Comparative study of biosurfactant produced by microorganisms isolated from formation water of petroleum reservoir

Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 124–130

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Comparative study of biosurfactant produced by microorganisms isolated from formation water of petroleum reservoir Wen-Jie Xia ∗ , Han-Ping Dong, Li Yu, Deng-Fei Yu Institute of Porous Flow & Fluid Mechanics, Chinese Academy of Sciences, Langfang 065007, China

a r t i c l e

i n f o

Article history: Received 29 June 2011 Received in revised form 31 August 2011 Accepted 30 September 2011 Available online 8 October 2011 Keywords: Biosurfactant Pseudomonas aeruginosa Bacillus subtilis Rhodococcus erythropolis Surface tension MEOR

a b s t r a c t Biosurfactant produced by Pseudomonas aeruginosa, Bacillus subtilis and Rhodococcus erythropolis that isolated from the formation water of Chinese petroleum reservoir has been compared in surface abilities and oil recovery. Maximum biosurfactant production reached to about 2.66 g/l and the surface tension of liquid decreased from 71.2 to 22.56 mN/m using P. aeruginosa. Three strains exhibited a good ability to emulsify the crude oil, and biosurfactant of P. aeruginosa attained an emulsion index of 80% for crude oil which was greater than other strains. Stability studies were carried out under the extreme environmental conditions, such as high temperature, pH, salinity and metal ions. Results showed an excellent resistance of all biosurfactants to retain their surface-active properties at extreme conditions. It was found that the biosurfactants from three isolated bacteria showed a good stability above pH of 5, but at lower pH (from 1 to 5) they will harmfully be affected. They were able to support the condition up to 20 g/l salinity. P. aeruginosa biosurfactant was even stable at the higher salinity. Regarding temperature, all produced biosurfactants demonstrated a good stability in the temperature up to 120 ◦ C. But stability of three biosurfactants was affected by monovalent and trivalent ions. Oil recovery experiments in physical simulation showed 7.2–14.3% recovery of residual oil after water flooding when the biosurfactant of three strains was added. These results suggest that biosurfactants of these indigenous isolated strains are appropriate candidates for enhanced oil recovery with a preference to biosurfactant of P. aeruginosa. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The surfactants produced by different family of microorganisms, such as bacteria, yeasts, and fungi, are known as biosurfactants. Biosurfactants reduce surface tension and interfacial tension in both aqueous and hydrocarbon mixtures. Low toxicity, high biodegradability and ecological acceptability are among the main characteristics of these surface active materials [1–6]. These favorable features make biosurfactants potential alternatives of chemically synthesized surfactants in a variety of applications [6,7]. Biosurfactants are widely used in different industries, such as cosmetics, special chemicals, food, pharmaceutics, agriculture, cleansers and microbial enhanced oil recovery (MEOR) [8–15]. The last mentioned application has attracted more attention because only 30% of oil present in reservoir can generally be recovered using primary and secondary recovery techniques [1]. MEOR is considered as a tertiary recovery technique that could recover the residual oil using microorganisms or their products

∗ Corresponding author at: Shenliu, Mailbox 44#, Langfang City, Hebei Province, China. Tel.: +86 10 69213741; fax: +86 10 69213362. E-mail address: [email protected] (W.-J. Xia). 0927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2011.09.044

(biosurfactants). However, the application of biosurfactants in microbial enhanced oil recovery depends on their stability at extreme conditions of temperature, salinity and pH, or surface activities [13]. Pseudomonas aeruginosa, Rhodococcus sp. and Bacillus subtilis are the well known bacteria for producing biosurfactants named rhamnolipid, trehalose lipids and surfactin that found application in MEOR [2,5,15,16]. Rhamnolipid, trehalose lipids and surfactin are the most effective biosurfactants with the ability to reduce the water surface tension and interfacial tension of water/oil system significantly even at the low concentration [17–26]. They also show excellent emulsifying activities with hydrocarbons and vegetable oils [21,24,26]. It is necessary to analyze and simulate their effects in the laboratory scale before their injection in the petroleum reservoir. In recent years, physical simulation test has been served as an excellent laboratory instrument to investigate and understand the mechanisms and performance of biosurfactant flooding in enhanced oil recovery. Physical simulation test even provide the opportunity to observe fluid flow within reservoirs. Several investigations have been carried out on the biosurfactant flooding [5,14,16,27,28]. The comparison between biosurfactants produced by different microorganisms in MEOR application could easily be realized by such instrument. The aims of this work are to study the biosurfactants produced by three screened isolates,

