The objectives of this paper are to review the wide range of kinetic models that have been introduced to describe the cometabolic oxidation of chlorinated solvents, to compare modeling approaches and associated experimental data, and to discuss knowledge gaps in the general topic of cometabolism kinetics. To begin, a brief description of the mechanism of oxygenase enzyme metabolism and its qualitative effects on cometabolic degradation kinetics is given. Next, a variety of kinetic expressions that have been used to describe cometabolism, ranging from adaptations of simple metabolic relationships to the development of complex equations that account for intracellular concentrations of key reaction species, are presented. A large number of kinetic coefficients published for a variety of oxygenase populations degrading a broad range of chlorinated solvents are categorized and compared. The discussion section of the paper contains an exploration of knowledge gaps that exist in our understanding of the kinetics of aerobic chlorinated solvent cometabolism. Specific topics covered include: • the use of half saturation constants (Ksc and Ksg) as estimates for inhibition constants (Kisc and Kisg) in saturation modeling expressions, • the specific nature of chlorinated solvent induced product toxicity and the capability for cells to recover from toxic effects, and • methods for incorporating reducing energy limitations into cometabolism models. Finally, the applicability of the broad range of kinetic modeling approaches to scale-up and field applications for in situ bioremediation of chlorinated solvents is discussed.

Kinetics of aerobic cometabolism of chlorinated solvents.

Cometabolism recognizes that microorganisms can transform non-growth-supporting substrates. The term "cometabolism" was first introduced over 30 years ago and has been redefined, criticized, and used widely ever since. In this review we have examined the aerobic cometabolism of chlorinated solvents, with a particular emphasis on the cometabolism of trichloroethylene. Monooxygenases or dioxygenases with relaxed substrate ranges initiate these transformations. The physiological role of the oxygenases is to initiate the metabolism of growth-supporting substrates (e.g., methane, propane, butane, toluene, ethylene, and ammonia). Diverse enzymes catalyze these oxidative reactions with chlorinated solvents. Synthesis of most of these enzymes is induced by the presence of the growth-supporting substrate and is largely regulated at the level of gene transcription. The genes that code for a given oxygenase are usually clustered together in a single operon and often share homology with counterparts that code for the subunits of related oxygenases in other bacteria. During cometabolism the growth-supporting and non-growth-supporting substrates can both bind to the oxygenase. Transformation of chlorinated solvents by these enzymes presents the cell with a new set of compounds. Some of these compounds are toxic to the cells, others are stable products that are expelled from the cell, and in a few cases the cells utilize the products. The combined effects of cometabolism can have a profound influence on a cell.

Molecular and cellular fundamentals of aerobic cometabolism of trichloroethylene.

Tetrachloroethylene (PCE) is thought to have no natural source, so it is one of the most difficult contaminants to degrade biologically. This common groundwater pollutant was thought completely nonbiodegradable in the presence of oxygen. Here we report that the wastewater bacterium Pseudomonas stutzeri OX1 degrades aerobically 0.56 μmol of 2.0 μmol PCE in 21 h (Vmax ≈ 2.5 nmol min−1 mg−1 protein and KM ≈ 34 μM). These results were corroborated by the generation of 0.48 μmol of the degradation product, chloride ions. This degradation was confirmed to be a result of expression of toluene-o-xylene monooxygenase (ToMO) by P. stutzeri OX1, since cloning and expressing this enzyme in Escherichia coli led to the aerobic degradation of 0.19 μmol of 2.0 μmol PCE and the generation of stoichiometric amounts of chloride. In addition, PCE induces formation of ToMO, which leads to its own degradation in P. stutzeri OX1. Degradation intermediates reduce the growth rate of this strain by 27%.

Aerobic degradation of tetrachloroethylene by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1.

The degradation of trichloroethylene (TCE) by toluene-oxidizing bacteria has been extensively studied, and yet the influence of environmental conditions and physiological characteristics of individual strains has received little attention. To consider these effects, the levels of TCE degradation by strains distinguishable on the basis of toluene and nitrate metabolism were compared under aerobic or hypoxic conditions in the presence and absence of nitrate and an exogenous electron donor, lactate. Under aerobic conditions with toluene-induced cells, strains expressing toluene dioxygenases (Pseudomonas putida F1, Pseudomonas sp. strain JS150, Pseudomonas fluorescens CFS215, and Pseudomonas sp. strain W31) degraded TCE at low rates, with less than 12% of the TCE removed in 18 h. In contrast, strains expressing toluene monooxygenases (Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1) degraded 36 to 67% of the TCE over the same period. Under hypoxic conditions (1.7 mg of dissolved oxygen per liter) or when lactate was added as an electron donor, the extent of TCE degradation by toluene-induced cells was generally lower. In the presence of lactate, degradation of TCE by denitrifying strain PKO1 was enhanced by nitrate under conditions in which dissimilatory nitrate reduction was observed. The results of experiments performed with strains F1, G4, PKO1, and KR1 suggested that TCE or an oxidation product induces toluene degradation and that TCE induces its own degradation in the monooxygenase strains. The role of TCE as an inducer of toluene oxygenase activity in PKO1 was confirmed by performing a promoter probe analysis, in which we found that TCE activates transcription from the PKO1 3-monooxygenase operon promoter.

Comparison of factors influencing trichloroethylene degradation by toluene-oxidizing bacteria.

Contamination by chloroethenes has a severe negative effect on both the environment and human health. This has prompted intensive remediation activity in recent years, along with research into the efficacy of natural microbial communities for degrading toxic chloroethenes into less harmful compounds. Microbial degradation of chloroethenes can take place either through anaerobic organohalide respiration, where chloroethenes serve as electron acceptors; anaerobic and aerobic metabolic degradation, where chloroethenes are used as electron donors; or anaerobic and aerobic co-metabolic degradation, with chloroethene degradation occurring as a by-product during microbial metabolism of other growth substrates, without energy or carbon benefit. Recent research has focused on optimising these natural processes to serve as effective bioremediation technologies, with particular emphasis on (a) the diversity and role of bacterial groups involved in dechlorination microbial processes, and (b) detection of bacterial enzymes and genes connected with dehalogenation activity. In this review, we summarise the different mechanisms of chloroethene bacterial degradation suitable for bioremediation and provide a list of dechlorinating bacteria. We also provide an up-to-date summary of primers available for detecting functional genes in anaerobic and aerobic bacteria degrading chloroethenes metabolically or co-metabolically.

Microbial degradation of chloroethenes: a review

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.

S Heald

Papers

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