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Application of Pseudomonas putida PpG 786 Containing P-450 Cytochrome Monooxygenase for Removal of Trace Naphthalene Concentrations Gail P. Kulisch and Vincent L. Vilker* Department of Chemical Engineering, University of California, Los Angeles, California 90024
This study explores the potential for a bacterial monooxygenase t o remove polynuclear aromatic hydrocarbons from aqueous solutions a t high rates. This is part of a larger effort t o test the versatility of the cytochrome P-450,, monooxygenase enzyme system for detoxification of industrial process wastewaters t h a t contain trace quantities of hazardous compounds like PAHs or halocarbons. T h e intracellular concentration of P-45OC, in washed, resting cell suspensions of Pseudomonoas putida PpG 786 t h a t were cultured on camphor was measured by adapting a spectrophotometric method used t o measure P-450 concentration in extracts of mammalian tissues. Naphthalene removal in the suspensions was measured as a function of incubation time, biomass concentration, starting naphthalene concentration (3-180 pmol/L) and in the presence of known P-450 inhibitors. Involvement of the P-45OC, system in the measured naphthalene disappearance was established by showing that while significant naphthalene removal occurred in camphor-grown biomass, no disappearance was observed in glutamate-grown biomass and that removal was turned off in the presence of the P-450 inhibitor metyrapone. T h e half-life of naphthalene removal for a 1ppm solution (7.8 pM)was about 18h. T h e fraction of naphthalene removed descreased rapidly as initial naphthalene concentration increased, and essentially no naphthalene was removed when the starting concentration exceeded 189 pmol/L (23 ppm).
Introduction There is an increasing demand for improved and more reliable wastewater treatment processes, especially in the area of industrial wastewater treatment. Biochemical treatment technologies possess several advantages over physicochemical processes such as lower cost, potential for retrofit to existing facilities, and minimal production of hazardous byproducts. However, implementation of these technologies will require, in particular, the development of biomass that is both highly active for detoxification reactions and is stable in mixed cultures under wastewater treatment environments. Conventional biotreaters can be made to meet these objectives when undefined biomass is acclimated to specific targeted contaminants. Often this strategy requires that the treatment process be augmented with a continuous feed of the contaminant or related compounds in order to maintain contaminant-degradation potential. Most often these inducing compounds need to be supplied at moderately high concentrations. However, Cardinal and Stenstrom (1991) have shown maintenance of biomass for degrading part per million levels of naphthalene through use of an enricher reactor that receives only low concentrations of naphthalene and salicylate; the later is a metabolic byproduct of naphthalene decomposition. Naphthalene degradation ceased soon after the inducing compounds were removed from the feed to the biotreater. The next generation of biochemical wastewater treatment processes will evolve from the new advances in modern microbiology and molecular biology. Development
* Corresponding author.
of technology data bases that include extent of contaminant conversion, reaction rate constants, active enzyme identification, and stability measurements will be needed to permit scaling, design, and construction of these biochemical processes. Polycyclic aromatic hydrocarbon (PAHs) are an important class of wastewater contaminants associated primarily with the production and transportation of liquid fuels such as refined oil and liquified coal. The potential for microorganisms associated with aerobic, anaerobic, and denitrification environments to degrade PAHs has been investigated (Mihelcic and Luthy, 1988; Bauer and Capone, 1985). Aerobic microorganisms give the most rapid degradation of PAHs by introducing oxygen into the ring structure, thereby allowing assimilation of the PAH into the general metabolism. However, the reported rates of aerobic degradation in nonacclimated microcosms or pure cultures are quite slow, of the order days to weeks (Heitkamp and Cerniglia, 1988;Heitkamp et al., 1988;Mihelcic and Luthy, 1988), compared to the time scale needed for efficient wastewater treatment processes. Mechanisms and metabolic pathways for the aerobic degradation of PAHs by microorganisms have been studied intensively. The critical first step in PAH conversion is hydroxylation of one aromatic ring by oxygenase enzymes (Cerniglia, 1984; Heitkamp et al., 1988). These enzymes, therefore, and the microorganisms in which they are present are logical starting points for designing biomass, or enzyme systems, that can be used in treating PAHcontaminated wastewaters. Our work has been focused on the monooxygenase enzyme system, cytochrome P-45OC,,, that is generated in Pseudomonas putida PpG
