective in situ microbial enhanced oil recovery
operation, bacteria that were compatible with the harsh conditions of a petroleum reservoir were
isolated from a crude oil sample. The application of an oil spreading technique showed that all of
the isolates were capable of producing biosurfactants from both the glucose and crude oil as carbon
sources. The secreted biosurfactants could at least reduce the surface tension 20 mN/m and for one
of the isolates; the surface tension value dropped below 40 mN/m. In addition, the contact angle
measurements revealed that the produced biosurfactants could effectively alter the wettability of the
oil saturated rock samples. At last, the effect of isolates and their biosurfactants on improving oil
production from oil saturated rock samples was investigated. It was observed that the presence of
bacteria in the system could increase the amount of produced oil in comparison with the case where
cell free biosurfactants were utilized.

Keywords: bacteria, biosurfactant, enhanced oil recovery, interfacial tension, wettability

INTRODUCTION

Indubitably, crude oil has been of great importance as a major energy source and raw feed to
petrochemical industries. To satisfy the growing world’s demand to this valuable material, the
recovery of oil from its limited reserves should be intensified. Consequently, several enhanced
oil recovery (EOR) techniques have been developed to increase the cumulative oil production
from the reservoirs. One of these methods involving surfactants application is surfactant flooding.
It is asserted that surfactants can lead to EOR in two different ways. First, by reducing the
interfacial tension between aqueous and nonaqueous phases existing in the porous media, the
capillary pressure will be lessened, and as a result, the trapped oil in the vuggy structure of the
porous rock would flow (Babadagli, 2002; Ayirala and Rao, 2004). Second, in oil-wet reservoirs
by altering the wettability of the reservoir formation rock from oil wet to water wet, the free

Address correspondence to D. Biria, Department of Biotechnology, Faculty of Advanced Sciences and Technologies,
University of Isfahan, Isfahan, Iran. E-mail: d.biria@ast.ui.ac.ir

1259
 1260 D. BIRIA ET AL.

imbibing process would be strengthened boosting the amount of oil production in a water-flooding
process (Austad and Standnes, 2003; Zhang et al., 2006; Gupta and Mohanty, 2011). This is
known as one of the most effective recovery mechanisms in the fractured reservoirs (Morrow and
Mason, 2001; Adibhatla and Mohanty, 2008). Although synthetic surfactants have widely been
used for environmental purposes and EOR, the application of their microbial counterparts—that
is, biosurfactants secreted by microorganisms has turned into a major trend in current years (Iqbal
et al., 1995; Mulligan et al., 2001; Kowalewski et al., 2006; Biria et al., 2007; Das and Mukhrejee,
2007). This change of focus has been due to the inherent merits of biosurfactants. As a case in
point, biosurfactants are less toxic, and as a consequence, they are not considered a threat to the
environment. In addition, it is possible to produce varieties of biosurfactants that are compatible
with such conditions as higher temperatures, salinity, and pH extremes and they can preserve their
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activity under such circumstances. Finally, their production is economical because it is possible to
synthesize them through fermentative processes (Fiechter, 1992; Rosenberg, 1993; Karanth et al.,
1999; Cha, 2000; Thavasi et al., 2008).
However, prior to the application of biosurfactants in an EOR process, their ability to intensify
the oil production from reservoir rock should be guaranteed through careful studies. The inter-
actions between the surface active molecules and oil-water interface and reservoir rock surface
determine the amount of oil which could be produced as a result of an EOR process. These
interactions have been quantified as the ratio of viscose to capillary forces in the form of capillary
number that is shown as
v
NC a D ; (1)
 cos 
where v and  are the velocity and viscosity of the displacing fluid,  is the oil water interfacial
tension and  is the contact angle that is considered as a measure of surface wettability. It has
been reported that an increase of four to six orders of magnitude in capillary number is required
to have an effective EOR operation (Klins, 1984). Unfortunately, such outcome is not satisfied
by many of the currently reported biosurfactants. In this work, the ability of several crude oil
isolated bacteria to produce suitable biosurfactants for EOR applications was examined through
a screening procedure. Then, the selected bacterium was used in oil production experiments.

