Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.     

Recovery of trichloroethylene removal efficiency through short-term toluene feeding in a biofilter enriched withPseudomonas putida F1

Trichloroethylene (TCE) is an environmental contaminant provoking genetic mutation and damages to liver and central nerve system even at low concentrations. A practical scheme is reported using toluene as a primary substrate to revitalize the biofilter column for an extended period of TCE degradation. The rate of trichloroethylene (TCE) degradation byPseudomonas putida F1 at 25°C decreased exponentially with time, without toluene feeding to a biofilter column (11 cm I.D.×95 cm height). The rate of decrease was 2.5 times faster at a TCE concentration of 970 μg/L compared to a TCE concentration of 110 μg/L. The TCE itself was not toxic to the cells, but the metabolic intermediates of the TCE degradation were apparently responsible for the decrease in the TCE degradation rate. A short-term (2 h) supply of toluene (2,200 μg/L) at an empty bed residence time (EBRT) of 6.4 min recovered the relative column activity by 43% when the TCE removal efficiency at the time of toluene feeding was 58%. The recovery of the TCE removal efficiency increased at higher incoming toluene concentrations and longer toluene supply durations according to the Monod type of kinetic expression. A longer duration (1.4∼2.4 times) of toluene supply increased the recovery of the TCE removal efficieny by 20% for the same toluene load.     

Cometabolism of trichloroethylene: concepts, limitations and available strategies for sustained biodegradation

Due to its toxicity and persistence in the environment, trichloroethylene (TCE) has become a major soil and groundwater contaminant in many countries. A group of aliphatic- and aromatic-degrading bacteria expressing nonspecific oxygenases have been reported to transform TCE through aerobic cometabolism in the presence of primary substrate such as methane, ammonia, propane, phenol, toluene or cumene. This paper reviews the fundamentals and results of TCE cometabolism from laboratory and field studies. The limitations associated with TCE cometabolism including the causes and effects of substrate and/or inducer utilization rate and depletion, enzyme inhibition and inactivation, and cytotoxicity during TCE oxidation among various TCE-degrading bacteria and enzymes are discussed. In addition, the potential strategies e.g. addition of primary substrate/inducer or external energy substrate, use of a two-stage reactor and application of cell immobilization for sustained TCE degradation are highlighted. The review summarizes important information on TCE cometabolism, which is necessary for developing efficient TCE bioremediation approaches.     

Use of starvation promoters to limit growth and selectively enrich expression of trichloroethylene- and phenol-transforming activity in recombinant Escherichia coli [corrected]

The expression of much useful bacterial activity is facilitated by rapid growth. This coupling can create problems in bacterial fermentations and in situ bioremediation. In the latter process, for example, it necessitates addition of large amounts of nutrients to contaminated environments, such as aquifers. This approach, termed biostimulation, can be technically difficult. Moreover, the resulting in situ bacterial biomass production can have undesirable consequences. In an attempt to minimize coupling between expression of biodegradative activity and growth, we used Escherichia coli starvation promoters to control toluene monooxygenase synthesis. This enzyme complex can degrade the environmental contaminants trichloroethylene (TCE) and phenol. Totally starving cell suspensions of such strains degraded phenol and TCE. Furthermore, rapid conversions occurred in the postexponential batch or very slow growth (dilution) rate chemostat cultures, and the nutrient demand and biomass formation for transforming a given amount of TCE or phenol were reduced by 60 to 90%. Strong starvation promoters have recently been clones and characterized in environmentally relevant bacteria like Pseudomonas species; thus, starvation promoter-driven degradative systems can now be constructed in such bacteria and tested for in situ efficacy.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments

The unsaturated subsurface (vadose zone) receives significant amounts of hazardous chemicals, yet little is known about its microbial communities and their capacity to biodegrade pollutants. Trichloroethylene (TCE) biodegradation occurs readily in surface soils; however, the process usually requires enzyme induction by aromatic compounds, methane, or other cosubstrates. The aerobic biodegradation of toluene and TCE by indigenous microbial populations was measured in samples collected from the vadose zone at unpolluted and gasoline-contaminated sites. Incubation at field moisture levels showed little activity on either TCE or toluene, so samples were tested in soil suspensions. No degradation occurred in samples suspended in water or phosphate buffer solution; however, both toluene and TCE were degraded in samples suspended in mineral salts medium. TCE degradation depended on toluene degradation, and little loss occurred under sterile conditions. Studies with specific nutrients showed that addition of ammonium sulfate was essential for degradation, and addition of other mineral nutrients further enhanced the rate. Additional studies with vadose sediments amended with nutrients showed similar trends to those observed in sediment suspensions. Initial rates of biodegradation in suspensions were faster in uncontaminated samples than in gasolinecontaminated samples, but the same percentages of chemicals were degraded. Biodegradation was slower and less extensive in shallower samples than deeper samples from the uncontaminated site. Two toluene-degrading organisms isolated from a gasoline-contaminated sample were identified as Corynebacterium variabilis SVB74 and Acinetobacter radioresistens SVB65. Inoculation with 10⁶ cells of C. variabilis ml^–1 of soil solution did not enhance the rate of degradation above that of the indigenous population. These results indicate that mineral nutrients limited the rate of TCE and toluene degradation by indigenous populations and that no additional benefit was derived from inoculation with a toluene-degrading bacterial strain. Correspondence to: K.M. Scow     

Effects of iron limitation on the degradation of toluene by Pseudomonas strains carrying the tol (pWWO) plasmid

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.     

Degradation of trichloroethylene by a growth-arrestedPseudomonas putida

A toluene-oxidizing strain ofPseudomonas mendocina KR1 containing toluene-4-mono-oxygenase (TMO) completely degrades TCE with the addition of toluene as a co-substrate in aerobic condition. In order to constructin situ bioremediation system for TCE degradation without any growth-stimulating nutrients or toxic inducers such as toluene, we used the carbon-starvation promoter ofPseudomonas putida MK1 (Kim, Y.et al., J. bacteriol., 1995). Upon entry into the stationary phase due to the deprivation of nutrients, this promoter is strongly induced without further cell growth. The TMO gene cluster (4.5 kb) was spliced downstream of the carbon starvation promoter ofPseudomona putida MK1, already cloned in pUC19. TMO under the carbon starvation promoter was not expressed inE. coli cells either in stationary phase or exponential phase. For TMO expression inPseudomonas strains,tmo and carbon starvation promoter region were recloned into a modified broad-host range vector pMMB67HES which was made from pMMB67HE (8.9 kb) by deletion oftac promoter andlacI ^q (about 1.5 kb). Indigo was produced by TMO under the carbon starvation promoter in aPseudomonas strain of post-exponential phase on M9 (0.2% glucose and 1mM indole) or LB. 18% of TCE was degraded in 14 hours after entering the stationary phase at the initial concentration of 6.6μ M in liquid phase.     

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

Trichloroethylene removal and oxidation toxicity mediated by toluene dioxygenase of Pseudomonas putida.

Whole cells of Pseudomonas putida containing toluene dioxygenase were able to remove all detectable trichloroethylene (TCE) from assay mixtures. The capacity of cells to remove TCE was 77 microM/mg of protein with an initial rate of removal of 5.2 nmol/min/ng of protein. TCE oxidation resulted in a decrease in the growth rate of cultures and caused rapid cell death. Addition of dithiothreitol to assay mixtures increased the TCE removal capacity of cells by up to 67% but did not prevent TCE-mediated cell death. TCE induced toluene degradation by whole cells to a rate approximately 40% of that induced by toluene itself.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil.

The biodegradation of trichloroethylene (TCE) and toluene, incubated separately and in combination, by indigenous microbial populations was measured in three unsaturated soils incubated under aerobic conditions. Sorption and desorption of TCE (0.1 to 10 micrograms ml-1) and toluene (1.0 to 20 micrograms ml-1) were measured in two soils and followed a reversible linear isotherm. At a concentration of 1 micrograms ml-1, TCE was not degraded in the absence of toluene in any of the soils. In combination, both 1 microgram of TCE ml-1 and 20 micrograms of toluene ml-1 were degraded simultaneously after a lag period of approximately 60 to 80 h, and the period of degradation lasted from 70 to 90 h. Usually 60 to 75% of the initial 1 microgram of TCE ml-1 was degraded, whereas 100% of the toluene disappeared. A second addition of 20 micrograms of toluene ml-1 to a flask with residual TCE resulted in another 10 to 20% removal of the chemical. Initial rates of degradation of toluene and TCE were similar at 32, 25, and 18 degrees C; however, the lag period increased with decreasing temperature. There was little difference in degradation of toluene and TCE at soil moisture contents of 16, 25, and 30%, whereas there was no detectable degradation at 5 and 2.5% moisture. The addition of phenol, but not benzoate, stimulated the degradation of TCE in Rindge and Yolo silt loam soils, methanol and ethylene slightly stimulated TCE degradation in Rindge soil, glucose had no effect in either soil, and dissolved organic carbon extracted from soil strongly sorbed TCE but did not affect its rate of biodegradation.     

