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The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1
Journal of Microbiological Methods
Journal of Microbiological Methods 40 (2000) 181–191
www.elsevier.com / locate / jmicmeth
The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1 Hae-jin Woo b
a,b ,1
a
, John Sanseverino , Chris D. Cox Gary S. Sayler b
a,b ,
*, Kevin G. Robinson a,b ,
a Center for Environmental Biotechnology, The University of Tennessee, Knoxville TN 37996, USA Department of Civil and Environmental Engineering, 223 Perkins Hall, The University of Tennessee, Knoxville TN 37996, USA
Received 18 September 1999; received in revised form 20 December 1999; accepted 20 December 1999
Abstract Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r 2 5 0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 1961.5 3 10 24 mg indigo (mg protein)21 min 21 was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.060.2 3 10 24 mg indigo (mg protein)21 min 21 . The half-life of whole-cell enzyme activity was estimated to be between 5 ? 5 and 8 h in both suspended and biofilm growth conditions. 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Toluene dioxygenase; Pseudomonas putida F1; TCE cometabolism
1. Introduction Toluene dioxygenase (Tod) of Pseudomonas putida F1 is a three component enzyme system that oxidizes toluene to cis-toluene dihydrodiol through
*Corresponding author. Tel.: 11-423-974-7729; fax: 11-423974-2669. E-mail address: [email protected] (C.D. Cox) 1 Present address: Institute for Environmental Technology and Industry, San 30 Changjeondong Kumjeongku, Pusan, Republic of Korea, 609-735.
the addition of both atoms of molecular oxygen to the aromatic nucleus. The mechanism by which Tod metabolizes toluene (Yeh et al., 1977) and its genetic regulation (Zylstra et al., 1988; Zylstra and Gibson, 1989) have been well characterized. The Tod system has a broad substrate range, including monoaromatics, certain halogenated aromatics, and various chlorinated aliphatics, including trichloroethylene (TCE) (Gibson et al., 1990). The cometabolic degradation of TCE by toluenedegrading bacteria may have practical applications in waste treatment and remediation (Fan and Scow,
0167-7012 / 00 / $ – see front matter 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0167-7012( 00 )00123-8
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H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
1993; Hopkins and McCarty, 1995). Cometabolic degradation of chlorinated aliphatic compounds by oxygenase enzymes is often hampered by (i) competitive inhibition between the primary and cometabolic substrates; (ii) energy limitations within the cell; and (iii) cell toxicity and enzyme inactivation by reactive intermediates of the cometabolite (Chang and Alvarez-Cohen, 1995). While much work has focused on the conditions that minimize competitive inhibition, there are no published reports of whole-cell enzyme activity in the presence of toluene and TCE. Our laboratory has been investigating the cometabolic degradation of TCE in biofilters (Cox et al., 1998; Cox et al., 1998; Shingleton et al., submitted). The goal of this work was to evaluate enzyme activity as a critical variable in the evaluation of TCE biofiltration efficiency by P. putida F1. Various techniques have been used to measure Tod activity. Yeh et al. (1977) measured the Tod activity in cell extracts by using [methyl– 14 C] toluene. Jenkins and Dalton (1985) measured the Tod activity
in cell extracts by measuring the rate of increase in absorbance of indoxyl at 400 nm by spectrophotometry. Toluene dioxygenase oxidizes indole to indigo via indoxyl, a soluble yellow dye (Fig. 1) (Ensely et al., 1983). However, in the present research, this method was found inadequate to measure enzyme activity of cells harvested from a biofilm due to interference by suspended biomass particles and also by the limited amount of biomass available. A fluorescence-based method for measuring Tod activity based on indoxyl formation was developed. The principle is similar to that used to quantify the nerve gas sodium perborate based on its reaction with indole to form the highly fluorescent compound indoxyl (Gehauf and Goldenson, 1957). The potential advantages of the fluorescence-based method compared to spectrophotometric methods include less interference by suspended biomass and greater sensitivity. NADH is a required co-factor for Tod-mediated oxidation reactions and has been hypothesized to
Fig. 1. Proposed pathway for indigo biosynthesis by toluene dioxygenase (Ensley et al. 1983).
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
become limiting in the degradation of TCE (Chang and Alvarez-Cohen, 1995). A second objective of this work is to monitor intracellular NADH concurrently with Tod enzyme activity. NADH can be monitored by fluorescence spectroscopy; NADH is excited at 340 nm and emits at 420 nm. Duysens and Amesz (1957) applied this property to quantify NADH levels in whole cells. Zabriskie and Humphrey (1978) used fluorescence to evaluate the concentration of cells by demonstrating a linear relationship between fluorescence response and biomass concentration. Li and Humphrey (1991) used the multiple excitation fluorometric system to monitor NAD(P)H, pyridoxine, tryptophan, and riboflavin in cells. They found that NADH fluorescence is a good indicator of the cellular metabolic state because the NADH fluorescence signal changed according to the utilization of ethanol and production of the metabolic intermediate, acetic acid. Angell et al. (1993) and Rice et al. (1995) reported that fluorescence measurement can be applied to monitor tryptophan and NAD(P)H of an actively growing bacterial biofilm. The primary objective of this research was to develop a fluorescence-based assay to measure intracellular Tod activity in a biofilm culture of P. putida F1 as a tool for assessing cometabolic degradation in fixed-film reactors. Additional objectives were to characterize whole-cell enzyme activity of P. putida F1 during growth, to determine if NADH is a potentially limiting factor in the expression of wholecell Tod enzyme activity, and to determine the halflife of the enzyme.