MATERIAL AND METHOD

Bacteria Isolation From Crude Oil

The isolation of microorganisms was carried out by putting Masjed-Soleyman (MS), a south-
western oil field in Iran, crude oil samples adjacent to Bushnell-Hass mineral culturing medium
enriched with 1.5 g/L yeast extract as an extra nitrogen source at 45ıC. The rationale for employing
a nitrogen rich medium was that it could help activate the spores of possibly spore forming species
existing in the sample. The incubation lasted for two weeks after which the isolates were plated
on the solid medium containing nutrient agar. Subsequently, separate morphologically distinct
colonies were formed as the result of a three stage linear cultivation on the solid medium. The
type of isolated microorganisms was preliminarily identified based on the colony morphology of
isolates, gram staining and microscopic observations. Moreover, motility, temperature, salinity,
and pH ranges of growth were also determined for each isolate. In particular, one of the species
that had the greatest tensioactive properties was selected for more analysis. Morphological and
physiological characteristics of this isolate were either studied on nutrient agar or in nutrient broth
plus 10% (w/v) NaCl. Gram reaction; motility; shape and color of colony; catalase, urease and
oxidase activities; nitrate reduction; tween 80 hydrolyses; Voges-Proskauer; and methyl red were
 ENHANCED OIL RECOVERY FROM OIL–WET CARBONATES 1261

checked as recommended by Smibert and Krieg (1994). Acid production from carbohydrates, sug-
ars, and carbon and nitrogen sources utilization were performed according to Ventosa et al. (1982).
The genomic DNA of the selected strain was extracted with the Genelute DNA extraction kit,
following the manufacturer’s recommended procedure. The 16S-rDNA genes were amplified using
8F (50-AAGAGTTTGATCATGGCTCAG-30) and 1541R (50-AGGAGGTGATCCAACCGCA-
30) universal primers.

Screening
Biosurfactant Production and Activity
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Each isolate was cultivated in a liquid medium containing 5 g/L glucose, 1 g/L KH2 PO4 , 0.5 g/L
K2 HPO4 , 0.5 g/L MgSO4 .7H2 O, 2 g/L NaCl, 1 g/L NH4 NO3 , and 0.5 g/L NaNO3 and incubated
for eight days at 45ıC. The cultivation was repeated for each isolate substituting the glucose in
the previous medium with 1% (v/v) crude oil as the carbon source. No agitation technique was
utilized in the process. After the period, the hydrocarbon top layer was decanted and the broth
was centrifuged (Hettiche-Rotina 38R) at 10,000 rpm for 20 min to obtain a cell free solution
containing the secreted biosurfactants. These solutions were both stored at 4ı C for subsequent
experiments.
Biosurfactant production capability of the isolates was quickly investigated by oil spreading
method (Youssef et al., 2004). In addition, the amount of secreted biosurfactant for each species
was measured on the basis of critical micelle dilution (McInerney et al., 2004). Surface tension
of biosurfactant solutions was measured using a k8 Kruss ring tensiometer. This was performed
for both cell free and the original broths.
Contact angle was used to investigate the effect of biosurfactant solution on the rock surface
wettability. The contact angle of a fluid with a solid surface can be regarded as an index of its
wettability (Shedid and Ghannam, 2004). The contact angle can be defined as the angle between
the tangent to the periphery of the point of fluid contact with the solid, and the surface of the
solid in the direction where the droplet exists as shown in Figure 1. Sessile drop method was
employed to measure contact angles. Briefly, the polished surface of Asmary outcrop rock sample
from MS oil field was used for measuring the contact angle. The oil saturated rock sample was
immersed in 5% brine for a period of 40 days at 70ıC. After this period, the rock was removed
from the solution, cleaned and dried. Then a drop of 5% brine was placed on the surface and
a digital camera was used to take high-resolution photos from the profile of the droplet until its
deformation stopped. Then the contact angle of the droplet was measured and this final value was
reported as the contact angle of the brine solution. The same procedure was repeated for each
biosurfactant solution. The concentration of biosurfactant solution used instead of the brine was
adjusted to 10 CMC.

FIGURE 1 The contact angle.

 1262 D. BIRIA ET AL.
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FIGURE 2 Schematic view of the oil production setup.

Improving Efficiency in EOR

To investigate the impact of isolates and their biosurfactants on oil production, the amounts of
produced oil from oil-saturated rock samples were measured. To achieve this end, the oil-saturated
rock samples were immersed in biosurfactant solution and the results were compared with a blank
test in which five percent brine was utilized instead of the biosurfactant solution. Figure 2 indicates
the setup used for these experiments.
In practice, the rock samples from MS outcrop with porosity of about 15% and permeability of
0.001 darcy were saturated with crude from the same reservoir at 70ıC. Then each saturated plug
was used in a relevant experiment. In other words, as represented in Figure 2, the containers in the
setup were filled with such solutions as biosurfactant solution (10 CMC) plus bacteria, cell free
biosurfactant with high (10 CMC) and low (1 CMC) concentration and 5% brine, respectively.
Then, each rock sample was immersed into one of these containers. The incubation of 45ıC lasted
for 40 days and the amount of produced oil was recorded every five days.

