Colloids and Surfaces B: Biointerfaces 114 (2014) 324–333
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Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil recovery Yahya Al-Wahaibi a,∗ , Sanket Joshi b , Saif Al-Bahry b , Abdulkadir Elshafie b , Ali Al-Bemani a , Biji Shibulal b a b
Petroleum and Chemical Engineering Department, Sultan Qaboos University, Oman Biology Department, Sultan Qaboos University, Oman
a r t i c l e
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Article history: Received 27 April 2013 Received in revised form 8 September 2013 Accepted 11 September 2013 Available online xxx Keywords: Biosurfactant Bacillus subtilis Surface and interfacial tension reduction Critical micelle dilution Emulsification index Microbial enhanced oil recovery
a b s t r a c t The fermentative production of biosurfactants by Bacillus subtilis strain B30 and the evaluation of biosurfactant based enhanced oil recovery using core-flood were investigated. Different carbon sources (glucose, sucrose, starch, date molasses, cane molasses) were tested to determine the optimal biosurfactant production. The isolate B30 produced a biosurfactant that could reduce the surface tension and interfacial tension to 26.63 ± 0.45 mN/m and 3.79 ± 0.27 mN/m, respectively in less than 12 h in both glucose or date molasses based media. A crude biosurfactant concentration of 0.3–0.5 g/l and critical micelle dilution (CMD) values of 1:8 were observed. The biosurfactants gave stable emulsions with wide range of hydrocarbons including light and heavy crude oil. The biosurfactants were partially purified and identified as a mixture of lipopeptides similar to surfactin, using high performance thin layer chromatography and Fourier transform infrared spectroscopy. The biosurfactants were stable over wide range of pH, salinity and temperatures. The crude biosurfactant preparation enhanced light oil recovery by 17–26% and heavy oil recovery by 31% in core-flood studies. The results are indicative of the potential of the strain for the development of ex situ microbial enhanced oil recovery processes using glucose or date molasses based minimal media. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Biosurfactants are biological surface active agents, produced by microorganisms, plants and animals including humans. Biosurfactants are classified by various methods – ionic nature, type, size [1]. Generally low molecular weight biosurfactants are well known for reducing surface tension (ST) and interfacial tension (IFT) between oil and water and thus enhancing oil recovery. Whereas, high molecular weight biosurfactant like emulsan are known for their emulsifying properties and thus enhancing the mobility of heavy-oil and its recovery [2]. Recently, biosurfactants have been the focus of extensive research because they have several advantages over chemical surfactants: lower toxicity, higher environmental compatibility, biodegradability, and synthesis from renewable raw materials [1,3]. Biosurfactants have the benefit of being biodegradable and relatively inexpensive; however, there are some limitations which prevent their practical applications in the field. The main limitation is the availability of the biosurfactants in bulk quantity as compared to the chemical surfactants, in addition to scale-up related complications in producing large
∗ Corresponding author. Tel.: +968 99358758. E-mail address: [email protected] (Y. Al-Wahaibi). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.022
amounts of biosurfactants for field applications. However, Torres et al. [4], reported the discovery of new biosurfactants and the development of new fermentation and recovery processes may allow more biosurfactants to be used for microbial enhanced oil recovery (MEOR). Currently the bulk availability of biosurfactants commercially is limited (except rhamnolipids, e.g. available at – http://www.rhamnolipid.com/) as the cost of producing biosurfactant in bulk is still high. Several researchers have suggested the application of statistical optimization methods or the use of cheaper raw material such as agro-industrial by-products to reduce the cost of production [5–12]. Depending on the ease of the availability, the selection of the raw material differs from country to country. In Oman, date molasses was proposed as a carbon sources, due to its ready availability and lower cost [5]. Bacillus group of bacteria are known for producing potent lipopeptide biosurfactants such as surfactins and lichenysins, using different raw materials, and have been widely studied because of their high surface activities and other properties [13]. The surfactins and lichenysins are also reported as strong bacterial biosurfactants suitable for enhancing oil recovery [14]. There is a need for sustainable and more efficient oil recovery due to the depletion of current world oil reserves and the increasing demand for liquid. Several enhanced oil recovery (EOR) processes are currently employed worldwide: thermal, chemical, physical, etc. [15]. These
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processes are successful, but also very expensive as well as environmentally harmful. The alternative is a biologically based EOR process, also known as MEOR. MEOR can be implemented in several ways: either as in situ process employing indigenous microorganisms or ex situ process where bioproducts are produced outside of oil wells and directly injected to enhance oil recovery [16]. For in situ MEOR, we need to identify the indigenous microbial population [17] and provide them with suitable nutrient media for growth and production of bioproducts which will eventually enhance the recovery factor. Unfortunately this process cannot be controlled completely and success ratios may be limited. Whereas, with ex situ MEOR we can produce microbial products outside the oil wells and can inject them directly for oil recovery enhancement. Perfumo et al. [16] studied several MEOR reports of both in situ or ex situ field studies and reported that we still lack accurate data on microbial growth and metabolism under field condition. We isolated Bacillus subtilis B30 from petroleum contaminated soil samples from a garage in Oman. The ability of the isolate to produce biosurfactant was studied. The produced biosurfactant was tested for stability under different conditions of temperature, pH and salinity, and was also checked for its effect on enhancing oil recovery. 2. Materials and methods 2.1. Chemicals and reagents All major chemicals and trace elements were purchased from Sigma–Aldrich Co., USA. Cane molasses and date molasses were purchased from the local market, Sultanate of Oman. Light and heavy crude oil was provided by Petroleum Development of Oman (PDO). 2.2. Microorganism and culture conditions Several bacteria were isolated from soil samples collected from petrol pump sites, garages and other sites exposed to crude oil, using oil spreading method as described by Youssef et al. [18]. Those isolates which showed good biosurfactant producing potential (based on a larger zone of clearance on oil layer) were selected for further experiments at shake flask level in Luria–Bertani (LB) broth. Isolates which reduced surface tension (ST) below 35 mN/m were selected further, as described by Joshi et al. [9]. One strain – B30 was selected for further screening, and was identified as B. subtilis by 16S rRNA gene sequencing (GenBank accession no. KC686715). The isolate was maintained aerobically on LB agar plates and was regularly transferred into fresh LB medium for shortterm storage. Stock cultures of all pure isolates were prepared in 30% glycerol and stored below −40 ◦ C, for long-term maintenance. 2.3. Production media and effect of different carbon sources on biosurfactant production The culture was grown for 15–16 h at 40 ◦ C (OD660 nm -1.0–1.2) in LB as a seed inoculum medium. The flasks containing 50 ml of different minimal production media in 250 ml Erlenmeyer flasks were inoculated with 2% (v/v) seed culture. The cultures were incubated on a temperature controlled incubator shaker at 160 rpm. The compositions of different media used in this study are (g/l): Medium ‘C’ [19] NH4 NO3 , 4.0; KH2 PO4 , 4.08; MgSO4 ·7H2 O, 0.2; FeSO4 ·7H2 O, 0.0011; MnSO4 ·4H2 O, 0.00067; CaCl2 , 0.00077 and Medium ‘M’ [20] NH4 NO3 , 3.3; K2 HPO4 , 2.2; KH2 PO4 , 0.14; MgSO4 ·7H2 O, 0.6; FeSO4 ·7H2 O, 0.2; CaCl2 , 0.04; NaCl, 0.01; Trace elements, 0.5 ml/l. Media were sterilized by autoclaving at 121 ◦ C for 15 min. Five different carbon sources: glucose, sucrose, starch, cane molasses or date molasses were added at 2% (w/v) final concentration to