W.-J. Xia et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 124–130

which belong to the families of Pseudomonas, Bacillus and Rhodococcus and to compare their stability at extreme conditions as agents for enhanced oil recovery. 2. Materials and methods

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of dichloro-methane. The solvent layer was harvested and evaporated [29,30]. The residue is not still a pure biosurfactant and can be considered as a semi-pure biosurfactant. To attain a highly pure biosurfactant, applying of other techniques such as thin layer chromatography (TLC) will be needed but for our purpose a relative degree of purification is sufficient.

2.1. Microorganisms Three strains used in this study were selected among the microorganisms that isolated from the formation water of Chinese oil reservoir and stored at −80 ◦ C in LB medium mixed with sterile glycerol at a final concentration of the 25% (v/v) in our lab. The isolates that show blood hydrolysis and/or oil spreading were selected and used for further experiments. At the end three microorganisms were selected due to their potential to cause highest surface tension decrease to lower than 30 mN/m. Primary specification and biochemical characterization were performed and afterward the strains were identified according to their 16S rRNA technology as P. aeruginosa, B. subtilis and Rhodococcus erythropolis, and named as WJ-1, H10, Z25. 2.2. Media and culture conditions All cultures were grown aerobically in liquid medium C that contained (KH2 PO4 , 1.5 g/l; K2 HPO4 , 3.5 g/l; crude oil, 20 g/l; NaCl, 10 g/l; yeast extract, 0.03 g/l; NaNO3 , 4 g/l; pH 6.9–7.2). This solution was autoclaved at 121 ◦ C for 20 min and after cooling 1% (v/v) of the trace salt solution was added to 500 ml of above medium. Trace salt solution contained 2.5 g/l of MgSO4 ; MnSO4 ·H2 O, 3 g/l; CaCl2 ·2H2 O, 0.1 g/l; ZnSO4 ·7H2 O, 0.1 g/l; FeSO4 ·7H2 O, 0.1 g/l; CuSO4 ·5H2 O, 0.01 g/l; Na2 MoO4 ·2H2 O, 0.01 g/l; boric acid, 0.01 g/l; Na2 SeO4 , 0.005 g/l; NiCl2 ·6H2 O, 0.003 g/l. Trace salt solution was filter sterilized. Chemicals used in the medium were with analytical grade and purchased from Beijing chemistry Co., Ltd. A loop of each bacterium, grown on a nutrient agar plate, was added to 100 ml LB medium in a 250 ml Erlenmeyer flask. The flask was then incubated for 24 h at 160 rpm and 37 ◦ C in a constant temperature shaker and used as preculture. Then, 10 ml of preculture was added to C medium to make 200 ml of solution in 500 ml flask and placed in shaking incubator at 250 rpm and 37 ◦ C. The strains were grown in C medium for 100 h separately. Fermentation was carried out for each bacterium in five 500 ml Erlenmeyer flasks containing 200 ml of medium. Samples were taken for analysis at irregular time intervals. The cultures were centrifuged at 10,000 rpm for 30 min. After separation of biomass and crude oil, further clarification was carried out by filtration of supernatant through 0.45 ␮m membrane. 2.3. Analytical methods

