- Email: [email protected]
interface properties and aggregation behavior of mono-rhamnolipid and di-rhamnolipid
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Comparative studies on the surface/interface properties and aggregation behavior of mono-rhamnolipid and di-rhamnolipid
T
⁎
Li-mei Wua, Lu Laia,b,c, , Qingye Lub, Ping Meia,c, Yan-qun Wanga, Li Chengd, Yi Liue,f,g a
College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, PR China Department of Chemical and Petroleum Engineering, University of Calgary, Calgary T2N 1N4, Canada c Hubei Cooperative Innovation Center of Unconventional Oil and Gas, Wuhan 430100, PR China d College of Petroleum Engineering, Yangtze University, Wuhan, 430100, PR China e State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China f College of Chemistry and Material Sciences, Guangxi Teachers Education University, Nanning 530001, PR China g Key Laboratory of Coal Conversion and Carbon Materials of Hubei Province, College of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rhamnolipid Dynamic surface tension High temperature resistance Aggregation behavior
The surface properties of mono-rhamnolipid (Rha-C10-C10, R1) and di-rhamnolipid (Rha-Rha-C10-C10, R2) were investigated after separation and purification. The effects of environmental factors on equilibrium surface tension of these surfactants were studied by changing the temperature, salinity, and pH. Results show that R1 possesses a better surface activity than R2, but both are stable at low pH values and high temperature. Moreover, the diffusion and adsorption processes of R1 and R2 were studied by dynamic surface tension measurements. The initial adsorption processes of R1 and R2 were diffusion-controlled, and the effective diffusion coefficient of R1 was higher than that of R2 at the same concentration. We also monitored the dynamic interfacial tension curves of R1 and R2 with or without aging at high temperature, revealing that both feature high temperature resistance, but R1 exhibited a better interfacial activity than R2. For aggregation behavior in the bulk phase, dynamic light scattering and UV ˗ vis spectrophotometry were used to measure and observe the aggregation of rhamnolipids R1 and R2 at different temperatures and pH values. Results show the vesicle-to-micelle transformation of R1 and R2 aggregates with decreasing pH. This result is attributed to the considerable influence of solution pH to the dissociation degree of rhamnolipids. Thus, pH values significantly influence particle size distribution.
1. Introduction Rhamnolipids are one of the most widely studied biosurfactants. Compared with chemical surfactants, rhamnolipids are highly biocompatible and biodegradable, and they have been proven to show potential applications in food, cosmetics, pesticides, detergents, pharmaceuticals, petroleum recovery, bioremediation and other industrial fields [1–4]. Rhamnolipids are the metabolites of microorganisms, and dozens of homologs are observed due to the different microbial strains, growth conditions and carbon sources used for its nutrition [5]. Rhamnolipids containing one hydrophilic headgroup are called monorhamnolipids, whereas rhamnolipids containing two hydrophilic headgroups are called di-rhamnolipids. The most common rhamnolipids homologs include Rha-C10-C10 (R1) and Rha-Rha-C10-C10 (R2) [6]. Previous reports indicated that rhamnolipids feature high surface
⁎
activity, which can reduce the surface tension from ∼72 mN/m to ∼30 mN/m [1,6,7]. In addition, other previous studies showed that rhamnolipid mixtures possess high resistance to temperature, salt, and pH [3,8,9]. These properties play important roles in bioremediation and petroleum recovery. Aparna et al. studied the effects of temperature on the surface activity in a wide temperature range (277–394 K), and observed that temperature caused no influence on the surface activity of biosurfactants rhamnolipids [10]. However, they only measured the effect of temperature (298–394 K) on the surface tension of crude rhamnolipid products. Meanwhile, the properties of rhamnolipids are related to the composition of its homologs. Therefore, studies should examine the influence of temperature, salinity, and pH on the surface/ interface properties of R1 and R2. Helvaci et al. investigated the effects of the salt concentrations of 0.05, 0.5, and 1 mol/L on the surface activities of R1 and R2 and noted that the electrolyte addition can
Corresponding author at: College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, PR China. E-mail address: [email protected] (L. Lai).
https://doi.org/10.1016/j.colsurfb.2019.06.012 Received 8 February 2019; Received in revised form 12 May 2019; Accepted 6 June 2019 Available online 07 June 2019 0927-7765/ © 2019 Published by Elsevier B.V.
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
form vesicles at a certain condition. However, to date, few people have systematically studied whether external factors can promote the transformation of R1 and R2 between vesicles and micelles after separation and purification. Because of its biocompatibility, rhamnolipid has a great application prospect in different industrial fields. The rhamnolipid produced by biological metabolism has a complex composition. In general, the structure of surfactants has a significant effect on the surface/interface properties. Understanding the surface/interface properties of biosurfactant homologs of different structures will be conducive to the application of biosurfactants. Especially, R2 has similar structures to Gemini surfactants. In this work, R1 and R2 were obtained by separation and purification. The equilibrium surface tension curves of R1 and R2 have been determined. Moreover, the dynamic surface activities of R1 and R2 were evaluated and well-explained from the perspective of differences in molecular structure. To gain insights into the resistance to high temperature, the dynamic interfacial activities of R1 and R2 with or without treatment at high temperature were studied. Finally, the aggregation behavior of R1 and R2 under different external environments was investigated by turbidity and dynamic light scattering (DLS) measurements.
improve the said property [11]. Nevertheless, almost no one has studied the changes in surface tension over a larger salt concentration range. Ozdemir et al. studied the effects of pH and reported the strong surface activity of R1 molecules at concentrations below critical micelle concentration (CMC), which is independent of pH values. However, they only studied the equilibrium surface tension at pH values of 5 and 6.8. Nevertheless, the pH range involved in practical applications may be larger. Thus, the effects of a large pH range on equilibrium surface tension of R1 and R2 must be investigated. Dynamic techniques allow us to measure the time dependence of the adsorption of surfactant molecules to an interface. This property plays an important role in the industry. However, almost no one studies the dynamic interfacial activity of rhamnolipids to date. Therefore, the dynamic interfacial activity of R1 and R2 was investigated in this work. In addition to interface activity, in recent years, studies on rhamnolipids mainly focused on the characteristics of adsorption, aggregation, emulsification, and micellation [12–15], which are also important parameters to evaluating the application effect of surfactant solubilization and transport capacity [16,17]. Thereinto, the aggregation behavior of rhamnolipids in solutions has been extensively studied [1,18,19]. Aggregates of surfactants feature a variety of microstructures, including spherical, rod micelles [19,20], spherical and irregular vesicles, tubular and irregular doublemembrane structures [14,21], and lamellar structures. Numerous researchers indicated that the morphology of surfactant aggregates depends not only on the chemical structure and concentration of the surfactant itself [14,22] but also on the influence of solution conditions, such as pH, temperature, and ionic strength. Guo et al. studied the effects of concentration on the aggregation behavior of mono- and dirhamnolipids [1]. They discovered that the diameter of the aggregates increased with increasing di-rhamnolipids concentration. Sánchez et al. reported that the aggregation behavior of di-rhamnolipids under different pH values and larger aggregates develop at concentrations above CMC [18]. However, in the present literature, only references [3] and [23] examined the surface and aggregation behavior of R1 and R2. Moreover, most of the above studies were conducted on rhamnolipids under conditions much higher than the CMC. In this work, the concentration of surfactant solution was close to CMC. Ikizler used the critical packing parameter (cpp) to characterize the molecule shape; this variable is defined as the ratio of the molecular volume times the cross-sectional area to the fully stretched length of the hydrocarbon groups [23]. When packing parameters are in the range 1/2 < cpp < 1, surfactants form bilayers. Aggregation gradually occurs with the initial formation of dimers at extremely low surface saturation [24]. The mechanism of dimer reaction is similar to micellations as the tail is sandwiched between hydrophilic groups, thus minimizing their free energy in aqueous environment. With the increase in total surfactant concentration, the dimers interact crosswise to form bilayers. Aside from fulfillment of cpp conditions, bilayer separation is a necessary condition for spontaneous formation of vesicles. The cpp value of R1 is 0.62, and that of R2 equals 0.73 [23]; thus, both can spontaneously
2. Experimental 2.1. Materials Rhamnolipid (50%) was purchased from REGE (Xi’an, China). Both decane and toluene were of analytical reagent grades. Toluene, decane, and diesel oil were used after purification. Both decane and diesel oil were purified by Florida Silica (Macklin, 60–100 mesh). The specific purification method was as follows: decane or diesel oil was mixed with Florida Silica at a ratio of 10 mL:1 g, stirred for 12 h at a rotational speed of 600 rpm and then separated by centrifugation to obtain the purified decane or diesel oil. Toluene was purified by distillation. All other reagents were of analytical reagent grade and used as received. The aqueous solution for DLS measurement was prepared with Milli-Q water, whereas the other solution was prepared directly with distilled water. 2.2. Separation and purification of rhamnolipids The main components of rhamnolipid are R1 and R2. Fig. 1 shows their structural formulas. R1 and R2 were separated and obtained by using a chromatography column according to the work of Guo et al. [1]. Specifically, the chromatography column (3.6 cm × 46 cm) was filled with a silica gel 60 purchased from Macklin Reagent (Shanghai, China, 200–300 mesh gel). Approximately 4 g of rhamnolipid was dissolved in CHCl3 and placed in the column. The column was washed by chloroform/methanol at 2:1 v/v. Each 15 ml sample was received by a test tube, followed by thin-layer chromatography (TLC) detection. R1 was
Fig. 1. Molecular structures of mono-rhamnolipid (R1, a) and di-rhamnolipid (R2, b) investigated in the current study. 594
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
eluted first, followed by R2. These two components were collected and dried in a rotary evaporator and finally dried in the vacuum drying oven at 338.15 K for 24 h. To confirm the molecular structure, the separated product was identified by proton nuclear magnetic resonance and Fourier transform infrared spectroscopy (Figures S1 and S2, respectively).
lgti = lgt * − 1/ n
(5)
lgtm = lgt * + 1/ n
(6)
In general, according to the calculations of Ward and Tordai, that is, Eq. (7) [26], the effective diffusion coefficient (Deff) at the start of the adsorption can be obtained. The Deff at the late stage of adsorption can also be calculated by Ward and Tordai’s formula, that is, Eq.(8) [26,30].
2.3. Equilibrium surface tension determination
γt → 0 = γ0 − 2nRTc0 The surface tensions of R1 and R2 were measured by K11 automatic tensiometer (Krüss, German) using the Wilhelmy plate method. The effects of temperature, salinity, and pH on the surface tension of R1 and R2 were also measured. In the range of 295–325 K, the temperature was controlled by a water bath connected with a tensiometer. In addition, the aqueous solutions of R1 and R2 were injected in a high-temperature and high-pressure reaction kettle. The reaction kettle was placed in a baking oven under different temperatures (353 K, 373 K, and 393 K) for 4 h. Then, the surface tension was measured after cooling. The change in salinity was achieved by dissolving R1 or R2 in NaCl solution at different concentrations (0, 20, 40, 80 and 100 g·L−1). The solution pH was adjusted with 1 mol/L HCl or 1 mol/L NaOH solution. The effects of salinity and pH on the surface tension of R1 and R2 were determined at 308.15 ± 0.1 K. According to the equilibrium surface tension curves, the adsorption capacity of surfactant at the air-water interface (Г) and the minimum area occupied by per surfactant molecule (Amin) were obtained using the Gibbs adsorption isothermal Eqs. (1) [25] and (2), respectively.
Γ=−
1 ⎛⎜ ∂γ ⎞⎟ 2.303nRT ⎝ ∂lgc ⎠T
Dt π
(7)
where c0 denotes the surfactant concentrations; R = 8.314 J mol−1 K−1; π = 3.142, and n = 2 for ionic surfactants [26]:
γt →∞ = γeq +
2 nRTΓeq
c
π 4Dt
(8)
where γeq indicates the equilibrium surface tension, and Γeq refers to the equilibrium surface exceeding the concentration calculated using the Gibbs adsorption (Eq. (1)); R is the gas constant (8.314 J mol−1 K−1), and T is absolute temperature (K). 2.5. Turbidity measurement To investigate the aggregation behavior of R1 and R2, turbidity measurements were performed to elucidate the transformation of the rhamnolipid aggregates. UV–vis spectrophotometer (UV-2450, Puxi Instruments Ltd., China) was used to measure sample absorbance at a wavelength of 600 nm (A600). Under different pH values (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5), the turbidities of R1 and R2 solutions at 5.0 × 10−4 mol/L concentration were examined at 298.15 K.
