Solubilization of Naphthalene and Methyl-Substituted Naphthalenes

For several years, oil dispersants and emulsifiers have been proposed as a means to remove oil from water surfaces. ... and dispersion of oil can rele...
0 downloads 0 Views 167KB Size
Environ. Sci. Technol. 1997, 31, 556-561

Solubilization of Naphthalene and Methyl-Substituted Naphthalenes from Crude Oil Using Biosurfactants SHAHRUKH A. KANGA, JAMES S. BONNER,* CHERYL A. PAGE, MARC A. MILLS, AND ROBIN L. AUTENRIETH Department of Civil Engineering, Texas A&M University, College Station, Texas 77843-3136

Glycolipids produced by Rhodococcus species H13-A and a representative synthetic surfactant Tween-80 (polyoxyethylene sorbitan monooleate) were used to demonstrate enhanced substrate “solubility” (aqueous-plusmicellar phase) in the presence of surfactants. Nascent concentrations of naphthalene and its methyl-substituted derivatives in crude oil were used as representative polycyclic aromatic hydrocarbons for the study. Both biosurfactant glycolipids from H13-A and Tween-80 lowered the surface tension of aqueous solutions from 72 to ∼30 dyn/ cm. The two-ring aromatics showed a substantial increase in their apparent solubilities in the presence of surfactants; the increase being significantly greater for the biosurfactant as compared to the synthetic surfactant. The aqueous phase solubility enhancement was greater for the highly substituted derivatives as compared to the lesser substituted compounds. Higher toxicity levels, as seen by the lower EC50 values, of the surfactant mixtures indicated enhanced partitioning of the petroleum contaminants in the aqueous phase. Higher initial EC50 values for the biosurfactant meant that they exhibit lesser aqueous toxicity as compared to the synthetic surfactant. When compared on a toxicity per mass of PAH basis, the end point Tween80 system was approximately 50% more toxic than the biosurfactant system.

Introduction For several years, oil dispersants and emulsifiers have been proposed as a means to remove oil from water surfaces. Successful application of dispersants causes surface oil to break into small droplets and become more bioavailable for degradation (1, 2). However, the dispersants themselves can be toxic to the microbial and aquatic community, and dispersion of oil can release more toxicants into the water body (3). Biosurfactants have advantages relative to synthetic surfactants for specific applications due to their structural diversity, biodegradability, and biocompatibility relative to synthetic surfactants (4). Surface-active agents concentrate at air-water or oilwater interfaces, thus reducing the physical forces that exist at these interfaces (5). Since surface tension is responsible for the resistance exerted by liquid to surface penetration, reducing it (surface tension) is an indicator of the effectiveness of surfactants (6). An increase in the solubility of petroleum components can be achieved by surfactants due to their * Corresponding author telephone: (409) 845-9770; fax: (409) 8623220; e-mail: [email protected].

556

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 2, 1997

abilities to form soluble colloidal groups of molecules called micelles and to lower surface and interfacial energies (7). Different types of bacteria, yeast, and fungi produce metabolic products or membrane components that behave similar to surfactants and are known as biologically produced surfactants or biosurfactants (7). Biosurfactants are usually produced by microorganisms growing on insoluble substrates. These metabolites or biosurfactants include products such as fatty acids, glycerides, phospholipids, lipopeptides, and antibiotics (8, 9). Glycolipids and phospholipids are two of the most common groups among biosurfactants. Most of the previous research has focused on either synthetic surfactants or purified biosurfactants with single substrates, under ideal laboratory conditions. This information, although valuable from the environmental science viewpoint, cannot be used to predict the solubilization potential or biodegradation capacity for complex substrates in natural conditions. The purpose of this research was to evaluate the effectiveness of biosurfactants in enhancing aqueous-plus-micellar phase solubility (or apparent solubility) of polycyclic aromatic hydrocarbons (PAHs) from weathered crude oil. The nascent content of the two-ring hydrocarbon, naphthalene, was used as the model hydrophobic constituent of crude oil. Though naphthalene is a minor component of refined petroleum products (10), it is abundant in coal and coal tar and is commonly found in soils, sediments, surface water, and groundwater. Solubility of naphthalene has been measured in a variety of synthetic surfactant solutions (11, 12). However, the influence of biologically produced surfactants on naphthalene solubilization is poorly understood. The extent to which biosurfactants produced during microbial activities affect the apparent solubility of naphthalene and its methyl-substituted derivatives is the main objective of this study. The experimental objectives were achieved by temporal monitoring of the aqueous-plus-micellar-phase (APMP) concentrations of the two-ring aromatics in the absence of surfactants and in the presence of a biosurfactant and synthetic surfactant. The apparent solubilities for naphthalene and its derivatives as a function of time were then fitted to a first-order saturation equation, and nonlinear regression analysis was used to estimate the saturation concentrations C*, aqueous-phase solubility enhancement factors K, and firstorder rate coefficients k for each target analyte. Parameters obtained by optimizing the model are good approximations of the solubilization kinetics for naphthalene and its derivatives. By comparing these results with those using a synthetic surfactant, the efficacy of the biosurfactant on the partitioning behavior of two-ring hydrocarbons was determined.