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both media after filter sterilization. Samples were withdrawn and, growth (OD660 ), pH, and biosurfactant production – surface tension (ST) and interfacial tension (IFT) were checked up to 72 h. 2.4. Analytical methods All measurements were made on cell-free broth after centrifugation (12,096 × g for 20 min) and analyzed at room temperatures. The experiments were performed in duplicate and the resulting reports are the mean of three independent experiments. 2.5. Biosurfactant analysis, contact angle measurements, and critical micelle dilution (CMD) Production of biosurfactant was analyzed by periodic measurement of ST and IFT of cell-free broth by means of pendant drop method using the Drop Shape Analyzing system – DSA 100 (KRÜSS, Germany). IFT was measured against Hexadecane. Contact angles of the untreated samples (un-inoculated media) and biosurfactants were measured using the Drop Shape Analysis System, DSA100 (Kruss, Germany), as described by Al-Sulaimani et al. [21]. All measurements were done in triplicates at ambient temperature (25 ± 2 ◦ C) and atmospheric pressure (1 atm) and the average values were reported. The biosurfactant concentration expressed in terms of critical micelle dilution (CMD) was estimated by measuring the ST for varying dilutions of the sample [9]. 2.6. Emulsification index (E24 ) determination The ability of the biosurfactants to emulsify liquid hydrocarbons, such as tridecane, pentadecane, tetradecane, hexadecane, heptamethyl nonane, heptane, hexane, methyl naphthalene, heavy crude oil (API 15.90◦ ), and light crude oil (API 36.51◦ ) was determined. The emulsification index (E24 ) was measured using a previously reported protocol with slight modifications [22–24]. E24 was determined by adding 2 ml of different hydrocarbons to 2 ml of cell free supernatant, vortex at high speed for 5 min, and the mixture was incubated at ambient temperature for 24 h. The E24 index is given as percentage of height of emulsified layer (mm) divided by total height of the liquid column (mm). 2.7. Biosurfactant extraction The biosurfactant was partially purified and extracted by the acid precipitation method [25]. Bacterial cells were separated by centrifuging at 12,096 × g for 20 min at 20 ◦ C (Beckman, JLA 16.250 rotor, USA). The pH was adjusted to 2.0 using 6 M HCl to precipitate biosurfactant. The solution was kept overnight at 4 ◦ C and the precipitated biosurfactant was collected by centrifuging the solution at 12,096 × g for 25 min at 4 ◦ C. The collected biosurfactant pellet was dissolved in distilled water and pH was adjusted to 8.0. Crude biosurfactant powder was collected by spray-drying at 100–160 ◦ C using Mini-Spray Dryer B-290 (BUCHI, Switzerland). Extracted crude biosurfactant was then tested for its stability and chemical characterization and in core-flooding experiments and other studies. 2.8. High performance thin layer chromatography (HPTLC) Biosurfactants were separated and analyzed using a completely automated HPTLC system (CAMAG, Switzerland) in the Central Analytical and Applied Research Unit (CAARU), Sultan Qaboos University, Oman. Twenty microliter samples were spotted onto a 10 cm × 10 cm precoated silica gel HPTLC plate (Merck, Germany) containing green fluorescent F254 . These samples were
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spotted under a flow of nitrogen gas using automatic TLC sampler 4 (ATS 4) spotting device (CAMAG, Switzerland) having a robotic arm in a closed chamber. The plates were developed using an automatic developing chamber ADC 2 (CAMAG, Switzerland) with remote operation from winCATS software, containing solvent systems – MP1: Butanol:Acetic acid:Water (8:2:2) and MP2: Chloroform:Methanol:Water (65:25:4). The documentation and evaluation of the TLC plate was done using TLC visualizer (CAMAG) under white light as well as with direct UV 254 and UV 366 nm light, capturing the images. The separated biosurfactants were also qualitatively detected by TLC scanner 4 (CAMAG) under Ultra Violet (UV) light (254 nm), and separated biosurfactant bands were compared. 2.9. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FT-IR) analysis was conducted to identify the structural groups of the biosurfactant. Infrared (IR) absorption spectra were obtained with a Perkin-Elmer grating 1000 IR (Norwalk, CT) in a dry atmosphere. In this test, 1 mg of crude biosurfactant was mixed with 100 mg of KBr and pressed with load for 30 s, to obtain translucent pellets. FTIR spectra were collected between 400 and 4000 wave numbers (cm−1 ). 2.10. Biosurfactant stability studies Stability of biosurfactant was studied under wide range of temperatures, pH and different salt concentrations to test the effect on the biosurfactant activity. Unless specified, the experiments were conducted at room temperature, pH 7.0 and 0% salt concentration. For the temperature stability test, the samples were kept in 10 ml serum bottles and were tightly sealed with butyl rubber stoppers and aluminum crimps. The range of temperatures tested were 40–100 ◦ C. The biosurfactant broth was also subjected to autoclave conditions (121 ◦ C, 15 psi for 30 min) in order to investigate the effect of such environment on the surface activity of the biosurfactant. For the salinity test, NaCl was added to the biosurfactant cell-free broth at different concentrations 0–15% (w/v). The stability of the biosurfactant was also determined by measuring the ST and IFT at different pH values (2.0–12.0) at room temperature. 2.11. Core-plugs and fluid samples A set of Berea sandstone cores (1.5 in. diameter × 3 in. long) with average porosity and permeability of 20–25% and 250–350 mD, respectively, were used for the core-flood experiments. The formation water and crude oil used in these experiments were obtained from an Omani oil field of interest which has an average reservoir temperature of 60 ◦ C. The salinity of the formation water was between 7% and 9% and its chemical composition was (kg/m3 ): Sodium, 25.083; Calcium, 3.762; Magnesium, 0.878; Iron, 0.045; Chloride, 47.722; Sulphate, 0.247; Bicarbonate, 0.079. Formation water was filtered prior to use, by Millipore Filtration Unit (0.45 m). The crude oils used for core-flood experiments were of API 36.51◦ and 15.90◦ . 2.12. Core-flood experiments For all core-flooding experiments, cores were cleaned by using the soxhlet extraction method, using chloroform and methanol as an azeotropic mixture in the proportion of 75:25 [26]. After cleaning, the core was dried at 65 ◦ C for 24 h before using. The core was saturated with filtered formation brine using vacuum desiccators for 24 h and pore volume (PV) was determined using the dry and wet weights of the core. The core was then flooded with crude oil at 24 cm3 /h until no more water was produced. The oil
initially in place (OIIP) was determined, which was indicated by the volume of water displaced. The core was subjected to waterflooding at 24 cm3 /h until no further oil was produced. The residual oil was calculated by measuring the amount of oil produced from the water-flood. Then, 5 PV of the cell-free supernatant (biosurfactant broth) was injected as a tertiary recovery stage and extra oil recovery was determined. All core floods were conducted at 60 ◦ C to mimic the average reservoir temperature of the field of interest. 3. Results and discussion 3.1. Carbon source optimization Several bacteria were isolated from different petroleum contaminated soil samples from Oman. Based on oil spreading and reduction in ST values, Bacillus B30 was selected for further studies. Two different reported minimal production media were used for carbon source optimization. We tested glucose, sucrose, starch, cane molasses and date molasses in both the minimal production media. Out of all the carbon sources tested, glucose in ‘C’ medium (CG) and date molasses in ‘M’ medium (MDM) gave better reduction in ST and IFT, as compared to rest of the media combinations (as per supplementary data). Media ‘CG’ and ‘MDM’ were further studied to find out the best time for biosurfactant production. Samples were harvested every 2 h for 72 h, and studied for growth and biosurfactant production. In medium ‘CG’, the lowest ST (25.56 ± 0.97 mN/m) and IFT (3.6 ± 0.66 mN/m) were observed at 8–10 h (Fig. 1A). Maximum growth was observed within 22 h and no change in pH was observed. In ‘MDM’ medium, maximum reduction in ST (26.31 ± 0.07 mN/m) and IFT (3.56 + 0.10 mN/m) was observed at around 12–14 h (Fig. 1B). Whereas, growth was better as compared to ‘CG’ medium at 22 h and no major changes in pH were observed. Lin et al. [27] have reported shake-flask studies for Bacillus licheniformis JF-2 lipopeptide biosurfactant production, which occurred during active growth but subsequently disappeared from the fermentation broth within 6 h. They have reported that the disappearance of the biosurfactant from the fermentation broth was due to its internalization by stationary-phase cells. However, Joshi and Desai [28] reported the biosurfactant production by 5 bacterial strains for up to 72 h and reported ST and IFT values in the range of 28–30 mN/m and 0.5–5.8 mN/m, respectively, with no changes in ST and CMD. Gojavand et al. [29] also reported the maximum production of biosurfactant by B. subtilis (PTCC 1696) during the growth phase (10 h), which remained constant even after the exponential phase. Al-Bahry et al. (2012) also reported similar phenomenon for biosurfactant production by B. subtilis B20 using date molasses within 10–12 h of fermentation and no changes thereafter till 72 h. We also observed similar trend for biosurfactant production by B. subtilis B30. Amongst all the carbon sources tested in present study, Date molasses is comparatively cheaper and available in abundant quantity here in Oman. So, it is recommended to be used as a suitable carbon source for higher biosurfactant scale-up production studies in Oman. 3.2. Contact angle determination A fluid is said to wet a solid surface if the contact angle is less than 90◦ (for surfactant containing liquids). If the contact angle is greater than 90◦ , the fluid is said to be non-wetting [30]. Recently, wettability alteration has been proposed as one of the mechanisms of MEOR where several studies reported the relation between IFT reduction and alteration of wetting conditions following microbial treatment [21,31–33]. It was suggested that the reduction in IFT may change the oil–rock contact causing an altered wettability. Studies reported that the change in wetting properties is dependent on the initial wetting condition where alterations from oil-wet toward more
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Fig. 1. Production profile (ST, IFT, OD660 , pH) of Bacillus B30 in ‘CG’ (A) and ‘MDM’ media (B). The error bars represents SD values of three independent experiments (n = 3).
