Cometabolic Degradation of Trichloroethene by Rhodococcus sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils

The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 μM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.Various bacteria have been reported to degrade trichloroethene (TCE) aerobically via cometabolic degradation with broad-substrate-specificity enzymes (2). However, TCE cometabolic degradation is considered an unsustainable process due to cytotoxicity, inhibition, or inactivation of TCE-degrading enzymes. These phenomena have been observed in studies using both whole cells and purified enzymes, including soluble methane monooxygenases from Methylosinus trichosporium OB3b (9) and Nitrosomonas europaea (13), toluene 2-monooxygenase from Burkholderia cepacia G4 (19, 27), toluene dioxygenase (TDO) from Pseudomonas putida F1 (15, 18), and butane-oxidizing bacteria, i.e., Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 (11). Nevertheless, the addition of an inducer or growth substrate can maintain TCE cometabolic degradation. For example, the TCE-degrading activity of P. putida F1 toluene dioxygenase was restored after adding benzene, cumene, or toluene to displace TCE and its reactive intermediates from the enzyme active site (18). Arp et al. (2) suggested that the rate of enzyme maintenance and recovery depended on the extent of inactivation and the balance of TCE and inducer/growth substrate concentrations.Plant essential oils and their components, such as citral, limonene, cumene, and cumin aldehyde, have been found to induce TCE degradation in Rhodococcus sp. L4 (24). However, the removal of TCE by this bacterium was effective only for a short period. The impacts of TCE on Rhodococcus spp. and their enzymes have not been studied in detail, even though many bacteria of this genus exhibited high TCE-degrading activities (i.e., Rhodococcus erythropolis JE 77, R. erythropolis BD2, Rhodococcus sp. Sm-1, and Rhodococcus sp. Wrink) (5, 6, 7, 16). This study therefore investigated the changes in TCE-degrading activity of Rhodococcus sp. L4 cells and TDO during exposure to TCE. Two enzyme maintenance approaches were evaluated, namely, repeated addition of essential oil components to the system and immobilization of the bacterial cells on plant material rich in essential oils. Immobilized microorganisms are generally capable of degrading pollutants at a higher initial concentration and for a longer period than those of free cells (21, 23), possibly because the microbial cells are protected from environmental stress and toxic compounds (3). In this study, the plant materials were used to provide a solid surface for bacterial attachment and a continuous source of essential oils for inducing TCE-degrading enzymes. Our results show that the repeated addition of limonene, cumene, or cumin aldehyde enhances TCE degradation and that bacteria immobilized on cumin seeds are able to maintain their TCE-degrading activity.     

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.     

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.     

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.     

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]⁻¹ h⁻¹). 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.     

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     

Isopropylbenzene (cumene) — a new substrate for the isolation of trichloroethene-degrading bacteria

Various bacterial isolates from enrichments with isopropylbenzene (cumene), toluene or phenol as carbon and energy sources were tested as to their potential to oxidize trichloroethene (TCE). In contrast to toluene and phenol, all isolates enriched on isopropylbenzene were able to oxidize TCE. Two isolates, strain JR1 and strain BD1, were identified as Pseudomonas spec. and as Rhodococcus erythropolis, respectively. TCE oxidation was accompanied by the liberation of stoichiometric amounts of chloride. Initial TCE oxidation rate increased proportional to the substrate concentration from 25 to 200 M TCE. Maximal initial TCE-degradation rates found here were 4 to 5 nmol · min^-1 · mg protein^-1. The TCE degradation rate decreased with time. The two isolates showed a temperature optimum for TCE degradation between 10 and 20 °C. In addition to TCE, R. erythropolis BD1 degraded only cis- and trans-dichloroethene whereas Pseudomonas spec. JR1 was able to oxidize also 1,1-dichloroethene, vinyl chloride, trichloroethane, and 1,2-dichloroethane.Abbrevations DMF dimethylformamide - TCE trichloroethene     

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.     

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.     

Cytotoxicity associated with trichloroethylene oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)) lost 95% of their acetate-dependent O(2) uptake activity (a measure of general respiratory activity), yet toluene-dependent O(2) uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 micromol (mg of cells(-1)) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)), up to 90% were Tol(-) variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.     

Toxicity of plant essential oils and their components against Lycoriella ingenua (Diptera: Sciaridae)

Plant essential oils from 20 plant species were tested for their insecticidal activity against larvae of Lycoriella ingenua (Dufour) (Diptera: Sciaridae) by using a fumigation bioassay. Good insecticidal activity (>90%) against larvae of L. ingenua was achieved with essential oils of caraway seed Carum carvi (L.)], lemongrass [Cymbopogon citratus (D.C.) Stapf.], mandarine (Citrus reticulate Blanco), nutmeg (Myristica fragrans Houtt), cade (Juniperus oxycedrus L.), spearmint (Mentha spicata L.), cumin (Cuminum cyminum L.), and thyme red [Thymus vulgaris (L.)] oils at 30 X 10-3 mg/1 air. Among them, caraway seed, spearmint, cumin, and thyme red essential oils were highly effective against L. ingenua at 20 x 10(-3) mg/ml air. Analysis by gas chromatography-mass spectrometry led to identification of 4, 9, 8, and 17 compounds from caraway seed, spearmint, cumin, and thyme red oils, respectively. These compounds were tested individually for their insecticidal activities against larvae of L. ingenua, and compared with the toxicity of dichlorvos. Carvacrol, thymol, linalool, cuminaldehyde, p-cymen, terpinen-4-ol, and carvone was effective at 10 x 10(-3) mg/l. The insecticidal activity of dichlorvos was 60% at 10 x 10(-3) mg/ml. Effects of four selected plant essential oils on growth of oyster mushroom, Pleurotus ostreatus, also were investigated.     

