Chapter 7. Enhanced Oil Recovery using crude biosurfactant. conditions

Transcription

1 Chapter 7 Enhanced Oil Recovery using crude biosurfactant preparation from B. subtilis K1 under laboratory conditions

2 7.1 Introduction Current worldwide market for surfactants is around $ 9.4 billion per annum. Almost all surfactants currently in use are chemically synthesized (Desai and Banat, 1997; Mukerjee, 2005). Biosurfactants are surface active amphiphilic molecules synthesized by microorganisms. These surface active molecules reduce surface tension as well as interfacial tension in aqueous solutions and hydrophobic mixtures (Banat, 1995). Most microbial surfactants are complex structurally diverse groups of molecules comprising lipopeptides, glycolipids, phospholipids, fatty acids and polysaccharide protein complexes (Cooper et al., 1980; Rosenberg, 1986; Desai and Banat, 1997). The important features of biosurfactants over chemically synthesized surfactants are their biodegradability, bioavailability, lower toxicity, higher foaming, high selectivity, specific activity at extreme ph, temperature and salinity (Rosenberg, 1986; Desai and Banat, 1997). These unique properties of biosurfactants are being increasingly used in place of chemically synthesized surfactants in industrial applications viz., as emulsifiers in food and pharmaceutical industries, as surfactants in laundry, as biological control agents, in mobilization of heavy oil spills, oil-pollution control, cleaning of oil sludge from storage facilities, bioremediation of oil contaminated soil and enhance oil recovery (EOR) (Kosaric, 1992; Banat, 1994; Mukherjee, 2005; Nitschke and Costa, 2007; Pattanathu et al., 2008; Brown, 2010). The recovery of left over oil from wells upon primary and secondary recovery processes, has to be recovered by specialized enhanced oil recovery techniques (EOR) (Morkes, 1993, Brown, 2010). The currently available chemical EOR processes are designed based on polymer flooding, surfactant flodding, alkaline flooding, injection of steam or in situ combustion (Brown, 2010). The use of chemical surfactants for EOR is undesirable as it is hazardous, costly and may leave undesirable residues, difficult to dispose off without adversely affecting the environment (Kosaric, 1992; Banat, 1995). The technique which uses microbes or their products to enhance oil recovery is known as microbially enhanced oil recovery (MEOR) and was first proposed by Beckman (1926). This MEOR technique has proved to be a better alternative to the currently available chemically EOR as microbes or microbial products are generally less toxic, 196

3 biodegradable as well as equally effective as chemical surfactant (Cooper et al., 1980; Rosenberg, 1986; Banat, 1995). Moreover, it requires lower capital and chemical or energy costs (Sarakar et al., 1989). The mechanisms of MEOR include: gas formation, acid production, degradation of lime stone matrices, solvent production, production of polymers, reduction in oil viscosity and interfacial tensions due to production of biosurfactants by microbes (Jack, 1988; Khire and Khan, 1994). Three main strategies have been developed for use of biosurfactants in MEOR viz., 1) injection of biosurfactant producing microbes into the oil reservoir accompanying multiplication of microorganisms through the reservoir rocks, (2) injection of selected nutrient into oil reservoir to stimulate the growth of indigenous biosurfactant producing microorganisms and (3) injection of ex situ produced biosurfactants (Shennan and Levi, 1987). The low molecular weight lipopeptide compounds produced by microorganisms are interesting class of amphiphilic membrane active biosurfactants. Among these lipopeptides, surfactins, iturins and fengycins produced by various Bacilli sp. have been characterized for their surface active properties, emulsifying activity; foaming property, hemolytic activity (Razafindralambo et al., 1998; Peypoux et al., 1999; Deleu et al., 1999; Heerklotz and Seeling, 2001) as well as for their potent antimicrobial activities (Winkelmann et al., 1983; Vanittankom et al., 1986; Vater et al., 2002; Sing and Cameotra, 2004). The interfacial and surface active properties of surfactin, iturin and fengycin produced by Bacillus subtilis have been compared by Deleu et al. (1999). The surfactins have been characterized to be better surface active agents in comparison to iturins and fengycins. The fengycins are 40 and 70 times less hemolytic in comparison surfactins and iturins, respectively. When surfactin is present together with iturin A, it acts synergistically to lyse red blood corpuscles. In this interaction, iturin A adsorbs and penetrated into surfactin monolayer and forms mixed micelles (Thimon, et al., 1992; Maget-Dana, et al., 1992). B. subtilis K1 produces heterogenous mixture of surfactins, iturins and fengycins, lowering the surface tension of broth to 27 mn/m. This property of biosurfactants is useful for their application in enhanced oil recovery (Ghojavand et al., 2008; Joshi et al., 2008; Pattanathu et al. 2008). This prompted us to evaluate the 197