water-wet and vice versa have been reported [33]. Microbial treatment has also shown to change wetting conditions of core samples, thus resulting in a change in the imbibition/drainage behavior [34]. The biosurfactants from ‘CG’ and ‘MDM’ media were studied for their effect on alteration of contact angle on a hydrophobic plate. The contact angles were reduced from 58.7 ± 0.85◦ of un-inoculated media to 28.4 ± 1.03◦ and 27.2 ± 0.72◦ in ‘CG’ and ‘MDM’ media, respectively (Fig. 2). Kowalewski et al. [33] reported simulation studies of a core-flood that reduced the residual oil saturation from 27% to 3%. Their objective was to investigate the mechanisms of MEOR focusing on IFT reduction and wettability alteration. The simulation results showed that both a gradual reduction in IFT and change in wettability can be used to observe the reduction in residual oil saturation. Al-Sulaimani et al. [21] reported that biosurfactant produced by B. subtilis W19 changed the contact angle of distilled water from 70.6 ± 0.3◦ to 25.32 ± 0.06◦ at 0.25% concentration and no further changes were observed while increasing the concentration of biosurfactant reported reduction in contact angle from 103.0 ± 0.36◦ to 82.7 ± 0.43◦ , by Bacillus I-19 biosurfactant, using crude oil as a carbon source. Karimi et al. [36] reported that microbial solutions can alter the wettability of 7 and 21 days aged glass surfaces to more water-wet conditions. In the current
study, wettability alteration caused by the Bacillus B30 biosurfactant was investigated by contact angle measurement, and it was observed that biosurfactant changed the wettability toward more water-wet, which could be quite helpful mechanism for improving oil recovery at field scale applications. 3.3. Biosurfactant yield and CMD determination The yield after acid precipitation and spray drying was 0.5 g/l for ‘CG’ medium and 0.3 g/l for ‘MDM’ medium. Generally CMD values are determined by carrying out ST or IFT measurements on a series of different dilutions of surfactant solutions and CMD is indirectly proportional to critical micelle concentration (CMC). The dilution at which the ST begins to increase is termed the CMD, which is the factor by which the effective biosurfactant concentration exceeds the critical micelle concentration [37]. Thus, higher the CMD – the more industrially promising is the surfactant. Estimating the CMD of the surfactant gives an indication on the optimum concentration to be used for maximum performance and minimum cost possible. Biosurfactant containing broths were used to determine CMD for both the biosurfactants. As shown in Fig. 3, ST and IFT decreased progressively with increasing biosurfactant concentration. The ST
Fig. 2. Contact angle determination from un-inoculated media 58.7 ± 0.85◦ (A); B30 biosurfactant from ‘CG’ medium 28.4 ± 1.03◦ (B) and B30 biosurfactant from ‘MDM’ medium 27.2 ± 0.72◦ (C).
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Fig. 4. Emulsification index against different hydrocarbons, for Bacillus B30 biosurfactants. The error bars represents SD values of three independent experiments (n = 3).
Fig. 3. CMD determination for biosurfactants produced by Bacillus B30 in ‘CG’ (A) and ‘MDM’ (B). The error bars represents SD values of three independent experiments (n = 3).
increased from an initial value of undiluted biosurfactant broth to 40.84 mN/m up to 1:8 times dilutions, and increased thereafter. The IFT increased following almost similar trend where the IFT began to increase at around 1:8 times dilution with an average of
11.86 mN/m. It was observed that, CMD for ‘CG’ was 8× and for ‘MDM’ were 8× (Fig. 3). Ghurye et al. [37] used activated sludge from the wastewater treatment as a source of microorganisms for biosurfactant production. For a pH controlled bioreactor containing 20 g/l molasses, yeast extract and other nutrients, the ST of the broth reduced to 40 dynes/cm within 48 h and maximum biosurfactant concentration of about 63× CMD in cell-free broth was achieved in 120 h. Joshi and Desai [28] reported CMD values of 75–100× for biosurfactants mixtures produced by different Bacilli strains. Makkar and Cameotra [38] have reported the production of biosurfactants by two B. subtilis strains at 45 ◦ C using molasses at the concentration of 2% total sugars, and minimal medium. Both strains reduced ST in the range of 30 dynes/cm with 10× CMD values – 35 to 40 dynes/cm and 100× CMD values – 50 to 65 dynes/cm after 96 h. We also observed CMD values of 8× (1:8 dilution), which is comparable to those reported by B. subtilis [38] and by microorganisms from activated sludge [37] under identical conditions.
Fig. 5. HPTLC plate showing separated biosurfactants from ‘CG’ and ‘MDM’ media, under (A) UV 254 nm and (B) UV 366 nm.
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Fig. 6. FTIR absorption spectra of biosurfactant produced by B. subtilis B30 from ‘CG’ medium (A) and ‘MDM’ medium (B).
3.4. Emulsification index The biosurfactant containing cell-free broth was able to emulsify many hydrocarbons. Biosurfactant containing cell free broth of ‘CG’ medium gave emulsions with various hydrocarbons such as, tridecane, tetradecane, hexadecane, methyl naphthalene, heavy crude oil and light crude oil (Fig. 4). Whereas, biosurfactant containing
cell-free broth of ‘MDM’ medium gave emulsions with all tested hydrocarbons, except heavy crude oil (Fig. 4). Somehow neither of biosurfactants gave >20% emulsion with pentadecane, after 24 h. Das et al. [22] reported biosurfactant production by marine Bacillus circulans in glycerol mineral salt medium and antracene supplemented glycerol mineral salt medium, which emulsified various hydrocarbons such as diesel, hexadecane, kerosene, benzene and
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Fig. 7. FTIR absorption spectra of standard Surfactin (Sigma Chemicals, USA).
petrol in the range of 30–80%. Das et al. [39] also reported biosurfactant production by a marine bacteria in mineral salt media containing different carbon sources (glucose, sucrose, starch or glycerol) with higher emulsification power of the biosurfactant obtained from glycerol and starch than that of the biosurfactant obtained from glucose and sucrose with E24 values in the range of 45–85%. Khopade et al. [23] reported almost 80% E24 against hydrocarbon, within 8–9 days by biosurfactant produced by a marine Nocardiopsis sp. B4. We observed higher emulsification (≥50%) with long chain hydrocarbons (Fig. 4) such as crude oil, which could most probably play an essential role in enhancing oil recovery.
