Appl Biochem Biotechnol DOI 10.1007/s12010-014-0928-9
First Report of a Lipopeptide Biosurfactant from Thermophilic Bacterium Aneurinibacillus thermoaerophilus MK01 Newly Isolated from Municipal Landfill Site Hakimeh Sharafi & Mahya Abdoli & Hamidreza Hajfarajollah & Nima Samie & Leila Alidoust & Habib Abbasi & Jamshid Fooladi & Hossein Shahbani Zahiri & Kambiz Akbari Noghabi
Received: 21 February 2014 / Accepted: 17 April 2014 # Springer Science+Business Media New York 2014
Abstract A biosurfactant-producing thermophile was isolated from the Kahrizak landfill of Tehran and identified as a bacterium belonging to the genus Aneurinibacillus. A thermostable lipopeptide-type biosurfactant was purified from the culture medium of this bacterium and showed stability in the temperature range of 20–90 °C and pH range of 5–10. The produced biosurfactant could reduce the surface tension of water from 72 to 43 mN/m with a CMC of 1.21 mg/mL. The strain growing at a temperature of 45 °C produces a substantial amount of 5 g/L of biosurfactant in the medium supplemented with sunflower oil as the sole carbon source. Response surface methodology was employed to optimize the biosurfactant production using sunflower oil, sodium nitrate, and yeast extract as variables. The optimization resulted in 6.75 g/L biosurfactant
Hakimeh Sharafi, Mahya Abdoli, and Hamidreza Hajfarajollah equally contributed to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0928-9) contains supplementary material, which is available to authorized users.
H. Sharafi : M. Abdoli : N. Samie : L. Alidoust : H. S. Zahiri : K. A. Noghabi (*) Division of Industrial & Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran e-mail: [email protected] H. Sharafi : J. Fooladi Department of Microbiology, Faculty of Sciences, Alzahra University, Vanak, Tehran, Iran M. Abdoli Faculty of Agriculture, Payam Noor University, Tehran 19569, Iran
H. Hajfarajollah Chemistry and Chemical Engineering Research Center of Iran, P.O. Box 14335-186, Tehran, Iran H. Abbasi Department of Chemical Engineering, Jundi-Shapur University of Technology, Dezful, Iran
Appl Biochem Biotechnol
production, i.e., 35 % improved as compared to the unoptimized condition. Thin-layer chromatography, FTIR spectroscopy, 1H-NMR spectroscopy, and biochemical composition analysis confirmed the lipopeptide structure of the biosurfactant. Keywords Thermophiles . Lipopeptide biosurfactant . Aneurinibacillus thermoaerophilus MK01 . Surface tension . Thin-layer chromatography . Response surface methodology
Introduction In the recent years, a wide range of investigations is being accomplished regarding the production of biosurfactants because of their potential applications in food, agriculture, pharmaceuticals, petroleum, and paper/pulp industries [1–3]. These amphiphilic compounds, which are produced by microbial cells [4], have several advantages, including low toxicity; high biodegradability; low irritancy to human skin; better environmental compatibility; higher foaming; specific activity at extreme temperatures, pH, and salinity; and the ability to be synthesized from renewable feedstocks [5, 6]. Biosurfactants have different structures. There are six major types of biosurfactants: glycolipids, lipopolysaccharides, lipoproteins–lipopeptides, phospholipids, hydroxylated and cross-linked fatty acids, and the complete cell itself [6]. Surfactin (lipopeptide) and rhamnolipid (glycolipid), from Bacillus subtilis and Pseudomonas aeruginosa, respectively, are the most extensively studied [7]. Information concerning biosurfactant production by thermophilic bacteria is relatively rare. Joshi et al. [8] reported biosurfactant production at 45 °C by a thermophilic species of Bacillus. The produced biosurfactant was stable up to 80 °C. Banat [9] has isolated a thermotolerant biosurfactant-producing Bacillus sp. that grew at temperatures up to 50 °C on a hydrocarbon-containing medium and is suitable for use in microbial enhanced oil recovery (MEOR) and oil-sludge cleanup. Bharali et al. [10] extracted a glycolipid biosurfactant by the thermophilic bacterium Alcaligenes faecalis isolated from the crude oil-contaminated soil. Mnif et al. [11] isolated three thermophile strains belonged to the genera Geobacillus, Bacillus, and Brevibacillus, which could produce biosurfactant in the range of 37–55 °C. Aneurinibacillus aneurinilyticus has been known as a lignin-degrading bacterium [12] and recently proved as a source of thermostable enzymes [13, 14]. The strain Aneurinibacillus sp. AM-1 produces a proline-specific aminopeptidase useful for collagen degradation [15]. A number of investigations have also been reported on the production of surface layer proteins by Aneurinibacillus thermoaerophilus [16]. However, there are no prior findings about biosurfactant production by Aneurinibacillus thermoaerophilus. Given the growing interest in biosurfactant-producing thermophiles, and following considerable current interest in emerging technology for the practical use of these useful natural products, identifying appropriate thermophile strains is of prime importance in developing biosurfactantbased technology. Here, we report for the first time the isolation and identification of a local strain of A. thermoaerophilus with considerable capability of biosurfactant production that may be appropriate for various industrial applications. This was accomplished by (1) comprehensive examinations of bacterial strains isolated from urban waste landfill for their ability to produce biosurfactant, (2) purification of biosurfactant and testing its stability at different temperatures and pH, (3) preliminary identification and characterization of the produced biosurfactant, and (4) product optimization using designed experiments.
