BIOLOGICAL SURFACTANT PRODUCTION BY PSEUDOMONAS AERUGINOSA ATCC 9027 AND PROBABLE APPLICATION IN MICROBIAL ENHANCED OIL RECOVERY (MEOR)

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1 International Journal of Civil Engineering and Technology (IJCIET) Volume 8, Issue 10, October 2017, pp , rticle ID: IJCIET_08_10_064 vailable online at ISSN Print: and ISSN Online: IEME Publication Scopus Indexed IOLOGICL SURFCTNT PRODUCTION Y PSEUDOMONS ERUGINOS TCC 9027 ND PROLE PPLICTION IN MICROIL ENHNCED OIL RECOVERY (MEOR) Rajesh Kanna ssistant Professor, Department of Petroleum Engineering, MET University, Chennai, Tamilnadu, India rajeshkanna.a@ametuniv.ac.in STRCT Microbial enhanced oil recovery (MEOR) is one of the potential technique which aids in improving oil recovery rate by producing bio-surfactant. io-surfactants produced by these microbes plays a critical role in extracting crude oil from the depleted formation soon after primary oil recovery and secondary process had been completed. Current research work mainly focused on the bio-surfactants production using bacteria Pseudomonas aeruginosa (TCC 9027) at both mesophilic and thermophilic conditions. The growth and bio-surfactant production was investigated by varying temperature as well as ph conditions which typically resembling to petroleum reservoir. Experiment was carried out at laboratory scale and analysis such as physical/biological parameters were studied to determine the optimal conditions for growth and bio-surfactant production. In addition to bio-surfactant production, analysis such as surface activities (surface tension and interfacial tension) have revealed that Pseudomonas aeruginosa (TCC 9027) has the potential to grow and produce maximum bio-surfactants which in-turn helped in reduction of interfacial tension from 73 mn/m to 33 mn/m at ph 8.0 and 30 C. However at temperature 55 C the strain could not produce bio-surfactant. lso the strain did not show much growth below ph of 5.0 and above ph of 8.0. Key words: bio-surfactant, MEOR, Interfacial tension, oil recovery, crude. Cite this rticle: Rajesh Kanna, iological Surfactant Production by Pseudomonas eruginosa TCC 9027 and Probable pplication in Microbial Enhanced Oil Recovery (MEOR). International Journal of Civil Engineering and Technology, 8(10), 2017, pp INTRODUCTION In current scenario, there are various sources of energies available globally, but the need for crude oil had been increased significantly during last five decades. Crude oil is extracted using conventional primary method (with native pressure) followed by secondary recovery editor@iaeme.com

2 iological Surfactant Production by Pseudomonas eruginosa TCC 9027 and Probable pplication in Microbial Enhanced Oil Recovery (MEOR) methods (water or gas injection). Huge amount of crude oil still remains as trapped in the cap rocks which are held by high interfacial tension between oil and water molecules. pproximately 55% of oil can be recovered using enhanced oil recovery (EOR) which is also known as tertiary recovery method. Injection of different agents like thermal, Polymers, Water alternative Gas, Chemical surfactants and Microbial surfactants is utilized to recover crude oil from trapped zone is known as enhanced oil recovery methods. Nearly 30 40% of oil remains trapped in the reservoir. Microbial enhanced oil recovery (MEOR) is one among the few techniques to improve oil recovery rate. Primary role of microbes EOR is utilized (metabolites generated by microbes) helps to recover oil by producing bio-surfactant and this technique is known as Microbial EOR [10]. In-order to recover the remaining residual oil, technology like Enhanced Oil Recovery (EOR) is being utilized which is also known as tertiary oil recovery [5]. Microbial enhanced oil recovery (MEOR) is considered a relatively cheap method to recover tertiary oil from reservoirs. In the recent years, increased interest for bio-surfactant production has stimulated