Constituents of an Organic Wood Preservative That Inhibit the

During several bioremediation studies, however, we observed that the fluoranthene degradative activity of strain EPA505 was inhibited by the presence ...
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Environ. Sci. Technol. 1997, 31, 3573-3580

Constituents of an Organic Wood Preservative That Inhibit the Fluoranthene-Degrading Activity of Sphingomonas paucimobilis Strain EPA505 S . E . L A N T Z , †,‡ M . T . M O N T G O M E R Y , § W. W. SCHULTZ,† P. H. PRITCHARD,| B . J . S P A R G O , | A N D J . G . M U E L L E R * ,† SBP Technologies, Inc., 1 Sabine Island Drive, Gulf Breeze, Florida 32561, GeoCenters, Inc., Washington, D.C. 20375, and Naval Research Laboratory, Washington, D.C. 20375

Sphingomonas paucimobilis strain EPA505 is capable of utilizing many components of coal tar creosote as sole sources of carbon and energy for bacterial growth, including fluoranthene and other polycyclic aromatic hydrocarbons (PAH). During several bioremediation studies, however, we observed that the fluoranthene degradative activity of strain EPA505 was inhibited by the presence of undefined creosote constituents. In practice, integration of a pretreatment step prior to inoculation with strain EPA505 was necessary to facilitate the biodegradation of high molecular weight (HMW) PAHs. Experiments were thus initiated to determine which compound classes in creosote inhibited fluoranthene metabolism by strain EPA505. Creosote was fractionated by solvent extraction at various pH, and three chemical classes were examined: acid (phenolics), base (N-heterocyclics), and neutral (PAH). The mineralization rate of 14C-labeled fluoranthene and cell viability were examined in the presence of these creosote fractions at a range of concentrations. These studies confirm that strain EPA505 has differing susceptibility to the effects of the three classes of creosote constituents. The observed order of toxicity/inhibition was basic fraction > acidic fraction > neutral fraction. These studies provide engineering guidelines and define contamination ranges under which strain EPA505 can be used most effectively as a catalyst in bioremediation (Figure 4).

Introduction Resulting from decades of wide-scale usage of organic wood preservatives, wood-preserving sites commonly have soil and groundwater contaminated with creosote and/or pentachlorophenol (PCP). Creosote is a complex mixture of hundreds of compounds that can be grouped into three broad chemical classes: polycyclic aromatic hydrocarbons (PAH, 85% by weight), phenolics (10% by weight), and heterocyclics (5% by weight) (1). Several of the high molecular weight (HMW) * Corresponding author present address: Dames & Moore, Inc., One Continental Towers, Suite 1000, Rolling Meadows, IL 60008; phone: 847-228-0707, ext 131; e-mail: [email protected]. † SBP Technologies, Inc. ‡ Present address: S. Lantz, Energy BioSystems Corp. § GeoCenters, Inc. | Naval Research Laboratory.

S0013-936X(97)00323-4 CCC: $14.00

 1997 American Chemical Society

PAHs (defined herein as containing four or more fused rings) are known or suspected carcinogens. Cleanup criteria for creosote-contaminated sites are often based on reducing the concentration of these compounds. Unfortunately, HMW PAHs may be quite persistent when subjected to conventional bioremediation measures (1). In an attempt to advance biotreatment technologies for creosote and similarly impacted sites, Sphingomonas paucimobilis strain EPA505 has been used in various ways to enhance biodegradation of HMW PAHs (2, 3). In so doing, we observed that the fluoranthene mineralizing activity of strain EPA505 (used as an indicator of HMW PAH-degrading capacity) was inhibited by certain undefined constituents of creosote. Our research has shown that this phenomenon can be mitigated through the integration of a pretreatment step whereby indigenous microorganisms or select pure culture(s) are used to biodegrade suspected inhibitory compounds prior to inoculation with strain EPA505; thus, enhancing biodegradation of the HMW PAHs (3, 4). It has been our position that bioprocess optimization and engineering modifications can facilitate the effective use of this powerful inoculant if the inhibitory constituents of creosote are identified. But, whereas a wealth of literature exists to describe the toxicity, bioaccumulation, and metabolism of individual PAH in higher organisms and recent literature addresses microbial consortia (5-7), few studies have examined the effect of complex chemical mixtures, such as creosote, on axenic bacterial cultures (8). Accordingly, the objective of these studies was to better define the chemical sensitivities of strain EPA505 by exposing it to [3-14C]fluoranthene in the presence of PCP, fractionated creosote, and synthetic mixtures simulating the creosote fractions. Concomitant measurements of [3-14C]fluoranthene mineralization and bacterial abundance identified those classes of compounds that reduced the ability of strain EPA505 to mineralize HMW PAHs (i.e., fluoranthene). Toxicity was observed in the form of reduced bacterial abundance. Inhibition of fluoranthene mineralization took several forms, either an extended lag in onset of mineralization or a decrease in the rate or extent of mineralization.

