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TECHNICAL REPORT
HEAVY METALS IN THE ENVIRONMENT

Stability Constants for the Complexation of Various Metals with a Rhamnolipid Biosurfactant

Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ 85721

Corresponding author (rmaier{at}ag.arizona.edu)

Received for publication May 3, 2000.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The presence of toxic metals in natural environments presents a potential health hazard for humans. Metal contaminants in these environments are usually tightly bound to colloidal particles and organic matter. This represents a major constraint to their removal using currently available in situ remediation technologies. One technique that has shown potential for facilitated metal removal from soil is treatment with an anionic microbial surfactant, rhamnolipid. Successful application of rhamnolipid in metal removal requires knowledge of the rhamnolipid–metal complexation reaction. Therefore, our objective was to evaluate the biosurfactant complexation affinity for the most common natural soil and water cations and for various metal contaminants. The conditional stability constant (log K) for each of these metals was determined using an ion-exchange resin technique. Results show the measured stability constants follow the order (from strongest to weakest): Al³⁺ > Cu²⁺ > Pb²⁺ > Cd²⁺ > Zn²⁺ > Fe³⁺ > Hg²⁺ > Ca²⁺ > Co²⁺ > Ni²⁺ > Mn²⁺ >Mg²⁺ > K⁺. These data indicate that rhamnolipid will preferentially complex metal contaminants such as lead, cadmium, and mercury in the presence of common soil or water cations. The measured rhamnolipid–metal stability constants were found in most cases to be similar or higher than conditional stability constants reported in the literature for metal complexation with acetic acid, oxalic acid, citric acid, and fulvic acids. These results help delineate the conditions under which rhamnolipid may be successfully applied as a remediation agent in the removal of metal contaminants from soil, as well as surface waters, ground water, and wastestreams.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
INCREASING accumulation of toxic metals in soil and freshwater environments represents a potential health hazard for humans (McKinney and Rogers, 1992). Consequently, the treatment of metal-contaminated soils and surface and subsurface water bodies has become a major focus of recent research. Because they are not degraded, metals in these sites must be either immobilized or removed. Both immobilization and removal are complex processes that require an understanding of the behavior of metals in the environment. The complexity arises because metal behavior is dependent on a variety of factors including speciation, complexation, precipitation, and sorption reactions. These reactions are defined by mineral composition and soil chemical properties, as well as variable environmental conditions such as pH, salinity, and redox.

Microorganisms and microbial products are receiving increased attention as alternative technologies for immobilization or removal of metals from soil (Miller, 1995). We have recently reported that monorhamnolipid (rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate), a biosurfactant produced by Pseudomonas aeruginosa, is an effective agent for complexation of metals such as Pb²⁺, Cd²⁺, and Zn²⁺ (Tan et al., 1994; Herman et al., 1995). However, in order to successfully apply rhamnolipids for in situ or ex situ cleanup of soil or wastestreams, an understanding is needed concerning the relative strength of rhamnolipid–metal complexes. Additionally, complexes may be formed between metals and other naturally occurring organic ligands (e.g., humic or fulvic acids), and between rhamnolipids and ions such as Ca²⁺, Mg²⁺, and K⁺. The strength of the complexation between organic ligands and metals is usually expressed in terms of a stability constant (Schnitzer and Skinner, 1967; Manunza et al., 1995; Cheam and Gamble, 1974). For example, stability constants for metal complexation with fulvic acids, humic acids, and sewage sludge have been reported by several different groups (Rhandhawa and Broadbent, 1965; Schnitzer and Skinner, 1966, 1967; Schnitzer and Hansen, 1970; Cheam and Gamble, 1974; Sposito et al., 1979; Stevenson, 1976; Gould and Genetelli, 1978; Saar and Weber, 1979, 1980; Pott et al., 1985; Breault et al., 1996).