2. Materials and methods
2.1. Bacterial strain and culture conditions Pseudomonas putida F1 was obtained from David T. Gibson (University of Iowa). P. putida F1 was inoculated from frozen stock cultures and grown in 50 ml of modified Hunters medium (Stanier et al., 1966). The phosphate buffer of Hunters medium was replaced with KH 2 PO 4 (2.15 g l 21 ) and K 2 HPO 4 (5.3 g l 21 ); the final pH was 7.0. Toluene (100 ml) was supplied in the vapor phase in a suspended glass bulb. Flasks were incubated at room temperature with shaking (200 rpm). When growth reached late
183
exponential phase (OD 600 5 0.8–1.0), 0.5 ml of culture was transferred to 50 ml fresh medium. Cells were harvested (OD 600 5 0.8) by centrifugation (10 000 3 g for 10 min at 48C), washed twice with phosphate buffer (pH 7.0), resuspended in Hunters medium to a cell density of 1.0, and placed on ice for batch experiments.
2.2. Analytical methodology Optical density (OD 600 ) and indigo production (A 600 ) were measured on a Beckman Spectrophotometer (Model DU-70; Fullerton, CA). NADH and indoxyl were measured by fluorescence (Model 240, Perkin-Elmer, Inc., Buckinghamshire, UK). Bovine serum albumin (BSA) (2 mg ml 21 in 0.9% sodium chloride and 0.05% sodium azide) was used as a protein standard. Protein was determined by the BCA protein assay (Pierce, Rockford, IL).
2.3. Determination of normalized toluene dioxygenase activity in whole cells 2.3.1. Spectrophotometric method The optical density of a 1 ml sample of the culture was determined; then the sample was immediately transferred to a microcentrifuge tube. The cells were harvested and washed with phosphate buffer solution (pH 5 7.2) by centrifugation (1 min at 14 000 rpm at 48C). The enzyme reaction was initiated by addition of indole (Jenkins and Dalton, 1985). Five microliters of 100 mM indole in N,N-dimethlylformamide was added to the cells and the formation of indigo was monitored spectrophotometrically at 600 nm over the reaction time against a control (resuspended cells without indole). The initial rate of indigo formation was determined by plotting the increase in indigo absorbance as a function of time. The enzyme activity was defined as the initial rate of indigo formation normalized to the protein content of the sample. 2.3.2. Fluorescence method The normalized enzyme activity in whole cells was determined by the rate of indoxyl formation, as quantified by fluorescence detection. Excitation and emission wavelengths were 365 and 470 nm, respectively. Cells were prepared as described above. Five
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microliters of 100 mM indole was added to the cells and the formation of indoxyl was measured after a given reaction time. Centrifugation and handling time for all samples was approximately 80 s.
2.4. Determination of intracellular NADH concentration One milliliter of sample culture was transferred to a microcentrifuge tube. The cells were harvested and washed with phosphate buffer solution (pH 5 7.2) as described above. A mixture of 100 ml of cells with 900 ml of deionized water was injected into the fluorescence detector; the excitation and emission wavelengths were 340 and 425 nm, respectively. The normalized NADH in whole cells was expressed as mmol NADH (mg protein)21 . Solutions of commercially available NADH (Sigma Chemical, St. Louis, MO) were used to prepare a standard curve.
2.5. Harvesting and preparation of cells from biofilms Tod assays and NADH and protein measurements were applied to P. putida F1 biofilm from two different biofilter reactors: a column reactor, 10 cm in diameter by 100 cm long (64 cm bed height) and a differential-volume reactor 10 cm diameter by 10 cm long (1 cm bed height). The differential volume reactor was operated such that toluene degradation was less than 5%. Under these conditions the toluene concentration across the 1 cm bed could be considered uniform. The biofilm in both reactors was supported on a bed of diatomaceous earth pellets (Celite Biocatalyst Carrier R-635, Manville, Denver, CO). The complete biofiltration apparatus, including systems for feeding liquid nutrients and humidified, toluene-contaminated air, is described elsewhere (Cox et al., 1998), as are the experimental procedures used to inoculate, operate, and sample the column. Tod activity, NADH and protein were determined in the biofilm by sampling two pellets from the biofiltration reactor operating at steady-state. Both pellets were mixed with 2 ml of sterile 0.1% Na 2 P2 O 7 and vortex mixed to detach cells from the
pellets. The cell mixture was centrifuged and washed with phosphate buffer solution (pH 5 7.2). The final cell pellets were resuspended in 5 ml of fresh medium. The cells were sonicated for 3 s, followed by a 30 s rest period; this cycle was repeated three times to thoroughly disperse the biofilm. The prepared cell culture was used for measurement of the Tod activity and NADH and protein concentrations as described above.
2.6. Data analysis With the exception of the spectrophotometricbased measurements of Tod activity, each data point in the following figures represents the mean of replicate samples. The standard deviation (S.D.) is shown by an error bar; many error bars are smaller than the data point symbols. All measurements of toluene concentration were made in triplicate. Protein, NADH, and fluorescence-based Tod activity measurements were made in duplicate. The data points for spectrophotometric measurements of Tod activity represent a single measurement because there was insufficient time between samples to process duplicates.