Correlation of TCE cometabolism with growth characteristics on aromatic substrates in toluene-degrading bacteria

We have constructed a bacterial library consisting of 97 strains of toluene-degrading bacteria from soil and activated sludge samples for examining their physiological properties in terms of cometabolism of TCE. Large variation of TCE degradation ability was observed in Gram-positive and Gram-negative strains, as well as diverse patterns of availability of aromatics as a growth substrate. No clear correlation was observed between the number of available substrates for growth and TCE degradation ability. However, the growth on some of the aromatics showed positive or negative correlations with TCE degradation ability. Kendall correlation constants (tau) for the growth on cumene, m-xylene, p-xylene, and m-cresol with TCE degradation ability were statistically significant (P < 0.001): their values were −0.44, −0.31, −0.26, 0.37, respectively. Among 12 of aromatics, only m-cresol showed positive correlation with TCE degradation ability. These findings would be useful for enrichment and isolation of the microbes that have TCE-cometabolism ability, which is a selective disadvantage through the toxicity or competitive inhibition against growth substrates.     

Phenol- and Toluene-Degrading Microbial Populations from an Aquifer in Which Successful Trichloroethene Cometabolism Occurred

We characterized the bacterial populations that grew in a Moffett Field, Calif., aquifer following three sequential field tests of phenol- or toluene-driven cometabolism of trichloroethene (TCE). Reducing the toluene and phenol concentrations in most-probable-number (MPN) tubes from 50 to 5 ppm increased the population density measured for these degraders by 1.5 and 1 log units, respectively, suggesting that natural populations might be quite sensitive to these substrates. Phenol and toluene degraders were isolated from the terminal MPN dilution tubes; 63 genetically distinct strains were identified among the 273 phenol- and toluene-degrading isolates obtained. TCE was cometabolized by 60% of the genetically distinct strains. Most strains (57%) grew on both phenol and toluene, and 78% of these strains hybridized to the toluene ortho-monooxygenase (TOM) probe. None of the strains hybridized to probes from the four other toluene oxygenase pathways. Gram-positive strains comprised 30% of the collection; all of these grew on phenol, and 47% of them also grew on toluene, but none hybridized to the TOM probe. Among the gram-negative strains, 86% of those that grew on both toluene and phenol hybridized to the TOM probe, while only 5% of those that were TOM-positive grew on toluene alone. A larger proportion of TCE degraders was found among gram-negative than gram-positive strains and among organisms that grew on phenol than those that grew on toluene. Hybridization of strains to the TOM probe was somewhat predictive of their TCE-cometabolizing ability, especially for strains isolated on toluene, but there was also a significant number (20%) of strains that hybridized to the TOM probe but were poor TCE cooxidizers. No Moffett Field isolates were as effective as Burkholderia cepacia G4 in cooxidizing TCE. Most of the aquifer strains ranged from moderately effective to ineffective in TCE cooxidation. Such populations, however, apparently accounted for the successful phenol- and toluene-stimulated TCE removal that occurred during the field assessment of this remediation process. This suggests that naturally occurring communities of only moderate TCE-cooxidizing ability may support successful TCE bioremediation as long as the phenol or toluene present is not limiting. This activity, however, may not be sustainable for the long term, because TCE-inactive populations that consumed toluene at rates equal to that of the best TCE degraders were present and hence would be expected to eventually dominate the community.     

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

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.     

A Gas Lift Bioreactor for Removal of Contaminants from the Vapor Phase

The cometabolic degradation of trichloroethylene (TCE) as a vapor by two aromatic-metabolizing pseudomonads was evaluated in an airlift reactor. These microorganisms were able to degrade 90 to 95% of TCE in air at concentrations at the reactor inlet of 300 to 4,000 μg/liter. Although exposure of the cells to high inlet concentrations of TCE (4 mg/liter) caused a decline in enzyme-specific activity and TCE removal efficiency, this loss in activity could be prevented or delayed by increasing the rate of cosubstrate addition. Under the appropriate operating conditions, the microorganisms were able to degrade even high concentrations of TCE and activity of the cells in the reactor could be maintained for periods of at least 2 weeks.     

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.     

Methyl tert-butyl ether biodegradation by microbial consortia obtained from soil samples of gasoline-polluted sites in Mexico

Microbial consortia obtained from soil samples of gasoline-polluted sites were individually enriched with pentane, hexane, isooctane and toluene. Cometabolism with methyl tert-butyl ether, (MTBE), gave maximum degradation rates of 49, 12, 32 and 0 mg g(-1)protein h(-1), respectively. MTBE was fully degraded even when pentane was completely depleted with a cometabolic coefficient of 1 mgMTBE mg(-1)pentane. The analysis of 16S rDNA from isolated microorganisms in the pentane-adapted consortia showed that microorganisms could be assigned to Pseudomonas. This is the first work reporting the cometabolic mineralization of MTBE by consortium of this genus.