Cometabolic transformation of 2-chlorophenol and 4-chlorophenol in the presence of phenol by Pseudomonas putida.

Loh, Kai-Chee ; Wu, Tingting

Biotransformation of 2-chlorophenol (2-cp) and 4-chlorophenol (4-cp) in the presence of phenol by Pseudomonas putida (ATCC 49451) was investigated. Strain ATCC 49451 was unable to utilize 2-cp and 4-cp as the sole carbon and energy source. In the presence of phenol as a growth substrate, 2-cp and 4-cp could be transformed through cometabolism. It was found, however, that cell growth and phenol degradation were strongly inhibited by the presence of 2-cp and 4-cp. A much longer lag phase (19 h versus 3 h) occurred with the mere addition of 40 mg/L 2-cp and 100 mg/L 4-cp. Further increase in 2-cp and 4-cp concentrations resulted in incomplete transformation: only 80% of the initial 100 mg/L 4cp and 50% of the initial 40 mg/L 2-cp could be degraded in the presence of 200 mg/L phenol. Interactions between substrates affected cell growth and substrates degradation significantly and both 2-cp and 4-cp were toxic to the cells. Kinetic models for cell growth as well as substrate transformation were established to simulate the experimental data. The form of the kinetic models and magnitude of the model parameters ([K.sub.2] = 5.62 mg/L > [K.sub.3] = 3.57 mg/L; [k.sub.d2] = 17.8 mg/L < [k.sub.d3] = 51.5 mg/L) indicate that 2-cp and 4-cp exhibited different inhibition and toxicity effects on the cells and their degradation capacities. Kinetics also revealed that the toxicity effect of the chlorophenols dominated over the competitive inhibition effect.

On a etudie la biotransformation du chlorophenol-2 (cp-2) et du chlorophenol-4 (cp-4) en presence de phenol par Pseudomonas putida (ATCC 49451). La souche ATCC 49451 a ete incapable d'utiliser le cp-2 et le cp-4 comme seules sources de carbone et d'energie. En presence de phenol comme substrat de croissance, le cp-2 et le cp-4 pourraient etre transformes par le cometabolisme. On a trouve cependant que la croissance des cellules et la degradation du phenol etaient fortement inhibees par la presence de cp-2 et de cp-4. Une phase de retard beaucoup plus longue (19 h au lieu de 3 h) survient avec le simple ajout de 40 mg/L de cp-2 et de 100 mg/L de cp-4. Une augmentation subsequente des concentrations de cp-2 et de cp-4 donne une transformation incomplete : seulement 80 % des 100 mg/L de cp-4 initial et 50 % des 40 mg/L de cp-2 initial ont pu etre degrades en presence de 200 mg/L de phenol. Des interactions entre les substrats influent de facon significative sur la croissance des cellules et la degradation des substrats, et tant le cp-2 que le cp-4 sont toxiques pour les cellules. On a etabli des modeles cinetiques pour la croissance des cellules et la transformation des substrats afin de simuler les donnees experimentales. La forme des modeles cinetiques et la grandeur des parametres de modeles ([K.sub.2] = 5,62 mg/L > [K.sub.3] = 3,57 mg/L; [k.sub.d2] = 17,8 mg/L < [k.sub.d3] = 51,5 mg/L) indiquent que le cp-2 et le cp-4 ont differents effets d'inhibition et de toxicite sur les cellules et leurs capacites de degradation. La cinetique revele egalement que l'effet de toxicite des chlorophenols l'emporte sur l'effet d'inhibition qui est en competition.

