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Characterization, production and optimization of lipopeptide biosurfactant by new strain Bacillus pumilus 2IR isolated from an Iranian oil field
Author’s Accepted Manuscript Characterization, production and optimization of lipopeptide biosurfactant by new strain Bacillus pumilus 2IR isolated from an Iranian oil field Tayebeh Fooladi, Nasrin Moazami, Peyman Abdeshahian, Abudukeremu Kadier, Hossein Ghojavand, Wan Mohtar Wan Yusoff, Aidil Abdul Hamid
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S0920-4105(16)30232-7 http://dx.doi.org/10.1016/j.petrol.2016.06.015 PETROL3506
To appear in: Journal of Petroleum Science and Engineering Received date: 12 July 2015 Revised date: 13 May 2016 Accepted date: 7 June 2016 Cite this article as: Tayebeh Fooladi, Nasrin Moazami, Peyman Abdeshahian, Abudukeremu Kadier, Hossein Ghojavand, Wan Mohtar Wan Yusoff and Aidil Abdul Hamid, Characterization, production and optimization of lipopeptide biosurfactant by new strain Bacillus pumilus 2IR isolated from an Iranian oil f i e l d , Journal of Petroleum Science and Engineering, http://dx.doi.org/10.1016/j.petrol.2016.06.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization, production and optimization of lipopeptide biosurfactant by new strain Bacillus pumilus 2IR isolated from an Iranian oil field Tayebeh Fooladi a, Nasrin Moazami b, Peyman Abdeshahian c, Abudukeremu Kadier d Hossein Ghojavand e, Wan Mohtar Wan Yusoff a, Aidil Abdul Hamid a,*
a
School of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia (Universiti Kebangsaan Malaysia), 43600 Bangi, Selangor, Malaysia b
Biotechnology center, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran c
Department of Microbiology, Masjed Soleyman Branch, Islamic Azad University, Masjed Soleyman, Iran d
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, National University of Malaysia (Universiti Kebangsaan Malaysia), 43600 Bangi, Selangor, Malaysia e
Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran [email protected] [email protected] [email protected] *
Corresponding author: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), Bangi43600, Selangor, Malaysia. . Tel: 0060193033094
Abstract Characterization and production of biosurfactant by a novel bacterial strain isolated from an Iranian oil field was investigated. Biosurfactant has wide applications in oil recovery. A number of ten strains of Bacillus sp. were isolated from oil-contaminated soil samples to study biosurfactant production. Among strains studied, the strain 2IR was selected as a potent biosurfactant-producing
1
2 bacterium. The identification of the strain 2IR was carried out using scanning electron microscopy, biochemical tests and partial 16S rRNA gene sequencing with generating a phylogenetic tree. Accordingly, the new strain was identified as Bacillus pumilus 2IR. The biosurfactant produced by the strain 2IR was characterized by high performance liquid chromatography, fourier transform infrared spectroscopy and proton nuclear magnetic resonance test. The tests results characterized the biosurfacant produced as a lipopeptide type. In order to attain the highest surface tension reduction of the lipopeptide biosurfactant, culture medium of the strain 2IR was optimized. Experimental results showed that glucose, crude oil, potassium nitrate and ammonium sulfate were pivotal components of the culture medium for biosurfactant synthesis by B.pumilus 2IR. Similar results revealed that optimum medium compositions were 30.31 g/L glucose, 0.8 % (v/v) crude oil, 2.88 g/L potassium nitrate and 2.4 g/L ammonium sulfate. A surface tension value of 30 mN/m was obtained in optimum conditions.
This research presents new indigenous strain B. pumilus 2IR for synthesis of
biosurfactant usable for possible in situ oil recovery.
Keywords: Lipopeptide biosurfactant; Bacillus pumilus 2IR; Surface tension; Oil field; Oil recovery
1. Introduction Biosurfactants are surface active agents that are mostly produced by microorganisms such as bacteria, yeast, fungi. They consists of both polar and non-polar domains and are capable of showing various surface activities which among other roles, help solubilize hydrophobic substances (Amodu et al., 2014; Sari et al., 2014). Lipopeptides are the most known types of biosurfactants which are normally produced by Bacillus spp. as a peptide linked to a fatty acid (Pereira et al., 2013).
3 Biosurfactants have shown more attractive utilization in industry compared to chemically synthetic surfactants since biosurfactants exhibit more environmentally-friendly characteristics and economically viable utilization (Ławniczak et al., 2013). Biosurfactant has been found wide applications in oil industry for oil recovery (Sarafzadeh et al., 2014). In this context, microbial enhanced oil recovery (MEOR) technology is referred to the utilization of microorganisms and their microbial products to help the mobilization of residual oil in an oil reservoir (Armstrong et al., 2015). The application of biosurfacatnt-producing microbes in oil industry could be taken into account by introducing externally produced biosurfactants, known as ex situ method or stimulating biosurfactan-producing microbes on-site, called in situ method (Ławniczak et al., 2013). One of the most critical bottlenecks related to the oil recovery is the high porosity of the media (rocks, sand, and earth). The oil generally surrounds the media and enters into inaccessible regions known as thief zones. The permeability of the media inhibits the extraction of the crude oil. In addition, the current technologies for the enhancement of crude oil yield are energy intensive and high economic costs with their own inherent risks due to use of toxic chemicals like ethylene oxide for the synthesis of nonionic surfactants (Patel et al., 2015). In this sense, MEOR represents a clean approach using biosurfactants with a further economic benefit to liberate the entrapped oil in the reservoirs. In MEOR technology, when indigenous bacteria are exploited they can grow in the oil reservoirs, so that they fill space and attach to the surface of substrates close to the place of growth. Consequently, microorganisms are capable of growing in the porous media which form a biofilm to aid in preventing more oil from being drawn into the porous regions. Indigenous microbial communities inside the oil wells can change crude oil properties allowing it to show enhanced flow features and elevated sweep efficiency which is done by both stabilization of the interfacial tension and decreased viscosity (Gaytán et al., 2015).
From a scientific aspect, the exogenous
4 microorganisms will out-compete those microbes are already adapted to the harsh conditions in the reservoirs (Patel et al., 2015). On the other hand, the use of exogenous microorganisms in ex situ methods of MEOR entails the increase of costs of the oil recovery technology (Pornsunthorntawee et al., 2008; Zheng et al., 2012). Indigenous microbes of interest are often stimulated with inexpensive substances to produce and release biosurfactants. In this view, it has been indicated that stimulating of indigenous populations of biosurfactant-producing bacteria would be amongst the most low-cost MEOR methods. Hence, indigenous microorganisms that exhibit the ability to tolerate harsh conditions in the oil reservoir such as high salinity, temperature and pH with the lack of oxygen would be advantageous for efficient MEOR (Patel et al., 2015).
One of the most important strategies for increasing biosurfactant production by microorganisms is the modification of the microbial culture medium in which the mixture of the hydrocarbon compounds, sugar-based carbon sources and nitrogen sources are optimized to enhance biosurfactant synthesis (Abouseoud et al., 2008; Mutalik et al., 2008). In this regard, the utilization of proper nutrients leads to in situ stimulation of microorganisms to produce surfactant in the oil reservoir (Patel et al., 2015). Indigenous microbes are therefore, capable of consuming crude oil existing in the reservoir to produce biosurfactants used in MEOR technology. Little information is available concerning the on-site application of biosurfactants in oil reservoirs and more research work should be conducted to evaluate the stimulation of the indigenous microorganisms for in situ production of biosurfacatnt rather than the utilization of exogenous microbial strains that could be outcompeted.
In the light of above, the current study was aimed at the isolation and determination of a new indigenous biosurfactant-producing
bacterium, namely Bacillus pumilus 2IR that was locally
isolated from the oil-contaminated soil in the Masjed-Soleyman oil field located in the southwest part
5 of Iran. The production and characterization of biosurfactant obtained by the strain was investigated to possibly be employed for the oil recovery in the oil industry. The utilization of the crude oil as the natural substance in the oil reservoirs for the enhancement of biosurfactant production was studied which could contribute to make the oil recovery technique more cost-effective and eco-friendly. Furthermore, the optimization of culture medium compositions was performed using a central composite design (Nalini et al., 2016).
2. Materials and methods 2.1. Microorganism isolation and culture medium For isolation of biosurfactant-producing bacteria, the samples of oil-contaminated soil were collected from an oil field in Masjed-Soleyman at the southwest part of Iran under aseptic condition. Accordingly, 5g of soil sample was inoculated in 50 ml of nutrient broth. The flask was incubated at 30°C for 72h. After incubation time, serially diluted culture suspension was done on nutrient agar plates, followed by transferring plates at the temperature of 30°C for 48h. Isolated single colonies were tested morphologically and a total number of ten strains of Bacillus species were further screened for production of biosurfactant. The isolated strains were maintained in nutrient agar slants containing glycerol 50% (v/v) at 20°C. Subculture of the strains was carried out by streaking the stock culture of the strains onto nutrient agar plates, followed by transferring plates in an incubator at the temperature of 30°C. Microbial growth was carried out in growth medium E using different solutions of A, B, C and a basal mineral solution. Solution A contained 25 g/L of MgSO4 and solution B contained 100 g/L of (NH4)2SO4. Solution C included following compositions (g/L): EDTA, 0.5; MnSO4·H2O, 3; NaCl, 1; CaCl2·2H2O, 0.1; ZnSO4·7H2O, 0.1; FeSO4·7H2O, 0.1; CuSO4·5H2O, 0.01; AlK (SO4)2, 0.01; Na2MoO4·2H2O, 0.01; boric acid, 0.01; Na2SeO4, 0.005; and NiCl2·6H2O, 0.003. Solutions A and B
6 and C were then sterilized. Growth medium E was prepared by transferring 10 ml of each solution A, B and C into the basal medium to make a total volume of 1 L. Growth medium E was then mixed well, sterilized and used in biosurfactant production experiments. The initial pH value of growth medium E was adjusted to 7.0 using 1 N NaOH. The basal mineral solution used was composed of the following components (g/L): KH2PO4, 2.7; K2HPO4, 13.9; sucrose, 10; NaCl, 50; yeast extract, 0.5 and KNO3, 1 (Ghojavand et al., 2008a; Youssef et al., 2004).
2.2. Biosurfactant production The production of biosurfactant was studied by transferring a single colony of the bacterial strains into a 200 ml Erlenmeyer flask containing 50 ml nutrient broth medium, followed by incubation at 30°C and 150 rpm agitation for 16 h to obtain an optical density of 0.5 at a wavelength of 600 nm using a spectrophotometer. Subsequently, 10 ml of prepared culture content was utilized to inoculate 500 ml of medium E in an Erlenmeyer flask, followed by the incubation of culture at 30°C on a rotary shaker at 150 rpm for 72 h. Samples were then collected to determine acid precipitated biosurfactant as described previously (Joshi et al., 2008a; Vaz et al., 2012).