2.3.3. Biomass analysis The conventional method of cell dry weight measurement was performed for biomass analysis. After centrifuging of sample and separation of supernatant from the biomass, the biomass was washed twice in distillated water and transferred into a prepared vial. The bacterial dry weight was determined after drying at 65 ◦ C for 48 h. 2.3.4. Determination of surface tension (ST), critical micelle concentration (CMC) and interfacial tension (IFT) The surface tension (ST), interfacial tension (IFT) and critical micelle concentration (CMC) were measured at 25 ◦ C by a digital tensiometer (Kruss, K10ST, Germany) using the ring method. IFT measurements were carried out against the crude oil. The critical micelle concentration (CMC) was determined by measuring surface tension at different concentrations of biosurfactant in distillated water up to a constant value of surface tension. The value of CMC was obtained from plot of surface tension versus the biosurfactant concentration [31]. For critical micelle dilution (CMD) measurements the produced biosurfactants of all cultures were diluted 10 times (CMD−1 ), 100 times (CMD−2 ) and 1000 times (CMD−3 ) respectively [13]. 2.3.5. Emulsification determination Emulsification activity of the biosurfactant solutions at CMC was determined by measuring the emulsion index (E24) at 25 ◦ C as described by Yeh et al. [32]. In general, 4 ml of crude oil was poured separately into a test tube containing 6 ml of biosurfactant solution. After being vigorously vortexed for 2 min, the test tube was kept still for 24 h and the heights of emulsion, oil and aqueous zones were measured. The emulsion index (E24) was then calculated from the ratio of the height of the emulsion zone to the total height of the oil, emulsion, and aqueous zones. Crude oil (crude oil viscosity 79.1 mPa·s, crude oil density 0.898 g/cm3 and API 14) used in this research was obtained from the Xinjiang reservoir, China. 2.3.6. Biosurfactant stability analysis 2.3.6.1. The effect of temperature. To study the effect of temperature, samples of biosurfactant at CMC were heated in a boiling water bath up to 24 h and cooled at temperature room. Besides, for 120 ◦ C, the samples were placed in an autoclave for 1 h, and cooled at room temperature. The changes in surface tension for each case were measured by the mentioned tensiometer (Kruss, K10ST, Germany).

2.3.1. Analysis of crude oil The remained concentration of crude oil was determined by Weighing-method [6,15]. Briefly, the solution of free-cell was filtrated by using the absorbent cotton that was washed three times by chloroform. Then washed the absorbent cotton by distilled water and dried to constant weight at 40 ◦ C, The difference value of the absorbent cotton before and after filtration is the remained concentration of the crude oil.

2.3.6.2. The effect of pH. To investigate the effect of pH on surface tension of supernatant, the pH of the biosurfactant solution at CMC was adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0 by using 3 M HCl and 3 M NaOH. Then the changes in surface tension for each case were measured.

2.3.2. Biosurfactant analysis After the separation of biomass/crude oil and further clarification, the pH of supernatant was adjusted to 2 by 6 M HCl to allow the precipitation of biosurfactant. The precipitates were collected by centrifugation to obtain a crude biosurfactant. For further purification, the crude biosurfactant was dissolved in double-distilled water and was then extracted three times with an equal volume

2.3.6.3. The effect of salinity and metal ions. To investigate the effect of salinity on surface tension, various amounts of NaCl up to 40 g/l were added to the biosurfactant solutions at CMC and mixed completely and incubated for 2 days at room temperature. Then the changes in surface tension for each case were measured. The same procedure was conducted for biosurfactant solutions mixed with different metal ions of K+ , Ca2+ , Mg2+ , Fe2+ and Al3+ 20,000 (mg/l).

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Table 1 The basic parameters of the testing core model. Model no. 1# 2# 3# 4#

Dimension (mm × mm) 25 × 200 25 × 200 25 × 200 25 × 200

Porosity volume (ml)

Porosity of model (%)

Permeability (␮m2 )

Initial saturated water (%)

Initial saturated oil (ml)

36.78 38.57 37.61 38.07

36.78 39.31 38.12 38.80

0.170 0.169 0.174 0.164

18.30 18.20 18.31 18.71

30.05 30.42 30.34 30.95

Note: The dates were average value of three parallel experiments.

Changes in the surface tension at 25 ◦ C for all mixtures of K+ , Ca2+ , Mg2+ , Fe2+ and Al3+ were also measured.

