Effect of Anionic Biosurfactant on Hexadecane Partitioning in Multiphase Systems Sreekala Thangamanit and Gina S. Shreve'** Department of Chemical Engineering, The University of Illinois at Chicago, Chicago, Illinois 60607 1, and Department of Chemical Engineering, Wayne State University, Detroit, Michigan 48202
The effectiveness of anionic rhamnolipid biosurfactant and a synthetic anionic surfactant in solubilizing hexadecane in multiphase systems is compared. The partitioning of hexadecane in multiphase systems containing aqueous, organic, and soil phases is examined in the presence of varying concentrations of rhamnolipid or alkyl benzyl sulfonate (ABS) surfactant. Partitioning of the rhamnolipid biosurfactant and the ABS surfactant between each of the phases is also examined. The critical micelle concentration (CMC) of each surfactant and the molar solubilization ratio (MSR) of each surfactant for hexadecane are experimentally determined. The MSR of the rhamnolipid is found to be approximately 20 times greater than the MSR of ABS for hexadecane. Differences in micellar structure between the rhamnolipid biosurfactant and the ABS syntheic surfactant are proposed to account for the increased ability of the rhamnolipid to solubilize hexadecane in the micellar phase.
While surfactants have been demonstrated to be effective in the desorption of hydrophobic contaminants from soil systems, their effect on bioremediation of soils and groundwater contaminated with petroleum hydrocarbons remains unclear. Biosurfactants are surface active agents produced by certain types of microorganisms during growth on insoluble substrates ( 2 , 3 ) . Biological surfactants have advantages relative to synthetic surfactants for specific applications due to their structural diversity, biodegradability, and effectiveness at extreme temperatures, pH, and salinity. Potential environmental advantages of such biologically based surfactants include their biocompatability and hence decreased likelihoodof cellular toxicity relative to synthetic surfactants. Microbial degradation of certain hydrocarbon contaminants has been demonstrated to be facilitated by the simultaneous production of biosurfactants (4-6). In contrast, synthetic surfactants have been shown to inhibit microbial activity when added to the environment in high concentrations (7).
Introduction
Contamination of aquifers by hazardous organic pollutants has become an increasing threat to the safety of many community sources of drinking water. Many of these pollutants, described as hydrophobic organic compounds (HOCs), are relatively insoluble in water. This low aqueous solubility affects their transport and localization in the environment. Most petroleum products are made up of a complex mixture of such hydrocarbons. These contaminants have entered the subsurface environment through waste disposal practices or chemical spills. These HOCs may be adsorbed onto the soil or present in the subsurface aquifer as a discrete organic phase mixture of nonaqueous phase liquids (NAPLs). Subsurface transport of these contaminants plays an important role in the remediation of soils and groundwater contaminated with petroleum hydrocarbons (1). Surfactant addition enhances the solubility of these HOCs in the aqueous phase. Such solubilized hydrocarbons are more available for conventional pump and treat strategies or for in situ biodegradation.