2.3. Biosurfactant determination To determine biosurfactant production, different screening methods including hemolytic activity (the ability to hydrolyze blood cells in blood agar), drop collapse (the ability to disperse on the oil) and oil spreading test (the ability to form a clear zone) were used as preliminary techniques. The bacterial isolates which showed a higher activity through the preliminary screening were subjected to complementary techniques. Surface tension activity and emulsification tests were the known methods to determine the biosurfactant produced by the strains.
Surface tension measurement is the most
known method to determine the biosurfactant produced by the bacterial strains. The surface tension measurement was conducted using a tensiometer at the room temperature. Critical micelle
7 concentration (CMC) is a relationship between biosurfactant concentration and surface tension; hence, it is an important property for various biosurfactant applications. In order to determine CMC, the surface tension of different concentrations of biosurfactant in distilled water was measured at the room temperature. On the other hand, critical micelle dilution (CMD) is defined as the dilution in which the surface tension starts to increase. CMD was determined by diluting culture supernatant in distilled water by 10-times and 100-times and subsequent measurements of the surface tension (Joshi et al., 2008b; Makkar and Cameotra, 2002; Vaz et al., 2012; White et al., 2013).
2.4. Screening and identification of the isolates A potent strain which could reduce the surface tension of medium to a lowest level and could show the highest emulsification activity (E24) was selected (Ghojavand et al., 2008a; Youssef et al., 2004). The identification of the isolated strain was carried out by scanning electron microscopy (SEM). Morphological and biochemical identification tests were also performed in accordance with Bergey’s Manual of Systematic Bacteriology. The sequence of the 16S rRNA gene was determined by Capillary electrophoresis and Dye-terminator sequencing at Bioneer Corporation Munpyeong-dong Daejeon, South Korea. The EzTaxon program was used to compare the 16S rRNA gene sequence results of the bacterial type strains (Chun et al., 2007).
2.5. Analytical methods The extracted components from the culture medium were further determined using high performance liquid chromatography (HPLC) technique. Fourier transform infrared spectroscopy (FTIR) was used to determine structural characterization of the biosurfactant extracted from the culture supernatant of the isolated strain. Proton nuclear magnetic resonance (1H NMR) was used for further characterization of biosurfactant produced.
8 2.6. Optimization of screened culture medium components A series of experiments of biosurfactant synthesis were designed based on the central composite design (CCD) in which culture medium (medium E) was supplemented with four efficient medium nutrients including glucose, crude oil, potassium nitrate and ammonium sulfate. Each variable was varied at different levels. The coded values and the actual levels of the medium nutrients tested are given in Table 1. The total number of experiments of the CCD generated was calculated according to Eq.(1): k
N=2 +2k + no
(1)
where k is the number of variables studied and no denotes the number of center points of the experimental design (Dashti et al., 2014). In this study, the total number of the fermentation runs for the center point was six treatments. The design matrix of the thirty experimental runs of the batch fermentation is given in Table S1. Experimental data from the mixture design (Table S1) were used to fit a second-order polynomial regression model (Eq. (2)) to CCD results to represent product formation as a function of variables tested: Y= ao +
ai Xi +
aii Xi2 +
aij Xi Xj
(2)
where Y is the measured response. Xi and Xj are the variables tested. ao represents the intercept. ai, aii and aij denote linear coefficient, quadratic coefficients and interaction coefficient, respectively (Arulmathi et al., 2015; Basak et al., 2014; Maran et al. 2013). The mathematical model generated for four independent variables studied was defined as Eq. (3): Y = ao + a1 X1 + a2 X2 + a3 X3 + a4 X4 + a11X12 + a22 X22 + a33 X32+ a44 X42 + a12 X1 X2 + a13 X1 X3 + a23 X2 X3 + a14 X1 X4+ a24 X2 X4+ a34 X3 X4
(3)
where Y is the measured surface tension activity. X1, X2, X3 and X4 represent the coded values of glucose concentration (g/L), crude oil (% v/v), potassium nitrate (g/L) and ammonium sulfate (g/L),
9 respectively. The experiments were conducted in 1000 ml Erlenmeyer flasks containing 250 ml medium E with pH 7.2, followed by incubation of the bacterial culture at 30°C on a rotary shaker at 150 rpm for 72 h. Statistical analysis of the data was performed using Design-Expert software (version 7.1 Stat-Ease, Inc). In order to find out the concurrent effects between variables tested on the response (surface tension activity), three dimensional (3D) graphs were constructed (Al-Shorgani et al., 2016; Betiku et al., 2015).
3. Results 3.1. Screening and identification of the selected biosurfactant-producing strains Table 2 shows the biochemical results obtained for ten strains tested in comparison to Bacillus subtilis ATCC 21332 as a known biosurfactant producer. As can be seen, the isolate 2IR showed the lowest surface tension value (32 mN/m) with the highest oil spreading (3.2 cm), crude oil emulsification (60 %) and hexadecane emulsification (68%) among strains screened. The positive results for emulsification assays (E24) suggested that the strain 2IR also produced emulsifier, which was notable compared to that from other isolates (Table 2). This finding indicated that results obtained for the strain 2IR were most consistent with those considered for B. subtilis ATCC 21332. Hence, the strain 2IR was selected as a potential biosurfactant-producing isolate with the highest efficiency in surface tension reduction. The strain 2IR was then studied based on morphological characteristics of the bacterial colonies and biochemical tests. Morphological observations revealed that the colonies formed were gray color, round and flat with an opaque appearance (Fig. 1A). The strain was subjected to the Gram staining method. Morphological observations by a light microscope showed a Gram-positive bacterium for the cells of the strain 2IR (Fig. 1B). Scanning electron microscope images showed the rod-shape of the bacterial cells with a diameter higher than 1 mm (Figs. 1C and 1D).
10 On the other hand, biochemical tests showed that the strain 2IR was a motile bacterium with a positive response to oxidase, catalase, nitrate reduction, citrate and esculin utilization tests (Table 3). Further identification was carried out using 16S rRNA ribotyping. 16S rRNA gene sequences (1478bp) of the strain 2IR demonstrated that the strain belonged to the species in genus of Bacillus as it was nearly identical with the strain Bacillus safensis FO-036bT and B. pumilus ATCC 7061T with similarity value of 98.11% and 97.97%, respectively. Comparison of biochemical tests for these strains demonstrated that strain 2IR and B. pumilus ATCC 7061T were identical in ability to hydrolyze esculin, while they are unable to show inositol fermentation. These findings confirmed that strain 2IR was B. pumilus. A phylogenetic tree was drawn based on a neighbor-joining analysis as shown in supplementary materials (Fig. S1). The strain characterized as Bacillus pumilus 2IR was deposited with the Microbial Culture Collection Unit (UNiCC) at the Institute of Bioscience at University Putra Malaysia under accession number UPMC 864. Moreover, the 16S rRNA gene sequence of strain Bacillus pumilus 2IR was deposited in Genbank under accession number KM 017130. 3.2. Profile of biosurfactant production by B. pumilus 2IR The profile of biosurfactant production by B. pumilus 2IR was studied using medium E as illustrated in Fig. 2. As can be seen, after a short lag phase, bacterial cells entered into the exponential growth phase, which continued up to 48 h of fermentation time and followed by the stationary phase. Biosurfactant production by the isolate 2IR was associated with bacterial cell growth so that an increase in the biomass concentration in exponential growth phase led to a high production of biosurfactant. In this regard, biosurfactant increased at exponential phase and reached up to 0.77 g/L at 72 h (stationary phase). Highest surface tension reduction (32 mN/m) was obtained at 30 h fermentation time (exponential phase) and did not change significantly afterwards.
3.3. Critical micelle concentration and critical micelle dilution
11 The experimental results of CMC measurement revealed that the surface tension of the medium decreased rapidly as the concentration of biosurfactant increased with a minimum surface tension of 31 mN/m (Fig. 3A). As shown in Fig. 3A, the surface tension remained constant for biosurfactant concentrations higher than 120 µg/ml and no further considerable changes occurred. This phenomenon was observed because of achieving CMC. Fig. 3B illustrates CMD results obtained from B. pumilus 2IR. As can be found, CMD-1 and CMD-2 were achieved after 30 h and 40 h of cultivation, respectively. Furthermore, maximum reduction in CMD-1 and CMD-2 indicated that biosurfactant had the highest activity even after 10 times and 100 times dilution of culture supernatant.
3.4. Characterization of the produced biosurfactant Acid precipitated biosurfactant extracted from isolate B. pumilus 2IR showed five main peaks at the retention time determined after 9.55 min using semi-preparative HPLC (Fig. 4A). In this study, the fraction N5 was collected and applied in 1H NMR and FTIR. The FTIR spectrum of the fifth compound (N5) from B. pumilus 2IR showed strong absorption bands, indicating the presence of a peptide component at 3290.78cm–1 resulting from N-H stretching mode (Fig. 4B). As shown, the stretching mode of a CO-N bond was observed at 1658.59 cm–1, while the deformation mode of the N-H bond combined with C-N stretching mode occurred at 1577.41cm–1. The presence of an aliphatic chain was determined by the C-H stretching modes at 2925 to 2850 cm–1 and 1429.46– 1387cm–1. These results strongly indicated that the biosurfactant contained aliphatic hydrocarbon combined with peptide moieties. Also, the band at 1734cm–1 was due to lactone carbonyl absorption. The IR spectra of the purified compound N5 displayed a significant similarity in IR adsorption obtained by other lipopeptide biosurfactant produced from Bacillus genus. The results from 1H NMR analysis was confirmed the lipopeptidic nature of the fragment N5 due to the presence of (CH3)2-CH group, indicating terminal branching in the fatty acid component at 0.96-0.81 ppm. The chemical
12 shift at (1.39-1.24 ppm) was consistent with a long aliphatic chain (CH2), a peptide backbone (N-H at 7.14-7.05 ppm) and an aliphatic carbon-hydrogen bond (4.9-3.9 ppm) (Fig. 4C). The intense singlet at 3.46 ppm (3.66 ppm for fraction N5) was similar to the 1H NMR spectrum of lipopeptide monoesters reported in literature, which indicated the presence of a methoxy group on the Glu or Asp amino residues. On the other hand, the presence of a lactone ring in the biosurfactant structure was confirmed by the detection of an ester carbonyl group at intense singlet of (5.1-4.9 ppm).