3.5. Partial identification using HPTLC and FTIR Out of two solvent systems used for HPTLC, MP1 gave better separation (Fig. 5) as compared to MP2 (supplementary file). As shown in Fig. 5, we loaded 160 g of biosurfactant from ‘MDM’ and 160 and 2400 g of biosurfactant from ‘CG’ medium. As the biosurfactant powder was dark brown, a lower concentration of biosurfactant from ‘MDM’ was loaded, in order to avoid any interference in separation. The separated biosurfactant bands from both glucose and date molasses containing media were almost similar with Rf values. The increase in the intensity of the bands for biosurfactant from glucose containing media was evident when we increased initial concentration of biosurfactant from 40 mg to 60 mg. Several researchers reported use of TLC or HPTLC as a tool for initial qualitative detection or quantitative analysis of different biosurfactants [20,39,40]. IR spectra of biosurfactants produced by Bacillus B30 are shown in Fig. 6(A and B). The IR spectrum in KBr showed bands characteristic of peptides at 3400 cm−1 (NH stretching mode) and at 1654 cm−1 (stretching mode of the CO N bond). The bands at 1406 cm−1 , 1246 cm−1 reflect aliphatic chains ( CH3, CH2 ) of the fraction [41,42]. These results imply the presence of aliphatic hydrocarbons as well as a peptide-like moiety in the biosurfactant. The peaks shown in Fig. 6 matched the standard surfactin (Sigma chemicals, USA, Fig. 7). The stretching modes of the NH, CO N bond and CH3, CH2 fractions fall within the same range of wave numbers which suggests the similarity in structure of the biosurfactant produced by Bacillus B30 with surfactin. Surfactins are lipopeptidal biosurfactant with very good surface activities and are reported as very good candidate with potential applications in enhancing oil recovery and petroleum industry [14].
3.6. Stability studies The use of partially purified biosurfactant for ex situ MEOR requires the biosurfactant to be stable within a range of high temperatures (around 50–80 ◦ C), wide range of pH and high salt concentrations to ensure wide applicability to induce oil recovery. The biosurfactant produced using either glucose or date molasses were quite stable over a wide range of temperatures from 40 to 100 ◦ C (Fig. 8A), and also was stable at 121 ◦ C (during autoclaving) and 160 ◦ C (during drying in spray drier). Khopade et al. [23] also reported the stability of biosurfactants under extreme conditions of temperature. Desai and Banat [1] reported that heat treatment on some biosurfactants caused no appreciable change in their properties, even after autoclaving at 120 ◦ C for 15 min. Similarly, Borodoli and Konwar [43] reported the biosurfactant produced by Pseudomonas aeruginosa strains to be stable at temperature of 100 ◦ C for different time periods of 5–60 min with respect to ST changes. Joshi et al. [9] reported biosurfactants produced by 4 Bacillus strains to be stable at 80 ◦ C for 9 days. The biosurfactants produced from ‘CG’ medium or ‘MDM’ were also stable in pH range of 6–12 and salt concentration up to 5% NaCl (Fig. 8B and C). Under highly acidic pH (pH 2.0 and 4.0) biosurfactants showed much less activity, since the biosurfactant is not soluble under highly acidic conditions and tends to precipitate. This higher instability of biosurfactants produced from some Lactobacilli in acidic conditions was described by some researchers to be related to the presence of negative charged groups at the polar ends of the molecules [44]. Several reports confirmed the stability of biosurfactant at different pH values, mostly in the alkaline medium [9,23,26,41,44,45]. Bacillus B30 biosurfactant showed stability under various extreme conditions as reported by other researchers.
3.7. Core-flooding for light and heavy oil recovery and future possibility of field application The pore volume of the core sample was 18–24 cm3 . Initial oil saturation (Soi ) was calculated to be 55–67% after oil flooding. It was found that after injecting 5–8 PV of water, no more oil was produced and residual oil saturation (Sor ) was about 25–38%. Extra oil recovery was observed after injecting 4–5 PV of biosurfactant solution from ‘CG’ medium or ‘MDM’ medium, where 17–26% of Sor was produced (Fig. 9A and B). Biosurfactant produced by ‘CG’ medium also showed 31% extra recovery (Fig. 9C) of heavy crude
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Fig. 8. Stability studies for biosurfactant produced by Bacillus B30 in ‘CG’ and ‘MDM’ media, under different temperature (A), pH (B) and salinity (C). The error bars represents SD values of three independent experiments (n = 3).
oil (API 15.90◦ ). Joshi et al. [28] reported 30.22–34.19% additional oil recovery over the water flood residual oil saturation using crude biosurfactant obtained from Bacillus strains by sand pack column studies. Darvishi et al. [46] reported 27.2% oil recovery efficiency due to the injection of the cell-free biosurfactant. Thomas et al. [47] reported enhancement of oil recovery from Berea sandstone core treated with cell-free metabolites from surfactant producing strain Bacillus sp. JF-2. In present study core flooding experiments were carried out using Berea cores which were treated with cell-free biosurfactant broths using glucose or date molasses as a carbon source, where an increase in oil recovery in ex situ MEOR experiments was detected following biosurfactant injection. This is similar to reports shown by other researchers [26,48,49]. The heavy oil recovery is the target for many EOR operations worldwide. The B30 biosurfactant showed additional oil recovery for both light and heavy oil, which
have quite promising applications in Oman. Especially as Oman has many oil fields with heavy oil. However real potential of microbial products (biosurfactants) in MEOR applications can only be fully assessed in field-scale applications. Several trials have been carried out during the past years and tentatively reviewed [16]. The real impact of biosurfactantbased MEOR trials however has never been tested at a larger-scale, because of lack of both quantitative information regarding microbial growth and metabolism in situ and insufficient data collection and processing. Recently Youssef et al. [50] reported a small fieldscale MEOR experiment (viola limestone formation) providing for the first time data of in situ bacterial metabolism and activities. Molecular biology techniques with traditional methods showed that Bacillus strains injected into oil wells were metabolically active, consuming the nutrients supplied (glucose, sodium nitrate
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Fig. 9. Cumulative oil recovery by B30 biosurfactant from ‘CG’ medium (A) and ‘MDM’ medium (B) with light oil (API 36.51◦ ) and for heavy oil recovery (API 15.90◦ ) with B30 biosurfactant from ‘CG’ medium (C). The error bars represents SD values of three independent experiments (n = 3).
and trace metals) and releasing CO2 , acids, solvents and lipopeptide biosurfactant thus releasing more oil from the formation. Their data demonstrated the technical feasibility of biosurfactant based MEOR future applications.
4. Conclusion Isolate B. subtilis B30 produced a potent biosurfactant (lipopeptide similar to Surfactin), using cheaper mineral salt media containing either glucose or date molasses, which is quite stable under harsh conditions; gives stable emulsions with wide range of hydrocarbons; and gave around 17–31% additional recovery of light or heavy crude oil. Further experiments should be done at higher scale.
