Interaction of the Biosurfactant, Surfactin with Betaines in Aqueous

Article pubs.acs.org/Langmuir

Interaction of the Biosurfactant, Surfactin with Betaines in Aqueous Solution Fang Liu,† Jingwen Xiao,† Vasil M. Garamus,§ László Almásy,‡ Regine Willumeit,§ Bozhong Mu,† and Aihua Zou*,† †

Shanghai Key Laboratory of Functional Materials Chemistry and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China ‡ Wigner Research Centre for Physics, Institute for Solid State Physics and Optics, Budapest 1525 POB 49, 11 Hungary § Helmholtz-Zentrum Geesthacht: Centre for Materials and Coast Research, Institute of Materials Research, Max-Planck-Str. 1, D-21502 Geesthacht, Germany ABSTRACT: The interactions between the lipopeptide Surfactin and four betaines, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SDDAB), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (STDAB), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SHDAB), and N-dodecyl-N,N-dimethyl-2-ammonio-acetate (C12BE) are studied by surface tension and small-angle neutron scattering (SANS). SDDAB, STDAB, and SHDAB have the same headgroup but different hydrophobic chains. C12BE has different headgroup but the same hydrophobic chain with SDDAB. According to the interfacial parameters calculated from surface tension, the synergism between Surfactin and betaine is relevant with the molecule structure of betaine and the mole ratio of them. For betaines, the optimum alkyl chain length (STDAB) and long enough separation between positive charge and negative charge in headgroup are responsible for highest synergetic interaction with Surfactin. The aggregates of individual Surfactin and the mixtures of Surfactin and sulfopropyl betaines are predicted to be spherical based on the packing parameter (pp) and the average packing parameter (Pav), which is in close qualitative agreement with SANS data analysis, while Surfactin/C12BE forms ellipsoidal micelles due to the smaller headgroup of C12BE. electrostatic attraction between the −N+(CH3)2 group in zwitterionic surfactant and the polar head in anionic surfactant is responsible for this synergistic effect.8 Surfactin consists of a cyclic heptapeptide (Glu-Leu-D-LeuVal-Asp-D-Leu-Leu) and the carbon number of the β-hydroxyl fatty acid moiety varies from 12 to 17.9−11 It is less toxic and easily biodegrades, and has powerful foaming and emulsification ability, thus making it a potential target for using in microbial enhanced oil recovery, bioremediation of the environmental pollutant and pharmacological research.12 It is an extensively studied and used anionic biosurfactant.13−16 In our previous work,17 we have investigated the effects of counterions on Surfactin-C16 micelle solution with its critical micelle concentration (CMC), microenvironment properties in micelles, micelle size distribution, and morphology. Counterions enhanced the surface activity of Surfactin-C16 and reduced the CMC. With the micellization of Surfactin-C16, it adopted a β-sheet conformation, and univalent counterions reduced micelle micropolarity, increased micelle microviscosity, and tended to cause formation of small and spherical micelles, while divalent counterions had a different effect. At low concentration

1. INTRODUCTION The various applications of surfactants, such as home and personal care products, usually involve mixtures of different surfactants. The mixtures of surfactants have attracted much more interest in recent years since mixtures provide a synergistic enhancement of performance and functionality which cannot take place in single surfactant systems.1−3 The adsorption behavior, micellar morphology, and rheological properties in mixed surfactants solution are usually different from that in a single surfactant system. Zwitterionic surfactant, containing both positive and negative charge groups, exhibit unique properties, such as mild functionality, low toxicity, high foam stability, excellent surface tension reducing properties, biodegradability and so on.4,5 Zwitterionic surfactants show a strong interaction with anionic surfactant in aqueous solution, thus the surface tension, electrical conductivity and viscoelastic properties will be altered.6 It is interesting and useful to design mixed zwitterionic/anionic surfactants system in which the advantages of either surfactant can be taken. Li et al. investigated the interaction between sodium dodecyl sulfonate (C12AS) and three alkyldimethylammoniopropanesulfonates of different alkyl chain lengths.7 The interactions between C12AS and alkyldimethylammoniopropanesulfonates became stronger as the alkyl chain length increases. It was revealed that the © 2013 American Chemical Society

Received: February 22, 2013 Revised: June 18, 2013 Published: July 18, 2013 10648

dx.doi.org/10.1021/la400683u | Langmuir 2013, 29, 10648−10657

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Figure 1. The chemical structure of Surfactin, which has the −COOH groups in L-Glu1 and L-Asp5.

pD = 0.929 pH meter reading + 0.42

of divalent cations, they showed strong interaction with Surfactin-C16 micelles and lead to formation of large micelle aggregates. In the present work, a systematic study of the interaction between Surfactin and different betaines at air/aqueous interface and in solution is investigated by surface tension and SANS, then the results are further analyzed according to regular solution theory (RST), aiming to provide a thorough physicochemical characterization of mixtures of Surfactin and conventional zwitterionic surfactants allowing to optimize formulations and product performance.