3.5. Optimization of culture medium components The preliminary study on the effect of the carbon and nitrogen sources tested on the biosurfactant production by B. pumilus 2IR reveled that glucose and crude oil as carbon sources with ammonium sulfate and potassium nitrate as nitrogen sources had notable effects on the synthesis of biosurfactant by the strain 2IR (data not shown). Hence, these nutrients were selected for further optimization of culture medium compositions. The experimental batch fermentations for the measurement of surface tension value were performed based on CCD with different levels of glucose (X1), crude oil (X2), potassium nitrate (X3) and ammonium sulfate (X4). The experimental results of CCD are shown in Table S1. As can be found, the highest reduction in surface tension was obtained in the treatments 11 and 29 with the value of 29 mN/m where similar 30 g/L glucose (0 as a coded value), 1% (v/v) crude oil (0 as a coded value) and 3 g/L potassium nitrate (0 as a coded value) were applied, while ammonium sulfate used in the treatments 11 and 29 was 3 g/L (0 as a coded value) and 1 g/L (-2 as a coded value), respectively. By applying multiple regression analysis to the test results, a secondorder polynomial equation was derived to represent surface tension as a function of glucose, crude oil, potassium nitrate and ammonium sulfate as shown in Eq. (4): Y= 30.83 -0.17 X1 +3.17 X2+0.42 X3+0.50 X4 +0.25 X1 X2 -0.13 X1 X3 +0.12 X1 X4 +0.50 X2 X3 +0.25 X2 X4 -0.38 X3 X4 +2.73 X12 +3.10 X22 +1.85 X32 + 0.48 X42
(4)
13 where Y is the measured surface tension value (mN/m). X1, X2, X3 and X4 are coded value for glucose, crude oil, potassium nitrate and ammonium sulfate, respectively. The statistical significance of the results was evaluated using the analysis of variance (ANOVA) for surface tension value (Table 4). As shown in Table 4, calculated model’s F value of 7.18 with a probability value (P>F) of 0.0002 suggested that the selected quadratic model was significant and fitted well to the experimental data (P<0.01). The F value for lack of fit (4.40) with a probability value of 0.0579 implied that the lack of fit for the model was insignificant and hence the model was valid for further studies. As can be observed from Table 4, the quadratic term of glucose, crude oil and potassium nitrate (X12 , X22 and X32) had a high significant effect on surface tension activity at 99% probability level (P<0.01), indicating that glucose, crude oil and potassium nitrate acted as critical factors and even a low change in their values could affect surface tension value to a remarkable level, while variations in ammonium sulfate had no significant effects on biosurfactant synthesis. The statistical analysis revealed that the optimum concentration for glucose, crude oil, potassium nitrate and ammonium sulfate were 30.31 g/L, 0.8 % (v/v), 2.88 g/L and 2.4 g/L, respectively. The statistical results suggested that the maximum reduction in surface tension would be 30.52 mN/m when the optimal concentrations of the variables were used. In this regard, a validation experiment was conducted using optimum concentrations of glucose, crude oil, potassium nitrate and ammonium sulfate by the consideration of C/N ratio of 12 as an optimum value. The experimental results showed that a surface tension value of 30 mN/m was obtained in optimum conditions, which was in good agreement with the suggested value (30.52 mN/m), indicating the acceptable reproducibility of results.
3.6. The simultaneous effect of carbon and nitrogen sources studied on surface tension value As can be seen from Fig. 5A, a rise in glucose concentration up to 30 g/L decreased surface tension value where crude oil was at a low concentration (0.5% v/v). Increasing crude oil concentration up to
14 1% (v/v) caused the high surface tension reduction, corroborating the significant quadratic effect of glucose and crude oil on the surface tension reduction (Table 4). The 3D plot for the concurrent effect of the crude oil and potassium nitrate showed that an exponential reduction in surface tension occurred when crude oil increased up to optimum level (1%) and potassium nitrate concentration was set at 3 g/L (Fig. 5B). The simultaneous effect of potassium nitrate and ammonium sulfate on surface tension value is illustrated in Fig. 5C. As shown, the surface tension value decreased concurrently with the increase in potassium nitrate concentration, whereas variations in ammonium sulfate had a minor shift in surface tension value. The 3D plots in Fig. 5 also indicated that a rise in surface tension value occurred when the concurrent effect of glucose, crude oil and potassium nitrate lied beyond their optimum levels, while ammonium sulfate showed insignificant effect on surface tension value.
4. Discussion According to the data obtained from this study, a direct relation between biosurfactant production and cell growth was observed. This indicated that biosurfactant production was consistent with cell growth which occurred at the exponential growth phase so that a decisive decrease in the surface tension occurred (Fig. 2). Similar pattern was observed for the production of lipopetide biosurfactant by Bacillus mojavensis (Ghojavand et al., 2011). Furthermore study on the biosurfactant by Bacillus strains revealed the growth-associated biosurfactant production for Bacillus mycoides and B. subtilis LB5 by high synthesis of lipopeptide biosurfactant at exponential growth phase (Najafi et al., 2010; Nitschke and Pastore, 2006). The CMC value of 120 µg/ml obtained by B. pumilus 2IR was consistent with CMC reported in literature with a wide range from 5 to 200 µg/ml (Chun et al., 2007; Ghojavand et al., 2008b). The CMD measured for the strain 2IR was in close agreement with that obtained by Nitschke and Pastore (2006) who observed that CMD-1 and CMD-2 were attained by Bacillus subtilis LB5 after 12 h
15 growth and remained constant along the surface tension reduction. The lipopeptidic nature of the produced biosurfactant by the strain 2IR was similar to the finding of Pereira et al. (2013) and Haddad et al. (2009) who observed the presence of lipopeptide in biosurfactant produced by Bacillus subtilis. The study fulfilled by Najafi et al. (2015) showed lipopetide nature of bisurfactant produced by Bacillus mycoides SH2, which was isolated from oil-contaminated samples. The data from 1H NMR analysis showed that the strain B. pumilus 2IR produced a mixture of biosurfactant components similar to Bacillus sp. which was reported elsewhere (Ghojavand et al., 2008b). Hence, experimental 1
H NMR and FTIR for fraction N5, revealed the lipopeptidic nature of biosurfactant produced by B.
pumilus 2IR. The current study showed that B. pumilus 2IR could better utilize glucose and sucrose to produce biosurfactant compared to other carbohydrates tested. In this regard, the production of biosurfactant by Pseudomonas aeruginosa from various sugars and hydrocarbons revealed that glucose was the best carbon source for bacterial cell growth and biosurfactant production, followed by fructose and sucrose (Lotfabad et al., 2009). The previous studies have shown that the utilization of non sugar-based hydrocarbons as the sole carbon source have inhibitory effects on the bacterial growth and biosurfactant production (Joshi et al., 2008b; Makkar and Cameotra, 2002). In the present study, it was found that the combination of sugar-based carbon source and a non sugar-based hydrocarbon had no deleterious effects on the biosurfactant production by B. pumilus 2IR. In this regard, as shown in Fig. 5A, the appropriate amount of crude oil 1% (v/v) showed a positive effect on the reduction of surface tension in relation to the optimal level of glucose and indicated the favorable effect of the combination of sugar-based and non sugar-based carbon source. In the same way, Mutalik et al. (2008) observed that the production of biosurfactant by Rhodococcus sp. increased up to 3.4-fold when combined mannitol and n-hexadecane were used. Apart from carbon source, nitrogen was the pivotal nutrient in the medium which could influence the microbial growth and
16 biosurfactant production. Although nitrate is a crucial factor for microbial growth, ammonium salts in the form of ammonium sulfate has also been used for the microbial growth. From the result shown in Fig. 5, it is evident that the surface tension value decreased when the concentration of the potassium nitrate increased from 1 g/L to 3 g/L under optimal concentration of carbon sources (glucose and crude oil). However, the excess concentration of the potassium nitrate more than 3 g/L resulted in a high increase in pH of the medium which in turn caused an inhibitory effect on the microbial cell growth and metabolic activity for surface tension value (Kiran et al., 2010; Khopade et al., 2012). This indicated the importance of nutrients for biosurfactant-mediated bioremediation where the insufficient carbon source and nutrients may inhibit the growth of biosurfactant-producing microbes (Ławniczak et al., 2013). The field study on biosurfactan-assisted oil recovery could elucidate the economical approach for the recovery of remarkable quantity of oil entrapped in the reservoirs. The study fuillfilled by Yussef et al. (2007) reveled that the in situ stimulation of two Bacillus strains in oil wells using nutrients including glucose, sodium nitrate, and trace metals resulted in to the production of lipopeptide biosurfactant that could mobilize entrapped oil from sandstone cores, demonstrating the point that on-site production of biosurfactant could be economically viable. Similar study showed that in situ stimulation of B. subtilis injected to the oil wells exhibited a cost-effective approach for the oil recovery by the production of lipopeptide biosurfactant (Yussef et al., 2013). Further research should focus on the various approaches of application of the biosurfactant in oil recovery to make this method economically competitive with the current strategies.
5. Conclusion The application of biosurfactants in oil reservoir is currently interesting in oil related research. In this regard, the key aspect is the efficient utilization of biosurfactants in the oil recovery with making this process economically feasible. This work showed that the new isolate B. pumilus 2IR was a
17 promising indigenous stain for the production of lipopeptide biosurfactant. The study also revealed that this strain was capable of consuming crude oil for the production of biosurfactant which can be advantageous to the in situ technology of the oil recovery from the abandoned oil reservoirs. The optimization of the culture medium indicated that glucose, crude oil and potassium nitrate had a significant effect on biosurfactant production by the strain 2IR with an optimum concentration value of 30.31 g/L glucose, 0.8 % (v/v) crude oil, 2.88 g/L potassium nitrate and 2.4 g/L ammonium sulfate.
Further research is recommended to conduct field study on the in situ production of
biosurfactant by this strain and to determine the efficiency of the strain for the successful MEOR.
Acknowledgment The authors sincerely acknowledge University Kebangsaan Malaysia (UKM), Malaysia and also would like to thank Department of Biotechnology, Iranian Research Organization Science and Technology (IROST), Tehran, Iran.
