Aggregation of low-concentration dirhamnolipid biosurfactant in electrolyte solution

College of Environmental Science and Enginee P. R. China. E-mail: zhonghua@email.arizon 126.com; yx2013@hnu.edu.cn; lzf1818200 yxz@hnu.edu.cn Key Laboratory of Environmental Biology a Ministry of Education, Changsha 410082, P Department of Soil, Water and Environm Tucson, AZ 85721, USA † Electronic supplementary information versus diRL concentration prole, typica Malvern DTS Nano soware, DLS diffusio prole, and the cryo-TEM images used f See DOI: 10.1039/c5ra16817a Cite this: RSC Adv., 2015, 5, 88578


Introduction
Biosurfactants are surfactants produced by microbes.Due to expanding applications of biosurfactants in many elds, e.g.[3] The aggregates have a variety of microstructures, including spherical, globular or cylindrical micelles, 1,4-8 spherical or irregular vesicles, 2,9,10 tubular or irregular bilayers, 11 and lamellar sheets. 3,12,13Also, the lyotropic liquid crystalline phases with lamellar, hexagonal and cubic aggregate morphologies are observed at high surfactant concentrations. 14The morphology of these aggregates has been demonstrated to be affected by surfactant concentration, 8,15 pH, 4,10,16 temperature, 12 counterions, 1,10,15 and ionic strength. 17hamnolipid 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 500-20 000 mg L À1 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. 18Champion 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 xed pH has also been reported. 19ll 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 signicantly 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. 16,20Hypothetically 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. 7,152][23][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 CMC st ) was investigated.The objective of this study is to examine whether rhamnolipid forms aggregate at concentrations below CMC st , and to explore the effect of solution conditions on aggregate formation at low rhamnolipid concentration range.

Determination of CMC st
The stock solution of the diRL were prepared in phosphate buffered saline solution (PBSS, NaNO 3 2 g L À1 , KH 2 PO 4 1.5 g L À1 , Na 2 HPO 4 $12H 2 O 1.5 g L À1 , MgSO 4 0.1 g L À1 , FeSO 4 $7H 2 O 0.01 g L À1 ).PBSS solutions of diRL in a series of concentrations were prepared using dilution method.Surface tension of diRL solutions was measured at 30 C with surface tensiometer (JZ-200A, Chengde, China) using the Du Noüy Ring method.CMC st of diRL was obtained from the relation of surface tension and diRL concentration using the scheme described by Yuan et al. 26 Electrical conductivity of diRL solutions was measured with DDS-11A Conductivity Meter (Shengci, Shanghai, China).

Hydrodynamic aggregate size and zeta potential
pH of PBSS solutions of diRL was adjusted to 8.0 with 20% NaOH solution using a capillary glass pipe.High concentration of NaOH was used to minimize change of solution volume during pH adjustment.These solutions were then ltered through a 0.22 mm lter (Millex-HV, Millipore, Billerica, Ma, US) to remove suspended solid particles that may interfere with the measurement.Results of preliminary test showed that the size of rhamnolipid aggregates is far less than 0.22 mm at pH 8.0, so they will not be retained by ltering.The solutions were allowed to stand still for 2 h.Then pH of the solutions was adjusted in sequence to 7.5, 7.0, 6.5, or 6.0 with 20% hydrochloric acid.For each sample, aggregate size and zeta potential were measured using Zetasizer Nano ZEN3600 (Malvern Instruments, U.K.).
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 soware (Malvern Instruments, U.K.) was used to represent the aggregate size of the sample.Also, the numberbased particle size distribution (number PSD) data generated by the soware were used for the statistical analysis of aggregate size.The diffusion coefficient of the aggregates was generated by the soware.
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. 274 Cryo-transmission electron microscopy test diRL solution or electrolyte solution in the absence of diRL in volume of 4 mL was placed on the grid with a holy polymer lm using a microsyringe, and then sent to a FEI Vitrobot sample plunger system (FEI, Hillsboro, Oregon).Excessive sample was removed by a lter paper.Then the sample grid was rapidly vitried in liquid ethane and transferred to a liquid nitrogen bath.The morphology of diRL aggregate were then viewed and photographed on a Tecnai F20 transmission electron microscope (FEI, Hillsboro, Oregon) at an acceleration voltage of 120 kV.Nano measurer 1.2.5 soware (Shanghai, Fudan University) was used to process the micrograph images.The program marked the recognized particles in an image with circles.The diameter of every circle was measured by pairing the circle to a screen ruler calibrated by the reference bar in the image and used as the size of the particle.In order to obtain statistically representative sample for aggregate size distribution analysis, the size data were collected on more than 100 or 200 particles from multiple images for rhamnolipid concentrations of 25 or 250 mM, respectively.