Acknowledgments acknowledge His Majesty Research Fund Authors (SR/SCI/BIOL/08/01) Sultan Qaboos University, Oman and the Petroleum Development of Oman (CR/SCI/BIOL/07/02) for the research grants.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.09.022.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil recovery Yahya Al-Wahaibi a,∗ , Sanket Joshi b , Saif Al-Bahry b , Abdulkadir Elshafie b , Ali Al-Bemani a , Biji Shibulal b a b
Petroleum and Chemical Engineering Department, Sultan Qaboos University, Oman Biology Department, Sultan Qaboos University, Oman
a r t i c l e
i n f o
Article history: Received 27 April 2013 Received in revised form 8 September 2013 Accepted 11 September 2013 Available online xxx Keywords: Biosurfactant Bacillus subtilis Surface and interfacial tension reduction Critical micelle dilution Emulsification index Microbial enhanced oil recovery
a b s t r a c t The fermentative production of biosurfactants by Bacillus subtilis strain B30 and the evaluation of biosurfactant based enhanced oil recovery using core-flood were investigated. Different carbon sources (glucose, sucrose, starch, date molasses, cane molasses) were tested to determine the optimal biosurfactant production. The isolate B30 produced a biosurfactant that could reduce the surface tension and interfacial tension to 26.63 ± 0.45 mN/m and 3.79 ± 0.27 mN/m, respectively in less than 12 h in both glucose or date molasses based media. A crude biosurfactant concentration of 0.3–0.5 g/l and critical micelle dilution (CMD) values of 1:8 were observed. The biosurfactants gave stable emulsions with wide range of hydrocarbons including light and heavy crude oil. The biosurfactants were partially purified and identified as a mixture of lipopeptides similar to surfactin, using high performance thin layer chromatography and Fourier transform infrared spectroscopy. The biosurfactants were stable over wide range of pH, salinity and temperatures. The crude biosurfactant preparation enhanced light oil recovery by 17–26% and heavy oil recovery by 31% in core-flood studies. The results are indicative of the potential of the strain for the development of ex situ microbial enhanced oil recovery processes using glucose or date molasses based minimal media. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Biosurfactants are biological surface active agents, produced by microorganisms, plants and animals including humans. Biosurfactants are classified by various methods – ionic nature, type, size [1]. Generally low molecular weight biosurfactants are well known for reducing surface tension (ST) and interfacial tension (IFT) between oil and water and thus enhancing oil recovery. Whereas, high molecular weight biosurfactant like emulsan are known for their emulsifying properties and thus enhancing the mobility of heavy-oil and its recovery [2]. Recently, biosurfactants have been the focus of extensive research because they have several advantages over chemical surfactants: lower toxicity, higher environmental compatibility, biodegradability, and synthesis from renewable raw materials [1,3]. Biosurfactants have the benefit of being biodegradable and relatively inexpensive; however, there are some limitations which prevent their practical applications in the field. The main limitation is the availability of the biosurfactants in bulk quantity as compared to the chemical surfactants, in addition to scale-up related complications in producing large
∗ Corresponding author. Tel.: +968 99358758. E-mail address: [email protected] (Y. Al-Wahaibi). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.022
amounts of biosurfactants for field applications. However, Torres et al. [4], reported the discovery of new biosurfactants and the development of new fermentation and recovery processes may allow more biosurfactants to be used for microbial enhanced oil recovery (MEOR). Currently the bulk availability of biosurfactants commercially is limited (except rhamnolipids, e.g. available at – http://www.rhamnolipid.com/) as the cost of producing biosurfactant in bulk is still high. Several researchers have suggested the application of statistical optimization methods or the use of cheaper raw material such as agro-industrial by-products to reduce the cost of production [5–12]. Depending on the ease of the availability, the selection of the raw material differs from country to country. In Oman, date molasses was proposed as a carbon sources, due to its ready availability and lower cost [5]. Bacillus group of bacteria are known for producing potent lipopeptide biosurfactants such as surfactins and lichenysins, using different raw materials, and have been widely studied because of their high surface activities and other properties [13]. The surfactins and lichenysins are also reported as strong bacterial biosurfactants suitable for enhancing oil recovery [14]. There is a need for sustainable and more efficient oil recovery due to the depletion of current world oil reserves and the increasing demand for liquid. Several enhanced oil recovery (EOR) processes are currently employed worldwide: thermal, chemical, physical, etc. [15]. These
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processes are successful, but also very expensive as well as environmentally harmful. The alternative is a biologically based EOR process, also known as MEOR. MEOR can be implemented in several ways: either as in situ process employing indigenous microorganisms or ex situ process where bioproducts are produced outside of oil wells and directly injected to enhance oil recovery [16]. For in situ MEOR, we need to identify the indigenous microbial population [17] and provide them with suitable nutrient media for growth and production of bioproducts which will eventually enhance the recovery factor. Unfortunately this process cannot be controlled completely and success ratios may be limited. Whereas, with ex situ MEOR we can produce microbial products outside the oil wells and can inject them directly for oil recovery enhancement. Perfumo et al. [16] studied several MEOR reports of both in situ or ex situ field studies and reported that we still lack accurate data on microbial growth and metabolism under field condition. We isolated Bacillus subtilis B30 from petroleum contaminated soil samples from a garage in Oman. The ability of the isolate to produce biosurfactant was studied. The produced biosurfactant was tested for stability under different conditions of temperature, pH and salinity, and was also checked for its effect on enhancing oil recovery. 2. Materials and methods 2.1. Chemicals and reagents All major chemicals and trace elements were purchased from Sigma–Aldrich Co., USA. Cane molasses and date molasses were purchased from the local market, Sultanate of Oman. Light and heavy crude oil was provided by Petroleum Development of Oman (PDO). 2.2. Microorganism and culture conditions Several bacteria were isolated from soil samples collected from petrol pump sites, garages and other sites exposed to crude oil, using oil spreading method as described by Youssef et al. [18]. Those isolates which showed good biosurfactant producing potential (based on a larger zone of clearance on oil layer) were selected for further experiments at shake flask level in Luria–Bertani (LB) broth. Isolates which reduced surface tension (ST) below 35 mN/m were selected further, as described by Joshi et al. [9]. One strain – B30 was selected for further screening, and was identified as B. subtilis by 16S rRNA gene sequencing (GenBank accession no. KC686715). The isolate was maintained aerobically on LB agar plates and was regularly transferred into fresh LB medium for shortterm storage. Stock cultures of all pure isolates were prepared in 30% glycerol and stored below −40 ◦ C, for long-term maintenance. 2.3. Production media and effect of different carbon sources on biosurfactant production The culture was grown for 15–16 h at 40 ◦ C (OD660 nm -1.0–1.2) in LB as a seed inoculum medium. The flasks containing 50 ml of different minimal production media in 250 ml Erlenmeyer flasks were inoculated with 2% (v/v) seed culture. The cultures were incubated on a temperature controlled incubator shaker at 160 rpm. The compositions of different media used in this study are (g/l): Medium ‘C’ [19] NH4 NO3 , 4.0; KH2 PO4 , 4.08; MgSO4 ·7H2 O, 0.2; FeSO4 ·7H2 O, 0.0011; MnSO4 ·4H2 O, 0.00067; CaCl2 , 0.00077 and Medium ‘M’ [20] NH4 NO3 , 3.3; K2 HPO4 , 2.2; KH2 PO4 , 0.14; MgSO4 ·7H2 O, 0.6; FeSO4 ·7H2 O, 0.2; CaCl2 , 0.04; NaCl, 0.01; Trace elements, 0.5 ml/l. Media were sterilized by autoclaving at 121 ◦ C for 15 min. Five different carbon sources: glucose, sucrose, starch, cane molasses or date molasses were added at 2% (w/v) final concentration to