(1)

2.2. Methods. 2.2.1. Surface Tension. The surface tension was measured at 25 °C by liquid measuring apparatus Dataphysics (DCAT21, Germany) using a Wilhelmy small platinum plate of 4 cm perimeter. The plate was first rinsed by deionized water and then burned to red before each measurement. The surface tension of deionized water was measured (72 ± 0.2 mN/m) at the beginning to check the instrument. The surface tension for aqueous solutions of pure Surfactin, SDDAB, STDAB, SHDAB, C12BE, and their mixtures of a wide range of mole fractions of Surfactin were measured, respectively, in order to get parameters like the CMC, the saturation adsorption value (Γmax) for each of them. Surface tension was measured three times and plotted as a function of the Surfactin concentration. The turning point in the plot corresponds to the CMC value. 2.2.2. Small-Angle Neutron Scattering, SANS. SANS measurements were performed on the Yellow Submarine instrument at the BNC in Budapest (Hungary).21 The overall range of scattering vectors q is from 0.09 to 3 nm −1. The samples were kept in Hellma quartz cells with a path length of 5 mm and placed in a T = 25.0 ± 0.5 °C thermostatic holder. The raw scattering patterns were corrected for sample transmission, background, and sample cell scattering.22 The 2dimensional scattering patterns were azimuthally averaged, converted to an absolute scale, and corrected for detector efficiency dividing by the incoherent scattering spectra of pure H2O, which was measured in a 1 mm path length quartz cell. The scattering from PBS buffer prepared in D2O was subtracted as the background.

2. MATERIALS AND METHODS 2.1. Materials. Surfactin was produced by Bacillus subtilis TD7 cultured in laboratory of East China University of Science and Technology.11,18 Surfactin-C15 isoform (Figure 1) was separated by extraction with anhydrous ether, isolated with normal pressure ODS C18 column and purified by the RP-HPLC (Jasco, Japan). Then the structure of the isolated lipopeptide was determined by the electrospray ionization-time-of-flight mass spectrometer (ESI-TOF MS/MS) and GC/MS. Betaines: SDDAB, STDAB, and SHDAB were purchased from Nanjing Robiot Co, Ltd., C12BE was from Zhixin Chemical Co. Ltd.. All of them were with over 99% purity and used as received. D2O (99.9% D): The deuterium oxide was 99.9% D2O from Sigma and used as received.

3. RESULTS AND DISCUSSION 3.1. Micellar and Interfacial Adsorption Parameters. The surface tensions for aqueous solutions of individual Surfactin, SDDAB, STDAB, SHDAB, C12BE, and their mixtures at different mole fractions of Surfactin (αSurfactin = 0.10, 0.33, 0.50, 0.67, 0.75, 0.80) are measured. The results are shown in Figure 3. Each surface tension-c curve shows a marked change when Surfactin is added. With the increasing mole fraction of Surfactin, the surface tension decreases and gradually approaches to that of Surfactin. The CMC values for each surfactant and mixtures are recorded in Table 1. The CMC values of SDDAB, STDAB, and SHDAB are 1.61, 0.34, and 2.95 × 10−2 mM, respectively, indicating that every two more carbon atoms added on hydrophobic chain will reduce the CMC by 1 order of magnitude in case the hydrophilic group is sulfopropyl. As to the CMC of SDDAB and C12BE, two of which have the same hydrophobic chain but different hydrophilic groups, from Table 1, it can be seen that