Appl Biochem Biotechnol
Materials and Methods Screening of Bacteria Samples were collected from urban wastes of the Kahrizak site in the south of Tehran. The samples were collected in plastic bags, immediately transported to the laboratory, and stored in an appropriate condition for further processing. For the screening of biosurfactant-producing bacteria, enrichment cultures utilizing hydrophobic compounds as the sole carbon source are more appropriate. Therefore, screening of the biosurfactant-producing microorganisms was carried out using sunflower oil as a sole carbon source. Samples were transferred to 50-mL tubes containing 7.5 mL of selective medium consisting of (g/L) NH4NO3 3.0, KH2PO4 0.3, MgSO4·7H2O 0.3, yeast extract 0.5, and sunflower oil 100, and incubated on a shaker– incubator (Kuhner, Germany) at 200 rpm and 45 °C for 7–10 days. When a nearly stable emulsion was produced, serial dilutions of cultures were prepared followed by spreading on nutrient agar plates. Purified single colonies were examined for their ability to produce biosurfactant using the blood agar hemolysis test, oil spreading, and emulsification activities [17]. Out of different strains, one strain, which was showing maximum competence for biosurfactant production, was selected and designated as the desired isolate and maintained on nutrient agar slants for further studies. Isolated colonies were cultured for biosurfactant production. Seed culture medium containing (g/L) glucose 40, NaNO3 3.0, KH2PO4 0.25, MgSO4·7H2O 0.25, and yeast extract 1.0 was prepared by overnight incubation on a rotary shaker at 200 rpm and 45 °C. The production medium was composed of (g/L) NaNO3 3.0, KH2PO4 0.25, MgSO4·7H2O 0.25, yeast extract 1.0, and sunflower oil 10 and incubated at 200 rpm and 45 °C for 4–7 days. However, for the response surface methodology (RSM) experiments, the amounts of some ingredients varied according to the experimental design. Biochemical and Molecular Identification of Isolated Strain Molecular identification of the selected bacterial isolate was referred for 16S rRNA gene sequence analysis. DNA extraction was performed from 2-mL bacterial cultures collected at the mid-exponential growth phase using the Roche Kit (Germany) according to the manufacturer’s instructions and run in triplicate through polymerase chain reaction (PCR). Three sets of primers: 27f (5′-AGAGTTTGATCCTGGCTCAG) and 1492r (5′ TACGGTTACCTTGTTA CGACTT) (Lane et al., 1991) and V3 and V6 primers were used to amplify the V3 and V6, the two most relevant hypervariable regions of the bacterial 16S rRNA gene, giving a product of 203 and 124 bp, respectively [18]. The reaction was carried out in a 25-μL volume containing 1× PCR buffer, 1.5 mM MgSO4, 2 mM dNTP mixture, 1 μM of each primer, 1 μL of Pfu DNA polymerase (Fermentas, St. Leon-Rot, Germany), and 1 ng of template DNA. PCR amplification was performed as follows: initial denaturation at 95 °C for 5 min, followed by 25 cycles each of 94 °C for 1 min, 55 °C of annealing for 45 s, and a 45-s extension at 72 °C. The 203- and 124-bp PCR products amplified from the bacterial isolates which appeared as a single band were purified using a High Pure PCR Product Purification Kit (Roche Applied Science, Germany) and sequenced on an ABI Prism 377 automatic sequencer (Applied Biosystems, CA, USA). Sequence homologies were examined using BLAST version 2.2.12 of the National Center for Biotechnology Information [19]. Multiple sequence alignments were carried out using ClastalW, and a consensus neighbor-joining tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software (version 4.0) [20].
Appl Biochem Biotechnol
Extraction of Biosurfactant and Physical Properties The produced biosurfactant was extracted from the culture broth with the method of acid precipitation followed by solvent extraction reported elsewhere [7]. Briefly, the culture broth was centrifuged (6,000×g, 10 min, 4 °C) to remove the cells. Then, pH was adjusted to 2 by 3 N HCl in order to precipitate the biosurfactant. After keeping it overnight, the crude biosurfactant was obtained by centrifugation (18,000×g, 30 min, 4 °C). The crude biosurfactant was purified using several times extraction by ethyl acetate. Physical and surface activity properties of the biosurfactant were determined in terms of surface tension (ST), interfacial tension (IFT), critical micelle concentration (CMC), oil spreading test (OST), and emulsion index (E24). ST and IFT were measured using a Krüss K100MK2 tensiometer (Krüss GmbH, Hamburg, Germany) using Wilhelm plate technique. The tensiometer was calibrated against double-distilled water. Before each measurement, the probe was rinsed several times with double-distilled water and acetone followed by flaming on a Bunsen burner. Interfacial tension was performed between water and three hydrophobes including hexadecane, isooctane, and cyclohexane. The CMC is a widely used index to evaluate surface activity. The crude biosurfactant was dissolved in distilled water, and the surface tension of the solution was measured with various concentrations of biosurfactant at 25 °C. CMC was measured from the breakpoint of surface tension versus biosurfactant concentration. Stabilization was allowed to occur until the standard deviation of five successive measurements was less than 0.9 mN/m. Each result was the average of at least five determinations after stabilization. The E24 of culture samples was determined by adding 6 mL of a hydrocarbon to 4 mL of culture, mixing with a vortex for 2 min, and leaving it standing for 24 h. The E24 index is given as the percentage of the height of the emulsified layer (mm) divided by the total height of the liquid column (mm) [21]. OST was performed according to the standard procedure [22]. The diameter of the clear zone after dropping 10 μL of culture broth on the surface of the oil was considered as a response. Growth Kinetics and Biosurfactant Production A 4 % cell suspension (in physiological saline) from an overnight seed culture medium was inoculated in 2,000-mL flasks containing 500 mL of medium and incubated at 45 °C while shaking at 200 rpm. Sunflower oil was used as carbon source. During fermentation, samples were aseptically retrieved from the liquid culture at different time intervals to monitor the kinetic parameters including cell growth, biosurfactant concentration, surface activity (diameter of clear zone (cm)), and pH of culture medium. Bacterial growth was determined by measuring the optical density of the culture broth at 600 nm with a UV spectrophotometer (PerkinElmer, Lambda 25, USA). Effect of Different Carbon Sources on the Surface Activity Various carbon sources from different origins including carbohydrates, hydrocarbons, and vegetable oils were used for evaluating the surface activity of the produced biosurfactant in terms of OST and E24. Characterization of Dried Biosurfactant Thin-layer chromatography (TLC) is a simple method for primary characterization of biosurfactants. TLC was applied using the solvent system of chloroform/methanol/water
Appl Biochem Biotechnol
