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Published online 1 March 2008
Published in J Environ Qual 37:509-514 (2008)
DOI: 10.2134/jeq2007.0258
© 2008 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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SHORT COMMUNICATIONS

Rhamnolipid Morphology and Phenanthrene Solubility at Different pH Values

Kyung-Hee Shina,c, Kyoung-Woong Kima,*, Ju-Yong Kima, Kyung-Eun Leeb and Sung-Sik Hanb

a Dep. of Environmental Science and Engineering, Gwangju Inst. of Science and Technology, Gwangju, Korea, 500-712
b School of Life Sciences and Biotechnology, Korea Univ., Seoul, Korea, 136-701
c present address: Environmental Assessment Group, Korea Environment Inst., 613-2, Bulgwang-Dong, Eunpyeong-Gu, Seoul, Korea

* Corresponding author (kwkim{at}gist.ac.kr).

Received for publication May 22, 2007.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
The effect of pH and rhamnolipids on the solubility of phenanthrene was investigated in a sand–water system. Batch phenanthrene solubilization experiments in this system showed that the highest phenanthrene solubility occurred at pH 5 in the presence of 240 and 150 mg L–1 rhamnolipids. As the pH was increased from 5 to 7, the apparent solubility of phenanthrene decreased and then stabilized from pH 7 to 8. At pH 4, a dramatic decrease in phenanthrene solubility was observed. This result is in contrast to previous findings obtained in an aqueous system without soil particles. To investigate the reason for this decrease, the critical micelle concentrations (CMCs) were measured in the presence or absence of sand particles, and the maximum amount of sorbed biosurfactant at each pH was calculated based on the differences of the two CMC values. More rhamnolipid molecules were lost by the sorption into sand particles at pH 4 than at other pH values; this explains the dramatic decrease of solubility at pH 4 in the sand–water system. To confirm the explanation for the different solubilizing capacity that results from the structural change of biosurfactant aggregate, cryo-transmission electron microscopy was used. Micrographs showed that the rhamnolipid morphology changed from large lamellar sheets, to vesicle, and then to micelle as the pH increased. The large and multilamellar vesicles at pH 5 were considered to be the most effective structure for the solubilization of phenanthrene.

Abbreviations: CMC, critical micelle concentration • cryo-TEM • cryo-transmission electron microscopy • HPLC, high-performance liquid chromatography • NAPL, non-aqueous–phase liquid • PAHs, polycyclic aromatic hydrocarbons


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
POLYCYCLIC aromatic hydrocarbons (PAHs) are ubiquitous in the environment and have been of great environmental concern. Their levels in the environment are regulated by government agencies because they are either known or suspected carcinogens or mutagens (Gerde et al., 2001; Mizesko et al., 2001; Tsai et al., 2001). Polycyclic aromatic hydrocarbons are hydrophobic, and most of them are practically insoluble in water, which contributes to their persistence in the environment. For example, their solubilities range from 32.5 mg L–1 for naphthalene to 0.14 mg L–1 for pyrene (Edwards et al., 1991). Conversely, PAHs often show strong tendencies to be sorbed onto soil and incorporated into soil micropores. The majority of chemical and biological remediation technologies require the transfer of contaminants from non-aqueous–phase liquids (NAPLs) into a mobile phase. However, the mass transfer rates of PAHs in contaminated soils are very low, and this low mass transfer rate limits the removal efficiency of PAHs in soil (Laha and Luthy, 1992; Fortin et al., 1997). To solubilize the hydrophobic contaminants, enhancing agents such as surfactants, which have an amphiphilic structure, are introduced by several researchers (Shonali and Richard, 1992; Josée et al., 1997).

Many microorganisms produce metabolic products or membrane components that behave similarly to surfactants and are known as biologically produced surfactants or biosurfactants. Glycolipids and phospholipids are two of the most common groups of biosurfactants (Kanga et al., 1997). Biosurfactants have several advantages over chemical surfactants, including lower toxicity (Van Dyke et al., 1991; Flasz et al., 1998), higher biodegradability (Zajic et al., 1977; Shoham et al., 1983; Oberbremer et al., 1990; Kesting et al., 1996), better environmental compatibility, and the ability to be synthesized from renewable feedstocks (Desai and Banat, 1997). They occur naturally in soils, which makes them acceptable from a social and ecological point of view. Their complete removal after treatment may not be necessary (Zhang and Miller, 1995; Lin, 1996; Wouter et al., 1998), making them ideal for environmental applications. There are many potentially useful biosurfactants, ranging from short fatty acids to large polymers, which results in a broad spectrum of potential industrial applications. In particular, enhanced oil recovery has been considered as one of the most important growth areas for biosurfactants for many years.

