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Applied and Environmental Microbiology, May 2004, p. 2830-2835, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2830-2835.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in Pseudomonas putida F1

Yuki Morono, Hajime Unno, Yasunori Tanji, and Katsutoshi Hori^*

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan

Received 26 October 2003/ Accepted 27 January 2004

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Trichloroethylene (TCE), a suspected human carcinogen, has been used extensively in commercial applications. Because of its widespread industrial use and improper disposal, TCE has become one of the most widespread groundwater contaminants. Aerobic degradation of TCE by bacteria via cometabolism, in which nonspecific oxygenases catalyze the initial transformation of this chemical, has been studied extensively; however, cytotoxicity of the degradation products results in the cessation of TCE degradation, which is a major drawback in the practical application of cometabolic TCE degradation to bioremediation.

As a result of TCE transformation, inactivation of methane monooxygenase and toluene 2-monooxygenase has been observed both in vitro (5, 8) and in vivo (9, 20). Although the exact nature of the destructive species remains unknown, highly reactive intermediates such as 2,2,2-trichloroacetaldehyde (chloral) and TCE-epoxide have been identified by in vitro experiments with methane monooxygenase (5) and toluene 2-monooxygenase (8). Furthermore, the degradation of TCE has also been reported to affect, both adversely and nonspecifically, more basic and general cellular functions such as respiratory and growth activities (3, 11, 15, 17, 20).

Owing to inactivation effects, the capacity of bacterial cells to degrade TCE is limited. Since Alvarez-Cohen and McCarty (2) first defined the term, the transformation capacity (Tc), which represents the maximum mass of cometabolized compounds that can be transformed per unit mass of resting cells, has been used as a scale to measure the TCE degradation ability of bacterial strains. The reported range of Tc values for pure wild-type strains varies from 0.05 to 2 µmol mg of cells^–1 (1, 6, 9, 12, 20), suggesting that there is diversity in the susceptibility of cells to damage by TCE degradation. For example, after oxidizing a quantity of TCE corresponding to the Tc, Methylosinus trichosporium OB3b cells did not recover even after 7 days of incubation (3). However, TCE degradation does not necessarily cause crucial damage that results in cell death in all bacterial strains. In some strains, the DNA repair mechanism (19) or de novo protein synthesis (11) can mediate the recovery of cellular activity after TCE degradation.

In this report, we show that the cessation of TCE degradation in Pseudomonas putida F1 is not the result of cytotoxic TCE degradation products. Almost no loss of toluene oxidation activity in the strain F1 was observed even though TCE degradation ceased, and TCE degradation activity could be restored simply by adding aromatic substrates to cultures. This restoration phenomenon can greatly improve the capacity of P. putida F1 to degrade TCE.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strain and culture conditions.
P. putida F1 was kindly provided by David Gibson (University of Iowa) and was maintained on basal salt (BS) (7) agar plates supplied with toluene vapor (0.2 mmol liter^–1). For the preparation of seed culture, a colony of P. putida F1 was inoculated into BS medium supplied with toluene as the sole carbon source and incubated overnight at 28°C.

For the preparation of toluene dioxygenase (TDO)-induced cell suspensions, 0.6 ml of the seed culture was inoculated into 60 ml of BS medium in a 300-ml flask with a side arm. Toluene vapor was supplied from a piece of cotton wool in the side arm of the flask that had soaked up 50 µl of toluene. Noninduced cell suspensions were prepared in the same manner, except that the carbon source was replaced by arginine (0.2%, wt/vol). After overnight incubation at 100 rpm and 28°C, the TDO-induced culture was supplied with another 50 µl of toluene and incubated for another 3 h to ensure adequate induction of the enzyme.

Cells were harvested by centrifugation (4,000 x g) at 28°C for 8 min and washed with nitrogen-free BS (BS-N) medium, prepared by replacing the (NH₄)₂SO₄ in BS medium with an equimolar amount of K₂SO₄. Finally, the washed cells were resuspended in BS-N medium at a final concentration of 20 mg of dry cells ml^–1.