(65:15:4v/v). Ten microliters of sample was put on the TLC plate (Silicagel 60, Merck, Germany) and developed with the solvent. After development, one of the plates was put into a jar saturated with iodine vapors to detect lipids [23]. Another plate was sprayed evenly with the ninhydrin reagent (0.5 g ninhydrin (Sigma-Aldrich) in 100 mL anhydrous acetone) and placed in an oven at 110 °C for 10 min to detect the presence of peptide as red spots [24]. The purified biosurfactant was analyzed using Fourier transform infrared (FTIR) spectroscopy with a PerkinElmer Spectrometer (FTIR GX 2000). Biosurfactant samples were dried first and then ground before performing FTIR analysis. The dried powder was finely mixed with a KBr matrix (Sigma). The spectra were in the range of 400–4,000/cm with a resolution of 8/cm. The biosurfactant was subjected to further analysis with nuclear magnetic resonance (NMR). 1H spectra were measured at 25 °C using a Bruker JNM-A500 spectrometer (Germany) equipped with a triple-resonance (1H, 13C, 15N) inverse probe with 5-mmdiameter tubes containing 600 μL of sample, with deuterated chloroform as a solvent. One hundred twenty-eight scans were collected (90° pulse, 7.3 μs; saturation pulse, 3 s; relaxation delay, 3 s; acquisition time, 4.679 s; 65,536 data points). A 1-Hz exponential line-broadening filter was applied before Fourier transformation, and a baseline correction was performed on spectra before integration using Bruker software (TopSpin 2.0). Biochemical Composition Analysis The carbohydrate content of the produced biosurfactant was determined by the method of phenol–sulfuric acid presented by Dubois et al. [25] using D-glucose as a standard. Briefly, a volume of 0.5 mL of cell supernatant was mixed with 0.5 mL of 5 % phenol solution and 2.5 mL of sulfuric acid and incubated for 15 min before measuring the absorbance at 490 nm. Lipid content was estimated according to Folch et al. [26]. The biosurfactant sample, 0.2 g, was blended with the chloroform–methanol mixture (2:1) and agitated acutely. Solvent phase was recovered by centrifuging at 10,000 rpm for 15 min. The extraction process was carried out three times. The whole solvent was collected, evaporated, and dried under vacuum. The lipid content was determined by gravimetric estimation. Protein content was determined by the method of Bradford [27] using Coomassie brilliant blue with bovine serum albumin as a standard. The absorbance of unknown samples was measured at 595 nm. Stability of Biosurfactant To evaluate the stability of the biosurfactant at different environmental conditions, 50 mg/mL solution of the biosurfactant was prepared and maintained at a temperature range of 20–90 °C for 120 h. After cooling to room temperature, ST was measured as an activity indicator of the biosurfactant. To determine the effect of pH on the biosurfactant activity, the ST was measured after adjusting the pH between 3 and 12 using 3 N NaOH or 3 N HCl. The effect of the addition of different concentrations of NaCl on the activity of the biosurfactant was also studied. Experimental Design for Optimization of Culture Condition RSM is a combination of mathematical and statistical techniques used for developing, improving, and optimizing the processes [28]. RSM was applied to study the effects of the various essential nutrients, namely sunflower oil, sodium nitrate, and yeast extract. This
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procedure was carried out to optimize the process conditions for the maximum production of the biosurfactant from A. thermoaerophilus MK01. In order to obtain the mutual interactions between the variables and to optimize these variables, a 23 factorial design based on central composite design (CCD) [28] with six replicates at the center points using a total number of 20 experiments was employed. A statistical program package, Minitab software version 16, was used for regression analysis of the data obtained and to estimate the coefficient of the regression equation. The range and the levels of the factors, which were varied according to the experimental design, are given in Table 1. The range of variables was chosen based on our earlier investigations for biosurfactant production in shake flasks. The statistical combinations of the critical media components along with the maximum biosurfactant concentrations in the broth are detailed in Table 2. The predicted biosurfactant concentration was calculated using the following second-order polynomial equation: Y ¼ β 0 þ β 1 A þ β 2 B þ β 3 C þ β 11 A A þ β 22 B B þ β 33 C C þ β 12 A B
ð1Þ
þ β 13 A C þ β 23 B C
where Y is the predicted response; β0 is the intercept; β1, β2, and β3 are linear coefficients; β11, β22, and β33 are squared coefficients; β12, β13, and β23 are interaction coefficients; and A, B, and C are independent variables. By using this equation, it is possible to evaluate the linear, quadratic, and interactive effects of the independent variables on the response appropriately. The significance of each coefficient was determined by the F factor and p values. The larger the magnitude of the F value and the smaller the p value, the more significant is the corresponding coefficient. Multiple correlation coefficient (R2) and adjusted R2 were used as quality indicators to evaluate the fitness of the second-order polynomial equation. The optimized amount of each factor was estimated using the Response Optimizer option of the Minitab software.
Results and Discussion Isolation and Characterization of Biosurfactant-Producing Isolate Among numerous bacterial strains, which were isolated from urban wastes and tested for biosurfactant production, one isolate with the best confirmatory results in OST, hemolytic activity, and emulsification activity was selected. Morphological and biochemical characteristics of the selected isolate showed that the selected isolate MK01 was Gram-positive, long, catalase-positive, and citrate utilization-, indole production-, and Voges–Proskauer reactionnegative. Results from 16S rRNA gene sequencing identified our strain as A. thermoaerophilus Table 1 Experimental range and levels of the independent variables Symbol
Factors
Levels −α
−1
0
1
+α
A
Sunflower oil (g/L)
11.59
15
20
25
28.4
B
Sodium nitrate (g/L)
2.16
2.5
3
3.5
3.84
C
Yeast extract (g/L)
0.65
1
1.5
2
2.34
Appl Biochem Biotechnol Table 2 Full factorial central composite design matrix of three variables in coded and actual units along with the observed responses Run order
1
Sunflower oil (g/L)
Sodium nitrate (g/L)
Yeast extract (g/L)
Coded value
Actual value
Coded value
Coded value
−1
Actual value
Actual value
Biosurfactant production (g/L)
15
1
3.5
1
2
4.75
2
1.68
28.409
0
3
0
1.5
6.4
3
0
20
0
3
0.65
4.05
4
1
25
−1
5
0
20
6
−1.68
−1.68
2.5
1
2
6.05
0
3
0
1.5
5.05
11.591
0
3
0
1.5
4.5
7
0
20
1.68
3.8409
0
1.5
5.45
8 9
−1 1
15 25
−1 1
2 1
4.5 6.1
10
0
20
0
3
1.68
2.3409
4.55
11
0
20
0
3
0
1.5
5.16
12
1
25
−1
2.5
−1
1
4.5
13
1
25
1
3.5
1
2
5.67
14
0
20
0
3
0
1.5
5.76
15
0
20
0
3
0
1.5
5.35
16 17
0 −1
20 15
2.1591 2.5
0 −1
1.5 1
4.35 3.67
−1
−1.68 −1
2.5 3.5
1 −1
18
−1
15
1
3.5
1
4.76
19
0
20
0
3
0
1.5
5.05
20
0
20
0
3
0
1.5
5.45
with 100 % similarity to A. thermoaerophilus strain L420-9 (Fig. 1). We tentatively labeled our strain A. thermoaerophilus MK01 and deposited it in GenBank with the accession number KF923810. Growth Kinetics of Biosurfactant Production A. thermoaerophilus MK01 was cultivated on sunflower oil as the sole carbon source. Figure 2 shows the pattern of growth kinetics and biosurfactant production. The results showed that biosurfactant production started early in the exponential growth phase and was considered to be predominantly parallel with the bacterial growth. The MK01 strain was able to synthesize up to 2 g/L of biosurfactant after 24 h of cultivation. The production of the biosurfactant was continued up to 96 h, and the maximum production of about 5 g/L was obtained after 120 h. The diameter of the clear zone (in the oil spreading test) was also increased parallel to the biosurfactant production. This observation is rational because the diameter of the clear zone has a direct correlation with biosurfactant concentration [21]. The crude biosurfactant reduces the surface tension of water from 72 to about 43 mN/m (Fig. 3). In a certain concentration, any further increase in the concentration of the biosurfactant was not accompanied by a decrease in surface tension. This concentration corresponds to the point where the biosurfactant first shows a stable low surface tension value
Appl Biochem Biotechnol
Fig. 1 Phylogenetic tree of Aneurinibacillus thermoaerophilus MK01. Numbers at the nodes indicate the bootstrap values on neighbor-joining analysis
termed CMC. Therefore, CMC was measured from the breakpoint of two lines as 1.21 mg/mL (Fig. 3). The solution of biosurfactant in water could also reduce the IFT between water and hexadecane from 41 to 19 mN/m, water and isooctane from 33 to 10 mN/m, and water and cyclohexane from 47 to 21 mN/m.