(1)
where γ refers to the surface tension of surfactant solution at a certain concentration (c); n is a constant that depends on the type of surfactant absorbed at the interface. n = 2 was employed for R1 and R2 [26–28]. T denotes the experimental temperature, and R is the gas constant (8.314 J mol−1 K−1).
2.6. DLS measurement
where Γmax corresponds to the maximum adsorption capacity, and NA is Avogadro’s constant.
DLS measurements were performed on a Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., UK). The scattering angle was 173°, and scattering intensity was recorded by the software supplied with the instrument to obtain the hydrodynamic diameter (DH). In the experiment, DH was represented by the number particle size distribution (PSD). The effects of temperature, salinity, and pH on the size distribution of self-aggregates of R1 and R2 were studied. The experiments were carried out at 298.15 ± 0.1 K.
2.4. Dynamic surface tension (DST) determination
2.7. Statistical analysis
A bubble profile tensiometer (Tracker, Teclis-IT Concept, France) was used to monitor the DST curves of R1 and R2 at 308.15 ± 0.1 K. Five microliters of air in a stainless reverse steel needle was contained in a gas-tight syringe to form the air-water interface. The bent needle was immersed vertically in the surfactant solution filled in a quartz cuvette. The bubble image was captured by a camera was analyzed by software employing the Laplace equation. The only difference between dynamic interfacial tension measurement and DST measurement is that a 5 μl organic phase was used in the stainless reverse steel needle attached to a gas-tight syringe to form an oil-water interface. The DST curves can be fitted by Rosen empirical formula (3) [29],
Some data presented in this study are the average ± SD of the experiments repeated at least three times. For each sample group, the homogeneity of variance was calculated using the paired t-test.
Amin =
1018 NA Γmax
(γ0 − γt )/(γt − γm ) = (t / t *)n
(2)
3. Results and discussion 3.1. Equilibrium surface properties of R1 and R2 3.1.1. CMC of R1 and R2 To determine the CMC, the equilibrium surface tension curves of R1 and R2 were measured by the Wilhelmy plate method. As shown in Fig. 2, with the increase in concentration, the surface tension gradually decreased until an inflection point occurred, which is defined as the surfactant CMC [4]. According to Fig. 2, the CMC values of R1 and R2 measured 4.0 × 10−4 mol/L and 4.6 × 10−4 mol/L, respectively. Both the CMC value and the equilibrium surface tension value of R1 were less than those of R2, indicating the superior surface activity of R1 compared with that of R2. Ikizler et al. reported that the CMC of R1 amounted to 1.0 × 10-4 mol/L, and that of R2 reached 1.5 × 10-4 mol/ L, that is, the CMC of R1 was more significant than that of R2 [23]; this finding coincides with our results. As shown in Fig. 1, R1 molecules feature more hydrophobicity than R2 molecules. Thus R1 molecules
(3)
where γ0 represents the surface tension of pure solvent; γt specifies the surface tension of the surfactant solution at interface age (t); γm indicates the meso-equilibrium surface tension, and n and t* are constants. n and t* can be obtained from the slope and intercept of plots of lg[(γ0 − γt )/(γt − γm )], respectively, versus lgt. The drop rate constant (R1/2) can be calculated by Eq. (4) [30]. Furthermore, parameters of ti and tm can be calculated by Eqs. (5) and (6), respectively [29].
R1/2 = (γ0 − γm )/2t *
(4) 595
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
of R1 and R2 are stable at the studied temperature range. To be used at the high temperature, the surfactant with high structure stability have been synthesized, e.g., sulfonate surfactants. However, these surfactants are challenging to be degraded in the environment. Biosurfactants R1 and R2 not only have resistance to a high temperature but also have the benefit to the environment. Thus, R1 and R2 can be used in the oil production process under high-temperature conditions. 3.1.3. Effects of salinity on the surface properties of R1 and R2 Fig. 4(a) and (b) illustrate the influence of salinity on the values of γ for R1 and R2, respectively. As cNaCl increased, the values of γ for R1 decreased firstly and then increased below CMC. By contrast, the values of γ for R2 decreased first and then stabilized. As shown in Table S1, when the surfactant concentration reached its CMC, NaCl addition caused no effect on the γ values for R1 and slightly affected those for R2. The result is attributed to the compressed double electric layer of ionic headgroups caused by NaCl addition. Thus, the electrostatic repulsion between hydrophilic headgroups decreased, and the number of surfactant molecules adsorbed to the surface increased. Moreover, as cNaCl increased, the hydrophilicity of the surfactant molecules decreased. As a result, the surfactant molecules can easily absorb to the surface. Moreover, NaCl addition will reduce the equilibrium surface tension. Helvac et al. discovered that the values of γ for R1 and R2 decreased when cNaCl increased from 0.05 mol/L to 1 mol/L [11]. According to the molecular structures of R1 and R2, NaCl addition will significantly affect the surface activity of R2, which includes two hydrophilic groups. Similarly, as indicated in Table S1, the CMC value decreased with the increase in cNaCl. Due to less hydrophilic headgroups, R1 molecules have a relatively small solubility that R2 molecules. At a high electrolyte concentration, R1 solubility decreased, causing an increase in surface tension.
Fig. 2. Variation of surface tension with surfactant concentration for R1 and R2 at 298.15 K. Table 1 Critical micelle concentration (cmc), value of surface tension at cmc (γcmc), minimum area occupied by per surfactant molecule (Amin), and maximum adsorption capacity (Гmax) of R1 and R2 obtained from surface tension curves at 298.15 K. Surfactant
cmc (mmol/ L)
γcmc (mN/ m)
Amin (nm2/ molecule)
Гmax (μmol/ m2)
R1 R2
0.40 0.46
27.44 31.27
1.10 1.38
1.52 1.21
aggregate into micelles at a lower concentration than R2. According to Eqs. (1) and (2), the adsorption capacity of the surfactant at the air-water interface (Г) and the minimum area occupied by per surfactant molecule (Amin) were obtained, respectively. Table 1 lists the calculation results. As shown in Table 1, the Amin of R1 is slightly less than that of R2, while the Γmax of R1 is greater than that of R2. Owing to the lesser number of hydrophilic headgroup, R1 is hydrophobic and can be easily absorbed to the surface. Moreover, as the molecular weight of R1 is smaller than that of R2, R1 molecules were packed more densely at the interface than those of R2. Amin of R1 is small, and its Γmax is larger than that of R2.
3.1.4. Effects of pH on the surface properties of R1 and R2 Fig. 5(a) and (b) depict the effects of pH on the surface properties of R1 and R2, respectively. When pH increased from 4.0 to 10.0, the values of γeq for R1 and R2 remained unchanged. When the pH was less than 4, the γ values for R2 decreased significantly compared with those of R1. This result is attributed to the enlarged hydrophobicity and solubility of surfactant molecules at pH less than 4. As shown in Fig. 1, both R1 and R2 molecules have carboxyl and hydroxyl groups. In general, carboxylate surfactant cannot be used in an acidic environment because of the protonation of carboxyl groups. The solubility of carboxylate surfactants will be sharply reduced in the acidic environment. However, the hydroxyl groups of R1 and R2 provide a large solubility at pH less than 4. As previously reported, the pKa values of mono- and dirhamnolipid are 5.5 and 5.6, respectively [31]. Below pH 4, the protonation of hydroxyl groups of R1 and R2 decreases the electrostatic repulsive of hydrophilic groups. Thus these surfactants can pack more closely at the air-water interface and exhibit a low value of surface tension. At the same time, the hydroxyl groups will interact with water molecules by hydrogen bonds. Biosurfactants R1 and R2 possess higher surfactant activities at strongly acidic environment than other anionic surfactants. When pH was greater than 10, both R1 and R2 exhibited a slight increase in equilibrium surface tension. Owing to two hydrophilic headgroups, the surface tension of R2 is sensitive to the pH values. Therefore, anionic surfactants R1 and R2 are highly deprotonated and hydrophilic above pH 10. Thus, their values of equilibrium surface tension increased.
3.1.2. Effects of temperature on the surface activity of R1 and R2 Considering high surface activity and biodegradability, rhamnolipids exhibit extensive application foreground in different industrial fields. In most cases, however, surfactants are used in environments with different temperatures, acidity, or salinity. Therefore, the stabilities of R1 and R2 at different temperatures, salinity, and pH values were studied. In this study, the effects of temperature (298–393 K) on the equilibrium surface tension of R1 and R2 were investigated. Considering that the surface tensiometer cannot be used at high temperatures, the effects of temperature on the surface tension of R1 and R2 were examined using two different methods according to the temperature ranges. In the range of 298–323 K, the values of surface tension were directly measured using the K11 surface tensiometer. Above 323 K, the R1 and R2 solutions were injected in a high-temperature and high-pressure reaction kettle. The kettle was placed in a baking oven at different temperatures (353, 373, and 393 K) for 4 h. Then, surface tension was measured after cooling. The results are shown in Fig. 3. According to Fig. 3(a), at 4.0 × 10−5 mol/L, the γ values for R1 reduced from 38.97 mN/m to 35.55 mN/m as the temperature increased from 298 K to 393 K. At the other two concentrations, the values of γ for R1 also decreased slightly with the increase in temperature. According to Fig. 3(b), for R2, at 4.6 × 10−5 mol/L, the values of γ decreased from 44.39 mN/m to 41.36 mN/m as the temperature increased from 298 K to 393 K. The results indicate that temperature exerts a negligible effect on the surface activity of R1 and R2. As a result, the molecule structures
3.2. DST of R1 and R2 3.2.1. DST curves of R1 and R2 DST curves were determined using drop-shape analysis techniques to understand the diffusion and adsorption of surfactant molecules to the interface. In general, a typical DST curve includes the induction, rapid fall, meso-equilibrium, and equilibrium regions [32]. Fig. 6 shows 596
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
Fig. 3. The values of the surface tension of R1 (a) and R2 (b) at different temperatures (298 K; 303 K; 308 K; 313 K; 318 K; 323 K; 353 K; 373 K; 393 K). Concentration of R1 (mol/L): 4.0 × 10−5; 2.0 × 10-4; 4.0 × 10-4. Concentration of R2 (mol/L): 4.6 × 10−5; 2.3 × 10-4; 4.6 × 10-4 mol/L. Error bars represent standard error of the mean. *P < 0.05 versus the group of 298 K using paired t-test.
Fig. 4. Effects of the addition of NaCl on the surface tension curves of R1 and R2 at 308.15 K. Concentration of NaCl (g/L): 0, 20, 40, 80, 100.
A high n value implies a substantial difference between the adsorption energy and desorption energy and easy adsorption of surfactant molecules adsorption to the surface. n and t* can be obtained from the slope and intercept of plots of lg[(γ0 − γt )/(γt − γm )], respectively, versus lgt as shown in Figure S3. The drop rate constant (R1/2) can be calculated by Eq. (4) [30]. Furthermore, parameters of ti and tm can be calculated by Eqs. (5) and (6) [29], respectively. Table 2 provides the calculated DST parameters of R1 and R2. With the increase in surfactant concentration, t*, n, and tm decreased, whereas R1/2 increased. This finding is explained as follows: for ionic surfactants, at the start of the adsorption (the process of surfactant molecules diffusion from the bulk phase to the subsurface), high concentration of the bulk phase indicated a rapid molecular diffusion, easy-to-reach meso-equilibrium region, and decreased t* and tm [13]. At the later stage of adsorption (surfactant adsorption from the subsurface to the surface), the surfactant concentration at the surface also increases with increased surfactant concentration in the bulk phase. This phenomenon results in increased required energy (adsorption
the DST curves of R1 and R2. The induction periods of R1 and R2 were extremely short, and the meso-equilibrium state was attained at approximately 1000s. This result indicates that both R1 and R2 feature excellent dynamic surface activities. At the same concentration, the value of equilibrium surface tension for R1 was smaller than that for R2, and this result is attributed to the additional hydrophilic headgroup of R2. Moreover, given the high steric adsorption resistance of R2, its maximum adsorption amount of R2 is less than that of R1. Thus, R2 exhibits a small surface tension value at equilibrium state. The DST curves of surfactant solutions have been fitted by the Rosen empirical formula (Eq. (3)) [29]. The t* values reflect the diffusion process of surfactant monomers from the bulk phase to the subsurface. Small t* indicates a short time required for surfactant molecules to diffuse from the bulk phase to the subsurface and easy achievement of the meso-equilibrium region. n is an empirical constant mainly related to the molecular structure of surfactants, and it reflects the difficulty of adsorption of monomers from the subsurface to the surface. n causes no considerable change in the range of a certain surfactant concentration.
