Hua
Zhong
*abc,
Xin
Yang
ab,
Fei
Tan
ab,
Mark L.
Brusseau
c,
Lei
Yang
ab,
Zhifeng
Liu
ab,
Guangming
Zeng
ab and
Xingzhong
Yuan
ab
aCollege of Environmental Science and Engineering, Hunan University, Changsha 410082, China. E-mail: yx2013@hnu.edu.cn; tanfei_2013@163.com; wlwyanglei@126.com; lzf18182002@163.com; zgming@hnu.edu.cn; yxz@hnu.edu.cn; zhonghua@hnu.edu.cn
bKey Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha, 410082, China
cDepartment of Soil, Water and Environmental Science, University of Arizona, Tucson, 1177 E 4th St., Arizona 85721, USA. E-mail: zhonghua@email.arizona.edu; brusseau@email.arizona.edu
First published on 7th December 2015
Solubilization of n-decane, dodecane, tetradecane and hexadecane by monorhamnolipid biosurfactant (monoRL) at concentrations near the critical micelle concentration (CMC) was investigated. The apparent solubility of all four alkanes increases linearly with increasing monoRL concentration either below or above the CMC. The capacity of solubilization presented by the molar solubilization ratio (MSR), however, is stronger at monoRL concentrations below the CMC. The MSR decreases following the order dodecane > decane > tetradecane > hexadecane at monoRL concentrations below the CMC. Formation of aggregates at sub-CMC monoRL concentrations was demonstrated by dynamic light scattering (DLS) and cryo-transmission electron microscopy. DLS-based size (d) and zeta potential of the aggregates decrease with increasing monoRL concentration. The surface excess (Γ) of monoRL calculated based on alkane solubility and aggregate size data increases rapidly with increasing bulk monoRL concentration and then asymptotically approaches the maximum surface excess (Γmax). The relationship between Γ and d indicates that the excess of monoRL molecules at the aggregate surface greatly impacts the surface curvature. The results demonstrate formation of aggregates for alkane solubilization at monoRL concentrations below the CMC, indicating the potential of employing low concentrations of rhamnolipid for enhanced solubilization of hydrophobic organic compounds.
Solubilization of HOCs by surfactants has been studied extensively at high surfactant concentrations, i.e. higher than the critical micelle concentration (CMC).7–14 Micelles are considered to be of spherical shape with three zones for solubilization: the core, the corona, and the core–corona interface.15,16 It is typically assumed that solubilization enhancement of hydrophobic compounds only occurs at surfactant concentrations higher than CMC.11,16,17
The results of some studies, however, showed that surfactants also solubilize HOCs at sub-CMC concentrations. For example, results of our prior study showed that synthetic surfactants SDBS and Triton X-100 enhanced solubilization of hexadecane at concentrations below the CMC based on an aggregate formation mechanism.18 There is evidence of similar behavior for biosurfactants, with Zhang and Miller reporting that solubility of octadecane was enhanced by rhamnolipid biosurfactant at sub-CMC concentrations. It is interesting to note that the enhancement was much more significant at concentrations below the CMC than at concentrations above.8 It was assumed that this sub-CMC enhancement of octadecane solubilization was due to the decrease of water–octadecane interfacial tension.8 In our prior study of hexadecane solubilization by rhamnolipids, similar results were also observed.19 Research is needed to delineate the mechanisms contributing to the sub-CMC solubilization capability observed for biosurfactants. This information is also relevant for the commercial application of biosurfactants in terms of their cost-effectiveness.
To date, rhamnolipid is the most extensively studied biosurfactant and has the greatest application potential. Solubilization of n-alkanes by rhamnolipid at concentrations near the CMC was investigated in this study, with a focus on solubilization behavior at concentrations lower than of CMC. Monorhamnolipid (monoRL), a rhamnolipid with one rhamnose ring and two alkyl chains (Fig. 1), was used in this study. MonoRL is selected because it is the class of species that always exists in a rhamnolipid mixture and is the precursor for biosynthesis of dirhamnolipid. Results of our prior study also showed that it appears to have stronger ability over dirhamnolipid and synthetic surfactants to enhance HOC solubilization at low concentrations.3,18 It is considered an anionic surfactant under the experimental conditions in this study due to the carboxyl group in the molecule (pKa = 5.6 under ambient temperature20). Four linear alkanes (n-decane, n-dodecane, n-tetradecane and n-hexadecane) with different chain lengths were selected to represent HOCs. In addition to n-alkane solubility, characterization of alkane–monoRL aggregates, such as measurement of aggregate size and zeta potential and cryo-TEM-based observation of aggregate morphology, was undertaken. Finally, surfactant interface partition theory, an assumption of spherical aggregates, and surfactant mass balance was used to interpret the sub-CMC solubilization of the alkanes by the rhamnolipid.