Fig. 2 Time course profile of biosurfactant production, cell growth, OST, and pH of Aneurinibacillus thermoaerophilus MK01 grown on sunflower oil as sole carbon source
Appl Biochem Biotechnol 70
65
ST (mN/m)
60
55
50
45
40
35 0
1
2
3
4
5
6
7
BS Concentration (mg/ml) Fig. 3 Variation of the surface tension (ST) of the biosurfactant versus concentration for the determination of CMC
Effect of Different Carbon Sources on Surface Activity The isolate was able to grow on the various carbon sources examined. However, the emulsification activity and diameter of the clear zone varied significantly. Figure 4 shows the effect of different carbon sources on the surface activity of the produced biosurfactant in terms of OST and E24. As shown, water-insoluble substrates were more effective in biosurfactant production. Glucose was the most effective amongst water-soluble carbon substrates with emulsification activity of more than 40 %. A similar observation has been reported by Abbasi et al. [29] on the production of rhamnolipids. Although strain MK01 grown on sucrose, maltose, and glycerol showed low emulsification activity, the clear zone diameter was zero, showing that the biosurfactant production on these carbon sources is insignificant. Maximum clear zone diameters were obtained on corn oil, canola oil, and sunflower oil. Therefore, it can be concluded that these carbon sources are more efficient in the production of biosurfactant by A. thermoaerophilus MK01. Overall, results showed that among all examined carbon substrates, vegetable oils were the most effective carbon sources in biosurfactant production. Characterization of Biosurfactant Primary characterization of the produced biosurfactant was carried out using TLC. After development, the plate that was in contact with iodine vapor showed a yellow spot indicating the presence of polar lipids. Treatment with ninhydrin solution revealed the presence of peptide as indicated by a red spot (Fig. 5a). This suggests the lipopeptide structure for the produced biosurfactant. Figure 5b shows the IR spectrum of the purified biosurfactant. As can be seen, characteristic absorption bands corresponding to functional groups characteristically forming part of the lipopeptide could be observed for the purified biosurfactant sample. The stretching
Appl Biochem Biotechnol 140 OST E24%
E24%, OST * 10 (cm)
120
100
80
60
40
20
0
se se se se se rol la ve er an rn nd e ut oil ne en en co no cro lto cto ce ano oli flow y be co lmo sam con ude eca tolu xyl n a se co cr ad glu ma su ma la gly c su so x he
carbon source
Fig. 4 Effect of various carbon sources on the surface activity of the biosurfactant
bands of the methylene and terminal methyl groups of the acyl chains between 2,850 and 2,930/cm and an asymmetric stretching strong peak which occurred at 1,745/cm suggest the presence of carboxyl groups (C=O in COOH) in the biosurfactant. Protein-related bonds, the vC=O amide I (1,646/cm) and δ NH/vC=O combination of the amide II bands (1,540–60/cm), were also observed for the sample. The absorption peaks at 1,250 and 1,064/cm could indicate the C–O stretching in ether or alcohol and methoxyl groups, respectively, although other groups also absorb in this region. Results obtained from 1H-NMR indicated that the produced biosurfactant has a lipopeptide structure. Almost all the backbone amide NH groups are in the region from 8.2 to 7.2 ppm downfield from tetramethylsilane. Alpha hydrogens of the amino acids come into resonance from 5.2 to 4 ppm for peptide moiety along with the fatty acid functional group as a single methyl-related peak (δ 0.8–0.88), –(CH2)n– (δ 1.1–1.45), C–CH2–CO (δ 1.6), and –C–OH (δ 2.2 ppm). An additional weak signal was also detected at δ 5.3 consistent with CHO of the alcohol moiety of an ester (Fig. 6). Biochemical composition analysis revealed that the produced biosurfactant is composed of 13 % peptide, 74 % lipid, and 3 % carbohydrate. The presence of a minor fraction of carbohydrate in the purified sample possibly arising from the residual cell debris in the broth coprecipitated with the biosurfactant during its extraction process. Biosurfactant Stability The biosurfactant produced by A. thermoaerophilus MK01 was shown to be thermostable (Fig. S1). Heating of the biosurfactant up to 90 °C caused no significant effect on the biosurfactant performance. Therefore, it can be concluded that the MK01 biosurfactant preserves its surface properties unaffected in the wide range of temperatures between 20 and 90 °C. This activity indicated the usefulness of the MK01 biosurfactant in food, pharmaceutical, and cosmetics industries as well as its application in high-temperature bioremediation.
Appl Biochem Biotechnol
(a)
Analytical TLC of MK01 biosurfactant. TLC was carried out on silica gel plate
using chloroform: methanol: water (65:15:4 v/v) as mobile phase. The plates were stained with ninhydrin (left) and iodine vapor (right).
1.2
1.0
Transmittance (%)
0.8 1556
1646
0.6
0.4 2854 1460
2925
1745
0.2
1160
0.0 3300
3000
2700
2400
2100
1800
1500
1200
900
600
-1 Wavenumber (cm )
(b)
FTIR spectrum of biosurfactant produced by A. thermoaerophilus MK01.
Fig. 5 a Analytical TLC of the MK01 biosurfactant. TLC was carried out on a silica gel plate using chloroform/ methanol/water (65:15:4v/v) as mobile phase. The plates were stained with ninhydrin (left) and iodine vapor (right). b FTIR spectrum of the biosurfactant produced by A. thermoaerophilus MK01
Recently, it was reported that bioremediation in the presence of biosurfactant at high temperatures can lead to better results [30]. The surface activity of the crude biosurfactant remained relatively stable over pH range 5–10. Optimum stability of biosurfactant was observed at NaCl concentrations below 10 % (Fig. S1).