Fig. 5. Effects of pH on the surface tension of R1 (a) and R2 (b) at 308.15 K. Concentration of R1 (mol/L): 4.0 × 10−5; 2.0 × 10-4; 4.0 × 10-4. Concentration of R2 (mol/L): 4.6 × 10−5; 2.3 × 10-4; 4.6 × 10-4 mol/L. The pH values: 2; 4; 6; 7; 8; 10; 12. Error bars represent standard error of the mean. **P < 0.01 and ***P < 0.001 versus the group of pH 7 using paired t-test.
597
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
Fig. 6. (a) Variation of surface tension with surface age (t) for R1 at 308.15 K; (b) Variation of surface tension with surface age (t) for R2 at 308.15 K. Concentration of R1 and R2 (mol/L): 4.6 × 10−6; 9.2 × 10−6; 1.0 × 10-5; 4.6 × 10-5.
energy barrier) for adsorption, difficulty in adsorption, and decreased n. Furthermore, with the increase in concentration in the bulk phase, the drop rate of the surface tension from the values of a pure solvent to meso-equilibrium values increased. The dynamic surface activity and R1/2 also increased. As shown in Table 2, t*, n and tm decreased, and R1/ 2 increased with increasing concentrations of R1 and R2. This result is consistent with the above conclusion. From the perspective of hydrophobicity, strong hydrophobicity of molecules indicates slow molecular diffusion in the bulk phase and large t* value. The increase in hydrophobicity of molecules allows easy adsorption of the subsurface to the surface. Thus, the value of n is high. The drop rate of the surface tension decreases, and R1/2 decreases [33]. According to the molecular structures of R1 and R2, the hydrophobicity of R1 is higher than that of R2. Thus, the values of n and t* should be large at the same concentration. These findings are consistent with experimental results. Table 2 also shows that with the increase in concentrations of R1 and R2, R1/2 increased, with the value of R1/2 for R2 being greater than that of R1. This outcome indicates the poorer dynamic surface activity of R1 than R2. In general, according to Ward and Tordai (Eq. (7)) [26], the Deff at the start of the adsorption process can be obtained from Figure S4. Moreover, the Deff at the late stage of adsorption can be calculated by Ward and Tordai’s formula (Eq. (8)) from Figure S5 [26,30]. When the surfactant concentration was lower than 4.6 × 10−5 mol/L, the starting point for the drop of surface tension approximated 70 mN/m, and t1/2 and γ were linear. These results indicate that the initial adsorption of R1 and R2 is diffusion-controlled. Table 3 shows the Deff for R1 and R2. By comparing the Deff of the late of adsorption of R1 and R2 under the same surfactant concentration, the Deff of R1 was greater than that of R2 at the same concentration. From the perspective of hydrophobicity, high hydrophobicity of molecules indicates a low requirement for energy barrier for adsorption, enabling the easy adsorption of surfactant, thus increasing the Deff value. From Table 3, at the late stage of adsorption, the Deff of R1 was greater than that of R2, consistent with their molecular structures.
Table 3 Effective diffusion coefficients of R1 and R2 at 308.15 K. System
R1 R1 R1 R1 R2 R2 R2 R2
c (mol L−1)
4.6 × 10−6 9.2 × 10−6 1.0 × 10−5 4.6 × 10−5 4.6 × 10−6 9.2 × 10−6 1.0 × 10−5 4.6 × 10−5
Long time t→∞ dγ/dt−1/2(mN m-1 s-1/2)
Deff (10−11 m2 s-1)
150.84 122.85 117.00 68.47 168.14 109.90 105.16 96.70
1.38 0.52 0.48 0.07 0.45 0.26 0.24 0.01
study. Figs. 7 shows the dynamic interfacial tension curves of R1 and R2. The equilibrium interfacial tension of R1 is lower than that of R2. At the surface, the period for surface tension attained a meso-equilibrium state at approximately 1000s, whereas approximately 500 s was required for the oil-water interface. These results indicate that the presence of the organic phase is conducive to the state of adsorption equilibrium. Furthermore, comparing the dynamic interfacial and surface tension curves, neither rhamnolipid presented an induction period. In other words, the dynamic surface and interfacial tension curves of R1 and R2 only included the rapid fall, meso-equilibrium, and equilibrium regions. Therefore, both R1 and R2 possess good dynamic interfacial activities. In this study, the effects of organic phases (decane, toluene, and diesel) on the dynamic interfacial tension curves of R1 and R2 were studied. As shown in Fig. 7(a), the values of decane/water interfacial tension for R1 and R2 decreased from 46 mN/m to 20 and 26 mN/m, respectively. In Fig. 7(b), the values of diesel/water interfacial tension for R1 and R2 decreased from 57 mN/m to 28 and 34 mN/m, respectively. Fig. 7(c) shows that the value of toluene/water interfacial tension for R1 reduced from 36 mN/m to 19 and 24 mN/m for R2. According to Figure S6, the kinetic parameters of interfacial processes were calculated (Table 4). For R1 and R2, the organic phase was toluene, and the shortest mesoscopic equilibrium time (tm) was achieved. Meanwhile, decane yielded the longest tm. Similarly, from Table 4, the n
3.2.2. Dynamic interfacial tension of R1 and R2 In addition to dynamic surface activity, the dynamic interfacial activity of R1 and R2 at 4.6 × 10−6 mol/L was also investigated in this Table 2 Parameters of dynamic surface tension of the R1 and R2 at 308.15 K. Surfactants
c (mol L−1)
γm (mN/m)
n
t*/s
tm/s
ti/s
R1/2 (mN m−1 s−1)
R1 R1 R1 R1 R2 R2 R2 R2
4.6 × 10−6 1.0 × 10−5 3.6 × 10−5 4.6 × 10−5 4.6 × 10−6 1.0 × 10−5 3.6 × 10−5 4.6 × 10−5
49.0 47.0 44.0 36.0 52.0 49.0 46.0 36.0
3.66 3.52 3.50 1.57 2.95 2.75 2.37 1.07
269.15 208.93 144.54 63.10 230.42 151.36 78.43 25.12
504.66 398.12 281.84 275.42 502.92 346.74 208.93 213.80
144.54 109.65 74.13 14.25 104.71 66.07 29.51 2.95
0.039 0.055 0.086 0.300 0.039 0.066 0.147 0.680
598
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
Fig. 7. (a) Variation of water/decane interfacial tension with surface age (t) for R1 and R2 solution at 298.15 K with and without high temperature ageing; (b) Variation of water/diesel oil interfacial tension with surface age (t) for R1 and R2 solution at 298.15 K with and without high temperature ageing; (c) Variation of water/toluene interfacial tension with surface age (t) for R1 and R2 solution at 298.15 K with and without high temperature ageing. Concentration of R1 and R2 (mol/L): 4.6 × 10−6.
Table 4 Parameters of dynamic interfacial tension of R1 and R2 solution at 298.15 K with and without high-temperature aging. System
γm (mN/ m)
n
t*/s
tm/s
ti/s
R1/2 (mN m−1 s−1)
R1 R1 R2 R2 R1 R2 R1 R2
20.0 20.8 26.1 25.9 27.8 35.1 19.6 23.2
1.14 1.11 1.08 1.06 1.70 1.65 2.20 2.10
386.99 409.84 375.04 401.58 210.62 177.83 208.93 182.77
3162.28 3235.94 3162.27 3523.71 812.83 707.95 588.84 616.60
51.28 51.49 43.65 44.77 53.70 43.25 72.44 61.66
0.033 0.031 0.041 0.038 0.069 0.062 0.039 0.035
+ decane Ageing + decane + decane Ageing + decane + diesel + diesel + toluene + toluene
Fig. 8. Variation of the size for R1 and R2 aggregates with concentration at 298.15 K. Concentration of R1 (mol/L): 2.0 × 10−5; 5.0 × 10−5; 1.0 × 10-4; 5.0 × 10-4; 1.0 × 10-3. Concentration of R2 (mol/L): 2.0 × 10−5; 5.0 × 10−5; 1.0 × 10-4; 5.0 × 10-4; 1.0 × 10-3. Error bars represent standard error of the mean. **P < 0.01 and ***P < 0.001 versus the group of 5.0×10-4 mol/L using paired t-test.
of R1 was slightly larger than that of R2 at the same organic phase as R1 is more hydrophobic than R2. The dynamic interfacial tensions of R1 and R2 aged at high temperature were also examined. High-temperature aging involves placing the surfactant solutions of R1 and R2 into a high-temperature and highpressure reaction kettle at 393.15 K for 4 h. Then, the dynamic interfacial tension curves were measured after cooling. Fig. 7 shows that the difference in dynamic interfacial tension curves of R1 and R2 with and without high-temperature aging reached less than ± 1 mN/m. Thus, high-temperature aging results in minimal influence on the dynamic interfacial activity of R1 and R2. Moreover, from Table 4, before and after high-temperature aging, a slight difference was observed in the dynamic interfacial parameters of R1 and R2. Thus, the molecule structures of R1 and R2 was stable in the studied temperature range. R1 and R2 can be used at high temperatures.
aggregation behavior of R1 and R2 were examined. As presented in Fig. 8, the size of R1 aggregations gradually increased with the increase in concentration. And the poly dispersity index (PDI) of R1 and R2 at the concentration of 2.0 × 10−5 mol/L, 5.0 × 10-5 mol/L, 1.0 × 10-4 mol/L, 5.0 × 10-4 mol/L and 1.0 × 10-3 mol/L was shown in Table S2. However, as the R2 concentration increased, the size of R2 aggregations decreased first and then increased, indicating that the increase in concentration is conducive to the formation of R1 aggregates. We mainly studied the influence of temperature and pH on the aggregation behavior of R1 and R2. The surfactant concentration was slightly higher than CMC when studying the influence of external factors on the aggregation behavior. Fig. 9 shows the influence of temperature on the aggregation behavior of R1 and R2. And the poly dispersity index (PDI) of R1 and R2 at the temperature of 293 K, 298 K, 303 K, 313 K, 318 K, and 323 K was shown in Table S3. At temperature less than 318 K, the sizes of R1 and R2 aggregates approximated 50 nm. When the temperature was greater than 318 K, the size of R1 aggregates was greater than 200 nm, whereas that of R2 aggregates was close to 200 nm. These observations indicate that 318 K may be a transition point of R1 and R2 aggregates as they changed from micelles to
3.3. Aggregation behavior of rhamnolipids To investigate the aggregation behavior of R1 and R2, DLS and turbidity measurements were performed to elucidate the transformation of the rhamnolipid aggregates. As the size distribution of intensity amplifies the incoming signal by approximately a million times, an extremely small amount of larger material in the solution will affect the experimental results. However, the number peak reflects the actual number of particles in each particle size. Therefore, DH was shown by the number PSD in this study. First, the effects of concentration on 599
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
transformation of R1 and R2 aggregates into vesicles. Fig. 10 (a) displays the influence of pH on the aggregation behavior of R1 and R2 at 5 × 10−4 mol/L concentration. And the poly dispersity index (PDI) of R1 and R2 at the pH of 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5 was shown in Table S4. With the increase in pH, a significant decrease in the size of surfactant aggregates was observed. For R1, with the increase in pH, the size of aggregates decreased from 350 nm to 40 nm, and pH = 3.5 was a notable break point. However, for R2, with the increase in pH, the size of aggregates decreased gradually from 450 nm to 50 nm. Such finding is attributed to the considerable influence of solution pH to the dissociation degree of hydrophilic headgroups. With the increase in pH, the number of ionized rhamnolipids increased. Increasing electrostatic repulsion results in a small micelle curvature, inducing the transformation of larger aggregates to small ones. Turbidity was also measured to clarify the transformations occurring in R1 and R2 aggregates. Figs. 10 (b) show the turbidity changes, whereas Fig. 10 (c) and (d) show the changes in physical appearance of R1 and R2. The changing trend of turbidity is consistent with that of aggregate diameter. Therefore, with the increase in pH, the size of rhamnolipid aggregates decreased at various concentrations. Larger particles transformed into small ones at the same time. Champion et al. showed that the aggregation morphology of mono-rhamnolipids solution with a high concentration (60 mmol/L) increased from 6.0 to 8.0 with pH value and changed from large double-layer capsule, large
Fig. 9. Variation of the size for R1 and R2 aggregates with temperatures. Concentration of R1 and R2 (mol/L): 5 × 10−4 mol/L. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the group of 293 K using paired t-test.