Keywords: cometabolic transformation, inhibition, toxicity, kinetic model, ternary substrate system

INTRODUCTION

Chlorophenols are on the United States Environmental Protection Agency's list of priority pollutants because they are toxic and persistent in the environment. More importantly, these compounds are carcinogenic, thus imposing a threat to human health (Goswami and Singh, 2002). Chlorinated phenols have been widely used as biocides and as precursors in the synthesis of other pesticides since the early 1930s (Hale et al., 1994). Pentachlorophenol (PCP) is the second most heavily used pesticide in the United States (Annachhatre and Gheewala, 1996). The annual production of PCP in 1984 was about 35 000 to 40 000 tons (Haggblom and Valo, 1995), not including the production in the former Eastern Block countries, and the annual production in 1970-1980 might have been close to 90 000 tons (Detrick, 1977; Dougherty, 1978). Although reliable data on the recent production levels of chlorophenols other than PCP are not available in open literature, it was reported that in 1975, the annual worldwide production of all chlorophenols was estimated to be 200 000 tons, of which approximately 80% was used by the wood-preserving industry (Ahlborg and Thunberg, 1980). More than half of these consisted of chlorophenols other than PCP--predominantly 2, 4-dichlorophenol (DCP), 2, 4, 5-trichlorophenol (TCP) and 2, 3, 4, 6-tetrachlorophenol (TTCP) (WHO, 1989). It was also reported that European production levels were 4.5 and 9.1 million kg for total monochlorophenols and 2, 4-DCP, respectively (Krijgsheld and Gen, 1986).

As a result of the multiple pathways that various chlorophenols can enter the environment, they have been detected in air, soil, surface waters and groundwaters and hence their fate in the environment is of great importance (Abrahamsson and Klick, 1991). Due to their inherent toxicity and persistence in the environment, the use of chlorophenols has recently been restricted or banned in several countries, such as Sweden, Finland, Germany and Singapore while they are still in use for wood-preservation in some other countries (Haggblom and Valo, 1995). Furthermore, the continued use of chlorophenols over the past several decades has frequently caused serious local contamination both during normal operation and after accidental spills (Renberg et al., 1983; Patterson and Liebscher, 1987; Lampi et al., 1990, 1992a, b).

Among the different methods used to treat chlorophenol contamination, biotransformation has been proved to be an effective and economical treatment technology. Usually, highly chlorinated phenols are degraded by anaerobic cultures while aerobic micro-organisms are effective for the degradation of mono- and di-chlorophenols (Piero et al., 1999). It has been shown that a big range of chlorinated solvents, including lower chlorophenols, can be degraded cometabolically under aerobic conditions (Alvarez-Cohen and Speitel, 2001). However, the necessity for the presence of a growth substrate for the transformation of the non-growth substrate makes cometabolism much more complicated. Moreover, chlorophenols commonly co-exist in the environment and the interactions between them can greatly influence the cell growth and biotransformation rates, hence the fate of these compounds in the environment. Since very often such mixtures of chlorinated phenols are the targets of biodegradation, and cell growth and substrates degradation have shown much different behaviour from that for single substrate, kinetics study, which focuses on the interactions among the substrates, is necessary and of great significance. For multiple substrates degradation systems, much research has reported the phenomena of competitive and uncompetitive inhibition, cell decay and death, enzyme inactivation and recovery (Ely et al., 1995; Chang and Alveraz-Cohen, 1995; Chang and Criddle, 1997; Aziz et al., 1999; Wang and Loh, 2000, 2001).

This research addressed quantitatively the cometabolic transformation of mixtures of phenol (growth substrate), 2-chlorophenol and 4-chlorophenol (both are non-growth substrates). Lower chlorinated phenols have shown carcinogenic traits (Colin et al., 1998). In addition, a lot of investigations showed that pentachlorophenol and other highly chlorinated phenols were degraded to mixtures of singly substituted ones (Abrahamsson and Klick, 1991). Cometabolism of monochlorinated phenols in the presence of phenol has been shown to be an effective treatment means of these toxic compounds (Saez and Rittmann, 1993; Loh and Wang, 1998; Kim and Hao, 1999; Hao et al., 2002). Considering the effects of -Cl and -OH on reactivity in the substituted aromatic ring, Menke and Rehm (1992) proposed that the order of monochlorophenols degradability could be phenol >4-cp >2-cp >3-cp. However, Dapaah and Hill (1992) observed quite differing behaviour in their report on the biodegradation of mixtures of the three monochlorophenols by Pseudomonas putida. They attributed the observations to different inhibition effect of the chlorine atom in the ortho position between the lag and log growth phases of the cells during biodegradation. The major objective of this study was to investigate the different inhibition and toxicity effects of the chlorinated isomers both experimentally and by the adaptation of earlier developed mathematical models based on the kinetics of microbial growth and substrate utilization.