Materials and Methods The Rhodococcus strain H13-A was selected as the representative biosurfactant-producing microorganism for the experiment. This strain produces extracellular glycolipid biosurfactants when grown on n-paraffins (5, 13, 14). Tween80 (polyoxyethylene sorbitan monooleate) was used as the representative synthetic surfactant to which the performance of the biosurfactant was compared. It is a well-characterized detergent with a hydrophile-lipophile balance (HLB) of 15, is effective in dispersing petroleum hydrocarbons (15-18), and was used as the representative synthetic surfactant to which the performance of the biosurfactant was compared. The evaluation was based on the extent of apparent solubilization of the two-ring PAHs and the initial and final toxicity of the surfactant solutions. Organisms and Culture Conditions. The strain H13-A was obtained from Dr. W. R. Finnerty (Finnerty Enterprises,

S0013-936X(96)00437-3 CCC: $14.00

 1997 American Chemical Society

TABLE 1. Experimental Aqueous-plus-Micellar Phase Concentrations for Naphthalene and Methyl-Substituted Naphthalenes after 16 Days, in the Absence and Presence of Surfactants weathered West Texas crude oil

aqueous solutions

biosurfactant solutions

Tween-80 solutions

compound

(mg/L)

(mg/L)

(mg/L)

(mg/L)

naphthalene C1-naphthalenes C2-naphthalenes C3-naphthalenes C4-naphthalenes

0.904 ( 0.006 2.383 ( 0.008 3.121 ( 0.024 2.864 ( 0.003 1.815 ( 0.010

0.179 ( 0.000 0.138 ( 0.001 0.041 ( 0.001 0.006 ( 0.000 0.00a ( 0.000

0.369 ( 0.025 0.683 ( 0.070 0.591 ( 0.103 0.484 ( 0.072 0.152 ( 0.031

0.331 ( 0.049 0.409 ( 0.070 0.205 ( 0.031 0.092 ( 0.018 0.006 ( 0.003

aDenotes

value below the detection limit, considered equal to zero.

