Hua
Zhong
*abc,
Lei
Yang
ab,
Xin
Yang
ab,
Guangming
Zeng
*ab,
Zhifeng
Liu
ab,
Yang
Liu
ab and
Xingzhong
Yuan
ab
aCollege of Environmental Science and Engineering, Hunan University, Changsha 410082, P. R. China. E-mail: zhonghua@email.arizona.edu; zgming@hnu.edu.cn; wlwyanglei@126.com; yx2013@hnu.edu.cn; lzf18182002@163.com; liuyang_feiyang@163.com; yxz@hnu.edu.cn
bKey Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082, P. R. China
cDepartment of Soil, Water and Environment Science, The University of Arizona, Tucson, AZ 85721, USA
First published on 12th October 2015
Dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM) tests demonstrated aggregate formation for dirhamnolipid biosurfactant (diRL) at concentrations lower than surface-tension-based critical micelle concentration (CMCst). An increase of diRL concentration and solution pH results in a decrease of the aggregate size at diRL concentrations below CMCst, whereas it has no influence at diRL concentrations above CMCst. The cryo-TEM micrographs show spherical morphology of the aggregates, and the logarithm of aggregate size follows Gaussian distribution. The aggregates are negatively charged. The zeta potential of the aggregates decreases with an increase of diRL concentration to CMCst, and stabilizes at diRL concentrations higher than CMCst. An increase of the solution pH causes a decrease of the zeta potential. A transitional state assumption is raised for the interpretation of the diRL aggregation behavior. The results demonstrate formation of aggregates at significantly low diRL concentrations, which is of importance for the cost-effective application of rhamnolipid biosurfactants.
Rhamnolipid is the most widely studied biosurfactant and its aggregates exhibit versatile structures at concentrations higher than the critical micelle concentration (CMC). For example, Ishigami et al. investigated the effect of solution pH on rhamnolipid aggregate structure at concentrations of 500–20000 mg L−1 in phosphate buffered saline solution. The result showed that the aggregates existing in form of bilayers vesicle at pH of 4.3–5.8, bilayer lamella with pH rising to 6.0–6.5, and micelles with further increase of pH to 6.8.18 Champion et al. determined the rhamnoliopid aggregate morphology at various pH at the concentration of 60 mM by cryo-TEM. The results show that aggregate phase transitioned in an order of bilayer lamella, large vesicles, small vesicles and micelle with the increase of pH.4 In addition, transformation of dirhamnolipid aggregations from large particles into small particles with the increase of concentration at fixed pH has also been reported.19
All these prior researches, however, were almost implemented with surfactant concentrations far higher than CMC. However, there are studies showing that rhamnolipid exhibited excellent HOC-solubilization activity at significantly low concentrations. For example, rhamnolipid can enhance the solubility of hexadecane and octadecane by 3–4 orders of magnitude at concentrations lower than CMC determined by surface tension method, and such solubilization efficiency is much higher than at concentrations above CMC.16,20 Hypothetically these HOC-solubilization activities of rhamnolipid surfactant may be related to its aggregation behavior at low concentrations, e.g. lower than CMC. Furthermore, signs of aggregate formation at concentrations lower than CMC for multi-component rhamnoliplids were observed using dynamic lighter scattering method.7,15 Formation of premicelles for a variety of surfactants also have been reported.21–24 These observations indicate the probability of sub-CMC aggregate formation for rhamnolipid, which still remains unexplored.
In this study, the aggregation behavior of dirhamnolipid in phosphate buffered electrolyte solution with concentrations near surface-tension-based CMC (or CMCst) was investigated. The objective of this study is to examine whether rhamnolipid forms aggregate at concentrations below CMCst, and to explore the effect of solution conditions on aggregate formation at low rhamnolipid concentration range.
The aggregate particle size was determined based on dynamic light scattering (DLS) mechanism using He–Ne laser at wavelength of 623 nm and working power of 4.0 mW. 1 ml of the sample was loaded to the DTS-0012 cell and measured at temperature of 30 °C. The scattered light was collected by receptor at angle of 173° from light path. A mean size provided by DTS Nano software (Malvern Instruments, U.K.) was used to represent the aggregate size of the sample. Also, the number-based particle size distribution (number PSD) data generated by the software were used for the statistical analysis of aggregate size. The diffusion coefficient of the aggregates was generated by the software.