4 applicability of crude lipopeptide preparation from B. subtilis K1 in enhanced oil recovery. The present study describes, 1) Surface active properties and stability of lipopeptide biosurfactants produced by B. subtilis K1 2) Application of lipopeptide biosurfactants produced by B. subtilis K1 in MEOR using sand pack column in laboratory. 7.2 Materials and methods Materials All the chemicals used in this study were of analytical grade. Four stroke engine oil from Honda (SAE 20W40) was procured form local market Medium An optimized glucose minimal salt medium with following constitutents in g/l: glucose, 50; KNO 3, 5.0; KH 2 PO 4.2H 2 O, 1.0; K 2 HPO 4.2H 2 O, 1.0; MgSO 4.7H 2 O, 0.2; CaCl 2.2H 2 O, 0.02) was used for production of biosurfactants. Medium was sterilized by autoclaving at 121º C for 15 minutes Profile of growth, surface tension as well as emulsifying activity of B. subtilis K1 The cells from a single colony of a B. subtilis K1 were inoculated in 50 ml of sterile Luria broth (LB) in 250 ml Erlenmeyer flask and incubated at 30 C for 12 h (O.D ) on orbital shaker (150 rpm) and used as inoculum. The appropriate aliquot of inoculum was added to 100 ml of sterile glucose minimal salt medium in 250 ml Erlenmeyer flasks to get an initial O.D. 600 nm The flasks were incubated on orbital shaker (150 rpm) at 30 C for 120 h and at regular interval of 24 h, one flask was removed, cells were separated by centrifugation (10,062 X g for 20 min.) and supernatant was monitored for surface tension and emulsifying activity. The cell pellet was used for measurement of growth by gravimetric method. 198

5 7.2.4 Surface tension measurement The surface tension (ST) was measured by DCAT-11digital surface-tensiometer (Dataphysics, instruments, Filderstadt, Germany) using Wilhemy plate. The surface tension was measured of the undiulted as well as different dilutions of culture supernatant prepared in distilled water (Vater, et al., 2002). All the measurements were made in triplicate and arithmetic averages and standard errors were calculated Emulsifying activity, stability and Emulsification index (E 24 ) The emulsifying activity of culture supernatant as well as methanolic crude extract obtained by acid precipitation followed by solubilization in methanol was determined as described in chapter 2. Similarly, the emulsifying activity of culture supernatant was also determined for tributurin, groundnut oil, dodecane and olive oil substrates. The emulsifying activity of some known chemical surfactants (Tween 20, Tween 40, Tween 60, Triton X 100 and SDS) was also determined by same method. The hexadecane emulsions prepared using (0.1 mg%, w/v) crude biosurfactant of B. subtilis K1 as well as different chemical surfactants, were allowed to stand for 10 min at room temperature (30 ºC ± 2) upon which absorbance at 600 nm was monitored after every 10 min interval upto 60 min. The log of the absorbance was plotted versus time and the slope of decay (decay constant, Kd) was expressed as the emulsion stability. For emulsification index (E 24 ), 6mL of hydrocarbon (hexane, heptane, octane, 4strok engine oil from Honda) with 4 ml of 48 h old culture supernatant were mixed at high speed for 5 minute. The emulsion thus formed was allowed to stand for 24 h and emulsification index was determined following formula; E 24 = (Height of emulsion layer/height of total liquid column) x Determination of critical micelle concentration (CMC) The stock solution of crude lipopeptide (10 mg per ml) dissolved in 10mM Tris. Cl (ph-8) was prepared. The ST was measured using automode of DCAT- 11digital surface-tensiometer at 25 ºC by automatic dilution of crude lipopeptide solution with distilled water till further increase in surface tension ceased. The 199

