Microbial oxidation and bioluminescence response for toluene and trichloroethylene

~

Pergamon

Wat.Sci. T~ch. Vol. 38. No. 7.pp.I-8.1998. IAWQ ~

PH: S0273-1223(98)00601-5

1998 Published by Elsevier Science Ltd.

Printedin GreatBotain.Allrightsreserved 0273- J223/98 519'00 + 0'00

MICROBIAL OXIDATION AND BIOLUMINESCENCE RESPONSE FOR TOLUENE AND TRICHLOROETHYLENE Kevin G. Robinson*, Jan G. Pieters**, John Sanseverino***, Chris D. Cox*, Charles L. Wright*, Catherine L. Cheng* and Gary S. Sayler*** '"Department of Civil & Environmental Engineering, The University ofTennessee, Knoxville, TN 37996, USA Department ofAgricultural Engineering, The University ofGhent. Belgium "''''''' Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN 37996. USA

"'*

ABSTRACf Two Pseudomonas strains, P. putida FI and P. putida TVA8, were used to evaluate biodegradation of toluene and cometabolism of trichloroethylene (TeE) in batch systems. Both organisms effectively utilized toluene although at different rates. The extent of TCE eometabolism was dependent upon the initial mass of toluene present. A relationship between toluenelTCE degradation and bioluminescence production for P. putida TVA8 was explored. A linear relationship between the total amount of toluene degraded and total bioluminescence was found. The slope of this straight line, however, was found to vary between experiments. To overcome this problem, a normalization method was proposed and successful tested. (C) 1998. Published by Elsevier Science Ltd. All rights reserved

KEYWORDS Bioremediation; Pseudomonas putida; toluene; trichloroethylene; bioluminescence. INTRODUCfION Trichloroethylene (TCE) is a widespread solvent used in a large variety of industrial processes. Since it is also readily mobile in the environment, contamination of soils and groundwater by TCE is a ubiquitous problem. Remediation is mostly based on removing the TCE and other volatile organic compounds (VOCs) by soil vapor extraction, air stripping, or air sparging, resulting in the release of high volumes of air Contaminated with low concentrations of VOCs. Trichloroethylene, however, is considered a dangerous (carcinogenic) air pollutant. Hence, subsequent treatment of these air streams is required. The most popular and least expensive biological technique for VOC treatment is biofiltration, in which the COntaminant is degraded by a biofilm supported on a porous medium, such as compost, peat, soil, ceramic or plastic packing material. However, biofiltration has not yet been applied on a large scale for control of chlorinated organics. This is due, in part, to the difficulty of accurately controlling the degradative process. Chlorinated compounds can be fortuitously degraded by aerobic bacteria which express non-specific

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K. G. ROBINSON et al.

