Modeling of the Cometabolic Biodegradation of Trichloroethylene by

Environ. Sci. Technol. 1997, 31, 3044-3052

Modeling of the Cometabolic Biodegradation of Trichloroethylene by Toluene-Oxidizing Bacteria in a Biofilm System JEAN-PIERRE ARCANGELI* AND ERIK ARVIN Department of Environmental Science and Engineering, Technical University of Denmark, Building 115, DK-2800 Lyngby, Denmark

Because of its intensive use in industry, trichloroethylene (TCE) is one of the most widespread contaminants in soil and groundwater. The aerobic biodegradation of TCE depends on the supplement of a primary carbon source, of which toluene appears to be the most efficient/practicable. For this reason, the cometabolic biodegradation of TCE was investigated in a continuously fed biofilm reactor with a mixed culture of toluene degraders. The interaction phenomena between toluene and TCE were studied and modeled in order to develop a kinetic model for the design of treatment processes. TCE degradation ([TCE] ) 40135 mg/L) was dependent upon the presence of toluene; however, if the latter was supplied at concentrations above 1 mg/L, TCE degradation was strongly inhibited. Similarly, TCE inhibits toluene degradation ([TCE] < 50 µg/L). A simple kinetic model which incorporates competitive inhibition between toluene and TCE, as well as the activation effect from toluene, was developed. A fair agreement between modeled and experimental data was found. However, the kinetic model was not able to predict the TCE removal in the absence of toluene (resting cells) or at very low toluene concentrations (i.e., below 0.1 mg/L). Parameter estimation yielded a maximum TCE degradation rate, kX(TCE), of 0.38 ( 0.11 gTCE gx day-1 and a half-saturation constant for TCE, KS(TCE), of 0.17 ( 0.1 mg/L. Furthermore, the model calculations suggested that the active biomass (toluene degraders) accumulated at the top of the biofilm in an active layer of ca. 120 µm. Finally, sensitivity analyses defined the model’s uncertainties to be (30-35% for TCE. The calibrated model is able to predict fairly well the removal of TCE for concentrations ranging from 0 to 5 mg/L.

Introduction Chlorinated aliphatic hydrocarbons (CAH’s) have a wide range of application, as solvents, degreasing agents, and intermediates in chemical synthesis. Therefore, it is not surprising that their extensive use has caused soil and groundwater contamination. These compounds migrate quickly through soils, and many are persistent under aerobic conditions. Furthermore, CAH’s are hazardous to human health, and most of them are confirmed or suspected carcinogens, even at very low concentrations (1). * Corresponding author present address: Membrane Extraction Technology Ltd., c/o Department of Chemical Engineering, Imperial College, London SW7 2BY; UK. Fax: (+44) 171 594 9603; E-mail: [email protected].

3044

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 11, 1997

TCE is the most frequently encountered CAH in contaminated soil and groundwater with concentrations ranging from 1 to 10 000 µg/L. TCE is usually biodegradable as a result of cometabolism in organisms expressing oxygenases. Methanotrophs and micro-organisms which oxidize aromatic compounds are among the micro-organisms capable of degrading TCE. The most extensively studied group of bacteria are the methanotrophs (e.g., refs 2-8), although it may be demonstrated that aromatic-utilizing bacteria are more efficient degraders (9, 10). At the Moffet Field groundwater test site (shallow aquifer), the highest removal efficiency for methanotrophs was 15-25% TCE, while over 90% removal was achieved when phenol stimulation was utilized (11). Similar results have been reported for chemostat experiments: the bacterium Pseudomonas cepacia G4 growing on toluene reached higher removal capacities and would maintain transformation at higher loading rates than would the methanotroph Methylosinus trichosporium OB3b (12). This suggests that toluene could be an attractive primary carbon source for the biological treatment of waste streams and bioremediation of TCE-contaminated sites. In practice, however, its application is hindered by the lack of understanding regarding how biotransformation rates can be optimized. Therefore, kinetic models which are capable of simulating cometabolism are required, in order to quantify cometabolic degradation and to design engineered treatment/ remediation systems. Within the last few years, many models have been proposed for cometabolic degradation by resting cells (e.g., refs 13 and 14) or by growing cells, which take competitive inhibition (15), lag time, and the deactivation of resting cells (16, 17) into account. However, all these kinetic studies focused on cometabolic degradation in batch systems with suspended cultures. Surprisingly, little literature can be found regarding cometabolic degradation in biofilm systems. Kinetic data obtained from batch experiments with suspended cultures often differ from that obtained from biofilm studies due to the matrix-like structure of the biofilm: the substrates have to diffuse into the biofilm, to react with the cells, and the metabolic products have to diffuse back through the biofilm and into the aqueous phase. Also, the exopolymeric substances (EPS) which hold bacteria together in the biofilm can contain exoenzymes that may play a role in the biodegradation of a substrate (18). It was the purpose of this work to investigate the cometabolic degradation of trichloroethylene (TCE) using a mixed culture of toluene-oxidizing bacteria in a biofilm system in order to quantify the influence of toluene on the TCE removal rate and the inhibition effect of TCE on the toluene removal rate. Preliminary experimental results have been discussed elsewhere (19).