Toxic effects of toluene on the growth of activated sludge bacteria

Two bacteria were isolated from the activated sludge sample of a wastewater treatment plant in Dublin by enrichment culture technique with toluene as the sole source of carbon and energy. They were identified as Aeromonas caviae (To-4) and Pseudomonas putida (To-5). The growth of these bacteria depended on the manner in which toluene was supplied. In general, growth was better when toluene was supplied in the vapour phase. When toluene was added directly to the growth medium it was found to be toxic to the organisms but the toxic effect could be alleviated in the presence of other carbon sources and by the acclimation of the cells. The growth of To-4 on toluene has never been previously reported.     

Anaerobic degradation of toluene by a denitrifying bacterium.

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Transformation capacities of chlorinated organics by mixed cultures enriched on methane, propane, toluene, or phenol

The degradation of trichloroethylene (TCE), chloroform (CF), and 1,2-dichloroethane (1,2-DCA) by four aerobic mixed cultures (methane, propane, toluene, and phenol oxidizers) grown under similar chemostat conditions was measured. Methane and propane oxidizers were capable of degrading both saturated and unsaturated chlorinated organics (TCE, CF, and 1,2-DCA). Toluene and phenol oxidizers degraded TCE but were not able to degrade CF, 1,2-DCA, or other saturated organics. None of the cultures tested were able to degrade perchloroethylene (PCE) or carbon tetrachloride (CC(4)). For the four cultures tested, degradation of each of the chlorinated organics resulted in cell inactivation due to product toxicity. In all cases, the toxic products were rapidly depleted, leaving no toxic residues in solution. Among the four tested cultures, the resting cells of methane oxidizers exhibited the highest transformation capacities (T(c)) for TCE, CF, and 1,2-DCA. The T(c) for each chlorinated organic was observed to be inversely proportional to the chlorine carbon ratio (Cl/C). The addition of low concentrations of growth substrate or some catabolic intermediates enhanced TCE transformation capacities and degradation rates, presumably due to the regeneration of reducing energy (NADH); however, addition of higher concentrations of most amendments reduced TCE transformation capacities and degradation rates. Reducing energy limitations and amendment toxicity may significantly affect T(c) measurements, causing a masking of the toxicity associated with chlorinated organic degradation. (c) 1995 John Wiley & Sons, Inc.     

Modeling trichloroethylene degradation by a recombinant pseudomonad expressing toluene ortho-monooxygenase in a fixed-film bioreactor

Burkholderia cepacia PR123(TOM23C), expressing constitutively the TCE-degrading enzyme toluene ortho-monooxygenase (Tom), was immobilized on SIRANtrade mark glass beads in a biofilter for the degradation and mineralization of gas-phase trichloroethylene (TCE). To interpret the experimental results, a mathematical model has been developed which includes axial dispersion, convection, film mass-transfer, and biodegradation coupled with deactivation of the TCE-degrading enzyme. Parameters used for numerical simulation were determined from either independent experiments or values reported in the literature. The model was compared with the experimental data, and there was good agreement between the predicted and measured TCE breakthrough curves. The simulations indicated that TCE degradation in the biofilter was not limited by mass transfer of TCE or oxygen from the gas phase to the liquid/biofilm phase (biodegradation limits), and predicts that improving the specific TCE degradation rates of bacteria will not significantly enhance long-term biofilter performance. The most important factors for prolonging the performance of biofilter are increasing the amount of active biomass and the transformation capacity (enhancing resistance to TCE metabolism). Copyright 1998 John Wiley & Sons, Inc.     

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.     

Effect of trichloroethylene (TCE) and toluene concentrations on TCE and toluene biodegradation and the population density of TCE and toluene degraders in soil.