catabolic oxygenase enzymes in the presence of a growth or inducer substrate. Nelson et al. (1987) reported that P. putida was capable of producing the toluene dioxygenase enzyme involved in degradation of toluene and co-metabol ism of TCE. Bioluminescent reporter organisms may represent a promising approach for biofiltration process control. Successful applications of bioluminescent bacteria for on-line monitoring of bioavailability have been described by Heitzer et al. (1994). Those researchers investigated naphthalene degradation in soil systems using light emission as a measure of catabolic activity. Recently, Applegate et al. (1997) inserted a tod-lux gene fusion into the chromosome of Pseudomonas putida F I, resulting in Pseudomonas putida TVA8. This organism metabolizes toluene, cometabolizes TCE, and produces light as an indicator of toluene dioxygenase production. Since gene expression of bioluminescence is under the same genetic control as toluene dioxygenase, light production may be used to monitor and provide feedback control for the degradation process. The overall goal of this research was to determine if bioluminescence can be used as a process control mechanism in the degradation of toluene and/or TCE. The specific objectives were to assess toluene and TCE degradation kinetics for P. putida FI and P. putida TVA8 and to determine if a qualitative and/or quantitative relationship exists between tolueneffCE degradation and bioluminescence production in P. putida TVA8. MATERIALS AND METHODS Microorl:anjsms The two bacterial strains used in this study, Pseudomonas putida FI and TVA8, differ in that lux genes (under the control of the tod promoter) were added to FI to create TVA8. This genetic construct results in light emission by TVA8 when the lux genes are expressed. These organisms were grown on a modified mineral salts buffer (MSB). The phosphate buffer of this medium was replaced with a buffer solution containing KH2P04 (2.15 gil) and K2HP04 (5.3 gil). Final pH was 7.0. Toluene (200 Ill) was supplied in the vapor phase in a suspended glass bulb. Once a week, cells were taken from a frozen stock (-80"C) and grown in a 250 ml Erlenmeyer flask containing 50 ml of MSB and toluene. Each day, a new subculture was prepared by transferring I ml of overnight culture into 50 ml of MSB and toluene. All cultures were incubated at 28"C at 200 rpm. All optical densities (00) were measured by a Beckman DU®-70 spectrophotometer at a wavelength of 546 nm. For each experiment, overnight cultures were centrifuged at 8000 rpm for 10 min, washed once in MSB and diluted to a final OD S46 of 1.0. Five ml of the diluted culture was added to a series of 20 ml vials. The vials were closed with Mininert® valves for the toluene experiments and with Teflon-faced silicone septa and open-top polypropylene caps for the TCE experiments. Toluene el(periments Toluene (0 to 100 ppm) was added to the cells as toluene-saturated MSB (saturated concentration about 515 ppm) at t 14 min, so that the first headspace sample for the determination of the toluene concentration was taken at t 15 min, all other samples being injected after the previous GC cycle had been completed, i.e. at approximately 2.5 min intervals. Bioluminescence readings were performed immediately following each GC injection. Each experiment also included a vial containing pure MSB, a vial containing 5 ml of the culture without toluene (the blank), and finally a killed control vial containing dead cells (cells were killed by adding 0.5 ml of 2 N H2S04)' The same amount of toluene-saturated MSB was injected in both the MSB and the killed control vials, allowing possible absorption of toluene by the cells or loss of toluene from the vials to be determined. The blank was used as a reference for the bioluminescence measurements . Experiments were run in triplicate. For the bioluminescence experiments in which toluene concentrat ions were not monitored, the same procedure was foIlowed except that experiments were run simultaneously instead of sequentially.

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Microbial oxidation bioluminescence response