The zeta potential measurement is based on the mechanism of particle electrophoresis in aqueous solution. 1 mL sample is loaded to DTS 1060 folded capillary cell and the electrophoretic mobility of the aggregate was measured at 30 °C under automatic voltage using a laser Doppler velocimetry with M3-PALS technique to avoid electroosmosis. The measured data was converted into corresponding zeta potential by applying the Helmholtz–Smoluchowski equation.27
Results of DLS-size measurement show that diRL aggregates were detected at diRL concentration both below and above CMCst. The number PSD profiles generated by Malvern DTS Nano software show only one peak for all the conditions of measurements (typical profiles are presented in Fig. S2, ESI†), indicating presence of only one type of aggregate. The influence of diRL concentration and solution pH on aggregate size is shown in Fig. 2a. The aggregate size is in a range of 8 to 72 nm. When the solution pH is not higher than 7.0, the aggregate size decreased with the increase of diRL concentration up to 100 μM. At diRL concentrations ranging from 10 to 100 μM (close to CMCst), the aggregate size decreases rapidly with increase of pH. When diRL concentration is higher than 100 μM, both diRL concentration and pH have no observable influence on the aggregate size. The relation between DLS diffusion coefficients and diRL concentrations is shown in Fig. S3, ESI.† The diffusion coefficient increases with increase of diRL concentration when the concentration is lower than CMCst. This result is in contrast to DLS diffusion coefficient for regular surfactants, for which an abrupt decrease of the coefficient is observed at CMC.28 This result, however, matches with the result of size measurement in that diffusion coefficient is larger for smaller particles.
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Fig. 2 DLS size (a) and zeta potential (b) of aggregates as a function of diRL concentration and solution pH. |
Aggregate zeta potential variation with diRL concentration and pH is presented in Fig. 2b. Because rhamnolipid is an anionic surfactant with carboxyl group in the hydrophilic moiety of the molecule, dissociation of the carboxyl groups yields negatively-charged aggregate surface and hence negative zeta potential of the aggregates. For all the pHs, the zeta potential decreases significantly the increase of diRL concentration from 25 μM to 100 μM. Further increase of concentration has minimal influence. For all the diRL concentrations tested, increase in solution pH causes decrease in aggregate zeta potential. Increase of solution pH results in enhanced dissociation of diRL carboxyl group, which in turn increases the aggregate surface charge density and lowers zeta potential (provided a dissociation equilibrium constant of 10−5.6 for rhamnolipid carboxyl group at room temperature,18 the dissociation rate of the rhamnolipid is 71.5, 88.8, 96.2, 98.8, 99.6% at pH of 6.0, 6.5, 7.0, 7.5, 8.0, respectively).
25 μM (below CMCst) and 250 μM (above CMCst) of diRL solution at pH of 6.0 or 8.0 were examined using cryo-TEM. Typical images of the aggregates are presented in Fig. 3. Aggregates are observed for all the four conditions, which is in contrast to the observation in the absence of diRL for which no aggregates are observed (Fig. S4a, ESI†). The morphology of the aggregates is spherical with minimal transparency, indicating micelle-type structure. Other aggregate structures reported in literatures at relatively high rhamnolipid concentrations, e.g. vesicles, lamella or microtubes,4,8,9,19 are not observed for any of the conditions tested. This is consistent with the result of DLS size measurement that only one type of aggregate is observed. The cryo-TEM result further confirms formation of diRL aggregates at concentrations below CMC. On the other hand, the DLS size and cryo-TEM results also shows that the aggregates are not premicelles, which are defined as dimers and low-aggregation number aggregates of surfactant molecules before micelle formation.21–24
All the cryo-TEM images used for aggregate size distribution analysis are shown in Fig. S4, ESI.† Gaussian distribution is commonly used to depict natural phenomena associated with real-valued random variables whose distributions are unknown. The distributions of aggregate sizes obtained with either DLS or cryo-TEM method appear to deviate from Gaussian distribution (data not shown), however, natural logarithm of the sizes follows Gaussian distribution very well for all the four conditions examined (Fig. 3). Values of the parameters for the fit are presented in Table 1. The mean of cryo-TEM size at diRL concentration of 25 μM (lower than CMCst) is larger than at diRL concentration of 250 μM (higher than CMCst), for pH of either 6.0 or 8.0. The cryo-TEM size at diRL concentration of 25 μM is larger for pH 6.0 than for pH 8.0, and they are identical at diRL concentration of 250 μM. These results show that change of the cryo-TEM size is similar to that of DLS size in terms of trend, indicating good consistency. The cryo-TEM sizes obtained at the condition of 25 μM diRL and pH 6.0 (24.9 nm) is significantly smaller than the DLS-based size (43.2 nm). The particle size obtained by DLS method is hydrodynamic diameter, which is the diameter of a sphere that has the same translational diffusion coefficient as the particle. This hydrodynamic size is usually larger than the real particle size.29 Either the DLS size or the cryo-TEM size obtained at high diRL concentration (0.5 mM) in our study is smaller than that measured at similar concentrations using similar methods in the study of Guo and Hu,8 in which formation of large vesicles was observed. The ionic strength of diRL solution in that study is approximately 10 mM, which is significantly lower than that in our study (55 mM with divalent ions). The hydrophilic head of diRL molecule contains a carboxylic group. At pH higher than 6.0, the majority of carboxylic groups are dissociated and negatively charged. Cations in the diRL electrolyte solution can easily bind with the carboxylate groups, resulting in the induction of the solvated groups and disfavours formation of large aggregates.9 Such a conversion of large vesicles to small ones was also observed when Cd2+ was introduced in solution of rhamnolipid solution.4 In addition, the dirhamnolipid used in the study of Guo and Hu contains higher ratio of long-chain species (Rha2C10C12:1 and Rha2C10C14:1. Rha2CxCy(:z) designates the diRL homologue with x and y as the carbon atom number of each aliphatic acid chain in the lipid moiety, and z as the number of unsaturated bonds in lipid moiety),8 which results in stronger hydrophobic interaction between molecules and thus favours formation of large vesicles.
diRL sample | DLS | cryo-TEM | ||||||
---|---|---|---|---|---|---|---|---|
μ a | σ 2 b | R 2 | d c (nm) | μ | σ 2 | R 2 | d (nm) | |
a Mean of ln![]() ![]() |
||||||||
25 μM, pH 6.0 | 3.77 | 0.031 | 1.00 | 43.2 | 3.22 | 0.033 | 0.96 | 24.9 |
250 μM, pH 6.0 | 2.05 | 0.030 | 0.97 | 7.8 | 2.61 | 0.053 | 0.98 | 13.7 |
25 μM, pH 8.0 | 2.80 | 0.046 | 0.98 | 16.5 | 3.05 | 0.033 | 0.97 | 21.2 |
250 μM, pH 8.0 | 2.06 | 0.027 | 0.98 | 7.8 | 2.61 | 0.044 | 1.00 | 13.6 |
The diRL used in this study is not a pure compound comprising one species of molecule. Instead, it is a rhamnolipid mixture consisting of three homologues which are the same in structure of polar moiety (double rhamnose rings and a carboxylic group) while different in length of aliphatic chains (Rha2C10C10, Rha2C10C12:1 and Rha2C10C12 with molar fractions of 0.70, 0.11 and 0.19, respectively).25 We speculate that this multi-component nature of the diRL results in formation of aggregates at concentrations below CMCst. The strength of hydrophobic interactions between diRL molecules with aliphatic chains of different lengths are not uniform, which may result in a transitional state for aggregation-related behavior, e.g. formation of aggregates in electrolyte solution before the solution surface is saturated with diRL (corresponding to diRL concentration of CMCst), and graduality in change of electrical conductivity increasing rate. In the transitional state, increase in diRL solution concentration may enhance partition of diRL molecules to aggregates and therefore increase the density of the molecules in aggregate. Increase of solution pH results in enhanced dissociation of diRL molecules. Both effects enhance the electrostatic repulsion between polar moieties of diRL molecules in aggregates and hence the curvature of aggregates. As a result, when diRL concentrations are lower than CMCst (the transitional state) the aggregate size decreases with increase of the concentration and solution pH.
Footnote |
† Electronic supplementary information (ESI) available: Electrical conductivity versus diRL concentration profile, typical number PSD profiles generated by Malvern DTS Nano software, DLS diffusion coefficient versus diRL concentration profile, and the cryo-TEM images used for aggregate size distribution analysis. See DOI: 10.1039/c5ra16817a |
This journal is © The Royal Society of Chemistry 2015 |