8756-7938/91/3007-0093$02.50/0 0 1991 American Chemical Society and American Institute of Chemical Engineers
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786 when the bacterium is cultured on a camphor substrate (Gunsalus and Wagner, 1978). We have previously shown that resting cultures of P. putida PpG 786 are capable of rapid biodegradation of volatile halocarbons a t trace concentrations (Lam and Vilker, 1987; Vilker and Khan, 1989). Although no clear evidence exists for the involvement of this particular oxygenase for conversion of PAHs, we were encouraged to investigate the possiblity for such activity in part because of reports showing rapid conversion of pyrene and benz[a]anthracene by a closely related type of cytochrome monooxygenase, rabbit liver microsomal P-45oLM (Imai, 1982). In this work, an initial assessment is made of the potential for the P-45OC,, monooxygenase enzyme to convert naphthalene a t trace concentrations in aqueous solutions. The measurements were carried out in resting cultures of P. putida PpG786 that were grown on camphor to induce expression of the P-450, enzyme system. Previous work with this organism/enzyme system investigated rates of biodehalogenation of halogenated alkanes, their byproducts, halide ion material balances, toxicity of halocarbon toward the host bacterium, and P-450 stability under reaction conditions (Lam and Vilker, 1987; Vilker and Khan, 1989). In this paper, we are exploring the capability of this system to detoxify trace levels of the molecularly simplest PAH, naphthalene. Naphthalene was selected for study in order to focus on intrinsic enzymatic activity of this monooxygenasefor PAH removal without the interference of competingrateprocesses such as PAH dissolution. Naphthalene removal rate, toxicity toward the host bacterium, P-450 stability, and inhibition in the presence of bactericides and P-450-specific inhibitors are evaluated.
Materials and Methods Materials. Naphthalene obtained from Aldrich Chemical Co. (98% purity, Milwaukee, WI) was used to make up a stock solution of 20 mM in ethanol. A single stock solution, maintained a t -20 "C for about 4 months, was used for all experiments. Hexane used in extracting naphthalene from the incubation media was pesticidegrade from Baxter Healthcare Corp., Burdick and Jackson Division, Muskegon, MI. Deionized water from a MilliQ system with an HPLCgrade organic scavenging cartridge was used for all media and incubation solutions. Chemicals used in the bacteria growth media were obtained from the following sources: NH4C1, K2HP04, KH2P04 (Fischer Scientific, Fair Lawn, NJ): MgS0~7H20, MnSOcH20, F e S 0 ~ 7 H z 0(Mallinckrodt Chemical Co., Paris, KY); CaC1~2H20,L-ascorbic acid, D-(+)-camphor, silicone antifoam, glutamic acid, sodium azide (Sigma Chemical Co., St. Louis, MO); bactoyeast extract, Bitek agar (Difco Laboratories, Detroit, MI). N,N-Dimethylformamide (DMF) used to make up a stock 3 M camphor solution for culturing bacteria containing P-450 enzyme was also purchased from Burdick and Jackson. Metyrapone was the kind gift of Dr. Arthur Cho of the UCLA Department of Pharmacology. Research-purity argon (99.9995% ) and carbon monoxide (99.99% ) were purchased from Matheson (Gloucester, MA) and each was passed separately through Alltech (No. 4004H) indicating oxy-traps before use in the enzyme assay. The sodium dithionite used in the assay was from Eastman Chemical Corp. (Rochester, NY). MicroorganismCultures. Pseudomonas putida strain PpG 786 (ATCC 29607) inocula were maintained at -80 "C until use. Storage and growth conditions were those
Biotechnol. Prog., 1991, Vol. 7, No. 2
described in Vilker and Khan (1989)except that the stocks were maintained in 1 mL each of glycerol buffer and L-broth. Harvested cells used for naphthalene incubations were washed twice with potassium monobasic buffer pH 7.4 in order to remove all carbon or energysources. These "resting" cells were resuspended in the same buffer a t 100 g of wet cells/L before use. Six separate preparations of P-450-containing biomass were used in the experiments discussed here. Biomass yields for these preparations ranged from 9 to 14 g of wet cells/L, and P-45OC, yield ranged from 50 to 100 nmol of P-450/g of wet cells. In some experiments, we used non-P-450 P. putida PpG 786 biomass as controls. These cells were grown as follows. P. putida (-80 "C stock) was quick-thawed in a 37 "C water bath for 30 s and transferred to a 125-mL Erlenmeyer flask containing 35 mL of L-broth enriched with an additional 0.35 mL of 20% glucose. After incubation for 16 h at 29 "C, 1.5-mL aliquots were transferred to each of four 2800-mL Fernbach flasks, each containing 400 mL of sterilized phosphate ammonium salt (PAS)solution (Lam and Vilker, 1987) and 0.8 g of L-glutamic acid as a 10-mM solution. After an 8-h incubation with agitation at 29 "C, another 1.6 g of L-glutamic acid was added, followed by a 15-h incubation. A final dose of 0.2 g of L-glutamic acid was added and incubation continued for a final 3 h. Cells were washed twice