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18 Armstrong, R.T., Wildenschild, D., Bay, 2015. The effect of pore morphology on microbial enhanced oil recovery. J. Petroleum. Sci. Eng. 130, 16-25. Arulmathi, P., Elangovan, G., Begum, A.F., 2015. Optimization of electrochemical treatment process conditions for distillery effluent using response surface methodology. Sci. World. J. 1-9. http://dx.doi.org/10.1155/2015/581463 Basak, N., Jana, A.K., Das, D., Saikia, D., 2014. Optimization of molecular hydrogen production byRhodobacter sphaeroides O.U.001 in the annular photobioreactor using response surface methodology. Int. J. Hydrogen. Energy. 2014; 39, 11889-11901. Betiku, E., Okunsolawo, S.S., Ajala, S.O., Odedele, O.S., 2015. Performance evaluation of artificial neural network coupled with generic algorithm and response surface methodology in modeling and optimization of biodiesel production process parameters from shea tree (Vitellaria paradoxa) nut butter. Renew. Energy. 76, 408-417. Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K., Lim, Y.W., 2007. EzTaxon: A web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst .Evol. Microbiol. 57, 2259-2261. Dashti, M.G., Abdeshahian, P., Yusoff, W.M.W., Kalil, M.S., Hamid, A.A., 2014. Repeated batch fermentation biotechnology for the biosynthesis of lipid and gamma-linolenic acid by Cunninghamella bainieri 2A1. BioMed. Res. Int. http://dx.doi.org/10.1155/2014/831783. Gaytán I, Mejía M.A, Hernández-Gama R, Torres L.G., Escalante, C.A., 2015. Effects of indigenous microbial consortia for enhanced oil recovery in a fragmented calcite rocks system. J. Petroleum. Sci. Eng. 128, 65-72. Ghojavand, H., Vahabzadeh, F., Azizmohseni, F., 2011. A halotolerant, thermotolerant, and facultative biosurfactant producer: identification and molecular characterization of a bacterium and evolution of emulsifier stability of a lipopeptide biosurfactant. Biotechnol. Bioprocess. Eng. 16, 7280 Ghojavand, H., Vahabzadeh, F., Mehranian, M., Radmehr, M., Shahraki, A.K.H., Zolfagharian, F., Emadi, M.A., Roayaei, E., 2008b. Isolation of termotolerant, halotolerant, facultative biosurfactantproducing bacteria. Appl. Microbiol. Biotechnol. 80, 1073-1085. Ghojavand, H., Vahabzadeh, F., Roayaei, E., Shahraki, A.K., 2008a. Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696). J. Colloid. Interface. Sci. 324, 172-176.
19 Haddad, N.I.A., Wang, J.I., Bozhong, M.U., 2009. Identification of a biosurfactant producing strain: Bacillus subtilis HOB2. Protein. Pept. Lett. 16, 7-137. Joshi, S., Bharucha, C., Desai, A.J., 2008a. Production of biosurfactant and antifungal compound by fermented food isolate Bacillus subtilis 20B. Bioresour. Technol. 99, 4603-4608. Joshi, S., Bharucha, C., Jha, S., Yadav, S., Nerurkar, A., Desai, A.J., 2008b. Biosurfactant production using molasses and whey under thermophilic conditions. Bioresour. Technol. 99, 195-199. Khopade, A., Liu, X.Y., Mahadik, K., Zhang, L., Kokare, C., 2012. Production and characterization of biosurfactant from marine Streptomyces species B3. J. Colloid. Inter. Sci. 367, 311-318. Kiran, S.G., Thomas, A.T., Selvin, J., Sabarathuhan, B., Lipton, A.P., 2010. Optimization and characterization of a new lipopeptide biosurfactant produced by marine Brevibacterium aureum MSA13 in solid state culture. Bioresour. Technol. 101, 2389–2396. Ławniczak, L., Marecik, R., Chrzanowski, Ł., 2013. Contributions of biosurfactants to natural or induced bioremediation. Appl. Microbiol. Biotechnol. 97, 2327-2339. Lotfabad, T.B., Shourian, M., Roostaazad, R., Najafabadi, A.R., Adelzadeh, M.R., Noghabi, K.A., 2009. An efficient biosurfactant-producing bacterium Pseudomonas aeruginosa MR01, isolated from oil excavation areas in south of Iran. Colloid. Surf . B: Biointerf. 69, 183–193. Makkar, R.S., Cameotra, S.S., 2002. Effects of various nutritional supplements on biosurfactant productions by a Strain of Bacillus subtilis at 45°C. J.Surfact. Deterg. 5, 11-17. Maran, J.P., Sivakumar, V., Thirugnanasambandham, K., Sridhar, R., 2013. Artificial neural network and response surface methodology modeling in mass transfer parameters predictions during osmotic dehydration of Carica papaya L. Alexandria. Eng. J. 52, 507-516. Mutalik S.R., Vaidya, B.K., Joshi, R.M., Desai, K.M., Nene, S.N., 2008. Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574. Bioresour. Technol. 99,7875-7880. Najafi, A.R., Rahimpour, M.R., Jahanmiri, A.H., Roostaazad, R., Arabian, D., Ghobadi, Z., 2010. Enhancing biosurfactant production from an indigenous strain of Bacillus mycoides by optimizing the growth conditions using a response surface methodology. Chemi. Eng. J. 163, 188-194. Najafi, A.R., Roostaazad, R., Soleimani, M., Arabian, D., Moazed, M.T., Rahimpour, M.R., Mazinani, S., 2015. Comparison and modification of models in production of biosurfactant for
20 Paenibacillus alvei and Bacillus mycoides and its effect on MEOR efficiency. J .Petroleum. Sci. Eng. 128, 177-183. Nalini, S., Parthasarathi, R., Prabudoss, V., 2016. Production and characterization of lipopeptide from Bacillus cereus SNAU01 under solid state fermentation and its potential application as antibiofilm agent. Biocatalysis. Agricultural. Biotechnol. 5, 123-132. Nitschke, M., Pastore, G.M., 2006. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresour. Technol. 97, 336-341. Patel, J., Borgohain S, Kumar M, Rangarajan V, Somasundaran P, Sen R., 2015. Recent developments in microbial enhanced oil recovery. Renew. Sustain. Energy. Rev. 52, 1539-1558. Pereira, J.F., Gudina, E.J., Costa, R., Vitorino, R., Teixeira, J.A., Coutinho, J.A., Rodrigues, L.R., 2013. Optimization and characterization of biosurfactant production by Bacillus subtilis isolates towards microbial enhanced oil recovery applications. Fuel. 111, 259-268. Pornsunthorntawee , O., Wongpanit P, Chavadej S, Abe M, Rujiravanit R., 2008. Structural and physicochemical characteriza tion of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil. Bioresour.Technol.99,1589–95. Sarafzadeh, P., Niazi, A., Oboodi, V., Ravanbakhsh, M., Hezave, A.Z., Ayatollahi, S.S., Raeissi, S., 2014. Investigating the efficiency of MEOR processes using Enterobacter cloacae and Bacillus stearothermophilus SUCPM#14 (biosurfactant-producing strains) in carbonated reservoirs. J. Petroleum. Sci. Eng. 113, 46-53. Sari, M., Kusharyoto, W., Artika, I.M., 2014. Screening for biosurfactant-producing yeast: confirmation of biosurfactant production. Biotechnol. 13, 106-111. Vaz, D.A., Gudina, E.J., Alameda, E.J., Teixeira, J.A., Rodrigues, L.R., 2012. Performance of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants. Colloid. Surf. B: Biointerf. 89, 167-174. White, D.A., Hird, L.C., Ali, S.D., 2013. Production and characterization of a trehalolipid biosurfactant produced by the novel marine bacterium Rhodococcus sp., strain PML026. J. Appl. Microbiol. 115, 744-755. Youssef, N.H., Duncan, K.E., Nagle, D.P., Savage, K.N., Knapp, R.M., McInerney, R.J., 2004. Comparison of methods to detect biosurfactant production by diverse microorganisms. J. Microbiol. Methods. 5, 339-347.
21 Youssef N, Simpsona, D.R., Duncan, K.E., McInerneya, M.J., Folmsbee, M., Fincher,T., Knapp, R.M., 2007. In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir. Appl. Environ. Microbiol. 73, 1239-1247. Youssef
N, Simpsona, D.R., McInerneya, M.J., Duncan, K.E., 2013. In-situ lipopeptide
biosurfactant production by Bacillus strains correlates with improved oil recovery in two oil wells approaching their economic limit of production. Int. Biodeterior. Biodegrad. 81, 127–32. Zheng, C.,Yu, L., Huang, L., Xiu, J., Huang, Z., 2012. Investigation of a hydrocarbon-degrading strain, Rhodococcus ruber Z25, for the potential of microbial enhanced oil recovery. J.Pet.Sci.Eng. 81, 49–56.
Fig. 1. The characteristics of Bacillus pumilus 2IR: (A) colony morphology; (B) light microscope image using Gram staining method; (C) scanning electron microscope image using 5 KX magnification and (D) scanning electron microscope image using 10 KX magnification Fig. 2. The profile of biosurfactant production by B. pumilus 2IR Fig. 3. The profile of surface tension in relation to acid precipitated biosurfactant concentration for B. pumilus 2IR: (A) critical micelle concentration (CMC) measured in biosurfactant production process and (B) critical micelle dilution (CMD) during the bacterial cell growth Fig. 4. Characterization of the biosurfactant produced by B. pumilus 2IR: (A) semi-preparative HPLC chromatogram of biosurfactant detected at 220 nm, (B) transmission FTIR spectrum of biosurfactant (fraction N5) obtained after purification by semi-prep HPLC and (C) proton NMR spectrum of the fraction N5 fractionated from semi-preparation HPLC Fig. 5. Three dimentional plots for showing the simultaneous effect of carbon and nitrogen sources tested on the surface tension (mNm-1) obtained from B. pumilus 2IR: (A) interaction effect between glucose (X1) and crude oil (X2); (B) interaction effect between crude oil (X2) and potassium nitrate (X3) and (C) interaction effect between potassium nitrate (X3) and ammonium sulfate (X4)
22 Table 1 Process variables and determined levels for central composite design (CCD) in biosurfactant production by B. pumilus 2IR.