Materials and Methods Experimental Setup. The experimental system is shown in Figure 1. It is a so-called Biodrum reactor system consisting of a drum rotating inside another drum (20). The biofilm grows on the surfaces of both the stator and the rotator. The rotation (200 rpm), as well as the recycling system (50 L/H), ensures total mixing of the bulk liquid and consequently promotes a relatively uniform biofilm thickness. The characteristics of the biofilm reactor and the operating conditions for the experiments are described in Table 1. The reactor was inoculated with a mixed culture obtained from a creosotecontaminated sandy aquifer (Frederiksborg, Denmark). The inoculation was accomplished by injecting 100 mL of this culture into the reactor in order to permit the biomass to attach to the walls of the rotator and the stator. Feeding of the reactor was initiated after 2 days. Three experimental

S0013-936X(96)00911-X CCC: $14.00

 1997 American Chemical Society

FIGURE 1. Experimental setup: (1) mineral medium, (2) toluene stock solution, (3) TCE stock solution, (4) aerator (oxygen supply), (5) biofilm reactor, (6) recirculation pump, (7) inlet sampling port, (8) outlet sampling port, (9) temperature and oxygen control, (10) outlet.

TABLE 1. Operational Data for the Biofilm Reactor during the Biofilm Growth surface area reactor volume average flow of water residence time toluene, infl conc oxygen, in the reactor nitrate, infl phosphate, infl temperature alkalinity pH in the reactor

unit

value

m3 L/h min mg/L mg/L mg N/L mg P/L °C mequiv/L

0.16 0.96 × 10-3 4.5 12.8 2.5 4-5 40 46.5 20-21 1.6 6.9-7.1

m2

runs were performed in an identical manner with the same mixed culture: bacteria from one reactor was used to inoculate the next reactor. All experiments were conducted under nonsterile conditions. Two peristaltic pumps were used to feed the substrate: one for the toluene stock solution (500 mg/L) and the other for the mineral solution. The reactor was protected from light in order to prevent growth of photosynthetic organisms. Due to the low influent toluene concentration, it was sufficient to saturate the mineral solution with atmospheric oxygen (9 mg O2/L at 20 °C). Thus, an aquarium aerator applied in the inlet (item 4, Figure 1) was sufficient to satisfy the oxygen demand. However, during kinetic experiments involving high concentrations of toluene, pure oxygen was injected in the mineral medium in order to avoid oxygen limitation and to keep the oxygen concentration in the reactor constant during the entire experiment. Substrate. Toluene was fed continuously to the reactor at a nearly constant concentration of 3.5 mg/L. The mineral

medium contained the following salts dissolved in distilled water (mg/L): NaNO3, 244; CaCl2, 73.5; NaHCO3 100; KCl, 4.7; FeCl3, 6H2O, 1.8; MgSO4, 7H2O, 25; KH2PO4, 688.6; and Na2HPO4, 12H2O, 879.5. The trace minerals were (mg/L) MnCl2, 4H2O, 992; CoCl2, 6H2O, 1192; CuCl2, 2H2O, 852; NaMoO4, 2H2O, 1208; NiCl, 6H2O, 476; and KI, 168. This microionic stock solution was diluted 10000-fold (2 mL of solution in 20 L of mineral medium). Final concentrations after dilution were (µg/L) Mn(II), 27.5; Co(II), 29.5; B, 5.5; Zn(II), 33; Cu(II), 32; Mo(VI), 48; Ni(II), 12; and I, 13. Nitrate was used as a nitrogen source in order to prevent the growth of nitrifying bacteria. Analytical Procedure. TCE and toluene were analyzed by membrane inlet mass spectrometry. This technique involves a membrane which is attached directly to the inlet port of the mass spectrometer. An aqueous sample flows across the membrane’s external surface (21), whereby a small fraction of the aqueous compounds diffuses through the membrane into the mass spectrometer source, where the compounds are detected. A considerable enrichment of hydrophobic and volatile organic compounds, relative to water, can be achieved using a silicone membrane (22, 23). Oxygen was monitored with a Clark-type oxygen electrode (WTW OXY 196). Biofilm Parameters. The biofilm thickness was measured on removable slides (located horizontally on the reactor stator wall) with a stereo microscope equipped with a microruler. The objective lens was lowered to the edge of the slide while focusing on the biofilm cross-section. Comparison of the biofilm cross-section with the microruler gave the biofilm thickness. In addition to this, a small area of the biofilm was scraped off in order to measure the biofilm dry-weight and the protein content. The biofilm dry-weight content was calculated as the attached dry biomass per unit of wet biofilm

VOL. 31, NO. 11, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3045

FIGURE 2. Kinetics of TCE absorption in the reactor (without biomass). volume. Proteins were assayed according to the method of Lowry et al. (24). Experimental Strategy. Prior to inoculation, a control experiment was performed to evaluate the TCE and toluene loss due to absorption in the reactor. The procedure involved supplying a given TCE concentration in the reactor and then measuring its absorption rate in the reactor after hydraulic equilibrium was reached, that is four hydraulic residence times (∼1 h). Afterward, the TCE inlet concentration was increased, and the corresponding TCE adsorption rate was measured as the new hydraulic equilibrium was reached in the reactor. This procedure was done stepwise for each experimental data plotted in Figure 2. Results show that the rate of TCE absorption in the reactor wall followed a first-order expression (Figure 2). Toluene was not absorbed. The biofilm was grown aerobically with toluene as the sole carbon source. Results of the biofilm growth are reported in Arcangeli and Arvin (25). Kinetic experiments on several levels of biofilm thickness were performed. They consisted of continuously supplying the reactor with toluene and TCE in order to assess substrate interaction between the primary substrate (toluene) and the cosubstrate (TCE). The kinetic study was based on two sets of investigations: (i) influence of the toluene concentration on the TCE removal rate and (ii) influence of the TCE concentration on the toluene removal rate. Kinetic Model. The kinetic model is based on a crosscompetitive inhibition between toluene and TCE. A similar model has already been discussed for the cometabolic degradation of o-xylene by toluene under nitrate-reducing conditions (26). The following assumptions are made: (i) the biofilm is homogeneous, and the liquid film diffusion is negligible; (ii) the transport of the substrate, toluene, and cosubstrate, TCE, into the biofilm takes place by diffusion only; (iii) only the substrate and cosubstrate are assumed to be rate limiting; and (iv) the loss of biofilm activity due to substrate toxicity is considered insignificant. Degradation by resting cells is important under circumstances where the primary substrate feeding is discontinued (e.g., refs 27 and 28). Since this work was carried out in a continuously fed biofilm reactor, it is assumed that the biodegradation of the cosubstrate by resting cells is inappropriate except for the case where the toluene was removed from the system. The reaction rate of toluene, which supports the biomass growth, can be described by eq 1, assuming a competitive inhibitory effect of the cosubstrate (29). The inhibition