Results and discussion
For all the pHs, surface tension of the solution decrease signicantly with the increase of rhamnolipid concentration at low surfactant concentrations, and then further increase of surfactant concentration has no signicant effect on surface tension (Fig. 1).Based on the method of Yuan et al., 26 the CMC st values obtained are 62, 78, 82, 83 and 82 mM for pH of 6.0, 6.5, 7.0, 7.5 and 8.0, respectively.The result showed that the increase of solution pH resulted in increase of diRL CMC st for pH not higher than 7.0.The electrical conductivity of diRL solution increased with the increase of diRL concentration for all pH conditions, however, the two-line prole with a distinguishable slope inection is not observed for any of the curves (Fig. S1a, ESI †).The plot of conductivity derivative versus diRL concentration is presented in Fig. S1b, ESI.† The conductivity derivative shows a gradual decrease at the concentration below CMC st , which is in contrast to an abrupt decrease at CMC generally for regular surfactants.
Results of DLS-size measurement show that diRL aggregates were detected at diRL concentration both below and above CMC st .The number PSD proles generated by Malvern DTS Nano soware show only one peak for all the conditions of measurements (typical proles are presented in Fig. S2, ESI †), indicating presence of only one type of aggregate.The inuence 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 mM.At diRL concentrations ranging from 10 to 100 mM (close to CMC st ), the aggregate size decreases rapidly with increase of pH.When diRL concentration is higher than 100 mM, both diRL concentration and pH have no observable inuence 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 CMC st .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.
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 signicantly the increase of diRL concentration from 25 mM to 100 mM.Further increase of concentration has minimal inuence.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 À5.6 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 mM (below CMC st ) and 250 mM (above CMC st ) 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 conrms formation of diRL aggregates at concentrations below CMC.][23][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 t are presented in Table 1.The mean of cryo-TEM size at diRL concentration of 25 mM (lower than CMC st ) is larger than at diRL concentration of 250 mM (higher than CMC st ), for pH of either 6.0 or 8.0.The cryo-TEM size at diRL concentration of 25 mM is larger for pH 6.0 than for pH 8.0, and they are identical at diRL concentration of 250 mM.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 mM diRL and pH 6.0 (24.9 nm) is signicantly 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. 29Either 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 signicantly 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. 9Such a conversion of large vesicles to small ones was also observed when Cd 2+ was introduced in solution of rhamnolipid solution. 4In addition, the dirhamnolipid used in the study of Guo and Hu contains higher ratio of long-chain species (Rha 2 C 10 C 12:1 and Rha 2 C 10 C 14:1 .Rha 2 C x C y(: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.
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

Fig. 1
Fig. 1 Surface tension versus diRL concentration in PBSS solution and determination of CMC st .

Fig. 2
Fig. 2 DLS size (a) and zeta potential (b) of aggregates as a function of diRL concentration and solution pH.

4 Conclusions
DLS and cryo-TEM methods were used to study aggregation behavior of low-concentration diRL and the results demonstrated formation of aggregates at concentrations below CMC st .The effect of diRL concentration and solution pH on aggregate size and zeta potential is signicant when diRL concentration is lower than the CMC st .The multicomponent nature of the diRL and consequently a transitional state are supposed to be responsible for these aggregation behaviors at low diRL concentrations.Also, results of the study indicate that the surface-tension-based CMC may not be used as the concentration dening aggregate formation for multicomponent biosurfactants.This work is of importance for cost-effective application of rhamnolipid.Further researches should be focused on validating the transitional state speculation and characterizing the rhamnolipid aggregates in transitional state in more detail.

Table 1
25ussian regression parameters for DLS and cryo-TEM aggregate size distribution of polar moiety (double rhamnose rings and a carboxylic group) while different in length of aliphatic chains (Rha 2 C 10 C 10 , Rha 2 C 10 C 12:1 and Rha 2 C 10 C 12 with molar fractions of 0.70, 0.11 and 0.19, respectively).25Wespeculate that this multi-component nature of the diRL results in formation of aggregates at concentrations below CMC st .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 CMC st ), 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 CMC st (the transitional state) the aggregate size decreases with increase of the concentration and solution pH.