with potassium monobasic buffer, pH 7.4, and resuspended in this buffer at 100 g of wet cells/L for short-term preservation. P-45OC, Measurements in Whole Cells. Cytochrome P-45OC, content in whole cells was measured by difference optical absorbance spectrophotometry using a Beckman DU-65 single-beam programmable spectrophotometer. Measurements were made as described (Vilker and Khan, 1989)except that here all measurements are for suspension densities of 25 g of wet cells/L, regardless of the suspension density at which naphthalene conversion was investigated with a sample of the same biomass. Intracellular P-450 loss was monitored during naphthalene conversion studies. This was done by sampling an incubation vessel that was like those described below but in which no naphthalene is added. One hundred milliliters of P-450 active P. putida suspension was placed in a glass-stoppered 500-mL Erlenmeyer flask and sampled at the same intervals as the naphthalene-containing incubation vessel. The spectrophotometric method described above was used to follow the decay of P-450 activity over the course of the 20+-h naphthalene conversion studies. Cell Viability. Cell viability was compared for resting cultures (100 mL of suspension at 25 mg of wet cells/mL in 500-mL Erlenmeyer flasks with ground glass stoppers) that were exposed to either no naphthalene (control), 7.8 pM naphthalene (1 ppm), or 180 pM naphthalene (23.5 ppm). Aliquots of 0.5 mL were withdrawn from each flask at predetermined intervals during incubation a t 25 "C for up to 18h. Serial dilutions (in 0.1 M monobasic potassium phosphate buffer, pH 7.4) down to 1 X lo-* were made from these aliquots. Fifty microliters of each of the 1 X and 1X dilutions was spread on agar plates (2 9% agar), in duplicate. The plates were fed with 0.2 mL of 2 M camphor in methylene chloride daily and incubated a t 29 "C until well-formed colonies were observed after about 4 days. Colony counting was performed with the assistance of an electric plate counter (Fischer Accu-lite colony counter 133-8002). Naphthalene Removal Kinetics. The rates of removal of naphthalene in resting batch cultures of P. putida were determined by following the disappearance of the parent
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compound. Cell concentrations were varied from 15 to 35 g of wet cell/L (2.3-5.4 g of dry wt/L). The experiments involved the addition of predetermined amounts of the 20 mM naphthalene-in-ethanol stock solution to 500-mL Erlenmeyer incubation flask with ground glass stoppers that contained either 100 mL of cell suspensions in monobasic potassium phosphate buffer (pH 7.4) or just 100 mL of the buffer. Initial naphthalene concentration was varied from 2.89 pM (370 ppb) to 180pM (23.5 ppm). Incubation was carried out in a New Brunswick Scientific controlled environment incubator a t 25 "C and gentle agitation. Incubation time ranged from 22 to 88 h. Aliquots of 5-mL volume were withdrawn from the incubation flasks to make the naphthalene concentration measurements. The first sample was withdrawn after only 2 min of mixing in order to establish the initial naphthalene concentration. The data from the buffer control flask was relied upon to confirm that the technique did not introduce unacceptably large volatilization losses of naphthalene. The 5-mL aliquots were transferred into ground glass stoppered 15-mLcentrifuge tubes that had been previously filled with 2 mL of hexane (as an extractant) and 0.25 mL of concentrated nitric acid (as a reaction quencher). The contents were mixed vigorously by vortexing followed by centrifugation in a Beckman GPR centrifuge at 3000 rpm for 30 min. Approximately 1mL of the hexane layer was transferred to a 4-mL glass screw-top GC storage vial fitted with aluminum foil on the underside of the tightly fitted screw cap. These were stored for up to several days at -20 "C. The hexane extracts (5-pL quantities injected slowly) were assayed for naphthalene with a Varian 3700 gas chromatograph fitted with a flame ionization detector and linked to a Hewlett-Packard 3390A integrator. The GC column was a J&W Scientific (Folsom, CA) DB-1 15 m X 0.53 mm megabore column. Nitrogen (99.999%) carrier gas was 10 mL/min through the column and makeup gas was 30 mL/min to the detector. Hydrogen (99.999 % ) a t 30 mL/min and air a t 300 mL/min went to the detector. The injector port temperature was 220 "C and the column temperature was programmed for 45 "C for 2 min, 80 "C/ min ramp, and 180 "C for 3 min. The detector temperature was 320 "C. Naphthalene retention time and concentration calibration both in pure hexane and in hexane extracts from known aqueous samples were established with authentic samples. In the course of this study, over 2000 chromatograms for naphthalene detection gave a retention time of 4.08 f 0.10 min. The GC area was linearly related to aqueous phase concentration over the range 2-200 pM naphthalene. Two sets of standards were run before every incubation data set in order to ensure retention time and area integration stability.