n-Alkanes (n-decane, n-dodecane, n-tetradecane and n-hexadecane) (purity >99%) were purchased from Sigma-Aldrich (St. Louis, Mo., U.S.A.). The selected properties of n-alkanes are listed in Table 1 and molecule structures are shown in Fig. 1. n-Octane (purity >95.0%) and HPLC grade ethanol were purchased from Damao Chemical Reagent Co. Ltd (Tianjin, China). All other chemicals were of analytical grade and used as received. Ultra-pure water with electrical resistivity of 18.2 MΩ cm produced by a UPT-II-40 (Ulupure, Chengdu, China) was used throughout the experiment. Phosphate buffer (PBS, 1.24 g L−1 KH2PO4 and 1.35 g L−1 K2HPO4·3H2O, pH 6.8) was used as the background electrolyte solution for monoRL solubilization. It provides a stable concentration of counterions, which is important for application of the Gibbs adsorption equation for monoRL with an ionic nature. In this PBS buffer, the degree of dissociation for the monoRL is 94% based on pKa of 5.6.20 Such a high degree of dissociation also supports the assumption in this study that the monoRL is anionic and resides only in the bulk solution or at the interface.
n-Alkane | Formula | Molecular weight (g mol−1) | Water solubilitya (μM, 25 °C) | log Kowb (25 °C) | Densityc (g cm−3, 25 °C) | CMCd (μM) | K (m3 mol−1) | Γ max (mol m−2) | A m (nm2) |
---|---|---|---|---|---|---|---|---|---|
a Solubilities of n-alkanes are reported by NCBI (ref. 25–28). b Octanol–water partition coefficient (Kow) values of n-alkanes from NCBI (ref. 25–28). c Relative density (water = 1) of n-alkanes from NCBI (ref. 25–28). d Critical micelle concentration (CMC) for monoRL biosurfactant in the presence of n-alkanes obtained by n-alkane-PBS interfacial tension measurement (CMC obtained by surface tension measurement in the absence of n-alkanes is 166 μM). | |||||||||
Decane | C10H22 | 142 | 0.37 | 5.01 | 0.73 | 150 | 0.98 × 103 | 3.1 × 10−6 | 0.54 |
Dodecane | C12H26 | 170 | 0.02 | 6.10 | 0.75 | 155 | 1.81 × 103 | 2.9 × 10−6 | 0.58 |
Tetradecane | C14H30 | 198 | 0.01 | 7.20 | 0.76 | 169 | 0.74 × 103 | 3.6 × 10−6 | 0.46 |
Hexadecane | C16H34 | 226 | 0.0004 | 8.25 | 0.77 | 152 | 0.57 × 103 | 4.1 × 10−6 | 0.41 |
The size and zeta potential of aggregate particles were measured using a ZEN3600 Zetasizer Nano (Malvern Instruments, U.K.). The particle size was determined based on the method of dynamic light scattering (DLS) at 633 nm with a He–Ne laser working at 4 mV power. 1 mL of sample was loaded to the DTS-0012 cell and maintained at 30 °C. The scattered light was collected by the receptor at an angle of 173° from the light path. The size of the aggregates was expressed in terms of hydrodynamic diameter, which was calculated using the software associated with the instrument. To obtain the zeta potential of the aggregates, approximately 1 mL of sample was loaded in the DTS1060 folded capillary cell and the electrophoretic mobility of the aggregate particles was measured at 30 °C under automatic voltage using laser Doppler velocimetry with the M3-PALS technique to avoid electroosmosis. The measured data were converted into the corresponding zeta potential by applying the Helmholtz–Smoluchowski equation.30
Fig. 2 (a) The air–PBS and n-alkane–PBS interfacial tension as a function of monoRL concentration. (b) Interfacial tension–concentration relation regression at monoRL concentrations below CMC using the Szyszkowski equation (eqn (3) in ref. 18). |
The interfacial tension data at sub-CMC monoRL concentration was well fit by eqn (3) in ref. 18 (Fig. 2b). The Langmuir adsorption constant (K), maximum interfacial access (Γmax), and minimum area per molecule (Am) obtained for the adsorption are summarized in Table 1. K decreases following the order dodecane > decane > tetradecane > hexadecane. Alkyl chain lengths in the monoRL are similar to those of dodecane and decane (Fig. 1), which may be favorable for hydrophobic interaction between monoRL and alkane molecules at the interface and hence lower the Gibbs energy, resulting in a stronger partitioning of monoRL at the interface in the cases of dodecane and decane. However, Γmax is large (Am is small) for tetradecane and hexadecane, showing that when the adsorption is saturated the monoRL molecules are more compacted at the interface for long-chain alkanes.