Inc., Athens, GA) and grown on hexadecane enrichment (19). The strain was grown at 25 °C by shaking at 300 rpm on a rotary shaker. Nutrient broth (0.8%)-yeast extract (0.5%) (Difco, Detroit) was used for growth, with the addition of 1.5% Bacto-agar for growth on solid medium. The basal salts minimal medium (BSE) used for growing the strain on hexadecane contained the following (in g/L): 10 g of K2HPO4; 5 g of NaH2PO4; 2 g of (NH4)2SO4; 200 mg of MgSO4‚7H2O; 1 mg of CaCl2‚2H2O; and 1 mg of FeSO4‚7H2O, pH 7.0, supplemented with 0.5% n-hexadecane and 0.05 mM thiamine (19). Surface Activity. The cultures were grown for biosurfactant production in 2000-mL batch reactors for 8 days. They were then centrifuged at 7500g for 15 min, and the supernatant was filtered through Whatman No. 1 filter paper (19). Surface tension of the spent culture broth was measured using the Fisher Autotensiomat recording du Nouy tensiometer. Reactor Setup. The spent-culture broth was autoclaved at 121 °C for 30 min to ensure elimination of trace active biomass without significantly destroying the surface activity of the extract. This surface-active solution, without further purification or processing, was used for the solubility experiment. The entire batch of this sterilized biosurfactant solution was homogenized, and 1000-mL batch reactors were set up (at room temperature of 25 °C) using an oil-water ratio of 1:10 with zero head space. The synthetic surfactant reactors used 0.1% Tween-80 solution. The crude oil used for this study was West Texas Crude obtained from Fina Petroleum, Port Arthur, TX, and was weathered by purging particle-free nitrogen for 48 h to eliminate the toxic volatile compounds. The nascent content of the PAHs in the crude oil (refer to column 1 in Table 1) served as the target analytes whose solubilization was monitored over time. Control reactors contained distilled deionized water and oil in the same ratio. The reactors were gently stirred at 100 rpm to ensure that no oil-in-water emulsion formed by maintaining the turbulence level below that necessary to separate oil particles from the oil layer. The three treatments (control, biosurfactant, and synthetic surfactant) were run in duplicate, and reactors were sacrificed for each sampling event. Sampling. Reactors were sacrificially sampled at 0, 6, 12, 24, and 48 h and 4, 8, and 16 days to determine the APMP concentration or water accommodated fraction (WAF). The reactors were allowed to stand for 5-10 min, enabling phase stabilization, before sampling from the port located at the bottom of the reactor. From each 1000-mL reactor, 250 mL was sampled for the solubility analysis, another 250 mL was sampled for toxicity measurements, and the balance was archived for future studies. Samples were collected in solventcleaned 250-mL amber bottles with zero head space and stored for 1 week at 4 °C until they were extracted. Extraction and Concentration. The 250-mL aliquot for the solubility study was spiked with surrogate recovery standards (20) and transferred to a separatory funnel for liquid extraction. The separatory funnel extraction used methylene chloride as the solvent and followed accepted procedures as per U.S. EPA Method 413.1. Caution was exercised to prevent the formation of stable emulsions. The sample extract was

then concentrated to 1 mL in hexane using a Kuderna-Danish (K-D) evaporative concentrator (20). Fractionation. Column chromatography was used to remove the biological interferences and to separate the sample into two hydrocarbon fractions, F1 and F2. The F1 fraction consisted of alkanes, branched alkanes, and cycloalkanes. The F2 fraction consisted of aromatics and polynuclear aromatics. The cleanup/fractionation column was prepared in methylene chloride with glass wool, fired sand, activated alumina (5 g), and activated silica gel (10 g), packed in that order (20). The column was then eluted with 20 mL of pentane and thereafter with 100 mL of 1:1 pentane to methylene chloride. The first fraction collected was the aliphatic (F1) fraction, and the second fraction was the aromatic (F2) fraction. The fractionated extracts were concentrated to 0.5 mL using the Kuderna-Danish (K-D) evaporative concentrator. The fractions were transferred to GC vials and brought up to 1 mL using methylene chloride. The consistency of the extraction, fractionation, and concentration procedures was verified by subsequent GC/MS analysis of the fractions. Target Compound Analysis. The analysis of the two-ring hydrocarbons was based on the NETAC petroleum analysis procedure (21) and U.S. EPA Methods for Wastewater Analysis Methods 610 and 625. The GC/MS analysis used variations of methods obtained from training sessions at the EPA Gulf Breeze Laboratories, Gulf Breeze, FL, and visits to Geochemical Environmental Research Group (GERG), College Station, TX. The samples were analyzed on a 5890II Hewlett-Packard gas chromatograph coupled to a HP5972A mass spectrophotometer integrated with HP MS Chemstation (HewlettPackard, Palo Alto, CA) (20). The separations were done using a 30 m × 0.25 mm PTE 5 fused silica capillary column with 0.25 µm film thickness. The injection temperature was 300 °C, and the helium gas had a linear velocity of 40 cm/s at 30 °C. The program was 10 min at 60 °C, to 300 °C at 5 °C per min, and 30 min at 300 °C. The 1-µL injections were done in the fast injection mode by an HP7673A autosampler (Hewlett-Packard, Palo Alto, CA), and compounds were identified using the scan mode by the mass spectrometer. Method detection limit for individual component concentration was 1 µg/mL of extract for a 250-mL sample and a final solvent volume of 1.0 mL. Solvent blanks, surrogate recovery, and internal standards were used throughout the GC/MS analysis to verify the integrity of the solvent and instrument, to assure proper extraction procedures, and to determine total method recoveries. The APMP concentrations of the target compounds over time, were statistically compared at 95% confidence to determine the “solubilization” potential of the biosurfactant (treatment B) and synthetic surfactant (treatment C), as compared to that of the control (absence of any surfactant or treatment A). Toxicity. The toxicity of the 250-mL aliquot of surfactant solution was evaluated using the Basic Test Microtox Toxicity Assay by Microbics (22). The results are reported as EC50 values or effective concentrations (given in percent of the original sample concentration) at which 50% of the test organisms die. The toxicity of a sample is inversely related