vesicles. Increasing temperature causes the surfactant molecules to be less hydrated and more hydrophobic. Therefore, the cpp values of R1 and R2 will increase. The lateral van der Waals interaction between R1 and R2 surfactant molecules is likely to form a bilayer structure. In other words, the increase in temperature is conducive to the
Fig. 10. (a) Variation of aggregate sizes of R1 and R2 with pH values at 298.15 K; (b) Variation of turbidity of R1 and R2 solution with pH values at 298.15 K; (c) The appearance of R1 solution at different pH values and 298.15 K; (d) The appearance of R2 solution at different pH values and 298.15 K. Concentration of R1 and R2 (mol/L): 5 × 10−4 mol/L. The pH values: 2.5; 3.5; 4.5; 5.5; 6.5; 7.5; 8.5; 9.5; 10.5. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the group of pH 7.5 using paired t-test. 600
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
single-layer capsule, and small single-layer capsules to micelles [34]. In this study, the size of R1 and R2 aggregates decreased with the increase in pH at low concentration (around CMC). This observation is consistent with the abovementioned findings. This result also indicates the similar effects of pH on the aggregation behavior of R2 and R1.
272 (2019) 19–25. [10] A. Aparna, G. Srinikethan, H. Smitha, Production and characterization of biosurfactant produced by a novel Pseudomonas sp 2B, Colloid Surf. B-Biointerfaces 95 (2012) 23–29. [11] S.S. Helvaci, S. Peker, G. Ozdemir, Effect of electrolytes on the surface behavior of rhamnolipids R1 and R2, Colloids Surf. B Biointerfaces 35 (2004) 225–233. [12] S.P. Verma, B. Sarkar, Simultaneous removal of Cd (II) and p-cresol from wastewater by micellar-enhanced ultrafiltration using rhamnolipid: flux decline, adsorption kinetics and isotherm studies, J. Environ. Manage. 213 (2018) 217–235. [13] S. Mohanty, S. Mukherji, Surfactant aided biodegradation of NAPLs by Burkholderia multivorans: comparison between Triton X-100 and rhamnolipid JBR-515, Colloid Surf. B-Biointerfaces 102 (2013) 644–652. [14] O. Pornsunthorntawee, S. Chavadej, R. Rujiravanit, Solution properties and vesicle formation of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa SP4, Colloid Surf. B-Biointerfaces 72 (2009) 6–15. [15] G. Ozdemir, U. Malayoglu, Wetting characteristics of aqueous rhamnolipids solutions, Colloids Surf. B Biointerfaces 39 (2004) 1–7. [16] A.A. Dar, G.M. Rather, S. Ghosh, A.R. Das, Micellization and interfacial behavior of binary and ternary mixtures of model cationic and nonionic surfactants in aqueous NaCl medium, J. Colloid Interface Sci. 322 (2008) 572–581. [17] L. Dai, S. Yang, Y. Wei, C. Sun, D.J. McClements, L. Mao, Y. Gao, Development of stable high internal phase emulsions by pickering stabilization: utilization of zeinpropylene glycol alginate-rhamnolipid complex particles as colloidal emulsifiers, Food Chem. 275 (2019) 246–254. [18] M. Sanchez, F.J. Aranda, M.J. Espuny, A. Marques, J.A. Teruel, A. Manresa, A. Ortiz, Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa in aqueous media, J. Colloid Interface Sci. 307 (2007) 246–253. [19] J.T. Champion, J.C. Gilkey, H. Lamparski, J. Retterer, R.M. Maier, Electron microscopy of rhamnolipid (biosurfactant) morphology, J. Colloid Interface Sci. 170 (1995) 569–574. [20] K. Matsuoka, K. Takagi, C. Honda, Micelle formation of sodium hyodeoxycholate, Chem. Phys. Lipids 172 (2013) 6–13. [21] Z.A. Raza, Z.M. Khalid, M.S. Khan, I.M. Banat, A. Rehman, A. Naeem, M.T. Saddique, Surface properties and sub-surface aggregate assimilation of rhamnolipid surfactants in different aqueous systems, Biotechnol. Lett. 32 (2010) 811–816. [22] R.J. Eismin, E. Munusamy, L.L. Kegel, D.E. Hogan, R.M. Maier, S.D. Schwartz, J.E. Pemberton, Evolution of aggregate structure in solutions of anionic monorhamnolipids: experimental and computational results, Langmuir 33 (2017) 7412–7424. [23] B. Ikizler, G. Arslan, E. Kipcak, C. Dirik, D. Celenk, T. Aktuglu, S.S. Helvaci, S. Peker, Surface adsorption and spontaneous aggregation of rhamnolipid mixtures in aqueous solutions, Colloids and Surfaces a-Physicochemical and Engineering Aspects 519 (2017) 125–136. [24] L. Leclercq, V. Nardello-Rataj, M. Turmine, N. Azaroual, J.-M. Aubry, Stepwise aggregation of Dimethyl-di-n-octylammonium chloride in aqueous solutions: from dimers to vesicles, Langmuir 26 (2010) 1716–1723. [25] S. Ghosh, Surface chemical and micellar properties of binary and ternary surfactant mixtures (Cetyl pyridinium chloride, Tween-40, and Brij-56) in an aqueous medium, J. Colloid Interface Sci. 244 (2001) 128–138. [26] J.G. Gobel, G.R. Joppien, Dynamic interfacial tensions of aqueous Triton x-100 solutions in contact with air, cyclohexane, n -heptane, and n -hexadecane, J. Colloid Interface Sci. 191 (1997) 30–37. [27] A. Abalos, A. Pinazo, M.R. Infante, M. Casals, F. García, A. Manresa, Physicochemical and antimicrobial properties of new rhamnolipids produced by pseudomonas a eruginosa AT10 from soybean oil refinery wastes, Langmuir 17 (2001) 1367–1371. [28] D. Manko, A. Zdziennicka, B. Janczuk, Composition of surface layer at the WaterAir Interface and Micelles of Triton x-100+Rhamnolipid mixtures, J. Solution Chem. 46 (2017) 1251–1271. [29] M.J. Rosen, X.Y. Hua, Dynamic surface tension of aqueous surfactant solutions: 2. Parameters at 1 s and at mesoequilibrium, J. Colloid Interface Sci. 139 (1990) 397–407. [30] S.-J. Li, L. Lai, P. Mei, Y. Li, L. Cheng, Z.-H. Ren, Y. Liu, Equilibrium and dynamic surface properties of cationic/anionic surfactant mixtures based on bisquaternary ammonium salt, J. Mol. Liq. 254 (2018) 248–254. [31] H. Abbasi, K.A. Noghabi, M.M. Hamedi, H.S. Zahiri, A.A. Moosavi-Movahedi, M. Amanlou, J.A. Teruel, A. Ortiz, Physicochemical characterization of a monorhamnolipid secreted by Pseudomonas aeruginosa MA01 in aqueous media. An experimental and molecular dynamics study, Colloid Surf. B-Biointerfaces 101 (2013) 256–265. [32] M.J. Rosen, L.D. Song, Dynamic Surface Tension of Aqueous Surfactant Solutions 8. Effect of Spacer on Dynamic Properties of Gemini Surfactant Solutions, J. Colloid Interface Sci. 179 (1996) 261–268. [33] H. Xi Yuan, M.J. Rosen, Dynamic surface tension of aqueous surfactant solutions: I. Basic paremeters, J. Colloid Interface Sci. 124 (1988) 652–659. [34] F.J. Aranda, M.J. Espuny, A. Marques, J.A. Teruel, A. Manresa, A. Ortiz, Thermodynamics of the interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with phospholipid membranes, Langmuir 23 (2007) 2700–2705.
4. Conclusion Rhamnolipids homologs R1 and R2 both possess good surface activity, but the surface activity of R1 is superior to that of R2. The values of γ for R1 and R2 decreased slightly with the increase in temperature. The results indicate that temperature barely affects the surface activity of R1 and R2. NaCl addition significantly influences the surface activity of R2, which contains two hydrophilic groups. However, at high electrolyte concentration, R1 solubility decreases, thus increasing the surface tension. Owing to two hydrophilic headgroups, the surface tension of R2 is sensitive to pH values. For dynamic surface/interfacial tension, the induction periods of R1 and R2 are notably short, and initial adsorption of R1 and R2 is diffusion-controlled. With the increase in R1 and R2 concentrations, R1/2 increases, and the value of R1/2 for R2 is higher than that of R1. This result indicates the better dynamic surface activity of R2 than R1. The dynamic interfacial curves of R1 and R2 after high-temperature aging slight differ from those without hightemperature aging, thus also confirming the high-temperature resistance of R1 and R2. Finally, we studied the effects of temperature and pH on the aggregation behavior of R1 and R2. The increase in temperature and the decrease in pH values are conducive to the transformation of R1 and R2 aggregates into vesicles. Turbidity and DLS measurements verify well the transformation of R1 and R2 aggregates. Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (grant no. 21403017, 21473125). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.06.012. References [1] Y.-P. Guo, Y.-Y. Hu, R.R. Gu, H. Lin, Characterization and micellization of rhamnolipidic fractions and crude extracts produced by Pseudomonas aeruginosa mutant MIG-N146, J. Colloid Interface Sci. 331 (2009) 356–363. [2] R.M. Maier, G. Soberón-Chávez, Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications, Appl. Microbiol. Biotechnol. 54 (2000) 625–633. [3] M.L. Chen, J. Penfold, R.K. Thomas, T.J.P. Smyth, A. Perfumo, R. Marchant, I.M. Banat, P. Stevenson, A. Parry, I. Tucker, I. Grillo, Solution self-assembly and adsorption at the air-water interface of the monorhamnose and dirhamnose rhamnolipids and their mixtures, Langmuir 26 (2010) 18281–18292. [4] D. Manko, A. Zdziennicka, B. Janczuk, Thermodynamic properties of rhamnolipid micellization and adsorption, Colloid Surf. B-Biointerfaces 119 (2014) 22–29. [5] S.G.V.A.O. Costa, M. Nitschke, F. Lepine, E. Deziel, J. Contiero, Structure, properties and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater, Process. Biochem. 45 (2010) 1511–1516. [6] T. Janek, M. Lukaszewicz, A. Krasowska, Identification and characterization of biosurfactants produced by the Arctic bacterium Pseudomonas putida BD2, Colloid Surf. B-Biointerfaces 110 (2013) 379–386. [7] J.L. Parra, J. Guinea, M.A. Manresa, M. Robert, M.E. Mercadé, F. Comelles, M.P. Bosch, Chemical characterization and physicochemical behavior of biosurfactants, J. Am. Oil Chem. Soc. 66 (1989) 141–145. [8] C. Chen, N. Sun, D. Li, S. Long, X. Tang, G. Xiao, L. Wang, Optimization and characterization of biosurfactant production from kitchen waste oil using Pseudomonas aeruginosa, Environ. Sci. Pollut. Res. - Int. 25 (2018) 14934–14943. [9] V.K. Gaur, A. Bajaj, R.K. Regar, M. Kamthan, R.R. Jha, J.K. Srivastava, N. Manickam, Rhamnolipid from a Lysinibacillus sphaericus strain IITR51 and its potential application for dissolution of hydrophobic pesticides, Bioresour. Technol.