3046

9


FIGURE 3. Concentration profile of toluene and TCE in a biofilm under circumstances where toluene is the limiting factor. In area 3 the TCE profile is linear due to the molecular diffusion in the biofilm. (- - -) TCE; (-‚-) toluene. coefficient of the competitive inhibitor is approximated by its single-substrate half-saturation coefficient, as proposed by Broholm et al. (15).

Stol rtol ) kX(tol)Xf STCE Stol + KS(tol) 1 + KS(TCE)

(

)

(1)

TCE cannot serve as a source of carbon for cell synthesis, but its transformation is possible when toluene is supplied in the reactor. It is hypothesized that the enzyme that initiates the transformation of TCE is synthesized only in the presence of toluene. Enzyme kinetics are described by a MichaelisMenten type equation (30). TCE degradation is described by eq 2, taking into account the competitive inhibition and the stimulating effect of toluene (i.e., likely the production of reducing equivalents).

rTCE ) kX(TCE)Xf

STCE STCE + KS(TCE)

)(

)

Stol Stol Stol + KS(tol) 1+ KS(tol) (2)

(

Hence, the optimum primary substrate concentration, Stol,opt, at which the cosubstrate degradation is maximized, can be found by differentiation of eq 2 with respect to Stol.

Stol(opt) )

x ( KS(tol)2

)

STCE + KS(TCE) KS(TCE)

(3)

The diffusion transport of both substrates into the biofilm must be considered in a biofilm system. As an example, the profiles of the primary substrate, toluene, and the cosubstrate, TCE, in a biofilm are shown schematically under circumstances in which the primary substrate does not fully penetrate the biofilm (Figure 3). Transformation of the cosubstrate occurs only in area 2. In area 1, the primary substrate is lacking, and in area 3, the primary substrate inhibits cosubstrate degradation almost completely. The concentration profiles of the toluene and the TCE can be calculated by integration of eqs 4 and 5, respectively.

d2Stol dz

2

)

kX(tol)Xf r Df(tol) tol

(4)

)

kX(TCE)Xf r Df(TCE) TCE

(5)

d2STCE dz

2

The Glossary follows the article. Finally, the kinetic model considers two biomasses: an active biomass, capable of degrading toluene and TCE, and an inactive biomass, which comprises dead cells and exopolymers (EPS) and, thus, is unable to degraded these compounds. The two biomasses are not, however, equally distributed in the biofilm. The active biomass accumulates in the top layer of the biofilm (31), and its concentration in the biofilm is assumed to be distributed linearly (i.e., the profile of active biomass in the biofilm is linear; see Figure 7). The thickness, Lfact, of this active layer is a function of the concentration of the primary substrate with which the biofilm grew. It can be determined by model calibration. The determination of the kinetic parameters and the thickness of the active layer are required to solve eqs 4 and 5. Analytical solutions are not possible since they are nonlinear. However, the equations can be solved numerically. For that purpose, the computer program AQUASIM developed by EAWAG in Switzerland was used (32). It is an interactive

program used to identify and simulate aquatic systems. The kinetic model used in this program is based on the formulation described by Wanner and Reichert (33). Model Parameters. The kinetic model consists of eqs 1 and 2 which express the degradation of TCE and toluene and the interaction between these two compounds. Since each kinetic experiment never lasted more than 24 h, the absorption of TCE in the reactor can be approximated by a first-order expression (Figure 2). Thus, a first-order absorption coefficient was estimated from a control experiment without biofilm. During the kinetic experiments (with a biofilm), the calibrated first-order expression was added to the kinetic model (as a third process) so that the absorption of TCE in the reactor could be taken into account. Eight of the model variables describe the experimental conditions. These are the influent and the effluent concentrations of toluene and TCE in the reactor, the biofilm thickness, the flow rate, and the volume and the surface area of the reactor. The solid-phase density parameters required in the kinetic model is a characteristic of the solid-phase, particulate component of the biofilm and is expressed as the mass of dry solids per unit solid-phase volume (i.e., the bacteria dryweight density). The value was estimated to be 168 000 g/m3. This calculation was based on a relationship proposed by Norland et al. (34), which correlates the dry matter and the volume of bacteria. The bacteria biovolume was determined as 0.67 µm3 (P. cepacia) according to the results of examination of pure cultures isolated from this toluene-utilizing mixed culture (9). The kinetic model assumed a liquid-volume fraction of 85.5 ( 3.5% estimated according to an average dry-weight content of 24 400 ( 5500 g/m3 and an average cell dry-weight of 0.113 pg/cell (34). This calculation assumes that all the cells in the biofilm have the same volume as P. cepacia. The substrate diffusion coefficients in water were estimated using the Wilke and Chang method (35). Values of 7.8 × 10-5 and 8.6 × 10-5 were found for TCE and toluene, respectively. The ratio of the diffusivity in biofilm by the diffusivity in water was set to 0.8 (36).