Figure 2. The chemical structures of sulfopropyl betaines, SDDAB (n = 11), STDAB (n = 13), SHDAB (n = 15), and carboxyl betaine C12BE. For the surface tension measurements, all the samples were prepared with 10 mM phosphate buffer solution (PBS, pH 7.4). Doubly distilled water was used throughout the PBS preparation. For SANS experiments, the samples were prepared with 10 mM deuterium phosphate buffer solution (pD 7.4). The pD value was from pH-meter readings of equivalent solutions in D2O which was mediated by eq 1, with the improvements made by Krezel et al.19,20 10649


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Figure 3. The γ-c curves of mixed surfactant systems at different mole fractions of Surfactin: (a) Surfactin/SDDAB; (b) Surfactin/STDAB; (c) Surfactin/SHDAB; and (d) Surfactin/C12BE.

aqueous interface. The surface excess (Γmax) is a measurement of the effectiveness of surfactant adsorption.28,29 It can be calculated from eq 2:30

the CMC value of SDDAB is higher than that of C12BE since the sulfopropyl group in SDDAB is more hydrophilic than the carboxyl group in C12BE. Moreover, the length of bridge chain, which connects positive charge and negative charge, is different in SDDAB and C12BE either. SDDAB has two more methylenes than C12BE on the bridge chain, which leads to increase of the dipole−dipole repulsion between the head groups, causing the increase of CMC.23−25 Hence, SDDAB has a higher CMC value than C12BE. As to the CMC values of the mixtures, Surfactin/SDDAB is smaller than Surfactin/C12BE with the increased molar ratio of Surfactin. There exists electrostatic attraction between the positive charge of betaine and the negative charge of Surfactin, while at the same time, there exists electrostatic repulsion between the negative charge of betaine and the negative charge of Surfactin. However, from the viewpoint of net charge, the whole C12BE molecule will exist as anion form in the solution (PBS, pH 7.4) because the pI value is 4.9,26 so there will be strong electrostatic repulsion between C12BE and Surfactin. While for SDDAB, it does not exhibit proton accepting or releasing ability in almost all of the pH range, and always exist as salt or zwitterionic form, so it has only one ionic form.27 Therefore, there exist electrostatic repulsion but also electrostatic attraction between SDDAB and Surfactin. Besides, the longer bridge chain makes SDDAB “soft and flexible”, thus the electrostatic attraction and hydrogen bonds between SDDAB and Surfactin will be stronger than that in C12BE. As a result, SDDAB has a stronger interaction with Surfactin. Consequently, the CMC values of Surfactin/SDDAB systems are smaller than those of Surfactin/C12BE with the increased molar ratio of Surfactin. 3.2. Interfacial Parameters at Air/Aqueous Interface. We present in Table 1 the interfacial adsorption parameters of individual surfactant and Surfactin/betaine solutions at air/

Γmax = −

⎛ ∂γ ⎞ 1 ⎟ × 1010mol/cm 2 σ ⎜ 2.303RT (1 + 2X1 ) ⎝ ∂log C ⎠ (2)

Xσ1

where (∂γ/∂ LogC), R, T, NA, and are the slope of the curve of surface tension versus the logarithm of surfactant concentration, ideal gas constant, temperature, Avogadro’s number, and monolayer composition of Surfactin. The average minimum area per molecule (Amin) is evaluated by equation: A min = (NA Γmax)−1 × 1016Å2

(3)

As seen from Table 1, the Amin value of Surfactin is larger than that of betaines because Surfactin has a larger peptide ring as headgroup. The Amin value of C12BE is smaller than sulfopropyl betaine mainly because the hydrophilic headgroup of C12BE is smaller than the headgroup of sulfopropyl betaine. When betaine is added into the Surfactin solution, the electrostatic attraction between the positive charge of betaine and the negative charge of Surfactin, together with the hydrophobic interaction and hydrogen bonds will make the surfactant molecules arranged closer at the interface (see in Figure 4), so the Amin values of Surfactin/sulfopropyl betaine decrease with the increasing hydrophobic chain length. The arrangements of Surfactin and betaine at air/aqueous interface are not close enough due to the large head ring of Surfactin. As a result, the Amin values of Surfactin/betaine systems increase with the increasing mole fraction of Surfactin. The Amin values of Surfactin/C12BE mixtures are higher than that of Surfactin/SDDAB. This can be attributed to the shorter 10650


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Table 1. CMC Values of Mixed Surfactin/SDDAB, Surfactin/STDAB, Surfactin/SHDAB, and Surfactin/C12BE Solutions with Different Mole Ratio of Surfactin αSurfactin

CMCexp/mM (±0.03)