Experimental Section Inoculum. The fluoranthene-utilizing bacterium Sphingomonas (formerly Pseudomonas) paucimobilis strain EPA505 (9) was grown to mid-log phase (48 h) in modified Luria Bertani broth (10 g/L tryptone, 5 g/L yeast extract, 2.5 g/L NaCl, 1 g/L glucose) on a rotary shaker (200 rpm, 30 °C). Cells were centrifuged, washed three times in potassium phosphate buffer (50 mM, pH 7.1), and resuspended in the same. The cell suspension was used to inoculate flasks to an optical density of 0.5 at a wavelength of 600 nm (5.0 × 108 cfu/mL). Standard plate count procedures were followed to determine bacterial abundance and to confirm inoculum purity (72 h, 30 °C). Chemicals. Chemicals used in the synthetic mixtures were >98% pure (Sigma Chemical Co., St. Louis, MO). [3-14C]Fluoranthene (Sigma Chemical Co., St. Louis, MO) was found to be 95% pure by preparative HPLC and liquid scintillation analysis and was used without further purification (data not shown). Commercial-grade creosote was obtained from Koppers Co., Inc. (Pittsburgh, PA). Triton X-100 was purchased from Aldrich Chemical Co. (Milwaukee, WI). Ultima Gold scintillation cocktail was purchased from Packard Instrument Co. (Meriden, CT). Microbiological media and reagents were purchased from Difco Laboratories (Detroit, MI). Source of Environmental Materials. Creosote (freeproduct) was collected from contaminated groundwater at

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TABLE 1. Composition of Synthetic Mixture Stock Solutions PAH (mg/100 mL CH2Cl2) naphthalene 134.2 acenaphthene 2-methylnaphthalene 145.1 acenaphthylene phenanthrene 127.2 fluoranthene anthracene 130.6 benz[a]anthracene 1-methylnaphthalene 67.4 chrysene biphenyl 82.7 pyrene fluorene 80.3 benzo[a]pyrene 2,3-dimethylnaphthalene 46.3 2-methylanthracene 2,6-dimethylnaphthalene 37.6 2,3-benzofluorene anthraquinone 9.4

40.1 32.8 41.6 16.2 18.6 35.5 9.8 8.9 9.4

Results

total 1073.7 mg/100 mL Heterocyclics (mg/100 mL CH2Cl2) quinoline 116.8 quinaldine isoquinoline 98.4 lepidine carbazole 99.4 benzo[b]thiophene 2,4-dimethylpyridine 95.9 dibenzothiophene acridine 49.4 dibenzofuran

60.0 46.0 100.5 101.4 100.0

total 867.8 mg/100 mL Phenolics (mg/100 mL CH2Cl2) phenol 204.1 o-cresol m-cresol 112.3 p-cresol 2,5-dimethylphenol 81.4 3,5-dimethylphenol 2,3-dimethylphenol 49.5 2,4-dimethylphenol 2,6-dimethylphenol 49.6 3,4-dimethylphenol 2,3,5-trimethylphenol 48.3