Appl Biochem Biotechnol
Fig. 6
1
H-NMR pattern of the biosurfactant produced by A. thermoaerophilus MK01
Response Surface Optimization Results The CCD design along with the corresponding experimental values of the biosurfactant production is given in Table 2. Analysis of variance (ANOVA) of the model and the corresponding p values, along with the parameter estimate for the biosurfactant production, are shown in Table 3. The ANOVA of the quadratic regression model indicated that the model was highly significant, as the F value for the model was 16.23 (p
First Report of a Lipopeptide Biosurfactant from Thermophilic Bacterium Aneurinibacillus thermoaerophilus MK01 Newly Isolated from Municipal Landfill Site Hakimeh Sharafi & Mahya Abdoli & Hamidreza Hajfarajollah & Nima Samie & Leila Alidoust & Habib Abbasi & Jamshid Fooladi & Hossein Shahbani Zahiri & Kambiz Akbari Noghabi
Received: 21 February 2014 / Accepted: 17 April 2014 # Springer Science+Business Media New York 2014
Abstract A biosurfactant-producing thermophile was isolated from the Kahrizak landfill of Tehran and identified as a bacterium belonging to the genus Aneurinibacillus. A thermostable lipopeptide-type biosurfactant was purified from the culture medium of this bacterium and showed stability in the temperature range of 20–90 °C and pH range of 5–10. The produced biosurfactant could reduce the surface tension of water from 72 to 43 mN/m with a CMC of 1.21 mg/mL. The strain growing at a temperature of 45 °C produces a substantial amount of 5 g/L of biosurfactant in the medium supplemented with sunflower oil as the sole carbon source. Response surface methodology was employed to optimize the biosurfactant production using sunflower oil, sodium nitrate, and yeast extract as variables. The optimization resulted in 6.75 g/L biosurfactant
Hakimeh Sharafi, Mahya Abdoli, and Hamidreza Hajfarajollah equally contributed to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0928-9) contains supplementary material, which is available to authorized users.
H. Sharafi : M. Abdoli : N. Samie : L. Alidoust : H. S. Zahiri : K. A. Noghabi (*) Division of Industrial & Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran e-mail: [email protected] H. Sharafi : J. Fooladi Department of Microbiology, Faculty of Sciences, Alzahra University, Vanak, Tehran, Iran M. Abdoli Faculty of Agriculture, Payam Noor University, Tehran 19569, Iran
H. Hajfarajollah Chemistry and Chemical Engineering Research Center of Iran, P.O. Box 14335-186, Tehran, Iran H. Abbasi Department of Chemical Engineering, Jundi-Shapur University of Technology, Dezful, Iran
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production, i.e., 35 % improved as compared to the unoptimized condition. Thin-layer chromatography, FTIR spectroscopy, 1H-NMR spectroscopy, and biochemical composition analysis confirmed the lipopeptide structure of the biosurfactant. Keywords Thermophiles . Lipopeptide biosurfactant . Aneurinibacillus thermoaerophilus MK01 . Surface tension . Thin-layer chromatography . Response surface methodology
Introduction In the recent years, a wide range of investigations is being accomplished regarding the production of biosurfactants because of their potential applications in food, agriculture, pharmaceuticals, petroleum, and paper/pulp industries [1–3]. These amphiphilic compounds, which are produced by microbial cells [4], have several advantages, including low toxicity; high biodegradability; low irritancy to human skin; better environmental compatibility; higher foaming; specific activity at extreme temperatures, pH, and salinity; and the ability to be synthesized from renewable feedstocks [5, 6]. Biosurfactants have different structures. There are six major types of biosurfactants: glycolipids, lipopolysaccharides, lipoproteins–lipopeptides, phospholipids, hydroxylated and cross-linked fatty acids, and the complete cell itself [6]. Surfactin (lipopeptide) and rhamnolipid (glycolipid), from Bacillus subtilis and Pseudomonas aeruginosa, respectively, are the most extensively studied [7]. Information concerning biosurfactant production by thermophilic bacteria is relatively rare. Joshi et al. [8] reported biosurfactant production at 45 °C by a thermophilic species of Bacillus. The produced biosurfactant was stable up to 80 °C. Banat [9] has isolated a thermotolerant biosurfactant-producing Bacillus sp. that grew at temperatures up to 50 °C on a hydrocarbon-containing medium and is suitable for use in microbial enhanced oil recovery (MEOR) and oil-sludge cleanup. Bharali et al. [10] extracted a glycolipid biosurfactant by the thermophilic bacterium Alcaligenes faecalis isolated from the crude oil-contaminated soil. Mnif et al. [11] isolated three thermophile strains belonged to the genera Geobacillus, Bacillus, and Brevibacillus, which could produce biosurfactant in the range of 37–55 °C. Aneurinibacillus aneurinilyticus has been known as a lignin-degrading bacterium [12] and recently proved as a source of thermostable enzymes [13, 14]. The strain Aneurinibacillus sp. AM-1 produces a proline-specific aminopeptidase useful for collagen degradation [15]. A number of investigations have also been reported on the production of surface layer proteins by Aneurinibacillus thermoaerophilus [16]. However, there are no prior findings about biosurfactant production by Aneurinibacillus thermoaerophilus. Given the growing interest in biosurfactant-producing thermophiles, and following considerable current interest in emerging technology for the practical use of these useful natural products, identifying appropriate thermophile strains is of prime importance in developing biosurfactantbased technology. Here, we report for the first time the isolation and identification of a local strain of A. thermoaerophilus with considerable capability of biosurfactant production that may be appropriate for various industrial applications. This was accomplished by (1) comprehensive examinations of bacterial strains isolated from urban waste landfill for their ability to produce biosurfactant, (2) purification of biosurfactant and testing its stability at different temperatures and pH, (3) preliminary identification and characterization of the produced biosurfactant, and (4) product optimization using designed experiments.