Variable Glucose (g/l) Crude oil (% v/v) Potassium nitrate (g/l) Ammonium sulfate (g/l)
Symbol Actual level X1 10 20 30
50
Coded value -2 -1 0
40
1
2
X2
0.1
0.5
1
1.5
2
-2
-1
0
1
2
X3
1
2
3
4
5
-2
-1
0
1
2
X4
1
2
3
4
5
-2
-1
0
1
2
Table 2 Screening of potential biosurfactant-producing strains using biochemical tests Strain
Blood a
Drop b
Oil
Surface
Emulsification
agar
collapse
spreading
tension
(E24)% Crude oil
lysis
(mm)
Emulsification (E24)% Hexadecane
-1
(cm)
(mNm )
1IR
-
-
1.2
48
52%
15%
2IR
++
+++
3.2
32
60%
68%
3IR
++
-
1.5
44
40%
65%
4IR
+++
++
0.8
37
40%
30%
5IR
+
+
1.5
40
35%
45%
6IR
+
+
0.8
38
40%
60%
7IR
+
-
1.0
43
35%
45%
8IR
++ +++
++ + +++
0.6 0.5 0.2 3.8
39 51 41 27
25% 10% 30% 70%
20% 20% 35% 75%
9IR 10IR Bacillus subtilis ATCC 21332
a
(-), no hemolysis; ( + ), incomplete hemolysis; (+ +) , complete hemolysis with a diameter of lysis
< 1 cm; (+ + +), complete hemolysis with a diameter of lysis between 1 to 3 cm b
(-) indicates the lack of biosurfactant production. Flat drops with signs (+), (+ +) and (+ + +) denote
the partial to complete spreading on the oil surface
23
Table 3 Biochemical testes conducted for the strain 2IR in comparison to most closely species Bacillus safensis FO-036bT
+
Bacillus pumilus ATCC 7061 T +
Motility
+
+
+
Oxidase
+
+
+
Catalase
+
+
+
Anaerobic growth
+
+
+
Voges- Proskauer reaction
+
+
+
Indol reaction
-
-
-
Utilization of Citrat
+
+
+
Nitrate reduction to Nitrite
-
-
-
Inositol
-
-
+
Starch
+
+
+
Casein
+
+
+
Tween 80
+
+
+
Esculine
+
+
+
Gelatine
-
-
+
Test/Characteristics
te 2IR
Endospore
+
Hydrolysis of :
(+): Positive (-): Negative
Table 4 Analaysis of variance (ANOVA) for the secod-order polynomial model of surface tension reduction produced by B. pumilus 2IR Sources Model
X1 X2 X3 X4 X1 X2 X1 X3 X1 X4
Sum of squares 710.88 0.67 240.67 4.17 6.00 1.00 0.25 0.25
DF 14 1 1 1 1 1 1 1
Mean square 50.78 0.67 240.67 4.17 6.00 1.00 0.25 0.25
F value 7.18 0.094 34.03 0.59 0.85 0.14 0.035 0.035
Prob>F 0.0002* 0.7630 < 0.0001* 0.4547 0.3716 0.7122 0.8534 0.8534
Coefficient estimated -0.17 3.17 0.42 0.50 0.25 -0.13 0.12
24 4.00
X2 X3 X2 X4 X3 X4 X12 X22 X32 X42
1.00
Residual Lack of fit Pure error *
2.25 204.30 264.30 94.30 6.30 106.08 95.25 10.83
1 1 1 1 1 1 1 15 10 5
4.00 1.00 2.25 204.30 264.30 94.30 6.30 7.07 9.53 2.17
0.57 0.14 0.32 26.89 37.37 13.33 0.89
0.4637 0.7122 0.5811 < 0.0001* < 0.0001* 0.0024* 0.3603
4.40
0.0579
0.50 0.25 -0.38 2.73 3.10 1.85 0.48
Statistically significant at 99% of probability level
X1, glucose (g/l); X2, crude oil (% v/v); X3, potassium nitrate (g/l); X4, ammonium sulfate (g/l) X1X2, X1 X3, X1 X4, X2 X3, X2 X4, X3 X4, the interaction terms, X12, X22, X32, X42 , the quadratic terms
Highlights ·
Biosurfactant-producing strains from oil fields in Iran were investigated.
·
Bacillus pumilus 2IR was identified as a promising lipopeptide biosurfactant producer.
·
Culture medium compositions were optimized by a central composite design.
·
Optimum conditions: 30.31g/L glucose; 0.8% crude oil; 2.88 g/L KNO3; 2.4g/L (NH4)2SO4
·
A surface tension value of 30 mN/m was obtained in optimum conditions.
Figure 5
A
X2
X1
B
X3
X2
C
X4
X3
Fig. 5
Figure 4
A N5
B
C
Fig. 4
Figure 3
75 Surface tension (mN/ m)
A
50
25
0
1
10
100
1000
Biosurfactant concentration (µg /ml)
Surface tension activity (m/ Nm ) CMD-1 , CMD -2
80
3
70
2.5
60 2
50 40
1.5
30
1
20 0.5
10 0
Absorbance (600 nm)
B
0 0
5
10
15
20
25 30 35 Time (h)
Surface Tension Activity(mN/m)
Fig. 3
CMD -1
40
45
50
CMD -2
55
60 Absorbance
Figure 2
30
20
10
0
0.8 2.5
2.0 2.0 1.5 1.5 1.0
0.6
0.4
1.0 0.2
0.5
0.5
0.0
0.0 0
6
12
18
24
30
48
56
72
Time (h) Growth (OD600nm) Biomass (g/L) Surface Tension (mN/m) Acid Precipitated Biosurfactant (g/L)
Fig. 2
80
96
0.0
Acid precipitated precipitated biosurfactant (g/L)(g /l) Acid biosurfactant
40
1.0
3.0
2.5
Growth (OD 600nm) Growth (OD 600nm)
Surface Surfacetension Tension(mN/m) (mN/m)
60
50
3.5
3.0
Biomass(g/l) (g/L) Biomass
70
Figure 1
A
B
C
Fig. 1
1
D
Graphical Abstract
Oil field
Sampling Isolation of Bacillus strains Isolation of Bacillus strains Oil-contaminated sample
Blood agar lysis
Drop-collapse test
Oil spreading test
Identification Identification
Fermentation
16S rRNA gene sequence
Electron microscope observation
Colony morphology
Precipitated biosurfactant
PII: DOI: Reference:
www.elsevier.com/locate/petrol
S0920-4105(16)30232-7 http://dx.doi.org/10.1016/j.petrol.2016.06.015 PETROL3506
To appear in: Journal of Petroleum Science and Engineering Received date: 12 July 2015 Revised date: 13 May 2016 Accepted date: 7 June 2016 Cite this article as: Tayebeh Fooladi, Nasrin Moazami, Peyman Abdeshahian, Abudukeremu Kadier, Hossein Ghojavand, Wan Mohtar Wan Yusoff and Aidil Abdul Hamid, Characterization, production and optimization of lipopeptide biosurfactant by new strain Bacillus pumilus 2IR isolated from an Iranian oil f i e l d , Journal of Petroleum Science and Engineering, http://dx.doi.org/10.1016/j.petrol.2016.06.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization, production and optimization of lipopeptide biosurfactant by new strain Bacillus pumilus 2IR isolated from an Iranian oil field Tayebeh Fooladi a, Nasrin Moazami b, Peyman Abdeshahian c, Abudukeremu Kadier d Hossein Ghojavand e, Wan Mohtar Wan Yusoff a, Aidil Abdul Hamid a,*
a
School of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia (Universiti Kebangsaan Malaysia), 43600 Bangi, Selangor, Malaysia b
Biotechnology center, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran c
Department of Microbiology, Masjed Soleyman Branch, Islamic Azad University, Masjed Soleyman, Iran d
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, National University of Malaysia (Universiti Kebangsaan Malaysia), 43600 Bangi, Selangor, Malaysia e
Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran [email protected] [email protected] [email protected] *
Corresponding author: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), Bangi43600, Selangor, Malaysia. . Tel: 0060193033094
Abstract Characterization and production of biosurfactant by a novel bacterial strain isolated from an Iranian oil field was investigated. Biosurfactant has wide applications in oil recovery. A number of ten strains of Bacillus sp. were isolated from oil-contaminated soil samples to study biosurfactant production. Among strains studied, the strain 2IR was selected as a potent biosurfactant-producing
1
2 bacterium. The identification of the strain 2IR was carried out using scanning electron microscopy, biochemical tests and partial 16S rRNA gene sequencing with generating a phylogenetic tree. Accordingly, the new strain was identified as Bacillus pumilus 2IR. The biosurfactant produced by the strain 2IR was characterized by high performance liquid chromatography, fourier transform infrared spectroscopy and proton nuclear magnetic resonance test. The tests results characterized the biosurfacant produced as a lipopeptide type. In order to attain the highest surface tension reduction of the lipopeptide biosurfactant, culture medium of the strain 2IR was optimized. Experimental results showed that glucose, crude oil, potassium nitrate and ammonium sulfate were pivotal components of the culture medium for biosurfactant synthesis by B.pumilus 2IR. Similar results revealed that optimum medium compositions were 30.31 g/L glucose, 0.8 % (v/v) crude oil, 2.88 g/L potassium nitrate and 2.4 g/L ammonium sulfate. A surface tension value of 30 mN/m was obtained in optimum conditions.
This research presents new indigenous strain B. pumilus 2IR for synthesis of
biosurfactant usable for possible in situ oil recovery.
Keywords: Lipopeptide biosurfactant; Bacillus pumilus 2IR; Surface tension; Oil field; Oil recovery
1. Introduction Biosurfactants are surface active agents that are mostly produced by microorganisms such as bacteria, yeast, fungi. They consists of both polar and non-polar domains and are capable of showing various surface activities which among other roles, help solubilize hydrophobic substances (Amodu et al., 2014; Sari et al., 2014). Lipopeptides are the most known types of biosurfactants which are normally produced by Bacillus spp. as a peptide linked to a fatty acid (Pereira et al., 2013).
3 Biosurfactants have shown more attractive utilization in industry compared to chemically synthetic surfactants since biosurfactants exhibit more environmentally-friendly characteristics and economically viable utilization (Ławniczak et al., 2013). Biosurfactant has been found wide applications in oil industry for oil recovery (Sarafzadeh et al., 2014). In this context, microbial enhanced oil recovery (MEOR) technology is referred to the utilization of microorganisms and their microbial products to help the mobilization of residual oil in an oil reservoir (Armstrong et al., 2015). The application of biosurfacatnt-producing microbes in oil industry could be taken into account by introducing externally produced biosurfactants, known as ex situ method or stimulating biosurfactan-producing microbes on-site, called in situ method (Ławniczak et al., 2013). One of the most critical bottlenecks related to the oil recovery is the high porosity of the media (rocks, sand, and earth). The oil generally surrounds the media and enters into inaccessible regions known as thief zones. The permeability of the media inhibits the extraction of the crude oil. In addition, the current technologies for the enhancement of crude oil yield are energy intensive and high economic costs with their own inherent risks due to use of toxic chemicals like ethylene oxide for the synthesis of nonionic surfactants (Patel et al., 2015). In this sense, MEOR represents a clean approach using biosurfactants with a further economic benefit to liberate the entrapped oil in the reservoirs. In MEOR technology, when indigenous bacteria are exploited they can grow in the oil reservoirs, so that they fill space and attach to the surface of substrates close to the place of growth. Consequently, microorganisms are capable of growing in the porous media which form a biofilm to aid in preventing more oil from being drawn into the porous regions. Indigenous microbial communities inside the oil wells can change crude oil properties allowing it to show enhanced flow features and elevated sweep efficiency which is done by both stabilization of the interfacial tension and decreased viscosity (Gaytán et al., 2015).
From a scientific aspect, the exogenous
4 microorganisms will out-compete those microbes are already adapted to the harsh conditions in the reservoirs (Patel et al., 2015). On the other hand, the use of exogenous microorganisms in ex situ methods of MEOR entails the increase of costs of the oil recovery technology (Pornsunthorntawee et al., 2008; Zheng et al., 2012). Indigenous microbes of interest are often stimulated with inexpensive substances to produce and release biosurfactants. In this view, it has been indicated that stimulating of indigenous populations of biosurfactant-producing bacteria would be amongst the most low-cost MEOR methods. Hence, indigenous microorganisms that exhibit the ability to tolerate harsh conditions in the oil reservoir such as high salinity, temperature and pH with the lack of oxygen would be advantageous for efficient MEOR (Patel et al., 2015).