MODEL DEVELOPMENT

Growth Kinetics

On the basis of the study of Wang and Loh (1999) and Gu and Korus (1995), the following expression that describes cell growth and death behaviour in a ternary substrate system is proposed as:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (1)

[S.sub.i0] is the initial substrate concentration and i = 1, 2 and 3 for phenol, 2-cp and 4-cp, respectively. The second term on the right-hand side of Equation (1) is a semi-empirical model accounting for the cell specific death rate attributed to 2-cp and 4-cp. The growth kinetics for the dual substrate systems (phenol - 2-cp or phenol - 4-cp) can be obtained through the simplification of Equation (1) by setting [S.sub.30] or [S.sub.20] = 0, respectively.

Biomass concentration (X) can be modelled by (Loh and Yu, 2000):

X = [X.sub.0][e.sup.[mu]xt] (2)

where [X.sub.0] represents the initial biomass concentration.

Substrate Degradation Kinetics

Wang and Loh (1999) have systematically investigated the biodegradation of phenol by P. putida ATCC 49451 in batch cultures and successfully simulated the phenol degradation profile by accounting for the role of the metabolic intermediates over a wide range of initial phenol concentrations (25-800 mg/L):

[q.sub.ph] = d[S.sub.1]/Xdt = 0.819[S.sub.1] / 2.19 + [S.sub.1] + [([S.sub.10]-[S.sub.1]).sup.2]/810 (3)

Equation (3) was used as a basis for our present study.

Dual substrate system of phenol - 2-cp and phenol - 4-cp

To account for the competitive inhibition effects between chlorophenols and phenol degradation, the specific degradation rate of phenol in the presence of 2-cp or 4-cp is described as (n = 2, 4):

[q.sub.ph,n-cp] = 0.819[S.sub.1] / 2.19 (1 + [S.sub.1]/[k.sub.I,n-cp]) + [S.sub.1] + [([S.sub.10]-[S.sub.1]).sup.2]/810 (4)

in which [S.sub.i] (i = 2, 3) is the substrate concentration of 2-cp and 4-cp, respectively.

An empirical Haldane-like equation is used to describe the cometabolic transformation of 2-cp/4-cp in the presence of phenol:

[q.sub.n-cp,ph] = [R.sub.mi][S.sub.i] / [K.sub.ci] (1 + [S.sub.1]/[k.sub.i,ph]) + [S.sub.i] + [S.sup.2.sub.1]/[K.sub.I,n-cp] (5)

Ternary substrate system of phenol, 2-cp and 4-cp

In the ternary substrate system, due to the analogous structures and non-specificity of the key enzymes for biodegradation, the consumptions of substrates is strongly interrelated and many kinds of interactions may occur during the degradation process. Development of a set of models that account for all the interaction factors can be quite laborious. Moreover, the excessive parameters and the possible propagation of errors during parameter estimation may render the models not only mathematically intractable, but also practically useless. Therefore, based on the model development in the dual substrate systems, we develop a semi-empirical model to describe the degradation of phenol in the ternary substrate system as:

[q.sub.ph,2-cp,4-cp] = 0.819[S.sub.1] / 2.19 (1 + [S.sub.2]/[k.sub.I,2-cp] + [S.sub.3]/[k.sub.I,4-cp] + [S.sub.1] + [([S.sub.10] - [S.sub.1]).sup.2]/810 (6)

Here, we incorporate both 2-cp and 4-cp inhibition effects to the phenol degradation model and neglect the interaction between 2-cp and 4-cp in the ternary substrate system. Such simplification is based on two reasons: (i) the interactions between phenol and the chlorophenols are more pronounced in the ternary substrate system; and (ii) unlike phenol, which is responsible for inducing the key degradative enzymes and providing reducing energy for cometabolism, the primary effect of the non-growth supporting chlorophenols is the toxicity toward cell growth, which has been incorporated in the cell growth kinetics. Therefore, the cometabolic transformation of chlorophenols can be described in the same form as Equation (5). The validity of this simplification is assessed in the last part of the Results and Discussion section.