107.0 108.4 80.0 45.7 54.4

total 940.7 mg/100 mL

the American Creosote Works facility (ACW) in Pensacola, FL (3). The sample was collected as a dense non-aqueous-phase liquid from an on-property monitoring well (MW-340) at a depth of 13 m below ground surface (groundwater table at 1.5 m bgs). Creosote Fractionation and Analysis. Creosote (commercial-grade and ACW MW-340 free-product) was fractionated by an acid/base/neutral pH extraction procedure modified from U.S. EPA Method 625 as described previously for extraction of contaminated soil (10). Gas chromatography (GC) coupled with flame ionization detection (FID) was used to analyze the composition of neutral and basic creosote fractions as described previously (10). The acid-extractable compounds were analyzed as their acetic acid derivatives, following reaction with acetic acid anhydride. PCP was analyzed without derivitization. All separations were carried out on a 25 m (0.32 mm i.d., 0.17 µm phase thickness; 5% phenyl-substituted methylpolysiloxane) HP-5 capillary column (Hewlett Packard). Mass spectra (Hewlett Packard 5971 detector) and atomic emission (Hewlett Packard 5921A detector) spectra were also used to classify and putatively identify creosote constituents. Simulated Creosote Fractions. Synthetic mixtures representing the creosote fractions were prepared in methylene chloride from the neat chemicals. The composition of these artificial fractions is presented in Table 1 and was based on literature values of the relative concentrations of the primary components of coal-tar creosote (1). Mineralization Studies. Biometer flasks (250 mL; Bellco Biotechnology, Vineland, NJ) were used to measure 14CO2 evolution from [3-14C]fluoranthene in a manner similar to that described by Bartha and Pramer (11) and Mueller et al. (12). 14CO2 was trapped in 1 N NaOH in the flask side arm and then analyzed by liquid scintillation to estimate fluoranthene mineralization. Fluoranthene mineralization was monitored, for 120 h, in the presence of the various creosote fractions. The incubation medium consisted of BushnellHaas minimal salts plus 0.03% Triton X-100, added as a dispersant, and fluoranthene (average 300 mg/L and 45 000 dpm). Fluoranthene and the synthetic mixtures were prepared as stock solutions in methylene chloride and added to

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sterile, dry flasks. The solvent was allowed to evaporate, minimal salts media was added, and the flasks were sonicated (5 min) in a water bath sonicator. Creosote, or a fraction thereof, was added via a glass gas-tight syringe following sonication. Excess fluoranthene crystals and creosote droplets were visible. Material additions were made based on the concentration of known chemicals in the fractions and were chosen experimentally to span the no-effect concentrations. The following biotic and abiotic controls accompanied each set of experiments: killed-cells (autoclaved), non-inoculated flasks, and inoculated flasks without a creosote test fraction.

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Mass Balancing. All test conditions were established in duplicate, and the results presented are averages. The addition of crude extracts was normalized for the concentration of the constituents in the synthetic mixtures (these are the analytes monitored by GC/FID). Test flasks and negative control flasks (uninoculated and killed-cell) were extracted and analyzed (GC/FID) at the conclusion of incubations. Abiotic losses were minimal (within the margin of error of the analysis), except where otherwise noted. Inorganic nutrient and oxygen concentrations were stoichiometrically sufficient to support complete mineralization of the added substrates. Chemical Composition of Neutral Fractions. The neutral extracts resulting from fractionation of creosote were primarily composed of PAHs. The major difference between the neutral creosote preparations (synthetic vs fractionated) was the presence of the heterocyclics dibenzofuran, dibenzothiophene, and carbazole in the extracts from both the commercialgrade and ACW MW-340 creosote (Figure 1). These compounds were not included in the simulated neutral fraction, and less than 50% of the weight of the neutral extracts was represented by PAHs in the simulated fraction (commercialgrade, 44 mg of PAHs/100 mg of extract; ACW MW-340, 24 mg of PAHs/100 mg of extract). Thus, both creosote neutral extracts contained unidentified compounds not represented in the synthetic PAH mixture, even though the major peaks in the chromatograms were represented. It was anticipated that the ACW MW-340 creosote would show extensive “weathering” expressed in a lesser concentration of LMW compounds, which are susceptible to volatilization and biodegradation. However, this expectation was not supported by chromatographic analyses, and the two creosotes appeared to be quite similar. Chemical Composition of Basic Fractions. GC/FID analysis of the basic fractions also showed similarity between the commercial-grade creosote and that recovered from ACW MW-340 (Figure 2). Predominant in both extracts were the N-heterocyclics quinoline, methylated quinolines, acridine, and carbazole. As mentioned previously, certain heterocyclics were extracted preferentially into the neutral fraction. Compounds in the simulated basic fraction accounted for only about 25% of the weight of the commercial-grade and ACW MW-340 creosote extracts. GC/MS analysis (supported by atomic emission spectral data) suggested that some of the unidentified compounds in the creosote extracts were HMW N-heterocyclics such as benzoquinoline, methylacridine, benzocarbazole, and related compounds (data not shown). Chemical Composition of Acidic Fractions. The acidic extracts were composed primarily of phenolic compounds (Figure 3). A notable difference between the commercialgrade creosote and the ACW MW-340 sample was the presence of PCP (average concentration 22.4 mg/L) in the ACW extract. Of the three simulated fractions that were prepared, the composition (distribution of compounds) of the phenolic mixture was the least representative of the creosote extracts. Even so, phenolics in the artificial mixture represented about