VOL. 31, NO. 2, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

557

to its measured EC50 value. The toxicity was monitored over time and correlated to the initial toxicity of the oil-free surfactant and the final toxicity due to partitioning of the contaminants into the surfactant solutions. Model Development. A simple first-order saturation equation was used to model the solubilization phenomenon of naphthalene and the methyl-substituted naphthalenes. The rate law frequently used is an empirically-based saturation kinetic model (23) and states that the dissolution is controlled by the rate of diffusion of contaminants from the solid phase, or the free-phase crude oil in this case. According to the rate law

dC/dt ) k(C* - C)

(1)

where C* is the saturation concentration of the target analyte (mg/L); C is the time-variable concentration of the target analyte (mg/L); and k is the first-order rate constant (h-1). The rate and extent of solubilization is altered in the presence of surfactant micelles and is represented as

dC/dt ) k (KC* - C)

(2)

where K is the aqueous solubility enhancement factor due to the presence of surfactants. It is defined as the ratio of the analyte concentration in surfactant solutions to that in aqueous solutions. Parameter estimation of the model provided the saturation concentrations (C*), rate coefficients (k), and solubility enhancement factors (K) for the two-ring hydrocarbons for the surfactant solutions.

Results and Discussions The strain H13-A was confirmed as a Rhodococcus species by GC-FID analysis of fatty acid methyl esters (FAME) by the Department of Plant Pathology, Texas A&M University. The strain was Gram-positive, exhibited rod or coccal morphogenesis, formed highly mucous colonies with a buff or orange pigmentation, and required thiamine for growth in chemically defined media. The biosurfactant synthesized by hexadecane-grown H13-A has been characterized by Singer et al. (5) to be a mixture of glycolipids, consisting of trehalose and a complex array of fatty acids and mycolic acids. The trehalose backbone is esterified with saturated and monounsaturated fatty acids, mycolic acids, 10-methyl hexadecanoic and 10methyl octadecanoic acids, and hexadecanoic and decanedioic acids, the latter accounting for the anionic character of the glycolipid. The glycolipids are stable to 120 °C at 18 psi (4), allowing steam sterilization to eliminate the trace viable biomass while preserving the surface activity of the spent-culture broth. Biosurfactant production increased several fold during the stationary growth phase, i.e., after 4-5 days (19), which resulted in the lowering of surface tension from 72 to approximately 30 dyn/cm. The surface tension remained constant after 5 days, indicating that the surfactant molecules had saturated the solution or reached its critical micelle concentration (cmc). Cultures were grown for 8 days to ensure that the spent-cell solution used for setting up the solubility reactors was above the cmc for the biosurfactant glycolipids. The Tween-80 solution used for the synthetic surfactant reactors was also above the cmc and resulted in an identical drop in surface tension. Decreases in the surface energies resulted in enhanced solubilization of the two-ring petroleum hydrocarbons, as seen from the GC/MS analysis (16-day sample) summarized in Table 1. Over the period of 16 days, naphthalene and methyl-substituted naphthalenes showed greater APMP concentrations in the presence of biosurfactants and synthetic surfactants than in the absence of surfactants. Concentrations in the presence of biosurfactants were found to be significantly greater (at 95% confidence) than in the presence of the