Appl Biochem Biotechnol
Materials and Methods Screening of Bacteria Samples were collected from urban wastes of the Kahrizak site in the south of Tehran. The samples were collected in plastic bags, immediately transported to the laboratory, and stored in an appropriate condition for further processing. For the screening of biosurfactant-producing bacteria, enrichment cultures utilizing hydrophobic compounds as the sole carbon source are more appropriate. Therefore, screening of the biosurfactant-producing microorganisms was carried out using sunflower oil as a sole carbon source. Samples were transferred to 50-mL tubes containing 7.5 mL of selective medium consisting of (g/L) NH4NO3 3.0, KH2PO4 0.3, MgSO4·7H2O 0.3, yeast extract 0.5, and sunflower oil 100, and incubated on a shaker– incubator (Kuhner, Germany) at 200 rpm and 45 °C for 7–10 days. When a nearly stable emulsion was produced, serial dilutions of cultures were prepared followed by spreading on nutrient agar plates. Purified single colonies were examined for their ability to produce biosurfactant using the blood agar hemolysis test, oil spreading, and emulsification activities [17]. Out of different strains, one strain, which was showing maximum competence for biosurfactant production, was selected and designated as the desired isolate and maintained on nutrient agar slants for further studies. Isolated colonies were cultured for biosurfactant production. Seed culture medium containing (g/L) glucose 40, NaNO3 3.0, KH2PO4 0.25, MgSO4·7H2O 0.25, and yeast extract 1.0 was prepared by overnight incubation on a rotary shaker at 200 rpm and 45 °C. The production medium was composed of (g/L) NaNO3 3.0, KH2PO4 0.25, MgSO4·7H2O 0.25, yeast extract 1.0, and sunflower oil 10 and incubated at 200 rpm and 45 °C for 4–7 days. However, for the response surface methodology (RSM) experiments, the amounts of some ingredients varied according to the experimental design. Biochemical and Molecular Identification of Isolated Strain Molecular identification of the selected bacterial isolate was referred for 16S rRNA gene sequence analysis. DNA extraction was performed from 2-mL bacterial cultures collected at the mid-exponential growth phase using the Roche Kit (Germany) according to the manufacturer’s instructions and run in triplicate through polymerase chain reaction (PCR). Three sets of primers: 27f (5′-AGAGTTTGATCCTGGCTCAG) and 1492r (5′ TACGGTTACCTTGTTA CGACTT) (Lane et al., 1991) and V3 and V6 primers were used to amplify the V3 and V6, the two most relevant hypervariable regions of the bacterial 16S rRNA gene, giving a product of 203 and 124 bp, respectively [18]. The reaction was carried out in a 25-μL volume containing 1× PCR buffer, 1.5 mM MgSO4, 2 mM dNTP mixture, 1 μM of each primer, 1 μL of Pfu DNA polymerase (Fermentas, St. Leon-Rot, Germany), and 1 ng of template DNA. PCR amplification was performed as follows: initial denaturation at 95 °C for 5 min, followed by 25 cycles each of 94 °C for 1 min, 55 °C of annealing for 45 s, and a 45-s extension at 72 °C. The 203- and 124-bp PCR products amplified from the bacterial isolates which appeared as a single band were purified using a High Pure PCR Product Purification Kit (Roche Applied Science, Germany) and sequenced on an ABI Prism 377 automatic sequencer (Applied Biosystems, CA, USA). Sequence homologies were examined using BLAST version 2.2.12 of the National Center for Biotechnology Information [19]. Multiple sequence alignments were carried out using ClastalW, and a consensus neighbor-joining tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software (version 4.0) [20].
Appl Biochem Biotechnol
Extraction of Biosurfactant and Physical Properties The produced biosurfactant was extracted from the culture broth with the method of acid precipitation followed by solvent extraction reported elsewhere [7]. Briefly, the culture broth was centrifuged (6,000×g, 10 min, 4 °C) to remove the cells. Then, pH was adjusted to 2 by 3 N HCl in order to precipitate the biosurfactant. After keeping it overnight, the crude biosurfactant was obtained by centrifugation (18,000×g, 30 min, 4 °C). The crude biosurfactant was purified using several times extraction by ethyl acetate. Physical and surface activity properties of the biosurfactant were determined in terms of surface tension (ST), interfacial tension (IFT), critical micelle concentration (CMC), oil spreading test (OST), and emulsion index (E24). ST and IFT were measured using a Krüss K100MK2 tensiometer (Krüss GmbH, Hamburg, Germany) using Wilhelm plate technique. The tensiometer was calibrated against double-distilled water. Before each measurement, the probe was rinsed several times with double-distilled water and acetone followed by flaming on a Bunsen burner. Interfacial tension was performed between water and three hydrophobes including hexadecane, isooctane, and cyclohexane. The CMC is a widely used index to evaluate surface activity. The crude biosurfactant was dissolved in distilled water, and the surface tension of the solution was measured with various concentrations of biosurfactant at 25 °C. CMC was measured from the breakpoint of surface tension versus biosurfactant concentration. Stabilization was allowed to occur until the standard deviation of five successive measurements was less than 0.9 mN/m. Each result was the average of at least five determinations after stabilization. The E24 of culture samples was determined by adding 6 mL of a hydrocarbon to 4 mL of culture, mixing with a vortex for 2 min, and leaving it standing for 24 h. The E24 index is given as the percentage of the height of the emulsified layer (mm) divided by the total height of the liquid column (mm) [21]. OST was performed according to the standard procedure [22]. The diameter of the clear zone after dropping 10 μL of culture broth on the surface of the oil was considered as a response. Growth Kinetics and Biosurfactant Production A 4 % cell suspension (in physiological saline) from an overnight seed culture medium was inoculated in 2,000-mL flasks containing 500 mL of medium and incubated at 45 °C while shaking at 200 rpm. Sunflower oil was used as carbon source. During fermentation, samples were aseptically retrieved from the liquid culture at different time intervals to monitor the kinetic parameters including cell growth, biosurfactant concentration, surface activity (diameter of clear zone (cm)), and pH of culture medium. Bacterial growth was determined by measuring the optical density of the culture broth at 600 nm with a UV spectrophotometer (PerkinElmer, Lambda 25, USA). Effect of Different Carbon Sources on the Surface Activity Various carbon sources from different origins including carbohydrates, hydrocarbons, and vegetable oils were used for evaluating the surface activity of the produced biosurfactant in terms of OST and E24. Characterization of Dried Biosurfactant Thin-layer chromatography (TLC) is a simple method for primary characterization of biosurfactants. TLC was applied using the solvent system of chloroform/methanol/water
Appl Biochem Biotechnol