One of the most important strategies for increasing biosurfactant production by microorganisms is the modification of the microbial culture medium in which the mixture of the hydrocarbon compounds, sugar-based carbon sources and nitrogen sources are optimized to enhance biosurfactant synthesis (Abouseoud et al., 2008; Mutalik et al., 2008). In this regard, the utilization of proper nutrients leads to in situ stimulation of microorganisms to produce surfactant in the oil reservoir (Patel et al., 2015). Indigenous microbes are therefore, capable of consuming crude oil existing in the reservoir to produce biosurfactants used in MEOR technology. Little information is available concerning the on-site application of biosurfactants in oil reservoirs and more research work should be conducted to evaluate the stimulation of the indigenous microorganisms for in situ production of biosurfacatnt rather than the utilization of exogenous microbial strains that could be outcompeted.
In the light of above, the current study was aimed at the isolation and determination of a new indigenous biosurfactant-producing
bacterium, namely Bacillus pumilus 2IR that was locally
isolated from the oil-contaminated soil in the Masjed-Soleyman oil field located in the southwest part
5 of Iran. The production and characterization of biosurfactant obtained by the strain was investigated to possibly be employed for the oil recovery in the oil industry. The utilization of the crude oil as the natural substance in the oil reservoirs for the enhancement of biosurfactant production was studied which could contribute to make the oil recovery technique more cost-effective and eco-friendly. Furthermore, the optimization of culture medium compositions was performed using a central composite design (Nalini et al., 2016).
2. Materials and methods 2.1. Microorganism isolation and culture medium For isolation of biosurfactant-producing bacteria, the samples of oil-contaminated soil were collected from an oil field in Masjed-Soleyman at the southwest part of Iran under aseptic condition. Accordingly, 5g of soil sample was inoculated in 50 ml of nutrient broth. The flask was incubated at 30°C for 72h. After incubation time, serially diluted culture suspension was done on nutrient agar plates, followed by transferring plates at the temperature of 30°C for 48h. Isolated single colonies were tested morphologically and a total number of ten strains of Bacillus species were further screened for production of biosurfactant. The isolated strains were maintained in nutrient agar slants containing glycerol 50% (v/v) at 20°C. Subculture of the strains was carried out by streaking the stock culture of the strains onto nutrient agar plates, followed by transferring plates in an incubator at the temperature of 30°C. Microbial growth was carried out in growth medium E using different solutions of A, B, C and a basal mineral solution. Solution A contained 25 g/L of MgSO4 and solution B contained 100 g/L of (NH4)2SO4. Solution C included following compositions (g/L): EDTA, 0.5; MnSO4·H2O, 3; NaCl, 1; CaCl2·2H2O, 0.1; ZnSO4·7H2O, 0.1; FeSO4·7H2O, 0.1; CuSO4·5H2O, 0.01; AlK (SO4)2, 0.01; Na2MoO4·2H2O, 0.01; boric acid, 0.01; Na2SeO4, 0.005; and NiCl2·6H2O, 0.003. Solutions A and B
6 and C were then sterilized. Growth medium E was prepared by transferring 10 ml of each solution A, B and C into the basal medium to make a total volume of 1 L. Growth medium E was then mixed well, sterilized and used in biosurfactant production experiments. The initial pH value of growth medium E was adjusted to 7.0 using 1 N NaOH. The basal mineral solution used was composed of the following components (g/L): KH2PO4, 2.7; K2HPO4, 13.9; sucrose, 10; NaCl, 50; yeast extract, 0.5 and KNO3, 1 (Ghojavand et al., 2008a; Youssef et al., 2004).
2.2. Biosurfactant production The production of biosurfactant was studied by transferring a single colony of the bacterial strains into a 200 ml Erlenmeyer flask containing 50 ml nutrient broth medium, followed by incubation at 30°C and 150 rpm agitation for 16 h to obtain an optical density of 0.5 at a wavelength of 600 nm using a spectrophotometer. Subsequently, 10 ml of prepared culture content was utilized to inoculate 500 ml of medium E in an Erlenmeyer flask, followed by the incubation of culture at 30°C on a rotary shaker at 150 rpm for 72 h. Samples were then collected to determine acid precipitated biosurfactant as described previously (Joshi et al., 2008a; Vaz et al., 2012).
2.3. Biosurfactant determination To determine biosurfactant production, different screening methods including hemolytic activity (the ability to hydrolyze blood cells in blood agar), drop collapse (the ability to disperse on the oil) and oil spreading test (the ability to form a clear zone) were used as preliminary techniques. The bacterial isolates which showed a higher activity through the preliminary screening were subjected to complementary techniques. Surface tension activity and emulsification tests were the known methods to determine the biosurfactant produced by the strains.
Surface tension measurement is the most
known method to determine the biosurfactant produced by the bacterial strains. The surface tension measurement was conducted using a tensiometer at the room temperature. Critical micelle
7 concentration (CMC) is a relationship between biosurfactant concentration and surface tension; hence, it is an important property for various biosurfactant applications. In order to determine CMC, the surface tension of different concentrations of biosurfactant in distilled water was measured at the room temperature. On the other hand, critical micelle dilution (CMD) is defined as the dilution in which the surface tension starts to increase. CMD was determined by diluting culture supernatant in distilled water by 10-times and 100-times and subsequent measurements of the surface tension (Joshi et al., 2008b; Makkar and Cameotra, 2002; Vaz et al., 2012; White et al., 2013).
2.4. Screening and identification of the isolates A potent strain which could reduce the surface tension of medium to a lowest level and could show the highest emulsification activity (E24) was selected (Ghojavand et al., 2008a; Youssef et al., 2004). The identification of the isolated strain was carried out by scanning electron microscopy (SEM). Morphological and biochemical identification tests were also performed in accordance with Bergey’s Manual of Systematic Bacteriology. The sequence of the 16S rRNA gene was determined by Capillary electrophoresis and Dye-terminator sequencing at Bioneer Corporation Munpyeong-dong Daejeon, South Korea. The EzTaxon program was used to compare the 16S rRNA gene sequence results of the bacterial type strains (Chun et al., 2007).
2.5. Analytical methods The extracted components from the culture medium were further determined using high performance liquid chromatography (HPLC) technique. Fourier transform infrared spectroscopy (FTIR) was used to determine structural characterization of the biosurfactant extracted from the culture supernatant of the isolated strain. Proton nuclear magnetic resonance (1H NMR) was used for further characterization of biosurfactant produced.
8 2.6. Optimization of screened culture medium components A series of experiments of biosurfactant synthesis were designed based on the central composite design (CCD) in which culture medium (medium E) was supplemented with four efficient medium nutrients including glucose, crude oil, potassium nitrate and ammonium sulfate. Each variable was varied at different levels. The coded values and the actual levels of the medium nutrients tested are given in Table 1. The total number of experiments of the CCD generated was calculated according to Eq.(1): k
N=2 +2k + no
(1)
where k is the number of variables studied and no denotes the number of center points of the experimental design (Dashti et al., 2014). In this study, the total number of the fermentation runs for the center point was six treatments. The design matrix of the thirty experimental runs of the batch fermentation is given in Table S1. Experimental data from the mixture design (Table S1) were used to fit a second-order polynomial regression model (Eq. (2)) to CCD results to represent product formation as a function of variables tested: Y= ao +
ai Xi +
aii Xi2 +
aij Xi Xj
(2)
where Y is the measured response. Xi and Xj are the variables tested. ao represents the intercept. ai, aii and aij denote linear coefficient, quadratic coefficients and interaction coefficient, respectively (Arulmathi et al., 2015; Basak et al., 2014; Maran et al. 2013). The mathematical model generated for four independent variables studied was defined as Eq. (3): Y = ao + a1 X1 + a2 X2 + a3 X3 + a4 X4 + a11X12 + a22 X22 + a33 X32+ a44 X42 + a12 X1 X2 + a13 X1 X3 + a23 X2 X3 + a14 X1 X4+ a24 X2 X4+ a34 X3 X4
(3)
where Y is the measured surface tension activity. X1, X2, X3 and X4 represent the coded values of glucose concentration (g/L), crude oil (% v/v), potassium nitrate (g/L) and ammonium sulfate (g/L),
9 respectively. The experiments were conducted in 1000 ml Erlenmeyer flasks containing 250 ml medium E with pH 7.2, followed by incubation of the bacterial culture at 30°C on a rotary shaker at 150 rpm for 72 h. Statistical analysis of the data was performed using Design-Expert software (version 7.1 Stat-Ease, Inc). In order to find out the concurrent effects between variables tested on the response (surface tension activity), three dimensional (3D) graphs were constructed (Al-Shorgani et al., 2016; Betiku et al., 2015).
3. Results 3.1. Screening and identification of the selected biosurfactant-producing strains Table 2 shows the biochemical results obtained for ten strains tested in comparison to Bacillus subtilis ATCC 21332 as a known biosurfactant producer. As can be seen, the isolate 2IR showed the lowest surface tension value (32 mN/m) with the highest oil spreading (3.2 cm), crude oil emulsification (60 %) and hexadecane emulsification (68%) among strains screened. The positive results for emulsification assays (E24) suggested that the strain 2IR also produced emulsifier, which was notable compared to that from other isolates (Table 2). This finding indicated that results obtained for the strain 2IR were most consistent with those considered for B. subtilis ATCC 21332. Hence, the strain 2IR was selected as a potential biosurfactant-producing isolate with the highest efficiency in surface tension reduction. The strain 2IR was then studied based on morphological characteristics of the bacterial colonies and biochemical tests. Morphological observations revealed that the colonies formed were gray color, round and flat with an opaque appearance (Fig. 1A). The strain was subjected to the Gram staining method. Morphological observations by a light microscope showed a Gram-positive bacterium for the cells of the strain 2IR (Fig. 1B). Scanning electron microscope images showed the rod-shape of the bacterial cells with a diameter higher than 1 mm (Figs. 1C and 1D).
10 On the other hand, biochemical tests showed that the strain 2IR was a motile bacterium with a positive response to oxidase, catalase, nitrate reduction, citrate and esculin utilization tests (Table 3). Further identification was carried out using 16S rRNA ribotyping. 16S rRNA gene sequences (1478bp) of the strain 2IR demonstrated that the strain belonged to the species in genus of Bacillus as it was nearly identical with the strain Bacillus safensis FO-036bT and B. pumilus ATCC 7061T with similarity value of 98.11% and 97.97%, respectively. Comparison of biochemical tests for these strains demonstrated that strain 2IR and B. pumilus ATCC 7061T were identical in ability to hydrolyze esculin, while they are unable to show inositol fermentation. These findings confirmed that strain 2IR was B. pumilus. A phylogenetic tree was drawn based on a neighbor-joining analysis as shown in supplementary materials (Fig. S1). The strain characterized as Bacillus pumilus 2IR was deposited with the Microbial Culture Collection Unit (UNiCC) at the Institute of Bioscience at University Putra Malaysia under accession number UPMC 864. Moreover, the 16S rRNA gene sequence of strain Bacillus pumilus 2IR was deposited in Genbank under accession number KM 017130. 3.2. Profile of biosurfactant production by B. pumilus 2IR The profile of biosurfactant production by B. pumilus 2IR was studied using medium E as illustrated in Fig. 2. As can be seen, after a short lag phase, bacterial cells entered into the exponential growth phase, which continued up to 48 h of fermentation time and followed by the stationary phase. Biosurfactant production by the isolate 2IR was associated with bacterial cell growth so that an increase in the biomass concentration in exponential growth phase led to a high production of biosurfactant. In this regard, biosurfactant increased at exponential phase and reached up to 0.77 g/L at 72 h (stationary phase). Highest surface tension reduction (32 mN/m) was obtained at 30 h fermentation time (exponential phase) and did not change significantly afterwards.