0 0.10 0.33 0.50 0.67 0.75 0.80 1

1.61 8.22 2.36 1.54 1.24 1.13 9.48 1.03

× × × × × × ×

10−2 10−2 10−2 10−2 10−2 10−3 10−2

0 0.10 0.33 0.50 0.67 0.75 0.80 1

0.34 5.38 2.65 1.29 1.12 9.51 1.06 1.03

× × × × × × ×

10−2 10−2 10−2 10−2 10−3 10−2 10−2

0 0.10 0.33 0.50 0.67 0.75 0.80 1

2.95 2.20 1.20 8.96 9.55 1.07 1.11 1.03

× × × × × × × ×

10−2 10−2 10−2 10−3 10−3 10−2 10−2 10−2

0 0.10 0.33 0.50 0.67 0.75 0.80 1

1.27 6.80 2.46 1.80 1.35 1.24 1.18 1.03

× × × × × × ×

10−2 10−2 10−2 10−2 10−2 10−2 10−2

CMCideal/mM (±0.03) system: Surfactin/SDDAB 1.61 9.74 × 10−2 3.08 × 10−2 2.05 × 10−2 1.53 × 10−2 1.37 × 10−2 1.29 × 10−2 1.03 × 10−2 system: Surfactin/STDAB 0.34 8.10 × 10−2 2.94 × 10−2 2.00 × 10−2 1.51 × 10−2 1.36 × 10−2 1.28 × 10−2 1.03 × 10−2 system: Surfactin/SHDAB 2.95 × 10−2 2.49 × 10−2 1.83 × 10−2 1.53 × 10−2 1.31 × 10−2 1.23 × 10−2 1.18 × 10−2 1.03 × 10−2 system: Surfactin/C12BE 1.27 9.60 × 10−2 3.07 × 10−2 2.04 × 10−2 1.53 × 10−2 1.37 × 10−2 1.28 × 10−2 1.03 × 10−2

Γmax × 1010/mol/cm2 (±0.27)

Amin/Å2 (±6.50)

0.98 1.63 1.33 1.51 1.78 1.50 2.34 1.47

56.61 70.45 82.22 92.17 87.09 101.09 60.87 112.85

1.05 1.72 1.62 1.64 2.00 1.65 2.01 1.47

52.83 59.47 78.07 83.77 73.86 81.13 92.27 112.85

1.33 1.71 2.49 1.64 2.05 1.67 1.69 1.47

41.50 53.46 46.28 76.84 69.27 90.35 91.21 112.85

1.56 1.89 2.08 1.44 2.02 1.28 1.64 1.47

35.58 56.21 73.61 96.26 85.80 114.72 88.80 112.85

3.3. Interfacial Parameters and Packing Parameter. To obtain ideal CMC values for the mixed surfactants systems, the regular solution theory (RST) is employed. The eq 4 below can be used to calculate the ideal CMC for the mixture (CMC12).31 α1 (1 − α1) 1 = + CMC12 f1 CMC1 f2 CMC2

(4)

where 1 and 2 refers to Surfactin and betaine respectively. α1 is the mole fraction of Surfactin in solution, f1 and f 2 are the activity coefficients of Surfactin and betaine in mixed micelles (for ideal mixing f1 = f 2 = 1). Figure 5 shows that the CMC values of mixtures decrease when the mole fraction of Surfactin increases and the experimental CMC are always smaller than the ideal CMC (expected values for ideal mixing). This indicates that there might be synergism pointing to the formation of mixed micelles in the Surfactin/betaine solutions. The synergism should arise from the screening of the electrostatic repulsion between the negatively charged head groups of Surfactin by inserting zwitterionic head groups. A smaller number of counterions are bound and there is entropy gaining in the formation of mixed micelles. The synergistic effect is also supported by the negative

Figure 4. Sketch of the mixed surfactants system at the air/aqueous interface and in the bulk solution.

carbon bridge connecting the positive and negative charge centers of C12BE, thus its negative charge center will overlap the positive charge center. Then the attraction of C12BE to the negative charge of the Surfactin molecule is not that strong as compared with SDDAB at air/aqueous interface. 10651


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Figure 5. Variation of experimental (filled circle) and ideal (empty circle) CMC12 values for mixed systems as a function of the mole fraction of Surfactin (αSurfactin) in the mixed solutions: (a) Surfactin/SDDAB; (b) Surfactin/STDAB; (c) Surfactin/SHDAB; and (d) Surfactin/C12BE.