Results Experimental Results. In the first experiment series (Figure 4, panels A and B), both toluene and TCE were supplied

FIGURE 4. Kinetic experiments with which the kinetic model was calibrated. Dark square, experimental data. Solid line, model. Dashed line, uncertainty of the model. (A and B) Influence of toluene on the TCE removal rate (TCE inlet concentration )135 µg/L). (C and D) Influence of TCE on toluene degradation rate (toluene inlet concentration ) 2.5 mg/L).


9

3047

TABLE 2. Estimated Parameter Valuesa KS(TCE) KS(tol) kX(TCE) kX(tol) Lfact a

unit

value

SD

mg/L mg/L gTCE gx-1 day-1 gtol gx-1 day-1 µm

0.173 0.027 0.377 1.53 122

(0.097 (0.017 (0.107 (0.153 (14

Abbreviations follow the article.

continuously to the reactor. The toluene concentration was varied in the range 0-12 mg/L, while the TCE inlet concentration was kept constant at 135 µg/L. The interaction between toluene and TCE is obvious. Figure 4A illustrates that toluene promoted the TCE degradation. The small removal of TCE that was observed in the absence of toluene can be explained by the combined effect of sorption and resting cells. In contrast, when toluene was provided in concentrations above 1.5-2 mg/L, degradation of TCE was inhibited. The maximum TCE surface removal rate of 0.045 g m-2 day-1 occurred with a toluene concentration ranging from 0.2 to 0.5 mg/L. At this optimum toluene concentration, the TCE transformation yield was approximately 0.05 g of TCE removed/g of toluene degraded. In the second experiment series (Figure 4, panels C and D), the TCE concentration was increased in the reactor, from 0 to 2 mg/L, while the toluene inlet concentration was kept constant at 2.3 mg/L (from 0.5 to 0.8 in the reactor). An inverse relationship was observed between toluene degradation and TCE concentration. The toluene removal rate decreased even at low TCE concentrations (below 50 µg/L). This inhibitory effect by TCE appears, however, to be reversible, as the toluene removal rate returned to its initial level when the TCE supply to the reactor was stopped (data not shown). A first-order reaction can be used as an approximation for the TCE transformation at TCE concentrations below 0.25 mg/L. The TCE transformation yield increased with an increasing TCE concentration up to 0.2 g of TCE removed/g of toluene degraded. Modeling and Estimation of Kinetic Parameters. An identifiability analysis was performed prior to parameter estimation which checks if the kinetic model parameters can be uniquely determined. The details of this procedure, as well as the numerical methods employed, are given by Reichert (32) and Reichert et al. (37). Results of this analysis (not shown) indicate that kinetic parameters and the depth of the active layer were individually identifiable. This requires, however, that a simultaneous fit be performed with the data from degradation experiments shown in Figure 4. Estimated values for the TCE and toluene kinetic parameters (both the half-saturation constant and the maximum degradation rate) as well as the thickness of the biofilm active layer are compiled in Table 2. The standard deviations were calculated during

the estimation procedure. A comparison of the experimental and the modeled data is shown in Figure 4. The observed data and the model account for the removal of TCE by biodegradation and by sorption. In general, the agreement is very good. The instability of the model shown in Figure 4D is due to the toluene inlet concentration which could not be maintained constant. The toluene degradation rate decreased for a toluene concentration above 4-5 mg/L (Figure 4B). This phenomenon of substrate inhibition could not be modeled, since it was not taken into account in the kinetic model. Variability Analysis. A sensitivity test was performed on this kinetic model which involved altering various model parameters independently to assess how much these variations affect the modeled curves. This task can be performed automatically by AQUASIM, providing that the standard deviations of each parameter are given. Factors investigated were kinetic parameters, substrate diffusivity, dry-weight density, and active biomass distribution in the biofilm. The standard deviations used for this test were those calculated in the estimation procedure (Table 2). A relative standard deviation of 20% was used for the biomass density and the effective diffusivity of substrates. The estimation of the diffusion coefficient for toluene and TCE in water leads to an average error of about 10% (35). The results, which are summarized in Table 3, indicate that the most influential factors are the solid-volume fraction, the active biomass depth, and the biomass density. The kinetic parameters related to toluene affect the toluene and TCE removal rate, whereas kx(TCE) and KS(TCE) only affect the TCE removal rate, and have a minor effect on toluene removal. The substrate diffusivity does not have a significant influence on the modeling. However, the model is more sensitive to the toluene diffusion coefficient than to that of TCE. All together, the uncertainty of the model is depicted in Figure 4: the envelopes defined with dashed lines describe the maximum variation of the model using the parameters and their respective standard deviations listed in Table 3. On average, the variability of the model ranges from 20 to 35%. The variability is somewhat higher for the modeling of TCE removal compared with the variability in the toluene removal modeling. Calibration of the Model. The kinetic model was calibrated using experimental data from Figure 4 representing the whole TCE and toluene concentration range, which is generally encountered in contaminated groundwater or industrial wastewater. However, before applying this kinetic model to design of treatment processes, it is important to assess the precision and the application range of the model. Therefore, the calibrated model was tested using other experimental data. Two series of experimental data were modeled. The first series (Figure 5) was obtained from a separate kinetic experiment conducted during the same biofilm growth campaign as the kinetic experiments which was used to