Our objective was to determine the biosurfactant complexation affinity for a variety of metals that are important either because of their natural prevalence in the environment or because they are potential contaminants of aquatic or soil systems. The biosurfactant used in this study was a monorhamnolipid produced by Pseudomonas aeruginosa ATCC 9027 (Zhang and Miller, 1992), which has been characterized as a mixture of four monorhamnolipids differing only in the number of carbons contained by the fatty acid tails. Monorhamnolipid has an average molecular weight of 504 (Zhang and Miller, 1994), a critical micelle concentration (cmc) of 40 mg L^-1 (Zhang and Miller, 1992), and a reported pK_a of 5.6 (Ishigami et al., 1987). The metals used in this study were Ca²⁺, Mg²⁺, and K⁺, three of the four most prevalent metal cations (other than Na⁺) in soil solution (Sparks, 1995, p. 81–97) and in most natural waters (Morel and Hering, 1993, p. 345–350). In addition, Al³⁺, Fe³⁺, Cd²⁺, Co²⁺, Pb²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Hg²⁺, and Mn²⁺ were chosen for study, most of which are among the twenty metals of major interest to the USEPA for their potential health risk (McKinney and Rogers, 1992).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Rhamnolipid Production
Pseudomonas aeruginosa ATCC 9027 was obtained from the American Type Culture Collection (Rockville, MD). Rhamnolipid production and purification have been described in detail previously (Zhang and Miller, 1992). Rhamnolipid concentration was estimated by surface tension measurement using a surface tensiomat (Model 21, Fisher Scientific, Fairlawn, NJ), which employs the Du Nouy ring method (Miller and Zhang, 1997). Rhamnolipid concentration was determined using a calibration curve relating surface tension (dyn cm^-1) to rhamnolipid concentration (mg L^-1). Calibration curves were prepared under conditions identical to those for each experiment.

Metals
Al(NO₃)₃, Fe(NO₃)₃, Pb(NO₃)₂, Cu(NO₃)₂, Zn(NO₃)₂, Co(NO₃)₂, Hg(NO₃)₂, Ni(NO₃)₂, Cd(NO₃)₂, Mn(NO₃)₂, Ca(NO₃)₂, Mg(NO₃)₂, and KNO₃ were purchased from Aldrich (Milwaukee, WI) with a purity from 98 to 100% and were used as received. Atomic absorption (AA) standard solutions for each metal were obtained from Fisher.

Measurement of Conditional Stability Constants
An ion-exchange technique was used to determine conditional stability constants for rhamnolipid with the various metals. This technique is suitable only for mononuclear complexes (Schnitzer and Hansen, 1970) and the number of moles of the organic ligand () combined with one mole of metal (M) must be an integer 1 (Clark and Turner, 1969; Smith et al., 1986). This technique was originally developed by Schubert (1948a)(b) and then applied to metal–soil organic matter complexes (Schnitzer and Skinner, 1966, 1967; Randhawa and Broadbent, 1965; Schnitzer and Hansen, 1970; Matsuda and Ito, 1970), to metal-activated sludge complexation (Cheng et al., 1975), and more recently to metal–biosurfactant complexation (Herman et al., 1995). All metal complexation experiments were performed in 0.01 M Pipes [piperazine-N,N'-bis(2ethane-sulfonic acid)] buffer solution (Sigma, St. Louis, MO) adjusted to pH 6.9 using 1 M NaOH. The resin used was sodium-saturated SP Sephadex C-25 (Pharmacia Biotech AB, Uppsala, Sweden), which was allowed to expand overnight in deionized distilled water, washed in deionized distilled water, washed several times in 0.01 M Pipes buffer, and then air-dried. The resin has a cation exchange capacity (CEC) of 2.6 mmol g^-1 as determined by the NH₄/Na acetate method at pH 7.0 (Rhoades, 1982).