Results The activity of cytochrome P-45OC,/P. putida PpG 786 biomass for removal of naphthalene was assessed by comparing naphthalene disappearance as a function of time in incubation flasks that contained P-450-active biomass, with disappearance in flasks that contained only naphthalene in the working buffer. The data of Figure 1 show that only the P-450-active biomass supported substantial naphthalene removal-half the initial naphthalene removed in about 18 h. The negligible loss of naphthalene from the buffer control suggests that abiotic losses of naphthalene from the incubation vessel (e.g., volatilization) were unimportant over this period. We have also analyzed the early time data for naphthalene disappearance in order to build greater quanti-
I A
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Figure 1. Naphthalene disappearance for cell suspension density 25 mg of wet cells/mL, initial concentration CO10.6 wmol of naphthalene/L (1.35 ppm); (A) buffer and naphthalene only, (A)camphor-grown P. putida PpG 786 cell mass that had an initial enzyme content of EO = 2.3 wmol of P-450/L of cell suspension. The linear regression through the initial data (0-16 h) gives first-order constant A = 1.35 X 10-l6 L/s.
tative understanding about the effects of biomass growth and incubation variables on the ability of this enzyme/ organism system to remove PAHs. Over 20 separate experiments covering a range of naphthalene concentrations and cell suspension concentrations were performed. Initial disappearance data is interpreted as pseudo-firstorder removal kinetics in these batch systems: In [C/C,]= -wAt where COis initial bulk naphthalene concentration (M), w is cell number/volume suspension ( w / ( p c V c ) )W , is dry cell mass/volume suspension (g/L), pc is single-cell density cm3), and (1.3 g/mL), V, is single-cell volume (3.4 X A is a kinetic parameter (L/s). This analysis, applied to the first 16 h of Figure 1, gives the smooth curve shown L/s. for which A = 1.35 X In Figure 2, we show results of experiments in which the decline of the intracellular enzyme level was followed over the same time course during which naphthalene disappearance was being followed. The incubation flask containing the biomass used in assessing enzyme decay did not receive naphthalene but was treated in every other respect the same as the removal study flask. Previously, we have shown that the spectrophotometric assay for intracellular P-450 level related directly to bioconversion activity toward DBCP (Vilker and Khan, 1989). We can wonder how much greater the naphthalene removal activity might be in Figure 2 if there existed a method for stabilizing the P-450 level a t the initial value. The role of the P-450 enzyme in naphthalene removal is further suggested by comparing the near lack of naphthalene disappearance in experiments that were run with P. putida in which the enzyme is absent (glutamic acid as carbon source during culturing), with the disappearance that occurs with biomass (at equivalent cell density) which was grown to have the enzyme (camphor carbon source). This comparison is shown in Figure 3. Also shown are data that indicate sodium azide and metyrapone, at concentrations of 1mM, are about equally effective in preventing naphthalene removal in the P-450 active systems. Measurements of the rate constant A taken as a function of increasing starting naphthalene concentration (Figure 4) indicate that the ability of the enzyme/organism system to remove naphthalene diminishes at the higher concen-
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Biotechnol. Prog., 1991, Vol. 7, No. 2
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Figure 3. Naphthalene removal in P. putida PpG 786 biomass that contains no P-450, enzyme (O),P.putida PpG 786 that contains P-450 (m,Eo = 2.1 pmol of P-450/L of cell suspension), biomass with P-450 and sodium azide (A,1 pM NaN3), and biomass with P-450 and metyrapone (A,1 mM metyrapone). For all experiments the cell density was 25 mg of wet cells/mL, and Co = 11pM naphthalene. The smooth curve through P-450data L/s. is for A = 1.36 X
,
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Figure 5. Comparison of P. putida PpG 786 cell viability for no exposure to naphthalene (striped bars) with viability after exposure (cross-hatchedbars). (A) Naphthalene exposure, 10.5 pM (1.35ppm); (B)Naphthalene exposure, 180 pM (23.5 ppm). Decline of intracellular P-450, levels for nonexposed cells is shown by open bars.