FIGURE 1. Creosote PAHs in extracted and synthetic fractions. Gas chromatograms illustrating the PAH components of neutral extracts from commercial-grade creosote (A), ACW MW-340 creosote (B), and a synthetic mixture of authentic PAH standards (C). Peaks identified in panel C correspond to the same peak in panels A and B of this 29-min GC/FID temperature-programmed run (see Experimental Section). DBF, dibenzofuran; DBT, dibenzothiophene. 35% of the weight of both creosote extracts (excluding PCP). Effect of Basic Fractions on EPA505 Activity. In the presence of 50 µL/L of the commercial-grade creosote extract (10 mg/L monitored heterocyclics), the rate of fluoranthene mineralization was reduced by >50%, and bacterial abundance was reduced slightly (Figure 4). At a concentration of 70 µL/L extract, mineralization was inhibited and abundance was reduced by 5 logs (% mineralization refers to the percentage of spiked [3-14C]fluoranthene evolved as 14CO2). The mineralization graphs for the ACW MW-340 basic fraction and the simulated synthetic fraction were qualitatively very similar to Figure 4. However, the ACW MW-340 fraction was 50% inhibitory at about half the concentration shown in Figure 4, and the simulated fraction was inhibitory at twice the concentration shown (21 mg/L heterocyclics caused a 50% reduction in fluoranthene mineralization rate and 50% but without further loss of viable cells. Similar results were obtained with the synthetic mixture where 67 mg/L phenolics caused a 50% reduction in the rate but not extent of fluoranthene mineralization, and 50% reduction in extent, and 1 log of killing. At double that concentration, complete inhibition and cell death were observed. Abiotic control experiments indicated that volatilization of phenolics was minimal. Beginning in the 1950s, pentachlorophenol was used in addition to creosote for wood preserving at the ACW site.

Hence, PCP was found in the free-product recovered from MW-340. PCP accounted for 84% of the identified phenolics in the ACW MW-340 acidic extract. Therefore, the extract that completely inhibited mineralization and killed the inoculum contained 26 mg/L total phenolics, but 20 mg/L was PCP. PCP was not included in the artificial phenolic fraction because it is not a component of creosote, but tests with PCP alone confirmed its toxicity to strain EPA505 at concentrations g9 mg/L (data not shown), and its general bactericidal effects have been well demonstrated (14, 15).

Discussion Sphingomonas paucimobilis strain EPA505 can degrade many constituents of creosote including PAHs and phenolics and, to a much lesser degree, heterocyclics, but it has not been shown to have activity toward PCP (3, 4, 9, 13). These studies confirm that strain EPA505 has differing susceptibility to the

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TABLE 3. PAH Concentration Remaining in Flasks following 120-h Incubation with 200 µL/L Neutral Commercial Creosote Extract killed cell control

live cell inoculum

% loss

naphthalene benzo[b]thiophenea 2-methylnaphthalene 1-methylnaphthalene biphenyl 2,6-dimethylnaphthalene 2,3-dimethylnaphthalene acenaphthylene acenaphthene dibenzofurana fluorene dibenzothiophenea phenanthrene anthracene carbazolea 2-methylanthracene anthraquinone fluorantheneb pyrene benzo[b]fluorene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene

1.06 0.15 2.93 1.23 1.78 0.75 0.33 0.50 9.18 6.31 7.13 2.28 21.86 1.67 1.44 1.31 1.45 na 7.61 1.84 3.19 1.45 0.99

nd 0.11 0.04 0.03 0.01 0.05 0.04 0.13 0.03 0.04 0.11 0.13 1.48 0.49 0.08 0.25 0.59 na 2.73 0.83 1.40 0.62 0.75

100 27 99 98 99 93 88 74 99 99 98 94 93 71 94 81 59 na 64 55 56 57 24

0.84

0.53

37

total

77.28

10.47

86

PAH (mg/L)

FIGURE 4. Mineralization of [3-14C]fluoranthene by strain EPA505 in the presence of the basic extract from a commercial-grade creosote. Percent mineralization refers to the percentage of the [3-14C]fluoranthene spike evolved as 14CO2. The concentration of heterocyclics refers to the sum of the heterocyclic analytes listed in Table 1. Error bars may be smaller than the symbols appear.