the attempt to enhance the present production rate of oil. Most of these io-surfactants are bio-degradable and less toxic than their chemically synthesized counterparts [3]. MEOR improves microscopic sweep through mechanisms like permeability profile modification (by microbial induction), stimulation of reservoir porosity along with reduction in permeability with microbial products like acids reduction of interfacial tension between oil and water and microbial bio-surfactants (reduction in capillary forces). combination of the above three mechanisms can also be used for improving macroscopic sweep efficiency to recover tertiary oil [7]. In aqueous surfactant where injected takes place in mature oil reservoir contacts the oil trapped in the pores of the reservoir rock which has the strength reduce interfacial tension (IFT) and thereby increase the capillary number, so that it mobilizes trapped oil [11]. Microbial EOR represents the one of the most possible technology to recover a substantial proportion of residual oil. acillus subtilis can reduce surface tension from 72 mn/m to 25 Mn/m [1]. acillus subtilis has been used to produce io-surfactants at both mesophilic and thermophilic conditions [9]. strain of acillus subtilis has been reported to be able to grow and produce bio-surfactant at 45 C [8]. Few reports suggest that the carbohydrates like sucrose helps to produce bio-surfactant and also increasing bio-surfactant concentrations are highly sensitive to ph compared to concentration in the reservoir, while at larger resident time and water saturation; the microbial and nutrient concentrations were lesser due to enhanced dispersion [6]. One of the recent report confirmed that bio-surfactant was produced at 40 C and ph 7.0 with consortium of Enterobacter cloacae and Pseudomonas sp. ERCPPI-2 [2]. Earlier work on bio-surfactant was studied using Pseudomonas putida (MTCC 2467) utilizing different source of carbon and nitrogen sources using minimal medium. esides this effect of initial ph were also studied and found that sucrose and ammonium sulphate as best carbon and nitrogen sources [4]. nother study revealed that acillus subtilis can grow and produce bio-surfactant with presence of different carbon and nitrogen sources under thermophilic conditions 45 C and reduced surface tension to 34 dynes cm 1 on 2% sucrose, and 32 dynes cm 1 on starch after 96 h of growth. Microbial surfactant activity was found to be unaltered even at elevated temperature of 100 C and wide ph range ( ). io-surfactant was also produced at mesophilic conditions (30 C) where nitrogen ions were supplied as the nitrogen source [9]. Current research work was attributed to bio-surfactants production from Pseudomonas aeruginosa (TCC 9027) at both mesophilic and thermophilic conditions editor@iaeme.com

3 Rajesh Kanna 2. MTERILS & METHODS 2.1. Microbe and Maintenance Conditions Pseudomonas aeruginosa (TCC 9027) was procured from merican Type Culture Collection centre was used for the present study. Culture was maintained in nutrient agar plates with following composition (g/l): Peptone, 5.0; beef extract, 1.0; yeast extract, 2.0; NaCl, 5.0; agar, 15.0; ph 7.0 ± 0.2, storage temperature 2 C - 8 C Media and Cultivation Conditions Nutrient broth was prepared with composition (g/l) was utilized for inoculum preparation. eef extract, 1.0; yeast extract, 2.0; peptone, 5.0; NaCl, 5.0. Pseudomonas aeruginosa (TCC 9027) grown in Nutrient broth for 8 10 hours at 30 o C (OD600nm ). It is used as inoculum at 2.0% (v/v) concentration. Mineral salt medium with the following composition (g/l) was used for bio-surfactant production: KNO 3, 0.3; NaCl, 0.001; MgSO 4, 0.06; CaCl 2, 0.004; Na 2 HPO 4, 0.2; KH 2 PO 4, 0.014; FeSO 4, along with the trace element for 0.1 (g/l) containing ZnSO 4.7H 2 O, 2.32; MnSO 4.4H 2 O, 1.78; H 3 O 3, 0.56; CuSO 4.5H 2 O, 1.0; Na 2 MoO 4.2H 2 O, 0.39; CoCl 2.6H 2 O, 0.42; EDT, 0.5; NiCl 2.6H 2 O, 0.004; KI, 0.66; Sucrose, 