558

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 2, 1997

FIGURE 1. (a) Aqueous-plus-micellar phase naphthalene concentration over time, in the absence and presence of surfactants. The experimental points are connected using theoretical curves generated by optimization of the first-order saturation model. (b) Solubilization kinetics for naphthalene over the first 48 h. synthetic surfactants. Figures 1 and 2 compare the concentrations of naphthalene and its derivatives for the three treatments studied. The points plotted are the averages of the data points for each sampling event, and the error bars indicate standard deviation or ranges in this case. The results reported here do not include aliphatics and higher molecular weight PAHs or any compounds that co-eluted with the target analytes. Figure 1a shows the enhancement in naphthalene concentration in the presence of the biosurfactant and Tween80 as compared to the control reactor. The regressed curve through the plotted points is the saturation kinetic model representation for naphthalene solubilization. Parameter estimations of the model variables for the three treatments are summarized in Table 2. Both biosurfactants and synthetic surfactants significantly enhanced the naphthalene solubility as compared to the WSF in the control sample. The synthetic surfactant had a lower solubilization potential than the biosurfactant, indicated by the smaller aqueous phase enhancement factor K. The first-order solubilization rate coefficient (k, h-1) was higher for the two-phase aqueous/oil systems (control) as compared to the three-phase aqueous/ surfactant/oil systems. Although biosurfactants had a higher solubilization potential, their rate coefficients were lower than that of the synthetic surfactants as statistically verified by the lesser slope values in Figure 1b. The differential rate (dC/dt) was greatest for biosurfactant system due to the larger magnitude of the solubility enhancement factor, K. Biosurfactant-enhanced aqueous systems were seen to take longer time periods to reach saturation than systems with synthetic surfactants. The correlation coefficients (R 2) calculated showed an acceptable closeness-of-fit between the model estimates and the experimental data. Similar trends were observed for the methyl-substituted derivatives as seen in

FIGURE 2. Aqueous-plus-micellar phase concentration of (a) C1-naphthalenes, (b) C2-naphthalenes, (c) C3-naphthalenes, and (d) C4naphthalenes over time, in the absence and presence of surfactants.

FIGURE 3. Aqueous solubility-enhancement factor (K), first-order rate coefficient (k), and time to reach 99% of the saturation concentration for the naphthalenes family, as calculated by optimizing the first-order saturation model. Figure 2 for the C1, C2, C3 and C4-naphthalenes, and the results are summarized in Table 2. Figure 3 compares the trends of the effect of biosurfactants and synthetic surfactants on partitioning of naphthalene and

methyl-substituted naphthalenes. Aqueous solubilities of the substituted naphthalenes decrease with an increase in methyl substitution (24). The figure indicates that in the presence of surfactants there was a direct correlation between the solubility enhancement factor K and the increase in methyl substitution. The potential for enhancing solubility was much greater in the case of biosurfactants as compared to synthetic surfactants, and these effects were more pronounced with increases in methyl substitution. The reason for the greater solubilization potential of biosurfactants could be the large micellar volume that is able to accommodate a greater amount of these substituted hydrocarbons. Preliminary experiments using light scattering techniques (or laser photometry) in our laboratories have indicated that biosurfactant micelles from H13-A are approximately four times in diameter and have a molecular weight of an order of magnitude greater than Tween-80 micelles (Paudel, manuscript in preparation). Glycolipid micelles from H13-A have been found to be larger than rhamnolipid micelles from Pseudomonas aeruginosa based on studies using molecular weight cutoff membranes (25, 26). In the absence of surfactants, the first-order rate coefficient k was observed to decrease rapidly with an increase in methyl substitution due to the low aqueous solubilities of these substituted compounds. In the case of biosurfactants, the differential rate dC/dt was higher but the rate coefficient was lower as compared to synthetic surfactants. Possible explanations could be that the two-ring aromatics take a longer time to align themselves in the glycolipid micellar core than in a micelle of the synthetic surfactant or mass transport phenomena based on geometric considerations. Another trend seen from Figure 3 is that the solubilization rate coefficients decreased with methyl substitution; this decrease was less pronounced for synthetic surfactants than the biosurfactants. This may be due to the limited accommodating potential of the synthetic surfactant micelles as compared to the relatively larger glycolipid micelles. For