TABLE 2. Heterocyclic Concentration Remaining in Flasks following 120-h Incubation with 50 µL/L Basic Commercial Creosote Extracta heterocyclic (mg/L)

killed cell control

live cell inoculum

2,4-dimethylpyridine benzo[b]thiophene quinoline isoquinoline quinaldine lepidine dibenzofuran dibenzothiophene acridine carbazole

0.08 nd 5.69 1.68 0.95 0.51 0.07 0.03 1.27 0.18

0.08 nd 6.09 1.72 0.96 0.53 0.04 0.02 1.30 0.11

10.46

10.85

total a

% loss (gain) 0 na 0 0 0 0 43 33 0 39

a

b

Heterocyclics extracted into the neutral fraction. Fluoranthene spike added for the mineralization assay; na, not applicable.

(3.7)

nd, not detected; na, not applicable.

FIGURE 6. Mineralization of [3-14C]fluoranthene by strain EPA505 in the presence of the neutral extract from ACW MW-340 creosote. Percent mineralization refers to the percentage of the [3-14C]fluoranthene spike evolved as 14CO2. The PAH concentration refers to the sum of the PAH analytes listed in Table 1. Error bars may be smaller than the symbols appear.

FIGURE 5. Mineralization of [3-14C]fluoranthene by strain EPA505 in the presence of the neutral extract from a commercial-grade creosote. Percent mineralization refers to the percentage of the [3-14C]fluoranthene spike evolved as 14CO2. The PAH concentration refers to the sum of the PAH analytes listed in Table 1. Error bars may be smaller than the symbols appear. toxic/inhibitory effects of these organic wood-preserving agents. The apparent order of toxicity/inhibition is PCP > basic extract (heterocyclics) > acidic extract (phenolics) > neutral extract (PAHs). Base-extractable creosote constituents

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(i.e., N-heterocyclics) depressed both the rate and extent of fluoranthene mineralization and significantly reduced bacterial abundance at concentrations in excess of 5-10 mg/L heterocyclics in the extracts (25-50 µL/L extract for ACW MW-340 and commercial-grade, respectively) or 21 mg/L heterocyclics in the artificial fraction. Similar inhibitory patterns were profiled by Dyreborg et al. (5, 6) where the effect of creosote components on toluene degradation by mixed microbial consortia showed that several N-,S-,Oheterocyclics and phenolics inhibited the degradation of toluene, while some aromatics slightly enhanced degradation.

FIGURE 7. Mineralization of [3-14C]fluoranthene by strain EPA505 in the presence of the acidic extract from a commercial-grade creosote. Percent mineralization refers to the percentage of the [3-14C]fluoranthene spike evolved as 14CO2. The phenolics concentration is the sum of the phenolic analytes listed in Table 1. Error bars may be smaller than the symbols appear.

TABLE 4. Phenolic Concentration Remaining in Flasks following 120-h Incubation with 100 µL/L Acidic Commercial Creosote Extracta phenolic (mg/L)

killed cell control

live cell inoculum

% loss

phenol o-cresol m-cresol p-cresol 2,6-dimethylphenol 2,5-dimethylphenol 2,4-dimethylphenol 3,5-dimethylphenol 2,3-dimethylphenol 3,4-dimethylphenol 2,3,5-trimethylphenol

7.06 3.32 7.88 3.57 0.91 1.30 2.51 4.01 0.45 0.77 1.47

1.22 0.19 0.49 0.24 nd nd nd 0.05 0.04 nd nd

83 94 94 93 100 100 100 99 91 100 100

33.25

2.23

93

total a

nd, not detected.

