Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.     

The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.     

Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida

Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, alpha and beta; the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the alpha subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase alpha subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the alpha subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.     

Biodegradation of ofloxacin,norfloxacin, and ciprofloxacin as single and mixed substrates by Labrys portucalensis F11

Fluoroquinolone (FQ) antibiotics are extensively used both in human and veterinary medicine, and their accumulation in the environment is causing an increasing concern. In this study, the biodegradation of the three most worldwide used FQs, namely ofloxacin, norfloxacin, and ciprofloxacin, by the fluoroorganic-degrading strain Labrys portucalensis F11 was assessed. Degradation occurred when the FQs were supplied individually or as mixture in the culture medium, in the presence of an easily degradable carbon source. Consumption of individual FQs was achieved at different extents depending on its initial concentration, ranging from 0.8 to 30 μM. For the lowest concentration, total uptake of each FQ was observed but stoichiometric fluoride release was not achieved. Intermediate compounds were detected and identified by LC-MS/MS with a quadrupole time of flight detector analyzer. Biotransformation of FQs by L. portucalensis mainly occurred through a cleavage of the piperazine ring and displacement of the fluorine substituent allowing the formation of intermediates with less antibacterial potency. FQ-degrading microorganisms could be useful for application in bioaugmentation processes towards more efficient removal of contaminants in wastewater treatment plants.     

Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p-xylene mixture

For the complete biodegradation of a mixture of benzene, toluene, and p-xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate-cis-glycol dehydrogenase in the tol pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis-glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis-glycol compounds by the action of toluate-cis-glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p-xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. (c) 1994 John Wiley & Sons, Inc.     

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.     

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.     

Addition of aromatic substrates restores trichloroethylene degradation activity in Pseudomonas putida F1

The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation. The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added. But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity. This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h. The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.     

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.     

Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1

Toluene dioxygenase oxidizes toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. This reaction is catalyzed by a multienzyme system that is induced in cells of Pseudomonas putida F1 during growth on toluene. One of the components of toluene dioxygenase has been purified to homogeneity and shown to be an iron-sulfur protein that has been designated ferredoxinTOL. The molecular weight of ferredoxinTOL was calculated to be 15,300, and the purified protein was shown to contain 2 g of atoms each of iron- and acid-labile sulfur which appear to be organized as a single [2Fe-2S]cluster. Solutions of ferredoxinTOL were brown in color and showed absorption maxima at 277, 327, and 460 nm. A shoulder in the spectrum of the oxidized protein was discernible at 575 nm. Reduction with sodium dithionite or NADH and ferredoxinTOL reductase resulted in a decrease in visible absorbance at 460 and 575 nm, with a concomitant shift in absorption maxima to 382 and 438 nm. The redox potential of ferredoxinTOL was estimated to be -109 mV. In the oxidized state, the protein is diamagnetic. However, upon reduction it exhibited prominent electron paramagnetic resonance signals with anisotropy in g values (gx = 1.81, gy = 1.86, and gz = 2.01). Anaerobic reductive titrations revealed that ferredoxinTOL is a one-electron carrier that accepts electrons from NADH in a reaction that is mediated by a flavoprotein (ferredoxinTOL reductase). The latter is the first component in the toluene dioxygenase system. Reduced ferredoxinTOL can transfer electrons to cytochrome c or to a terminal iron-sulfur dioxygenase (ISP-TOL) which catalyzes the incorporation of molecular oxygen into toluene and related aromatic substrates.     

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.     

Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).     