(65:15:4v/v). Ten microliters of sample was put on the TLC plate (Silicagel 60, Merck, Germany) and developed with the solvent. After development, one of the plates was put into a jar saturated with iodine vapors to detect lipids [23]. Another plate was sprayed evenly with the ninhydrin reagent (0.5 g ninhydrin (Sigma-Aldrich) in 100 mL anhydrous acetone) and placed in an oven at 110 °C for 10 min to detect the presence of peptide as red spots [24]. The purified biosurfactant was analyzed using Fourier transform infrared (FTIR) spectroscopy with a PerkinElmer Spectrometer (FTIR GX 2000). Biosurfactant samples were dried first and then ground before performing FTIR analysis. The dried powder was finely mixed with a KBr matrix (Sigma). The spectra were in the range of 400–4,000/cm with a resolution of 8/cm. The biosurfactant was subjected to further analysis with nuclear magnetic resonance (NMR). 1H spectra were measured at 25 °C using a Bruker JNM-A500 spectrometer (Germany) equipped with a triple-resonance (1H, 13C, 15N) inverse probe with 5-mmdiameter tubes containing 600 μL of sample, with deuterated chloroform as a solvent. One hundred twenty-eight scans were collected (90° pulse, 7.3 μs; saturation pulse, 3 s; relaxation delay, 3 s; acquisition time, 4.679 s; 65,536 data points). A 1-Hz exponential line-broadening filter was applied before Fourier transformation, and a baseline correction was performed on spectra before integration using Bruker software (TopSpin 2.0). Biochemical Composition Analysis The carbohydrate content of the produced biosurfactant was determined by the method of phenol–sulfuric acid presented by Dubois et al. [25] using D-glucose as a standard. Briefly, a volume of 0.5 mL of cell supernatant was mixed with 0.5 mL of 5 % phenol solution and 2.5 mL of sulfuric acid and incubated for 15 min before measuring the absorbance at 490 nm. Lipid content was estimated according to Folch et al. [26]. The biosurfactant sample, 0.2 g, was blended with the chloroform–methanol mixture (2:1) and agitated acutely. Solvent phase was recovered by centrifuging at 10,000 rpm for 15 min. The extraction process was carried out three times. The whole solvent was collected, evaporated, and dried under vacuum. The lipid content was determined by gravimetric estimation. Protein content was determined by the method of Bradford [27] using Coomassie brilliant blue with bovine serum albumin as a standard. The absorbance of unknown samples was measured at 595 nm. Stability of Biosurfactant To evaluate the stability of the biosurfactant at different environmental conditions, 50 mg/mL solution of the biosurfactant was prepared and maintained at a temperature range of 20–90 °C for 120 h. After cooling to room temperature, ST was measured as an activity indicator of the biosurfactant. To determine the effect of pH on the biosurfactant activity, the ST was measured after adjusting the pH between 3 and 12 using 3 N NaOH or 3 N HCl. The effect of the addition of different concentrations of NaCl on the activity of the biosurfactant was also studied. Experimental Design for Optimization of Culture Condition RSM is a combination of mathematical and statistical techniques used for developing, improving, and optimizing the processes [28]. RSM was applied to study the effects of the various essential nutrients, namely sunflower oil, sodium nitrate, and yeast extract. This
Appl Biochem Biotechnol
procedure was carried out to optimize the process conditions for the maximum production of the biosurfactant from A. thermoaerophilus MK01. In order to obtain the mutual interactions between the variables and to optimize these variables, a 23 factorial design based on central composite design (CCD) [28] with six replicates at the center points using a total number of 20 experiments was employed. A statistical program package, Minitab software version 16, was used for regression analysis of the data obtained and to estimate the coefficient of the regression equation. The range and the levels of the factors, which were varied according to the experimental design, are given in Table 1. The range of variables was chosen based on our earlier investigations for biosurfactant production in shake flasks. The statistical combinations of the critical media components along with the maximum biosurfactant concentrations in the broth are detailed in Table 2. The predicted biosurfactant concentration was calculated using the following second-order polynomial equation: Y ¼ β 0 þ β 1 A þ β 2 B þ β 3 C þ β 11 A A þ β 22 B B þ β 33 C C þ β 12 A B
ð1Þ
þ β 13 A C þ β 23 B C
where Y is the predicted response; β0 is the intercept; β1, β2, and β3 are linear coefficients; β11, β22, and β33 are squared coefficients; β12, β13, and β23 are interaction coefficients; and A, B, and C are independent variables. By using this equation, it is possible to evaluate the linear, quadratic, and interactive effects of the independent variables on the response appropriately. The significance of each coefficient was determined by the F factor and p values. The larger the magnitude of the F value and the smaller the p value, the more significant is the corresponding coefficient. Multiple correlation coefficient (R2) and adjusted R2 were used as quality indicators to evaluate the fitness of the second-order polynomial equation. The optimized amount of each factor was estimated using the Response Optimizer option of the Minitab software.
Results and Discussion Isolation and Characterization of Biosurfactant-Producing Isolate Among numerous bacterial strains, which were isolated from urban wastes and tested for biosurfactant production, one isolate with the best confirmatory results in OST, hemolytic activity, and emulsification activity was selected. Morphological and biochemical characteristics of the selected isolate showed that the selected isolate MK01 was Gram-positive, long, catalase-positive, and citrate utilization-, indole production-, and Voges–Proskauer reactionnegative. Results from 16S rRNA gene sequencing identified our strain as A. thermoaerophilus Table 1 Experimental range and levels of the independent variables Symbol
Factors
Levels −α
−1
0
1
+α
A
Sunflower oil (g/L)
11.59
15
20
25
28.4
B
Sodium nitrate (g/L)
2.16
2.5
3
3.5
3.84
C
Yeast extract (g/L)
0.65
1
1.5
2
2.34
Appl Biochem Biotechnol Table 2 Full factorial central composite design matrix of three variables in coded and actual units along with the observed responses Run order
1
Sunflower oil (g/L)
Sodium nitrate (g/L)
Yeast extract (g/L)
Coded value
Actual value
Coded value
Coded value
−1
Actual value
Actual value
Biosurfactant production (g/L)
15
1
3.5
1
2
4.75
2
1.68
28.409
0
3
0
1.5
6.4
3
0
20
0
3
0.65
4.05
4
1
25
−1
5
0
20
6
−1.68
−1.68
2.5
1
2
6.05
0
3
0
1.5
5.05
11.591
0
3
0
1.5
4.5
7
0
20
1.68
3.8409
0
1.5
5.45
8 9
−1 1
15 25
−1 1
2 1
4.5 6.1
10
0
20
0
3
1.68
2.3409
4.55
11
0
20
0
3
0
1.5
5.16
12
1
25
−1
2.5
−1
1
4.5
13
1
25
1
3.5
1
2
5.67
14
0
20
0
3
0
1.5
5.76
15
0
20
0
3
0
1.5
5.35
16 17
0 −1
20 15
2.1591 2.5
0 −1
1.5 1
4.35 3.67
−1
−1.68 −1
2.5 3.5
1 −1
18
−1
15
1
3.5
1
4.76
19
0
20
0
3
0
1.5
5.05
20
0
20
0
3
0
1.5
5.45
with 100 % similarity to A. thermoaerophilus strain L420-9 (Fig. 1). We tentatively labeled our strain A. thermoaerophilus MK01 and deposited it in GenBank with the accession number KF923810. Growth Kinetics of Biosurfactant Production A. thermoaerophilus MK01 was cultivated on sunflower oil as the sole carbon source. Figure 2 shows the pattern of growth kinetics and biosurfactant production. The results showed that biosurfactant production started early in the exponential growth phase and was considered to be predominantly parallel with the bacterial growth. The MK01 strain was able to synthesize up to 2 g/L of biosurfactant after 24 h of cultivation. The production of the biosurfactant was continued up to 96 h, and the maximum production of about 5 g/L was obtained after 120 h. The diameter of the clear zone (in the oil spreading test) was also increased parallel to the biosurfactant production. This observation is rational because the diameter of the clear zone has a direct correlation with biosurfactant concentration [21]. The crude biosurfactant reduces the surface tension of water from 72 to about 43 mN/m (Fig. 3). In a certain concentration, any further increase in the concentration of the biosurfactant was not accompanied by a decrease in surface tension. This concentration corresponds to the point where the biosurfactant first shows a stable low surface tension value
Appl Biochem Biotechnol
Fig. 1 Phylogenetic tree of Aneurinibacillus thermoaerophilus MK01. Numbers at the nodes indicate the bootstrap values on neighbor-joining analysis
termed CMC. Therefore, CMC was measured from the breakpoint of two lines as 1.21 mg/mL (Fig. 3). The solution of biosurfactant in water could also reduce the IFT between water and hexadecane from 41 to 19 mN/m, water and isooctane from 33 to 10 mN/m, and water and cyclohexane from 47 to 21 mN/m.