3. Results and discussion
3.1. Determination of optimum emission wavelength for indoxyl The excitation and emission wavelengths established by Gehauf and Goldenson (1957) for indoxyl were confirmed. Indoxyl was generated from a pure culture of toluene-grown P. putida, because a commercial standard was not available. Cell supernatants from the indole assay were subjected to excitation and emission scanning. The emission scan ranged from 400 to 700 nm with a fixed excitation at 365 nm. A peak was observed in the range of 460 to 490 nm, consistent with that reported by Gehauf and Goldenson (1957). The maximum emission occurred at 470 nm; the method was further developed using this wavelength. Indigo and indole did not fluoresce under these conditions.
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
3.2. Comparison of enzyme activity assay To demonstrate the effectiveness of the indoxyl assay, indoxyl formation was compared to indigo formation. The increase in absorbance at 600 nm and the increase in fluorescence intensity at 470 nm is shown in Fig. 2 over a period of 10 min. Fluorescence increased to approximately 75% of its maximum intensity within 2 min; thereafter, the rate of increase in fluorescence intensity decreased. Therefore, the Tod activity was calculated as the rate of increase in fluorescence during the first 2 min after addition of indole. All fluorescence readings of cell blanks were less than 10 mV. Since indoxyl is not available commercially, a standard curve relating fluorescence to indoxyl concentration could not easily be developed; therefore, a correlation between fluorescence and indigo production was developed. Fig. 3 shows the whole cell Tod activity in P. putida F1, as measured by fluorescence (Efluorescence, mV min 21 ) correlated with the enzyme activity as given by the rate of indigo production (Eindigo, mg indigo min 21 ) measured spectrophotometrically. Efluorescence can be expressed as the rate of indigo production by the following correlation (r 2 5 0.90) derived from Fig. 3:
Efluorescence Eindigo 5 ]]] 2280
185
(1)
Subsequent measurements of Tod activity in suspended growth cells and biofilms were made using the spectrophotometric method and fluorescencebased methods, respectively.
3.3. Specific enzyme activity and NADH concentration profiles during growth phase Normalized whole-cell Tod enzyme activity (Fig. 4a) and NADH (Fig. 4b) profiles were correlated with growth of P. putida F1 on toluene vapor. Tod activity was observed to vary between 3.8 and 7.73 10 24 mg indigo (mg protein)21 min 21 during the log growth phase (OD 600 ,0.9). During the stationary growth phase (OD 600 .0.9), the enzyme activity increased to between 9.0 and 20310 24 mg indigo (mg protein)21 min 21 . Hugouvieux-Cotte-Pattat et al. (1990) reported a similar growth-phase-dependent expression of the TOL plasmid pWW0 catabolic genes by P. putida. Although they were unable to specify a mechanism, they reported that genes were poorly induced during the early-exponential-growth phase but became strongly induced during the late-
Fig. 2. Absorbance and fluorescence intensities as a function of time for indole oxidation by whole cells of Pseudomonas putida F1. Error bars for fluorescence data represent S.D. with n52.
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H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
Fig. 3. Correlation of Tod activity as measured by indoxyl and indigo formation in whole cells of Pseudomonas putida F1. Error bars for fluorescence data represent S.D. with n52.
exponential-growth phase. The specific intracellular NADH concentration (Fig. 4b) maintained a constant value of 0.160.02 mmol (mg protein)21 during the log growth phase (OD 600 ,0.9) and increased linearly after the early-stationary-growth phase (OD 600 . 0.9) to a maximum value of 0.2960.02 mmol (mg 21 protein) .
3.4. Estimation of normalized Tod activity half-life in whole cells Toluene dioxygenase enzyme activity naturally decays over time in the absence of toluene. There are no published reports of Tod half-life either as a purified enzyme or in whole cells. P. putida F1 was grown to OD 600 of 1.0 as previously described. Fifty milliliters of cell suspension was dispensed into two 300 ml flasks after determining the initial enzyme activity of the cells. Only one flask was provided with toluene. Both flasks were placed on a shaker (180 rpm) at room temperature. Five-milliliter sam-
ples were taken periodically and the normalized whole-cell enzyme activity and OD 600 determined. The decrease in normalized whole-cell enzyme activity in both the absence and presence of toluene is shown in Fig. 5. In the absence of toluene, the normalized enzyme activity decreased from 1.623 10 23 to 0.77310 23 mg indigo (mg protein)21 min 21 over a period of 7 h (Fig. 5a). Over the same time period, the normalized whole cell activity in the flask supplied with toluene remained relatively constant at about 2310 23 mg indigo (mg protein)21 min 21 . The rate of normalized whole-cell enzyme decay in the absence of toluene was described by the first order equation: Ex.t 5 Exo e 2kt
(2)
where Ex.t and Exo are the final and initial normalized whole-cell enzyme activities, respectively, t is the incubation time, and k is the decay constant. The value of k was 0.123 h 21 as determined by nonlinear regression (r 2 50.92) with a corresponding half-life of 5?6 h. To confirm the half-life value
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
187
Fig. 4. Measured response of Pseudomonas putida F1 during growth. (a) Normalized Tod activity; (b) normalized NADH concentration; error bars representing S.D. with n52 were smaller than the symbols for all data points.
determined, a second experiment was conducted over a longer period (14.5 h) (Fig. 5b). The normalized whole-cell Tod activity decreased from 8.54310 24 to 2.63310 24 mg indigo (mg protein)21 min 21 in the absence of toluene. Eq. (2) was fitted to the data
and a value of 0.0869 h 21 was determined (r 2 5 0.94); the corresponding half-life was 8 h. With toluene present, the activity remained relatively constant at about 7.5310 24 mg indigo (mg protein)21 min 21 .