Biodegradation kinetics of stored wastewater substrates by a mixed microbial culture

The anaerobic accumulation of several organic pollutants from industrial wastewaters, as storage substrates, and their subsequent aerobic biodegradation using a wastewater treatment mixed microbial culture for biological nutrient removal has been studied. The amount and the kinetics of substrate accumulation in the anaerobic stage depended on the characteristics of the wastewater fed to the anaerobic stage. Depending on the substrate used, levels of between 27 and 86% of storage polymers were accumulated with respect to the level obtained on feeding with acetate. The biodegradation kinetics were studied by modelling respirometry results. During the aerobic stage, oxygen-consumption data obtained in the respirometric tests were fitted to a model using a non-linear fitting estimation method. The simulation data obtained correlated well with the experimental oxygen-consumption data. The estimated kinetic parameters obtained indicate that each storage polymer was degraded at a different rate. However, the values obtained for the storage polymer half-saturation coefficient, K_S: 16 mg COD l⁻¹, and for the coefficient for endogenous respiration, b: 0.008 h⁻¹, were similar in all the experiments. The results indicate that each substrate produces the synthesis of a specific storage polymer that is degraded at a different rate.     

A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1

A two-step model is developed for the aerobic biodegradation of benzene,toluene, and p-xylene (BTX) by Pseudomonas putida F1. The model contains three unique features. First, an initial dioxygenation step transforms BTX into their catechol intermediates, but does not support biomassgrowth. Second, the benzene or toluene intermediates are mineralized, which supports biomass synthesis. Third, BTX exhibit competitive inhibition on each other's transformation, while toluene and benzenenoncompetitively inhibit the mineralization of their catechol intermediate. A suite of batch and chemostat experiments is used to systematically measure the kinetic parameters for the two-step transformations and the substrate interactions.     

Biodegradation of benzene, toluene and naphthalene in soil-water slurry microcosms

Aerobic biodegradation of benzene, toluene andnaphthalene was studied in pre-equilibrated soil-waterslurry microcosms. The experiments were designed tosimulate biodegradation at waste sites where sorptionreaches equilibrium before biodegradation becomesimportant. Rates of biodegradation were reduced by thepresence of soil. For example, nearly completenaphthalene biodegradation (1.28 mg/L) by indigenoussoil bacteria occurred within 60 hours in aqueoussolution (soil-free) while it took two weeks todegrade the same amount in the presence of 0.47 kgsoil/L of water. The rate of biodegradation wasobserved to decrease with increasing organic compoundhydrophobicity, soil/water ratio, soil particle size,and soil organic carbon content. These resultsclearly indicate that the rate of biodegradation isaffected by both the extent and rate of sorption. Further analysis suggests that mass transfer couldcontrol the performance of in situ bioremediation forhighly hydrophobic organic contaminants which exhibita large extent of sorption and slow rate ofdesorption.     

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.     

New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites

The influence of toluene concentration on the specific growth rate, cellular yield, specific CO₂, and metabolite production by Pseudomonas putida F1 (PpF1) was investigated. Both cellular yield and specific CO₂ production remained constant at 1.0 ± 0.1 g biomass dry weight (DW) g⁻¹ toluene and 1.91 ± 0.31 g CO₂ g⁻¹ biomass, respectively, under the tested range of concentrations (2–250 mg toluene l⁻¹). The specific growth rate increased up to 70 mg toluene l⁻¹. Further increases in toluene concentration inhibited PpF1 growth, although inhibitory concentrations were far from the application range of biological treatment processes. The specific ATP content increased with toluene concentration up to toluene concentrations of 170 mg l⁻¹. 3-Methyl catechol (3-MC) was never detected in the cultivation medium despite being an intermediary in the TOD pathway. This suggested that the transformation from toluene to 3-MC was the limiting step in the biodegradation process. On the other hand, benzyl alcohol (BA) was produced from toluene in a side chain reaction. This is, to the best of our knowledge, the first reported case of methyl monoxygenation of toluene by PpF1 not harboring the pWW0 TOL plasmid. In addition, the influence of 3-MC, BA, and o-cresol on toluene degradation was investigated respirometrically, showing that toluene-associated respiration was not significantly inhibited in the presence of 10–100 mg l⁻¹ of the above-mentioned compounds.