Degradation experiments.
To avoid cell growth during a degradation assay, we prepared resting cells by suspending the cells in BS-N medium. Assays for the degradation of TCE and aromatic chemicals by TDO-induced and noninduced P. putida were conducted in 123-ml vials containing 19.5 ml of BS-N medium and sealed with Teflon-lined butyl rubber septa. When necessary, chloramphenicol was added to the vials at a final concentration of 0.17 mg ml^–1 to inhibit de novo protein synthesis. This concentration of chloramphenicol was experimentally confirmed to inhibit cellular growth effectively. Prior to cell inoculation, aromatic chemicals or an aqueous solution saturated with TCE was added to the vials. The vials were then incubated at 28°C with shaking at 100 rpm for at least 1 h to attain equilibration of the volatile chemicals between the gas and liquid phases. Degradation reactions were initiated by using a microsyringe to inject an aliquot of concentrated P. putida F1 grown on toluene or arginine into the vials. The optical density at 660 nm of the reaction mixture containing the cells was adjusted to 1.5 for TCE degradation, 0.5 for toluene degradation, or 0.1 for degradation of a mixture of toluene, benzene, and cumene. Periodically, 50 µl of the headspace gas was analyzed by gas chromatography (GC) as described below. The abiotic loss of substrates was confirmed to be less than 5% of the initial values by monitoring control vials incubated in the absence of bacterial cells.

Analytical methods.
TCE was analyzed by a GC system equipped with an electron capture detector and a DB-624 capillary column (30 m x 0.32 mm inside diameter; Agilent Technologies). Other hydrocarbons were analyzed by a GC system equipped with a flame ionization detector and a ULBON HR-1 capillary column (50 m x 0.25 mm inside diameter; Shinwa Chemical Industries, Kyoto, Japan). The aqueous concentrations of TCE and toluene in BS-N medium at 28°C were calculated using dimensionless Henry's constants of 0.447 and 0.315, respectively, which were experimentally determined.

Assays for the activity of TDO, uptake of O₂ in whole cells, and toluene degradation in the presence of catechols.
To investigate the effect of the addition of catechols, indigo formation, the uptake of O₂, and toluene degradation were measured in whole cells in the presence of 3-methylcatechol or catechol. As an indicator of the activity of TDO in whole cells, the formation of indigo from indole was determined (18). Toluene-grown cells were harvested by centrifugation (8 min at 4,000 x g at 28°C), washed, and resuspended in phosphate buffer solution (pH = 7.0). The enzyme reaction was initiated by the addition of indole. Fifteen microliters of 100 mM indole in N,N-dimethylformamide was added to 3 ml of the cell suspension, and the formation of indigo was monitored spectrophotometrically at 600 nm over the reaction time against a cell suspension without indole. The concentration of indigo was calculated by using a molar extinction coefficient at 600 nm of 3,530 cm^–1 M^–1, which was experimentally determined. The initial rate of indigo formation was determined by plotting the increase in indigo concentration as a function of time.

As an indicator of basic cellular activity independent of toluene metabolism, arginine-dependent O₂ uptake rates were analyzed with a Clark-type O₂ electrode (HACH Company) mounted in an Erlenmeyer flask (50 ml). Arginine-grown cells were harvested by centrifugation (8 min at 4,000 x g at 28°C), washed, and resuspended in BS-N medium. The reaction flask was filled with BS-N medium, and cells were added with arginine (to a final concentration of 10 mM) to determine the arginine-stimulated O₂ uptake rate. From this rate, a basal respiratory rate, which was measured in the absence of arginine with cells pregrown on arginine, was subtracted to obtain the arginine-dependent O₂ uptake rate.

Toluene degradation activity was measured as described above. To analyze the effect of the addition of 3-methylcatechol or catechol on each of these activities, the catechols were added to the reaction solution, except in control reactions.

Viable cell count.
Viable cells were assessed by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which specifically stains respiring cells (13). To a sterile tube containing 900 µl of cell suspension, 100 µl of 50 mM CTC staining solution and 10 µl of 100 g liter^–1 arginine solution were added and then mixed. The tube was incubated with shaking in the dark at 28°C for 2 h. The stained cells were then appropriately diluted with BS-N medium and trapped on a black polycarbonate membrane filter with 0.22-µm pores. The membrane filter was air dried and transferred to a slide glass, and then one drop of immersion oil was put onto the preparation before it was covered with a coverslip. Viable cells stained with bright intracellular red formazan (CTC stain) were enumerated using a fluorescence microscope equipped with a polarizing filter (U-MWBV; band-pass, 400 to 440 nm; Olympus Optical, Tokyo, Japan).