of 1.1 X to 3.7 X This is likely to be the range of reproducibility for our methods, and no trends in A with biomass concentration were revealed. Finally, in Figure 5, the viability of resting cells is examined over the time frame that the naphthalene removal was studied. The control cells (no naphthalene inoculation) show a rapid decline in viability during the first 6 h but then maintain a reduced level of activity, about 10-1374 of initial activity, through the 18-h study period. The spectrophotometric indication of intracellular P-450 levels shows a much smaller relative decline in the enzyme concentration over this period. For cells that received an inoculation of a solution containing 1.35 pM naphthalene, Figure 5A shows that only about 5 5% of the initial viability was retained after 18 h. In Figure 5B, essentially no viability was detected after 8 h for inoculations at initial naphthalene concentration of 180 pM.
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Figure 2. Time course of (0) naphthalene disappearance (25 mg of wet cells/mL, CO= 10.6 pM naphthalene) and decay of cytochrome P-450,concentration (.,Eo = 2.3 pmolofenzyme/L of cell suspension) in a separate incubation flask without
-G
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-6
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Figure 4. Decrease of first-orderkineticconstant for naphthalene
disappearancewith increasein initial naphthaleneconcentration.
trations. Above 180 pM naphthalene, removal capacity is nearly gone. Also in Figure 4 are the results of several experiments performed at COof 10.5 pM naphthalene in which cell mass concentration was varied over the range of 2.3-5.4 g of dry wt/L. The results show a range for A
Our results show removal of naphthalene from laboratory-scale buffer solutions by P. putida that contains the cytochrome P-450, monoxygenase enzyme system. Two important questions about these results are (i) is naphthalene being biochemically converted by enzymatic activity of the P-450,, and (ii) do these results suggest potential for application of this microorganism and/or the P-45OC, monooxygenase system to industrial wastewater treatment processes? Regarding bioconversion of naphthalene by P-450,, a definitive conclusion can only be reached by studying the
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byproducts of the biochemical reaction. We have such studies underway in cell-free reconstituted enzyme incubations using the three purified proteins that make up the P-450,, monooxygenase system. The goal is to identify the hydroxylation products resulting from the first step in naphthalene degradation without the interference of other possible intracellular enzymatic activity. However, the results presented here do provide inferential evidence that P-450 mediated degradation of naphthalene is occurring in our incubations. This evidence is (i) the lack of naphthalene removal by P. putida PpG 786 cell mass that was not grown on camphor, (ii) the inhibition of naphthalene removal when the P-450 inhibitor metyrapone is added to P450-active cell mass, and (iii) substrate/ enzyme turnover calculations. P. putida PpG 786 that has been grown on glutamic acid as sole carbon source does not possess P-450, enzyme as shown by our standard spectrophotometric assay of whole cells [measurement of the 446-nm absorption peak produced by the dithionite/CO reaction (Vilker and Khan, 1989)]. Figure 3 shows one of many experimental results that indicate this glutamic acid grown cell mass has little or no capacity for removing naphthalene. Metyrapone inhibition of naphthalene removal, also shown in Figure 3, further suggests that P-450-catalyzed degradation occurs in our systems when active uninhibited P-450 is present. Metyrapone is an established inhibitor of P-450c,, activity (Peterson et al., 1971; Brewer and Peterson, 1986). Cooxidation mechanisms for naphthalene conversion are not considered likely due to the fact that the incubations were performed with resting cell mass (i.e., no added carbon or energy sources) and because metyrapone was able to inhibit degradation. Finally, we have estimated substrate/enzyme turnover ratios in order to examine more carefully the action of the P-450 enzyme during naphthalene removal. These calculations were made for the initial period of naphthalene removal when a first-order description of removal kinetics could be applied (see linear regression in Figure 1). A complication in making these calculations was the effect of the declining enzyme activity due to thermal denaturation as exemplified by E/Eo data in Figure 2. An average enzyme concentration (spectrophotometric measurement) for the time period was used; all turnover calculations can be off by about a factor of 2 due to this approximation. The results shown in Table I indicate