VOL. 31, NO. 2, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

559

TABLE 2. Parameter Estimates for Aqueous-plus-Micellar Phase Solubilization, Based on First-Order Saturation Model treatment strategy model parameter

control

biosurfactant

Saturation Concentration, C* (mg/L) naphthalene 0.185 0.339 C1-naphthalenes 0.142 0.628 C2-naphthalenes 0.043 0.555 C3-naphthalenes 0.006 0.492 C4-naphthalenes 0.002 0.182 naphthalene C1-naphthalenes C2-naphthalenes C3-naphthalenes C4-naphthalenes

Rate Coefficient, k (h-1) 0.239 0.075 0.225 0.025 0.188 0.021 0.041 0.010 0.030 0.005

Tween-80 0.294 0.345 0.177 0.079 0.004 0.123 0.097 0.086 0.078 0.075

Solubility Enhancement Factor, K naphthalene 1.8 C1-naphthalenes 4.4 C2-naphthalenes 12.8 C3-naphthalenes 88.0 C4-naphthalenes >86.0

1.6 2.4 4.1 14.2 >2.0

Time To Reach 99% Saturation, t (h) naphthalene 19.3 61.3 C1-naphthalenes 20.5 183.3 C2-naphthalenes 24.5 224.6 C3-naphthalenes 112.2 477.4 C4-naphthalenes 151.1 974.3

37.4 47.6 53.8 59.3 61.5

Model Correlation Coefficient, R 2 naphthalene 0.996 0.985 C1-naphthalenes 0.994 0.985 C2-naphthalenes 0.995 0.988 C3-naphthalenes 0.953 0.999 C4-naphthalenes 0.415 0.985

0.960 0.926 0.944 0.936 0.666

substituted naphthalenes and other larger PAHs (2), the aqueous/surfactant/oil systems did not reach a saturation state within the experimental run of 16 days. The time to reach 99% of the saturation concentration C* was calculated by optimizing the first-order saturation model for minimum residual values, and these are plotted in Figure 3. The substituted naphthalenes took a longer time to reach saturation, and the time was significantly greater for biosurfactants as compared to synthetic surfactants. With regards to the highly substituted naphthalenes, the model determined longer equilibration times for the control reactors as compared to the synthetic surfactants since concentrations were near the detection limit of the GC/MS. To summarize, biosurfactants had higher solubility enhancement potential but lower rate coefficients as compared to the synthetic surfactants. The toxicity effects due to enhanced solubilization of the two-ring PAHs are summarized in Figure 4. The time zero points effectively served as the controls for both surfactant systems, since no exposure to the oil occurred. They are not plotted on the EC50 graph (Figure 4a), as the values would be offscale. Both solutions were determined to be nontoxic. Since naphthalene and its substituted derivatives were the major constituents of the aqueous fraction (27), their concentrations were summed and plotted as “total-naphthalenes” along the y-axis. There was a 2.5-fold increase in total naphthalenes concentration in the aqueous fraction in the presence of biosurfactants as compared to synthetic surfactants. At 6 h, the biosurfactant system demonstrated relatively higher EC50 values than the Tween-80 (Figure 4a), thereby suggesting that biologically produced surfactants have lower aqueous toxicity. Moreover, when the 6-h toxicity data are divided by the total naphthalenes concentration and compared, the Tween-80 system was found to be approximately 50% more toxic than the H13-A system, on a toxicity per mass of PAH basis.