601
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Comparative studies on the surface/interface properties and aggregation behavior of mono-rhamnolipid and di-rhamnolipid
T
⁎
Li-mei Wua, Lu Laia,b,c, , Qingye Lub, Ping Meia,c, Yan-qun Wanga, Li Chengd, Yi Liue,f,g a
College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, PR China Department of Chemical and Petroleum Engineering, University of Calgary, Calgary T2N 1N4, Canada c Hubei Cooperative Innovation Center of Unconventional Oil and Gas, Wuhan 430100, PR China d College of Petroleum Engineering, Yangtze University, Wuhan, 430100, PR China e State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China f College of Chemistry and Material Sciences, Guangxi Teachers Education University, Nanning 530001, PR China g Key Laboratory of Coal Conversion and Carbon Materials of Hubei Province, College of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rhamnolipid Dynamic surface tension High temperature resistance Aggregation behavior
The surface properties of mono-rhamnolipid (Rha-C10-C10, R1) and di-rhamnolipid (Rha-Rha-C10-C10, R2) were investigated after separation and purification. The effects of environmental factors on equilibrium surface tension of these surfactants were studied by changing the temperature, salinity, and pH. Results show that R1 possesses a better surface activity than R2, but both are stable at low pH values and high temperature. Moreover, the diffusion and adsorption processes of R1 and R2 were studied by dynamic surface tension measurements. The initial adsorption processes of R1 and R2 were diffusion-controlled, and the effective diffusion coefficient of R1 was higher than that of R2 at the same concentration. We also monitored the dynamic interfacial tension curves of R1 and R2 with or without aging at high temperature, revealing that both feature high temperature resistance, but R1 exhibited a better interfacial activity than R2. For aggregation behavior in the bulk phase, dynamic light scattering and UV ˗ vis spectrophotometry were used to measure and observe the aggregation of rhamnolipids R1 and R2 at different temperatures and pH values. Results show the vesicle-to-micelle transformation of R1 and R2 aggregates with decreasing pH. This result is attributed to the considerable influence of solution pH to the dissociation degree of rhamnolipids. Thus, pH values significantly influence particle size distribution.
1. Introduction Rhamnolipids are one of the most widely studied biosurfactants. Compared with chemical surfactants, rhamnolipids are highly biocompatible and biodegradable, and they have been proven to show potential applications in food, cosmetics, pesticides, detergents, pharmaceuticals, petroleum recovery, bioremediation and other industrial fields [1–4]. Rhamnolipids are the metabolites of microorganisms, and dozens of homologs are observed due to the different microbial strains, growth conditions and carbon sources used for its nutrition [5]. Rhamnolipids containing one hydrophilic headgroup are called monorhamnolipids, whereas rhamnolipids containing two hydrophilic headgroups are called di-rhamnolipids. The most common rhamnolipids homologs include Rha-C10-C10 (R1) and Rha-Rha-C10-C10 (R2) [6]. Previous reports indicated that rhamnolipids feature high surface
⁎
activity, which can reduce the surface tension from ∼72 mN/m to ∼30 mN/m [1,6,7]. In addition, other previous studies showed that rhamnolipid mixtures possess high resistance to temperature, salt, and pH [3,8,9]. These properties play important roles in bioremediation and petroleum recovery. Aparna et al. studied the effects of temperature on the surface activity in a wide temperature range (277–394 K), and observed that temperature caused no influence on the surface activity of biosurfactants rhamnolipids [10]. However, they only measured the effect of temperature (298–394 K) on the surface tension of crude rhamnolipid products. Meanwhile, the properties of rhamnolipids are related to the composition of its homologs. Therefore, studies should examine the influence of temperature, salinity, and pH on the surface/ interface properties of R1 and R2. Helvaci et al. investigated the effects of the salt concentrations of 0.05, 0.5, and 1 mol/L on the surface activities of R1 and R2 and noted that the electrolyte addition can
Corresponding author at: College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, PR China. E-mail address: [email protected] (L. Lai).
https://doi.org/10.1016/j.colsurfb.2019.06.012 Received 8 February 2019; Received in revised form 12 May 2019; Accepted 6 June 2019 Available online 07 June 2019 0927-7765/ © 2019 Published by Elsevier B.V.
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
form vesicles at a certain condition. However, to date, few people have systematically studied whether external factors can promote the transformation of R1 and R2 between vesicles and micelles after separation and purification. Because of its biocompatibility, rhamnolipid has a great application prospect in different industrial fields. The rhamnolipid produced by biological metabolism has a complex composition. In general, the structure of surfactants has a significant effect on the surface/interface properties. Understanding the surface/interface properties of biosurfactant homologs of different structures will be conducive to the application of biosurfactants. Especially, R2 has similar structures to Gemini surfactants. In this work, R1 and R2 were obtained by separation and purification. The equilibrium surface tension curves of R1 and R2 have been determined. Moreover, the dynamic surface activities of R1 and R2 were evaluated and well-explained from the perspective of differences in molecular structure. To gain insights into the resistance to high temperature, the dynamic interfacial activities of R1 and R2 with or without treatment at high temperature were studied. Finally, the aggregation behavior of R1 and R2 under different external environments was investigated by turbidity and dynamic light scattering (DLS) measurements.
improve the said property [11]. Nevertheless, almost no one has studied the changes in surface tension over a larger salt concentration range. Ozdemir et al. studied the effects of pH and reported the strong surface activity of R1 molecules at concentrations below critical micelle concentration (CMC), which is independent of pH values. However, they only studied the equilibrium surface tension at pH values of 5 and 6.8. Nevertheless, the pH range involved in practical applications may be larger. Thus, the effects of a large pH range on equilibrium surface tension of R1 and R2 must be investigated. Dynamic techniques allow us to measure the time dependence of the adsorption of surfactant molecules to an interface. This property plays an important role in the industry. However, almost no one studies the dynamic interfacial activity of rhamnolipids to date. Therefore, the dynamic interfacial activity of R1 and R2 was investigated in this work. In addition to interface activity, in recent years, studies on rhamnolipids mainly focused on the characteristics of adsorption, aggregation, emulsification, and micellation [12–15], which are also important parameters to evaluating the application effect of surfactant solubilization and transport capacity [16,17]. Thereinto, the aggregation behavior of rhamnolipids in solutions has been extensively studied [1,18,19]. Aggregates of surfactants feature a variety of microstructures, including spherical, rod micelles [19,20], spherical and irregular vesicles, tubular and irregular doublemembrane structures [14,21], and lamellar structures. Numerous researchers indicated that the morphology of surfactant aggregates depends not only on the chemical structure and concentration of the surfactant itself [14,22] but also on the influence of solution conditions, such as pH, temperature, and ionic strength. Guo et al. studied the effects of concentration on the aggregation behavior of mono- and dirhamnolipids [1]. They discovered that the diameter of the aggregates increased with increasing di-rhamnolipids concentration. Sánchez et al. reported that the aggregation behavior of di-rhamnolipids under different pH values and larger aggregates develop at concentrations above CMC [18]. However, in the present literature, only references [3] and [23] examined the surface and aggregation behavior of R1 and R2. Moreover, most of the above studies were conducted on rhamnolipids under conditions much higher than the CMC. In this work, the concentration of surfactant solution was close to CMC. Ikizler used the critical packing parameter (cpp) to characterize the molecule shape; this variable is defined as the ratio of the molecular volume times the cross-sectional area to the fully stretched length of the hydrocarbon groups [23]. When packing parameters are in the range 1/2 < cpp < 1, surfactants form bilayers. Aggregation gradually occurs with the initial formation of dimers at extremely low surface saturation [24]. The mechanism of dimer reaction is similar to micellations as the tail is sandwiched between hydrophilic groups, thus minimizing their free energy in aqueous environment. With the increase in total surfactant concentration, the dimers interact crosswise to form bilayers. Aside from fulfillment of cpp conditions, bilayer separation is a necessary condition for spontaneous formation of vesicles. The cpp value of R1 is 0.62, and that of R2 equals 0.73 [23]; thus, both can spontaneously
2. Experimental 2.1. Materials Rhamnolipid (50%) was purchased from REGE (Xi’an, China). Both decane and toluene were of analytical reagent grades. Toluene, decane, and diesel oil were used after purification. Both decane and diesel oil were purified by Florida Silica (Macklin, 60–100 mesh). The specific purification method was as follows: decane or diesel oil was mixed with Florida Silica at a ratio of 10 mL:1 g, stirred for 12 h at a rotational speed of 600 rpm and then separated by centrifugation to obtain the purified decane or diesel oil. Toluene was purified by distillation. All other reagents were of analytical reagent grade and used as received. The aqueous solution for DLS measurement was prepared with Milli-Q water, whereas the other solution was prepared directly with distilled water. 2.2. Separation and purification of rhamnolipids The main components of rhamnolipid are R1 and R2. Fig. 1 shows their structural formulas. R1 and R2 were separated and obtained by using a chromatography column according to the work of Guo et al. [1]. Specifically, the chromatography column (3.6 cm × 46 cm) was filled with a silica gel 60 purchased from Macklin Reagent (Shanghai, China, 200–300 mesh gel). Approximately 4 g of rhamnolipid was dissolved in CHCl3 and placed in the column. The column was washed by chloroform/methanol at 2:1 v/v. Each 15 ml sample was received by a test tube, followed by thin-layer chromatography (TLC) detection. R1 was
Fig. 1. Molecular structures of mono-rhamnolipid (R1, a) and di-rhamnolipid (R2, b) investigated in the current study. 594
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
eluted first, followed by R2. These two components were collected and dried in a rotary evaporator and finally dried in the vacuum drying oven at 338.15 K for 24 h. To confirm the molecular structure, the separated product was identified by proton nuclear magnetic resonance and Fourier transform infrared spectroscopy (Figures S1 and S2, respectively).
lgti = lgt * − 1/ n
(5)
lgtm = lgt * + 1/ n
(6)
In general, according to the calculations of Ward and Tordai, that is, Eq. (7) [26], the effective diffusion coefficient (Deff) at the start of the adsorption can be obtained. The Deff at the late stage of adsorption can also be calculated by Ward and Tordai’s formula, that is, Eq.(8) [26,30].
2.3. Equilibrium surface tension determination
γt → 0 = γ0 − 2nRTc0 The surface tensions of R1 and R2 were measured by K11 automatic tensiometer (Krüss, German) using the Wilhelmy plate method. The effects of temperature, salinity, and pH on the surface tension of R1 and R2 were also measured. In the range of 295–325 K, the temperature was controlled by a water bath connected with a tensiometer. In addition, the aqueous solutions of R1 and R2 were injected in a high-temperature and high-pressure reaction kettle. The reaction kettle was placed in a baking oven under different temperatures (353 K, 373 K, and 393 K) for 4 h. Then, the surface tension was measured after cooling. The change in salinity was achieved by dissolving R1 or R2 in NaCl solution at different concentrations (0, 20, 40, 80 and 100 g·L−1). The solution pH was adjusted with 1 mol/L HCl or 1 mol/L NaOH solution. The effects of salinity and pH on the surface tension of R1 and R2 were determined at 308.15 ± 0.1 K. According to the equilibrium surface tension curves, the adsorption capacity of surfactant at the air-water interface (Г) and the minimum area occupied by per surfactant molecule (Amin) were obtained using the Gibbs adsorption isothermal Eqs. (1) [25] and (2), respectively.
Γ=−
1 ⎛⎜ ∂γ ⎞⎟ 2.303nRT ⎝ ∂lgc ⎠T
Dt π
(7)
where c0 denotes the surfactant concentrations; R = 8.314 J mol−1 K−1; π = 3.142, and n = 2 for ionic surfactants [26]:
γt →∞ = γeq +
2 nRTΓeq
c
π 4Dt
(8)
where γeq indicates the equilibrium surface tension, and Γeq refers to the equilibrium surface exceeding the concentration calculated using the Gibbs adsorption (Eq. (1)); R is the gas constant (8.314 J mol−1 K−1), and T is absolute temperature (K). 2.5. Turbidity measurement To investigate the aggregation behavior of R1 and R2, turbidity measurements were performed to elucidate the transformation of the rhamnolipid aggregates. UV–vis spectrophotometer (UV-2450, Puxi Instruments Ltd., China) was used to measure sample absorbance at a wavelength of 600 nm (A600). Under different pH values (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5), the turbidities of R1 and R2 solutions at 5.0 × 10−4 mol/L concentration were examined at 298.15 K.