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both media after filter sterilization. Samples were withdrawn and, growth (OD660 ), pH, and biosurfactant production – surface tension (ST) and interfacial tension (IFT) were checked up to 72 h. 2.4. Analytical methods All measurements were made on cell-free broth after centrifugation (12,096 × g for 20 min) and analyzed at room temperatures. The experiments were performed in duplicate and the resulting reports are the mean of three independent experiments. 2.5. Biosurfactant analysis, contact angle measurements, and critical micelle dilution (CMD) Production of biosurfactant was analyzed by periodic measurement of ST and IFT of cell-free broth by means of pendant drop method using the Drop Shape Analyzing system – DSA 100 (KRÜSS, Germany). IFT was measured against Hexadecane. Contact angles of the untreated samples (un-inoculated media) and biosurfactants were measured using the Drop Shape Analysis System, DSA100 (Kruss, Germany), as described by Al-Sulaimani et al. [21]. All measurements were done in triplicates at ambient temperature (25 ± 2 ◦ C) and atmospheric pressure (1 atm) and the average values were reported. The biosurfactant concentration expressed in terms of critical micelle dilution (CMD) was estimated by measuring the ST for varying dilutions of the sample [9]. 2.6. Emulsification index (E24 ) determination The ability of the biosurfactants to emulsify liquid hydrocarbons, such as tridecane, pentadecane, tetradecane, hexadecane, heptamethyl nonane, heptane, hexane, methyl naphthalene, heavy crude oil (API 15.90◦ ), and light crude oil (API 36.51◦ ) was determined. The emulsification index (E24 ) was measured using a previously reported protocol with slight modifications [22–24]. E24 was determined by adding 2 ml of different hydrocarbons to 2 ml of cell free supernatant, vortex at high speed for 5 min, and the mixture was incubated at ambient temperature for 24 h. The E24 index is given as percentage of height of emulsified layer (mm) divided by total height of the liquid column (mm). 2.7. Biosurfactant extraction The biosurfactant was partially purified and extracted by the acid precipitation method [25]. Bacterial cells were separated by centrifuging at 12,096 × g for 20 min at 20 ◦ C (Beckman, JLA 16.250 rotor, USA). The pH was adjusted to 2.0 using 6 M HCl to precipitate biosurfactant. The solution was kept overnight at 4 ◦ C and the precipitated biosurfactant was collected by centrifuging the solution at 12,096 × g for 25 min at 4 ◦ C. The collected biosurfactant pellet was dissolved in distilled water and pH was adjusted to 8.0. Crude biosurfactant powder was collected by spray-drying at 100–160 ◦ C using Mini-Spray Dryer B-290 (BUCHI, Switzerland). Extracted crude biosurfactant was then tested for its stability and chemical characterization and in core-flooding experiments and other studies. 2.8. High performance thin layer chromatography (HPTLC) Biosurfactants were separated and analyzed using a completely automated HPTLC system (CAMAG, Switzerland) in the Central Analytical and Applied Research Unit (CAARU), Sultan Qaboos University, Oman. Twenty microliter samples were spotted onto a 10 cm × 10 cm precoated silica gel HPTLC plate (Merck, Germany) containing green fluorescent F254 . These samples were
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spotted under a flow of nitrogen gas using automatic TLC sampler 4 (ATS 4) spotting device (CAMAG, Switzerland) having a robotic arm in a closed chamber. The plates were developed using an automatic developing chamber ADC 2 (CAMAG, Switzerland) with remote operation from winCATS software, containing solvent systems – MP1: Butanol:Acetic acid:Water (8:2:2) and MP2: Chloroform:Methanol:Water (65:25:4). The documentation and evaluation of the TLC plate was done using TLC visualizer (CAMAG) under white light as well as with direct UV 254 and UV 366 nm light, capturing the images. The separated biosurfactants were also qualitatively detected by TLC scanner 4 (CAMAG) under Ultra Violet (UV) light (254 nm), and separated biosurfactant bands were compared. 2.9. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FT-IR) analysis was conducted to identify the structural groups of the biosurfactant. Infrared (IR) absorption spectra were obtained with a Perkin-Elmer grating 1000 IR (Norwalk, CT) in a dry atmosphere. In this test, 1 mg of crude biosurfactant was mixed with 100 mg of KBr and pressed with load for 30 s, to obtain translucent pellets. FTIR spectra were collected between 400 and 4000 wave numbers (cm−1 ). 2.10. Biosurfactant stability studies Stability of biosurfactant was studied under wide range of temperatures, pH and different salt concentrations to test the effect on the biosurfactant activity. Unless specified, the experiments were conducted at room temperature, pH 7.0 and 0% salt concentration. For the temperature stability test, the samples were kept in 10 ml serum bottles and were tightly sealed with butyl rubber stoppers and aluminum crimps. The range of temperatures tested were 40–100 ◦ C. The biosurfactant broth was also subjected to autoclave conditions (121 ◦ C, 15 psi for 30 min) in order to investigate the effect of such environment on the surface activity of the biosurfactant. For the salinity test, NaCl was added to the biosurfactant cell-free broth at different concentrations 0–15% (w/v). The stability of the biosurfactant was also determined by measuring the ST and IFT at different pH values (2.0–12.0) at room temperature. 2.11. Core-plugs and fluid samples A set of Berea sandstone cores (1.5 in. diameter × 3 in. long) with average porosity and permeability of 20–25% and 250–350 mD, respectively, were used for the core-flood experiments. The formation water and crude oil used in these experiments were obtained from an Omani oil field of interest which has an average reservoir temperature of 60 ◦ C. The salinity of the formation water was between 7% and 9% and its chemical composition was (kg/m3 ): Sodium, 25.083; Calcium, 3.762; Magnesium, 0.878; Iron, 0.045; Chloride, 47.722; Sulphate, 0.247; Bicarbonate, 0.079. Formation water was filtered prior to use, by Millipore Filtration Unit (0.45 m). The crude oils used for core-flood experiments were of API 36.51◦ and 15.90◦ . 2.12. Core-flood experiments For all core-flooding experiments, cores were cleaned by using the soxhlet extraction method, using chloroform and methanol as an azeotropic mixture in the proportion of 75:25 [26]. After cleaning, the core was dried at 65 ◦ C for 24 h before using. The core was saturated with filtered formation brine using vacuum desiccators for 24 h and pore volume (PV) was determined using the dry and wet weights of the core. The core was then flooded with crude oil at 24 cm3 /h until no more water was produced. The oil
initially in place (OIIP) was determined, which was indicated by the volume of water displaced. The core was subjected to waterflooding at 24 cm3 /h until no further oil was produced. The residual oil was calculated by measuring the amount of oil produced from the water-flood. Then, 5 PV of the cell-free supernatant (biosurfactant broth) was injected as a tertiary recovery stage and extra oil recovery was determined. All core floods were conducted at 60 ◦ C to mimic the average reservoir temperature of the field of interest. 3. Results and discussion 3.1. Carbon source optimization Several bacteria were isolated from different petroleum contaminated soil samples from Oman. Based on oil spreading and reduction in ST values, Bacillus B30 was selected for further studies. Two different reported minimal production media were used for carbon source optimization. We tested glucose, sucrose, starch, cane molasses and date molasses in both the minimal production media. Out of all the carbon sources tested, glucose in ‘C’ medium (CG) and date molasses in ‘M’ medium (MDM) gave better reduction in ST and IFT, as compared to rest of the media combinations (as per supplementary data). Media ‘CG’ and ‘MDM’ were further studied to find out the best time for biosurfactant production. Samples were harvested every 2 h for 72 h, and studied for growth and biosurfactant production. In medium ‘CG’, the lowest ST (25.56 ± 0.97 mN/m) and IFT (3.6 ± 0.66 mN/m) were observed at 8–10 h (Fig. 1A). Maximum growth was observed within 22 h and no change in pH was observed. In ‘MDM’ medium, maximum reduction in ST (26.31 ± 0.07 mN/m) and IFT (3.56 + 0.10 mN/m) was observed at around 12–14 h (Fig. 1B). Whereas, growth was better as compared to ‘CG’ medium at 22 h and no major changes in pH were observed. Lin et al. [27] have reported shake-flask studies for Bacillus licheniformis JF-2 lipopeptide biosurfactant production, which occurred during active growth but subsequently disappeared from the fermentation broth within 6 h. They have reported that the disappearance of the biosurfactant from the fermentation broth was due to its internalization by stationary-phase cells. However, Joshi and Desai [28] reported the biosurfactant production by 5 bacterial strains for up to 72 h and reported ST and IFT values in the range of 28–30 mN/m and 0.5–5.8 mN/m, respectively, with no changes in ST and CMD. Gojavand et al. [29] also reported the maximum production of biosurfactant by B. subtilis (PTCC 1696) during the growth phase (10 h), which remained constant even after the exponential phase. Al-Bahry et al. (2012) also reported similar phenomenon for biosurfactant production by B. subtilis B20 using date molasses within 10–12 h of fermentation and no changes thereafter till 72 h. We also observed similar trend for biosurfactant production by B. subtilis B30. Amongst all the carbon sources tested in present study, Date molasses is comparatively cheaper and available in abundant quantity here in Oman. So, it is recommended to be used as a suitable carbon source for higher biosurfactant scale-up production studies in Oman. 3.2. Contact angle determination A fluid is said to wet a solid surface if the contact angle is less than 90◦ (for surfactant containing liquids). If the contact angle is greater than 90◦ , the fluid is said to be non-wetting [30]. Recently, wettability alteration has been proposed as one of the mechanisms of MEOR where several studies reported the relation between IFT reduction and alteration of wetting conditions following microbial treatment [21,31–33]. It was suggested that the reduction in IFT may change the oil–rock contact causing an altered wettability. Studies reported that the change in wetting properties is dependent on the initial wetting condition where alterations from oil-wet toward more
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Fig. 1. Production profile (ST, IFT, OD660 , pH) of Bacillus B30 in ‘CG’ (A) and ‘MDM’ media (B). The error bars represents SD values of three independent experiments (n = 3).
