Interfacial properties rhamnolipids from an extract produced by a strain of <i>Pseudomonas aeruginosa</i> were analyzed in this study. The extract of rhamnolipid was characterized by surface tension in different conditions; interfacial tension with different hydrocarbons; critical micelle concentration under different pH and temperatures; particle size and emulsification capacity using laser light profiling. It was observed that the rhamnolipids extract are sensitive to variations in pH, thermostable and function as good emulsificant for emulsification of methyl methacrylate. The emulsion stability order in function of the oil phase was methyl methacrylate > emulsions of castor oil > emulsion n-heptane > emulsion toluene > emulsion hexadecane > octane emulsion. The data presented show that rhamnolipid extracts may be used to formulate stable emulsions of methyl methacrylate. This process can be used to do nano/microsphere of polymethyl methacrylate.
Rhamnolipids are amphiphilic molecules composed of a hydrophobic fatty acid moiety (with C8-C14 carbon atoms, which may or may not be saturated) and a hydrophilic portion composed of one or two rhamnose residue [
The presence of others minor rhamnolipids homologues involving C8, C10, C12, and C14 3-hydroxy fatty acids have been described in the literature [
Several recent studies on rhamnolipids were focused on their adsorption, aggregation, and micellization properties, which are also the important parameters for estimating surfactant in process of solubilization, suspension, and dispersion [
In fact, surface properties and micellization behavior of a single chemical and separated rhamnolipid, or even rhamnolipidic crude mixture, were reported to be variable [
In this study, a rhamnolipid mixture was produced by the P. aeruginosa strain PA1 and then isolated by partitioning with chloroform-ethanol. The composition of the rhamnolipid mixture was chemically characterized, and the interfacial properties and micellization behavior were comparatively investigated. The analysis of the data shown in this article can provide a better understanding of the functions, characteristics and behavior of mixtures of rhamnolipids for use in polymerization processes, biochemical processes, and pharmaceutical and environmental remediation.
Pseudomonas aeruginosa PA1 was inoculated in Erlenmeyer flasks containing 600 mL of culture medium (NaNO3 1.0 g/L; KH2PO4 3.0 g/L; K2HPO4 7.0 g/L; MgSO4∙7H2O 0.2 g/L; 0.5% yeast extract; peptone 0.5% and 3% glycerol). The Erlenmeyers were placed in a rotary shaker at 170 rpm for 24 hours, at 30˚C. The cell culture was centrifuged (10,000 g, 30 minutes) and then dispersed again in distilled water. The cells were then centrifuged, recovered and used as inoculums [
The cell concentration in the suspension of P. aeruginosa was calculated as dry mass (PS) (g/L), by measuring the absorbance at 600 nm and using a calibration curve of dry weight (ABS PS = 1.2595 (g/L) − R2 = 0.989). To determine the concentration of rhamnolipids, the method described by Dubois et al. was used [
The cells were separated by centrifugation of the culture medium at 10,000 × g for 20 minutes and autoclaved. The culture medium, free of cells, containing the biosurfactant was subjected to a reverse osmosis process for concentration by water removal using a reverse osmosis system containing N2 at a constant pressure of 300 psi (20 kgf/cm2), a reverse osmosis membrane (model: BW30-2540-DOW FILMTEC) and a magnetic stirring system. Rhamnolipid purification was carried out by extraction, using a chloroform/methanol/culture medium mixture, in the proportion of 2:1:1 and a separating funnel (see
The surface tension measurements were carried on a tensiometer Krüss K100 using the Wilhelmy plate method. The rhamnolipids rich extract (RLe) was diluted in deionized water at a concentration of 0.15 g/L. The samples were then analyzed for 20 minutes at each temperature of 4˚C, 25˚C, 60˚C and 80˚C, then cooled from 80˚C to 4˚C following the same path. The sample was stored in the refrigerator and analyzed again in the following day.
To measure the surface tension at different pH, the rhamnolpid extract was diluted in 1 g/L with water deionized with different pH: alkaline medium (pH = 10.8), adjusted with a 1 M NaOH or KOH solutions; acid medium (pH = 3.4), adjusted with a 1 M HCl solution; and without any adjustment of pH (pH about 6.3). During the whole titration process, the pH value was monitored.