TABLE 3. Sensitivity Analysis. Effect of Kinetic Factors on the Toluene and TCE Removal Ratea run 1

KS(TCE) KS(tol) kX(TCE) kX(tol) Lfact FXf Solid f D(tol) D(TCE) a

run 2

unit

value

SD

toluene deg

TCE deg

toluene deg

TCE deg

mg/L mg/L gTCE gx-1 day-1 gtol gx-1 day-1 µm g/m3 m2/d m2/d

0.173 0.027 0.377 1.53 122 168 000 0.145 0.8 8.6 × 10-5 7.8 × 10-5

(0.097 (0.017 (0.107 (0.153 (14 (33400 (0.035 (0.16 (9 × 10-6 (8 × 10-6

+ + ++ ++ ++ ++ + + -

++ ++ ++ + ++ ++ ++ + + +

++ ++ + ++ + ++ ++ ++ ++ -

++ ++/+ ++ + ++ ++ ++ + +

-, no significant effect; +, moderate effect; ++, significant effect. Abbreviations follow the article.

3048

9


FIGURE 5. Verification of the model. Effect of toluene on TCE transformation rate (TCE inlet concentration, 40 µg/L). Dark square, experimental data. Solid line, model. Dashed line, uncertainty of the model.

FIGURE 6. Verification of the model. In this histogram two bars are represented. The first depicts the calibrated model with the uncertainty, the second shows the experimental data. On the abscissa, two lines are shown: the upper one is the toluene concentration and the other one is the TCE concentration. calibrate the model (Figure 4). As described previously, the toluene concentration in the reactor was increased stepwise, whereas the TCE concentration was maintained constant (ca. 40 µg/L). The agreement between the model and the experimental data is poor (Figure 5). The reason may be that the TCE inlet concentration in this kinetic experiment was outside the concentration range with which the model was calibrated. Nevertheless, several of the experimental data are included in the variability “envelope” defined by the dashed lines. Furthermore, the trend of the model is in agreement with the experimental data. The second series of experimental data to be modeled was obtained from a separate kinetic experiment conducted with another biofilm. The biofilm grew under conditions

similar to those described above (temperature and nutrient concentration) and with the same mixed culture of tolueneoptimizing bacteria. As the biofilm reached a thickness of about 600 µm, TCE was supplied to the reactor. Various concentration levels of toluene and TCE were investigated. The surface removal rate of TCE is plotted in a histogram (Figure 6). The comparison between the kinetic model and the kinetic experiment is fairly good. A better fit of the experimental data was achieved by adjusting the kinetic parameters. However, it was of interest to evaluate the accuracy of the calibrated model. Therefore, only input parameters such as the liquid flow in the reactor, the biofilm thickness, and the inlet concentration of TCE and toluene were adjusted in order to match the experimental conditions.


9

3049

TABLE 4. Values Reported in the Literature for TCE Degraders Growing of Toluene as Carbon Sourcesa T (°C) Pseudomonas cepacia G4 Pseudomonas cepacia G4

chemostat fed-batch reactor Pseudomonas putita F1 batch Pseudomonas putita F1 biofilm mixed culture batch this study biofilm Methylosinus trichlosporium OB3b batch

kX(Tol) (gtol gx-1 day-1) KS(Tol) (mg/L) kX(TCE) (gTCE gx-1 day-1) KS(TCE) (mg/L) ref

28 28 30 30 26-28 20 30

9.27 0.207 16.4c 0.44 1.53 8.36f

2.3

13.8c 1.02 0.027 1.47g

0.95 0.083 0.68b,e 0.0362d,e 0.17 0.377 56.6

0.79

8.64 0.173 19.6

12 45 43 44 51 39

a

For comparison, the last row shows kinetic parameters related to a well know methanotroph, Methylosinus trichosporium OB3. b Assuming 50% protein in the cells. c Measured in chemostat 30 °C. d Estimated from a TCE degradation rate of 867.5 g/day m-3 of biofilm and assuming a dry weight density of 24 400 g/m3 of biofilm, TCE concentration 80 µmol. e Initial TCE degradation rate observed with a TCE conc of 10.5 mg/L. f kx(CH4) (gCH4 gx-1 day-1). g KS(CH4) (mg/L).