In earlier studies, Ishigami et al. (1987) and Champion et al. (1995) observed the rhamnolipid structure to be strongly pH dependent, changing from large lamellar sheets to vesicles and then to micelles. They reported that the pKa for rhamnolipid is 5.6 (Ishigami et al., 1987), and as the pH is increased from 5.5 to 8.0, the repulsion between the more negatively charged head groups effectively creates a larger head diameter, causing the changes in morphology from lamellar to vesicles and then to micelles (Champion et al., 1995). Zhang and Miller (1992) also observed the surface tension and dispersion of octadecane to be significantly affected by pH. Moreover, Bai et al. (1998) investigated the effect of pH on the solubilization and mobilization of NAPL and found that a decrease in the pH caused a decrease in the interfacial tension between the rhamnolipid solution and a NAPL, resulting in the mobilization of residual NAPL. These results indicate that pH control could be an important factor in enhancing the removal efficiency of anionic biosurfactant applications.

Even though several studies have considered the pH effect, a greater understanding of the pH effect on the solubilization of PAHs under various conditions is still required because, for example, the presence of soil particles can significantly affect the solubilization mechanisms of surfactants.

In this study, the effect of pH on the effectiveness of rhamnolipid in enhancing the apparent solubility of phenanthrene was examined, with the specific objectives of examining the effects of pH on (i) the solubilization of phenanthrene in a sand–water system and (ii) surface tension variation by rhamnolipid addition. Furthermore, electron microscopic analysis of a rhamnolipid-phenanthrene aggregate was investigated to confirm the morphology variation with changing pH.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results and Discussion
 Conclusions
 REFERENCES
 
Chemicals
Phenanthrene (purity >98%) was purchased from Aldrich Chemical Co. (Milwaukee, WI), with a molecular weight of 178.24 and an aqueous solubility reported as 1290 µg L–1. Dichloromethane (high-performance liquid chromatography [HPLC] grade, used to dissolve phenanthrene), methanol (HPLC grade), acetonitrile (HPLC grade), and water (HPLC grade) were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Phenanthrene-Contaminated Sand
The solid phase used in this study to provide a simplified system mimicking the effect of soil particles was Chumunjin standard sand, which was purchased from Dongyang Science Co. (Gwangju, Korea). Before each experiment, the sand was rinsed five times with deionized water to remove possible residual salts or impurities and air-dried. The sand was artificially contaminated with phenanthrene using the spiking method described below to create homogeneous conditions. Phenanthrene (400 mg) was dissolved in 500 mL of dichloromethane to prepare the spiking solution. In a 2-L beaker, 200 g of dried sand and 50 mL of spiking solution were added. This procedure was repeated for spiking up to 1000 g of sand with a final concentration of phenanthrene of approximately 200 mg kg–1. The contaminated sand was placed in an extractor hood for 7 d to evaporate the dichloromethane.

Biosurfactant
The biosurfactant used in this study was a rhamnolipid solution, purchased from the Jeneil Biosurfactant Company (Saukville, WI). The Jeneil product JBR425, with a mono- to di-rhamnolipid ratio of 1:1 (w/w), was used and was supplied as a 25% (w/v) aqueous solution. Selected characteristics of the biosurfactant are summarized in Table 1 .


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  		Table 1. Characteristics of rhamnolipids.

 
Solubilization of Phenanthrene by Rhamnolipid Biosurfactant
The solubilities of phenanthrene in the rhamnolipid solutions at various pH values were determined in the sand–water systems using batch tests. Two biosurfactant concentrations, 240 ({cong}4.3 x critical micelle concentration [CMC] at pH 7) and 150 mg L–1 ({cong}2.7 x CMC at pH 7), were applied. In each experiment, 2 g of phenanthrene-contaminated sand was added, and the initial pH values were adjusted to 4, 5, 6, 7, and 8 by the addition of 0.1 N HCl or 0.1 N NaOH solution, as necessary. The final sand/solution ratio was 2:10 (w/v). Triplicate samples at each pH were placed on a rotary shaker for 48 h at room temperature (24 ± 1°C). The pH did not change during this period. Afterward, the samples were centrifuged at 4000 rpm for 10 min, and the supernatants were analyzed for phenanthrene by HPLC. The filtration step was not applied because filtration may deform the rhamnolipid-phenanthrene aggregates in solution.