5

Bacillus subtilis Pseudomonas aeruginosa Rhodococcus erythropolis

i) All columns were sterilized at 121 ◦ C for 2 h. Each sandstone core was evacuated for about 2 h. After this operation, the cores were saturated with Xinjiang brine for porosity and permeability determination. ii) Saturation of the sand pack with brines: The column packed with sand was evacuated and nitrogen gas was passed through its one end for 5 min, using a syringe to remove oxygen from the column. To ensure removal of all the gases from the column, the flow of nitrogen was stopped and vacuum was held for 2 min at the other end of the column. The column was then flooded with brines at a pressure of 8–10 kg/cm2 . Pore volume of the column was calculated by measuring the volume of brine required to saturate the column. Subsequently, four pore volumes of brines were passed through the column to ensure its 100% saturation with brines. iii) Saturation of the sand pack with oil: Crude oil (density 898 g/l) of Xinjiang reservoir was used in all the experiments. The oil, filled in a tank, was passed under pressure into the sand pack column, in the same way as brines, until residual brine saturation was reached. As oil entered into the column, brines was displaced and discharged from the pack through a tubing inserted into the bottom end of the column. Initial oil saturation was calculated by measuring the volume of brines displaced by oil saturation. iv) Brines flooding (the first water flooding): The sand pack was again flooded with brines until there was no oil coming in the effluent, i.e. residual oil saturation was reached. Approximately, 6–9 pore volumes of brines were sufficient to reduce the pack nearly to its residual oil saturation. The amount of crude oil retained in the sand pack was determined volumetrically. Residual oil saturation was calculated by measuring the volume of displaced oil. v) Biosurfactant flooding: This was done in a manner similar to brine floods. 0.5 pore volume (PV) of biosurfactant solution (1 g/l) of P. aeruginosa, B. subtilis and R. erythropolis were respectively passed through the 1#, 2# and 3# columns at a flow rate of approximately 1.0 ml/min and stayed for 1 day. 4# was control experiment with no biosurfactant. Then, the columns were again flooded with Xinjiang brine (the second water flooding). Discharges from the column were collected to measure the amount of oil recovered (the difference between the first and the second water flooding). These experiments were used to check their oil

3 2 1 0

0

4

8

16

20

30

40

50

60

80

100

Time(h) Fig. 1. Time courses of cell growth during fermentations of Bacillus subtilis, Pseudomonas aeruginosa, and Rhodococcus erythropolis at 250 rpm and 37 ◦ C.

recovery efficiency and done in triplicates, and the results presented were the average data. The basic parameters of the model reservoirs were listed in Table 1. 3. Results and discussion 3.1. Growth and biosurfactant production The profiles of biomass concentration, biosurfactant production, changes in surface tension and substrate concentration versus time have been presented in Figs. 1–3. All of three microorganisms were able to grow in C medium using crude oil as carbon source. Fig. 1 shows that P. aeruginosa WJ1 reached to a higher concentration of biomass of about 4.76 g/l. The growth in all cases continued until 80 h where the crude oil has not been consumed (Fig. 2). Figs. 1 and 2 showed the relations between crude oil consumption and cell growth, indicating that the biomass concentration has been increased with time and crude oil concentration has been decreased with cell growth before 50 h. However, after 50 h three isolated strains have a small increase without the consumption of crude oil. Possibly the growth of strains after 50 h was due to utilization of some metabolites produced by themselves when using some available fractions of crude oil as carbon source.

12

Crude oil concentration(g/L)

2.3.7. Physical simulation experiments in the oil recovery To evaluate the potential application of three strains in enhanced oil recovery (EOR), the core flooding system was employed. A standard core flooding equipment used was similar to that described by Lotfabada et al. [14]. Three stainless steel columns of 200 mm in length and 25 mm in diameter, named 1#–4#, were packed with sandstone. The sandstone with particle size distribution of 380 ␮m (m/m, 35%), 120–109 ␮m (m/m, 30%), 80–75 ␮m (m/m, 35%) was free of organic contaminants, and dried for 24 h at 120 ◦ C and used throughout the study. Operation of oil recovery simulation was done as follows:

Biomass(g/L)

4

Bacillus subtilis

10

Pseudomonas aeruginosa 8

Rhodococcus erythropolis

6 4 2 0

0

20

40

60

80

100

120

Time(h) Fig. 2. Time courses of crude oil concentration during fermentations of three isolated bacterium in a shaker-incubator at 250 rpm and 37 ◦ C.