From previous experiments (data not shown) in the absence of biosurfactant, a resin concentration of 10 mg mL^-1 bound most (99%) of the metal added (0.5 mM). Thus, for each experiment, 100 mg of air-dried sodium-saturated resin was transferred into triplicate 20-mL polypropylene scintillation vials containing 5 mL of Pipes buffer solution. Metal stock solutions were freshly prepared less than 0.5 h before use. Appropriate aliquots of a 20.0 mM rhamnolipid stock solution and a 1 mL aliquot of a 5.0 mM metal stock solution were added to give a final concentration of 0 to 5.0 mM rhamnolipid and 0.5 mM metal in 10 mL 0.01 M Pipes buffer. Samples were shaken at 100 rotations per minute on a rotary shaker for 2 h at room temperature to allow the mixture to reach equilibrium and then allowed to settle for at least 1 h. Metal concentration in the supernatant was determined by atomic absorption (AA) on an Instrument Laboratory Video 12 aa/ae spectrophotometer (Allied Analytical Systems, Waltham, MA). A calibration curve was prepared for each metal using AA standard solutions.

Calculation of the Stability Constant
The principle of ion-exchange equilibrium (Schubert, 1948a,b) was used to investigate the complex composition and the relative affinity of a number of metals to complex with rhamnolipid. The equilibrium reactions of a metal with an organic ligand and an ion-exchange resin are (Schubert, 1948a,b; Cheng et al., 1975):

[1]

[2]

where M_L is the free metal in solution at equilibrium in an organic ligand-containing system (mol L^-1), M_R is the free metal in solution at equilibrium in an organic ligand-free system (mol L^-1), is the number of moles of organic ligand that combine with one mole of metal ion (mol mol^-1), L is the soluble organic ligand (mol L^-1), ML is the complexed metal–organic ligand in solution at equilibrium (mol L^-1), R is the ion-exchange resin (kg L^-1), and MR is the metal bound to the ion-exchange resin at equilibrium by a unit weight (mol kg^-1).

From the complex formation reaction (Eq. [1]), the equilibrium constant, called a stability constant, K, can be determined using the equation:

[3]

At equilibrium, the metal distribution ratio between MR and M_R in the absence of a complexing agent is a constant, _o:

[4]

and the distribution constant in the presence of a complexing agent, , is

[5]

An experimental determination of the metal distribution constants (Eq. [4] and [5]) can be carried out and a conditional stability constant, log K, can be obtained through the linear relationship

[6]

The unknown values of and log K can be determined separately for each metal from the slope and intercept, respectively, of a plot of log [(_o/) - 1] versus log L, where L is the rhamnolipid concentration.

The above relationships can be applied only if the organic ligand is not bound by the ion exchanger and the metal solution concentration is small compared with that of the complexing agent (Randhawa and Broadbent, 1965). Previous investigation has shown that rhamnolipid does not bind to the ion-exchange resin (Herman et al., 1995).

	RESULTS AND DISCUSSION

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The effect of rhamnolipid concentration on complexation of 13 metals was determined. Results for copper and cobalt are shown in Fig. 1 . In the absence of rhamnolipid, approximately 99% of each metal was bound to the resin at equilibrium leaving 1% in the aqueous phase. As increasing amounts of rhamnolipid were added, increasing amounts of both metals were observed in the aqueous phase as a result of complexation with rhamnolipid. Copper was complexed much more readily than cobalt (e.g., at 5.0 mM rhamnolipid, 99.5% of Cu²⁺) but only 11% of Co²⁺ was found in the aqueous phase. Complexation efficiency can be expressed in terms of conditional stability constants. Therefore, the measured data were used to calculate the molar ratio () and conditional stability constant (log K) for rhamnolipid complexation of each metal as shown for copper in Fig. 2 . The molar ratio and conditional stability constant values obtained for rhamnolipid complexation with the remaining 12 metals tested were determined similarly (Table 1). Comparing the variability for log K and among all metals tested through the coefficient of variation (CV) [(std. error/log K or ) 100%], we found that for log K and the CV ranged from 3.6 to 15.9% and from 4.2 to 19.3%. The greatest variability was associated with Pb²⁺ (13.2 and 15.8%), Fe³⁺ (13.8 and 19.3%), Ca²⁺ (15.5 and 15.9%), and Mn²⁺ (15.9 and 16.7%). Coefficients of determination (r²) and F values from regressions indicate that all linear regressions provided a good fit and were significant at the 1% level.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Distribution of Cu2+ and Co2+ between ion-exchange resin and the aqueous phase as rhamnolipid concentration increases from 0 to 5 mM. Each point represents the mean and standard deviation of triplicate samples