560

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 2, 1997

FIGURE 4. Comparison of EC50 and total naphthalenes (i.e., naphthalene plus methyl-substituted naphthalenes) concentration for the three treatment strategies. The enhanced solubilization of total naphthalenes in the presence of surfactants contributed to the higher toxicity of the surfactant solutions, as indicated by lowering EC50 values over time. At the end point (16 days), the EC50 values for both surfactant solutions were approximately the same. However, when also compared on a toxicity per mass of PAH basis, the Tween-80 solution was again determined to be more toxic (approximately 55%) than the biosurfactant solution. Increasing trends in APMP concentrations of naphthalene and its derivatives were observed for synthetic surfactants as in the case of biosurfactants. Compounds with relatively higher solubilities, such as BTEX (benzene, toluene, ethylbenzene, xylene) and naphthalene, were less affected due to the presence of surfactants. Alternatively, those compounds with lower solubilities and higher octanol-water partitioning coefficients, such as the methyl-substituted naphthalenes and the larger PAHs (27), were significantly affected. The phase containing the aromatic contaminants, in this case the crude oil, also influences the effectiveness of surfactants with regard to their solubilization potential (28). The crude oil phase consists of compounds that are less polar and provide a better organic phase for the partitioning of the hydrophobic tworing compounds. Hence these contaminants preferred

staying in the crude oil phase rather than partitioning into the aqueous phase, as confirmed by their higher concentrations in the crude oil as compared to the solubilized fraction (Table 1). Surfactants play a critical role in driving these less polar petroleum constituents from the oil phase to the aqueous phase, making them readily available to the microorganisms for degradation. Enhanced solubility of the two-ring aromatics is most likely due to the physical association of these compounds within the biosurfactant aggregates or micelles. The hydrocarbon solubilization is highest at pH 7.0 (29), which was maintained throughout the experiment. It was seen that a large fraction of the solubilized contaminant preferred to stay associated within the aggregates. This was a major problem during the bulk production of surfactants for use in the solubility experiments. Hexadecane, the substrate on which H13-A was grown for biosurfactant production, remained entrapped within the micellar core and could have contributed to cosolvency effects during the solubilization studies. The 102>104% increase in the APMP concentrations for naphthalene and its substituted derivatives need not imply an equally significant increase in biodegradation, since these compounds may still not be freely available to the microbial community. Biosurfactants form micelles with a lesser likelihood of being spherical, and they exhibit less dense packing of the monomers due to the larger size of the hydrophilic group (29, 30). These large volume, low density micelles could therefor accommodate a greater amount of the solubilized PAHs and hence be the reason for increased solubilization potential. The substituted naphthalenes have large molecular dimensions, and hence solubilization of these compounds into the smaller synthetic surfactant micelles is very limited. However, the larger glycolipid micelle is more favorable to accommodate these higher molecular weight substituted derivatives. An advantage of the synthetic surfactant Tween-80 is its nonionic nature; it does not react to the pH variations and hence does not affect the solubilization of the target contaminants. This is not the case for the anionic glycolipid biosurfactant synthesized by Rhodococcus H13-A. To summarize, the biosurfactants were more effective than synthetic surfactants in solubilizing the two-ring aromatics over time. This was also verified by the elevated levels of toxicity of the surfactant solutions over the duration of the experiment. The research is an on-going effort to study the application of biosurfactants in biodegradation of other highly insoluble wastes such as coal tar, methyl parathion, TNT, and others.

(3)

(4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)

(22) (23) (24) (25) (26) (27) (28)

Acknowledgments

(29)

We thank Texas General Land Office, Texas Higher Education Coordinating Board, and the U.S. Department of Defense for funding this research. We also would like to thank Dr. W. R. Finnerty (Finnerty Enterprises, Inc., Athens, GA) for providing the strain H13-A and Ms. Bea Lambert for her valuable contribution during the toxicity studies of the surfactants.