RESULTS AND DISCUSSION

Isolation and Characterization

Six different morphological colonies were obtained from the isolation stage. They were all rod
shaped bacteria and except for one, the others were all gram positive. The fact that the majority of
the isolates were gram positive was interesting because as is often the case the bacteria separated
from hydrocarbon contaminations have been reported as gram negative. It has been claimed that
the gram-negative feature of the bacteria is closely related to their survival capability under
sever conditions (Bicca et al., 1999). All the isolates were motile and remained active at high
temperatures, pH, and salinities. These bacteria were identified as facultative capable of forming
spores. Moreover, none of the species were rated as sulfate-reducing bacteria (SRB). Table 1
indicates details of bacterial characterizations.
The results of the screening revealed that isolate no. 3 showed a better performance in the
production of biosurfactant compared with the others. Its primary biochemical characteristics are
also appended to Table 1. The 840-bp of 16S rDNA gene of the strain no. 3 has been deposited
in GenBank under the accession number EU715326. Alignment of this sequence with described
 ENHANCED OIL RECOVERY FROM OIL–WET CARBONATES 1263

TABLE 1
Phenotypic Characteristics of the Isolated Bacteria

Temperature Salinity
Gram pH Upper Limit, Upper Limit,
ı
No. Morphology Spore Motility Reaction Range C % SRB

1 Bacilli C C C 6–9 75 22 —
2 Bacilli C C C 5–10 70 26 —
3 Bacilli C C C 5–10 80 25 —
4 Bacilli C C C 6–9 65 17 —
5 Bacilli C C 6–10 75 25 —
6 Bacilli C C C 6–9 75 20 —
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Biochemical characteristics of strain no. 3 (B. licheniformis MS3)

Acid production from: L-arabinose, Amylase C, Protease C, Utilizing Inulin, D-fructose,

D-fructose, D-galactose, Gelatinase , Urease C, D-galactose, D-glucose, Starch,
D-glucose, maltose, D-mannitol, Catalase C, Oxidase C, Nitrate Sucrose, Lactose, D-mannose,
D-mannose, sucrose, D-salicin reduction C, Dnase , capable D-salicin
of hydrolysis of tween 80

species showed more than 99% similarity to Bacillus licheniformis and it was named Bacillus
licheniformis MS3 strain.

Biosurfactant Production and Activity

The results of oil spreading method indicated that all isolates under investigation were capable of
producing biosurfactants. However, when the crude was used as the carbon source, the amount of
secreted biosurfactant was comparatively higher for the crude than that of glucose. In fact, CMD
of the biosurfactants from crude containing medium was about 40 CMC while it was about 15–20
CMC for that of glucose. Evidently, such difference reflected the significant role of the crude as
the carbon source in improving the secretion of biosurfactants by these bacteria (Peihui et al.,
2001; Ilori et al., 2005). Ultimately, the surface tension of the biosurfactants was measured, the
results of which are indicated in Table 2. As can be seen, the produced biosurfactant decreased the
surface tension of the solution at least 20 mN/m and the presence of the bacteria in the broth did

TABLE 2
Biosurfactant Production of the Isolates and Their Properties

Surface Max. CMD in the Max. CMD in Contact

Tension, Crude Oil Glucose Medium Angle,
No. mN/m Medium (CMC) (CMC) ı

1 53 40 ˙ 1 17 ˙ 2 46 ˙ 5
2 50 32 ˙ 2 20 ˙ 1 54 ˙ 6
3 38 39 ˙ 1 16 ˙ 1 48 ˙ 6
4 48 37 ˙ 1 12 ˙ 1 39 ˙ 4
5 52 35 ˙ 1 16 ˙ 2 44 ˙ 5
6 43 35 ˙ 1 18 ˙ 1 45 ˙ 7
Brine 71 — — 85 ˙ 3
 1264 D. BIRIA ET AL.
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FIGURE 3 Molecular structure for licheniformin.

not produce a considerable effect on the reduction of surface tension. It was noted that a rise in the
solution concentration or in the extension of the incubation time did not influence surface tension
values since they were related to the CMC concentration of biosurfactants. Physical characteristics
and chemical structure of the biosurfactant produced by strain no. 3 (Bacillus licheniformis MS3)
was determined and it was identified as a new lipopeptide biosurfactant called licheniformin (Biria
et al., 2010). The proposed chemical structure for licheniformin is shown in Figure 3.
Table 2 indicates contact angles belonging to different biosurfactant solutions. It is observed
that for all biosurfactants the contact angle has been reduced in comparison with that of brine
used as blank test. In some cases, biosurfactants had not considerably reduced the surface tension;
however, they possessed a higher contact angle reduction. Figure 4 indicates such inconsistency
in more detail.
A note of caution is in order here. Although the rock samples employed had similar porous
characteristics, small variations in the results are inevitable due to the varied petrophysical nature of
the rock samples. Furthermore, the variation observed in the values of contact angle measurement
was indicative of the fact that surface roughness cannot be totally removed. Consequently, such
roughness, even though insignificant, could influence the results. In addition, liquid absorption
kinetics was not uniform causing fluctuations in the results.