that multiple turnovers of the P-450 enzyme are involved in causing the extent of naphthalene removal that we measured in the incubations. This suggests that P-450 is doing more with naphthalene than simply removing it from solution by irreversible binding. The high end of the turnover numbers shown in the table is about 0.12 X lO-3/s. This is about 200 times smaller than the turnover number reported by Imai (1982) for the binding and conversion of pyrene by microsomal P-450. The potential for using P-450-active P. putida PpG 786 to treat PAH-containing wastewater could be realized in a biotreatment process like extended aeration, where wastewater residence time is 15-30 h and biomass concentration is around 4.5 g/L (Sundstrom and Klei, 1979). An estimate of naphthalene half-life, from our data, under these conditions would be around 36 h. This is about 5-15 times faster than the half-life found in aerobic acclimated microcosms (Heitkamp and Cerniglia, 1988; Heitkamp et al., 1988),but about 20 times slower than the rates found for an experimental conventional activated sludge system that was acclimated to naphthalene and maintained on a reduced level of naphthalene in the
Table I. Turnover Num'ber for Naphthalene Removal Presented in Order of Increasing Initial Naphthalene Concentration naphnaphavg enzyme thalene thalene concn, concn, turnover no., concn, expt initial, consumed, unexposed molecules/ molecules/ site sample, pM (sitees) X lo5 pM no. pM 27 2.89 1.36 1.95 2.42 0.700 2.11 14 4.30 3.77 1.79 4.18 13 7.42 5.16 1.85 3.51 2.79 17 7.81 5.26 1.85 3.59 2.84 9 8.60 4.73 1.35 5.14 3.52 23 10.50 5.10 2.94 3.01 1.73 22 10.50 4.70 1.79 4.57 2.64 19 10.94 3.29 1.68 5.44 1.96 18 10.94 5.11 0.760 7.79 6.72 21 11.72 6.92 0.822 14.6 8.42 8 11.72 7.59 1.36 8.23 5.62 10 12.50 6.69 1.16 6.97 5.77 29 12.50 5.19 0.795 9.07 6.54 25 17.19 7.86 1.69 6.46 4.64 15 21.88 11.58 1.85 7.900 6.27 16 25.00 16.99 1.72 10.5 9.85 24 54.69 19.72 1.50 11.4 13.18 26 109.38 14.56 1.69 12.0 8.62
wastewater feed (Cardinal and Stenstrom, 1991). It is likely that the degradation rate in the P-45O/P. putida system would be about as fast as the rate in the experimental acclimated activated sludge if the P. putida biomass could be continuously challenged with the inducing substrate (camphor) over the time of degradation measurements. It is also important to consider the naphthalene biodegradation rate in relation to the rates for other processes that lead to loss of the contaminant from the biotreatment system. Cardinal and Stenstrom (1991) determined that the primary competing process for removal of trace levels of naphthalene (concentrations below the solubility limit of 20-33 ppm) from laboratory-scale activated sludge reactors was volatilization. By combining their observed laboratory degradation rate with liquid-phase control mass transfer for naphthalene volatilization, they estimated the volatilization loss of naphthalene from a commercial-scale unit. The analysis showed that less than 5% of the naphthalene would be lost by volatilization in a conventional domestic activated sludge unit for which wastewater residence time is 6 h and aeration rate is 2 m3 of air/ (m3 of reactor vol-h). For a P-45O/P. putida extended aeration using higher cell densities and the naphthalene degradation rates that we measured, the analysis of Cardinal and Stenstrom would predict about a 50/50 split in the removal of naphthalene by biodegradation and by volatilization. Newer industrial wastewater biotreatment processes are likely to be enclosed and use vapor recycle in order to prevent volatilization losses. The potential for using P-450,- monooxygenase in an advanced wastewater treatment process for PAH removal may be greater than the potential for using the natural host (P. putida PpG 786) as biomass. This conclusion follows from seeing that whole-cell kinetics give reasonable removal rates relative to conventional treatment processes like extended aeration activated sludge but that cell viability declines much faster than P-450 enzyme concentration (Figure 5). It is likely that the spectrophotometric indication of P-450,, concentration level, which decays rather slowly, is directly related to P-450, biochemical activity; such relationships have been examined previously for microsomal P-450 (Wiseman and Gondal, 1975), and by us for biodehalogenation reactions by P-45OC,, (Vilker and Khan, 1989). If the P-450,
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system should become more attractive as an agent for detoxifying PAH-contaminated wastewater, it is likely that modern genetic engineering techniques could be used to further enhance this protein’s thermal stability. The major challenge that will remain is development of inexpensive substrates to drive the detoxification activity of this oxidoreductase enzyme.