The solubilization capacity of a surfactant for a HOC is presented by the molar solubilization ratio (MSR), which is defined as the increase of solubilized HOC concentration (mol L−1) per unit increase of surfactant concentration (mol L−1) in the solution or the slope of the linear solubilization curve.32 The MSR for the four alkanes are listed in Table 2. MSR for all four alkanes are significantly higher at monoRL concentrations below the CMC than above the CMC. Similar results were observed for octadecane solubilization by monoRL8 and hexadecane solubilization by SDBS (also an anionic surfactant).18
n-Alkane | MSR | |
---|---|---|
Below CMC | Above CMC | |
Decane | 5.73 | 0.29 |
Dodecane | 8.28 | 2.91 |
Tetradecane | 3.27 | 0.94 |
Hexadecane | 2.55 | 0.89 |
These observations indicate a difference in the modes of alkane solubilization below and above the CMC. The MSR decreases in the order dodecane > decane > tetradecane > hexadecane at monoRL concentrations below the CMC (Table 2), which is the same as the order for K. This indicates a relationship between alkane solubilization and interfacial partitioning of monoRL. It can be noted that the MSR for hexadecane solubilization by the monoRL at sub-CMC concentrations (2.55) is larger than that for SDBS (0.84) and Triton X-100 (1.90),18 indicating higher solubilization efficiency of the biosurfactant monoRL over synthetic surfactants. This is probably due to the presence of two alkyl chains in the monoRL molecule.
Fig. 4 Number distribution of aggregate particles for solubilization of dodecane and hexadecane by monoRL at concentrations of 30 μM and 750 μM. |
For all four alkanes, the DLS particle size first decreases rapidly with increase of C0, and then stabilizes with increase of C0 above the CMC (Fig. 6). By amongst the alkanes, it is observed that the aggregates size at monoRL concentration equivalent to the CMC decreases in the order decane ≈ dodecane > tetradecane > hexadecane. This order is in contrast to the order of Γmax for these four alkanes, which increases in the order decane ≈ dodecane < tetradecane < hexadecane (Table 1).
Fig. 6 DLS aggregate size (diameter, d) versus the total monoRL concentration (C0) for n-alkane solubilization. Error bars show mean ± standard deviation. |
Zeta potentials of the aggregates are shown in Fig. 7. The aggregates are negatively charged. The change of zeta potential with increase of C0 exhibits a similar trend for all four alkanes. It decreases rapidly with increase of C0 to the CMC, and then stabilizes or decreases slowly with further increase of monoRL concentration.
Fig. 7 Zeta potential of aggregates versus the monoRL total concentration (C0) for n-alkane solubilization. Error bars show mean ± standard deviation. |
For all four alkanes, a linear relationship between the apparent solubility of alkane, Calk, and Cw is observed with increase of Cw to the CMC (Fig. 8a). This is similar to the relationship between Calk and C0 (the total monoRL concentration in solution) (Fig. 3). By comparing the slopes of the Calk–C0 profiles at C0 below the CMC with those of the Calk–Cw profiles (5.7 versus 7.5 for decane, 8.3 versus 10.8 for dodecane, 3.3 versus 5.3 for tetradecane, and 2.55 versus 6.3 for hexadecane), the percentage of the aggregate-associated monoRL is calculated to be 24%, 23%, 38%, and 59% of the total for decane, dodecane, tetradecane, and hexadecane, respectively. Note that the aggregate size for hexadecane is significantly smaller than that for the other three alkanes at C0 lower than CMC. The higher surface area for smaller particles is responsible for the enhanced partition of monoRL to the aggregates, in spite of the fact that the K and Calk for hexadecane are the smallest among the four alkanes.
The dependence of monoRL surface excess (Γ) and molecule area (A) versus Cw are presented in Fig. 8b. A rapid increase of Γ and decrease of A with increasing Cw are observed when Cw is low. Further increase of Cw causes asymptotic approach of Γ and A to Γmax and Am, respectively. More significant change of Γ and A is observed for the long-chain alkanes (tetradecane and hexadecane). Based on eqn (2) in ref. 18, Γ is more sensitive to change of Cw with a smaller K. K for the four alkanes follows the order dodecane > decane > tetradecane > hexadecane (Table 1). Thus, the most significant change of Γ and A over the broadest range of Cw occurred for hexadecane.
As shown in Fig. 9, for all four alkanes, aggregate size, d, decreases with the increase of monoRL surface excess in the aggregates, such that d approaches the stabilized minimum aggregate size (dmin) as Γ approaches Γmax. This result indicates that the curvature of the aggregate surface increases with increasing surface excess of monoRL molecules. Because monoRL is anionic and 94% of the monoRL molecules dissociate in PBS, the presence of monoRL causes a negative aggregate surface charge. Enhancement in electrostatic repulsion induces unequal rate of approach for polar and hydrophobic moieties between molecules, and therefore an increase in aggregate surface curvature (Fig. 10). Thus, the aggregate size, d, decreases with increasing Γ. Zeta potential is a function of both particle size and surface charge density.30,33,34 Therefore, it is essentially a function of Γ and its change also exhibits an asymptotic pattern of decrease at concentrations lower than the CMC (see Fig. 7).
Fig. 9 Aggregate diameters (d) versus surface excess of monoRL (Γ) at monoRL bulk concentration (Cw) below the CMC. Error bars show mean ± standard deviation. |
Fig. 10 Schematic diagram of alkane–monoRL aggregate formation at monoRL concentrations below the CMC. |
When monoRL concentration in a bulk solution (Cw) is higher than the CMC, Γ at the aggregate surface reaches Γmax and the size of aggregates reaches the minimum, giving low efficiency for alkane solubilization. As a result, the MSR at monoRL concentrations above the CMC is significantly smaller than that for monoRL concentrations below the CMC.
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