FIGURE 4 Contact angle and surface tension reduction of the isolates and the brine. (color figure available
online)
 ENHANCED OIL RECOVERY FROM OIL–WET CARBONATES 1265

Improving the Efficiency in EOR

Bacillus licheniformis MS3, which showed the greatest potential to be used in EOR (i.e., surface
tension and contact angle reduction), was selected to examine its practical capability in improving
the production of oil from porous media. As Figure 5 represents, the amount of produced oil in the
rock sample containing biosurfactant was considerably higher than that of the brine. In general,
such rise is directly proportional with the increase in biosurfactant concentration. It was observed
that the increase in biosurfactant concentration from zero in brine solution to 1 CMC and then
to 10 CMC could lead to higher oil production from the rock samples. However, the slope of
the incremental trend was lessened because of an increase in the biosurfactant concentration. In
fact, the concentration of biosurfactant after CMC did not affect the surface or interfacial tension
anymore; however, it could influence the alteration of the wettability of the rock surface to a
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more water wet state which was more favorable to oil production. Hammond and Unsal (2010)
studied the effects of surfactant diffusion on the displacement of the two-phase meniscus in an
oil-wet capillary. Their analysis showed that although the surfactant concentration did not have a
significant effect on the speed of meniscus advancement under differential pressures greater than
the capillary threshold, for lower pressures, there was a critical surfactant concentration below
which the interface was not able to advance into the capillary even under positive differential
pressure. This means that in a surfactant flooding operation, the displacing fluid should be supplied
by the lower limit of concentration to freely imbibe to the porous structure of the sample rock
and simultaneously push the oil phase out of the capillaries. Therefore, the observed increase in
the amount of produced oil from the rock sample by increasing the biosurfactant concentration
could be interpreted by the ability of the biosurfactant to alter the wettability of the surface rock
at higher concentrations.
Arguably, the presence of the bacteria in the system has a positive influence on oil production.
This improvement can be justified based on bacteria motility and their chemotaxis nature. Some
motile microorganisms have the ability of sensing the chemical gradient within the environment
and are attracted to nutrients and repelled by the harmful agents. This phenomenon, known as
chemotaxis, might cause the hydrocarbon assimilating bacteria used in this study to swim into the
pores of the rock, which are saturated by the crude oil. In fact, by producing a cross sectional cut
in the rock sample, the microscopic bacterial count revealed that the number of viable bacteria in

FIGURE 5 Cumulative oil production by MS3 strain and its biosurfactant. (color figure available online)
 1266 D. BIRIA ET AL.

the porous medium saturated by crude were by far larger than those of the liquid surrounding the
rock. Normally, the diffusion of bacteria in the rock and secretion of biosurfactants in the pores
may result in a dramatic rise in the local biosurfactant concentration and subsequent improvement
of oil production.
It is worth to note that the effects of interfacial tension reduction and wettability alteration on
oil production could not be measured separately because both of these phenomena simultaneously
influence the system and it is not possible to segregate their effects in such an interactive process.
In fact, the reduction of the interfacial tension facilitates the flow of hydrocarbons out of the
pore throats, and at the same time, results in lowering the capillary pressure, which in turn would
strengthen the diffusion of biosurfactant into the canals of the porous media where the wettability
alteration would occur. This alteration stimulates the free imbibing mechanism and the resulting
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counter current flow of aqueous and nonaqueous phases in the canals would enhance the oil
production (Standnes and Austad, 2000). The higher interfacial tension reduction induced by
MS3 strain, however, prepares much better circumstances for the wettability alteration leading to
more cumulative oil production in this case.

CONCLUSION

To sum up, six rod-shaped and motile bacteria were isolated from a crude oil sample. Their
capability in producing biosurfactants was proved by utilizing the oil spreading technique. Two
of them were able to reduce the surface tension of the water below 40 mN/m. The contact angle
measurements revealed their potential for altering the wettability preference of the rock surface
to a more water wet state. The isolate no. 3, which had better tensioactive characteristics, was
selected to 16S-rRNA analysis. The results of the analysis divulged that it was a new Bacillus
licheniformis strain. The EOR experiments ensured the capability of MS3 strain on improving the
efficiency of oil production through both interfacial tension reduction and wettability alteration
mechanisms. It also brought to view that the presence of bacteria in the system positively affected
the cumulative oil production.

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