Fig. 2 Time course profile of biosurfactant production, cell growth, OST, and pH of Aneurinibacillus thermoaerophilus MK01 grown on sunflower oil as sole carbon source
Appl Biochem Biotechnol 70
65
ST (mN/m)
60
55
50
45
40
35 0
1
2
3
4
5
6
7
BS Concentration (mg/ml) Fig. 3 Variation of the surface tension (ST) of the biosurfactant versus concentration for the determination of CMC
Effect of Different Carbon Sources on Surface Activity The isolate was able to grow on the various carbon sources examined. However, the emulsification activity and diameter of the clear zone varied significantly. Figure 4 shows the effect of different carbon sources on the surface activity of the produced biosurfactant in terms of OST and E24. As shown, water-insoluble substrates were more effective in biosurfactant production. Glucose was the most effective amongst water-soluble carbon substrates with emulsification activity of more than 40 %. A similar observation has been reported by Abbasi et al. [29] on the production of rhamnolipids. Although strain MK01 grown on sucrose, maltose, and glycerol showed low emulsification activity, the clear zone diameter was zero, showing that the biosurfactant production on these carbon sources is insignificant. Maximum clear zone diameters were obtained on corn oil, canola oil, and sunflower oil. Therefore, it can be concluded that these carbon sources are more efficient in the production of biosurfactant by A. thermoaerophilus MK01. Overall, results showed that among all examined carbon substrates, vegetable oils were the most effective carbon sources in biosurfactant production. Characterization of Biosurfactant Primary characterization of the produced biosurfactant was carried out using TLC. After development, the plate that was in contact with iodine vapor showed a yellow spot indicating the presence of polar lipids. Treatment with ninhydrin solution revealed the presence of peptide as indicated by a red spot (Fig. 5a). This suggests the lipopeptide structure for the produced biosurfactant. Figure 5b shows the IR spectrum of the purified biosurfactant. As can be seen, characteristic absorption bands corresponding to functional groups characteristically forming part of the lipopeptide could be observed for the purified biosurfactant sample. The stretching
Appl Biochem Biotechnol 140 OST E24%
E24%, OST * 10 (cm)
120
100
80
60
40
20
0
se se se se se rol la ve er an rn nd e ut oil ne en en co no cro lto cto ce ano oli flow y be co lmo sam con ude eca tolu xyl n a se co cr ad glu ma su ma la gly c su so x he
carbon source
Fig. 4 Effect of various carbon sources on the surface activity of the biosurfactant
bands of the methylene and terminal methyl groups of the acyl chains between 2,850 and 2,930/cm and an asymmetric stretching strong peak which occurred at 1,745/cm suggest the presence of carboxyl groups (C=O in COOH) in the biosurfactant. Protein-related bonds, the vC=O amide I (1,646/cm) and δ NH/vC=O combination of the amide II bands (1,540–60/cm), were also observed for the sample. The absorption peaks at 1,250 and 1,064/cm could indicate the C–O stretching in ether or alcohol and methoxyl groups, respectively, although other groups also absorb in this region. Results obtained from 1H-NMR indicated that the produced biosurfactant has a lipopeptide structure. Almost all the backbone amide NH groups are in the region from 8.2 to 7.2 ppm downfield from tetramethylsilane. Alpha hydrogens of the amino acids come into resonance from 5.2 to 4 ppm for peptide moiety along with the fatty acid functional group as a single methyl-related peak (δ 0.8–0.88), –(CH2)n– (δ 1.1–1.45), C–CH2–CO (δ 1.6), and –C–OH (δ 2.2 ppm). An additional weak signal was also detected at δ 5.3 consistent with CHO of the alcohol moiety of an ester (Fig. 6). Biochemical composition analysis revealed that the produced biosurfactant is composed of 13 % peptide, 74 % lipid, and 3 % carbohydrate. The presence of a minor fraction of carbohydrate in the purified sample possibly arising from the residual cell debris in the broth coprecipitated with the biosurfactant during its extraction process. Biosurfactant Stability The biosurfactant produced by A. thermoaerophilus MK01 was shown to be thermostable (Fig. S1). Heating of the biosurfactant up to 90 °C caused no significant effect on the biosurfactant performance. Therefore, it can be concluded that the MK01 biosurfactant preserves its surface properties unaffected in the wide range of temperatures between 20 and 90 °C. This activity indicated the usefulness of the MK01 biosurfactant in food, pharmaceutical, and cosmetics industries as well as its application in high-temperature bioremediation.
Appl Biochem Biotechnol
(a)
Analytical TLC of MK01 biosurfactant. TLC was carried out on silica gel plate
using chloroform: methanol: water (65:15:4 v/v) as mobile phase. The plates were stained with ninhydrin (left) and iodine vapor (right).
1.2
1.0
Transmittance (%)
0.8 1556
1646
0.6
0.4 2854 1460
2925
1745
0.2
1160
0.0 3300
3000
2700
2400
2100
1800
1500
1200
900
600
-1 Wavenumber (cm )
(b)
FTIR spectrum of biosurfactant produced by A. thermoaerophilus MK01.
Fig. 5 a Analytical TLC of the MK01 biosurfactant. TLC was carried out on a silica gel plate using chloroform/ methanol/water (65:15:4v/v) as mobile phase. The plates were stained with ninhydrin (left) and iodine vapor (right). b FTIR spectrum of the biosurfactant produced by A. thermoaerophilus MK01
Recently, it was reported that bioremediation in the presence of biosurfactant at high temperatures can lead to better results [30]. The surface activity of the crude biosurfactant remained relatively stable over pH range 5–10. Optimum stability of biosurfactant was observed at NaCl concentrations below 10 % (Fig. S1).
Appl Biochem Biotechnol
Fig. 6
1
H-NMR pattern of the biosurfactant produced by A. thermoaerophilus MK01
Response Surface Optimization Results The CCD design along with the corresponding experimental values of the biosurfactant production is given in Table 2. Analysis of variance (ANOVA) of the model and the corresponding p values, along with the parameter estimate for the biosurfactant production, are shown in Table 3. The ANOVA of the quadratic regression model indicated that the model was highly significant, as the F value for the model was 16.23 (p