3.3. Critical micelle concentration and critical micelle dilution
11 The experimental results of CMC measurement revealed that the surface tension of the medium decreased rapidly as the concentration of biosurfactant increased with a minimum surface tension of 31 mN/m (Fig. 3A). As shown in Fig. 3A, the surface tension remained constant for biosurfactant concentrations higher than 120 µg/ml and no further considerable changes occurred. This phenomenon was observed because of achieving CMC. Fig. 3B illustrates CMD results obtained from B. pumilus 2IR. As can be found, CMD-1 and CMD-2 were achieved after 30 h and 40 h of cultivation, respectively. Furthermore, maximum reduction in CMD-1 and CMD-2 indicated that biosurfactant had the highest activity even after 10 times and 100 times dilution of culture supernatant.
3.4. Characterization of the produced biosurfactant Acid precipitated biosurfactant extracted from isolate B. pumilus 2IR showed five main peaks at the retention time determined after 9.55 min using semi-preparative HPLC (Fig. 4A). In this study, the fraction N5 was collected and applied in 1H NMR and FTIR. The FTIR spectrum of the fifth compound (N5) from B. pumilus 2IR showed strong absorption bands, indicating the presence of a peptide component at 3290.78cm–1 resulting from N-H stretching mode (Fig. 4B). As shown, the stretching mode of a CO-N bond was observed at 1658.59 cm–1, while the deformation mode of the N-H bond combined with C-N stretching mode occurred at 1577.41cm–1. The presence of an aliphatic chain was determined by the C-H stretching modes at 2925 to 2850 cm–1 and 1429.46– 1387cm–1. These results strongly indicated that the biosurfactant contained aliphatic hydrocarbon combined with peptide moieties. Also, the band at 1734cm–1 was due to lactone carbonyl absorption. The IR spectra of the purified compound N5 displayed a significant similarity in IR adsorption obtained by other lipopeptide biosurfactant produced from Bacillus genus. The results from 1H NMR analysis was confirmed the lipopeptidic nature of the fragment N5 due to the presence of (CH3)2-CH group, indicating terminal branching in the fatty acid component at 0.96-0.81 ppm. The chemical
12 shift at (1.39-1.24 ppm) was consistent with a long aliphatic chain (CH2), a peptide backbone (N-H at 7.14-7.05 ppm) and an aliphatic carbon-hydrogen bond (4.9-3.9 ppm) (Fig. 4C). The intense singlet at 3.46 ppm (3.66 ppm for fraction N5) was similar to the 1H NMR spectrum of lipopeptide monoesters reported in literature, which indicated the presence of a methoxy group on the Glu or Asp amino residues. On the other hand, the presence of a lactone ring in the biosurfactant structure was confirmed by the detection of an ester carbonyl group at intense singlet of (5.1-4.9 ppm).
3.5. Optimization of culture medium components The preliminary study on the effect of the carbon and nitrogen sources tested on the biosurfactant production by B. pumilus 2IR reveled that glucose and crude oil as carbon sources with ammonium sulfate and potassium nitrate as nitrogen sources had notable effects on the synthesis of biosurfactant by the strain 2IR (data not shown). Hence, these nutrients were selected for further optimization of culture medium compositions. The experimental batch fermentations for the measurement of surface tension value were performed based on CCD with different levels of glucose (X1), crude oil (X2), potassium nitrate (X3) and ammonium sulfate (X4). The experimental results of CCD are shown in Table S1. As can be found, the highest reduction in surface tension was obtained in the treatments 11 and 29 with the value of 29 mN/m where similar 30 g/L glucose (0 as a coded value), 1% (v/v) crude oil (0 as a coded value) and 3 g/L potassium nitrate (0 as a coded value) were applied, while ammonium sulfate used in the treatments 11 and 29 was 3 g/L (0 as a coded value) and 1 g/L (-2 as a coded value), respectively. By applying multiple regression analysis to the test results, a secondorder polynomial equation was derived to represent surface tension as a function of glucose, crude oil, potassium nitrate and ammonium sulfate as shown in Eq. (4): Y= 30.83 -0.17 X1 +3.17 X2+0.42 X3+0.50 X4 +0.25 X1 X2 -0.13 X1 X3 +0.12 X1 X4 +0.50 X2 X3 +0.25 X2 X4 -0.38 X3 X4 +2.73 X12 +3.10 X22 +1.85 X32 + 0.48 X42
(4)
13 where Y is the measured surface tension value (mN/m). X1, X2, X3 and X4 are coded value for glucose, crude oil, potassium nitrate and ammonium sulfate, respectively. The statistical significance of the results was evaluated using the analysis of variance (ANOVA) for surface tension value (Table 4). As shown in Table 4, calculated model’s F value of 7.18 with a probability value (P>F) of 0.0002 suggested that the selected quadratic model was significant and fitted well to the experimental data (P<0.01). The F value for lack of fit (4.40) with a probability value of 0.0579 implied that the lack of fit for the model was insignificant and hence the model was valid for further studies. As can be observed from Table 4, the quadratic term of glucose, crude oil and potassium nitrate (X12 , X22 and X32) had a high significant effect on surface tension activity at 99% probability level (P<0.01), indicating that glucose, crude oil and potassium nitrate acted as critical factors and even a low change in their values could affect surface tension value to a remarkable level, while variations in ammonium sulfate had no significant effects on biosurfactant synthesis. The statistical analysis revealed that the optimum concentration for glucose, crude oil, potassium nitrate and ammonium sulfate were 30.31 g/L, 0.8 % (v/v), 2.88 g/L and 2.4 g/L, respectively. The statistical results suggested that the maximum reduction in surface tension would be 30.52 mN/m when the optimal concentrations of the variables were used. In this regard, a validation experiment was conducted using optimum concentrations of glucose, crude oil, potassium nitrate and ammonium sulfate by the consideration of C/N ratio of 12 as an optimum value. The experimental results showed that a surface tension value of 30 mN/m was obtained in optimum conditions, which was in good agreement with the suggested value (30.52 mN/m), indicating the acceptable reproducibility of results.
3.6. The simultaneous effect of carbon and nitrogen sources studied on surface tension value As can be seen from Fig. 5A, a rise in glucose concentration up to 30 g/L decreased surface tension value where crude oil was at a low concentration (0.5% v/v). Increasing crude oil concentration up to
14 1% (v/v) caused the high surface tension reduction, corroborating the significant quadratic effect of glucose and crude oil on the surface tension reduction (Table 4). The 3D plot for the concurrent effect of the crude oil and potassium nitrate showed that an exponential reduction in surface tension occurred when crude oil increased up to optimum level (1%) and potassium nitrate concentration was set at 3 g/L (Fig. 5B). The simultaneous effect of potassium nitrate and ammonium sulfate on surface tension value is illustrated in Fig. 5C. As shown, the surface tension value decreased concurrently with the increase in potassium nitrate concentration, whereas variations in ammonium sulfate had a minor shift in surface tension value. The 3D plots in Fig. 5 also indicated that a rise in surface tension value occurred when the concurrent effect of glucose, crude oil and potassium nitrate lied beyond their optimum levels, while ammonium sulfate showed insignificant effect on surface tension value.
4. Discussion According to the data obtained from this study, a direct relation between biosurfactant production and cell growth was observed. This indicated that biosurfactant production was consistent with cell growth which occurred at the exponential growth phase so that a decisive decrease in the surface tension occurred (Fig. 2). Similar pattern was observed for the production of lipopetide biosurfactant by Bacillus mojavensis (Ghojavand et al., 2011). Furthermore study on the biosurfactant by Bacillus strains revealed the growth-associated biosurfactant production for Bacillus mycoides and B. subtilis LB5 by high synthesis of lipopeptide biosurfactant at exponential growth phase (Najafi et al., 2010; Nitschke and Pastore, 2006). The CMC value of 120 µg/ml obtained by B. pumilus 2IR was consistent with CMC reported in literature with a wide range from 5 to 200 µg/ml (Chun et al., 2007; Ghojavand et al., 2008b). The CMD measured for the strain 2IR was in close agreement with that obtained by Nitschke and Pastore (2006) who observed that CMD-1 and CMD-2 were attained by Bacillus subtilis LB5 after 12 h
15 growth and remained constant along the surface tension reduction. The lipopeptidic nature of the produced biosurfactant by the strain 2IR was similar to the finding of Pereira et al. (2013) and Haddad et al. (2009) who observed the presence of lipopeptide in biosurfactant produced by Bacillus subtilis. The study fulfilled by Najafi et al. (2015) showed lipopetide nature of bisurfactant produced by Bacillus mycoides SH2, which was isolated from oil-contaminated samples. The data from 1H NMR analysis showed that the strain B. pumilus 2IR produced a mixture of biosurfactant components similar to Bacillus sp. which was reported elsewhere (Ghojavand et al., 2008b). Hence, experimental 1
H NMR and FTIR for fraction N5, revealed the lipopeptidic nature of biosurfactant produced by B.