* Corresponding author;e-mail address: gshrevea eng.wayne.edu. The University of Illinois at Chicago. Wayne State University. 0013-936X/94/0928-1993$04.50/0
0 1994 American Chemlcal Society
To date, biologicalsurfactants remain untested for their ability to mobilize contaminants in multiphase environments. They have also not been directly compared to chemical surfactantsfor waste treatment applications. This work examines the effectiveness of microbially produced surfactants in solubilizing alkane HOCs in multiphase systems. While alkane hydrocarbons are not particularly recalcitrant in the environment, they are useful as model compounds for examining the role of biological surfactants in HOC mobilization and microbial degradation. Biological Surfactants
Microbial biosurfactants are comprised of a wide variety of chemical structures including glycolipids, lipopeptides, phospholipids, fatty acids, neutral lipids, and certain polysaccharide-protein complexes. Biosurfactants include a hydrophilic and a water-insoluble structural component. The hydrophilic component generally consists of either a carbohydrate, a hydrophilic amino acid such as glutamate, aspartate, lysine, or arginine, or a hydrophilic peptide. The water-insoluble portion frequently consists of either a lipid structure, an isoprenoid structure such as a cholesterol, or a hydrophobic amino acid or peptides including amino acids such as phenylalanine, leucine, isoleucine, valine, or alanine. The range of biological Envlron. Sci. Technol., Vol. 28, No. 12, 1994
1993
elements which may be combined to yield the amphiphilic biosurfactant structure is enormous, and new biosurfactant molecules are still being discovered that incorporate previously undescribed structural elements. Biosurfactant molecules are cell wall associated and are also secreted into the surrounding media. Such extracellular and cell wall associated molecules have the potential to promote cellular attachment to hydrophobic surfaces, to affect the distribution of cells between oil and water phases, to emulsify water-insoluble substances, and to mediate transport of hydrophobic substrates into the cell. Biosurfactant production is enhanced by growth of the microorganism on certain water-insoluble carbon substrates including alkane hydrocarbons (4-8) and vegetable oils (9, 10). In most of these cases, growth of the organism on insoluble substrate is stimulated by the presence of biosurfactant, and in some cases biosurfactant is required for growth. Biological surfactant synthesis is also influenced by environmental conditions such as nitrogen availabilty and divalent cation concentration. Biosurfactant production in certain Pseudomonas species is enhanced when cells are grown under low nitrogen conditions (11). Production of biosurfactant is also stimulated under conditions where multivalent cation is limiting (12). Biosurfactant synthesis in several systems is subject to catabolite repression. Biosurfactant production in certain Acinetobacter species (13, 14) and Pseudomonas aeruginosa (15, 16) has been shown to decrease when glucose is added in addition to alkane substrate. Mutants which are deficient in biosurfactant production are also available for rhamnolipid-producing Pseudomonas species (17) and trehalose ester-producing Rhodococcusspecies (18). Such mutants have been useful in providing evidence that biosurfactant production plays a general role in cellular growth on water-insoluble substrates and is important for cellular differentiation for some microbial species. There is also evidence that certain biosurfactants are specific in the uptake of a particular alkane substrate in some microbial species (19). Such mutants provide important tools for determining the key elements controlling synthesis of biosurfactant in the cell and the function of biosurfactant in uptake of insoluble hydrocarbon substrates. Experimental Procedures
Chemicals. n-Hexadecane was purchased from Aldrich Chemical Co. The synthetic surfactant used in these studies is alkyl benzene sulfonate (ABS). ABS was purchased in powdered form from Aldrich Chemical Co. Purified rhamnolipid was obtained as a purified lyophilysate from Othmar Kappeli of the BATS Institute in Switzerland. Both surfactants were used directly without further purification. Each of the organic phase constituents, namely, benzene, xylene (a mixture of 0-,m-, and p-isomers), and toluene were obtained from Aldrich Chemicals Co. The soil used in this study was obtained from the Environmental Protection Agency Environmental Research Laboratory in Athens, GA, and is designated as EPA-22. EPA-22 is a sediment (21.2% clay, 52.7% silt, and 26.1 % sand) possessing an organic carbon content of 1.67 % Aqueous phase media chemical components were purchased from Sigma Chemical Co. and Aldrich Chemical co. Measurement of Surface Tension. Measurements of the surface tension of surfactant solutions in water were
.