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Restoration of TCE degradation by toluene addition.
As in previous papers (6, 16), the rate of TCE degradation by resting cells of P. putida F1 gradually decreased and eventually stopped within 1.5 h (Fig. 1a). This loss of activity has been considered to be due to bacterial cell damage caused by the oxidation products of TCE. To confirm that the cells had lost their toluene-metabolizing activity, 9.4 µmol of toluene was added to the culture after the cessation of TCE degradation. Unexpectedly, the added toluene was degraded immediately without a lag period (Fig. 1b). Furthermore, immediately after the toluene was exhausted, degradation of TCE restarted at a rate similar to the first degradation (Fig. 1a).

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FIG. 1. Time course of TCE (a) and toluene (b) degradation by P. putida F1. Cells (optical density of 1.5 at 660 nm) were incubated at 28°C. TCE degradation with no toluene addition is represented by diamonds. At 1.7 h, toluene was added (indicated by an arrow) to vials without (squares) and with (triangles) chloramphenicol (0.17 mg ml–1). Circles represent the abiotic control. All data displayed are the means (symbols) ± standard deviations (error bars) obtained from three independent experiments, although some error bars are hidden under key symbols.

The immediate degradation of the added toluene without a lag period and the subsequent degradation of TCE, at a rate similar to the first degradation, by resting cells of P. putida F1 in nitrogen-free medium do not seem to be due to de novo protein synthesis. The existence of a lag period for synthesizing TDO was confirmed in toluene degradation experiments using toluene-induced and noninduced cells. As shown in Fig. 2, the toluene-induced cells consumed the added toluene rapidly. In contrast, the noninduced cells grown on arginine showed a lag period before toluene degradation began. Therefore, the rapid degradation of toluene without a lag period seen in Fig. 1 precludes the possibility that the restoration of TCE degradation by strain F1 was due to newly synthesized TDO. In addition, we stopped protein synthesis by adding chloramphenicol to the reaction mixture containing the resting cells. The same profiles of both toluene degradation without a lag period and restoration of TCE degradation were observed even in the presence of chloramphenicol (Fig. 1). These results clearly show that TCE degradation does not affect the toluene oxidation activity of TDO, and TDO molecules that are present before the addition of toluene are responsible for the immediate degradation of the added toluene. Because restoration of nearly 100% of the initial TCE degradation activity was observed, the loss of TCE transformation activity after a short period of TCE degradation can be considered as "pseudoinactivation."

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FIG. 2. Time course of toluene degradation by TDO-induced and noninduced resting cells. TDO-induced cells pregrown on toluene (diamonds) and noninduced cells pregrown on arginine (squares) were incubated (optical density of 0.5 at 660 nm) in nitrogen-free medium at 28°C with shaking at 100 rpm. Circles represent the abiotic control. All data displayed are the means (symbols) ± standard deviations (error bars) obtained from three independent experiments, although some error bars are hidden under key symbols.

Effect of other substrates on the restoration of TCE degradation.
We tested whether other chemicals can restore the degradation of TCE by P. putida F1. Figure 3 shows the amounts of TCE degraded by equimolar (10 µmol) amounts of chemicals added once TCE degradation had ceased. Arginine, which is a typical growth substrate for the cultivation of P. putida F1, did not restore any TCE degradation activity. In addition, tryptophan and phenylalanine, which induce the TCE degradation pathway in Burkholderia cepacia G4 and Ralstonia pickettii PKO1, respectively, showed no TCE degradation restoration, although they sustained the growth of P. putida F1 (data not shown). Furthermore, typical NADH-producing substrates such as citrate and formate (14) did not restore activity, although these substrates were consumed by the bacteria, as confirmed by high-performance liquid chromatography and increased oxygen consumption (data not shown). These results imply that the restoration of TCE degradation activity cannot only be due to NADH or energy supplied to the cells by the added toluene, although NADH is required as an electron donor for TDO-mediated catalysis (4).