pumilus 2IR. The current study showed that B. pumilus 2IR could better utilize glucose and sucrose to produce biosurfactant compared to other carbohydrates tested. In this regard, the production of biosurfactant by Pseudomonas aeruginosa from various sugars and hydrocarbons revealed that glucose was the best carbon source for bacterial cell growth and biosurfactant production, followed by fructose and sucrose (Lotfabad et al., 2009). The previous studies have shown that the utilization of non sugar-based hydrocarbons as the sole carbon source have inhibitory effects on the bacterial growth and biosurfactant production (Joshi et al., 2008b; Makkar and Cameotra, 2002). In the present study, it was found that the combination of sugar-based carbon source and a non sugar-based hydrocarbon had no deleterious effects on the biosurfactant production by B. pumilus 2IR. In this regard, as shown in Fig. 5A, the appropriate amount of crude oil 1% (v/v) showed a positive effect on the reduction of surface tension in relation to the optimal level of glucose and indicated the favorable effect of the combination of sugar-based and non sugar-based carbon source. In the same way, Mutalik et al. (2008) observed that the production of biosurfactant by Rhodococcus sp. increased up to 3.4-fold when combined mannitol and n-hexadecane were used. Apart from carbon source, nitrogen was the pivotal nutrient in the medium which could influence the microbial growth and
16 biosurfactant production. Although nitrate is a crucial factor for microbial growth, ammonium salts in the form of ammonium sulfate has also been used for the microbial growth. From the result shown in Fig. 5, it is evident that the surface tension value decreased when the concentration of the potassium nitrate increased from 1 g/L to 3 g/L under optimal concentration of carbon sources (glucose and crude oil). However, the excess concentration of the potassium nitrate more than 3 g/L resulted in a high increase in pH of the medium which in turn caused an inhibitory effect on the microbial cell growth and metabolic activity for surface tension value (Kiran et al., 2010; Khopade et al., 2012). This indicated the importance of nutrients for biosurfactant-mediated bioremediation where the insufficient carbon source and nutrients may inhibit the growth of biosurfactant-producing microbes (Ławniczak et al., 2013). The field study on biosurfactan-assisted oil recovery could elucidate the economical approach for the recovery of remarkable quantity of oil entrapped in the reservoirs. The study fuillfilled by Yussef et al. (2007) reveled that the in situ stimulation of two Bacillus strains in oil wells using nutrients including glucose, sodium nitrate, and trace metals resulted in to the production of lipopeptide biosurfactant that could mobilize entrapped oil from sandstone cores, demonstrating the point that on-site production of biosurfactant could be economically viable. Similar study showed that in situ stimulation of B. subtilis injected to the oil wells exhibited a cost-effective approach for the oil recovery by the production of lipopeptide biosurfactant (Yussef et al., 2013). Further research should focus on the various approaches of application of the biosurfactant in oil recovery to make this method economically competitive with the current strategies.
5. Conclusion The application of biosurfactants in oil reservoir is currently interesting in oil related research. In this regard, the key aspect is the efficient utilization of biosurfactants in the oil recovery with making this process economically feasible. This work showed that the new isolate B. pumilus 2IR was a
17 promising indigenous stain for the production of lipopeptide biosurfactant. The study also revealed that this strain was capable of consuming crude oil for the production of biosurfactant which can be advantageous to the in situ technology of the oil recovery from the abandoned oil reservoirs. The optimization of the culture medium indicated that glucose, crude oil and potassium nitrate had a significant effect on biosurfactant production by the strain 2IR with an optimum concentration value of 30.31 g/L glucose, 0.8 % (v/v) crude oil, 2.88 g/L potassium nitrate and 2.4 g/L ammonium sulfate.
Further research is recommended to conduct field study on the in situ production of
biosurfactant by this strain and to determine the efficiency of the strain for the successful MEOR.
Acknowledgment The authors sincerely acknowledge University Kebangsaan Malaysia (UKM), Malaysia and also would like to thank Department of Biotechnology, Iranian Research Organization Science and Technology (IROST), Tehran, Iran.
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21 Youssef N, Simpsona, D.R., Duncan, K.E., McInerneya, M.J., Folmsbee, M., Fincher,T., Knapp, R.M., 2007. In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir. Appl. Environ. Microbiol. 73, 1239-1247. Youssef
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Fig. 1. The characteristics of Bacillus pumilus 2IR: (A) colony morphology; (B) light microscope image using Gram staining method; (C) scanning electron microscope image using 5 KX magnification and (D) scanning electron microscope image using 10 KX magnification Fig. 2. The profile of biosurfactant production by B. pumilus 2IR Fig. 3. The profile of surface tension in relation to acid precipitated biosurfactant concentration for B. pumilus 2IR: (A) critical micelle concentration (CMC) measured in biosurfactant production process and (B) critical micelle dilution (CMD) during the bacterial cell growth Fig. 4. Characterization of the biosurfactant produced by B. pumilus 2IR: (A) semi-preparative HPLC chromatogram of biosurfactant detected at 220 nm, (B) transmission FTIR spectrum of biosurfactant (fraction N5) obtained after purification by semi-prep HPLC and (C) proton NMR spectrum of the fraction N5 fractionated from semi-preparation HPLC Fig. 5. Three dimentional plots for showing the simultaneous effect of carbon and nitrogen sources tested on the surface tension (mNm-1) obtained from B. pumilus 2IR: (A) interaction effect between glucose (X1) and crude oil (X2); (B) interaction effect between crude oil (X2) and potassium nitrate (X3) and (C) interaction effect between potassium nitrate (X3) and ammonium sulfate (X4)
22 Table 1 Process variables and determined levels for central composite design (CCD) in biosurfactant production by B. pumilus 2IR.
Variable Glucose (g/l) Crude oil (% v/v) Potassium nitrate (g/l) Ammonium sulfate (g/l)
Symbol Actual level X1 10 20 30
50
Coded value -2 -1 0
40
1
2
X2
0.1
0.5
1
1.5
2
-2
-1
0
1
2
X3
1
2
3
4
5
-2
-1
0
1
2
X4
1
2
3
4
5
-2
-1
0
1
2
Table 2 Screening of potential biosurfactant-producing strains using biochemical tests Strain
Blood a
Drop b
Oil
Surface
Emulsification
agar
collapse
spreading
tension
(E24)% Crude oil
lysis
(mm)
Emulsification (E24)% Hexadecane
-1
(cm)
(mNm )
1IR
-
-
1.2
48
52%
15%
2IR
++
+++
3.2
32
60%
68%
3IR
++
-
1.5
44
40%
65%
4IR
+++
++
0.8
37
40%
30%
5IR
+
+
1.5
40
35%
45%
6IR
+
+
0.8
38
40%
60%
7IR
+
-
1.0
43
35%
45%
8IR
++ +++
++ + +++
0.6 0.5 0.2 3.8
39 51 41 27
25% 10% 30% 70%
20% 20% 35% 75%
9IR 10IR Bacillus subtilis ATCC 21332
a
(-), no hemolysis; ( + ), incomplete hemolysis; (+ +) , complete hemolysis with a diameter of lysis
< 1 cm; (+ + +), complete hemolysis with a diameter of lysis between 1 to 3 cm b
(-) indicates the lack of biosurfactant production. Flat drops with signs (+), (+ +) and (+ + +) denote
the partial to complete spreading on the oil surface
23
Table 3 Biochemical testes conducted for the strain 2IR in comparison to most closely species Bacillus safensis FO-036bT
+
Bacillus pumilus ATCC 7061 T +
Motility
+
+
+
Oxidase
+
+
+
Catalase
+
+
+
Anaerobic growth
+
+
+
Voges- Proskauer reaction
+
+
+
Indol reaction
-
-
-
Utilization of Citrat
+
+
+
Nitrate reduction to Nitrite
-
-
-
Inositol
-
-
+
Starch
+
+
+
Casein
+
+
+
Tween 80
+
+
+
Esculine
+
+
+
Gelatine
-
-
+
Test/Characteristics
te 2IR
Endospore
+
Hydrolysis of :
(+): Positive (-): Negative
Table 4 Analaysis of variance (ANOVA) for the secod-order polynomial model of surface tension reduction produced by B. pumilus 2IR Sources Model
X1 X2 X3 X4 X1 X2 X1 X3 X1 X4
Sum of squares 710.88 0.67 240.67 4.17 6.00 1.00 0.25 0.25
DF 14 1 1 1 1 1 1 1
Mean square 50.78 0.67 240.67 4.17 6.00 1.00 0.25 0.25
F value 7.18 0.094 34.03 0.59 0.85 0.14 0.035 0.035
Prob>F 0.0002* 0.7630 < 0.0001* 0.4547 0.3716 0.7122 0.8534 0.8534
Coefficient estimated -0.17 3.17 0.42 0.50 0.25 -0.13 0.12
24 4.00
X2 X3 X2 X4 X3 X4 X12 X22 X32 X42
1.00
Residual Lack of fit Pure error *
2.25 204.30 264.30 94.30 6.30 106.08 95.25 10.83
1 1 1 1 1 1 1 15 10 5
4.00 1.00 2.25 204.30 264.30 94.30 6.30 7.07 9.53 2.17
0.57 0.14 0.32 26.89 37.37 13.33 0.89
0.4637 0.7122 0.5811 < 0.0001* < 0.0001* 0.0024* 0.3603
4.40
0.0579
0.50 0.25 -0.38 2.73 3.10 1.85 0.48
Statistically significant at 99% of probability level
X1, glucose (g/l); X2, crude oil (% v/v); X3, potassium nitrate (g/l); X4, ammonium sulfate (g/l) X1X2, X1 X3, X1 X4, X2 X3, X2 X4, X3 X4, the interaction terms, X12, X22, X32, X42 , the quadratic terms
Highlights ·
Biosurfactant-producing strains from oil fields in Iran were investigated.
·
Bacillus pumilus 2IR was identified as a promising lipopeptide biosurfactant producer.
·
Culture medium compositions were optimized by a central composite design.
·
Optimum conditions: 30.31g/L glucose; 0.8% crude oil; 2.88 g/L KNO3; 2.4g/L (NH4)2SO4
·
A surface tension value of 30 mN/m was obtained in optimum conditions.
Figure 5
A
X2
X1
B
X3
X2
C
X4
X3
Fig. 5
Figure 4
A N5
B
C
Fig. 4
Figure 3
75 Surface tension (mN/ m)
A
50
25
0
1
10
100
1000
Biosurfactant concentration (µg /ml)
Surface tension activity (m/ Nm ) CMD-1 , CMD -2
80
3
70
2.5
60 2
50 40
1.5
30
1
20 0.5
10 0
Absorbance (600 nm)
B
0 0
5
10
15
20
25 30 35 Time (h)
Surface Tension Activity(mN/m)
Fig. 3
CMD -1
40
45
50
CMD -2
55
60 Absorbance
Figure 2
30
20
10
0
0.8 2.5
2.0 2.0 1.5 1.5 1.0
0.6
0.4
1.0 0.2
0.5
0.5
0.0
0.0 0
6
12
18
24
30
48
56
72
Time (h) Growth (OD600nm) Biomass (g/L) Surface Tension (mN/m) Acid Precipitated Biosurfactant (g/L)
Fig. 2
80
96
0.0
Acid precipitated precipitated biosurfactant (g/L)(g /l) Acid biosurfactant
40
1.0
3.0
2.5
Growth (OD 600nm) Growth (OD 600nm)
Surface Surfacetension Tension(mN/m) (mN/m)
60
50
3.5
3.0
Biomass(g/l) (g/L) Biomass
70
Figure 1
A
B
C
Fig. 1
1
D
Graphical Abstract
Oil field
Sampling Isolation of Bacillus strains Isolation of Bacillus strains Oil-contaminated sample
Blood agar lysis
Drop-collapse test
Oil spreading test
Identification Identification
Fermentation
16S rRNA gene sequence
Electron microscope observation
Colony morphology
Precipitated biosurfactant