1994 Envlron. Scl. Technol., Vol. 28, No. 12, 1994
made using a Fisher 215 surface tensiometer. All measurements are made at room temperature. The Fisher Autotensiomat surface tension analyzer operates on the principles of the du Nuoy ring and Wilhelmy plate methods. To measure surface tension, the Autotensiomat employs a silicon strain gage. The ring and glassware used for measurement were cleaned with methanol and flamed. Surface tension values were taken when stable readings were obtained for a given concentration of surfactant in water, as indicated by at least two consecutive measurements having nearly the same value. The critical micelle concentration (CMC) was determined by measuring surfactant concentration over a wide concentration range and noting the inflection in the plot of surface tension versus surfactant concentration. Measurement of Hexadecane and Surfactant Concentration. The multiphase systems studied combined either organic and aqueous; soil and aqueous; or soil, aqueous, and organic phases. These multiphase systems were chosen to simulate the typical environment in a contaminated subsurface aquifer. The organic phase consisted of 10 mL of a 1:l:l mixture of benzene, toluene, and xylene. The aqueous phase consisted of 80 mL of the bacteriological media described by Guerra-Santos et al. (12). Soil-containing systems contained 2 g of EPA-22 soil. Batch multiphase experimental systems were constructed in 100-mL borosilicate glass serum bottles. Ten pL of a 100 ppm dilution of hexadecane in hexane was added to each of the experimental bottles. Surfactant was added to the batch systems at concentrations both above and below the CMC ranging from concentrations of surfactant which were roughly 25% of the CMC to approximately five times the CMC for a given surfactant. The experimental multiphase mixtures were then agitated on an orbital shaker a t 200 rpm overnight. Aliquots of the aqueous and organic phases were taken and directly analyzed on the HPLC for surfactant. Seventy-five mL of the aqueous phase was extracted with 5 mL of hexane. The hexane was evaporated off, and the hexadecane was resuspended in 10 pL of hexane. The BTX organic phase was evaporated down, and the remaining hexadecane was resuspended in 100 pL of hexane. The soil was washed with 10 mL of hexane from which hexadecane was extracted in a similar manner to the organic phase. Gas chromatograph analysis of the resulting sample was then performed. Contaminant material balances were assessed for each microcosm. Each series of multiphase systems evaluated included eight microcosms containing surfactant solutions at a range of concentrations both above and below the CMC and included a control microcosm containing no surfactant. Analytical Methods. High-performance liquid chromatography (HPLC) was used for the quantification of the alkyl benzene sulfonate and rhamnolipid biosurfactant. A Waters LC Module 1 HPLC with Millenium 2010 software was used with a C-18 Novapak column. The mobile phase for the ABS analysis consisted of 40% tetrahydrofuran (THF) and 60 % water containing paired ion chromatography (PIC A) reagent. PIC A reagent was obtained from Waters Chromatography. For the rhamnolipid HPLC analysis, the mobile phase consisted of 45 % THF and 55 % water containing the PIC A reagent. The surfactants in the organic phase were analyzed on the HPLC using a mobile phase of 60% THF and 40% water containing PIC A reagent. The HPLC pump flow rate
Rhamnolipid 1
Rharnnolipid 3
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ICH2), CH3
Alkyl Benzene Sulfonate S03Na
CH3(CH2)1 Flgure 1. Structures of rhamnolipid and alkyl benzene sulfonate surfactants.
was 1 mL/min. The ABS and rhamnolipid surfactant concentrations were measured using a UV detector set at a wavelength of 254 nm. The concentration of hexadecane in the organic, aqueous, and soil phases was determined by gas chromatography on a Hewlett Packard 5750 system using a RT,1 megabore column with a flame ionization detector. The instrumental method used included three sequential temperature stages. In the first stage, the column temperature was increased from 42 to 80 OC at a rate of 40 OC/min. In the second stage, the column was heated from 80 to 100 OC at 45 "C/min. The final stage incorporated a temperature increase of 10 OC/min from 100to 165 OC. Ten mL of the organic phase was evaporated and resuspended in 100 mL of hexane. Ten p L of the resulting sample was then injected onto the gas chromatograph. Standard experimental errors were evaluated from triplicate GC analysis of samples. Surfactant Molar Solubilization Ratios