Critical Micelle Concentration
A surface tension method was used to determine the maximum sorption of the surfactant into sand. Surface tension experiments in this study were conducted with a NIMA tensiometer (Model 9002; NIMA Technology, Coventry, England) at room temperature (22 ± 1°C) to evaluate the CMC values of the rhamnolipids. The measurements were made in the presence or absence of sand. When the sand was included, the sand/water ratio was 2:10 (w/v); this ratio was the same as that used in the sand–water solubilization experiment. Rhamnolipid solutions of varying concentrations were made from the stock solution and diluted with deionized water, and the pH was adjusted using 0.1 N NaOH and 0.1 N HCl to five pH values (4, 5, 6, 7, and 8). The applied rhamnolipid concentrations were (in g L–1) 1 x 10°, 3 x 10–1, 1 x 10–1, 3 x 10–2, 1 x 10–2, 3 x 10–3, 1 x 10–3, 3 x 10–4, 1 x 10–4, 3 x 10–5, and 1 x 10–5. The sand–water suspensions were placed on a rotary shaker for 48 h at room temperature. The batch test samples were then quiescently settled for 48 h, after which the supernatant was free of suspended soil particles and ready for measurement (Zheng and Obbard, 2002). The rhamnolipid solutions were allowed to equilibrate for approximately 2 h before measurements were made (Edwards et al., 1991). All glassware was cleaned with 10% nitric acid and deionized water before the measurements, and the tensiometer's ring (Du Nouy ring with 20 mm diameter) was heated to redness in a flame for each measurement. The measurement was performed three times to ensure consistent readings were obtained.

Analytical Methods
Aqueous samples were analyzed for phenanthrene using an HPLC instrument equipped with a Waters model 717 Plus autosampler, a Waters model 600 pump, a M720 absorbance detector (Young-In, Korea), and a Novapak column C18 (Waters, MA). The HPLC analysis was performed isocratically using a mobile phase of 35% water and 65% acetonitrile, at a flow rate of 1 mL min–1, with UV detection of the phenanthrene at a wavelength of 254 nm. The injection volume used was 10 µL. The low concentrations of phenanthrene were measured using a M474 fluorescence detector (Waters, MA) at excitation and emission levels set at 254 and 390 nm, respectively. The mobile phase was a 35:65 mixture of acetonitrile and water (Chang et al., 2002).

Cryo-TEM Analysis
Sample Preparation
Samples of 26 mmol L–1 rhamnolipid were adjusted to the appropriate pH using 1 N NaOH or 0.1 N HCl solution and filtered with a 0.45-µm filter. The sample pH remained stable over the duration of the experiment.

Grid Formation
Formvar/chloroform solution with 10% glycerol was sonicated to produce a holey film, and a slide glass was coated with the solution. The holey film was floated on clean water, and a cryo grid was placed on one side of holey film. Coated grids were washed with ethanol to remove the glycerol, and carbon was evaporated onto the formvar-coated side of cryo grid. A drop of sample (about 7 µL) was placed on the grid and blotted at the back of grid. The grid was immediately plunged into a bath of liquid ethane and transferred to a liquid nitrogen bath. The sample was stored in a GATAN model 630 cryotransfer (Gatan, Inc., Warrendale, PA) under liquid nitrogen at approximately –185°C.

Cryo-transmission Electron Microscopy
The samples were observed at approximately –170°C using a Tecnai 12 electron microscope (Philips, Eindhovenm, the Netherlands) at 120 kV, and the images were acquired with a Multiscan 600W CCD camera (Gatan, Inc., Warrendale, PA).


   Results and Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
Conclusions
 REFERENCES
 
Effect of pH on Phenanthrene Solubilization with Rhamnolipids
To examine the effect of pH on the solubility of phenanthrene due to rhamnolipid in the sand–water system, two biosurfactant concentrations, 240 ({cong}4.3 x CMC at pH 7) and 150 mg L–1 ({cong}2.7 x CMC at pH 7), were applied.

