Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1.

Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strains derived from P. putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.     

Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1

Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strains derived from P. putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.     

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.     

Metabolism of Chlorotoluenes by Burkholderia sp. Strain PS12 and Toluene Dioxygenase of Pseudomonas putida F1: Evidence for Monooxygenation by Toluene and Chlorobenzene Dioxygenases

The degradation of toluene by Pseudomonas putida F1 and of chlorobenzenes by Burkholderia sp. strain PS12 is initiated by incorporation of dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenzenes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown cells of Burkholderia sp. strain PS12 were found to monooxygenate the side chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols. Further metabolism of these products was slow, and the corresponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp. strain PS12 was metabolized further. Escherichia coli cells containing the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols as major products, demonstrating that this enzyme is responsible for the observed side chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes were formed as minor products from 2- and 3-chlorotoluene, whereas a chloro- and methyl-substituted cyclohexadiene was the only product formed from 4-chlorotoluene. The toluene dioxygenase of P. putida F1 and chlorobenzene dioxygenase from Burkholderia sp. strain PS12 are the first enzymes described that efficiently catalyze the oxidation of 2-chlorotoluene.     

Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid-degrading bacteria: chromogenic reactions for cloning genes encoding dioxygenases that act on aromatic acids.

The p-cumate-degrading strain Pseudomonas putida F1 and the m- and p-toluate-degrading strain P. putida mt-2 transform indole-2-carboxylate and indole-3-carboxylate to colored products identified here as indigo, indirubin, and isatin. A mechanism by which these products could be formed spontaneously following dioxygenase-catalyzed dihydroxylation of the indolecarboxylates is proposed. Indolecarboxylates were employed as chromogenic substrates for identifying recombinant bacteria carrying genes encoding p-cumate dioxygenase and toluate dioxygenase. Dioxygenase gene-carrying bacteria could be readily distinguished as dark green-blue colonies among other colorless recombinant Escherichia coli colonies on selective agar plates containing either indole-2-carboxylate or indole-3-carboxylate.     

Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor

Pseudomonas putida GS1 is able to convert limonene to perillic acid (up to 64 mM,(11 g/l) when the bacteria is cultivated in fed-batch culture with non-limiting amounts of glycerol. ammonium, and limonene. P. putida GS1 can use p-cymene as a single source of carbon and energy, and the enzymes that are responsible for the conversion of limonene to perillic acid belong to the degradation pathway of p-cymene. The p-cymene pathway of P putida GS1 is very similar, if not identical, to the cym pathway of P. putida F1. The latter strain, and a recombinant Escherichia coli strain that carried the genes of the cym pathway of P. putida Fl, also converted limonene to perillic acid. However, the final concentrations that were obtained in batch cultures with these two strains were lower than those obtained with P. putida GS1.     

Trichloroethylene and chloroform degradation by a recombinant pseudomonad expressing soluble methane monooxygenase from Methylosinus trichosporium OB3b.

Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate.     

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria

By complementing cell-free extracts of Pseudomonas putida F1/pSMMO20 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMMO20 and pSMMO50 into various bacterial species including pseudomonads and alpha-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO20 (resulting in pSMMO40) and found to enhance plasmid stability in P. putida F1 and R. meliloti 102F34 (first report of hok/sok in Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(II), Ni(II), and Zn(II)] and chloramphenicol from the medium and by supplementing with an iron source (3.6 muM of ferrous ammonium sulfate). Using chemostat-grown P. putida F1/pSMMO40, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity. (c) 1996 John Wiley & Sons, Inc.     

Recruitment and expression of toluene/trichloroethylene biodegradation genes in bacteria native to deep-subsurface sediments.

Four plasmids, each encoding a combination of either an Escherichia coli or Pseudomonas putida promoter and either toluene dioxygenase or toluene monooxygenase, were electroporated into five bacterial strains isolated from sediments found at depths of 91 to 295 m. Four of these engineered bacterial strains demonstrated both toluene and trichloroethylene degradation activities.     

Oxygenation Reactions of Various Tricyclic Fused Aromatic Compounds Using Escherichia coli and Streptomyces lividans Transformants Carrying Several Arene Dioxygenase Genes

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and ¹H and ¹³C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-di-hydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.     

Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes.

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.     

Chloroform mineralization by toluene-oxidizing bacteria.

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.     

Cloning of the Arthrobacter globiformis fcbA gene for dehalogenase and construction of a hybrid pathway of 4-chlorobenzoic acid degradation in Pseudomonas putida

The Artrobacter globiformis KZT1 fcbA gene responsible for dehalogenase (4-chlorobenzoate-4-hydroxylase) activity was cloned in Escherichia coli and Pseudomonas putida cells. The character of the fcbA gene expression was studied. Notwithstanding amplification of the gene dose and control of the inducible Plac promoter, the level of substrate dehalogenation by recombinant E. coli strains was lower, as compared with that in the original KZT1 strain. Cloning of the fcbA gene in P. putida KZ6R cells utilizing 4HBA resulted in a recombinant pathway of 4CBA degradation, which proved more effective for substrate consumption, in comparison with the original KZT1 strain.     

Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83.

Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13. P. putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA). Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source. Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs. Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83. Gas chromatography-mass spectrometry analysis of the metabolites of P. putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid. These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE). The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19. The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol.     

Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83

Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13. P. putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA). Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source. Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs. Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83. Gas chromatography-mass spectrometry analysis of the metabolites of P. putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid. These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE). The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19. The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol.     

Quantification of toluene dioxygenase induction and kinetic modeling of TCE cometabolism by Pseudomonas putida TVA8.

As measured by the toluene-induced bioluminescent response of Pseudomonas putida TVA8 in batch experiments, toluene dioxygenase (Tod) enzyme activities are dependent on toluene concentration between 0 and 30 mg/L. To provide a measure of the Tod activity for use in Michaelis-Menten competitive-inhibition kinetics, a correlation between toluene concentration and induced Tod activity as measured by an induced bioluminescent response of P. putida TVA8 is presented as a nondimensional Tod activity parameter. A packed-bed, radial-flow bioreactor (RFB) using the bioreporter P. putida TVA8A serves as the model system for studying the effect of the enzyme activity parameter on model predictions of vapor-phase toluene oxidation and trichloroethylene (TCE) cometabolism. Mass balances were performed on a differential section of the RFB to describe the radial transport of vapor-phase toluene and TCE through a bulk gas phase and the concomitant biological reaction in a stationary biofilm phase. The finite-element Galerkin weak-statement formulation with first-order basis functions was used to find the optimum solution to the highly nonlinear, coupled equations. For this RFB system with toluene concentrations less than 1 mg/L in the bulk gas phase, the Tod activity parameter enables accurate predictions of steady-state TCE degradation rate (0.27 microg TCE/min).