20; K 2 SO 4, 3. Growth studies were carried out separately for 30 o C and 45 o C in a rotatory shaker at 180 rpm. Growth analysis and bio-surfactant production were done at phs ranging from iomass Calculation 10ml samples were collected at regular time interval of fermentation (12 h), centrifuged at rmp for 30 minutes. The pellet was dried at 50 C for overnight and the cell dry weight was determined Surface Tension ctivity Liquid broth which is cell free was extracted by centrifuging the cultures at rpm for 30 minutes was used for the analysis of Surface tension and Interfacial tension. Tensiometer (Data Physics Scientific Company) was used for interfacial and surface tension analysis. 10ml of the sample were taken for analysis. Wilhelmy plate method was used to determine the surface tension. Thin plate (perimeter about 30 mm) is immersed to the liquid surface and the downward force to the plate is measured. Surface tension is the force divided by the perimeter of the plate. The plate must be completely wetted prior to the measurement so that the contact angle between the plate and the liquid remains zero. The plate position must be maintained constant so that the lower end of the plate is actually maintained same level than that of the liquid surface Isolation & Purification of io-surfactant Sample was centrifuged at rpm to remove the cells (bacteria). The supernatant was subjected to acid precipitation where the ph is maintained as 2.0 with 6 N HCl at 4 C. Formed precipitate was pelleted out by centrifugation at rpm for 30 minutes, was again re-suspended in double distilled H 2 O, ph was adjusted to 7.0, freeze dried and weighed. The dried surfactant was extracted with Dichloromethane. Final extract was dried using rotary evaporator under vacuum. This bio-surfactant was further utilized for analysis purposes Effect of Produced io-surfactant on Oil Viscosity and queous phases Main aim of the experiment is to determine the effects of bio-surfactant produced due to viscosity of oil and aqueous phases, 2.0 ml aliquot of solution of purified bio-surfactant (0.1% w/v) produced by Pseudomonas aeruginosa TCC 9027 were subjected to emulsification editor@iaeme.com

4 iological Surfactant Production by Pseudomonas eruginosa TCC 9027 and Probable pplication in Microbial Enhanced Oil Recovery (MEOR) with a volume of 4ml crude oil. t the same time purified bio-surfactant powder with concentration of 0.1% (w/v) was used to emulsify fixed volume of 4ml. Viscosity was measured using a standard viscometer (rookfield metek, DV1 digital viscometer, Russia) Effect of io-surfactant in Oil Recovery Using Sand Packed Column Experiment Sand packed column was designed to evaluate the efficiency of produced bio-surfactant to recover oil. The sand column was fabricated with plastic material such that length of the column is 10 cm with 2.5 cm diameter. The column was packed with fine sand material. Fine sand was initially saturated with brine followed by oil so that it replicate typical porous petroleum reservoir. Sand column was injected again with brine until no more oil recovred at the bottom. 0.5 PV (Pore Volume) of Pseudomonas aeruginosa TCC 9027 (OD = 0.22) in mineral salt medium was injected into the column. The column was flooded with water (Secondary flooding) followed Tween separately (Chemical flooding). Similarly biosurfactant produced at different temperature conditions (37 C and 55 C) was injected into the sand column to check how efficiently oil recovery can be improved. The oil collected from the outlet of the column gives the amount of oil recovery. 