3

TQlueneplus TCE e3periments The toluene plus TCE experiments were conducted in basically the same way as the toluene experiments. However. TCE (0 to 10 J.1g) was injected as TCE-saturated MSB (saturated concentration of 1100 ppm) immediately following the injection of the toluene-saturated MSB. Bioluminescence was measured every 2 to 3 minutes. In addition to the controls described above, a treatment with toluene only was included. Analytical procedures Headspace analysis of toluene and TCE was performed using a Hewlett Packard 5890 Series II gas chromatograph, equipped with a HP 624 capillary column (0.S3 mm inner diameter, 3.0 J.1m film thickness, 30 m length). Gas sample injections (25 J.11) were made manually. Toluene was detected by a flame ionization detector (FID), while TCE was detected by an electron capture detector (ECD). Helium was the carrier gas (10 ml/min), and nitrogen was the make-up gas (25 ml/rnin), Injection and detector temperatures were 150·C and 250'C, respectively. Injector split ratio was 4: I. The capillary column contained a splitter which directed equal carrier gas flow to the FlO and the ECD simultaneously. FQr experiments where both toluene and TCE concentrations where measured, temperature programming was applied: the oven was operated at 40·C for 2 min, after which the temperature was increased by 25·CJmin until a final temperature of 140·C was reached. One measurement took about 10 min. FQr experiments where only the toluene concentration was to be determined, the total time needed for one measurement was reduced to about 2.5 min by operating the oven isothermally at 102"C. Bioluminescence was measured with the use of an Oriel digital display with a photomultiplier connected to a flexible liquid light pipe and collimating beam probe, Since the photon flux was converted in an electric current, bioluminescence was read in nanoamperes [nA]. RESULTS AND DISCUSSION TQluene de~radatiQn assays Both P. putida FI and P. putida TVA8 were efficient in degrading toluene; both strains removed all the added toluene although at different rates. Figure 1 shQWS the toluene degradation rates for both F1 and IVA8 as a function of the initial concentrations in the liquid phase over a range of 20 to 85 ppm, corresponding to injected masses Qf toluene between 0.1 and 0.5 mg. Rates are expressed with respect to disappearance of toluene from the headspace in the vials and not from the liquid phase. Under these experimental conditions, toluene degradation rates for P. putida IVAS increased from 100 ppmv/min for 20 ppm initial toluene concentration to approximately 130-140 ppmv/min for an initial tQluene concentration of 50 ppm. Degradation rates remained almost constant for higher toluene concentratlons up to 85 ppm. FQr P. putida FI, the apparent increase in degradation rate as a function of tQluene concentration was statistically insignificant. The maximum degradation rate was approximately 100 Ppmv/min. From the control vials, statistically insignificant absorbance or loss from the vial occurred, therefore the disappearance of toluene from the headspace was due primarily to biodegradation. B.iQlumjnescence Figure 2 shQWS the bioluminescence response of IVAS to the addition of three different amounts of toluene, As a reference. the toluene concentration in the headspace is plotted on the same graph. In most experiments, the injection of toluene at t 14 min resulted in an almost immediate increase in bioluminescence . It was also observed that during active toluene degradation, bioluminescence remained relatively constant. Only after most of the toluene had been biodegraded did the bioluminescence increase by a factor of tWQ or more, as can be seen in Figs 2b and 2c. It was also observed that for different amounts of toluene injected, the time interval to maximum bioluminescence changed. Lower levels of toluene gave

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K. G. ROBINSON et al.

rise to shorter intervals before peak light; higher levels gave rise to longer intervals. The time elapsed between toluene injection and peak bioluminescence increased from about 10 min for 0.10 mg of toluene injected to about 30 min. 50 min, and more than 80 min for 0.18, 0.26 and 0.52 mg of toluene, respectively. These time intervals were reproducible for discrete toluene doses.

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Figure I. Toluene degradationrates for FI and TVAS as a functionof the initial amountof toluene.

In contrast to the high reproducibility of the elapsed time of maximum bioluminescence, bioluminescence levels and the total bioluminescence produced for each experiment showed more scalier. This was clearly demonstrated by the results of Fig. 2. For these experiments, which were all conducted (in triplicate) on different days. no relationship between these different levels and the initial toluene concentration could be found. Throughout several days of experiments. however, it was observed that the bioluminescence at the start of the experiment, i.e. before toluene was injected, varied from day to day, while much less variance was found during one day. Based on this observation, results were normalized using the following expression:

B n = E[bIOJ .

B --E,,[b,oJ

Bn: normalized total bioluminescence [C] B: total bioluminescence calculated (C] b lO: bioluminescence determined at t 10 min. i.e. at the last measurement point before injection of the toluene [Al E[bJo]: expected value of bJ()t determined as the average of all bJOvalues measured over all days [Al EJblOl: expected value of bl()t determined as the average of all blO values measured on day d [Al

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This normalization method was applied to the experiments described above. By means of linear regression. prediction of the untreated (B) and the normalized (Bn> values of the total bioluminescence for an injection of 0.52 mg (100 ppm) of toluene was attempted. The predicted values were compared to the results of two series of 0.52 mg injections obtained on two different days. with different EJblOl values. It was concluded that the data for absolute bioluminescence showed no regular pattern. while the normalized data are situated on a straight line. The coefficients of determination for the two regression lines were 0.502 and 0.995, respectively. Furthermore, the untreated data obtained for the two series in which 0.52 mg of toluene was injected are found to be far from one another. while their error bars do not overlap. In both cases, the data points were situated far above the corresponding regression line. After normalization of the total bioluminescence, however. the results are almost coincidental and situated slightly below the regression lines.