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Table 2 Comparison of the yield of biosurfactant on biomass Yp/x, the production yield of Yp/s and overall production rate during fermentation of three strains. Microorganism

Surface tension (mN/m)

Biosurfactant yield, Yp/x (g/g)

Production yield, Yp/s (g/g)

Overall production rate (mg/l/h)

Pseudomonas aeruginosa Bacillus subtilis Rhodococcus erythropolis

22.56 27.12 29.45

0.58 0.46 0.57

0.26 0.18 0.16

44.3 21.8 20.0

3.2. Effect of biosurfactants on surface tension and emulsification activity The biosurfactants produced by P. aeruginosa, B. subtilis and R. erythropolis were able to reduce the surface tension of supernatant significantly. As seen in Fig. 4, the surface tension of supernatant in all cultures has been drastically decreased from 71.2 to about 22–30 mN/m. The decrease of the surface tension has been happened by the early taken samples, it showed that the production of biosurfactant has taken place at early stage of the cultivation. For further researches, CMC values of biosurfactants were determined. The presence of biosurfactants reduced the surface tension, which was proportional to biosurfactant concentration in solutions, until they reached the CMC. The profiles of changes in surface tension versus concentration have been presented in Fig. 5. As shown in

80 Bacillus subtilis

Surface tension (mN/m)

For P. aeruginosa, B. subtilis and R. erythropolis, the production of biosurfactant was proportional to the cell growth, representing biosurfactant as a growth associated product (Figs. 1 and 3). Maximum biosurfactant production reached to about 2.66 g/l using P. aeruginosa. In addition, for an initial crude oil concentration of 10 g/l, the concentration of biosurfactant was reached to its maximum at about 60th hour of fermentation where the maximal cell concentration was observed for P. aeruginosa. These results were in agreement with other research works [13,33,34]. However the concentration of biosurfactant was reached to its maximum at about 60th hour of fermentation while the maximal cell concentration was observed at 80th hour for B. subtilis and R. erythropolis, possible because the available fractions of crude oil for three strains were different in composition and content. The comparison between the amounts of biosurfactant produced by three microorganisms, presented in Fig. 3, shows P. aeruginosa as the best candidate for the highest yield. It produced the highest amount of biosurfactant while R. erythropolis produced the lowest amount (1.56 g/l). For a better comparison between these bacteria, some parameters such as yield of biosurfactant on biomass Yp/x (g biosurfactant/g biomass), the production yield Yp/s (g biosurfactant/g crude oil) and overall production rate (mg/l/h) were calculated and presented in Table 2. As seen, the higher production yields belong to Pseudomonas aerugenous.

70

Rhodococcus erythropolis Pseudomonas aerugenous

60 50 40 30 20 20

0

40

60

80

100

Time (h) Fig. 4. Changes in surface tension of culture supernatant during fermentations of three isolated bacteria.

Fig. 5, the CMC of the biosurfactants produced by P. aeruginosa, B. subtilis and R. erythropolis were almost 30, 50, 70 mg/l, respectively. Parameters such as IFT are important factors in oil recovery because capillary number is defined as the ratio of inertial to capillary forces. Capillary number increases with decreases in interfacial forces. Increases in capillary number lower the residual oil saturation in the core and increase residual oil recovery. Therefore the profiles of changes in IFT versus biosurfactant concentration have been presented in Fig. 5. This figure shows the minimum IFT of the biosurfactants produced by P. aeruginosa, B. subtilis and R. erythropolis were 1.85, 2.87, 4.45 mN/m, respectively. So, we could think due to the low amount of biosurfactants’ CMC value, the production of biosurfactant after 10th hour (Fig. 5) could no more be evaluated by decreasing the surface tension. However, the lowest surface tension was not observed at 10th hour (Fig. 4) because of the purity between the culture and purified biosurfactant from the culture. As described previously, emulsification activity of the biosurfactant solutions (at CMC) was determined by measuring the emulsion index (E24). The emulsification ability of biosurfactants produced by these microorganisms has been compared in Fig. 6. The results indicated that the biosurfactants produced by these strains show the great potential of emulsification capacity. The crude oil

80

2.5 2 1.5 Bacillus subtilis Pseudomonas aeruginosa Rhodococcus erythropolis

1 0.5 0

IFT:Pseudomonas aerugenosa IFT:Bacillus subtilis IFT:Rhodococcus erythropolis ST:Pseudomonas aerugenosa ST:Bacillus subtilis ST:Rhodococcus erythropolis

70

Surface and interfacial tension (mN/m)

Biosurfactant yield(g/L)

3

0

20

40

60

80

100

60 50 40 30 20 10

120

Time(h) Fig. 3. Time courses of biosurfactant concentration during fermentations of three isolated bacterium when using Xinjiang crude oil as carbon source.