Microbial rhamnolipid is an anionic biosurfactant which is produced in four different structural forms with a primary structure linking a rhamnose sugar to I-/3hydroxydecanoic acid. The four structural forms combine either one or two rhamnose sugar units with either one or two hydroxydecanoic acid units (Figure 1). An average molecular mass of 526 Da was estimated for the rhamnolipid. Alkyl benezene sulfonate (ABS), the anionic synthetic surfactant used in these studies, is a linear dodecyl benzene sulfonate with a molecular mass of 377. Alkyl benzene sulfonate was chosen as the most readily available synthetic surfactant possessing several structural elements which are common to the rhamnolipid and ABS. Both the ABS and rhamnolipid surfactants contain straight chain hydrocarbon groups: dodecane in the case of ABS and two heptane moieties in the case of rham-
nolipids 1 and 3. Both ABS and the rhamnolipid surfactants contain six-membered rings, and both contain anionic functional groups. For ABS, the sulfate group of the benzyl sulfate group is anionic. The terminal carboxylic acid of the rhamnolipid hydroxydecanoic acid group is primarily present in anionic form at neutral pH since the pK, of the terminal carboxylic acid hydrogen is expected to be similar to those of other alkanoic acids ranging from 4.4 to 4.9 for fatty acids possessing between 6 and 10 carbon length tails (20). Also, the surface tension at the CMC was similar for the ABS and the rhamnolipid biosurfactant. This provides a basis for comparison of two surfactants sharing common structural elements and possessing a similar CMC surface tension. The concentration at which the solution surfactant concentration is sufficient to accommodate the aggregation of surfactant molecules into ordered micelle structures is known as the critical micelle concentration. At the critical micelle concentration, an abrupt change in certain solution properties including the lowering of the liquid surface tension is observed. This observed abrupt change in surface tension may be employed to determine the solution CMC. It should be noted, however, that the CMC determined for a surfactant varies to some extent depending on the solution property measured and the evaluation method applied. The CMC values for the ABS and rhamnolipid surfactants were determined through a conventional plot of the surface tension versus the logarithm of the surfactant concentration (Figure 2). In the plot, the concentration at which a pronounced break in the slope occurs corresponds to the CMC. The CMC of the ABS synthetic surfactant in water was found to be 160 mg/L or 0.424 mM (Figure 2). The CMC of the rhamnolipid biosurfactant in water was determined to be approximately 18 mg/L or 0.0342 mM for rhamnolipid (Figure 1). This is within reasonable agreement with the value of about 60 mg/L measured for the purified R-1 Envlron. Scl. Technol., Vol. 28, No. 12, 1994 1995
7c
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,
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of moles of organic compound solubilized per mole of surfactant added to solution has been previously defined as the molar solubilization ratio (MSR) (22). The MSR for hexadecane may be calculated as
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Figure 2. Aqueous phase surface tension at varying surfactant concentrations (8)rhamnolipid; (6) alkyl benzene sulfonate.
rhamnolipid structure (19). The surface tension measured at the CMC was approximately 32 dyn/cm for ABS and 33.5 dyn/cm for the rhamnolipid. The value of 33.5 obtained is similar to that of 30 dyn/cm obtained by Fiechter et al. for an uncharacterized mixture of rhamnolipid isomers in culture liquid (21). Hexadecane Partitioning and Solubilization in Two-Phase Systems. Hexadecane partitioning in twophase systems consisting of an organic and an aqueous phase or a soil and an aqueous phase was examined. The effect of the addition of synthetic or biological surfactants on contaminant partitioning and solubilization into the aqueous phase was assessed. The solubilization of hexadecane into the aqueous phase from the organic or soil phase in the presence of ABS and rhamnolipid was compared. Hexadecane partitioning in aqueous and organic twophase systems was examined first. Hexadecane is essentially insoluble in water. Addition of the BTX organic phase enhances the hexadecane solubility in the aqueous mol/L presumably phase to approximately 4.7 X through the cosolvency effect of dissolved aqueous phase BTX molecules. The quantity of hexadecane in the aqueous phase was dramatically enhanced by the addition of sufficient quantities of surfactant. This surfactantmediated micellar solubilization represents the dissolution of the compound by reversible interaction and solubilization within the micellar phase of the surfactant solution. For experimental systems containing either the ABS or rhamnolipid surfactant, the onset of transport of