Nonchlorinated phenolics were less toxic/inhibitory than the heterocyclics, but PCP was highly toxic at 9 mg/L. The presence of PCP in the ACW MW-340 acidic extract greatly increased the toxicity of this fraction as compared to the acidic extract from commercial-grade creosote or to the simulated fraction without PCP. Because of the presence of PCP (11 mg/L), only 13 mg/L total phenolics in the ACW MW-340 acidic extract were as inhibitory as 70 mg/L total phenolics in either the commercial-grade creosote extract or the artificial phenolic fraction. PAHs were tolerated at concentrations greatly exceeding their aqueous solubility and were the least toxic/inhibitory fraction. This is not surprising since this bacterium was isolated from a soil enrichment for PAH-degrading organisms. Interestingly, we have evidence from experiments with artificially weathered (heated) creosote and whole creosote that LMW PAH are more inhibitory than HMW PAH (data not shown). Also, it was previously reported that at higher concentrations strain EPA505 could grow on naphthalene and 1- and 2-methylnaphthalene supplied in the vapor phase but not in liquid (9). Considering the data presented herein, it is our position that the synthetic mixture was more toxic than the extracts as it contained higher concentrations of two-ring PAHs. Bouchez et al. (8) reported that naphthalene was toxic to all of their PAH-degrading strains not specifically isolated on naphthalene. And in inhibition studies with pairs of PAH, they commonly found inhibition when the co-substrate was

more water soluble than the primary substrate. They also observed both enhanced degradation and inhibition when PAH degraders were fed certain pairs of PAH. A very interesting observation was that inhibition could be relieved by addition of a second strain capable of degrading the inhibitory substrate. All creosote fractions tested elicited some sublethal effects, but the concentrations that completely inhibited mineralization of fluoranthene were always accompanied by several logs of killing. In some cases, extended lags in the onset of mineralization were observed as the concentration of the test fraction was increased. This was not always accompanied by a decrease in the rate or extent of mineralization after the lag or by significant cell death. It was not the intent of this study to determine the mechanism of inhibition. But the lags may have been due to some combination of the following: (1) metabolism of competitive substrates in a complex mixture, (2) competitive substrate interactions (7, 20), (3) initial inhibition that diminished as volatile substrates partitioned to the head space, (4) an adaptation period (1619), or (5) cell death. Also, in batch cultures, co-metabolism may result in accumulation of toxic dead-end products manifested in inhibition and reduced bacterial abundance. Physicochemical parameters such as pH, nutrients, and oxygen were not considered to be limited. We also recognize that biodegradation rates can be limited by the rate of transfer of hydrophobic compounds into the aqueous phase. Thus, Triton X-100 was used as a dispersant to increase chemical dissolution rate. But surfactants can be toxic to bacterial cells, particularly at or above the critical micelle concentration (cmc), and this toxicity can be manifested as a decrease in the rate and extent of carbon mineralization (21, 22). Triton X-100 toxicity to strain EPA505 at 0.03% (cmc ) 0.01%; 0.2 M) was investigated. Control experiments showed no effect on mineralization under the conditions used in these assays (data not shown). Nevertheless, Triton X-100 was present in all incubations so the data are effectively normalized. We investigated the use of synthetic mixtures to simulate the effects of the extracted fractions. Although the mixtures were composed of the primary constituents of creosote, in relative concentrations based on literature values, some were better representations than others (either analytically or predictively). The artificial phenolic fraction least represented the composition of the extract (acidic) it was intended to mimic, but its influence on mineralization was very similar to that of the commercial-grade creosote extract. Thus, the unidentified compounds in this extract appeared to exert little influence on fluoranthene mineralization. Conversely, the PAH synthetic mixture was the most representative of the creosote extracts in composition, but it was significantly more toxic at lower concentrations of total PAHs than either extract, presumably due to the influence of higher concentrations of LMW PAH. Extract PAHs may have been less bioavailable due to lower partitioning into the aqueous phase from creosote oil droplets than from crystalline PAHs in the synthetic fraction. Compounds in the simulated basic fraction did not represent approximately 75% of the weight of the basic extracts, and the extracts were twice as toxic as the simulation, so the unknown chemicals in these extracts did influence mineralization. These results suggest that when concentrations of specific creosote fractions or PCP exceed defined concentrations, a pretreatment phase may be necessary before strain EPA505 can be expected to enhance HMW PAH degradation. Stimulating the catabolic activity of indigenous organisms in contaminated soil and groundwater has the potential to serve as the pretreatment phase by reducing the concentration of inhibitory compounds (4). Use of these guidelines should facilitate more effective use of strain EPA505 as a catalyst in bioremediation applications.