water-wet and vice versa have been reported [33]. Microbial treatment has also shown to change wetting conditions of core samples, thus resulting in a change in the imbibition/drainage behavior [34]. The biosurfactants from ‘CG’ and ‘MDM’ media were studied for their effect on alteration of contact angle on a hydrophobic plate. The contact angles were reduced from 58.7 ± 0.85◦ of un-inoculated media to 28.4 ± 1.03◦ and 27.2 ± 0.72◦ in ‘CG’ and ‘MDM’ media, respectively (Fig. 2). Kowalewski et al. [33] reported simulation studies of a core-flood that reduced the residual oil saturation from 27% to 3%. Their objective was to investigate the mechanisms of MEOR focusing on IFT reduction and wettability alteration. The simulation results showed that both a gradual reduction in IFT and change in wettability can be used to observe the reduction in residual oil saturation. Al-Sulaimani et al. [21] reported that biosurfactant produced by B. subtilis W19 changed the contact angle of distilled water from 70.6 ± 0.3◦ to 25.32 ± 0.06◦ at 0.25% concentration and no further changes were observed while increasing the concentration of biosurfactant reported reduction in contact angle from 103.0 ± 0.36◦ to 82.7 ± 0.43◦ , by Bacillus I-19 biosurfactant, using crude oil as a carbon source. Karimi et al. [36] reported that microbial solutions can alter the wettability of 7 and 21 days aged glass surfaces to more water-wet conditions. In the current
study, wettability alteration caused by the Bacillus B30 biosurfactant was investigated by contact angle measurement, and it was observed that biosurfactant changed the wettability toward more water-wet, which could be quite helpful mechanism for improving oil recovery at field scale applications. 3.3. Biosurfactant yield and CMD determination The yield after acid precipitation and spray drying was 0.5 g/l for ‘CG’ medium and 0.3 g/l for ‘MDM’ medium. Generally CMD values are determined by carrying out ST or IFT measurements on a series of different dilutions of surfactant solutions and CMD is indirectly proportional to critical micelle concentration (CMC). The dilution at which the ST begins to increase is termed the CMD, which is the factor by which the effective biosurfactant concentration exceeds the critical micelle concentration [37]. Thus, higher the CMD – the more industrially promising is the surfactant. Estimating the CMD of the surfactant gives an indication on the optimum concentration to be used for maximum performance and minimum cost possible. Biosurfactant containing broths were used to determine CMD for both the biosurfactants. As shown in Fig. 3, ST and IFT decreased progressively with increasing biosurfactant concentration. The ST
Fig. 2. Contact angle determination from un-inoculated media 58.7 ± 0.85◦ (A); B30 biosurfactant from ‘CG’ medium 28.4 ± 1.03◦ (B) and B30 biosurfactant from ‘MDM’ medium 27.2 ± 0.72◦ (C).
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Fig. 4. Emulsification index against different hydrocarbons, for Bacillus B30 biosurfactants. The error bars represents SD values of three independent experiments (n = 3).
Fig. 3. CMD determination for biosurfactants produced by Bacillus B30 in ‘CG’ (A) and ‘MDM’ (B). The error bars represents SD values of three independent experiments (n = 3).
increased from an initial value of undiluted biosurfactant broth to 40.84 mN/m up to 1:8 times dilutions, and increased thereafter. The IFT increased following almost similar trend where the IFT began to increase at around 1:8 times dilution with an average of
11.86 mN/m. It was observed that, CMD for ‘CG’ was 8× and for ‘MDM’ were 8× (Fig. 3). Ghurye et al. [37] used activated sludge from the wastewater treatment as a source of microorganisms for biosurfactant production. For a pH controlled bioreactor containing 20 g/l molasses, yeast extract and other nutrients, the ST of the broth reduced to 40 dynes/cm within 48 h and maximum biosurfactant concentration of about 63× CMD in cell-free broth was achieved in 120 h. Joshi and Desai [28] reported CMD values of 75–100× for biosurfactants mixtures produced by different Bacilli strains. Makkar and Cameotra [38] have reported the production of biosurfactants by two B. subtilis strains at 45 ◦ C using molasses at the concentration of 2% total sugars, and minimal medium. Both strains reduced ST in the range of 30 dynes/cm with 10× CMD values – 35 to 40 dynes/cm and 100× CMD values – 50 to 65 dynes/cm after 96 h. We also observed CMD values of 8× (1:8 dilution), which is comparable to those reported by B. subtilis [38] and by microorganisms from activated sludge [37] under identical conditions.
Fig. 5. HPTLC plate showing separated biosurfactants from ‘CG’ and ‘MDM’ media, under (A) UV 254 nm and (B) UV 366 nm.
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Fig. 6. FTIR absorption spectra of biosurfactant produced by B. subtilis B30 from ‘CG’ medium (A) and ‘MDM’ medium (B).
3.4. Emulsification index The biosurfactant containing cell-free broth was able to emulsify many hydrocarbons. Biosurfactant containing cell free broth of ‘CG’ medium gave emulsions with various hydrocarbons such as, tridecane, tetradecane, hexadecane, methyl naphthalene, heavy crude oil and light crude oil (Fig. 4). Whereas, biosurfactant containing
cell-free broth of ‘MDM’ medium gave emulsions with all tested hydrocarbons, except heavy crude oil (Fig. 4). Somehow neither of biosurfactants gave >20% emulsion with pentadecane, after 24 h. Das et al. [22] reported biosurfactant production by marine Bacillus circulans in glycerol mineral salt medium and antracene supplemented glycerol mineral salt medium, which emulsified various hydrocarbons such as diesel, hexadecane, kerosene, benzene and
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Fig. 7. FTIR absorption spectra of standard Surfactin (Sigma Chemicals, USA).
petrol in the range of 30–80%. Das et al. [39] also reported biosurfactant production by a marine bacteria in mineral salt media containing different carbon sources (glucose, sucrose, starch or glycerol) with higher emulsification power of the biosurfactant obtained from glycerol and starch than that of the biosurfactant obtained from glucose and sucrose with E24 values in the range of 45–85%. Khopade et al. [23] reported almost 80% E24 against hydrocarbon, within 8–9 days by biosurfactant produced by a marine Nocardiopsis sp. B4. We observed higher emulsification (≥50%) with long chain hydrocarbons (Fig. 4) such as crude oil, which could most probably play an essential role in enhancing oil recovery.