more contaminant into the aqueous phase due to the addition of surfactant occurred after the system had attained the CMC and was proportional to the volume of the aqueous micellar phase. There was not much change in the amount of aqueous phase contaminant at concentrations below the critical micelle level when no micellar phase exists. Below the CMC, the hexadecane concentration in the aqueous phase remained constant at approximately 4.7 X l@gmol/L. A t concentrations above the CMC, hexadecane was increasingly solubilized into the aqueous micellar phase. Hexadecane solubilization showed a linear dependence on ABS concentration with a slope of 6.2 X mol of hexadecane partitioned into the aqueous phase per mole of ABS added (Figure 3). This ratio of the number l9SS
Environ. Sci. Tschnol., Vol. 28, No. 12, 1994
whereSm,micis the total apparent aqueous phase solubility of the hexadecane (in mol/L) in micellar solution at a , ,the ~ ~apparent particular surfactant concentration; S H D is solubility of hexadecane compound (in mol/L) at the CMC; and CBurfis the surfactant concentration at which S H D , ~ ~ , is evaluated. At concentrations above the CMC of 18 mg of rhamnolipid/L, the hexadecane concentration in the aqueous phase increased in proportion to the amount of rhamnolipid added yielding an MSR of 1.35 X 10-5 moles of hexadecane solubilized into the aqueous phase per mole of rhamnolipid added (Figure 4). A similar effect was observed in the soil and aqueous two-phase systems. For both surfactant systems, at concentrations below the CMC the aqueous phase hexadecane concentration was approximately constant at 4.5 X 10-9 mg/mL. As the surfactant concentration increased, increasing amounts of hexadecane were mobilized from the soil to the aqueous phase. This linear dependence of the aqueous hexadecane concentration on increasing concentrations of ABS yielded a MSR of 7.9 X 10-7 for ABS (Figure 5). The MSR value obtained in the soil and aqueous two-phase systems to which rhamnolipid was (Figure 6). The solubilizing ability added was 1.33 X of the ABS and rhamnolipid surfactants was compared by evaluating the MSR for the alkyl benzene sulfonate and the rhamnolipid for the multiphase systems examined (Table 1). The magnitude of the MSR value reflects the effectiveness of a particular surfactant in solubilizing the contaminant. The molar solubilization ratio of the rhamnolipid was determined to be approximately 20 times greater than that of the alkyl benzene sulfonate. This indicates that on a per mole basis the rhamnolipid is capable of solubilizing about 20 times more hexadecane than the synthetic surfactant. Hexadecane Partitioning and Solubilization in Three-phase Systems. The partitioning of the hexadecane into the aqueous phase in the ABS or rhamnolipid containing three-phase systems is shown in Figures 7 and 8, respectively. As in the two-phase systems, the first point in these graphs represents the solubility of the contaminant in the absence of surfactant. Contaminant solubility shows a linear dependence on surfactant concentration a t surfactant concentrations above the CMC. There is not a significant change in the amount of contaminant in the aqueous phase at concentrations below the CMC. The amount of contaminant solubilized again shows a linear dependence on the amount of surfactant added in excess of the quantity needed to attain the CMC. Figures 9 and 10 represent the amount of contaminant desorbed from the soil in the presence of the ABS or rhamnolipid, respectively. The hexadecane MSR obtained for the rhamnolipid in the three-phase systems was 1.38 X The hexadecane MSR of ABS was 5.9 X in the three-phase system. Thus, the MSR of the rhamnolipid was approximately 23 times higher than the MSR of the ABS in the three phase systems. The surfactant concentrations in Figures 3-6 represent the total surfactant concentration present in the multiphase system. Since different surfactants are
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known to partition differently between phases, it was necessary to determine what the actual aqueous phase surfactant concentration was to ascertain whether differences in the partitioning of the synthetic and biological surfactants between the various phases might result in
differences between the actual aqueous phase surfactant concentration and the overall surfactant concentration in the system. Such differences in ABS and rhamnolipid partitioning could account for the observed greater effectiveness of the biological surfactant. In fact, the Environ. Sci. Technol., Voi. 28, No. 12, 1994
1997
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Flgure 5. Aqueous phase hexadecane concentration for varying concentrations of ABS surfactant added to the soil/aqueous two-phase system.