The interfacial tension analyses were performed in DSA100 tensiometer, using the pendant drop method. The data were analyzed using the software Drop Shape Analysis System DSA100. The interfacial tension between water (with and without the extract of rhamnolipids) and different hydrocarbons was measured at room temperature (25˚C).
The zeta potential and particle size was measured on a Nano ZS-Malvern Instruments equipment. Rhamnolipids extract solutions were prepared at pH 3.4, 10.8 and 6.3 at different concentrations at a concentration of 5 g/L. The samples were pre-filtered in a 0.45 μm membrane filter and added to a cuvette for measurements.
The emulsion stability was analyzed at room temperature by laser profiling using the Turbiscan TLAB from Formulaction®. This technique allows the scanning of transmitted and scattered light of flasks containing emulsions or suspensions in various positions along the sample height. The measurement result is the light transmission and backscattering profile from the sample in function of the height of the tube (mm), from the bottom to the top of the tube [
The emulsions were prepared by mixing 50% water rhamnolipid solution (150 mg/L) and 50% of hydrocarbon (w/w). The samples were sonicated by an ultrasound probe (Sonics model VCX750) for 4 minutes with amplitude of 40%. Then, 20 ml of sample was added into a flask for analysis of the stability of the emulsion in the Turbiscan TLAB.
A comparison of the surface tension of solutions (culture medium RLb and rhamnolipid extract RLe at different concentrations) shows that the rhamnolipid extract (RLe) reduces the surface tension of water at a lower concentration than RLb, as expected by an increase in the rhamnolipid concentration due to the purification process. At the same time the CMC of the RLb and 198.1 mg/L while of the RLe is 25.7 mg/L, resulting in a difference of almost eight times (
The SDS reduced the surface tension less than the rhamnolipid (~27 mN/m-rhamnolipids against ~36 mN/m- SDS). Interestingly, it is only required about 2.6 g of SDS, compared to 26 mg of RLe (value 100 times smaller), to achieve CMC. This shows that in relation to surface tension reduction, rhamnolipids are more effective and have a much larger molecular area at air/liquid interface.
Industrial applications, such as surfactant in polymerization reaction, require surfactant thermal stability at up to 80˚C. Many surfactants are sensitive to temperature, thus, RLe solutions were heated and cooled to assess thermal stability (
Environmental factor such as pH, salinity and temperature play a crucial role in influencing the effectiveness of rhamnolipids [
Many surfactants can tolerate extremes pH, but rhamnolipids are sensitive to pH changes, the variation of surface tension under different pH is shown in
According to Lebrón-Paler et al. (2006), rhamnolipids are weak acids due to the presence of the carboxylic acid moiety, known to undergo aggregation in solution and to have a pKa of about 5.6 [
A similar result, with different concentrations of di-rhamnolipids, was reported by Sánchez et al. [
At concentrations above the CMC, rhamnolipids form micelles, vesicles, or lamella depending on the solution pH, concentration, and presence of electrolytes [
The micelles or bi-layers formed by self assembly of surfactants in CMC may also aggregate and generate vesicles in aqueous solution as bulk [
The variation of pH is an important factor to increase or decrease the stability of emulsions with rhamnolipids [
Analysis of zeta potential and particle size can help to understand the behavior of rhamnolipids in solutions (
A significant proportion of large aggregates (distribution mode about 200 nm) was found in all pHs indicating the formation of aggregates with relatively large hydrodynamic radius. At pH 10.8, a size distribution mode about 7 nm was observed, suggesting that when the rhamnolipid is in the charged state electrostatic repulsion existing between headgroups leads to small micelles. This confirms that micelle growth, due to the intermolecular or intermicellar aggregation, or aggregate shape transition may occurs when a change of pH (or concentration) in the bulk happen.
The previous experiments were important to demonstrate the effects of various parameters, such as: temperature, pH and salts in the properties of solutions containing the extract RLe. Previous works demonstrate the ability of rhamnolipids to reduce aqueous surface tension [
These results demonstrate that RLe can reduce the interfacial tension between water and hydrocarbons with
different polarities and structures using just a small amount of surfactant. Furthermore, the highest interfacial tension reduction were observed in the systems with linear alkanes.