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Fig. 5. Decay of normalized enzyme activity in whole Pseudomonas putida F1 cells in suspend batch culture: (a) 7 h experiment, (b) 14.5 h experiment.
3.5. Enzyme activity and NADH concentration in the biofilm Biofiltration experiments were conducted to examine the profiles of toluene, normalized Tod enzyme activity, protein, and normalized NADH concentrations along the column (Fig. 6). Air containing 1000 mg l 21 of toluene was fed continuously to the column at a rate of 500 ml min 21 , corresponding to an empty-bed contact time (EBCT) of 10.0 min. At steady-state, toluene concentration, protein content,
NADH concentration and normalized whole-cell Tod activity were measured along the length of the column. Toluene was consumed in the upper half of the column; its concentration was below detectable levels in the lower half of the column. As expected, Tod enzyme activity and cellular protein decreased with decreasing toluene concentration along the length of the column. The normalized NADH concentration was relatively constant over the entire length of the column at approximately 0.4 mmol (mg protein)21 .
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
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Fig. 6. Toluene, protein, NADH and enzyme activity profiles of Pseudomonas putida F1 biofilms from a biofiltration column. Error bars for toluene represent S.D. with n53. All other error bars represent S.D. with n52.
Fig. 7. Decay of normalized enzyme activity in whole Pseudomonas putida F1 cells from a differential-volume biofilm reactor. Error bars represent S.D. with n52.
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3.6. Estimation of the half-life of whole-cell Tod activity in biofilms Air contaminated with toluene (1000 mg / l) was continuously fed to the differential-volume biofilm reactor at a flow rate of 500 ml min 21 , yielding an EBCT of 9.4 s. After steady-state conditions had been established, toluene-free air was fed to the reactor and the normalized whole cell enzyme activity measured as a function of time (Fig. 7). The initial normalized enzyme activity decreased from 4.43 10 24 to 1.27310 24 mg indigo (mg protein)21 min 21 over a period of 12.3 h. Eq. (2) was fitted to the data (r 2 50.92) and a k value of 0.0982 h 21 determined. The corresponding half-life was 7.1 h, consistent with the whole-cell enzyme half-life determined under suspended-growth conditions.
4. Conclusion A method of measuring the whole-cell Tod activity in P. putida F1 based on the fluorescent properties of indole was described. Compared to the spectrophotometric method, it was found to be less susceptible to interference by suspended biomass. Application of the assay for both suspended and biofilm cells was demonstrated and the enzyme half-life in each mode of growth determined. Coupled with simultaneous determination of intracellular NADH concentrations, the assay can be used to elucidate factors controlling the biokinetics of metabolic and cometabolic degradation processes in fixed film reactors. Future work will focus on the role of Tod enzyme activity in the cometabolic degradation of TCE during biofiltration.
References Angell, P., Arrage, A.A., Mittelman, M.W., White, D.C., 1993. On line, non-destructive biomass determination of bacteria biofilms by fluorometry. J. Microbiol. Methods 18, 317–327. Chang, H.L., Alvarez-Cohen, L., 1995. Model for the cometabolic biodegradation of chlorinated organics. Environ. Sci. Technol. 29, 2357–2367. Cox, C.D., Robinson, K.G., Woo, H.J., Wright, C.L., Sanseverino, J., 1998. Cometabolic biofiltration of TCE using biolumines-
cent reporter bacteria. In: Wickramanayake, G.B., Hinchee, R.E. (Eds.), Bioremediation and Phytoremediation, Battelle Press, Columbus, OH, pp. 221–226. Cox, C.D., Woo, H.J., Robinson, K.G., 1998. Cometabolic biodegradation of trichloroethylene (TCE) in the gas phase. Water Sci. Tech. 37, 97–104. Duysens, L.N.M., Amesz, J., 1957. Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the near-ultraviolet and visible region. Biochim. Biophys. Acta 24, 19–26. Ensely, B.D., Ratzkin, B.J., Osslund, T.D., Simon, M.J., Wackett, L.P., Gibson, D.T., 1983. Expression of naphthalene oxidation genes in Escherichia coli: Results in the biosynthesis of indigo. Science 222, 167–169. Fan, S., Scow, K.M., 1993. Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil. Appl. Environ. Microbiol. 59, 1911–1918. Gehauf, B., Goldenson, J., 1957. Detection and estimation of nerve gases by fluorescence reaction. Anal. Chem. 29, 276– 278. Gibson, D.T., Zylstra, G.J., Chauhan, S., 1990. Biotransformations catalyzed by toluene dioxygenase from Pseudomonas putida F1. In: Silver, S., Chakrabarty, A.M., Iglewski, B., Kaplan, S. (Eds.), Pseudomonas: Biotransformations, Pathogenesis, and Emerging Biotechnology, American Society for Microbiology Press, Washington, pp. 121–132. Hopkins, G.D., McCarty, P.L., 1995. Field evaluation of in situ aerobic cometabolism of trichloroethylene and three dichloroethylene isomers using phenol and toluene as the primary substrates. Environ. Sci. Technol. 29, 1628–1637. Hugouvieux-Cotte-Pattat, N., Kohler, T., Rekik, M., Harayama, S., 1990. Growth-phase-dependent expression of the Pseudomonas putida TOL plasmid pWW0 catabolic genes. J. Bacteriol. 172, 6651–6660. Jenkins, R.O., Dalton, H., 1985. The use of indole as a spectrophotometric assay substrate for toluene dioxygenase. FEMS Microbiol. Lett. 30, 227–231. Li, J., Humphrey, A.E., 1991. Use of fluorometry for monitoring and control of a bioreactor. Biotechnol. Bioeng. 37, 1043– 1049. Rice, J.F., Fowler, R.F., Flemming, J.T., Arrage, A.A., White, D.C., Sayler, G.S., 1995. Effects of external stimuli on environmental bacteria strains harboring an algD-lux bioluminescent reporter plasmid for the study of corrosive biofilms. J. Ind. Microbiol. 15, 318–328. Shingleton, J.T., Sayler, G.S., Bienkowski, P.R. Model of toluene dioxygenase enzyme activity and TCE co-oxidation by Pseudomonas putida TVA8 in a radial flow bioreactor. Biotechnol. Bioeng, submitted. Stanier, R.Y., Palleroni, N.J., Doudoroff, M., 1966. The aerobic Pseudomonads: a taxonomic study. J. Gen. Microbiol. 43, 159–271. Yeh, W.K., Gibson, D.T., Liu, T.N., 1977. Toluene dioxygenase: A multicomponent enzyme system. Biochem. Biophys. Res. Commun. 78, 401–410. Zabriskie, D.W., Humphrey, A.E., 1978. Estimation of fermentation biomass concentration by measuring culture fluorescence. Appl. Environ. Microbiol. 35, 337–343.