0

0

10 20 30 40 50 60 70 80 90 100 120 140 160 180 200

Biosurfactant concentration (mg/L) Fig. 5. Changes in surface tension (ST) and interfacial tension (IFT) versus biosurfactant concentration.

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35

80 30

70

Surface tension(mN/m)

Emulsification index (%)

90

60 50 40 30 20 10 0 Pseudomonas aeruginosa

Bacillus subtilis

Rhodococcus erythropolis

Control

25 20 15

Pseudomonas aeruginosa Bacillus subtilis Rhodococcus erythropolis

10 5 0

Bacterium

20

0

40

60

80

100

120

140

Temperature( ºC )

Fig. 6. Emulsion index of crude oil for biosurfactants of three isolated bacteria at CMC.

Fig. 8. Changes in surface tension of solutions containing biosurfactants of three isolated bacteria at CMC after heat treatment at different temperatures.

emulsifications were evaluated after 1 day of experiment for all samples. E24 ranged from 74 to 80% for crude oil. The highest emulsification (80%) was obtained for biosurfactant of P. aeruginosa. The culture medium without microorganisms was used as control solution and no emulsification of crude oil was observed with this control. On the base of the emulsion index after 24 h it can be concluded that the biosurfactants of these three bacteria were appropriate candidates for enhanced oil recovery but the biosurfactant of P. aeruginosa was preferred one. 3.3. Study of stability 3.3.1. Effect of pH The effect of pH on biosurfactant activity at CMC was shown in Fig. 7. It shows that when pH of biosurfactants decreased, the surface tensions increased. It could be the result of biosurfactant precipitation in acidic condition. It was found that from pH 1 to 5 biosurfactant of three strains got precipitated but the sample of R. erythropolis has been precipitated more than the samples of B. subtilis and P. aeruginosa. Its surface tension increased more than the others. On the other hand, the surface tension of biosurfactant produced by P. aeruginosa behaves differently with others after pH7, possibly due to the solubility of rhamnolipid. Some literatures [6,11,20–22,34] have reported that the alkaline solution was conducive to the solubility. In common biosurfactant activity is determined by surface tension measurements in samples diluted to 1:10, 1:100 and 1:1000, the value of them remained at low point (35 mN/m). Therefore the effect of pH on CMD−1 , CMD−2 and CMD−3 (these parameters were measured for produced biosurfactants of all cultures by taking samples after 100 h of

fermentation), showed that biosurfactants have an effective surface activity from about pH = 5 to about pH = 12.0. In other words, CMD values remained stable at this range. 3.3.2. Effect of temperature As seen in Fig. 8, studies on effect of heat treatment showed that some changes in biosurfactants surface activities have occurred and there was some difference in surface tension before and after this treatment in all cases. As temperature increases, the surface tension increased from 22.9 mN/m to 27.21 mN/m for P. aeruginosa, from 26.7 mN/m to 32.5 mN/m for B. subtilis and from 28.9 mN/m to 31.9 mN/m for R. erythropolis. This maybe due to the strength of hydrogen bond or hydration decreased with temperature increasing. However the surface tension of three biosurfactant remained at low value (<32 mN/m) with temperature increasing from 4 ◦ C to 120 ◦ C. When placed to autoclave (120 ◦ C for 2 h) the surface activities were also maintained. So they could be interpreted as a good heat-resistance of biosurfactants produced by evaluated strains and could be used in extreme temperature conditions in the oil reservoir. 3.3.3. Effect of salinity As described various amounts of NaCl were added to biosurfactants at CMC and mixed completely and then surface tension was measured. The results of Fig. 9 showed that the biosurfactants produced by these strains resist well against salt concentration up to 20 g/l. However the sample of B. subtilis was more affected than the others in the salt concentration range from 20 to 40 g/l, and sample of P. aeruginosa was more stable than the others in the salt 60

70 Pseudomonas aeruginosa

50

Bacillus subtilis

Surface tension(mN/m)

Surface tension (mN/m)

60

Rhodococcus erythropolis

50 40 30 20

30 20

Pseudomonas aeruginosa Bacillus subtilis

10

10 0

40

0

2

4

6

8

10

12

14

pH Fig. 7. Relationship between surface tension and pH of biosurfactant solutions at CMC of three isolated bacterium.