In a more hydrophilic system, such as castor oil and methyl-metacrylate, the ramnolipid decreases slightly the interfacial tension. Comparatively, RLe was more effective than SDS (interfacial tension reduction per weight of surfactant), suggesting that the RLe can be used in various processes (especially those required to reduce the in-
Hydrocarbons | Surfactant | |||
---|---|---|---|---|
Water | RLe (0.15 g/L) | RLe (1 g/L) | SDS (10 g/L) | |
Toluene | 32.50 ± 0.37 | 3.95 ± 0.47 | - | 2.63 ± 0.14 |
n-heptane | 43.23 ± 1.35 | 3.54 ± 0.16 | - | 1.33 ± 0.03 |
Octane | 47.17 ± 0.13 | 1.11 ± 0.03 | - | 1.31 ± 0.05 |
Hexadecane | 47.95 ± 0.87 | 0.70 ± 0.11 | - | 1.85 ± 0.89 |
Methyl metacrilate | 11.64 ± 1.01 | 9.21 ± 0.18 | 5.22 ± 0.06 | 6.78 ± 0.17 |
Castor oil | 18.75 ± 0.32 | 10.27 ± 0.65 | 7.60 ± 1.3 | 9.54 ± 0.86 |
Petroleum | 21.21 ± 0.08 | 1.40 ± 0.15 | - | 7.19 ± 3.12 |
terfacial tension between water and an organic phase), and can replace the petrochemical surfactants. Another favorable point of rhamnolipids is that they are biodegradable, while SDS compounds have low biodegradability [
The SDS and RLe reduced the interfacial tension between crude oil and water as seen in
Figures 6-11 show the emulsion stability behavior of emulsions of water solutions with 0.15 g/L of RLe and the hydrocarbons which were evaluated for interfacial tension. This characterization is important to evaluate how the RLe can assist in the stabilization of emulsions with different types of hydrocarbons.
Emulsions are not stable and are subject to various phenomena, such as flocculation, Otswald ripening (diffusional degradation) and coalescence [
Emulsions were prepared in a 50/50 water:hydrocarbon ratio, 0.15 g/L of RLe was dissolved in water. Figures 6-11 show the emulsions light transmission and backscattering profiles. During the first hour, none of these emulsions showed changes in the transmission, but an increase in transmission (
Through the backscattering is possible to identify different phenomena that occur in emulsions, such as: a clarification, creaming, flocculation and coalescence [
The backscattering of the octane emulsion (
The hexadecane emulsion (
significant changes in the droplet size, indicated by significant changes in the backscattering profile. Thus, RLe in concentration of 0.15 g/L is able to maintain a good stability of methyl methacrylate/water emulsions. This is not suprisingly because the rhamnolipid was not able to reduce water/MMA interfacial tension significantly. The size of the micelles formed in the emulsions methyl methacrylate was between 80 nm and 90 nm in 24 hours.
The profile of the backscattering emulsions can be classified by the degree of stability as follows: emulsion methyl methacrylate (
ever, for emulsions with octane, the use of RLe is not recommended.
Rhamnolipids are thermostable surfactants. Two heating treatments at a temperature of 80˚C for a period of one
hour did not affect the ability of the rhamnolipid extract to reduce the interfacial tension. This information is important to demonstrate that the extract can be reused in systems that require the use of relatively high temperatures and pH can interfere in the critical micellar concentration and size of the rhamnolipid micelle.
The emulsion stability did not follow the trend suggested by the interfacial tension. Emulsions of methyl
methacrylate and water containing rhamnolipid are stable, nevertheless the reduction in the interfacial tension in this system due to rhamnolipid is very modest emphasizing, that to estimate the ability of a surfactant to stabilize an emulsion it is necessary to use a different parameter. Thus, this paper proposes the possibility of using rhamnolipids in processes of nano/micropheres formulations of poly-methyl methacrylate that require using biosurfactants.
The authors wish to thank to Prof. Denise Freire for donating the strain of Pseudomonas. Brigida Orioli and Davyson Moreira helpful discussions and suggestions. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES).