Vol. 58, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1992, p. 2237-22440099-2240/92/072237-08$02.00/0Copyright © 1992, American Society for Microbiology

Biodegradation of Mixtures of Substituted Benzenes byPseudomonas sp. Strain JS150

BILLY E. HAIGLER, CHARLES A. PETTIGREW,t AND JIM C. SPAIN*

Air Force Civil Engineering Support Agency, Tyndall Air Force Base, Flonida 32403-6001

Received 10 February 1992/Accepted 21 April 1992

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 thatcontains the genes for the degradative pathways of a wide range of substituted aromatic compounds.Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate,p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experi-ments to determine the conditions required for induction of the individual pathways and to determine whethermultiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells andenzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavagepathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a

substrate range similar to that found in strains ofPseudomonas putida. The presence of the dioxygenase alongwith multiple pathways for metabolism of substituted catechols allows facile extension of the growth range byspontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-growncells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene,and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cellsdegraded a mixture of phenol, 2-chloro-, 3-chloro-, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol.These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits thebiodegradation of complex mixtures of aromatic compounds.

Contaminated ecosystems typically contain heteroge-neous mixtures of organic compounds. Although numerousstudies have demonstrated the biodegradation of individualsubstituted benzenes, our understanding of the microbialdegradation of complex mixtures of organic compounds islimited. Some insight into this issue has been gained throughprevious studies involving cometabolism (25), preexposureto other aromatic hydrocarbons (2, 5), enzyme inactivationor inhibition (4, 27), diauxie versus simultaneous substrateutilization (22, 39), and the simultaneous degradation ofchloro- and methyl-substituted aromatic substrates (33).However, the ability to predict the fate of individual com-ponents of mixtures of organic compounds in natural andengineered systems is restricted (28, 40).

Biochemical studies of the biodegradation of aromaticcompounds have traditionally involved the use of singlesubstrates. This approach has usually resulted in the char-acterization of pure cultures that degrade only a narrowrange of closely related compounds. Although recently at-tention has focused on genetic engineering as a means ofincreasing the substrate range of single organisms, the re-lease of such microorganisms into the environment is tightlyregulated. We have isolated a strain of Pseudomonas thatdegrades a wide range of substituted aromatic compounds,including benzene, toluene, ethylbenzene, benzoate, p-hy-droxybenzoate, chlorobenzene, p-dichlorobenzene, phenol,salicylate, and naphthalene. Strain JS150 is capable of syn-thesizing at least four ring-fission pathways and three sepa-rate initial dioxygenases when grown on the appropriatesingle substrates. We designed experiments to determinewhether the pathways would operate with complex mixtures

* Corresponding author.t Present address: Environmental Safety Department, The

Procter & Gamble Co., Cincinnati, OH 45217.

of these substrates. A preliminary report of this work hasbeen presented previously (19).

MATERIALS AND METHODS

Materials. Chlorobenzene and toluene were purchasedfrom Fisher Scientific Co., Fairlawn, N.J. 3-Methylcatecholwas from Pfaltz & Bauer, Inc., Waterbury, Conn. Catecholwas from Aldrich Chemical Co., Inc., Milwaukee, Wis.Gentisic acid was from Sigma Chemical Co., St. Louis, Mo.3-Chlorocatechol was prepared biologically from chloroben-zene by the action of Pseudomonas putida Fl (14, 16).3,6-Dichlorocatechol was synthesized from 3,6,6-trichloro-2-hydroxycyclohex-2-en-1-one (31, 32). 4-Chlorocatecholwas a generous gift from David Gibson, University of Iowa.Aminodinitrotoluenes were provided by Ronald Spanggord.3-Nitrocatechol was prepared from catechol as described byAstle and Stephenson (3). Catechols were examined forpurity by using high-performance liquid chromatography(HPLC) and gas chromatography (GC)-mass spectrometryand further purified by HPLC when necessary. All otherchemicals were of the highest purity available.Organisms and culture conditions. Pseudomonas sp. strain

JS1 is an encapsulated bacterium originally isolated for itsability to degrade p-dichlorobenzene (44, 45). A nonencap-sulated derivative, designated strain JS150, was obtainedafter ethyl methanesulfonate mutagenesis (11) and selectionon the basis of a nonmucoid colony morphology.

Cultures were grown at 30°C in a minimal salts medium(MSB) (46). For agar plates and small liquid cultures, volatilesubstrates were supplied as previously described (20).Larger cultures of toluene- or chlorobenzene-induced cellswere grown in a 2-liter benchtop bioreactor (model C32;New Brunswick Scientific Co., Inc., Edison, N.J.) operatedin the batch mode. The bioreactor contained 1.3 liters ofMSB and was agitated at 500 rpm. Substrates were supplied

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TABLE 1. Oxygen consumption by whole cells of Pseudomonas sp. strain JS150

,umol of oxygen consumed/min/mg of protein after growth with:Substrate

Toluene Benzene Chlorobenzene Benzoate Naphthalene Salicylate Glucose Phenol

Toluene 0.291 0.228 0.158 0.002 0.015 <0.001 <0.001 0.076Benzene 0.008 0.045 0.009 0.001 0.010 0.009 <0.001 0.050Chlorobenzene 0.023 0.045 0.192 <0.001 0.002 0.010 <0.001 0.015Benzoate <0.001 <0.001 <0.001 0.287 0.012 0.019 <0.001 <0.001Naphthalene 0.116 0.051 0.024 0.003 0.445 0.667 0.009 0.010Salicylate <0.001 <0.001 <0.001 <0.001 0.056 0.063 <0.001 <0.0011,4-Dichlorobenzene 0.016 0.013 0.093 <0.001 0.005 0.006 0.003 0.006Phenol 0.006 0.007 0.016 <0.001 <0.001 <0.001 <0.001 0.4091-Methylnaphthalene <0.001 <0.001 <0.001 0.004 0.120 0.189 NDa ND2-Methylnaphthalene ND ND 0.012 0.007 0.298 0.453 ND ND1-Chloronaphthalene <0.001 <0.001 0.018 0.006 0.211 0.279 ND ND2-Chloronaphthalene ND ND 0.011 ND 0.229 0.319 ND NDEthylbenzene ND 0.177 0.121 <0.001 0.005 0.012 ND 0.069Catechol 0.058 0.064 0.172 0.667 0.015 0.025 <0.001 0.5193-Methylcatechol 0.077 0.233 0.397 0.038 0.217 0.220 <0.001 0.4283-Chlorocatechol <0.001 <0.001 0.184 0.009 0.031 0.032 <0.001 0.0084-Chlorocatechol ND 0.009 0.197 0.075 0.029 0.026 <0.001 0.0783,6-Dichlorocatechol ND <0.001 0.104 0.014 0.040 0.014 ND 0.017

a ND, not determined.

by a syringe pump (model 975; Harvard Apparatus, SouthNatick, Mass.) at 42 p.l/h, and the culture was sparged withair at 1 liter/h. Benzene-induced cells were grown in a similarmanner, with influent air at 24 liters/h and benzene influxinitially at 19 p.l/h. As the culture grew and benzene was nolonger detected in the effluent air, the feed rate of thesubstrate was increased to 42 ul/h. The cultures wereharvested during early log phase. Benzoate-, naphthalene-,or salicylate-induced cells were grown in the bioreactor withthe substrate at a concentration of 0.1% and an airflow rateof 1 liter/h. Cells were grown on phenol under continuous-flow conditions with phenol (5 x 10-' M) supplied in theMSB at a dilution rate of 0.05 h- 1. Growth on glucose was asdescribed previously (20).For the biodegradation of mixtures of substituted ben-

zenes, the bioreactor was operated as a chemostat by usingsimilar operating conditions. MSB was added continuouslyby a peristaltic pump (Harvard Apparatus). Water-solublearomatic compounds were dissolved in the MSB, and theconcentrations of these compounds and those of their metab-olites were determined by HPLC. Insoluble compounds ingas samples and pentane extracts of aqueous samples weremeasured by GC.

Additional continuous-flow experiments were done with acolumn (50 by 5 cm) of Manville Celite R-635 as a supportmatrix connected in series to the chemostat effluent port. Asthe aqueous and gaseous effluent passed down the column,cells of strain JS150 adhered to the support matrix. Thecolumn served as a secondary treatment step that removedtraces of aromatic compounds that remained in the aqueousand gas effluent from the chemostat.

Respirometry and enzyme assays. Cells grown on glucose,toluene, chlorobenzene, or benzene were harvested andwashed prior to respirometry as described previously (44).Cells grown on benzoate, salicylate, or naphthalene exhib-ited low activities when washed. Therefore, they wereharvested at 10°C, suspended in 0.02 M sodium phosphatebuffer (pH 7.0), and sparged with air until endogenous levelsof oxygen consumption were lowered. Oxygen uptake wasmeasured polarographically at 25°C with a Clark-type oxy-gen electrode (44). Reaction mixtures contained i0-4 M

substrate, cells (0.20 to 0.55 mg of protein), and 0.02 Msodium phosphate buffer (pH 7.0) to a final volume of 1.8 ml.

Cell extracts for enzyme assays were prepared as previ-ously described (44). Catechol 1,2-dioxygenase and catechol2,3-dioxygenase were measured as described before (44).Cell extracts for gentisate oxygenase assays were preparedin 0.1 M potassium phosphate buffer (pH 7.0) containing 1mM dithiothreitol, and the assays were as previously de-scribed (9). Protein determinations were done by using themethod of Smith et al. (42).

Analytical methods. HPLC was performed on a ,uBonda-pak C18 column (3.9 mm by 30 cm) (Waters Associates, Inc.,Milford, Mass.) with methanol-water-phosphoric acid (370:630:1) or acetonitrile-13.6 mM trifluoroacetic acid (60:40) asthe mobile phase at a flow rate of 1.5 ml min-1. Compoundswere detected by their UVA210, A254, andA280 with a model1040 diode array detector (Hewlett Packard Co., Palo Alto,Calif.). GC was done on a 30-m capillary column (DB-5; J &W Scientific, Folsom, Calif.) in a Hewlett Packard 5890series II gas chromatograph equipped with a flame ionizationdetector. Samples were analyzed by using a 3-ml/min flowrate and 30-ml/min split injection. The carrier gas washigh-purity helium maintained at a column head pressure of7.0 lb/in2. The injection temperature was 220°C, and theoven temperature was held at 30°C for 2 min and thenincreased at a rate of 25°C min-1 to a final temperature of175°C. The detector temperature was held at 250°C.

RESULTS

Oxidation of aromatic compounds by cell suspensions. Cellsgrown on toluene and chlorobenzene rapidly oxidized tolu-ene, catechol, and 3-methylcatechol (Table 1). In addition,chlorobenzene-grown cells oxidized chlorobenzene, 1,4-dichlorobenzene, ethylbenzene, and all of the substitutedcatechols tested. The pattern suggests that chlorobenzeneinduces a nonspecific initial dioxygenase and catechol oxy-genase. Benzene-grown cells oxidized toluene, benzene,chlorobenzene, catechol, and 3-methylcatechol. Benzoate-grown cells rapidly oxidized benzoate, catechol, and 4-chlo-rocatechol. Suspensions of naphthalene- or salicylate-grown

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BIODEGRADATION OF SUBSTITUTED BENZENES 2239

TABLE 2. Enzyme activities in cell extracts of Pseudomonas sp. strain JS150

Enzyme assayed and assay Sp act (timol/min/mg of protein) after growth with:substrate Toluene Benzene Chlorobenzene Benzoate Naphthalene Salicylate Phenol

Catechol 2,3-dioxygenaseCatechol 0.289 0.258 0.058 <0.001 0.023 0.011 0.0643-Methylcatechol 0.620 0.530 0.184 0.003 0.165 0.134 0.132

Catechol 1,2-dioxygenaseCatechol 0.009 0.046 0.237 0.424 <0.001 <0.001 0.8153-Methylcatechol 0.014 0.034 0.360 0.025 0.017 0.005 0.2793-Chlorocatechol <0.001 0.001 0.285 0.003 <0.001 0.002 0.0014-Chlorocatechol NDa 0.005 0.214 0.026 <0.001 <0.001 0.0323,6-Dichlorocatechol <0.001 0.001 0.151 0.001 <0.001 0.001 ND

a ND, not determined.

cells gave similar results and rapidly oxidized naphthalene,salicylate, 3-methylcatechol, and the chloro- and methyl-substituted naphthalenes. Phenol-grown cells rapidly oxi-dized phenol, catechol, and 3-methylcatechol. Toluene, ben-zene, ethylbenzene, and 4-chlorocatechol were oxidizedmore slowly by phenol-grown cells. Glucose-grown cells didnot oxidize any of the aromatic compounds at appreciablerates, which indicates that all of the enzymes for metabolismof aromatic compounds are inducible.Enzyme activities in cell extracts. Catechol 2,3-dioxygenase

activity was detected in cell extracts of strain JS150 grownon all of the aromatic substrates tested except benzoate(Table 2). Extracts from benzene-, phenol-, or benzoate-grown cells contained catechol 1,2-dioxygenase activity thatwas specific for catechol and 3-methylcatechol. Extractsfrom chlorobenzene-grown cells contained the nonspecificchlorocatechol 1,2-dioxygenase (12, 30), which attacked allof the substituted catechols tested. In separate experiments,cell extracts of naphthalene- and salicylate-grown cells oxi-dized gentisate at rates of 0.266 and 0.444 p.mol of oxygenconsumed per min per mg of protein, respectively. Thesesame cell extracts exhibited catechol 2,3-dioxygenase activ-ity for catechol at rates of 0.011 and 0.026 ,umol/min/mg ofprotein. Because glucose-grown cells did not oxidize any ofthe catechols tested, cell extracts were not tested (Table 1).

Substrates transformed by Pseudomonas strain JS150. Cellsof strain JS150 transformed a variety of substituted benzenesafter growth on chlorobenzene (Table 3). Toluene-growncells of strain JS150 often accumulated toxic levels ofsubstituted catechols when exposed to substituted benzenes.No products were detected by HPLC from the metabolismof 1,2- and 1,3-dichlorobenzene and the chloro- and meth-ylphenols. These results indicate that the aromatic ring wascleaved and suggest complete metabolism of these com-pounds.Spontaneous mutants that grew on 4-chlorotoluene, 4-bro-

motoluene, m-toluate, 3-chlorobenzoate, and 4-chloroben-zoate were readily isolated after extended exposure to thesecompounds. Strains of JS150 that grow on 4-chlorotolueneand 4-bromotoluene appear to use the same degradativepathway used by another derivative of JS1, Pseudomonassp. strain JS21, when it grows on 4-chlorotoluene (20). Theability to readily isolate strains of JS150 that grow on novelsubstrates suggests that the growth range can be furtherextended if the appropriate pathways can be induced.

Metabolism of substrate mixtures by JS150. The presenceof multiple pathways for the degradation of substitutedbenzenes in strain JS150 suggested that it could grow on

mixtures if the appropriate enzyme sequences could beinduced. Chlorobenzene was provided as the primary sub-strate in experiments involving halogenated aromatic com-pounds, because it led to induction of the modified orthocleavage pathway (Table 2) (12, 30). Cells were grown tosteady state on chlorobenzene, and then a mixture of chlo-robenzene, benzene, toluene, 4-chlorotoluene, 1,4-dichlo-robenzene, and naphthalene was provided (Fig. 1). Theoptical density of the culture remained constant, and morethan 96% of each compound was removed. When 10% of thechlorobenzene was replaced with trichloroethylene, cellwashout occurred.

In another experiment, JS150 was grown to steady stateand a mixture of chloro- and methyl-substituted benzenes,naphthalene, and trichloroethylene was provided (Table 4).After steady-state growth was achieved, JS150 metabolizedmore than 90% of each substrate, with the exception of thedichlorobenzenes and trichloroethylene. Passage of theaqueous and gaseous effluent from the chemostat through atrickling filter column resulted in the removal of 99 to 100%of each substrate.

In a subsequent experiment, JS150 grown to steady stateon chlorobenzene degraded more than 90% of each compo-nent of a complex mixture of halogenated benzenes (Fig. 2).When 10% of the chlorobenzene was replaced with 1,2,4-trichlorobenzene, cell washout occurred. In a separate ex-periment, strain JS150 degraded a mixture of chlorobenzene(9.774 M) and 1,2,4-trichlorobenzene (0.040 M), which sug-gested that lower concentrations of 1,2,4-trichlorobenzenemight be tolerated in complex mixtures.

After steady-state growth on phenol, strain JS150 de-graded a mixture of chloro- and methyl-substituted phenols(Table 5). There was no evidence for accumulation ofintermediate metabolites under steady-state conditions.Cells grown on phenol were able to adapt quickly to growthon both chloro- and methyl-substituted phenols.Both trichloroethylene and trichlorobenzene were toxic at

high concentrations (1.15 and 0.85 M, respectively) but notat the lower of the two concentrations tested. The resultssuggest that mixtures with more components could be de-graded, but analytical constraints prevented calculation ofrigorous mass balances with more complex mixtures.

DISCUSSION

The presence of multiple pathways for the degradation ofsubstituted catechols and a broad-substrate toluene dioxy-genase accounts for the broad substrate range of strain

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TABLE 3. Substrates oxidized by Pseudomonas sp. strain JS150

Substrate Growtha Transformationb Major product(s) Reference or source

BenzeneTolueneEthylbenzeneChlorobenzeneBromobenzeneIodobenzeneFluorobenzeneNitrobenzene4-Chlorotoluene4-Bromotoluene2-Nitrotoluene3-Nitrotoluene4-Nitrotoluene1,2,4-Trinitrotoluene1,2-Dichlorobenzene1,3-Dichlorobenzene1,4-Dichlorobenzene1,4-Dibromobenzene1,4-Bromochlorobenzene1,4-BromofluorobenzeneNaphthaleneSalicylatePhenol2-Chlorophenol3-Chlorophenol4-Chlorophenol2-Methylphenol3-Methylphenol2,5-Dichlorophenol3-NitrophenolIndanIndeneIndolem-ToluateBenzoatep-Hydroxybenzoate3-Chlorobenzoate4-ChlorobenzoateTrichloroethylenem-Xylenep-XyleneAnthranilate

+

+c

+ Unidentified+ 3-Nitrocatechol

2-Nitrobenzyl alcohol3-Nitrobenzyl alcohol, 3-nitrobenzoate3-Methyl-6-nitrocatechol, 2-methyl-5-nitrophenol2-Amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene

+ 4-Carboxymethylene-2-fluorobut-2-en-4-olided

+c

+c

3-NitrocatecholUnidentifiedUnidentifiedIndigoe

+ Unidentified+ Unidentified+ Unidentified

a +, growth; -, nio growth.h Cells were grown on chlorobenzene prior to transformation experiments. +, transformation.c Spontaneous mutants that grew on these compounds were isolated.d Tentative identification is based on the mass spectrum of the isolated product.e Tentative identification is based on the characteristic pigment formation by colonies on agar plates exposed to indole vapors (13).

JS150. In a previous investigation, we showed that toluene-grown cells of JS150 catalyzed an initial dioxygenase attackon nitrobenzene (21). Toluene- and chlorobenzene-growncells of JS150 convert substituted benzenes to the samesubstituted catechols (21, 37; also unpublished data). Theseresults indicate that chlorobenzene- or toluene-grown JS150contains toluene dioxygenase and dihydrodiol dehydroge-nase enzymes with substrate specificities similar to those inthe toluene-degradative pathway of other derivatives ofstrain JS1, Pseudomonas species strains JS6 and JS21 (20,43, 45) (Fig. 1) and P. putida Fl (15, 16). The presence ofcatechol 2,3-dioxygenase but not catechol 1,2-dioxygenasein toluene-grown cells of strain JS150 indicates that tolueneis degraded by the meta cleavage pathway, as previouslydescribed for P. putida Fl and the other derivatives of JS1(Fig. 3) (15, 20). We have previously shown that strain JS6can also degrade toluene by the modified ortho pathway, butwith toluene as the sole carbon source, the modified ortho

pathway is not induced (33). After growth on chlorobenzene,strain JS150 contained both catechol 1,2-oxygenase andcatechol 2,3-oxygenase. The ability of the catechol 1,2-oxygenase to attack halogenated catechols indicates that it issimilar to the nonspecific chlorocatechol 1,2-dioxygenase(12, 30). The pathway for degradation of chlorobenzene byJS150 appears to be identical to that described previously forstrain JS6 (44) and for the strain studied by Reineke andKnackmuss (35) (Fig. 3).Our unpublished observations suggest that the naphtha-

lene oxygenase induced during growth of JS150 on eithernaphthalene or salicylate has a broad substrate specificitysimilar to that of the naphthalene dioxygenase studied byGibson and his colleagues (15, 18). Salicylate-grown cellshad the same pattern of substrate oxidation and enzymeactivities as naphthalene-grown cells, which suggests thatsalicylate induces the naphthalene-degradative pathwaywhich proceeds via salicylate. The oxidation of gentisate by

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BIODEGRADATION OF SUBSTITUTED BENZENES 2241

1.2

1.0

Ec

a0

(00

.00

.0

0.8

0.6

0.4

Ec

0

(0

0

.0

1-

0

.0

c

4

0.2

0.0 . *20 40 60 80

Hours

FIG. 1. Growth of JS150 on substituted benzenes. The chemostatwas operated at a constant dilution rate of 0.2 h-1 with the influent airat 1 liter h-1. Cells were grown to steady state on chlorobenzene andthen switched at 44 h to a mixture of compounds. The substratemixture consisted of chlorobenzene (5.07 M), benzene (1.17 M),toluene (0.97 M), 4-chlorotoluene (0.87), 1,4-dichlorobenzene (0.70M), and naphthalene (0.81 M). The substrate mixture was added at aconstant rate of 84 pul/h. At steady state, 1.7% of the chlorobenzene,0.9% of the benzene, 2.2% of the toluene, 3.7% of the 4-chlorotolu-ene, 2.8% of the 1,4-dichlorobenzene, and 3.0% of the naphthalene inthe influent mixture were recovered in the effluent. At 66 h, 10% ofthe chlorobenzene was replaced with trichloroethylene (1.15 M).

crude cell extracts from naphthalene- and salicylate-growncells was an order of magnitude greater than the activity forcatechol, which indicates that naphthalene and salicylate aremetabolized via gentisate. This result agrees with observa-tions by Callicotte et al. which indicate that salicylate isdegraded via gentisate (10). These results indicate that JS150degrades naphthalene and salicylate as in previously pro-posed pathways (48) (Fig. 3).Growth of JS150 on benzoate or phenol induced the type

TABLE 4. Biodegradation of substituted benzenes andtrichloroethylene by JS150'

Influent % RemovalbCompound (mo - 1(mmol h1) Chemostat Column

Steady-state (100% chloroben- 0.98 95 100zene) chlorobenzene

Steady-state (90% chloroben-zene mixture):

Chlorobenzene 0.90 92 99Benzene 0.02 92 100Toluene 0.02 90 100o-Dichlorobenzene 0.01 76 99p-Dichlorobenzene 0.02 83 99Naphthalene 0.02 93 100Trichloroethylene 0.01 83 100a The chemostat was operated at a constant dilution rate of 0.1 h', with the

influent air at 6 liters h-1. The substrate mixture was added at a constant rateof 100 p1l/h. The aqueous and gaseous effluent from the chemostat immediatelyentered the trickling filter column.

b Data represent the combination of measurements of each aqueous andgaseous compound exiting the chemostat or column as percent removal of thecompound entering the bioreactor.

1.0 Chlorobenzene + 1,2,4-Trichlorobenzene

0.8 j

0.6 ChlorobenzeneBromobenzene \1 ,3-Dichlorobenzene

0.4 1,4-Dichlorobenzene

1 ,4-Bromochlorobenzene

0.0 .2 0 4 0 6 0 80

HoursFIG. 2. Growth of strain JS150 on halogen-substituted benzenes.

The conditions of the chemostat operation and substrate additionwere as described in the legend to Fig. 1. Cells were grown to steadystate on chlorobenzene and then switched at 40 h to a mixture ofcompounds. The substrate mixture consisted of chlorobenzene (6.23M), bromobenzene (1.01 M), 1,3-dichlorobenzene (0.93 M), 1,4-dichlorobenzene (0.72 M), and 1,4-bromochlorobenzene (0.55 M).Prior to the addition of 1,2,4-trichlorobenzene, 1.3% of the chlo-robenzene, 1.7% of the bromobenzene, 5.2% of the 1,3-dichloroben-zene, 2.7% of the 1,4-dichlorobenzene, and 3.9% of the 1,4-bromo-chlorobenzene in the influent mixture were recovered in the effluent.At 65 h, 10% of the chlorobenzene was replaced with 1,2,4-trichlorobenzene (0.85 M).

I catechol 1,2-dioxygenase (12, 30). Phenol-grown cellscontained a catechol 2,3-dioxygenase in addition to thecatechol 1,2-dioxygenase. These results and comparison tothe pathways described for benzoate and phenol degradationin other bacteria (29) indicate that JS150 degrades thesesubstrates as depicted in Fig. 3.

It is not clear whether benzene-grown cells contain abenzene oxygenase that is specific for benzene. Benzene-grown cells oxidize toluene rapidly, but toluene- or chlo-robenzene-grown cells oxidize benzene very slowly. Thissuggests that the benzene dioxygenase is distinct from thetoluene dioxygenase induced after growth on toluene orchlorobenzene. Benzene-grown cells of JS150 containedboth catechol 1,2- and 2,3-dioxygenases. Pseudomonas sp.strain JS150 appears to degrade benzene by the same path-way as that described for other bacteria (Fig. 3) (15).The absence of detectable metabolites from mixtures of

substituted aromatic compounds and the ability to maintainthe density of the culture (Fig. 1 and 2) suggested complete

TABLE 5. Biodegradation of phenolic compounds byPseudomonas sp. strain JS150a

Compound Influent concentration %(mM) Removalb

Phenol 5.0 1002-Chlorophenol 0.1 1003-Chlorophenol 0.1 1002,5-Dichlorophenol 0.1 982-Methylphenol 0.1 993-Methylphenol 0.1 91

a The chemostat was operated at a constant dilution rate of 0.05 h1 at 30°C,with the influent air at 1 liter h-1.

b Data represent the averages of two samples obtained at 48-h intervalsduring steady-state operation.

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CIOH COOH

/ l

aOH

'OH

a; COOH

HOOCOH ciH

~~~~H

OH

COOH

CCOOH

I

OH

OH

I

COOH

COOH

FIG. 3. Proposed pathways for the degradation of toluene, naphthalene, salicylate, benzene, phenol, benzoate, and chlorobenzene byPseudomonas sp. strain JS150.

metabolism of some of the compounds that could not serve

as growth substrates when provided alone. Chloro- andmethyl-substituted phenols, 1,3-dichlorobenzene, and4-chlorotoluene were in this category. The results in Table 3indicate that chlorobenzene-grown cells readily metabolizethese compounds with no accumulation of transformationproducts. This result agrees well with earlier observationsthat Pseudomonas sp. strain JS6 completely degraded4-chlorotoluene after growth on p-dichlorobenzene (20) and2,5-dichlorophenol (43) after growth on chlorobenzene.

It has been held that bacteria have two general optionswhen confronted with metabolizable organic compounds(47). They could use a compound as the sole source ofcarbon and energy (growth substrate), or they could carryout partial degradation leading to the accumulation of trans-formation products (cometabolism). In the latter case, thecell is presumed to derive no carbon or energy from theprocess. Our results and similar findings by Hernandez et al.(24) and Ramos et al. (34) illustrate a third scenario in whichenzymes induced by one substrate allow the organism toderive carbon and energy from a second compound thatcannot serve as a growth substrate. This may be common innatural ecosystems in which a variety of related moleculesare present at low concentrations because of decompositionof organic matter. In such situations, evolution of a specificinduction system for the degradation of each compoundwould be inefficient, whereas induction of a nonspecificenzyme sequence by one component of the mixture wouldallow the organism to derive carbon and energy from a suiteof related compounds. If this phenomenon proves to bewidespread, growth of microorganisms on a single carbonsource might be considered a laboratory artifact. Evaluationof the above hypothesis requires a greater understanding notonly of induction, but also of metabolic pathways and theirinteractions. It may be less confusing in the interim to placeless emphasis on "cometabolism" and to use the term"metabolism" to describe all three of the situations de-scribed above (45).The recent attention to bioremediation as a means of

alleviating environmental pollution has resulted in a varietyof investigations involving the biodegradation of heteroge-

neous mixtures of organic compounds. Since few bacterialstrains have the genetic diversity of a heterogeneous popu-lation, studies involving degradation of mixtures of com-

pounds have involved mixed bacterial cultures that may beundefined, partially defined, or defined (2, 7, 8, 17, 38).Recent studies with isolated strains have demonstrated theabilities of various bacteria to degrade simple mixtures ofcompounds. Hutchinson and Robinson demonstrated thesimultaneous metabolism of phenol and cresol by P. putida(26). Alvarez and Vogel examined the substrate interactionsof mixtures of benzene, toluene, and p-xylene in two purecultures and a mixed culture (1). Their findings stressed theimportance of efficient enzyme inducers in the degradationof such mixtures. Bacteria that metabolized either 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyaceticacid were inefficient at degrading mixtures of these herbi-cides, presumably because of the accumulation of toxicintermediates (23). A constructed strain containing indepen-dent degradative pathways for these compounds metabo-lized the mixtures (23). Smith et al. described a Pseudomo-nas species that grew on mixtures of biphenyl orethylbenzene with 3- or 4-hydroxybenzoate but not whenboth biphenyl and ethylbenzene were together in the mixture(41). The researchers attributed this to a phenomenon theycalled synergistic inhibition. An aqueous mixture of phenol;o-, m-, andp-cresols; and 4-chlorophenol was degraded by astrain of P. putida in a continuous-flow fluidized bed biore-actor (6). These studies demonstrate that isolated strains candegrade mixtures of compounds when the problems ofsubstrate and/or product toxicity can be overcome throughinduction of the appropriate degradative pathways.

In cultures of JS150 exposed to mixtures containing vola-tile halogenated aromatic compounds, chlorobenzene was

always maintained in excess to ensure adequate induction ofthe nonspecific catechol oxygenase essential for degradationof substituted catechols. Since the bioreactor used in thesestudies was not designed for high-efficiency removal ofvolatile compounds, the addition of a column of immobilizedcells was necessary for the complete removal of the aromaticsubstrates in one of the solvent mixtures. Such a column

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BIODEGRADATION OF SUBSTITUTED BENZENES 2243

might not be necessary in an appropriately designed biore-actor.

ACKNOWLEDGMENTS

We thank David Gibson for providing 4-chlorocatechol and P.putida Fl, Howard Mayfield and Mike Henley for GC-mass spec-trometry analysis, and Shirley Nishino and Matt Bonzani for tech-nical assistance. We also thank Shirley Nishino for reviewing themanuscript.

This research was supported in part by an appointment to theResearch Participation Program at the Air Force Civil EngineeringSupport Agency Laboratory administered by Oak Ridge AssociatedUniversities through an interagency agreement between the U.S.Department of Energy and Tyndall Air Force Base.

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