3. RESULTS & DISCUSSION 3.1. Growth and io-surfactant Production Profiles of cell weight and bio-surfactant produced have been presented in Figs. 1 & 2. Pseudomonas aeruginosa (TCC 9027) was able to grow in mineral salt medium both at 37 C and 55 C. Fig. 1 shows that Pseudomonas aeruginosa (TCC 9027) of ph 8 at 84 th hour gave maximum of 1.98 g/l, followed by ph 7 at 96 th hour, the dry cell weight was 1.8 g/l. s the ph had been reduced the maximum yield was only about g/l. Fig. 1 shows that Pseudomonas aeruginosa with ph 7 at 96 th hour gave maximum of 1.8 g/l followed by ph 6.0 at 108 th hour gave maximum of 1.8 g/l. ph 8 and ph 5 gave only 1.6 and 1.7 g/l after 108 th hour of fermentation. Figure 1 () Time vs Cell dry weight at 37 C with varying ph, () Time vs Cell dry weight at 55 C with varying ph Fig. 2 and 2 indicates the relationship between sucrose consumption as time proceeds at varying ph with temperature maintained at 37 o C and 55 o C respectively. The figure shows that concentration of sucrose has been decreased with cell growth with time. Fig. 2 shows that at ph 7 the maximum reduction of sugar concentration was achieved at 120 hours. Fig. 2 shows that at ph 5 the maximum reduction of sugar concentration was achieved at 120 hours as 3 g/l. ph 8 gave the least amount of sugar reduction of 8 g/l at 120 hours of growth editor@iaeme.com

5 Rajesh Kanna Our earlier work which were we conducted both experimental and performed simulation and found that both laboratory measured and predicted value for bio-surfactant was 2.7 g/l and 2.8 g/l respectively using strain Pseudomonas putida MTCC 2467 [12]. This result was slightly higher that result with our current work. Figure 2 () Time vs reducing sugar for varying ph at 37 C, () Time vs reducing sugar for varying ph at 55 C acteria Pseudomonas aeruginosa (TCC 9027), the production of bio-surfactant was proportional to the growth rate, which represents bio-surfactant as a growth associated product. The results obtained by optimizing with varying ph at both 37 C and is 55 C shown in figure 3 and 3 respectively. mong all, ph 8 gave maximum bio-surfactant yield of 1.2 g/l at 84 th hour of growth at 37 C. Figure 3 () Time vs io-surfactant concentration for varying ph at 37 C, () Time vs iosurfactant concentration for varying ph at 55 C 3.2. Effect of io-surfactant on Surface tension and Interfacial tension Surface activities are the most important factors to be considered during oil recovery process. io-surfactant produced by bacteria Pseudomonas aeruginosa has the capability to lower surface tension of the oil significantly. From figure 4 it can be noted that the surface tension has been reduced from 66, 67 and 68 mn/m to 45, 48 and 41 mn/m at ph 5, 6 and 7 respectively. However growth at ph 8 showed a maximum reduction of surface tension from 71 to 34 mn/m. Figure 4 shows that reduction of surface tension was found to be maximum from 70 to 42 mn/m at ph 7 and 45 C. Our previous work on bio-surfactant produced by editor@iaeme.com

6 iological Surfactant Production by Pseudomonas eruginosa TCC 9027 and Probable pplication in Microbial Enhanced Oil Recovery (MEOR) Pseudomonas putida MTCC 2467 exhibited higher surface activity of mn/m which is in par with current result [4]. Figure 4 () Time vs Surface Tension for varying ph at 37 C, () Time vs Surface Tension for varying ph at 55 Interfacial tension is one of the key parameter, as there is an increase in capillary number there will be reduction in interfacial tension which lowers the residual oil saturation in the core and increases the residual oil recovery rate. Figure 5 and 5 gives the profiles changes in IFT (interfacial tension) with respect to time at both 37 C and 55 C. The maximum reduction in IFT at 30 C was 8 mn/m at ph 8.0. Temperature at 55 C, the reduction in IFT was 11 mn/m at ph 6.0. Figure 5 () Time vs Interfacial Tension for varying ph at 37 C, () Time vs Interfacial Tension for varying ph at 55 C 3.3. Effect of io-surfactant on Emulsification The result on bio-surfactant stability using strain P. aeruginosa were analyzed and it was observed that the surface activity of purified bio-surfactant aliquots was 27% than the cell free broth (Figure 6). This result is in par with report by [2], where the emulsification index is between 55 and 60 C editor@iaeme.com

