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Application of Biosurfactants toWettability Alteration and IFT Reductionin Enhanced Oil Recovery From Oil–WetCarbonatesD. Biria a , E. Maghsoudi b & R. Roostaazad ca Department of Biotechnology, Faculty of Advanced Sciences andTechnologies , University of Isfahan , Isfahan , Iranb Department of Civil Engineering , École Polytechnique University ,Montreal , Canadac Chemical and Petroleum Engineering Department , SharifUniversity of Technology , Tehran , IranPublished online: 24 May 2013.

To cite this article: D. Biria , E. Maghsoudi & R. Roostaazad (2013) Application of Biosurfactantsto 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 FromOil–Wet Carbonates

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

1Department of Biotechnology, Faculty of Advanced Sciences and Technologies,University of Isfahan, Isfahan, Iran

2Department of Civil Engineering, École Polytechnique University, Montreal, Canada3Chemical 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 productionfrom 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, thecapillary 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

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1260 D. BIRIA ET AL.

imbibing process would be strengthened boosting the amount of oil production in a water-floodingprocess (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—thatis, 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 theenvironment. 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

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 surfacedetermine 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

NCa D

v�

� cos �; (1)

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 hasbeen 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 througha 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 speciesexisting 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 ofisolates, 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 brothplus 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

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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

Each isolate was cultivated in a liquid medium containing 5 g/L glucose, 1 g/L KH2PO4, 0.5 g/L

K2HPO4, 0.5 g/L MgSO4.7H2O, 2 g/L NaCl, 1 g/L NH4NO3, and 0.5 g/L NaNO3 and incubatedfor 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 solutioncontaining the secreted biosurfactants. These solutions were both stored at 4ıC 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 specieswas 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 thesolid 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 removedfrom 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 eachbiosurfactant solution. The concentration of biosurfactant solution used instead of the brine was

adjusted to 10 CMC.

FIGURE 1 The contact angle.

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1262 D. BIRIA ET AL.

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-saturatedrock 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 of0.001 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 separatedfrom 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 formingspores. 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 arealso 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

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ENHANCED OIL RECOVERY FROM OIL–WET CARBONATES 1263

TABLE 1

Phenotypic Characteristics of the Isolated Bacteria

No. Morphology Spore Motility

Gram

Reaction

pH

Range

Temperature

Upper Limit,ıC

Salinity

Upper Limit,

% 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 —

Biochemical characteristics of strain no. 3 (B. licheniformis MS3)

Acid production from: L-arabinose,

D-fructose, D-galactose,

D-glucose, maltose, D-mannitol,

D-mannose, sucrose, D-salicin

Amylase C, Protease C,

Gelatinase �, Urease C,

Catalase C, Oxidase C, Nitrate

reduction C, Dnase �, capable

of hydrolysis of tween 80

Utilizing Inulin, D-fructose,

D-galactose, D-glucose, Starch,

Sucrose, Lactose, D-mannose,

D-salicin

species showed more than 99% similarity to Bacillus licheniformis and it was named Bacilluslicheniformis 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 ofsecreted 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

No.

Surface

Tension,

mN/m

Max. CMD in the

Crude Oil

Medium (CMC)

Max. CMD in

Glucose Medium

(CMC)

Contact

Angle,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

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1264 D. BIRIA ET AL.

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 brineused 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 porouscharacteristics, 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, suchroughness, 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)

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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. Infact, 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

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 anoil-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 belowwhich 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 inthe 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 thepores 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)

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1266 D. BIRIA ET AL.

the porous medium saturated by crude were by far larger than those of the liquid surrounding therock. 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 onoil 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 wouldstrengthen 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

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 byMS3 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. Theircapability 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, wasselected to 16S-rRNA analysis. The results of the analysis divulged that it was a new Bacilluslicheniformis 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 affectedthe cumulative oil production.

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