6 surface tension was plotted against the concentration of biosurfactant and CMC was determined (Ghojavand, et al., 2008) Stability of crude biosurfactant preparation The stability of crude biosurfactant at 100 C, over a ph range from 2.0 to 12.0 and salinity was investigated. The temperature stability was investigated by monitoring ST of crude biosurfactant before and after incubation at 100 ºC for 2 h (allowed to cool to ambient temperature before monitoring ST). For ph stability, the crude biosurfactant preparation with varying ph (ph 2.0, 4.0, 6.0, 8.0, 10.0 and12.0) adjusted using 1N HCl or 1N NaOH was incubated for 2 days at 30 º C and then monitored for surface tension. The surface tension of crude biosurfactant was monitored before and after incubation in solutions with varying salinity (5%, 10% and 15% (w/v) NaCl) for 2 days in order to determine its salt stability Enhanced oil recovery using sand pack column Sand pack column preparation and oil recovery experiment was carried out with minor modification in the sand pack method described by Suthar et al. (2008). A glass column (25 X 500 mm) was packed with 100 g of acid washed sand (100mesh size). The brine solution (5% NaCl, w/v) was then passed through the column and pore volume (PV) was determined by measuring the volume required to make the sand matrix wet in brine solution. To ensure 100% saturation three PVs of brine was passed through the column. After saturating column with brine solution, four stroke engine oil (Honda) was passed through the column under pressure until the column got saturated with oil. Once, oil entered in the column, discharge of brine solution was observed from the matrix of sand. The discharged volume of brine from sand pack column was collected and measured to calculate initial oil saturation (Soi). The oil saturated column was washed with 4-6 PV of brine solution until no further oil was discharged in the effluent. The oil retained i.e. residual oil saturation (Sor) after brine solution wash was calculated on the basis of oil loaded and oil discharged in the effluent from column. The 0.6 PV of 48 h old cell free fermentation broth containing lipopeptide biosurfactants was 200

7 then passed through the oil saturated sand pack column and allowed to stand for 24 h. The amount of oil recovered upon incubation for 24 h was measured by collecting effluent in 10ml of quantities. This experiment was repeated thrice to evaluate the reproducibility and efficiency B. subtilis K1 crude biosurfactant enhanced oil recovery using sand pack column. The percentage oil recovery was calculated as described by Suthar et al. (2008). The percentage oil recovery was calculated as follows: Pore volume (PV) (ml) = Volume of brine required to saturate the column. Original oil in place (OOIP) (ml) = Amount of brine solution discharged upon displacement by oil sand pack column. Sorwf (ml) = Oil retained after brine flooding. Sorbf (ml) = Oil released after the feeding of sand pack column, saturated with residual oil, with 0.6 PV of biosurfactant preparation. Where x = pore volume - volume of brine displaced after injection of oil in sand pack column Where xi = OOIP - Volume of oil displaced after water flooding 201