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191 Zylstra, G.J., Gibson, D.T., 1989. Toluene degradation by Pseudomonas putida F1: Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli. J. Biol. Chem. 264, 14940–14946.
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Zylstra, G.J., McCombie, W.R., Gibson, D.T., Finette, B.A., 1988. Toluene degradation by Pseudomonas putida F1: Genetic organization of the tod operon. Appl. Environ. Microbiol. 54, 1498–1503.
Journal of Microbiological Methods 40 (2000) 181–191
www.elsevier.com / locate / jmicmeth
The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1 Hae-jin Woo b
a,b ,1
a
, John Sanseverino , Chris D. Cox Gary S. Sayler b
a,b ,
*, Kevin G. Robinson a,b ,
a Center for Environmental Biotechnology, The University of Tennessee, Knoxville TN 37996, USA Department of Civil and Environmental Engineering, 223 Perkins Hall, The University of Tennessee, Knoxville TN 37996, USA
Received 18 September 1999; received in revised form 20 December 1999; accepted 20 December 1999
Abstract Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r 2 5 0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 1961.5 3 10 24 mg indigo (mg protein)21 min 21 was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.060.2 3 10 24 mg indigo (mg protein)21 min 21 . The half-life of whole-cell enzyme activity was estimated to be between 5 ? 5 and 8 h in both suspended and biofilm growth conditions. 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Toluene dioxygenase; Pseudomonas putida F1; TCE cometabolism
1. Introduction Toluene dioxygenase (Tod) of Pseudomonas putida F1 is a three component enzyme system that oxidizes toluene to cis-toluene dihydrodiol through
*Corresponding author. Tel.: 11-423-974-7729; fax: 11-423974-2669. E-mail address: [email protected] (C.D. Cox) 1 Present address: Institute for Environmental Technology and Industry, San 30 Changjeondong Kumjeongku, Pusan, Republic of Korea, 609-735.
the addition of both atoms of molecular oxygen to the aromatic nucleus. The mechanism by which Tod metabolizes toluene (Yeh et al., 1977) and its genetic regulation (Zylstra et al., 1988; Zylstra and Gibson, 1989) have been well characterized. The Tod system has a broad substrate range, including monoaromatics, certain halogenated aromatics, and various chlorinated aliphatics, including trichloroethylene (TCE) (Gibson et al., 1990). The cometabolic degradation of TCE by toluenedegrading bacteria may have practical applications in waste treatment and remediation (Fan and Scow,
0167-7012 / 00 / $ – see front matter 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0167-7012( 00 )00123-8
182
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
1993; Hopkins and McCarty, 1995). Cometabolic degradation of chlorinated aliphatic compounds by oxygenase enzymes is often hampered by (i) competitive inhibition between the primary and cometabolic substrates; (ii) energy limitations within the cell; and (iii) cell toxicity and enzyme inactivation by reactive intermediates of the cometabolite (Chang and Alvarez-Cohen, 1995). While much work has focused on the conditions that minimize competitive inhibition, there are no published reports of whole-cell enzyme activity in the presence of toluene and TCE. Our laboratory has been investigating the cometabolic degradation of TCE in biofilters (Cox et al., 1998; Cox et al., 1998; Shingleton et al., submitted). The goal of this work was to evaluate enzyme activity as a critical variable in the evaluation of TCE biofiltration efficiency by P. putida F1. Various techniques have been used to measure Tod activity. Yeh et al. (1977) measured the Tod activity in cell extracts by using [methyl– 14 C] toluene. Jenkins and Dalton (1985) measured the Tod activity
in cell extracts by measuring the rate of increase in absorbance of indoxyl at 400 nm by spectrophotometry. Toluene dioxygenase oxidizes indole to indigo via indoxyl, a soluble yellow dye (Fig. 1) (Ensely et al., 1983). However, in the present research, this method was found inadequate to measure enzyme activity of cells harvested from a biofilm due to interference by suspended biomass particles and also by the limited amount of biomass available. A fluorescence-based method for measuring Tod activity based on indoxyl formation was developed. The principle is similar to that used to quantify the nerve gas sodium perborate based on its reaction with indole to form the highly fluorescent compound indoxyl (Gehauf and Goldenson, 1957). The potential advantages of the fluorescence-based method compared to spectrophotometric methods include less interference by suspended biomass and greater sensitivity. NADH is a required co-factor for Tod-mediated oxidation reactions and has been hypothesized to
Fig. 1. Proposed pathway for indigo biosynthesis by toluene dioxygenase (Ensley et al. 1983).