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FIG. 3. Restorability of TCE degradation by various chemicals. The amount of TCE degraded after the addition of 10 µmol of each chemical is indicated. All data displayed are the means (symbols) ± standard deviations (error bars) obtained from three independent experiments. TMB, 1,3,5-trimethylbenzene.

Among aromatic compounds, benzene and cumene, which are both substrates of TDO although only benzene can sustain the growth of P. putida F1 as the sole carbon source, showed high restorability of TCE degradation activity: benzene restored TCE degradation to a level similar to that of toluene, whereas cumene showed more than 1.5 times the restorability of toluene (Fig. 3). Like toluene, benzene and cumene were degraded immediately after they were added without a lag period (Fig. 4). The degradation rate of cumene was slower than that of toluene, whereas benzene was degraded at a rate similar to toluene degradation. Degradation of TCE restarted before benzene and cumene had been completely exhausted. When little of the added benzene remained, degradation of TCE restarted at a slower rate than was observed after the addition of toluene. When cumene was added, degradation of TCE restarted immediately after cumene addition and proceeded simultaneously with the degradation of cumene. Cumene restarted the degradation of TCE at a slower rate compared with that of toluene or benzene; however, the TCE degradation activity was sustained for a longer period until the added cumene was completely exhausted, resulting in the complete degradation of all TCE contained in the vial bottle. Therefore, the high restorability of cumene, which is shown as TCE degraded by activity restoration in Fig. 3, may actually be an underestimate.

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FIG. 4. Time course of TCE (a) and toluene, benzene, and cumene (b) degradation by P. putida F1. Cells (optical density of 1.5 at 660 nm) were incubated at 28°C. TCE degradation with no addition of aromatics is represented by circles. At 1.8 h (arrow), 10 µmol of toluene (squares), benzene (triangles), or cumene (diamonds) was added to vials. All data displayed are the means (symbols) ± standard deviations (error bars) obtained from three independent experiments, although many error bars are hidden under key symbols.

The restart time of TCE degradation and the amount of TCE degraded after the addition of toluene, benzene, and cumene reflect the relative affinity of TDO for these aromatics, which was deduced, from the degradation profiles of a mixture, to follow the order cumene < benzene < toluene (Fig. 5). Due to the high affinity of TDO for toluene, TCE degradation can restart only after toluene is exhausted; in addition, because the added toluene is exhausted before the degradation of TCE restarts, the cycle of pseudoinactivation and restoration goes round only once. In the case of the low-affinity substrate cumene, degradation of TCE can restart simultaneously with cumene degradation, and therefore the pseudoinactivation and restoration can go through many cycles. In contrast, 1,3,5-trimethylbenzene, which is not a substrate of TDO, showed almost no restoration of TCE degradation (Fig. 3).

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FIG. 5. Time course of the degradation of toluene (squares), benzene (diamonds), and cumene (triangles) by P. putida F1 in a mixture of the three chemicals. Cells (optical density of 0.1 at 660 nm) were incubated at 28°C. All data displayed are the means (symbols) ± standard deviations (error bars) obtained from three independent experiments, although some error bars are hidden under key symbols.

A possible explanation of our results is that some of the TCE degradation products, or TCE itself, might remain in the active site of TDO during the TCE degradation process, thereby inactivating the enzyme; subsequently, aromatic substrates such as toluene, benzene, and cumene could then displace these inhibitors from the active site of TDO, thereby restoring enzyme activity. Because the number of TDO molecules released from this pseudoinactivation at any one time would be affected by the affinity of the activity-restoring compound for TDO, and thus would be less for cumene than for toluene or benzene, the degradation rate of TCE would be slower after cumene addition than after toluene or benzene addition, as we observed.