8 7.3 Results and discussion: Profile of growth, surface tension and emulsifying activity of B. subtilis K1. The surface tension and emulsifying activity of B. subtilis K1 culture supernatant was monitored along with growth during batch fermentation. The surface tension of the culture supernatant was found to reduce from mn/m to 27.8 mn/m within 24 h of incubation and remained constant thereafter upto 120 h of fermentation (Fig. 7.1). Since surface tension of a solution is expected to remain constant for all surfactant concentrations above their critical micelle concentration, ST of 10- and 100-fold diluted culture supernatant in distilled water was monitored as well. The ST values of 10- as well as 100 fold diluted culture supernatant remained constant upon 48 hours of incubation, suggesting that B. subtilis K1 produced and secreted biosurfactant upto 48 h in batch culture on glucose minimal salt medium. The emulsifying activity of culture supernatant increased along with accumulation of biosurfactant upto 48 h, in batch culture of B. subtilis K1, upon which it started decreasing with further incubation period (Fig. 7.1). The accumulation of biosurfactants in media during the early exponential phase upto mid or late logarithmic growth phase has been reported for several strains of Bacillus sp. and Pseudomonas sp. (Yakimov et al., 1995; Lee et al., 2006; Joshi et al., 2008; Ghojavand et al., 2008) Emulsification index and emulsion stability The culture supernatant of B. subtilis K1 was used to prepare emulsion of hexane, hepatane and octane, which were found to remain stable up to 2 days while emulsion formed with four stroke engine oil (Honda) remained stable for more than two weeks (Table 7.1). The emulsification index of culture supernatant was found to be least with hexane (33.3 %) and maximum with four stroke engine oil (93.3 %). Furthermore it can be seen in table 7.1 that emulsification index increase with increase in chain length of hydrocarbons. Abu-Ruwaida et al. (1991) reported that emulsification index of surfactant produced by Rhodococcus ST-5 was lower for short chain hydrocarbons. The emulsification index values of culture supernatant of B. subtilis K1 were found to be lower than that reported for B. subtilis LB5a (66.6% for hexane and heptanes) (Nitschke and Pastore, 2006). 202

9 Higher emulsification index of B. subtilis K1 culture supernatant with four stroke oil suggests its applications forming emulsions for oil recovery. 80 Surface tension (mn/m): ST/ ST(1:10) / ST(1:100) Biomass yield (g/l) Emulsifying activity (U/mL) Time (h) 0 ST ST(1:10) ST(1:100) E.A Biomass yield Figure 7.1: Profile of biomass yield and production of extracellular biosurfactant antifungal as well as emulsifying activity by B. subtilis K1 Table 7.1: Emulsification index (E 24 (%)) and emulsion stability of biosurfactant produced by Bacillus subtilis K1 Hydrocarbons Hexane Heptane Octane Honda engine oil E 24 (%) 33.3±0.5 40± ± ±0.7 Stability(Days) >15 203

10 Emulsification activity and emulsion stability of crude biosurfactant preparation from B. subtilis K1 The emulsifying activity of crude biosurfactant from B. subtilis K1 was found to be maximum with hexadecane and decreased with decrease in hydrocarbon chain length (Table 7.2). Significantly higher emulsification activity was observed with olive oil, indicating its application in preparation of olive oil based cosmetics. Furthermore, the emulsification activity of biosurfactant from B. subtilis K1 was slightly better than Tween 20, Tween 40, Tween 60 and Triton X100 except for the SDS (Table 7.3). However, the stability of hexadecane emulsion prepared using biosurfactant of B. subtilis K1 and SDS was found to be same while it was lower in comparison to stability of emulsion formed by non-ionic surfactants used in this study. The stability of emulsion of hexadecane formed by biosurfactant of B. subtilis K1 is lower than reported for emulsion stability (soyabean oil) of for surfactant produced by B. subtilis A8-8, by Lee et al. (2006). However, since the substrates used for emulsion in both cases are different, such comparison does not explain the superiority of the biosurfactant. Table 7.2 Emulsifying activity of various substrates by crude biosurfactant preparation from B. subtilis K1 Substrate Emulsification activity (O.D 600nm ) (U/ml) Hexadecane Dodecane Olive oil Groundnut oil Tributurin

11 Table 7.3 Comparison of emulsification activity of crude biosurfactant preparation from B. subtilis K1 and emulsion stability using hexadecane as substrate with chemical surfactants Surfactant Critical micelle concentration (CMC) The critical micelle concentration (CMC) of crude lipopeptide biosurfactant produced by B. subtilis K1 was found to be 20.5 μg/ml (Figure 7.2). The CMC value for the crude lipopeptide biosurfactant is similar to the CMC of lichenysin B (20 μg/ml) biosurfactant produced by B. licheniformis JF-2 (Janneman et al., 1983) and 1.5 fold (30 μg/ml) less than lipopeptide biosurfactant produced by B. subtilis A8-8 (Lee et al, 2006). 80 Emulsification activity (O.D 600nm ) (U/ml) Decay constant (Kd10-3 ) Tween Tween Tween Triton X SDS Biosurfactant Surface tension ST(mN/m) Surface tension Concentration of biosurfactant(ug/ml) Figure 7.2: Surface tension of various concentrations of crude lipopeptide biosurfactant from B. subtilis K1 205