MATERIALS AND METHODS

Organism and Culture Conditions

The bacterium Pseudomonas putida ATCC 49451, which is able to grow on phenol and cometabolize 2-cp and 4-cp, was used throughout this work. Stock cultures of P. putida were maintained by periodic sub-transfer on nutrient agar (Oxoid, U.K.) slants, which were stored at 4[degrees]C in the refrigerator. All batch cultures were performed in 500 mL Erlenmeyer flasks fitted with a cotton plug at 50% medium volume. The culture media composed of basal mineral salt medium and carbon substrates. The mineral salt medium was prepared as described by Loh and Wang (1998). The concentration of carbon substrates added, i.e., phenol, 2-cp and 4-cp, were varied for different experiments.

All media (except phenol, 2-cp and 4-cp), pipette tips, and Erlenmeyer flasks fitted with cotton plugs were autoclaved at 121[degrees]C for 20 min for sterilization before using. Culture transfers and sampling were conducted aseptically around a Bunsen burner in a biological safety cabinet (GELMAN, U.S.A.) to minimize contamination. Unless otherwise stated, prior to inoculation for each experiment, cells were induced by transferring a loop of stock culture maintained on nutrient agar slant to the mineral medium and adding 200 mg/L of phenol as the sole carbon source. The resulting cell suspension (2.5 mL) from the late exponential growth phase of the induced cells was used as an inoculum and transferred to each flask. After inoculation, phenol, 2-cp and 4-cp were added from stock solutions (at concentrations of 10 000 mg/L; 5 000 mg/L and 10 000 mg/L, respectively) to give the desired initial concentrations. Cells were grown in flasks on a rotary shaker at 30[degrees]C and 160 rpm. All experiments were performed at least in duplicates.

Analytical Methods

Samples were withdrawn periodically for analysis. Cell density, concentrations of phenol, 2-cp and 4-cp were monitored.

Cell density measurement

A 4 mL sample from each flask was taken for determination of biomass. Cell density was monitored spectrophotometrically by measuring the absorbance at a wavelength of 600 nm using a Shimadzu model UV-1601 spectrophotometer with 1-cm path quartz cuvettes. When 2-cp was transformed, to exclude the dark colouration effect on the OD reading, the OD of the sample was taken before and after filtration through 0.45 [micro]m syringe filter and the difference in values taken as an indication of the real cell density. The validity of this analysis was ascertained by determining the dry cell weight of the samples. The comparison of the weights of neat dry samples (without filtration) and the residue on the filter paper (from the proposed filtration method) showed that the filtration method works fine (data not shown).

Phenol, 2-cp and 4-cp analysis

Concentrations of phenol, 2-cp and 4-cp in the samples were analyzed by HPLC. Culture samples (3 mL) for HPLC analysis were filtered through a 0.45 [micro]m syringe filter (Millipore, U.S.A.). The cell-free samples were stored at -20[degrees]C until required for analysis. For analysis, a 25 [micro]L aliquot was injected into the HPLC system (Waters, Milford, U.S.A.) equipped with a 4.6 x 100 mm Chromolith C18e column (Merck, Darmstadt, Germany). The solvents used for the HPLC system were solvent A, methanol (HPLC grade, Merck, Darmstadt, Germany) and solvent B, 1% acetic acid (HPLC grade, Merck, Darmstadt, Germany). The elution rate used was 3.0 mL/min and the volume ratio of A to B was 40% to 60% over 3 min. Phenol, 2-cp and 4-cp were monitored by UV detection at 275 nm, and the retention times, in minutes for phenol, 2-cp and 4-cp were 0.9, 1.5 and 2.1, respectively.