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(e) Figure 2. Hcadspacetolueneconcentration [tol.] and bioluminescence responses[b] of1VA8 to injectionsof (a) 0.10 mg. (b) 0.18 mg. and (c) 0.26 mg of toluene.

Basedon the previous findings. it was hypothesized that the total bioluminescence of P. putida TVAS as a function of the injected mass of toluene measured on any day could be described by a straight line through the origin. while the slope of the line would be proportional to the EJblOl value. This hypothesis was verified by means of simultaneous bioluminescence only experiments. In these experiments, the bioluminescence response of P. putida TVAS to seven different concentrations of toluene was measured in triplicate. An exampleof the results from such an experiment is given in Fig. 3. In this experiment a linear relationship was found (r2 =0.958)between bioluminescence produced vs. toluene concentration. Applegate et al. (1997) also observed this relationship. Bioluminescent experiments performed on different days did give different light levels and different slopes. These differences may be due to minor variations in time of

6

K.G. ROBINSON tt al,

cell harvest, temperature at which the experiment is conducted (20 ± Z'C), etc. The biochemistry of bioluminescence is complicated and may be affected by a number of subtle factors. Normalization, as described above, will minimize such variability. For all experiments, application of the normalization method gave rise to a slope that was statistically the same as for all other days, even though the maximum slope calculated for the untreated (B) data were more than 10 times higher than the minimum slope.

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Toluene plus TCE

de~radatiQn

assays

The main purpose of these assays was to investigate and describe the behavior of P. putida TVA8 when exposed to toluene and TCE. These results are only preliminary. TCE degradation data for P. putida TVA8 are summarized in Table I. Toluene degradation data were not included since biological utilization was essentially complete after 20 min for the concentrations tested. In control vials, headspace TCE concentrations above dead cells were not lower than TCE concentrations above MSB. The disappearance of TCE from the headspace of the vials was concluded to be the consequence of biodegradation only. Table I shows that the majority of TCE degraded occurred in the first hour. For 100 I!g of toluene, TCE removal efficiencies decreased with increasing TCE concentrations. This was expected since the amount of toluene dioxygenase produced will be proportional to the amount of toluene injected. Since the enzyme has only a limited half-life and assuming that the TCE degradation is not purely substrate limited, higher amounts of TCE will give rise to lower final degradation efficiencies. It was also observed that TCE degradation was not detectable during the first 10 min after TCE injection, corresponding to the time needed to degrade all the toluene. At a higher toluene concentration (260 ug), higher amounts of TCE were degraded; 62% after I hand 74% after 3 h. Although it took more time to degrade the larger amount of toluene and thus to start TCE degradation, the relative TCE removal was much higher than for the corresponding experiment at a lower toluene concentration. This is probably due to higher amounts of toluene dioxygenase being produced by the increased amount of toluene present. Absolute amounts of toluene dioxygenase in the cells were. not measured. It is important to note that TCE is not degraded until the majority of toluene has been removed. This is likely due to competitive inhibition. Toluene will outcompete TCE for the active site of the toluene dioxygenase enzyme. Once toluene is removed, gene expression ceases. Any TCE removed is dependent on any toluene dioxygenase remaining in the cell. Bjoluminescence Figure 4 shows the bioluminescence response of P. putida TVA8 to 100 I!g of toluene and to 100 I!g of toluene plus 10 I!g of TCE. The addition of TCE did not significantly affect bioluminescence. A decrease in