12 7.3.4 Temperature, ph and salinity stability of biosurfactant produced by B. subtilis K1 The surface tension of culture supernatant remained constant even after incubation of 2 h at 100 C, indicating its thermostability (Table 7.4). The surface tension of culture supernatant as well remained unaltered within a ph range 6-12 even upon incubation for 2 days at corresponding ph (Fig. 7.3). At acidic ph, lipopeptides started precipitating and as a resulted the ST would found to increase as expected. Table 7.4 Thermostability of biosurfactant produced by B. subtilis K1 at 100 C for 2 h. Temperature (ºC) ST (mn/m) ST(1:10) (mn/m) ST(1:100) (mn/m) ± ± ± ± ± ±0.03 Surface tension(mn/m): ST/ ST(1:10)/ ST(1:100) ST ST(1:10) ST(1:100) ph Figure 7.3 : ph stability of biosurfactant produced by B. subtilis K1 for 2 days 206

13 The ST of culture supernatant of B. subtilis K1 in absence of NaCl was 27.55mN/m, which decrease slightly with increase in NaCl concentration upto 10 g/100 ml. The ST of 10-fold and 100-fold diluted culture supernatant also decreased with increase in NaCl concentration upto 5 g/100 ml, which further increased with higher NaCl concentrations. Thus the results indicate that surfactant of B. subtilis K1 exhibited maximum surface activity at around 5 g NaCl/100 ml. Similar properties of stability at high temperature, over wide ph range and salinity have been reported for surfactants produced by several Bacilli spp. (Vater et al., 2002; Nitschke et al., 2006; Joshi et al., 2008; Ghojavand et al., 2008).The surface active property and emulsifying activity of biosurfactants produced B. subtilis K1 along with their stability towards extreme conditions suit to its application in enhanced oil recovery (MEOR). Surface tension (mn/m) : ST/ ST(1:10)/ ST(1:100) ST ST(1:10) ST(1:100) NaCl concentration (g %, w/v) Figure 7.4: Effect of NaCl concentrations on surface activity of biosurfactant produced by B. subtilis K1 207

14 7.3.5 Enhanced oil recovery using crude biosurfactant produced by B. subtilis K1 To evaluate the efficiency of crude biosurfactant produced by B. subtilis K1 in oil recovery process, sand pack column as a laboratory scale technique was employed. Table 7.3 shows the values for the various parameters viz., PV, OOIP, Sorwf, Sorbf, Swi(%), Soi(%), Sor(%), Aor(%) monitored for determination oil recovery repeated thrice. The value for PV, OOIP and Sorwf observed for the three replicates varied from ml, ml and ml, respectively. Upon flooding of column with brine, % oil was recovered from the oil saturated column. This brine flooding step can be considered analogous to the water flooding operation in oil well during secondary phase oil recovery (Suthar et al., 2008.) The remaining to 67.5 % of oil in column was trapped in matrix of sand. The trapping of oil in sand pack column mimics the situation in oil well, where the oil gets trapped in the form of oil ganglia in pores of rocks (Shah, 1981). To recover the oil trapped in the sand pack column, 0.6 pore volume of 48 h old cell free fermentation broth of B. subtilis K1 as a source of biosurfactant was passed in the oil saturated sand column. Upon 24 h of incubation, the oil displaced from the sand matrix was recovered and percentage additional oil recovery (AOR) was calculated. By using cell free fermentation broth of B. subtilis K1, 43.33±0.87% of AOR was achieved. This AOR obtained in this study was comparable with the recovery achieved by the crude biosurfactant (43.1±3.3%) produced by B. licheniformis K125 (Suthar et al., 2008). The strain B. mojavensis JF2 was isolated from the water injection brine of water-flood operation in Carter County, Oklahoma oil reserve and it has been reported for their efficient oil recovery in core flood studies as well as from actual oil reservoirs (Maudgalya, 2005; McInerney et al., 1999). B. mojavensis JF2 was used as standard strain for evaluation of MEOR by Suthar et al., (2008), and with this strain, 29.4±5.6% additional oil over Sorwf was achieved. The AOR value obtained by cell free fermentation broth of B. subtilis K1 was 13.9 % higher then the AOR obtained using culture supernatant of. B. mojavenis JF2. Several Bacillus strains have been studied for their ability of biosurfactant production (Vater et al., 2002; Nitschke et al., 2006; Ghojavand et al., 2009) and also have 208