(30)

Management IV; Tedder, D. W., Pohland, F. G., Eds.; ACS Symposium Series 554; American Chemical Society: Washington, DC, 1994; pp 460-463. Ruel, M.; Ross, S. L.; Nagy, E.; Sprague, J. B. In Guidelines on the Use and Acceptability of Oil Spill Dispersants; Environmental Emergency Branch Report EPS 1-EE-73-1; Environment Canada: Ottawa, 1973. Singer, M. E. Int. Bioresour. J. 1985, 1, 9-38. Vogt Singer, M. E.; Finnerty, W. R.; Tunelid, A. Can. J. Microbiol. 1990, 36, 746-750. Kouloheris, A. P. Chem. Eng. 1989, 96, 130-136. Falatko, D. M. M.S. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA, 1991. Cooper, D. G.; Zajic, J. E.; Gerson, D. F. Appl. Environ. Microbiol. 1979, 37, 4-10. Cooper, D. G.; Zajic, J. E. Adv. Appl. Microbiol. 1980, 26, 229253. Guerin, W. F.; Boyd, S. A. Appl. Environ. Microbiol. 1992, 58, 1142-1152. Morisue, T.; Moroi, Y.; Shibata, O. J. Phys. Chem. 1994, 98, 1299513000. Moroi, Y.; Mitsunobu, K.; Morisue, T.; Kadobayashi, Y.; Sakai, M. J. Phys. Chem. 1995, 99, 2372-2376. Georgiou, G.; Lin, S. C.; Sharma, M. M. Biotechnol. Bioeng. 1992, 10, 60-65. Finnerty, W. R.; Singer, M. E. Dev. Ind. Microbiol. 1984, 25, 3140. Janiyani, K. L.; Wate, S. R.; Joshi, S. R. J. Chem. Tech. Biotechnol. 1993, 56, 305-308. Rosen, M. J. In Surfactants and Interfacial Phenomenon; John Wiley & Sons, Inc.: New York, 1978. Porter, M. R. In Handbook of Surfactants; Chapman & Hall: New York, 1991. Jafvert, C. T.; Van Hoof, P. L.; Heath, J. K. Water Res. 1994, 28, 1009-1017. Vogt Singer, M. E.; Finnerty, W. R. Can. J. Microbiol. 1990, 36, 741-745. Mills, M. A. M.S. Thesis, Texas A&M University, College Station, TX, 1994. NETAC. Draft Oil Spill Bioremediation Products Testing Protocol Methods Manual; National Environmental Technology Applications Corporation, University of Pittsburgh Trust: Pittsburgh, PA, 1992. Microbics Corporation. In Microtox Manual: A Toxicity Testing Handbook; Microbics Corporation: Carlsbad, CA, 1992. Walton, A. G. In The Formation and Properties of Precipitates; Wiley-Interscience: New York, 1967. Verschueren, K. In Handbook of Environmental Data on Organic Chemicals; Van Nostrand Reinhold Company: New York, 1977. Bryant, F. O. Appl. Environ. Microbiol. 1990, 56, 1494-1496. Mulligan, C. N.; Gibbs, B. F. J. Chem. Technol. Biotechnol. 1990, 47, 23-29. Kanga, S. A. M.S. Thesis, Texas A&M University, College Station, TX, 1995. Falatko, D. M.; Novak, J. T. Water Environ. Res. 1992, 64, 163169. Zhang, Y.; Miller, R. M. Appl. Environ. Microbiol. 1992, 58, 32763282. Thangamani, S.; Shreve, G. S. Environ. Sci. Technol. 1994, 28, 1994-2000.

Received for review May 20, 1996. Revised manuscript received September 25, 1996. Accepted October 4, 1996.X ES9604370

Literature Cited (1) Kile, D. E.; Chiou, C. T. Environ. Sci. Technol. 1989, 23, 832-838. (2) Kanga, S.; Page, C.; Lambert, B.; Mills, M.; Simon, M.; Bonner, J.; Autenrieth, R. In Emerging Technologies in Hazardous Waste

X

Abstract published in Advance ACS Abstracts, December 15, 1996.

VOL. 31, NO. 2, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

561