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Figure 6. Aqueous phase hexadecane concentration for varying concentrations of rhamnolipid surfactant added to the sol/aqueous two-phase system.
apparently greater effectiveness of the rhamnolipid for solubilizing hexadecane could be due to the fact that more of the biological surfactant was present in the aqueous phase compared to the aqueous phase concentration of the synthetic surfactant. To determine if such differences in surfactant partitioning were a factor in these studies, we examined the distribution of ABS and rhamnolipid surfactant in the two-phase systems studied. Distribution Coefficient of the ABS and Rhamnolipid Surfactants in Two-Phase Systems. The amount of surfactant present in the aqueous and organic phases was determined by HPLC analysis of samples from each of the aqueous and organic liquid phases. The amount of surfactant present in the soil was calculated as the difference between the total surfactant added to the system and the quantity present in the aqueous phase (Table 2). The average distribution coefficient measured across the ABS concentration range examined was 0.215, indicating that approximately 17.5% of the ABS added partitioned lSS8 Environ. L :i. Technol., Vol. 28, No. 12, 1994
into the aqueous phase with the remainder partitioning into the organic phase. In the soil and aqueous phase system, the average distribution coefficient measured was 1.22 corresponding to 55% of the ABS partitioning into the aqueous phase with the remaining 45 % localizing on the soil. In the aqueous and organic two-phase systems to which rhamnolipid was added, the average distribution coefficient measured was 0.286, indicating that approximately 22.5 % of the rhamnolipid localized into the aqueous phase and 77.5% into the organic phase. In the soil and aqueous phase systems, the average distribution coefficient measured for the rhamnolipid was 1.54 corresponding to approximately 60.5 % of the rhamnolipid partitioning into the aqueous phase with 39.5 % localizing on the soil. These results indicate that both the biological and the synthetic surfactant were most soluble in the organic phase and that at the concentrations examined precipitation of either surfactant onto the soil was not excessive. Comparison of the MSRs of the synthetic ABS surfactant and the rhamnolipid biosurfactant on the basis of the aqueous phase surfactant concentration yielded the same trend on a per mole basis. In general, the rhamnolipid was approximately 20 times more effective than the synthetic surfactant (Table 1).
Discussion Most studies and generalizations of micellar assembly have been developed using straight chain hydrocarbon amphiphiles. The ability of these models to predict rhamnolipid micelle formation is limited. Detailed physical analysis and spectroscopic studies will undoubtedly be required to develop similar models which will be applicable to more complex biosurfactant structures. However,application of the known generalizations to ABS surfactant micelles leads to the following conclusions. At the temperature and ionic strength conditions used in these studies, sodium dodecyl sulfate (SDS) would be expected to form spherical micelles. This may be determined by applying the critical condition for spherical micelle formation as described by Israelachvilli et al. (23). Calculation of the ratio of the micelle hydrocarbon core volume to the product of the optimum surface area and the critical length of the hycarbon chain yields a value of approximately 0.005 using an aggregation number of 112 and a total surface area of 34.5 nm2 for SDS (24). This is considerably less than the critical value of 0.333 at which the transition from spherical to cylindrical micelles begins. The similarity in structure between ABS and SDS indicates that ABS probably also forms spherical micelles under the conditions used in these studies. The rhamnolipid structure is expected to form a micelle with a lower likelihood of being spherical and which possesses a less dense packing of surfactant monomer due to the larger size of the surface group, which includes the rhamnose sugar moities and the two alkyl groups on the rhamnolipid 1 and 3 isomers. This would provide for a large volume, low density micelle that is able to accommodate a greater amount of hexadecane. This type of micelle is more characteristic of nonionic surfactant micelles than anionic surfactant micelles. Hence, the MSR obtained with the rhamnolipid may be closer to those obtained with nonionic surfactants than anionic surfactants. In fact, in experimental systems containing much greater hexadecane concentrations, the MSRs obtained for both rhamnolipid and ABS are several orders of magnitude higher and comparable to values published for certain nonionic
Table 1. Hexadecane Solubilization by Synthetic Surfactant and Biosurfactant.
organic/aqueous
soil/aqueous
organiciaqueous soil
5.9 x lo-' 7.9 x 10-7(1.5 x 10-8) 6.2 X 10-7 (3.58 X 10-8) alkyl benzyl sulfonate 1.33 X (2.13 X lod) 1.38 X 1od 1.35 X 106 (6.02 X lo3) rhamnolipid "Molar solubilization ratio based on total surfactant in the system (MSR based on aqueous phase surfactant). Units are in moles of mol of ABS/g of ABS. To hexadecane per mole of surfactant. To convert to moles of hexadecane per gram of ABS, multiply by 2.65 X convert units to moles of hexadecane per gram of rhamnolipid, multiply by 1.9 X lo3 mol of rhamnolipid/g of rhamnolipid.