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Acknowledgments The authors wish to acknowledge the contributions of Beat Blattmann, Charylene Godwin, Pam Hollenbeck, and Peter Chapman. This research was funded by the Strategic Environmental Research and Development Program (SERDP-030) under contract to the U.S. NAVY (SBP Contract NOOO1494-C-2160) in collaboration with the U.S. EPA NHEERL, Gulf Breeze, FL (FTTA CRADA 0017-B-92).

Literature Cited (1) Mueller, J. G.; Chapman, P. J.; Pritchard, P. H. Environ. Sci. Technol. 1989, 23, 1197-1201. (2) Mueller, J. G.; Cerniglia, C. E.; Pritchard, P. H. In Bioremediation of Environments Contaminated by Polycyclic Aromatic Hydrocarbons; Crawford, R., Crawford, D., Eds.; Cambridge University Press: Cambridge, 1996; pp 125-194. (3) Mueller, J. G.; Lantz, S. E.; Ross, D.; Colvin, R. J.; Middaugh, D. P.; Pritchard, P. H. Environ. Sci. Technol. 1993, 27, 691-698. (4) Mueller, J. G.; Lantz, S. E.; Pritchard, P. H. U.S. Patent 08/308,483, 1996. (5) Dyreborg, S.; Arvin, E.; Broholm, K. Biodegradation 1996, 6, 97107. (6) Dyreborg, S.; Arvin, E.; Broholm, K. Biodegradation 1996, 7, 191201. (7) Millette, D.; Barker, J. F.; Comeau, Y.; Butler, B. J.; Frind, E. O.; Clement, B.; Samson, R. Environ. Sci. Technol. 1995, 29, 194452. (8) Bouchez, M.; Blanchet, D.; Vandecasteele, J. P. Appl. Microbiol. Biotechnol. 1995, 43, 156-164.

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(9) Mueller, J. G.; Chapman, P. J.; Blattmann, B. O.; Pritchard, P. H. Appl. Environ. Microbiol. 1990, 56, 1079-1086. (10) Mueller, J. G.; Lantz, S. E.; Blattmann, B. O.; Chapman, P. J. Environ. Sci. Technol. 1991, 25, 1045-1055. (11) Bartha, R.; Pramer, D. Soil Sci. 1965, 100, 68-70. (12) Mueller, J. G.; Resnick, S. M.; Shelton, M. E.; Pritchard, P. H. Ind. Microbiol. 1992, 10, 95-102. (13) Ye, D.; Siddiqi, M. A.; MacCubbin, A. E.; Kumar, S.; Sikka, H. C. Environ. Sci. Technol. 1996, 30, 136-142. (14) Rao, K. R. Pentachlorophenol: Chemistry, pharmacology and environmental toxicology; Plenum Press: New York, 1978. (15) Trevors, J. T.; Mayfield, C. I.; Inniss, W. E. Bull. Environ. Contam. Toxicol. 1981, 26, 433-439. (16) Heipieper, H. J.; de Bont, J. A. M. Appl. Environ. Microbiol. 1994, 58, 1847-1852. (17) Sikkema J.; de Bont, J. A. M.; Poolman, B. Microbiol. Rev. 1995, 59, 201-222. (18) Ramos, J. L.; Duques, E.; Rodriguez-Hervas, J.-J.; Godoy, P.; Fernandez-Barrero, A. J. Biol. Chem. 1997, 272, 3887-3890. (19) Weber, F. J.; Isken, S.; de Bont, J. A. M. Appl. Environ. Microbiol. 1994, 59, 3502-3504. (20) Kompala, D. S.; Ramkrishna, D.; Jansen, N. B.; Tsao, G. T. Biotechnol. Bioeng. 1986, 28, 1044-1055. (21) Laha, S.; Luthy, R. G. Biotechnol. Bioeng. 1992, 40, 1367-1380. (22) Thiem, A. Appl. Environ. Microbiol. 1994, 60, 258-263.

Received for review April 10, 1997. Revised manuscript received September 15, 1997. Accepted September 15, 1997.X ES9703234 X

Abstract published in Advance ACS Abstracts, November 1, 1997.