H.-j. Woo et al. / Journal of Microbiological Methods 40 (2000) 181 – 191
become limiting in the degradation of TCE (Chang and Alvarez-Cohen, 1995). A second objective of this work is to monitor intracellular NADH concurrently with Tod enzyme activity. NADH can be monitored by fluorescence spectroscopy; NADH is excited at 340 nm and emits at 420 nm. Duysens and Amesz (1957) applied this property to quantify NADH levels in whole cells. Zabriskie and Humphrey (1978) used fluorescence to evaluate the concentration of cells by demonstrating a linear relationship between fluorescence response and biomass concentration. Li and Humphrey (1991) used the multiple excitation fluorometric system to monitor NAD(P)H, pyridoxine, tryptophan, and riboflavin in cells. They found that NADH fluorescence is a good indicator of the cellular metabolic state because the NADH fluorescence signal changed according to the utilization of ethanol and production of the metabolic intermediate, acetic acid. Angell et al. (1993) and Rice et al. (1995) reported that fluorescence measurement can be applied to monitor tryptophan and NAD(P)H of an actively growing bacterial biofilm. The primary objective of this research was to develop a fluorescence-based assay to measure intracellular Tod activity in a biofilm culture of P. putida F1 as a tool for assessing cometabolic degradation in fixed-film reactors. Additional objectives were to characterize whole-cell enzyme activity of P. putida F1 during growth, to determine if NADH is a potentially limiting factor in the expression of wholecell Tod enzyme activity, and to determine the halflife of the enzyme.
2. Materials and methods
2.1. Bacterial strain and culture conditions Pseudomonas putida F1 was obtained from David T. Gibson (University of Iowa). P. putida F1 was inoculated from frozen stock cultures and grown in 50 ml of modified Hunters medium (Stanier et al., 1966). The phosphate buffer of Hunters medium was replaced with KH 2 PO 4 (2.15 g l 21 ) and K 2 HPO 4 (5.3 g l 21 ); the final pH was 7.0. Toluene (100 ml) was supplied in the vapor phase in a suspended glass bulb. Flasks were incubated at room temperature with shaking (200 rpm). When growth reached late
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exponential phase (OD 600 5 0.8–1.0), 0.5 ml of culture was transferred to 50 ml fresh medium. Cells were harvested (OD 600 5 0.8) by centrifugation (10 000 3 g for 10 min at 48C), washed twice with phosphate buffer (pH 7.0), resuspended in Hunters medium to a cell density of 1.0, and placed on ice for batch experiments.
2.2. Analytical methodology Optical density (OD 600 ) and indigo production (A 600 ) were measured on a Beckman Spectrophotometer (Model DU-70; Fullerton, CA). NADH and indoxyl were measured by fluorescence (Model 240, Perkin-Elmer, Inc., Buckinghamshire, UK). Bovine serum albumin (BSA) (2 mg ml 21 in 0.9% sodium chloride and 0.05% sodium azide) was used as a protein standard. Protein was determined by the BCA protein assay (Pierce, Rockford, IL).
2.3. Determination of normalized toluene dioxygenase activity in whole cells 2.3.1. Spectrophotometric method The optical density of a 1 ml sample of the culture was determined; then the sample was immediately transferred to a microcentrifuge tube. The cells were harvested and washed with phosphate buffer solution (pH 5 7.2) by centrifugation (1 min at 14 000 rpm at 48C). The enzyme reaction was initiated by addition of indole (Jenkins and Dalton, 1985). Five microliters of 100 mM indole in N,N-dimethlylformamide was added to the cells and the formation of indigo was monitored spectrophotometrically at 600 nm over the reaction time against a control (resuspended cells without indole). The initial rate of indigo formation was determined by plotting the increase in indigo absorbance as a function of time. The enzyme activity was defined as the initial rate of indigo formation normalized to the protein content of the sample. 2.3.2. Fluorescence method The normalized enzyme activity in whole cells was determined by the rate of indoxyl formation, as quantified by fluorescence detection. Excitation and emission wavelengths were 365 and 470 nm, respectively. Cells were prepared as described above. Five
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microliters of 100 mM indole was added to the cells and the formation of indoxyl was measured after a given reaction time. Centrifugation and handling time for all samples was approximately 80 s.