RESULTS AND DISCUSSION

Batch experiments of the dual substrate systems (phenol - 2-cp and phenol - 4-cp) were first performed to study the cell growth and substrates transformation behaviour, elucidate the possible degradation pathway and also to determine the parameters in the kinetics models. Experiments of the ternary substrate system were then carried out to examine the substrate interactions and the consequential effects on cell growth and substrates degradation. Quantitative effects of inhibition and toxicity of 2-cp and 4-cp were then obtained and explained. All biodegradation experiments performed in this research are summarized in Table 1.

Cell Growth and Degradation in Dual Substrate Systems

Typical results of phenol degradation in the presence of 2-cp are exemplified in Figures 1a and 1b for experiments D7 and D10, respectively. It was found that as the initial concentration of 2-cp increased, the time required for the complete degradation of phenol (200 mg/L) was prolonged: 9.5 h (phenol alone, figure not shown); 11 h (D7) and 20 h (D10). The average degradation rates of phenol were calculated to be 40 mg/L-h (phenol alone), 29 mg/L-h (D7) and 23 mg/L-h (D10). For comparison, in the presence of 100 mg/L 4-cp (D18), the phenol degradation rate was 30 mg/L-h. These indicate a stronger negative effect of 2-cp on phenol degradation than that of 4-cp. From Figure 1, it can be seen that rapid transformation of 2-cp only occurred when a large part of phenol had been degraded clearly demonstrating competitive inhibition between phenol and 2-cp. Similar observations have been reported for phenol and 4-cp by Loh and Wang (1998). When 2-cp concentration was increased to 50 mg/L (Figure 1b), only 35% of it was transformed at the end of the experiment. This incomplete transformation was due to the toxicity of 2-cp on cell growth and consequently degradation ability.

[FIGURE 1 OMITTED]

Kinetics of cell growth on phenol in the presence of 2-cp/4-cp was modelled by the simplified form of Equation (1). [[mu].sub.m] (0.9[h.sup.-1]), [K.sub.1] (6.93 mg/L) and [K.sub.11] (284 mg/L) were obtained from the kinetics for cell growth on phenol alone (Wang and Loh, 1999), while the inhibition parameters from the effect of 4-cp were obtained as [K.sub.3] (3.57 mg/L) and [k.sub.d3] (51.5 mg/L) (Wang and Loh, 2000). [k.sub.d0], which represents the cell endogenous decay coefficient, is usually very small and negligible (Klecka and Maier, 1988). In this study, the value of [k.sub.d0] was assumed to be 0.002[h.sup.-1], which is within the range reported in the literature (Wang et al., 1979). The only two parameters in the growth kinetics model, [K.sub.2] and [k.sub.d2], were determined by curve fitting to the cell growth data obtained from the dual system of phenol and 2-cp. It was found that [K.sub.2] = 5.62 mg/L and [k.sub.d2] = 17.8 mg/L with a correlation coefficient of [r.sup.2] = 0.98. Thus the cell growth kinetics for phenol and 2-cp can be written as:

[mu] = 0.900[S.sub.10] / [S.sub.10] + 6.93 (1 + [S.sub.20]/5.62) + [s.sup.2.sub.10]/284 - 0.002Exp ([S.sub.20]/17.8) (7)

Figure 2 shows the excellent agreement of the model to the experimental data. It is important to note that the model parameters were determined based on data from experiments D1-D5 and D11-D15. The data obtained in experiments D6-D10 were used for model validation, which in Figure 2 shows very good corroboration.

[FIGURE 2 OMITTED]

Phenol degradation and chlorophenols transformation in the dual substrates system were modelled by Equations (4) and (5), respectively. In order to estimate the model parameters, two sets of experimental data (D6 and D9) and (D16 and D18) were each used for curve fitting. The parameters for substrate degradation are summarized in Table 2. Figures 3 and 4 shows that the model fitted very well with the experimental data. In all cases, the correlation coefficient, [r.sup.2], was in excess of 0.98.