Toluene is one of several cosubstrates able to support the cometabolism of trichloroethylene (TCE) by soil microbial communities. Indigenous microbial populations in soil degraded TCE in the presence, but not the absence, of toluene after a 60- to 80-h lag period. Initial populations of toluene and TCE degraders ranged from 0.2 x 10(3) to 4 x 10(3) cells per g of soil and increased by more than 4 orders of magnitude after the addition of 20 micrograms of toluene and 1 microgram of TCE per ml of soil solution. The numbers of TCE and toluene degraders and the percent removal of TCE increased with an increase in initial toluene concentration. As the initial TCE concentration was increased from 1 to 20 micrograms/ml, the numbers of toluene and TCE degraders and the rate of toluene degradation decreased, and no TCE degradation occurred. No toluene or TCE degradation occurred at a TCE concentration of 50 micrograms/ml.     

Impact of trichloroethylene and toluene on nitrogen cycling in soil.

The effects of trichloroethylene (TCE) and toluene on soil nitrogen-cycling activities were examined. Ammonium oxidation potential (AOP) was reduced after incubation with as little as 1 microgram of TCE ml-1, and the effects were generally greater when toluene was present and increased with longer exposure. Arginine ammonification potential and denitrification enzyme activity were constant regardless of TCE concentration or the presence of toluene, while nitrite oxidation potential (NOP) exhibited variable sensitivity. KCl-extractable ammonium levels increased dramatically after exposure to 30 and 60 micrograms of TCE ml-1 in the presence of toluene, whereas gamma-irradiated or sodium azide-treated soil incubated with the same concentrations of TCE and toluene showed no increase. Alfalfa-amended soils showed similar decreases in AOP and increases in extractable ammonium during incubation with 60 micrograms of TCE ml-1 and 20 micrograms of toluene ml-1, although most probable number estimates of the ammonium oxidizer population showed no difference between exposed and unexposed soil. AOP and extractable ammonium returned slowly to control levels after 28 days of incubation in the presence of TCE and toluene. Activity assays to which various TCE and toluene concentrations were added indicated that AOP and NOP were relatively more sensitive to these compounds than was arginine ammonification potential. These results indicate that the soil microbial populations responsible for nitrogen cycling exhibit different sensitivities to TCE and toluene and that they may be more susceptible to adverse effects than previously thought.     

Effect of ethanol, acetate, and phenol on toluene degradation activity and tod-lux expression in Pseudomonas putida TOD102: evaluation of the metabolic flux dilution model

The reporter strain Pseudomonas putida TOD102 (with a tod-lux fusion) was used in chemostat experiments with binary substrate mixtures to investigate the effect of potentially occurring cosubstrates on toluene degradation activity. Although toluene was simultaneously utilized with other cosubstrates, its metabolic flux (defined as the toluene utilization rate per cell) decreased with increasing influent concentrations of ethanol, acetate, or phenol. Three inhibitory mechanisms were considered to explain these trends: (1) repression of the tod gene (coding for toluene dioxygenase) by acetate and ethanol, which was quantified by a decrease in specific bioluminescence; (2) competitive inhibition of toluene dioxygenase by phenol; and (3) metabolic flux dilution (MFD) by all three cosubstrates. Based on experimental observations, MFD was modeled without any fitting parameters by assuming that the metabolic flux of a substrate in a mixture is proportional to its relative availability (expressed as a fraction of the influent total organic carbon). Thus, increasing concentrations of alternative carbon sources "dilute" the metabolic flux of toluene without necessarily repressing tod, as observed with phenol (a known tod inducer). For all cosubstrates, the MFD model slightly overpredicted the measured toluene metabolic flux. Incorporating catabolite repression (for experiments with acetate or ethanol) or competitive inhibition (for experiments with phenol) with independently obtained parameters resulted in more accurate fits of the observed decrease in toluene metabolic flux with increasing cosubstrate concentration. These results imply that alternative carbon sources (including inducers) are likely to hinder toluene utilization per unit cell, and that these effects can be accurately predicted with simple mathematical models.     

Kinetics of BTEX biodegradation by a coculture of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> under hypoxic conditions

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500mgl^–1 of benzene, 600mgl^–1 of o-xylene, and 1000mgl^–1 of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO₂ and H₂O without producing any identifiable intermediate metabolites.