(1)
where γ refers to the surface tension of surfactant solution at a certain concentration (c); n is a constant that depends on the type of surfactant absorbed at the interface. n = 2 was employed for R1 and R2 [26–28]. T denotes the experimental temperature, and R is the gas constant (8.314 J mol−1 K−1).
2.6. DLS measurement
where Γmax corresponds to the maximum adsorption capacity, and NA is Avogadro’s constant.
DLS measurements were performed on a Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., UK). The scattering angle was 173°, and scattering intensity was recorded by the software supplied with the instrument to obtain the hydrodynamic diameter (DH). In the experiment, DH was represented by the number particle size distribution (PSD). The effects of temperature, salinity, and pH on the size distribution of self-aggregates of R1 and R2 were studied. The experiments were carried out at 298.15 ± 0.1 K.
2.4. Dynamic surface tension (DST) determination
2.7. Statistical analysis
A bubble profile tensiometer (Tracker, Teclis-IT Concept, France) was used to monitor the DST curves of R1 and R2 at 308.15 ± 0.1 K. Five microliters of air in a stainless reverse steel needle was contained in a gas-tight syringe to form the air-water interface. The bent needle was immersed vertically in the surfactant solution filled in a quartz cuvette. The bubble image was captured by a camera was analyzed by software employing the Laplace equation. The only difference between dynamic interfacial tension measurement and DST measurement is that a 5 μl organic phase was used in the stainless reverse steel needle attached to a gas-tight syringe to form an oil-water interface. The DST curves can be fitted by Rosen empirical formula (3) [29],
Some data presented in this study are the average ± SD of the experiments repeated at least three times. For each sample group, the homogeneity of variance was calculated using the paired t-test.
Amin =
1018 NA Γmax
(γ0 − γt )/(γt − γm ) = (t / t *)n
(2)
3. Results and discussion 3.1. Equilibrium surface properties of R1 and R2 3.1.1. CMC of R1 and R2 To determine the CMC, the equilibrium surface tension curves of R1 and R2 were measured by the Wilhelmy plate method. As shown in Fig. 2, with the increase in concentration, the surface tension gradually decreased until an inflection point occurred, which is defined as the surfactant CMC [4]. According to Fig. 2, the CMC values of R1 and R2 measured 4.0 × 10−4 mol/L and 4.6 × 10−4 mol/L, respectively. Both the CMC value and the equilibrium surface tension value of R1 were less than those of R2, indicating the superior surface activity of R1 compared with that of R2. Ikizler et al. reported that the CMC of R1 amounted to 1.0 × 10-4 mol/L, and that of R2 reached 1.5 × 10-4 mol/ L, that is, the CMC of R1 was more significant than that of R2 [23]; this finding coincides with our results. As shown in Fig. 1, R1 molecules feature more hydrophobicity than R2 molecules. Thus R1 molecules
(3)
where γ0 represents the surface tension of pure solvent; γt specifies the surface tension of the surfactant solution at interface age (t); γm indicates the meso-equilibrium surface tension, and n and t* are constants. n and t* can be obtained from the slope and intercept of plots of lg[(γ0 − γt )/(γt − γm )], respectively, versus lgt. The drop rate constant (R1/2) can be calculated by Eq. (4) [30]. Furthermore, parameters of ti and tm can be calculated by Eqs. (5) and (6), respectively [29].
R1/2 = (γ0 − γm )/2t *
(4) 595
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
of R1 and R2 are stable at the studied temperature range. To be used at the high temperature, the surfactant with high structure stability have been synthesized, e.g., sulfonate surfactants. However, these surfactants are challenging to be degraded in the environment. Biosurfactants R1 and R2 not only have resistance to a high temperature but also have the benefit to the environment. Thus, R1 and R2 can be used in the oil production process under high-temperature conditions. 3.1.3. Effects of salinity on the surface properties of R1 and R2 Fig. 4(a) and (b) illustrate the influence of salinity on the values of γ for R1 and R2, respectively. As cNaCl increased, the values of γ for R1 decreased firstly and then increased below CMC. By contrast, the values of γ for R2 decreased first and then stabilized. As shown in Table S1, when the surfactant concentration reached its CMC, NaCl addition caused no effect on the γ values for R1 and slightly affected those for R2. The result is attributed to the compressed double electric layer of ionic headgroups caused by NaCl addition. Thus, the electrostatic repulsion between hydrophilic headgroups decreased, and the number of surfactant molecules adsorbed to the surface increased. Moreover, as cNaCl increased, the hydrophilicity of the surfactant molecules decreased. As a result, the surfactant molecules can easily absorb to the surface. Moreover, NaCl addition will reduce the equilibrium surface tension. Helvac et al. discovered that the values of γ for R1 and R2 decreased when cNaCl increased from 0.05 mol/L to 1 mol/L [11]. According to the molecular structures of R1 and R2, NaCl addition will significantly affect the surface activity of R2, which includes two hydrophilic groups. Similarly, as indicated in Table S1, the CMC value decreased with the increase in cNaCl. Due to less hydrophilic headgroups, R1 molecules have a relatively small solubility that R2 molecules. At a high electrolyte concentration, R1 solubility decreased, causing an increase in surface tension.
Fig. 2. Variation of surface tension with surfactant concentration for R1 and R2 at 298.15 K. Table 1 Critical micelle concentration (cmc), value of surface tension at cmc (γcmc), minimum area occupied by per surfactant molecule (Amin), and maximum adsorption capacity (Гmax) of R1 and R2 obtained from surface tension curves at 298.15 K. Surfactant
cmc (mmol/ L)
γcmc (mN/ m)
Amin (nm2/ molecule)
Гmax (μmol/ m2)
R1 R2
0.40 0.46
27.44 31.27
1.10 1.38
1.52 1.21
aggregate into micelles at a lower concentration than R2. According to Eqs. (1) and (2), the adsorption capacity of the surfactant at the air-water interface (Г) and the minimum area occupied by per surfactant molecule (Amin) were obtained, respectively. Table 1 lists the calculation results. As shown in Table 1, the Amin of R1 is slightly less than that of R2, while the Γmax of R1 is greater than that of R2. Owing to the lesser number of hydrophilic headgroup, R1 is hydrophobic and can be easily absorbed to the surface. Moreover, as the molecular weight of R1 is smaller than that of R2, R1 molecules were packed more densely at the interface than those of R2. Amin of R1 is small, and its Γmax is larger than that of R2.
3.1.4. Effects of pH on the surface properties of R1 and R2 Fig. 5(a) and (b) depict the effects of pH on the surface properties of R1 and R2, respectively. When pH increased from 4.0 to 10.0, the values of γeq for R1 and R2 remained unchanged. When the pH was less than 4, the γ values for R2 decreased significantly compared with those of R1. This result is attributed to the enlarged hydrophobicity and solubility of surfactant molecules at pH less than 4. As shown in Fig. 1, both R1 and R2 molecules have carboxyl and hydroxyl groups. In general, carboxylate surfactant cannot be used in an acidic environment because of the protonation of carboxyl groups. The solubility of carboxylate surfactants will be sharply reduced in the acidic environment. However, the hydroxyl groups of R1 and R2 provide a large solubility at pH less than 4. As previously reported, the pKa values of mono- and dirhamnolipid are 5.5 and 5.6, respectively [31]. Below pH 4, the protonation of hydroxyl groups of R1 and R2 decreases the electrostatic repulsive of hydrophilic groups. Thus these surfactants can pack more closely at the air-water interface and exhibit a low value of surface tension. At the same time, the hydroxyl groups will interact with water molecules by hydrogen bonds. Biosurfactants R1 and R2 possess higher surfactant activities at strongly acidic environment than other anionic surfactants. When pH was greater than 10, both R1 and R2 exhibited a slight increase in equilibrium surface tension. Owing to two hydrophilic headgroups, the surface tension of R2 is sensitive to the pH values. Therefore, anionic surfactants R1 and R2 are highly deprotonated and hydrophilic above pH 10. Thus, their values of equilibrium surface tension increased.
3.1.2. Effects of temperature on the surface activity of R1 and R2 Considering high surface activity and biodegradability, rhamnolipids exhibit extensive application foreground in different industrial fields. In most cases, however, surfactants are used in environments with different temperatures, acidity, or salinity. Therefore, the stabilities of R1 and R2 at different temperatures, salinity, and pH values were studied. In this study, the effects of temperature (298–393 K) on the equilibrium surface tension of R1 and R2 were investigated. Considering that the surface tensiometer cannot be used at high temperatures, the effects of temperature on the surface tension of R1 and R2 were examined using two different methods according to the temperature ranges. In the range of 298–323 K, the values of surface tension were directly measured using the K11 surface tensiometer. Above 323 K, the R1 and R2 solutions were injected in a high-temperature and high-pressure reaction kettle. The kettle was placed in a baking oven at different temperatures (353, 373, and 393 K) for 4 h. Then, surface tension was measured after cooling. The results are shown in Fig. 3. According to Fig. 3(a), at 4.0 × 10−5 mol/L, the γ values for R1 reduced from 38.97 mN/m to 35.55 mN/m as the temperature increased from 298 K to 393 K. At the other two concentrations, the values of γ for R1 also decreased slightly with the increase in temperature. According to Fig. 3(b), for R2, at 4.6 × 10−5 mol/L, the values of γ decreased from 44.39 mN/m to 41.36 mN/m as the temperature increased from 298 K to 393 K. The results indicate that temperature exerts a negligible effect on the surface activity of R1 and R2. As a result, the molecule structures
3.2. DST of R1 and R2 3.2.1. DST curves of R1 and R2 DST curves were determined using drop-shape analysis techniques to understand the diffusion and adsorption of surfactant molecules to the interface. In general, a typical DST curve includes the induction, rapid fall, meso-equilibrium, and equilibrium regions [32]. Fig. 6 shows 596
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
Fig. 3. The values of the surface tension of R1 (a) and R2 (b) at different temperatures (298 K; 303 K; 308 K; 313 K; 318 K; 323 K; 353 K; 373 K; 393 K). Concentration of R1 (mol/L): 4.0 × 10−5; 2.0 × 10-4; 4.0 × 10-4. Concentration of R2 (mol/L): 4.6 × 10−5; 2.3 × 10-4; 4.6 × 10-4 mol/L. Error bars represent standard error of the mean. *P < 0.05 versus the group of 298 K using paired t-test.
Fig. 4. Effects of the addition of NaCl on the surface tension curves of R1 and R2 at 308.15 K. Concentration of NaCl (g/L): 0, 20, 40, 80, 100.
A high n value implies a substantial difference between the adsorption energy and desorption energy and easy adsorption of surfactant molecules adsorption to the surface. n and t* can be obtained from the slope and intercept of plots of lg[(γ0 − γt )/(γt − γm )], respectively, versus lgt as shown in Figure S3. The drop rate constant (R1/2) can be calculated by Eq. (4) [30]. Furthermore, parameters of ti and tm can be calculated by Eqs. (5) and (6) [29], respectively. Table 2 provides the calculated DST parameters of R1 and R2. With the increase in surfactant concentration, t*, n, and tm decreased, whereas R1/2 increased. This finding is explained as follows: for ionic surfactants, at the start of the adsorption (the process of surfactant molecules diffusion from the bulk phase to the subsurface), high concentration of the bulk phase indicated a rapid molecular diffusion, easy-to-reach meso-equilibrium region, and decreased t* and tm [13]. At the later stage of adsorption (surfactant adsorption from the subsurface to the surface), the surfactant concentration at the surface also increases with increased surfactant concentration in the bulk phase. This phenomenon results in increased required energy (adsorption
the DST curves of R1 and R2. The induction periods of R1 and R2 were extremely short, and the meso-equilibrium state was attained at approximately 1000s. This result indicates that both R1 and R2 feature excellent dynamic surface activities. At the same concentration, the value of equilibrium surface tension for R1 was smaller than that for R2, and this result is attributed to the additional hydrophilic headgroup of R2. Moreover, given the high steric adsorption resistance of R2, its maximum adsorption amount of R2 is less than that of R1. Thus, R2 exhibits a small surface tension value at equilibrium state. The DST curves of surfactant solutions have been fitted by the Rosen empirical formula (Eq. (3)) [29]. The t* values reflect the diffusion process of surfactant monomers from the bulk phase to the subsurface. Small t* indicates a short time required for surfactant molecules to diffuse from the bulk phase to the subsurface and easy achievement of the meso-equilibrium region. n is an empirical constant mainly related to the molecular structure of surfactants, and it reflects the difficulty of adsorption of monomers from the subsurface to the surface. n causes no considerable change in the range of a certain surfactant concentration.