Microbialoxidationbioluminescence response

7

bioluminescence was observed when lower amounts of TCE (between 3 and 10 ug) were injected with toluene (data not shown). Future experiments will evaluate whether this might be explained by two opposite effects of TCE on the cells: inhibition versus solvation effects, the latter giving rise to a continuous destruction of the cell membrane with subsequent stimulation of metabolism for membrane repair. Heitzer et al. (1994) showed that perturbing cells with organic solvents will generate an increase in bioluminescence. This is due to increased cell cycling of fatty acids (a required substrate for bacterialluciferase) and not due to increased gene expression. Table I. Toluene!fCE degradation data for P. putida TVA8 initial TCEconc. TCE after I cone. hour [11&] [ppmv) [ppmv] 24 3 I 17 (40 min)" (50 min)" 17 100 22 I 40 12 5 52 90 10 49 82 34 90 260 10 • Time after which all TCE has disappeared .. Linearly extrapolated value amount of toluene hlg)

amount ofTCE

TCE removed after 1 hr [ppmv) 23 (25)" (20)" 21 28 38 33 56

TCE removal after 1 hr {Oft] 97 100 100 94 70 42 40 62

TCE removal after 3 hrs [0/0] 100 100 100 100 87 52 58 74

-·-(TeE) - - b(tol. + TCE) -.- b(tol.) -.- b(bIank)

Figure 4. HeadspaceTCE concentrationand bioluminescence response [b] ofTYA8l0 the injectionof 100 I.lg toluene plus 10I.lg ofTCE (101. + TCE) and 100I1g of toluene (101.)compared 10 the bioluminescence ofliving cells without tolueneand TCE (blank).

SUMMARY ANDCONCLUSIONS A genetically engineered organism, P. putida TVAS was derived from P. putida F1 by inserting the lux operon behind the tod promoter. P. putida TVA8 was tested in liquid batch assays to determine its toluene and TCE degradation capabilities. For P. putida TVA8, toluene degradation rates increased from about 100 ppmv/min for a toluene injection of 0.10 mg to about 130-140 ppmv/min for a toluene injection of 0.26 mg.

8

K. G. ROBINSON et al.

while they remained almost constant for higher toluene injections of up to 0.52 mg. For the same experimental conditions, toluene degradation rates for P. putida FI were shown to be lower. With respect to TCE degradation in the presence of toluene, it was found that lower values of the tolueneffCE ratio gave rise to lower degradation efficiencies after three hours. This was explained by the fact that relatively high toluene concentrations lead to relatively large amounts of dioxygenase being produced, resulting in quicker TCE removal during the limited lifetime of the enzymes. Results suggested that TCE degradation took place only after all the toluene had been degraded. The data were analyzed for a qualitative and/or quantitative relationship between the toluene and TCE degradation processes and light production by P. putida TVA8. A linear relationship between the total amount of toluene degraded and total bioluminescence was found. The slope of this straight line, however, was found [Q vary between experiments. To overcome this problem, a normalization method in which total bioluminescence was divided by the background bioluminescence before toluene injection was proposed and successfully tested. Due to the limited number of experiments. no decisive conclusions could be drawn at this time with respect to the effect of TCE on the bioluminescence of TVA8. Changes in bioluminescence were observed during experiments in which amounts of TCE between 3 and 10 ~g were injected; higher amounts of TCE giving rise to larger increases in bioluminescence. Such variations must be investigated further if bioluminescence is to be used as a reliable process control parameter for tolueneffCE degradation. REFERENCES Applegate. B. M.• Kehrmeyer, S. R. and Sayler. G. S. (1997). Generation of a chromosomally-based too-lux reporter for detection and quantification of BTEX compounds in aqueous solutions. J. Bacteriol., (submitted). Heitzer, A.• Malachowsky. K.• Thonnard, J. E.• Bienkowski, P. R.• White. D. C. and Sayler. G. S. (1994). Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium. AppL Environ. Mlcrobiol .• 60, 1487-1494. Nelson. M. J. K., Montgomery , S. 0 .• Mahaffey, W. R. and Pritchard, P. H. (1987). Biodegradation of Trichloroethylene and involvement of an aromatic biodegradative pathway. Appl. Environ . Microbiol., 53,949-954.