Discussion The experimental results shown in Figures 4-6 suggests that this mixed culture was active in degrading TCE. A maximum degradation rate of 0.25 g of TCE/g of biomass day-1 and a transformation yield up to 0.2 g of TCE removed/g of toluene degraded were found. For comparison, the values reported in the literature for pure and mixed cultures are compiled in Table 4. In general, the maximum transformation rates of TCE, kx(TCE), are within the same order of magnitude, although only few studies have been conducted. However, compared to the maximum transformation rate achieved with the methanotroph M. trichosporium OB3b (38, 39), the mixedculture transformation rate is much lower. The low measured values of the half-saturation constant, KS(TCE), indicate that toluene degraders may have an affinity to degrade TCE at low concentration similar to the Methylosinus strain. Indeed, a first-order rate constant, kx(TCE)/KS(TCE), of 2.2 m3 day-1 gx-1 was found in this study, whereas Oldenhuis et al.’s (38) reported value of 2.9 m3 day-1 gx-1 for the M. trichosporium OB3b. Unlike the experiments reported in the literature, this study was carried out with a mixed culture of toluene-oxidizing bacteria. Therefore, attempts were made to characterize this culture. It appears from the work of Jensen (9) that at least seven different strains were present in the mixed culture. Among these strains, however, five isolates were able to grow on toluene and only two isolates were capable of degrading TCE. These latter were identified as P. cepacia and an Acinetobacter strain. The Pseudomonas strain was the most active in terms of TCE transformation. A detailed kinetic study showed that the TCE transformation rate was strongly affected by the concentration of the primary substrate, toluene: toluene promotes the TCE degradation, on the other hand, if provided in excess, the toluene inhibits the TCE transformation. Consequently, an optimum toluene concentration is required in order to sustain the biomass growth and to maximize the TCE transformation. This work suggests that this optimum concentration ranges from 0.2 to 0.5 mg/L. This is in agreement with the results reported by Jensen (9): in batch experiments, the transformation of TCE was delayed until the toluene concentration was below 0.2 mg/L. Toluene degradation was inhibited only to a small degree by the TCE. Competitive inhibition is probably responsible of this interaction phenomenon, assuming that TCE and toluene are degraded by the same enzyme, and that this enzyme has a lower affinity for TCE than for toluene. Although we did not investigate the enzyme activity of this mixed culture, it has been shown that toluene mono-oxygenase (40-42) and/or toluene dioxygenase (43) are responsible for the oxidation of both toluene and TCE. Similarly, Landa et al. (12) described the cometabolic degradation of TCE with P. cepacia G4 by a MichaelisMenten-type equation, assuming a competitive inhibition between the substrate (toluene) and the contaminant (TCE). They reported maximum utilization rate for toluene and TCE higher than those found in this study, presumably the result

3050

9


of temperature differences (Table 4). However, their kinetic model does not include an activation term as described in eq 2. This could lead at an overestimation of the TCE removal rate when very low concentrations of toluene are encountered. From an investigation of TCE degradation with P. putita F1, Reardon et al. (44) also showed that toluene competitively inhibits TCE degradation and reported a TCE degradation rate of 0.036 gTCE gx-1 day-1 (see footnote in Table 4). They worked with a biofilm reactor continuously fed with toluene and TCE. This is a surprisingly low value when compared with that reported by Wakett and Gibson (43) for the same strain (Table 4). However, the latter worked with resting cells whereas Reardon et al. (44) maintained the toluene concentration at a level of 6.5 mg/L. The inhibition of toluene was minimized by operating through pulse-feeding of toluene, leading to an increase of 35% in the TCE transformation (44). Apparently, TCE was not lethal for this mixed culture: the inhibition of toluene removal due to the presence of TCE was reversible. Nevertheless, the stability of this mixed culture on a long-term basis is unknown. Landa et al. (12) demonstrated that P. cepacia can steadily and continuously degrading toluene and TCE in a chemostat without any inhibition. However, under nongrowth conditions, the same strain appeared to be sensitive to TCE (45) since the presence of TCE led to a 4-fold increase in the maintenance energy demand and mutants appeared which were no longer able to grow on toluene or to degraded TCE. Modeling. A simple kinetic model satisfactorily described the cometabolic transformation of TCE with toluene as a primary substrate. The model was calibrated and could be used to simulate other kinetic experiments with a reasonable agreement (Figure 5 and 6). However, the model could not fit the amount of active biomass in the biofilm, if this term is expressed in terms of proteins. The concentration of protein in the biofilm was measured as 4042 g/m3 of wet biofilm. Assuming that a dry bacteria is made up of 50% protein (46), the amount of active biomass in the biofilm can be estimated as 33% (using a measured dry-weight density of 24 400 g/m3). However, according to the model calculation, the amount of active biomass in the biofilm was hardly above 10% (Figure 7). Such a discrepancy may be explained by the fact that the active biomass calculated by the model is that involved in the degradation of toluene and TCE only. Other types of bacteria growing on hydrolysis products may coexist in the biofilm. This hypothesis is consistent with the work of Jensen (9), who characterized this mixed culture. It appears from her investigation that toluene degraders constitute 90% of the mixed culture. However, among the toluene degraders which were isolated, only a fraction of about 30% were also efficient TCE degraders. Finally, proteins are also present in the extracellular matrix, comprising up to 10-15% of the matrix’s total mass (47). Consequently, expressing the active biomass in term of proteins leads to an overestimation of the active cells. Among the parameters listed in Table 3, the active biomass thickness, Lfact, is the most difficult parameter to assess

FIGURE 7. Simulated biomass concentration profile in the biofilm for the kinetic experiment shown in Figure 4. The concentration of the total biomass was 24 400 g/m3 (input data). The concentration of active biomass was approx. 2700 g/m3 (calculated data). Solid line, inactive biomass; dashed line: active biomass.