3.5. Partial identification using HPTLC and FTIR Out of two solvent systems used for HPTLC, MP1 gave better separation (Fig. 5) as compared to MP2 (supplementary file). As shown in Fig. 5, we loaded 160 g of biosurfactant from ‘MDM’ and 160 and 2400 g of biosurfactant from ‘CG’ medium. As the biosurfactant powder was dark brown, a lower concentration of biosurfactant from ‘MDM’ was loaded, in order to avoid any interference in separation. The separated biosurfactant bands from both glucose and date molasses containing media were almost similar with Rf values. The increase in the intensity of the bands for biosurfactant from glucose containing media was evident when we increased initial concentration of biosurfactant from 40 mg to 60 mg. Several researchers reported use of TLC or HPTLC as a tool for initial qualitative detection or quantitative analysis of different biosurfactants [20,39,40]. IR spectra of biosurfactants produced by Bacillus B30 are shown in Fig. 6(A and B). The IR spectrum in KBr showed bands characteristic of peptides at 3400 cm−1 (NH stretching mode) and at 1654 cm−1 (stretching mode of the CO N bond). The bands at 1406 cm−1 , 1246 cm−1 reflect aliphatic chains ( CH3, CH2 ) of the fraction [41,42]. These results imply the presence of aliphatic hydrocarbons as well as a peptide-like moiety in the biosurfactant. The peaks shown in Fig. 6 matched the standard surfactin (Sigma chemicals, USA, Fig. 7). The stretching modes of the NH, CO N bond and CH3, CH2 fractions fall within the same range of wave numbers which suggests the similarity in structure of the biosurfactant produced by Bacillus B30 with surfactin. Surfactins are lipopeptidal biosurfactant with very good surface activities and are reported as very good candidate with potential applications in enhancing oil recovery and petroleum industry [14].
3.6. Stability studies The use of partially purified biosurfactant for ex situ MEOR requires the biosurfactant to be stable within a range of high temperatures (around 50–80 ◦ C), wide range of pH and high salt concentrations to ensure wide applicability to induce oil recovery. The biosurfactant produced using either glucose or date molasses were quite stable over a wide range of temperatures from 40 to 100 ◦ C (Fig. 8A), and also was stable at 121 ◦ C (during autoclaving) and 160 ◦ C (during drying in spray drier). Khopade et al. [23] also reported the stability of biosurfactants under extreme conditions of temperature. Desai and Banat [1] reported that heat treatment on some biosurfactants caused no appreciable change in their properties, even after autoclaving at 120 ◦ C for 15 min. Similarly, Borodoli and Konwar [43] reported the biosurfactant produced by Pseudomonas aeruginosa strains to be stable at temperature of 100 ◦ C for different time periods of 5–60 min with respect to ST changes. Joshi et al. [9] reported biosurfactants produced by 4 Bacillus strains to be stable at 80 ◦ C for 9 days. The biosurfactants produced from ‘CG’ medium or ‘MDM’ were also stable in pH range of 6–12 and salt concentration up to 5% NaCl (Fig. 8B and C). Under highly acidic pH (pH 2.0 and 4.0) biosurfactants showed much less activity, since the biosurfactant is not soluble under highly acidic conditions and tends to precipitate. This higher instability of biosurfactants produced from some Lactobacilli in acidic conditions was described by some researchers to be related to the presence of negative charged groups at the polar ends of the molecules [44]. Several reports confirmed the stability of biosurfactant at different pH values, mostly in the alkaline medium [9,23,26,41,44,45]. Bacillus B30 biosurfactant showed stability under various extreme conditions as reported by other researchers.
3.7. Core-flooding for light and heavy oil recovery and future possibility of field application The pore volume of the core sample was 18–24 cm3 . Initial oil saturation (Soi ) was calculated to be 55–67% after oil flooding. It was found that after injecting 5–8 PV of water, no more oil was produced and residual oil saturation (Sor ) was about 25–38%. Extra oil recovery was observed after injecting 4–5 PV of biosurfactant solution from ‘CG’ medium or ‘MDM’ medium, where 17–26% of Sor was produced (Fig. 9A and B). Biosurfactant produced by ‘CG’ medium also showed 31% extra recovery (Fig. 9C) of heavy crude
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Fig. 8. Stability studies for biosurfactant produced by Bacillus B30 in ‘CG’ and ‘MDM’ media, under different temperature (A), pH (B) and salinity (C). The error bars represents SD values of three independent experiments (n = 3).
oil (API 15.90◦ ). Joshi et al. [28] reported 30.22–34.19% additional oil recovery over the water flood residual oil saturation using crude biosurfactant obtained from Bacillus strains by sand pack column studies. Darvishi et al. [46] reported 27.2% oil recovery efficiency due to the injection of the cell-free biosurfactant. Thomas et al. [47] reported enhancement of oil recovery from Berea sandstone core treated with cell-free metabolites from surfactant producing strain Bacillus sp. JF-2. In present study core flooding experiments were carried out using Berea cores which were treated with cell-free biosurfactant broths using glucose or date molasses as a carbon source, where an increase in oil recovery in ex situ MEOR experiments was detected following biosurfactant injection. This is similar to reports shown by other researchers [26,48,49]. The heavy oil recovery is the target for many EOR operations worldwide. The B30 biosurfactant showed additional oil recovery for both light and heavy oil, which
have quite promising applications in Oman. Especially as Oman has many oil fields with heavy oil. However real potential of microbial products (biosurfactants) in MEOR applications can only be fully assessed in field-scale applications. Several trials have been carried out during the past years and tentatively reviewed [16]. The real impact of biosurfactantbased MEOR trials however has never been tested at a larger-scale, because of lack of both quantitative information regarding microbial growth and metabolism in situ and insufficient data collection and processing. Recently Youssef et al. [50] reported a small fieldscale MEOR experiment (viola limestone formation) providing for the first time data of in situ bacterial metabolism and activities. Molecular biology techniques with traditional methods showed that Bacillus strains injected into oil wells were metabolically active, consuming the nutrients supplied (glucose, sodium nitrate
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Fig. 9. Cumulative oil recovery by B30 biosurfactant from ‘CG’ medium (A) and ‘MDM’ medium (B) with light oil (API 36.51◦ ) and for heavy oil recovery (API 15.90◦ ) with B30 biosurfactant from ‘CG’ medium (C). The error bars represents SD values of three independent experiments (n = 3).
and trace metals) and releasing CO2 , acids, solvents and lipopeptide biosurfactant thus releasing more oil from the formation. Their data demonstrated the technical feasibility of biosurfactant based MEOR future applications.
4. Conclusion Isolate B. subtilis B30 produced a potent biosurfactant (lipopeptide similar to Surfactin), using cheaper mineral salt media containing either glucose or date molasses, which is quite stable under harsh conditions; gives stable emulsions with wide range of hydrocarbons; and gave around 17–31% additional recovery of light or heavy crude oil. Further experiments should be done at higher scale.
Acknowledgments acknowledge His Majesty Research Fund Authors (SR/SCI/BIOL/08/01) Sultan Qaboos University, Oman and the Petroleum Development of Oman (CR/SCI/BIOL/07/02) for the research grants.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.09.022.
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