Fig. 5. Effects of pH on the surface tension of R1 (a) and R2 (b) at 308.15 K. Concentration of R1 (mol/L): 4.0 × 10−5; 2.0 × 10-4; 4.0 × 10-4. Concentration of R2 (mol/L): 4.6 × 10−5; 2.3 × 10-4; 4.6 × 10-4 mol/L. The pH values: 2; 4; 6; 7; 8; 10; 12. Error bars represent standard error of the mean. **P < 0.01 and ***P < 0.001 versus the group of pH 7 using paired t-test.
597
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
Fig. 6. (a) Variation of surface tension with surface age (t) for R1 at 308.15 K; (b) Variation of surface tension with surface age (t) for R2 at 308.15 K. Concentration of R1 and R2 (mol/L): 4.6 × 10−6; 9.2 × 10−6; 1.0 × 10-5; 4.6 × 10-5.
energy barrier) for adsorption, difficulty in adsorption, and decreased n. Furthermore, with the increase in concentration in the bulk phase, the drop rate of the surface tension from the values of a pure solvent to meso-equilibrium values increased. The dynamic surface activity and R1/2 also increased. As shown in Table 2, t*, n and tm decreased, and R1/ 2 increased with increasing concentrations of R1 and R2. This result is consistent with the above conclusion. From the perspective of hydrophobicity, strong hydrophobicity of molecules indicates slow molecular diffusion in the bulk phase and large t* value. The increase in hydrophobicity of molecules allows easy adsorption of the subsurface to the surface. Thus, the value of n is high. The drop rate of the surface tension decreases, and R1/2 decreases [33]. According to the molecular structures of R1 and R2, the hydrophobicity of R1 is higher than that of R2. Thus, the values of n and t* should be large at the same concentration. These findings are consistent with experimental results. Table 2 also shows that with the increase in concentrations of R1 and R2, R1/2 increased, with the value of R1/2 for R2 being greater than that of R1. This outcome indicates the poorer dynamic surface activity of R1 than R2. In general, according to Ward and Tordai (Eq. (7)) [26], the Deff at the start of the adsorption process can be obtained from Figure S4. Moreover, the Deff at the late stage of adsorption can be calculated by Ward and Tordai’s formula (Eq. (8)) from Figure S5 [26,30]. When the surfactant concentration was lower than 4.6 × 10−5 mol/L, the starting point for the drop of surface tension approximated 70 mN/m, and t1/2 and γ were linear. These results indicate that the initial adsorption of R1 and R2 is diffusion-controlled. Table 3 shows the Deff for R1 and R2. By comparing the Deff of the late of adsorption of R1 and R2 under the same surfactant concentration, the Deff of R1 was greater than that of R2 at the same concentration. From the perspective of hydrophobicity, high hydrophobicity of molecules indicates a low requirement for energy barrier for adsorption, enabling the easy adsorption of surfactant, thus increasing the Deff value. From Table 3, at the late stage of adsorption, the Deff of R1 was greater than that of R2, consistent with their molecular structures.
Table 3 Effective diffusion coefficients of R1 and R2 at 308.15 K. System
R1 R1 R1 R1 R2 R2 R2 R2
c (mol L−1)
4.6 × 10−6 9.2 × 10−6 1.0 × 10−5 4.6 × 10−5 4.6 × 10−6 9.2 × 10−6 1.0 × 10−5 4.6 × 10−5
Long time t→∞ dγ/dt−1/2(mN m-1 s-1/2)
Deff (10−11 m2 s-1)
150.84 122.85 117.00 68.47 168.14 109.90 105.16 96.70
1.38 0.52 0.48 0.07 0.45 0.26 0.24 0.01
study. Figs. 7 shows the dynamic interfacial tension curves of R1 and R2. The equilibrium interfacial tension of R1 is lower than that of R2. At the surface, the period for surface tension attained a meso-equilibrium state at approximately 1000s, whereas approximately 500 s was required for the oil-water interface. These results indicate that the presence of the organic phase is conducive to the state of adsorption equilibrium. Furthermore, comparing the dynamic interfacial and surface tension curves, neither rhamnolipid presented an induction period. In other words, the dynamic surface and interfacial tension curves of R1 and R2 only included the rapid fall, meso-equilibrium, and equilibrium regions. Therefore, both R1 and R2 possess good dynamic interfacial activities. In this study, the effects of organic phases (decane, toluene, and diesel) on the dynamic interfacial tension curves of R1 and R2 were studied. As shown in Fig. 7(a), the values of decane/water interfacial tension for R1 and R2 decreased from 46 mN/m to 20 and 26 mN/m, respectively. In Fig. 7(b), the values of diesel/water interfacial tension for R1 and R2 decreased from 57 mN/m to 28 and 34 mN/m, respectively. Fig. 7(c) shows that the value of toluene/water interfacial tension for R1 reduced from 36 mN/m to 19 and 24 mN/m for R2. According to Figure S6, the kinetic parameters of interfacial processes were calculated (Table 4). For R1 and R2, the organic phase was toluene, and the shortest mesoscopic equilibrium time (tm) was achieved. Meanwhile, decane yielded the longest tm. Similarly, from Table 4, the n
3.2.2. Dynamic interfacial tension of R1 and R2 In addition to dynamic surface activity, the dynamic interfacial activity of R1 and R2 at 4.6 × 10−6 mol/L was also investigated in this Table 2 Parameters of dynamic surface tension of the R1 and R2 at 308.15 K. Surfactants
c (mol L−1)
γm (mN/m)
n
t*/s
tm/s
ti/s
R1/2 (mN m−1 s−1)
R1 R1 R1 R1 R2 R2 R2 R2
4.6 × 10−6 1.0 × 10−5 3.6 × 10−5 4.6 × 10−5 4.6 × 10−6 1.0 × 10−5 3.6 × 10−5 4.6 × 10−5
49.0 47.0 44.0 36.0 52.0 49.0 46.0 36.0
3.66 3.52 3.50 1.57 2.95 2.75 2.37 1.07
269.15 208.93 144.54 63.10 230.42 151.36 78.43 25.12
504.66 398.12 281.84 275.42 502.92 346.74 208.93 213.80
144.54 109.65 74.13 14.25 104.71 66.07 29.51 2.95
0.039 0.055 0.086 0.300 0.039 0.066 0.147 0.680
598
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
Fig. 7. (a) Variation of water/decane interfacial tension with surface age (t) for R1 and R2 solution at 298.15 K with and without high temperature ageing; (b) Variation of water/diesel oil interfacial tension with surface age (t) for R1 and R2 solution at 298.15 K with and without high temperature ageing; (c) Variation of water/toluene interfacial tension with surface age (t) for R1 and R2 solution at 298.15 K with and without high temperature ageing. Concentration of R1 and R2 (mol/L): 4.6 × 10−6.
Table 4 Parameters of dynamic interfacial tension of R1 and R2 solution at 298.15 K with and without high-temperature aging. System
γm (mN/ m)
n
t*/s
tm/s
ti/s
R1/2 (mN m−1 s−1)
R1 R1 R2 R2 R1 R2 R1 R2
20.0 20.8 26.1 25.9 27.8 35.1 19.6 23.2
1.14 1.11 1.08 1.06 1.70 1.65 2.20 2.10
386.99 409.84 375.04 401.58 210.62 177.83 208.93 182.77
3162.28 3235.94 3162.27 3523.71 812.83 707.95 588.84 616.60
51.28 51.49 43.65 44.77 53.70 43.25 72.44 61.66
0.033 0.031 0.041 0.038 0.069 0.062 0.039 0.035
+ decane Ageing + decane + decane Ageing + decane + diesel + diesel + toluene + toluene
Fig. 8. Variation of the size for R1 and R2 aggregates with concentration at 298.15 K. Concentration of R1 (mol/L): 2.0 × 10−5; 5.0 × 10−5; 1.0 × 10-4; 5.0 × 10-4; 1.0 × 10-3. Concentration of R2 (mol/L): 2.0 × 10−5; 5.0 × 10−5; 1.0 × 10-4; 5.0 × 10-4; 1.0 × 10-3. Error bars represent standard error of the mean. **P < 0.01 and ***P < 0.001 versus the group of 5.0×10-4 mol/L using paired t-test.
of R1 was slightly larger than that of R2 at the same organic phase as R1 is more hydrophobic than R2. The dynamic interfacial tensions of R1 and R2 aged at high temperature were also examined. High-temperature aging involves placing the surfactant solutions of R1 and R2 into a high-temperature and highpressure reaction kettle at 393.15 K for 4 h. Then, the dynamic interfacial tension curves were measured after cooling. Fig. 7 shows that the difference in dynamic interfacial tension curves of R1 and R2 with and without high-temperature aging reached less than ± 1 mN/m. Thus, high-temperature aging results in minimal influence on the dynamic interfacial activity of R1 and R2. Moreover, from Table 4, before and after high-temperature aging, a slight difference was observed in the dynamic interfacial parameters of R1 and R2. Thus, the molecule structures of R1 and R2 was stable in the studied temperature range. R1 and R2 can be used at high temperatures.
aggregation behavior of R1 and R2 were examined. As presented in Fig. 8, the size of R1 aggregations gradually increased with the increase in concentration. And the poly dispersity index (PDI) of R1 and R2 at the concentration of 2.0 × 10−5 mol/L, 5.0 × 10-5 mol/L, 1.0 × 10-4 mol/L, 5.0 × 10-4 mol/L and 1.0 × 10-3 mol/L was shown in Table S2. However, as the R2 concentration increased, the size of R2 aggregations decreased first and then increased, indicating that the increase in concentration is conducive to the formation of R1 aggregates. We mainly studied the influence of temperature and pH on the aggregation behavior of R1 and R2. The surfactant concentration was slightly higher than CMC when studying the influence of external factors on the aggregation behavior. Fig. 9 shows the influence of temperature on the aggregation behavior of R1 and R2. And the poly dispersity index (PDI) of R1 and R2 at the temperature of 293 K, 298 K, 303 K, 313 K, 318 K, and 323 K was shown in Table S3. At temperature less than 318 K, the sizes of R1 and R2 aggregates approximated 50 nm. When the temperature was greater than 318 K, the size of R1 aggregates was greater than 200 nm, whereas that of R2 aggregates was close to 200 nm. These observations indicate that 318 K may be a transition point of R1 and R2 aggregates as they changed from micelles to
3.3. Aggregation behavior of rhamnolipids To investigate the aggregation behavior of R1 and R2, DLS and turbidity measurements were performed to elucidate the transformation of the rhamnolipid aggregates. As the size distribution of intensity amplifies the incoming signal by approximately a million times, an extremely small amount of larger material in the solution will affect the experimental results. However, the number peak reflects the actual number of particles in each particle size. Therefore, DH was shown by the number PSD in this study. First, the effects of concentration on 599
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
transformation of R1 and R2 aggregates into vesicles. Fig. 10 (a) displays the influence of pH on the aggregation behavior of R1 and R2 at 5 × 10−4 mol/L concentration. And the poly dispersity index (PDI) of R1 and R2 at the pH of 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5 was shown in Table S4. With the increase in pH, a significant decrease in the size of surfactant aggregates was observed. For R1, with the increase in pH, the size of aggregates decreased from 350 nm to 40 nm, and pH = 3.5 was a notable break point. However, for R2, with the increase in pH, the size of aggregates decreased gradually from 450 nm to 50 nm. Such finding is attributed to the considerable influence of solution pH to the dissociation degree of hydrophilic headgroups. With the increase in pH, the number of ionized rhamnolipids increased. Increasing electrostatic repulsion results in a small micelle curvature, inducing the transformation of larger aggregates to small ones. Turbidity was also measured to clarify the transformations occurring in R1 and R2 aggregates. Figs. 10 (b) show the turbidity changes, whereas Fig. 10 (c) and (d) show the changes in physical appearance of R1 and R2. The changing trend of turbidity is consistent with that of aggregate diameter. Therefore, with the increase in pH, the size of rhamnolipid aggregates decreased at various concentrations. Larger particles transformed into small ones at the same time. Champion et al. showed that the aggregation morphology of mono-rhamnolipids solution with a high concentration (60 mmol/L) increased from 6.0 to 8.0 with pH value and changed from large double-layer capsule, large
Fig. 9. Variation of the size for R1 and R2 aggregates with temperatures. Concentration of R1 and R2 (mol/L): 5 × 10−4 mol/L. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the group of 293 K using paired t-test.