FIGURE 8. Simulation of TCE removal rate versus toluene concentration for various TCE inlet concentrations: (O) 0.1 mg/L; (∆) 0.5 mg/L; (×) 1 mg/L; (0) 2.5 mg/L; (]) 5 mg/L. experimentally. From model calibration, Lfact was estimated to be 122 µm. This is approximately one-fifth of the total biofilm thickness measured experimentally. A possible explanation of this thin active thickness is that the biofilm grew with a low concentration of toluene. This resulted in a partly penetrated biofilm as the thickness exceeded a certain value. Then, the toluene degraders accumulated in the top of the biofilm where the substrate was not limiting. Therefore, if the substrate was maintained constant during the entire period of biofilm growth, the thickness of this active biofilm layer would be similar to the substrate penetration in the biofilm. Hence, as a first approximation, the toluene penetration can be estimated using half-order degradation kinetics in a biofilm (48):

toluene penetration )

x

Stol 2Df,tol kX,tolXf

where Df,tol is the diffusion coefficient in the biofilm (m2/

day), kX,tol is the maximum degradation rate constant (gtol/gx day-1), Xf is the concentration of total biomass in the biofilm (gx/m3), and Stol is the toluene concentration in the bulk (g/ m3). The maximum degradation rate constant, kX,tol, is expressed per gram of total biomass since the total biomass in the biofilm can easily be measured (kX,tol ) 0.17 gtol/gx day-1). In this work, the biofilm was grown with an average toluene concentration in the reactor of 0.6 g/m3. From the above equation, the substrate penetration is estimated to be approximately 160 µm. This is in reasonable agreement with the result of the estimation procedure, 122 µm (Table 2). Simulation. Following the calibration of the proposed kinetic model, a series of simulations were performed. The goal of these simulations was to predict the concentration of the toluene in the reactor and thus, the amount of toluene which has to be amended in the reactor in order to maximize the degradation of a given TCE concentration. Computer simulations showed that in order to treat an influent TCE concentration ranging from 0.1 to 5 mg/L the optimal


9

3051

concentration of toluene is 0.3-0.5 mg/L (Figure 8). This is of practical interest, because the maximum USEPA drinking water concentration (MCL) permitted for toluene is 1 mg/L (49). For comparison the MCL for TCE is 0.005 mg/L. Furthermore, toluene is a readily biodegradable substrate, especially under aerobic condition (i.e., refs 25 and 50). This suggests that using toluene as a primary substrate to enhance the biodegradation of TCE is a feasible technique. On the basis of this study, it is concluded that a bioreactor using a toluene-oxidizing biofilm can be implemented to treat TCE-contaminated groundwater. The kinetic model that was developed is able to predict the removal of TCE with a standard deviation of (30-35% for TCE concentrations ranging from 0 to 5 mg/L. However, further work is needed in order to characterize the biofilm (i.e., biomass density, solid-volume fraction, and the depth of the active biofilm) in order to improve the accuracy of the model when it is used for reactor design.

Acknowledgments This study was funded by the Commission of the European Communities. Contract EV5V-CT92-0239; BIODEC project.

Glossary Stol STCE Stol (opt) kX(tol) KS(tol) kx(CH4) KS(CH4) KS(TCE) kX(TCE) Df(tol) Df(TCE) f Lfact FXf Solid

toluene concentration (g/m3) TCE concentration (g/m3) optimum toluene concentration (g/m3) maximum toluene utilization rate (gtol gx-1 day-1) toluene half-saturation constant (g/m3) maximum methane utilization rate (gCH4 gx-1 day-1) methane half-saturation constant (g/m3) TCE half-saturation constant (g/m3) maximum TCE utilization rate (gTCE gx-1 day-1) diffusion coefficient of toluene into the biofilm (m2/day) diffusion coefficient of TCE into the biofilm (m2/ day) effective diffusion coefficient (-) thickness of the biofilm active layer (mm) dry weight density of the bacteria (Pseudomonas cepacia) (g/m3) solid-volume fraction in the biofilm (-)

Literature Cited (1) Eder, E. Chemosphere 1991, 23, 1783. (2) Fogel, M. M.; Taddeo, A. R.; Fogel, S. Appl. Environ. Microbiol. 1986, 51, 720. (3) Little, C. D.; Palumbo, A. V.; Herbes, S. E.; Lindstrom, M. E.; Tyndall, R. L.; Gilmer, P. J. Appl. Environ. Microbiol. 1988, 54, 951. (4) Janssen, D. B.; Grobben, G.; Hoekstra, R.; Oldenhuis, R.; Witholt, B. Appl. Microbiol. Biotechnol. 1988, 29, 392. (5) Oldenhuis, R.; Oedzes, J. Y.; Van der Waarde, J. J.; Janssen, D. B. Appl. Environ. Microbiol. 1991, 57, 7. (6) Tsien, H. C.; Brusseau, G. A.; Hanson, R. S.; Wackett, L. P. Appl. Environ. Microbiol. 1989, 55, 3155. (7) Broholm, K.; Christensen, T. H.; Jensen, B. K. Water Res. 1993, 27, 215. (8) Oldenhuis, R.; Janssen, D. B. In Microbial growth on C1 compounds; Murrel, J. C., Kelly, D. P., Eds.; Intercept Ltd.: Andover, United Kingdom, 1993; pp 121-133. (9) Jensen, H. M. Ph.D. Dissertation, The Technical University of Denmark, 1994. (10) Bielefeldt, A. R.; Stensel, H. D.; Strand, S. E. J. Environ. Eng. 1995, 121, 791. (11) Hopkins, G. D.; Semprini, L.; McCarty P. L. Appl. Environ. Microbiol. 1993, 59, 2277. (12) Landa, A. S.; Sipkema, E. M.; Weijma, J.; Beenackers, A. C. M.; Dolfing, J.; Janssen, D. B. Appl. Environ. Microbiol. 1994, 60, 3368.