3-Methylcatechol and catechol, which are the respective intermediates of the toluene and benzene metabolic pathways, exhibited partial restoration even though they are not substrates of TDO. TCE degradation restarted immediately after the addition of these two catechols (data not shown). As reported previously (10), the catechols were cytotoxic: arginine-dependent O₂ uptake was depressed by these chemicals, independently of toluene metabolism (Table 1). In addition, toluene degradation was more severely inhibited, suggesting that such toxic intermediates negatively regulate the activity of TDO at the protein level. A direct interaction between these chemicals and TDO was also indicated by indigo formation, which has been used as an indicator of TDO activity (18): the addition of low concentrations (0.1 and 1 mM) of 3-methylcatechol and catechol largely enhanced indigo formation, although at 10 mM these chemicals greatly inhibited it. It is possible that conformational changes in TDO caused by 3-methylcatechol and catechol may destabilize toluene in the active site but may enhance accessibility of the larger indole molecules to the active site. Such a conformational change might cause the inhibitory TCE or its degradation products to fall away from some of the enzyme molecules.

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TABLE 1. Effect of 3-methylcatechol and catechol on rates of toluene degradation, arginine-dependent oxygen uptake, and indigo formation

Repetitiveness of the restoration of TCE degradation.
To examine the repetitiveness of the restoration phenomenon, we added toluene to the vial each time that TCE degradation stopped, in total making up to six additions over 30 h (Fig. 6). In this experiment, the initial concentration of TCE was kept the same to preclude the possibility of dose-dependent substrate inhibition. Therefore, the same amount of TCE was resupplied to the vials immediately after the previous amount had been exhausted (Fig. 6a). Each aliquot of toluene added was degraded instantaneously (Fig. 6b), followed by the degradation of TCE (Fig. 6a). Thus, the repetitiveness of the restoration phenomenon was clearly demonstrated for up to at least six additions of toluene. As a result of the six cycles of restoration, the total amount of degraded TCE reached 0.20 µmol mg of cells^–1, corresponding to about eight times the amount degraded in the first 1.5 h (0.027 µmol mg of cells^–1) and being significantly more than the reported Tc value of 0.05 µmol mg of cells^–1 for this species (6). Retention of a high rate of TCE degradation over the experimental period was shown by the value at 20.3 h, which was similar to those at 2.8 and 10.2 h (Table 2).

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FIG. 6. Effect of repeated additions of toluene on bacterial activity. (a) Time course of TCE degradation. (b) Time course of toluene degradation. TCE and toluene were added at the time points indicated by the dashed and solid arrows, respectively. (c) Cell viability was assessed by counting the number of respiring cells stained with CTC. All data displayed are the means (symbols) ± standard deviations (error bars) obtained from three independent experiments, although some error bars are hidden under key symbols.

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TABLE 2. Maximum rate of TCE degradation observed in each restoration cycle

To examine the nonspecific cytotoxic effects of TCE degradation on whole cells, we monitored the number of viable cells during TCE degradation by detecting respiratory activity with CTC staining (Fig. 6c). The number of viable cells decreased slightly over the first few hours and then remained nearly constant throughout the experimental period. This result implies that, for P. putida F1, TCE degradation has very little effect not only on toluene oxidation activity but also on cell respiration. The stable number of active cells remaining after six cycles of toluene addition, which was also confirmed by counting viable cells on agar plates (data not shown), holds the promise that repeated additions of toluene will make this strain highly effective in TCE degradation. If we consider the process of in situ biostimulation by the injection of toluene, the populations of various kinds of toluene degraders, including F1-type strains, may be increased by the stimulation. Repetitive injection with proper intervals is expected to realize the full capacity of such strains in a microbial community. If we consider the degradation of TCE in a reactor integrated into waste treatment, a constant supply of cumene at a low concentration will become an effective method for the continuous degradation of TCE over long periods because the TCE degradation activity of cells is maintained in the presence of cumene.

In summary, our results demonstrate a new way of restoring TCE degradation that has not been previously reported, either for P. putida F1 possessing TDO or for other bacteria possessing other kinds of oxygenase. It is uncertain whether our findings in P. putida F1 are common phenomena among TCE cometabolizing bacteria. The real cause of the cessation of TCE degradation may depend on the bacterial strain, which ultimately governs the potential for restoration.

 
We thank David T. Gibson for providing P. putida F1.

 
* Corresponding author. Present address: Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan. Phone: 81-52-735-5214. Fax: 81-52-735-5255. E-mail: hori.katsutoshi{at}nitech.ac.jp.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, May 2004, p. 2830-2835, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2830-2835.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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