0

Rhodococcus erythropolis 0

10

20

30

40

50

NaCl concentration(g/L) Fig. 9. Influence of NaCl on surface tension of biosurfactant solutions at CMC of three isolated bacteria.

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Table 3 EOR parameters of physical simulation experiment. Model no.

Microorganism

First water flooding (%)

Second water flooding (%)

Enhanced oil recovery (%)

1# 2# 3# 4#

Pseudomonas aeruginosa Bacillus subtilis Rhodococcus erythropolis Control

61.09 61.22 60.59 61.06

76.09 72.32 68.49 61.76

15.0 11.1 7.9 0.7

Note: The dates were average value of three parallel experiments.

concentration range up to 40 g/l. Little changes also were observed on CMD values with the addition of up to 20 g/l NaCl. So they could be interpreted as a good salt-resistance of biosurfactants produced by evaluated strains.

3.3.4. Effect of metal ions Biosurfactant solutions at CMC were adjusted with different metal ions of K+ , Ca2+ , Mg2+ , Fe2+ and Al3+ (concentrations 20,000 mg/l). At 25 ◦ C the surface tension of different K+ , Ca2+ , Mg2+ , Fe2+ and Al3+ solutions were measured. The results (Fig. 10) showed that no differences in surface tension before and after this treatment when using bivalent ion (Ca2+ , Mg2+ , Fe2+ ). However the surface tension was obviously increased when the biosurfactant solution was added with monovalent ion (K+ ) especially when using trivalent ions (Al3+ ). This maybe due to the association degree of the biosurfactant by different metal ions, and this study need further investment.

3.4. Physical simulation of oil recovery

Surface tension(mN/m)

35 30 25 20 15 Pseudomonas aeruginosa Bacillus subtilis Rhodococcus erythropolis

5 0

K+

Ca2+

Mg2+

Three biosurfactants produced by bacterial isolates from reservoir formation water, B. subtilis, P. aeruginosa, and R. erythropolis, were studied when using crude oil as carbon source and compared from diverse points of view. Among these bacteria, P. aeruginosa could be positioned well, by taking into account the overall biosurfactant production rate, resistance and stability at extreme conditions and influence on oil recovery. The biosurfactant produced by P. aeruginosa also attained an emulsion index of 80% for crude oil. It showed good surface activity with a decrease on surface tension of medium from 71.2 to 22.56 mN/m. The results of biosurfactant flooding experiment using P. aeruginosa showed 14.3% oil recovery after water flooding. This work was another demonstration of usefulness of traditional screening and isolation of indigenous microorganisms for the industrial applications, especially it paves the way to further exploit the potential of this strain and its biosurfactant in microbial enhanced oil recovery (MEOR) technology. Acknowledgements

Biosurfactant showed a higher potential for MEOR applications, it was used for oil displacement analysis. Results in Table 3 have been showed that the biosurfactant of P. aeruginosa in the oil recovery experiment was 14.3% against the control after water flooding which more than that observed in the rests by using the biosurfactant of B. subtilis (10.4%) and R. erythropolis (7.2%). The results indicated that biosurfactant mobilized oil in the micromodel and should have a significant role in enhanced oil recovery. Therefore, this microorganism produces biosurfactant that can lower oil–water interfacial tension. The drop in interfacial tension increases the capillary number. The increased capillary numbers are associated with reduction of residual oil saturation. Based on the analysis of the results in this study, biosurfactant produced by P. aeruginosa showed good properties for use in enhanced oil recovery.

10

4. Conclusions

Fe2+

AL3+

Metal ions Fig. 10. Influence of metal ions on surface tension of biosurfactant solutions at CMC of three isolated bacteria.

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