2.4. Determination of intracellular NADH concentration One milliliter of sample culture was transferred to a microcentrifuge tube. The cells were harvested and washed with phosphate buffer solution (pH 5 7.2) as described above. A mixture of 100 ml of cells with 900 ml of deionized water was injected into the fluorescence detector; the excitation and emission wavelengths were 340 and 425 nm, respectively. The normalized NADH in whole cells was expressed as mmol NADH (mg protein)21 . Solutions of commercially available NADH (Sigma Chemical, St. Louis, MO) were used to prepare a standard curve.
2.5. Harvesting and preparation of cells from biofilms Tod assays and NADH and protein measurements were applied to P. putida F1 biofilm from two different biofilter reactors: a column reactor, 10 cm in diameter by 100 cm long (64 cm bed height) and a differential-volume reactor 10 cm diameter by 10 cm long (1 cm bed height). The differential volume reactor was operated such that toluene degradation was less than 5%. Under these conditions the toluene concentration across the 1 cm bed could be considered uniform. The biofilm in both reactors was supported on a bed of diatomaceous earth pellets (Celite Biocatalyst Carrier R-635, Manville, Denver, CO). The complete biofiltration apparatus, including systems for feeding liquid nutrients and humidified, toluene-contaminated air, is described elsewhere (Cox et al., 1998), as are the experimental procedures used to inoculate, operate, and sample the column. Tod activity, NADH and protein were determined in the biofilm by sampling two pellets from the biofiltration reactor operating at steady-state. Both pellets were mixed with 2 ml of sterile 0.1% Na 2 P2 O 7 and vortex mixed to detach cells from the
pellets. The cell mixture was centrifuged and washed with phosphate buffer solution (pH 5 7.2). The final cell pellets were resuspended in 5 ml of fresh medium. The cells were sonicated for 3 s, followed by a 30 s rest period; this cycle was repeated three times to thoroughly disperse the biofilm. The prepared cell culture was used for measurement of the Tod activity and NADH and protein concentrations as described above.
2.6. Data analysis With the exception of the spectrophotometricbased measurements of Tod activity, each data point in the following figures represents the mean of replicate samples. The standard deviation (S.D.) is shown by an error bar; many error bars are smaller than the data point symbols. All measurements of toluene concentration were made in triplicate. Protein, NADH, and fluorescence-based Tod activity measurements were made in duplicate. The data points for spectrophotometric measurements of Tod activity represent a single measurement because there was insufficient time between samples to process duplicates.
3. Results and discussion
3.1. Determination of optimum emission wavelength for indoxyl The excitation and emission wavelengths established by Gehauf and Goldenson (1957) for indoxyl were confirmed. Indoxyl was generated from a pure culture of toluene-grown P. putida, because a commercial standard was not available. Cell supernatants from the indole assay were subjected to excitation and emission scanning. The emission scan ranged from 400 to 700 nm with a fixed excitation at 365 nm. A peak was observed in the range of 460 to 490 nm, consistent with that reported by Gehauf and Goldenson (1957). The maximum emission occurred at 470 nm; the method was further developed using this wavelength. Indigo and indole did not fluoresce under these conditions.
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3.2. Comparison of enzyme activity assay To demonstrate the effectiveness of the indoxyl assay, indoxyl formation was compared to indigo formation. The increase in absorbance at 600 nm and the increase in fluorescence intensity at 470 nm is shown in Fig. 2 over a period of 10 min. Fluorescence increased to approximately 75% of its maximum intensity within 2 min; thereafter, the rate of increase in fluorescence intensity decreased. Therefore, the Tod activity was calculated as the rate of increase in fluorescence during the first 2 min after addition of indole. All fluorescence readings of cell blanks were less than 10 mV. Since indoxyl is not available commercially, a standard curve relating fluorescence to indoxyl concentration could not easily be developed; therefore, a correlation between fluorescence and indigo production was developed. Fig. 3 shows the whole cell Tod activity in P. putida F1, as measured by fluorescence (Efluorescence, mV min 21 ) correlated with the enzyme activity as given by the rate of indigo production (Eindigo, mg indigo min 21 ) measured spectrophotometrically. Efluorescence can be expressed as the rate of indigo production by the following correlation (r 2 5 0.90) derived from Fig. 3:
Efluorescence Eindigo 5 ]]] 2280
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(1)
Subsequent measurements of Tod activity in suspended growth cells and biofilms were made using the spectrophotometric method and fluorescencebased methods, respectively.
3.3. Specific enzyme activity and NADH concentration profiles during growth phase Normalized whole-cell Tod enzyme activity (Fig. 4a) and NADH (Fig. 4b) profiles were correlated with growth of P. putida F1 on toluene vapor. Tod activity was observed to vary between 3.8 and 7.73 10 24 mg indigo (mg protein)21 min 21 during the log growth phase (OD 600 ,0.9). During the stationary growth phase (OD 600 .0.9), the enzyme activity increased to between 9.0 and 20310 24 mg indigo (mg protein)21 min 21 . Hugouvieux-Cotte-Pattat et al. (1990) reported a similar growth-phase-dependent expression of the TOL plasmid pWW0 catabolic genes by P. putida. Although they were unable to specify a mechanism, they reported that genes were poorly induced during the early-exponential-growth phase but became strongly induced during the late-
Fig. 2. Absorbance and fluorescence intensities as a function of time for indole oxidation by whole cells of Pseudomonas putida F1. Error bars for fluorescence data represent S.D. with n52.