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Figure 7. Aqueous phase hexadecane concentration for varying concentrations of ABS surfactant added to the organlc/aqueous/soil three-phase system; (0)f l SD.
Figure 9, Soil phase hexadecane concentration for varying concentrations of ABS surfactant added to the organic/aqueous/soii threephase system; (0)f l SD. 101
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surfactants, although the rhamnolipid MSR remains approximately 1 order of magnitude higher than that of ABS. Since the alkyl chains on both the rhamnolipid and the ABS are longer than six carbon atoms, the character of both micelles should be predominantly deep palisades possessing a micellar phase with an organic character (25). The addition of aromatic hydrocarbons to the experimental system may affect the organic character of the hydrophobic core. Since hexadecane is fully miscible in the benzene, xylene, and toluene mixture, their presence may have more of an effect on hexadecane uptake through their occupation of micellar space than through a change in the chemical environment of the micelle hydrophobic core.
A B S concentration (mgiL)
Figure 10. Soil phase hexadecane concentration for varying concentrations of rhamnollpld surfactant added to the organic/aqueous/ soil three-phase system; (0)flSD.
Table 2. Surfactant Distribution in Two-Phase Systems. Kaqueouslorganie
~aqusoualsoil
alkyl benzyl sulfonate 0.215 1.22 rhamnolipid 0.286 1.54 Distribution coefficient. Mass of surfactant in phase I/mass of surfactant in phase I1 ( g / g ) .
The greater capacity of the rhamnolipid to solubilize the hexadecane contaminant is most likely due to the greater volume of the micellar phase in the rhamnolipid systems, which allow for increased hexadecane solubilization. Also since the chain length of the hexadecane is significantly greater than that of dodecane, solubilization Environ. Sci. Technol., Vol. 28, No. 12, 1994 1999
of hexadecane into a spherical ABS micelle may be very limited whereas a much larger elliptical rhamnolipid micelle may more favorably accommodate the hexadecane molecule. Also with a much lower CMC the rhamnolipid monomer is more insoluble, thus more of the biosurfactant exists in the micellular phase and more micelles or micelles with higher aggregation numbers are formed. Further studies characterizing the physical size of the rhamnolipid micelle, in particular, determination of either the biosurfactant micelle aggregation number or the diameter of the biosurfactant micelle, will be useful to confirm these conclusions. It will also be of interest to evaluate the ability of such biological surfactants to solubilize additional contaminants of environmental interest. Acknowledgments
The authors would like to thank William Finnerty and Othmar Kappeli for supplying rhamnolipids. This work was supported by the Environmental Protection Agency's Office of Exploratory Research under Research Grant R820 399-01-0. Literature Cited McCarthy, J. F.; Zachara, J. M. Environ. Sci. Technol. 1989, 23,496-502. Desai, J. D. J. Sci. Ind. Res. 1987, 46, 440-449. Georgiou, G.;Lin, S. C.; Sharma, M. M. Biotechnology 1992, IO, 60-65. Suzuki, T.; Tanaka, K.; Matsubara, I.; Kinoshita, S. Agric. Biol. Chem. 1969, 33 (ll),1619-1627. Schmidt, M.; Passeri, A.; Schulz, D.; Lang, S.A.; Wagner; Poremba, K.; Gunkel, W.; Wray,V. DechemaBiotechnology Conferences 3; VCH Verlagsgesellshaff Braunschwig, F.R.G, 1989. Martin, M.; Bosch, P.; Parra, J. L. Carbohydr. Res. 1991, 220, 93-100. Laha, S.; Luthy, R. G. Biotechnol. Bioeng. 1992,40,13671380.
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Received for review July 26,1993. Revised manuscript received February 7, 1994. Accepted August 3, 1994.' Abstract published in Advance ACS Abstracts, September 1, 1994. @