The phenanthrene solubilities in the rhamnolipid solutions at each pH are presented in Fig. 1 . For both concentrations, the highest solubility was detected for the pH 5 rhamnolipid solution. As the pH was increased from 5 to 7, the apparent solubility of the phenanthrene decreased. A further pH increase from 7 to 8 did not significantly affect the solubility. The apparent phenanthrene solubility at pH 5 in the presence of 150 mg L–1 rhamnolipid solutions was 4.7 times greater than that at pH 7. The aqueous phenanthrene solubility in absence of rhamnolipids was 0.46~0.64 mg L–1 at pH from 4 to 8.


Figure 1
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  		Fig. 1. The effect of pH on the phenanthrene solubility with rhamnolipid solutions in the sand–water system. Symbols equal the average of triplicate vials. Error bars represent ±1 SD.

 
The trends in the solubility changes at various pHs were similar to the previous results obtained in absence of soil particle (Shin et al., 2004), with the exception of that at pH 4. When the rhamnolipid solution was used in the sand–water system, the solubility dramatically decreased at pH 4, which might have been due to the loss of rhamnolipid molecules. A possible reason for the loss of rhamnolipid at pH 4 may have been sorption of surfactant onto the sand particles. The sorption of surfactant onto soil may result in much of the surfactant being unavailable for micellar solubilization of hydrophobic organic compounds. In addition, the presence of sorbed and dissolved surfactant changes the hydrophobic organic compound sorption behavior, with the surfactant sorption onto soil particles affecting the transport of these compounds in the soil (Liu et al., 1992).

In this study, the CMC values of the rhamnolipid at various pHs were determined from plots of the surface tension measured by the tensiometer versus the concentration of the rhamnolipid solution. These data allow the determination of the CMC as the surfactant concentration denoted by the intersection of two linear portions of a curve that shows the variation in the surface tension as a function of the logarithm of the surfactant concentration. The calculated CMC values in the presence or absence of sand particles are shown in Table 2 . The CMC values in the absence of sand at pH 6 and 7 were 8.3 x 10–2 and 1.0 x 10–1 mmol L–1, respectively. These values were similar to previous literature values (Zhang and Miller, 1992). However, under slightly acidic conditions, the CMC decreased indicating that, at higher pH values, a greater amount of surfactant must be added to the system to decrease the surface tension by a specific amount.


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  		Table 2. Measured critical micelle concentration (CMC) values of rhamnolipid solutions at various pH values from surface tension experiments.

 
To determine the maximum sorption of surfactant into the sand, the surface tension in a suspension system (sand/water = 2.0 g/10 mL) was measured (Table 2). The CMC of the sand/water system was generally elevated at each pH, which may have been due to the surfactant sorption onto the sand particles (Zheng and Obbard, 2002; Chen et al., 2005). Based on the differences of the two CMC values, the maximum amount of sorbed biosurfactant can be predicted; more biosurfactant was sorbed at pH 4 than at other pH values. This result supports the finding of the solubilization experiment for the sand–water system. As shown in Fig. 1, a large decrease in the solubility was detected at pH 4, which also showed the highest amount of sorption, as based on the CMC experiment.

Cryo-TEM Analysis
One possible explanation for the greater solubilizing capacity of the biosurfactants at pH 5 and 6 may be differences in the structure and size of the biosurfactant aggregate. An anionic surfactant, such as rhamnolipid, can undergo changes in the diameter of the head group depending on the protonation state of the carboxyl group (Champion et al., 1995). To confirm the effect of pH on the rhamnolipid morphology in this study, cryo-transmission electron microscopy (cryo-TEM) was introduced. Cryo-transmission electron microscopy is uniquely suited for viewing surfactant morphology because the samples are instantaneously vitrified in their hydrated state without dyes, fixatives, or buffers. The structures have minimal deformation, and many include micelles that can be distinguished (Champion et al., 1995).

The cryo-TEM micrographs showed that large lamellar sheets dominated at pH 4, with the presence of tubular and irregular bilayered structures (Fig. 2a and 2b ). At pH 5, large lamellar sheets predominated with the existence of vesicles. Multilamellar vesicles and large unilamellar were frequently found (Fig. 2c and 2d). Generally, large structures were dominant at pH 4 and 5. Vesicles were observed at pH 6, but bilayered vesicles rarely existed (Fig. 2e and 2f). Compared with the structures at pH 5, the number of small vesicles increased, and spherical micelles were seen. At pH 7, small micellar structures were found (Fig. 2g and 2h), but no vesicles were observed.