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Fig. 3. Correlation of Tod activity as measured by indoxyl and indigo formation in whole cells of Pseudomonas putida F1. Error bars for fluorescence data represent S.D. with n52.
exponential-growth phase. The specific intracellular NADH concentration (Fig. 4b) maintained a constant value of 0.160.02 mmol (mg protein)21 during the log growth phase (OD 600 ,0.9) and increased linearly after the early-stationary-growth phase (OD 600 . 0.9) to a maximum value of 0.2960.02 mmol (mg 21 protein) .
3.4. Estimation of normalized Tod activity half-life in whole cells Toluene dioxygenase enzyme activity naturally decays over time in the absence of toluene. There are no published reports of Tod half-life either as a purified enzyme or in whole cells. P. putida F1 was grown to OD 600 of 1.0 as previously described. Fifty milliliters of cell suspension was dispensed into two 300 ml flasks after determining the initial enzyme activity of the cells. Only one flask was provided with toluene. Both flasks were placed on a shaker (180 rpm) at room temperature. Five-milliliter sam-
ples were taken periodically and the normalized whole-cell enzyme activity and OD 600 determined. The decrease in normalized whole-cell enzyme activity in both the absence and presence of toluene is shown in Fig. 5. In the absence of toluene, the normalized enzyme activity decreased from 1.623 10 23 to 0.77310 23 mg indigo (mg protein)21 min 21 over a period of 7 h (Fig. 5a). Over the same time period, the normalized whole cell activity in the flask supplied with toluene remained relatively constant at about 2310 23 mg indigo (mg protein)21 min 21 . The rate of normalized whole-cell enzyme decay in the absence of toluene was described by the first order equation: Ex.t 5 Exo e 2kt
(2)
where Ex.t and Exo are the final and initial normalized whole-cell enzyme activities, respectively, t is the incubation time, and k is the decay constant. The value of k was 0.123 h 21 as determined by nonlinear regression (r 2 50.92) with a corresponding half-life of 5?6 h. To confirm the half-life value
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Fig. 4. Measured response of Pseudomonas putida F1 during growth. (a) Normalized Tod activity; (b) normalized NADH concentration; error bars representing S.D. with n52 were smaller than the symbols for all data points.
determined, a second experiment was conducted over a longer period (14.5 h) (Fig. 5b). The normalized whole-cell Tod activity decreased from 8.54310 24 to 2.63310 24 mg indigo (mg protein)21 min 21 in the absence of toluene. Eq. (2) was fitted to the data
and a value of 0.0869 h 21 was determined (r 2 5 0.94); the corresponding half-life was 8 h. With toluene present, the activity remained relatively constant at about 7.5310 24 mg indigo (mg protein)21 min 21 .
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Fig. 5. Decay of normalized enzyme activity in whole Pseudomonas putida F1 cells in suspend batch culture: (a) 7 h experiment, (b) 14.5 h experiment.
3.5. Enzyme activity and NADH concentration in the biofilm Biofiltration experiments were conducted to examine the profiles of toluene, normalized Tod enzyme activity, protein, and normalized NADH concentrations along the column (Fig. 6). Air containing 1000 mg l 21 of toluene was fed continuously to the column at a rate of 500 ml min 21 , corresponding to an empty-bed contact time (EBCT) of 10.0 min. At steady-state, toluene concentration, protein content,
NADH concentration and normalized whole-cell Tod activity were measured along the length of the column. Toluene was consumed in the upper half of the column; its concentration was below detectable levels in the lower half of the column. As expected, Tod enzyme activity and cellular protein decreased with decreasing toluene concentration along the length of the column. The normalized NADH concentration was relatively constant over the entire length of the column at approximately 0.4 mmol (mg protein)21 .
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Fig. 6. Toluene, protein, NADH and enzyme activity profiles of Pseudomonas putida F1 biofilms from a biofiltration column. Error bars for toluene represent S.D. with n53. All other error bars represent S.D. with n52.
Fig. 7. Decay of normalized enzyme activity in whole Pseudomonas putida F1 cells from a differential-volume biofilm reactor. Error bars represent S.D. with n52.
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3.6. Estimation of the half-life of whole-cell Tod activity in biofilms Air contaminated with toluene (1000 mg / l) was continuously fed to the differential-volume biofilm reactor at a flow rate of 500 ml min 21 , yielding an EBCT of 9.4 s. After steady-state conditions had been established, toluene-free air was fed to the reactor and the normalized whole cell enzyme activity measured as a function of time (Fig. 7). The initial normalized enzyme activity decreased from 4.43 10 24 to 1.27310 24 mg indigo (mg protein)21 min 21 over a period of 12.3 h. Eq. (2) was fitted to the data (r 2 50.92) and a k value of 0.0982 h 21 determined. The corresponding half-life was 7.1 h, consistent with the whole-cell enzyme half-life determined under suspended-growth conditions.
4. Conclusion A method of measuring the whole-cell Tod activity in P. putida F1 based on the fluorescent properties of indole was described. Compared to the spectrophotometric method, it was found to be less susceptible to interference by suspended biomass. Application of the assay for both suspended and biofilm cells was demonstrated and the enzyme half-life in each mode of growth determined. Coupled with simultaneous determination of intracellular NADH concentrations, the assay can be used to elucidate factors controlling the biokinetics of metabolic and cometabolic degradation processes in fixed film reactors. Future work will focus on the role of Tod enzyme activity in the cometabolic degradation of TCE during biofiltration.
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