values of Rubingh’s interaction parameter (βm),32 which is related to the strength of interaction between two surfactants in mixed micelles. A negative βm value indicates that the attraction between different types of surfactants is stronger than that between the same surfactants. The existence of synergism in binary surfactants mixtures has been shown to depend on not only the strength of intermolecular interaction (measured by the values of βm parameter), but also the relevant properties of the individual component of the mixture.33 The necessary conditions for existence of synergism in mixture are as follows: m (a) β should be negative; (b) | βm | > | ln(Cm 1 /C2 ) |. The values of βm and Xm can be calculated based on RST using the 1 numerical solution of eqs 5 and 6. m (X1m)2 ln[(α1C12 /X1mC1m)⎤⎦ m (1 − X1m)2 ln[(1 − α1)C12 /(1 − X1m)C2m]

βm =

Table 2. Molecular Interaction and Synergism Parameters for Surfactin/SDDAB, Surfactin/STDAB, Surfactin/SHDAB, and Surfactin/C12BE Mixtures in Solution

=1 (5)

m ln(α1C12 /X1mC1m)

(1 − X1m)2

(6)

where α1 is the mole fraction of Surfactin in solution, Xm 1 is the m m mole fraction of Surfactin in the mixed micelle. Cm 1 , C2 , and C12 is the molar concentrations of pure Surfactin, pure betaines and mixed surfactants in solution, respectively. m m In Table 2, the values of βm, Xm 1 , and | ln(C1 /C2 ) | are presented for solutions of different compositions. It can be seen that all βm values are negative. To meet the condition of | βm | > m | ln(Cm 1 /C2 ) |, in Surfactin/SDDAB, Surfactin/STDAB and Surfactin/SHDAB systems, synergism arises when αSurfactin ≥0.67, αSurfactin ≥0.50, and 0.33≤αSurfactin ≤ 0.67, respectively. This indicates that the hydrophobic interaction between Surfactin and sulfopropyl betaine gets stronger with the increasing length of the carbon chain. However, as an exception 10652

α1

βm

0.1 0.33 0.5 0.67 0.75 0.8

−1.81 −3.98 −4.91 −5.12 −5.36 −6.67

0.1 0.33 0.5 0.67 0.75 0.8

−2.02 −1.24 −4.16 −4.09 −4.91 −3.92

0.1 0.33 0.5 0.67 0.75 0.80

−0.62 −1.71 −2.42 −1.87 −1.12 −0.70

0.1 0.33 0.5 0.67 0.75 0.8

−2.71 −3.40 −3.24 −4.52 −4.58 −4.65

X1m

Xideal

system: Surfactin/SDDAB 0.8372 0.9456 0.8387 0.9872 0.8433 0.9936 0.8744 0.9969 0.8842 0.9979 0.8521 0.9984 system: Surfactin/STDAB 0.6599 0.7867 0.8677 0.9424 0.7742 0.9708 0.8254 0.9854 0.8167 0.9901 0.8752 0.9925 system: Surfactin/SHDAB 0.2921 0.2414 0.5466 0.5852 0.6184 0.7412 0.7196 0.8533 0.8109 0.8957 0.8721 0.9197 system: Surfactin/C12BE 0.7658 0.9320 0.8504 0.9838 0.9016 0.9920 0.9105 0.9960 0.9259 0.9973 0.9348 0.9980

| ln(C1m/C2m) | 5.05

3.50

1.24

4.81


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for SHDAB, there is no synergism between SHDAB and Surfactin when αSurfactin ≥ 0.75. This can be attributed to that the hydrophobic chain of SHDAB is so long that it is much easier to be self-aggregated. As reported before,10 Surfactin is also prone to be self-aggregated due to the strong intramolecular hydrogen bond. There is no synergism between Surfactin and C12BE for all of the measured compositions, which is consistent with the Amin values and can be explained by good screening of charge in C12BE headgroup. The mixed micellar regular solution theory can be extended to the mixed monolayer. From the analogy with the derivation of Rubingh equation, the mole fraction of surfactant 1, α1, in solution is related to its mole fraction in mixed monolayer, X1σ, by the equation, (1 −

0 (X1σ )2 ln⎡⎣(α1C12 /X1σ C10)⎤⎦ σ 2 0 X1 ) ln⎡⎣(1 − α1)C12 /(1 − X1σ )C20⎤⎦

βσ =

C01,

0 ln(α1C12 /X1σ C10) (1 − X1σ )2

C02,

Table 3. Molecular Interaction and Synergism Parameters for Surfactin/SDDAB, Surfactin/STDAB, Surfactin/SHDAB and Surfactin/C12BE Mixtures at the Interface

0.1 0.33 0.5 0.67 0.75 0.8 0.1 0.33 0.5 0.67 0.75 0.8

=1 (7)

(8)

0.1 0.33 0.5 0.67 0.75 0.8

C012

where and are the molar concentrations of Surfactin, betaine, and the mixture, respectively, at a given interfacial tension (40 mN/m) in the solution phase. α1 and Xσ1 are the mole fraction of Surfactin in solution and the mole fraction of Surfactin in the mixed monolayer at the interface respectively. The superscript “0” refers to the bulk solution, and “α” to the interface. In comparison to | βm |, | βσ | is smaller, which indicates that the attractive interaction between surfactants in the mixed adsorption film at the air/aqueous interface is weaker than that in the mixed micelles. Besides, the interaction types at interface and in the bulk phase are different. In Surfactin/ betaine systems, the interactions include: (1) electrostatic attraction between −N+(CH3)2 of betaine and −COO− of Surfactin; (2) hydrophobic interaction between hydrophobic groups; (3) steric hindrance between bulky groups. (4) hydrogen bond between Surfactin and betain. At air/aqueous interface, when the mole fraction of betaine is high, the electrostatic attraction and the hydrogen bonds between a small amount of Surfactin and betaine, and the association between the hydrophobic chains bring in synergistic effect, but as the mole fraction of Surfactin increases, the steric hindrance and the intramolecular hydrogen bonds of Surfactin weaken the electrostatic attraction, so the synergistic effect disappears. In the bulk phase, the behavior is different: when the mole fraction of betaine is high, betaine molecules form smaller micelles. It is difficult to form mixed micelles because Surfactin with a large headgroup is hard to insert (Figure 4). When the mole fraction of Surfactin is high, it is easier for betaine molecule with a smaller headgroup to insert into the Surfactin micelle, thus a synergistic effect emerges (see in Figure 4). The shape of the surfactant aggregates in solution is determined by the packing parameter (pp) which is defined as pp =Vc/Amin lc, where Vc is the alkyl chain molecular volume, lc is the extended chain length, and Amin is the area of the headgroup.34 The variables lc and Vc can be calculated by Tanford’s formula, eqs 9 and 10.35 lc = (0.154 + 0.127nc)nm Vc ≈ (0.0274 + 0.0269nc)nm 3

βσ

α1

0.1 0.33 0.5 0.67 0.75 0.8

X1σ

system: Surfactin/SDDAB −2.73 0.5212 −2.68 0.6775 −2.60 0.7504 −1.60 0.8721 −2.07 0.8751 −2.18 0.8897 system: Surfactin/STDAB −1.88 0.4306 −1.31 0.6406 −1.09 0.7513 −1.09 0.8365 −1.29 0.8607 −0.38 0.9376 system: Surfactin/SHDAB −1.45 0.3267 −1.12 0.5421 −1.42 0.6397 −0.71 0.7817 −0.58 0.8419 −0.21 0.8990 system: Surfactin/C12BE −2.01 0.4573 −1.25 0.6721 −1.75 0.7364 −0.79 0.8772 −1.76 0.8489 −0.20 0.9553

| ln(C10/C20 | 2.40

1.66

0.97

1.85

very effective in predicting the shape of surfactant aggregates. Surfactants with the pp value of less than 1/3 are supposed to form spherical micelles. When the pp value is between 1/3 and 1/2, it generally corresponds to rod-shaped micelles, whereas when the pp value is between 1/2 and 1, it indicates that the surfactant is to form a monolayer or multilayer vesicles. For mixed micelles, the shape can be predicted in a rough approximation by the average packing parameter (Pav)36 calculated by eq 11: Pav =

∑i Vc , iXl , i ∑i lc , iXl , i ∑i A min , i Xl , i

(11)

Vc,i, lc,i, Xl,im,

where and Amin,i are the volume, extended chain length, mole fraction in the mixed micelle, and the area of the headgroup of the component in mixed surfactant solution, respectively. The pp value for pure Surfactin and Pav values for mixed systems are listed in Table 4. The shape of a micelle can be predicted from pp, but it does not provide any information about the size. It is found that the pp value of Surfactin and Pav value of mixed surfactants are all below 1/3, suggesting that they formed spherical micelles in solution. To further investigate this issue, we have used SANS to determine the structure of the aggregates. 3.4. Small-Angle Neutron Scattering, SANS. SANS data are shown in Figure 6. We analyzed the data by employing the Indirect Fourier Transformation (IFT) method developed by Glatter37 and using the version reported by Pedersen.38 At the same concentration, the scattering intensities are proportional to the mass of aggregates and also to the square of

(9) (10)

where nc is the number of methylene groups on the hydrocarbon chain of the surfactant. The parameter pp is 10653


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Table 4. Results of SANS Data of 0.24 mM Pure Surfactin and 0.24 mM Equimolar Surfactin/Betaine Systems: Analyzed by a Model-Independent Approach of IFT sample pure Surfactin Surfactin/SDDAB Surfactin/STDAB Surfactin/SHDAB Surfactin/C12BE a

Dmax (nm)

Rg (nm)

Io (cm−1)

5.5 5.5 6.5 6.0 7.0

± ± ± ± ±

± ± ± ± ±

1.8 1.9 2.0 2.0 2.2

0.1 0.2 0.1 0.1 0.4

0.010 0.007 0.015 0.013 0.003

0.001 0.001 0.001 0.001 0.001

Pav 0.185 0.201 0.210 0.244 0.199

a

Rm (nm)

Vs (nm3)

Nagg

2.26 2.41 2.63 2.53 2.79

1.72 1.54 1.47 1.31 1.60

28 38 52 52 26

Packing parameter for pure Surfactin.

Figure 6. Small-angle neutron scattering data of solutions of 0.24 mM pure Surfactin and 0.24 mM equimolar Surfactin/betaine systems: Surfactin (empty circle); Surfactin/betaines (filled circle).

form, so it has only one ionic form.27 Thus the synergism can arise from the stronger electric attraction between the −N+(CH3)2 group of sulfopropyl betaine and the −COO− group of Surfactin rather than C12BE. In the mixed Surfactin/ sulfopropyl betaine micelles, the electrostatic attraction between SDDAB, STDAB, and SHDAB with Surfactin is almost the same, while the hydrophobic interactions are different. As the length of hydrophobic chain increases, the hydrophobic interaction is stronger, so is the synergism. But the hydrophobic chain of SHDAB is very long and leads to selfaggregation. As a result, the interaction between Surfactin and STDAB is the strongest. The pair distance distribution function of scattering excess P(r) is shown in Figure 7, and the key parameters are listed in Table 4. The distance distribution function of pure Surfactin in PBS buffer exhibits a nearly local homogeneous spherical shape because the shape of P(r) function is nearly symmetric. We obtain a first estimate of the diameter from the maximum distance of approximately 55 Å, which is comparable to the result obtained using electron cryo-microscopy by Knoblich et

the contrast of neutron scattering length densities of the aggregates and the solvent.37 Figure 6 reveals that for four equimolar mixtures, compared with Surfactin, the intensity of Surfactin/SDDAB is similar to it, while the intensity of Surfactin/STDAB and Surfactin/SHDAB are both higher, and the scattering intensity of Surfactin/C 12BE is reduced. Furthermore, the intensities of Surfactin/STDAB are higher than that of Surfactin/SHDAB. Therefore, it can be concluded that for equimolar mixed systems, the interaction strength between Surfactin and betaine follows the order of Surfactin/ STDAB > Surfactin/SHDAB > Surfactin/SDDAB > Surfactin/ C12BE. For the zwitterionic betaines, the positive charge is close to the core of the micelle. For C12BE, as previously mentioned, the whole molecule exists as an anion form in solution (pH = 7.4) since the pI value is 4.9,26 there is strong electrostatic repulsion between C12BE and Surfactin. However, the headgroup of sulfopropyl betaines, −N+(CH3)2−CH2−CH2−CH2− SO3−, is as long as 6 Å,39 so the negative charge center would hardly overlap with the positive charge center. Besides, sulfopropyl betaine always exists as a salt or zwitterionic 10654


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Figure 7. P(r) function of 0.24 mM pure Surfactin and 0.24 mM equimolar Surfactin/betaine systems: Surfactin (empty circle); Surfactin/betaines (filled circle).

al.40 The radius of gyration increases when betaine is added into Surfactin solution, indicating that the size of mixed micelles increases. The Dmax of the aggregate of Surfactin/STDAB (αSurfactin = 0.5) is larger than that of pure Surfactin and the other mixed surfactant systems, with the exception of Surfactin/ C12BE. For mixed systems, the results of P(r) functions suggest that there are some almost homogeneous sphere-like aggregates in Surfactin/SDDAB, Surfactin/STDAB, and Surfactin/ SHDAB systems and ellipsoidal aggregates in Surfactin/ C12BE system. If the surfactant composition at interface is nonuniform, with each dominating in regions of preferred curvature, then the spherical Surfactin micelles can be deformed into prolate ellipsoids as C12BE is added to the system. The aggregation number (Nagg) of micelles defines the total number of surfactant molecules forming a pure or mixed micelle. Nagg from SANS measurements has been calculated by the following relation:

Nagg =

Vm Vs

where a is major axis, and b is minor axis. The volume of prolate ellipsoid micelle is calculated from Vm = 4π(abc)/3. Here prolate ellipsoid of revolution is used, so b = c and Vm = 4π(ab2)/3. For a binary mixed micelle, the volume is related with the mole fractions of two surfactants in micelle. Relevant parameters are listed in Table 4. The Nagg of pure Surfactin is 28, and this is consistent with the literature.42 The value is relatively small because Surfactin has a large peptide ring, the steric hindrance reduces the monomers in the Surfactin micelle. For a binary mixture of equimolar Surfactin/sulfopropyl betaine, the Nagg is higher than that of pure Surfactin, and the Nagg values of Surfactin/STDAB and Surfactin/SHDAB are higher than that of Surfactin/SDDAB. This is because the electrostatic attraction and the hydrophobic association involve betaine and Surfactin to form mixed micelle, in addition, betaine with a smaller headgroup will bring a larger aggregation number. The synergism in Surfactin/STDAB and Surfactin/ SHDAB are stronger than in Surfactin/SDDAB as described before, so the aggregation numbers of Surfactin/STDAB and Surfactin/SHDAB are larger than that of Surfactin/SDDAB. For equimolar Surfactin/C12BE, the aggregation number is the smallest, Nagg = 26, indicating that the interaction between Surfactin and C12BE is weak.

(12) 3

where Vm is the micellar volume given by Vm = 4πRm /3 when the micelle is spherical. Rm is the radius of the micelle. Vs is the volume of the surfactant. For a spherical micelle, Rm can be obtained from Rg by equation:9 R g2 =

3 2 Rm 5

4. CONCLUSIONS In this work, we study the interfacial and micellar physiochemical properties of binary systems formed by Surfactin and four betaine surfactants, respectively. At interface, the synergy between Surfactin and betaine emerges when the mole fraction of Surfactin is lower than in in the bulk phase. The interaction between Surfactin and C12BE is weakest among the four measured surfactants because the carbon bridge which

(13)

where Rg is the radius of gyration of the scattering object. But for prolate ellipsoid micelle, the following equation can be used:41

R g2 =

1 2 (a + 2b2) 5

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connects the positive and negative charge centers is shorter which leads to weak electrostatic attraction between C12BE and Surfactin the head groups. The synergism arises in Surfactin/ sulfobetaine systems at a specific fraction of Surfactin. From the SANS results, it can be concluded that for equimolar mixed systems, the strength of synergetic interaction between Surfactin and betaines follows the order of Surfactin/STDAB > Surfactin/SHDAB > Surfactin/SDDAB > Surfactin/C12BE. The optimum alkyl chain length (STDAB) and long enough separation between charges in headgroup of sulfopropyl are responsible for highest synergetic interaction with Surfactin.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-21-64252231; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.Z. gratefully acknowledges the support of this work by the Alexander von Humboldt Foundation. We gratefully acknowledge the support of this work by Chinese National Natural Science Foundation (201003047) and Fundamental Research Funds for the Central Universities. The SANS measurements have been performed under the support of the European Commission (Grant No. 283883-NMI3).

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REFERENCES

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Interaction of the Biosurfactant, Surfactin with Betaines in Aqueous

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