Figure 2
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  		Fig. 2. Representative cryo-transmission electron micrographs of the morphology of rhamnolipid in solution at (a) and (b) pH 4, (c) and (d) pH 5, (e) and (f) pH 6, and (g) and (h) pH 7. L, multilamellar vesicle; M, micelle; T, tubule; V, vesicle. Bar = 200 nm.

 
The structure formed by aggregation of surfactant monomers is dependent on the ratio of the effective diameter of the non-polar tails. If this ratio is close to 1, the surfactant monomers line up side by side to form planer bilayers. As this ratio increases, the bilayer structures curve to form vesicles. If the ratio continues to increase, micelles are formed. In this study, rhamnolipid, which is an anionic surfactant, can undergo changes in the diameter of its head group depending on the protonation state of the carboxyl group. The reported pKa for rhamnolipid is 5.6 (Ishigami et al., 1987; Lebron-Paler et al., 2006), so as the pH increases, the negative charge of the polar head of the rhamnolipid increases. As the head group becomes more charged, the charge repulsion between adjacent polar heads effectively creates a larger head diameter, causing the observed progression in morphology from lamellar to vesicles and finally to micelles. Using fluorescent microscopy, Ishigami et al. (1987) reported that rhamnolipids form liposome-like vesicles at low pH values (≤6.0). Between pH 6.0 and 6.6, the rhamnolipids formed lamella-like structures or lipid aggregates; above pH 6.8, micelles were formed when the rhamnosyl moiety was negatively charged. However, with the recent use of cryo-transmission electron microscopy, Champion et al. (1995) observed the rhamnolipid structure decreasing in size with increasing pH from 5.5 to 8.0, going from large lamellar sheets to vesicles and finally to micelles.

Our cryo-TEM analysis showed structural changes of the rhamnolipid, which were similar to those reported by Champion et al. (1995). In summary, larger structures (e.g., lamellar sheets, multilamellar vesicles, and large unilamellar vesicles) were more frequently seen under slightly acidic conditions. Moreover, these results seem to correlate to the size-distribution results of Shin et al. (2004). They found that the largest fraction of the phenanthrene was in the MW range >300,000 at all pH values, whereas the fraction between 30,000 and 300,000 increased by increasing the pH from 5 to 8. This indicated a trend toward smaller structures with increasing pH, as shown in the cryo-TEM analysis.

Zhang and Miller (1992) observed a decrease in octadecane dispersion from pH 5.5 to 6.0, but this was followed by an increase from pH 6.0 to 7.0 and a subsequent decrease in dispersion from pH 7.0 to 8.0, with no further pH effect from 8.0 to 10.0. This result indicated that micelles were more effective for the dispersion of octadecane. Their results are different from our findings that a larger structure more effectively solubilized the phenanthrene than a spherical micelle. These differences may be due to differences in the structures between the normal alkane and aromatic organics.


   Conclusions
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
REFERENCES
 
pH variation can significantly affect the rhamnolipid morphology, and these structural changes induce variations in the CMC and phenanthrene solubility. The highest solubility was detected at pH 5 for the rhamnolipid solution in the sand–water system, indicating that lamellar or large vesicles are more effective for phenanthrene solubilization, as based on the cryo-TEM micrographs. Therefore, it is clear that anionic surfactants can be more powerful for contaminant solubilization when solution pH control is used.


   ACKNOWLEDGMENTS
 
This paper is based on work supported by the Korea Science and Engineering Foundation (KOSEF) through the Advanced Environmental Monitoring Research Center at GIST and by the Brain Korea 21 Program of the Ministry of Education, Korea.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 





This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
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Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shin, K.-H.
Right arrow Articles by Han, S.-S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Shin, K.-H.
Right arrow Articles by Han, S.-S.
Agricola
Right arrow Articles by Shin, K.-H.
Right arrow Articles by Han, S.-S.
Related Collections
Right arrow Remediation
Right arrow Sorption/Exchange
Right arrow Soil pH
Right arrow Organic Compounds
Right arrow Soil Pollution

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