15 been evaluated for the enhanced oil recovery using biosurfactants produced by them (Makkar and Cameotra, 1997; Janneman et al., 1984; Banat et al., 1991; Joshi et al., 2008; Suthar et al., 2008). The biosurfactants produced by B. subtilis MTCC 1427 and B. subtilis MTCC 2423 were shown to recover 56 and 62% of oil from sand pack columns (Makkar and Cameotra, 1997). The biosurfactant of B. subtilis 20B was demonstrated to enhance recovery of % oil from sand pack column (Joshi et al., 2008). Thus, the crude biosurfactant obtained from culture supernatant of B. subtilis K1 is a potential emulsifying agent and can be useful for enhanced oil recovery from oil reserves. Table 7.3: Parameters for oil recovery using water flood and lipopeptide biosurfactant from B. subtilis K1 in sand pack glass columns Parameters Sand pack column(spc) SPC SPC-1 SPC-2 SPC-3 Mean± S.E. PV(mL) ±0.57 OOIP(mL) ± 0.66 Sorwf(mL) ±0.00 Sorbf(mL) ±0.16 Swi(%) ±0.71 Soi(%) ±0.71 Sor(%) ±0.87 Orecwf(%) ±0.83 AOR(%) ± Summary and Conclusion: The biosurfactants produced by B. subtilis K1 showed good emulsification activity, with high stability and significantly good surface active properties. It was found to be stable over wide range of ph, high temperature and highly salinity, thus making it suitable for various industrial applications. The application of crude biosurfactant preparation in enhanced oil recovery using sand pack column in laboratory was successfully demonstrated with 43.3% oil recovery. 209

16 7.5 References Abu-Ruwaida, AS., Banat, IM., Haditirto, S., Salem, A., Kadri, M. (1991) Isolation of biosurfactant producing bacteria. Product characterization and evaluation. Acta Biotechnology, 11, Banat, I.M. (1995) Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: A review. Bioresource Technology, 51, Banat, IM., Samarah, N., Murad, M., Horne, R. and Banerjee, S. (1991) Biosurfactant production and use in oil tank clean-up. World Journal of Microbiology and Biotechnology, 7, Brown, L. (2010) Microbial enhanced oil recovery (MEOR). Current Opinion in Microbiology, 13: Cooper, DG., Goldenberg, BG. (1987) Surface active agents from two Bacillus species. Applied and Environmental Microbiology, 53, Deleu, MH., Razafindralambo, H., Popineau, Y., Jacques, P., Thonart, P., Paquot, M. (1999) Interfacial and emulsifying properties of lipopeptides from Bacillus subtilis. Colloids Surface A Physicochemical Engineering Aspects, 152, Desai, J.D., Banat, I.M. (1997) Microbial production of surfactants and their commercial potentials. Microbiology and Molecular Biology Review, 61, Ghojavand, H., Vahabzadeh, F., Roayaei, E., Shahraki, AK. (2008) Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696). Journal of Colloid and Interface Science, 324, Heerklotz, H., Seelig, J. (2001) Detergent-like action of antibiotic peptide surfactin on lipid membranes. Biophysical Journal, 81, Jack, TR. (1988) Microbially enhanced oil recovery. Biorecovery,1, Jenneman, GE., Knapp, RM., Mclnerney, MJ., Menzie, DE., Revus, DE. (1984) Experimental studies of in situ microbial enhanced oil recovery. Society of Petroleum Engineering Jounral,

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