7 Rajesh Kanna Figure 6 Effect of temperature on the stability of bio-surfactant produced by Pseudomonas aeruginosa TCC 9027 at incubation time of 90 min 3.4. Effect of io-surfactant to Recover Oil Using Sand Packed Column Oil recovered using bio-surfactant shown in Figure 7 and 7 at 37 C and 55 C respectively. With water injection, oil recovered was 32%. With tween injection the oil recovery was additional 3% and 4% It was absorbed that with bio-surfactant produced by maintaining temperature 37 C and 55 C the oil recovery was increased to 7% and 8.5% respectively. MEOR injection was carried out by injecting pure bio-surfactant produced by the metabolites using strain P. aeruginosa. The result obtained was compared to our previous work where using water flooding 27% of oil was recovered. Tween injection gave additional oil recovery of 2.6% [5]. The obtained was in par with our previous work where instead of tween, triton (2.8% additional oil recovery) was used to compare the efficiency of biosurfactant produced using Pseudomonas.sp [6]. Figure 7 () % oil recovery using sand packed column at 37 C, () % oil recovery using sand packed column at 55 C editor@iaeme.com

8 iological Surfactant Production by Pseudomonas eruginosa TCC 9027 and Probable pplication in Microbial Enhanced Oil Recovery (MEOR) 4. CONCLUSIONS Current research has revealed that Pseudomonas aeruginosa TCC 9027 has the potential to produce bio-surfactant at both 37 C and 55 C. acteria (Pseudomonas aeruginosa TCC 9027) showed a tremendous reduction of surface tension from 73 mn/m to 34 mn/m and interfacial tension form 41 to 9 mn/m at 37 C which is higher than growth at 55 C. The potential use of this bacterium is highly suitable for MEOR applications where additional oil recovery of 7 8.5% after water injection. io-surfactant stability was tested for emulsification and found to be stable at different temperature ranges, which shows produced bio-surfactant is thermostable. Hence, the strain Pseudomonas aeruginosa TCC 9027 can be used for extreme environmental conditions and in situ operations for microbial enhanced oil recovery. REFERENCES [1] mani H, Sarrafzadeh MH, Haghihi M, Mehrina MR. Comparative study of biosurfactant producing bacteria in MEOR applications, J Pet Sci Eng. 2010; 75, doi: /j.petrol [2] Darvishi P, yatollahi S, Mowla D, Niazi. iosurfactant production under extreme environmental conditions by an efficient microbial consortium, ERCPPI-2. J Col Surf; 2011,4 (2), doi: /j.colsurfb [3] Desai J, Patel RM, Desai J. dvances in the production of io-surfactants and their commercial applications. J Sci Ind Res; 1994, 53, [4] Kanna R, Kumar GS, Gummadi SN. io-surfactant production and its application in microbial EOR. Int J iol Vet gri Food Eng; 2014, 8 (10), [5] Kanna R, Gummadi SN, Kumar GS. Production and characterization of biosurfactant by Pseudomonas putida MTCC 2467.J iol Sci; 2014,14 (6), doi: /jbs [6] Kanna R, Gummadi SN, Kumar GS. Evaluation of bio-surfactant on microbial EOR using sand packed column. iotechnology and iochemical Engineering, Springer Publications, 2016, pp doi: / _13. [7] Lazar I, Petrisor IG, Yen TF. Microbial Enhanced Oil Recovery (MEOR), Pet Sci and Technol. 2007; 25, doi: / [8] Makkar RS, Cameotra SS. io-surfactant production by a thermophilic acillus subtilis strain. J Ind Microbiol iotechnol;1997, 18, [9] Makkar RS, Cameotra SS. Production of bio-surfactant at mesophilic and thermophilic conditions by a strain of acillus subtilis, J Ind Microbiol iotechnol. 1998; 20, [10] Qingxin Li, Kang C, Wang H, Liu C, Zhang C. pplication of microbial enhanced oil recovery technique daqing oilfield. J iochem Eng; 2002,11, [11] Sen R. iotechnology in petroleum recovery: The Microbial EOR, Prog Energy Combust Sci. 2008;34, [12] Sivasankar, Kanna R, Gummadi SN, G Kumar, Experimental and numerical modeling on microbial enhanced oil recovery. ioresour Technol; 2016, 211, doi: /j.bioetech editor@iaeme.com