Acknowledgment We thank Ms. A. Bramblett and F. Khan for assistance with the experiments. We thank the National Science Foundation Engineering Research Center for Hazardous Substance Control Research, the University of California Program for Research in Engineering and Systems Analysis for the Control of Toxic Substances (ESACT) for financial support, and the U S . Coast Guard for fellowship support for G.P.K.
Literature Cited Bauer, J. E.; Capone, D. G. Degradation and Mineralization of the Polycyclic Aromatic Hydrocarbons Anthracene and Naphthalene in Intertidal Marine Sediments. Appl. Environ. Microbiol. 1985,50,81-90. Brewer, C. B.;Peterson, J. A. Single Turnover Studies with OxyArch. Biochem. Biophys. 1986,249,515cytochrome P-450,. 521. Cardinal, L.J.; Stenstrom, M. K. Enhanced Biodegradation of Polyaromatic Hydrocarbons in the Activated Sludge Process. Res. J. Water Pollut. Control Fed. 1991,in press. Cerniglia, C. E. Microbial Transformation of Aromatic Hydrocarbons. In Petroleum Microbiology; Atlas, R. M., Ed.; Macmillan: New York, 1984; Chapter 3.
Gunsalus, I. C.; Wagner, G. C. Bacterial P-450, Methylene Monooxygenase Components: Cytochrome m, Putidaredoxin, and Putidaredoxin Reductase. In Methods in Enzymology;Fleischer, S., Packer, L., Eds.; Academic Press: New York, 1978; Vol. 52,pp 166-188. Heitkamp, M. A,; Cerniglia, C. E. Mineralization of Polycyclic Aromatic Hydrocarbons By a Bacterium Isolated from Sediment Below an Oil Field. Appl. Environ. Microbiol. 1988,54, 1612-1614. Heitkamp, M. A,; Freeman, J. P.; Miller, D. W.; Cerniglia, C. A. Pyrene Degradation by a Mycobacterium sp.: Identification of Ring Oxidation and Ring Fission Products. Appl. Environ. Microbiol. 1988,54, 2556-2565. Imai, Y.Interaction of Polycyclic Hydrocarbons with Cytochrome P-450. I. Specific Binding of Various Hydrocarbons to P-488one. J. Biochem. (Tokyo) 1982,92,57-66. Lam, T.; Vilker, V. L. Biodehalogenation of Bromotrichloromethane and 1,2-dibromo-3-chloropropaneby Pseudomonas putida PpG 786. Biotechnol. Bioeng. 1987,29,151-159. Mihelcic, J. R.; Luthy, R. G. Degradation of Polycyclic Aromatic Hydrocarbon under Various Redox Conditions. Appl. Environ. Microbiol. 1988,54, 1182-1187. Peterson, J. A.; Volker, U.; Hildebrandt, A. G. Metyrapone Interaction with Pseudomonas putida Cytochrome P-450. Arch. Biochem. Biophys. 1971,145, 531-542. Sundstrom, D. W.; Klei, H. E. Wastewater Treatment;PrenticeHall, Inc.: Englewood Cliffs, NJ, 1979;p 155. Vilker, V. L.;Khan, F. Stability of Cytochrome P-450 Enzyme and the Dehalogenation Activity in Resting Cultures of Pseudomonas putida PpG 786. Biotechnol. Prog. 1989, 5, 70-74. Wiseman, A,; Gondal, J. A. Protection of Cytochrome P-450 by Substrates Against Tween 80 Assisted Thermal Denaturation. Biochem. SOC.Trans. 1975,3,977-980. Accepted January 24, 1991.