View larger version (18K): [in this window] [in a new window]   Fig. 2. Determination of the Cu2+ complexation constant and molar rhamnolipid to Cu ratio by the ion-exchange equilibrium method

The order of stabilities for rhamnolipid–metal complexation (Table 1) differs from that reported previously for fulvic acids (Schnitzer and Skinner, 1967; Schnitzer and Hansen, 1970); however, it is in general agreement with values found in a literature search (Table 2). In terms of , which indicates the molar ratio of ligand to metal, the results in Table 1 indicate that varied from 0.57 to 2.48 depending on the metal tested. A similar range of values have been reported for metal–organic matter complexes in which the ion-exchange equilibrium method has been employed (Randhawa and Broadbent, 1965; Schnitzer and Skinner, 1966, 1967). It should be noted that the organic complexing agents usually found in soils are large polyvalent particles and the equilibrium reactions shown in Eq. 1 and 2 are for monomer–metal mononuclear complex formation. Thus, for a surfactant such as rhamnolipid, at concentrations at and above the critical micelle concentration (cmc), the formation of rhamnolipid micelles or vesicles will affect metal complex formation. In addition, the failure to obtain nonintegral organic ligand–metal ratios can be largely attributed to analytical errors (Schnitzer and Hansen, 1970). These factors may contribute to the fact that varied over a wide range. If the values of are rounded off (see * in Table 1), it can be seen for many of the metals tested, that approximately one mole of rhamnolipid complexed one mole of metal. The exceptions to this are Al³⁺, Cu²⁺, Pb²⁺, Cd²⁺, and Zn²⁺, for which approximately 2 moles of rhamnolipid were required to bind one mole of metal.

There is little consensus on how to predict the order of stabilities for metal–organic complexes (Pott et al., 1985). However, a general rule is that trivalent metals form stronger bonds than divalent and monovalent ones (Morel and Hering, 1993, p. 345–350; Snoeyink and Jenkins, 1980, Chapter 5), although the amount of the trivalent form found in nature may be largely limited by speciation into the corresponding oxides and hydroxides (Morel and Hering, 1993, p. 345–350). Our data shows that of the two trivalent metals tested, Al³⁺ formed the most stable complex with rhamnolipid (log K = 10.3, Table 1). Thus, the presence of Al³⁺ could be a major constraint in application of rhamnolipid since it may compete effectively with divalent metals. Examples of sites that could contain high levels of Al³⁺ include highly chemically weathered soils or sites affected by acid mine drainage. The second trivalent metal tested was Fe³⁺, which has a measured conditional stability constant of 5.16 and a molar ratio of 1.2. The stability constant for Fe³⁺ is lower than the stability constants for Cu²⁺ (9.27), Pb²⁺ (8.58), Cd²⁺ (6.89), and Zn²⁺ (5.62), suggesting that there would be little interference from Fe³⁺ in the complexation of these metals, particularly for the former three metals.