3052

9


(13) Alvarez-Cohen, L.; McCarty P. L. Appl. Environ. Microbiol. 1991, 57, 228. (14) Flyvbjerg, J.; Jørgensen, C.; Arvin, E.; Jensen, B. K.; Olsen, S. K. Appl. Environ. Microbiol. 1993, 59, 2286. (15) Broholm, K.; Christensen, T. H.; Jensen. B. K. Water Res. 1992, 26, 1177. (16) Criddle, C. S. Biotechnol. Bioeng. 1992, 41, 1048. (17) Chang, M. K.; Voice, V. C.; Criddle, C. S. Biotechnol. Bioeng. 1993, 41, 1057. (18) Wilderer, P. A.; Characklis, W. G. In Structure and Function of Biofilms (Dahlem Workshop reports); Characklis, W. G., Wilderer, P. A., Eds.; Life Science Research Report 46; Wiley: New York, 1989; pp 5-17. (19) Arcangeli, J. P.; Arvin, E. Jensen, H. M. In Bioremediation of Chlorinated Solvents; Hinchee, R. E., et al., Eds.; Battelle Press: Columbus, Richland, 1995; pp 203-211. (20) Kristensen, G. H.; Jansen, J. l. C. Technical report 80-58; Department of Environmental Engineering, Technical University of Denmark, 1980. (21) Lauritsen, F. R. Int. J. Mass Spectrom. Ion Processes 1990, 95, 259. (22) Kotiaho, T.; Lauritsen, F. R.; Choudhury, T. K.; Cooks, R. G.; Tsao, G. T. Anal. Chem. 1991, 63, 875A. (23) Lauritsen, F. R.; Lloyd, D. In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C. Ed.; Symposium Series 541, American Chemical Society 1994; pp 91-106. (24) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (25) Arcangeli, J. P.; Arvin, E. Appl. Microbiol. Biotechnol. 1992, 37, 510. (26) Arcangeli, J. P.; Arvin, E. Biodegradation. 1995, 6, 19-27. (27) Alvarez-Cohen, L.; McCarty, P. L. Environ. Sci. Technol. 1991, 25, 1381. (28) Sae´z, P. B.; Rittmann, B. E. Biodegradation 1993, 4, 3. (29) Bailey, J. E.; Ollis, D. F. Biochemical engineering fundamentals; International student edition; McGraw-Hill: Tokyo, 1977. (30) Lehninger, A. L. Biochemistry; Worth Publishers Inc.: New York, 1972. (31) Anderson, J. E.; McCarty, P. L. J. Environ. Eng. 1994, 30, 379. (32) Reichert, P. Water Sci. Technol. 1994, 30, 21. (33) Wanner, O.; Reichert, P. Biotechnol. Bioeng. 1996, 49, 172. (34) Norland, S.; Heldal, M.; Tumyr, O. Microb. Ecol. 1987, 13, 95. (35) Perry, R. H.; Green, D. Perry’s Chemical Engineers’ Handbook, 6th ed.; McGraw-Hill International Editions: Singapore, 1987. (36) Christensen, B. E.; Characklis, W. G. In Biofilms; Characklis, W. G., Marshall, K. C. Eds.; Wiley: New York, 1990; pp 93-130. (37) Reichert, P.; von Schulthess, R.; Wild, D. Water Sci. Technol. 1995, 31, 135. (38) Oldenhuis, R.; Vink, R. L. j. M.; Janssen, D. B.; Witholt, B. Appl. Environ. Microbiol. 1989, 55, 2819. (39) Oldenhuis, R.; Oedzes, J. Y.; Van der Waarde, J. J.; Janssen, D. B. Appl. Environ. Microbiol. 1991, 57, 7. (40) Shields, M. S.; Montgomery, S. O.; Chapman, P. J.; Cuskey, S. M.; Pritchard P. H. Appl. Environ. Microbiol. 1989, 55, 1624. (41) Shields, M. S.; Montgomery, S. O.; Cuskey, S. M.; Chapman, P. J.; Pritchard, P. H. Appl. Environ. Microbiol. 1991, 57, 1935. (42) Shields, M. S.; Reargin, M. J.; Gerger, R. R.; Campbell, R.; Sommerville, C. Appl. Environ. Microbiol. 1991, 61, 1352. (43) Wackett, L. P.; Gibson. D. T. Appl. Environ. Microbiol. 1988, 54, 1703. (44) Reardon, K. F.; Conuel, J. R.; Mosteller, D. C. In Proceedings of the 9th Conference on Hazardous Waste Remediation; Erickson, L. E., Ed.; EPA, 1994. (45) Mars, A. E.; Houwing, J.; Dolfing, J.; Janssen, D. B. Appl. Environ. Microbiol. In press. (46) Sperandio, A.; Pu ¨ chner, P. Wasser-Abwasser (Munich) 1993, 134, 482. (47) Ford, T.; Sacco, E.; Black, J.; Kelley, T.; Goodacre, R.; Berkeley, R. C. W.; Mitchell, R. Appl. Environ. Microbiol. 1991, 57, 1595. (48) Harremoe¨s, P. In Water Pollution Microbiology; Mitchell, R., Ed.; John Wiley: New York, 1978; Vol. 2, pp. 82-109. (49) Pontuis F. W. J. Am. Water Works Assoc. 1995, February, 48. (50) Nielsen, P. H.; Bjerg, P. L.; Nielsen, P.; Smith, P.; Christensen, T. H. Environ. Sci. Technol. 1996, 30, 31. (51) Chang, H. L.; Alvarez-Cohen, L. Biotechnol. Bioeng. 1995, 45, 440.

Received for review October 24, 1996. Revised manuscript received April 4, 1997. Accepted July 15, 1997.X ES9609112 X

Abstract published in Advance ACS Abstracts, September 1, 1997.

Modeling of the Cometabolic Biodegradation of Trichloroethylene by

Recommend Documents