vesicles. Increasing temperature causes the surfactant molecules to be less hydrated and more hydrophobic. Therefore, the cpp values of R1 and R2 will increase. The lateral van der Waals interaction between R1 and R2 surfactant molecules is likely to form a bilayer structure. In other words, the increase in temperature is conducive to the
Fig. 10. (a) Variation of aggregate sizes of R1 and R2 with pH values at 298.15 K; (b) Variation of turbidity of R1 and R2 solution with pH values at 298.15 K; (c) The appearance of R1 solution at different pH values and 298.15 K; (d) The appearance of R2 solution at different pH values and 298.15 K. Concentration of R1 and R2 (mol/L): 5 × 10−4 mol/L. The pH values: 2.5; 3.5; 4.5; 5.5; 6.5; 7.5; 8.5; 9.5; 10.5. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the group of pH 7.5 using paired t-test. 600
Colloids and Surfaces B: Biointerfaces 181 (2019) 593–601
L.-m. Wu, et al.
single-layer capsule, and small single-layer capsules to micelles [34]. In this study, the size of R1 and R2 aggregates decreased with the increase in pH at low concentration (around CMC). This observation is consistent with the abovementioned findings. This result also indicates the similar effects of pH on the aggregation behavior of R2 and R1.
272 (2019) 19–25. [10] A. Aparna, G. Srinikethan, H. Smitha, Production and characterization of biosurfactant produced by a novel Pseudomonas sp 2B, Colloid Surf. B-Biointerfaces 95 (2012) 23–29. [11] S.S. Helvaci, S. Peker, G. Ozdemir, Effect of electrolytes on the surface behavior of rhamnolipids R1 and R2, Colloids Surf. B Biointerfaces 35 (2004) 225–233. [12] S.P. Verma, B. Sarkar, Simultaneous removal of Cd (II) and p-cresol from wastewater by micellar-enhanced ultrafiltration using rhamnolipid: flux decline, adsorption kinetics and isotherm studies, J. Environ. Manage. 213 (2018) 217–235. [13] S. Mohanty, S. Mukherji, Surfactant aided biodegradation of NAPLs by Burkholderia multivorans: comparison between Triton X-100 and rhamnolipid JBR-515, Colloid Surf. B-Biointerfaces 102 (2013) 644–652. [14] O. Pornsunthorntawee, S. Chavadej, R. Rujiravanit, Solution properties and vesicle formation of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa SP4, Colloid Surf. B-Biointerfaces 72 (2009) 6–15. [15] G. Ozdemir, U. Malayoglu, Wetting characteristics of aqueous rhamnolipids solutions, Colloids Surf. B Biointerfaces 39 (2004) 1–7. [16] A.A. Dar, G.M. Rather, S. Ghosh, A.R. Das, Micellization and interfacial behavior of binary and ternary mixtures of model cationic and nonionic surfactants in aqueous NaCl medium, J. Colloid Interface Sci. 322 (2008) 572–581. [17] L. Dai, S. Yang, Y. Wei, C. Sun, D.J. McClements, L. Mao, Y. Gao, Development of stable high internal phase emulsions by pickering stabilization: utilization of zeinpropylene glycol alginate-rhamnolipid complex particles as colloidal emulsifiers, Food Chem. 275 (2019) 246–254. [18] M. Sanchez, F.J. Aranda, M.J. Espuny, A. Marques, J.A. Teruel, A. Manresa, A. Ortiz, Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa in aqueous media, J. Colloid Interface Sci. 307 (2007) 246–253. [19] J.T. Champion, J.C. Gilkey, H. Lamparski, J. Retterer, R.M. Maier, Electron microscopy of rhamnolipid (biosurfactant) morphology, J. Colloid Interface Sci. 170 (1995) 569–574. [20] K. Matsuoka, K. Takagi, C. Honda, Micelle formation of sodium hyodeoxycholate, Chem. Phys. Lipids 172 (2013) 6–13. [21] Z.A. Raza, Z.M. Khalid, M.S. Khan, I.M. Banat, A. Rehman, A. Naeem, M.T. Saddique, Surface properties and sub-surface aggregate assimilation of rhamnolipid surfactants in different aqueous systems, Biotechnol. Lett. 32 (2010) 811–816. [22] R.J. Eismin, E. Munusamy, L.L. Kegel, D.E. Hogan, R.M. Maier, S.D. Schwartz, J.E. Pemberton, Evolution of aggregate structure in solutions of anionic monorhamnolipids: experimental and computational results, Langmuir 33 (2017) 7412–7424. [23] B. Ikizler, G. Arslan, E. Kipcak, C. Dirik, D. Celenk, T. Aktuglu, S.S. Helvaci, S. Peker, Surface adsorption and spontaneous aggregation of rhamnolipid mixtures in aqueous solutions, Colloids and Surfaces a-Physicochemical and Engineering Aspects 519 (2017) 125–136. [24] L. Leclercq, V. Nardello-Rataj, M. Turmine, N. Azaroual, J.-M. Aubry, Stepwise aggregation of Dimethyl-di-n-octylammonium chloride in aqueous solutions: from dimers to vesicles, Langmuir 26 (2010) 1716–1723. [25] S. Ghosh, Surface chemical and micellar properties of binary and ternary surfactant mixtures (Cetyl pyridinium chloride, Tween-40, and Brij-56) in an aqueous medium, J. Colloid Interface Sci. 244 (2001) 128–138. [26] J.G. Gobel, G.R. Joppien, Dynamic interfacial tensions of aqueous Triton x-100 solutions in contact with air, cyclohexane, n -heptane, and n -hexadecane, J. Colloid Interface Sci. 191 (1997) 30–37. [27] A. Abalos, A. Pinazo, M.R. Infante, M. Casals, F. García, A. Manresa, Physicochemical and antimicrobial properties of new rhamnolipids produced by pseudomonas a eruginosa AT10 from soybean oil refinery wastes, Langmuir 17 (2001) 1367–1371. [28] D. Manko, A. Zdziennicka, B. Janczuk, Composition of surface layer at the WaterAir Interface and Micelles of Triton x-100+Rhamnolipid mixtures, J. Solution Chem. 46 (2017) 1251–1271. [29] M.J. Rosen, X.Y. Hua, Dynamic surface tension of aqueous surfactant solutions: 2. Parameters at 1 s and at mesoequilibrium, J. Colloid Interface Sci. 139 (1990) 397–407. [30] S.-J. Li, L. Lai, P. Mei, Y. Li, L. Cheng, Z.-H. Ren, Y. Liu, Equilibrium and dynamic surface properties of cationic/anionic surfactant mixtures based on bisquaternary ammonium salt, J. Mol. Liq. 254 (2018) 248–254. [31] H. Abbasi, K.A. Noghabi, M.M. Hamedi, H.S. Zahiri, A.A. Moosavi-Movahedi, M. Amanlou, J.A. Teruel, A. Ortiz, Physicochemical characterization of a monorhamnolipid secreted by Pseudomonas aeruginosa MA01 in aqueous media. An experimental and molecular dynamics study, Colloid Surf. B-Biointerfaces 101 (2013) 256–265. [32] M.J. Rosen, L.D. Song, Dynamic Surface Tension of Aqueous Surfactant Solutions 8. Effect of Spacer on Dynamic Properties of Gemini Surfactant Solutions, J. Colloid Interface Sci. 179 (1996) 261–268. [33] H. Xi Yuan, M.J. Rosen, Dynamic surface tension of aqueous surfactant solutions: I. Basic paremeters, J. Colloid Interface Sci. 124 (1988) 652–659. [34] F.J. Aranda, M.J. Espuny, A. Marques, J.A. Teruel, A. Manresa, A. Ortiz, Thermodynamics of the interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with phospholipid membranes, Langmuir 23 (2007) 2700–2705.
4. Conclusion Rhamnolipids homologs R1 and R2 both possess good surface activity, but the surface activity of R1 is superior to that of R2. The values of γ for R1 and R2 decreased slightly with the increase in temperature. The results indicate that temperature barely affects the surface activity of R1 and R2. NaCl addition significantly influences the surface activity of R2, which contains two hydrophilic groups. However, at high electrolyte concentration, R1 solubility decreases, thus increasing the surface tension. Owing to two hydrophilic headgroups, the surface tension of R2 is sensitive to pH values. For dynamic surface/interfacial tension, the induction periods of R1 and R2 are notably short, and initial adsorption of R1 and R2 is diffusion-controlled. With the increase in R1 and R2 concentrations, R1/2 increases, and the value of R1/2 for R2 is higher than that of R1. This result indicates the better dynamic surface activity of R2 than R1. The dynamic interfacial curves of R1 and R2 after high-temperature aging slight differ from those without hightemperature aging, thus also confirming the high-temperature resistance of R1 and R2. Finally, we studied the effects of temperature and pH on the aggregation behavior of R1 and R2. The increase in temperature and the decrease in pH values are conducive to the transformation of R1 and R2 aggregates into vesicles. Turbidity and DLS measurements verify well the transformation of R1 and R2 aggregates. Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (grant no. 21403017, 21473125). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.06.012. References [1] Y.-P. Guo, Y.-Y. Hu, R.R. Gu, H. Lin, Characterization and micellization of rhamnolipidic fractions and crude extracts produced by Pseudomonas aeruginosa mutant MIG-N146, J. Colloid Interface Sci. 331 (2009) 356–363. [2] R.M. Maier, G. Soberón-Chávez, Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications, Appl. Microbiol. Biotechnol. 54 (2000) 625–633. [3] M.L. Chen, J. Penfold, R.K. Thomas, T.J.P. Smyth, A. Perfumo, R. Marchant, I.M. Banat, P. Stevenson, A. Parry, I. Tucker, I. Grillo, Solution self-assembly and adsorption at the air-water interface of the monorhamnose and dirhamnose rhamnolipids and their mixtures, Langmuir 26 (2010) 18281–18292. [4] D. Manko, A. Zdziennicka, B. Janczuk, Thermodynamic properties of rhamnolipid micellization and adsorption, Colloid Surf. B-Biointerfaces 119 (2014) 22–29. [5] S.G.V.A.O. Costa, M. Nitschke, F. Lepine, E. Deziel, J. Contiero, Structure, properties and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater, Process. Biochem. 45 (2010) 1511–1516. [6] T. Janek, M. Lukaszewicz, A. Krasowska, Identification and characterization of biosurfactants produced by the Arctic bacterium Pseudomonas putida BD2, Colloid Surf. B-Biointerfaces 110 (2013) 379–386. [7] J.L. Parra, J. Guinea, M.A. Manresa, M. Robert, M.E. Mercadé, F. Comelles, M.P. Bosch, Chemical characterization and physicochemical behavior of biosurfactants, J. Am. Oil Chem. Soc. 66 (1989) 141–145. [8] C. Chen, N. Sun, D. Li, S. Long, X. Tang, G. Xiao, L. Wang, Optimization and characterization of biosurfactant production from kitchen waste oil using Pseudomonas aeruginosa, Environ. Sci. Pollut. Res. - Int. 25 (2018) 14934–14943. [9] V.K. Gaur, A. Bajaj, R.K. Regar, M. Kamthan, R.R. Jha, J.K. Srivastava, N. Manickam, Rhamnolipid from a Lysinibacillus sphaericus strain IITR51 and its potential application for dissolution of hydrophobic pesticides, Bioresour. Technol.
601