The low log K and values obtained for Fe³⁺ may be ascribed, in part, to the precipitation of Fe³⁺ in the ferric nitrate stock solution used in this experiments, which in turn could have caused a decrease in the concentration of the ferric hydrolyzed soluble species available to complex with rhamnolipid. In fact, the stock solutions were slightly turbid indicating such precipitation may have occurred. Thus, the determination of log K and for Fe³⁺ may have been underestimated. Unlike Fe³⁺, precipitation of the aluminum nitrate stock solution was not observed prior to performing the experiment. Another possible explanation for the difference in the stability constants of the two trivalent metals tested in this study may be that these values actually represent an average of the different complexation constants of several dissolved species. Schnitzer and Skinner (1963) and Khan (1969), based on potentiometric titrations, found that soil organic matter and humic acids formed stable complexes with Al³⁺ and Fe³⁺ up to approximately pH 8. At pH 8, Al(OH)₃ and Fe(OH)₃ become the predominant metal forms, which due to low solubility do not readily form metal–organic ligand complexes. Within the pH range of 3 to 8, the dominant aluminum and iron species found in Al³⁺– and Fe³⁺–organic matter complexes were Al(OH)²⁺ and to some extent Al⁺₂, and FeAl⁺₂ (Schnitzer and Skinner, 1963). These same ferric and aluminum species were reported by Scheffer and Ulrich (1960) as the predominant species in solutions in equilibrium with Fe(OH)₃ and Al(OH)₃ at pH above 3.0 and 4.5, respectively. This led us to hypothesize that, under our experimental conditions, aluminum was primarily present as a divalent species, Al (OH)²⁺, and iron was primarily present as the monovalent species FeAl⁺₂. As a result, it would be expected that the and log K values for Al³⁺ would be greater than for Fe³⁺. This is confirmed by the results in Table 1, which show that the stability constants were 10.3 and 5.16 for Al³⁺ and Fe³⁺, respectively. The molar ratios were approximately 2 for Al³⁺ and 1 for Fe³⁺.

To further confirm these findings, we used the ground water model SURFEQL, an improved version of MINEQL (Westall et al., 1976), to predict the dominant aluminum and iron species that would be present in solution under the conditions used in this study. SURFEQL predicted the predominant aluminum species to be Al(OH)²⁺ and the dominant iron species to be the neutral trihydroxide Fe(OH)₃ with a limited amount of the monovalent species, Fe⁺₂. Thus, these modeling results also support the finding that aluminum would be expected to have a higher stability constant than iron.

The following order of stabilities was formed between rhamnolipid and the 10 divalent metal ions studied: Cu²⁺ > Pb²⁺ > Cd²⁺ > Zn²⁺ > Hg²⁺ > Ca²⁺ > Co²⁺ Ni²⁺ > Mn²⁺ > Mg²⁺. The results show that the stability of complexes of divalent metal ions for the first and third transition series followed the order Pb > Hg and Cu > Zn > Co Ni > Mn, respectively, which also correspond to the order of the Irving–Williams series (Irving and Williams, 1948). Some researchers have found this order to be followed closely in complexation of divalent metals by organic matter in soil (Beckwith, 1955; Khanna and Stevenson, 1962; Khan, 1969), while others have found significant deviation (Schnitzer and Skinner, 1966, 1967).

In summary, the purpose of this investigation was to determine stability constants for common soil cations and metal contaminants found in aquatic or soil systems. Stability constants determined in this study suggest that soil cations such as Ca²⁺, Mg²⁺, and K⁺ (log K = 4.10, 2.66, and 0.96, respectively) will not interfere with the removal of metal contaminants such as Cu²⁺, Pb²⁺, Cd²⁺, and Zn²⁺ (log K = 9.27, 8.58, 6.89, and 5.62, respectively) unless they are present in much greater concentration than the contaminant metals. Of the remaining metals studied, aluminum has a very high stability constant (log K = 10.30), and could be expected to interfere with contaminant metal complexation. However, high aluminum concentrations only occur in acidic soils (pH < 5), which due to the low pH contain few metal contaminants. Further, this technique is not suitable for acidic soils since rhamnolipid (pK_a = 5.6) precipitation occurs at acidic pH. The results obtained provide a basis for comparing the metal complexing abilities of rhamnolipid, a naturally ocurring organic ligand with other metal complexation agents both natural and synthetic.

	ACKNOWLEDGMENTS

 
Our thanks to Dr. David Hendricks for his help with atomic absorption analyses. This work was supported in part by Grant Number 2 P42 ESO4940-11 from the National Institute of Environmental Health Sciences, NIH with funds from the USEPA, and in part by Grant Number DE-FGD3-97ER62470 from the U.S. Department of Energy.

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