[FIGURES 3-4 OMITTED]

During the experiments for cometabolic transformation of 2-cp, it was observed that the colour of the culture medium changed from colourless to greenish yellow before turning brown, which persisted in the medium. It has been reported that the greenish yellow colour was due to the formation of 2-hydroxy muconic acid semialdehyde, an intermediate of the meta cleavage pathway of phenol degradation (Wang, 1997) as shown in Figure 5a. The brown colouration in the medium was reported to be a common observation when chloroaromatics were degraded via 3-chlorocatechol (Haller and Finn, 1979; Adams et al., 1992). Farrell and Quilty (1999) reported that the brown pigment could be attributed to the build-up of 3-chlorocatechol, which polymerized due to autoxidation when 2-cp was transformed via the meta-pathway (Figure 5c). It has been proposed that the meta-cleavage product of 3-chlorocatechol, a highly reactive acylchloride could act as a suicide compound, binding irreversibly to the meta-cleavage enzyme with a subsequent release of chloride and the destruction of metabolic activity. This negative effect that 3-chlorocatechol has on the meta-cleavage enzyme resulted in the accumulation of chlorocatechol. It is noteworthy that in our experiments, very negligible chloride ions were found in the culture medium.

[FIGURE 5 OMITTED]

Cell Growth and Degradation in Ternary Substrate System

Figure 6a shows a typical profile for biodegradation and cell growth for the case of complete degradation of 2-cp and 4-cp in the presence of phenol. The data presented correspond to experiment T2 when 2-cp concentration was only 20 mg/L and 4-cp concentration was 50 mg/L. In this case, after a lag phase of about 6 h, the cells began to grow exponentially and phenol was the first substrate to be degraded. When phenol was almost completely depleted, both 2-cp and 4-cp were simultaneously transformed. Among the three compounds, phenol was completely degraded in the shortest time (11.5 h), followed by 4-cp (12.5 h) and finally 2-cp (16 h). Figure 6b shows a typical multiple substrates degradation profile for the experiments that exhibited incomplete 2-cp and 4-cp degradations. The data shown correspond to experiment T14 when 2-cp concentration was high at 40 mg/L and 4-cp concentration was 100 mg/L. It was found that the cells went into exponential phase only after a long lag phase of 19 h. During the following 11 h, phenol was completely degraded. However, only 80% of the initial 4-cp and 50% of the initial 2-cp were degraded. The maximum cell density achieved in T14 was also 30% lower than that of T2. In cometabolism, the consumption of the growth substrate (phenol) was used for cell synthesis, maintenance, as well as to overcome substrate inhibitions. With the increase of 2-cp and 4-cp concentrations in the culture, the energy requirement for these and hence active cell mass needed concomitantly increased. It is also postulated that the toxicities associated with high 4-cp and 2-cp concentrations resulted in enzyme inactivation and poor recovery with incomplete transformation of these substrates being the net effect (Ely et al., 1995). Figure 7 shows the effect of 2-cp and 4-cp on cell growth in the ternary substrate systems. It can be seen that with the increase of 2-cp/4-cp concentration, the highest cell densities as well as the specific growth rate were decreased, while the lag phase was prolonged. All of these can be ascribed to the toxicity pressure exerted by 2-cp and 4-cp on the cells.

[FIGURES 6-7 OMITTED]

Equation (1) was used to describe cell growth on these three substrates. All the parameters in Equation (1) except [K.sub.23.sup.*] and [k.sub.d] have been previously obtained. By correlating the experimental data to the proposed model, these two parameters were determined as [K.sub.23.sup.*] = 202 mg/L and [k.sub.d] = 35.7 mg/L. Table 3 presents a summary of all the parameters determined. Figure 8 shows that the model represented the experimental data very well ([r.sup.2] = 0.99). Again, experiments T1-T5 and T11-T15 were used for parameter estimation while T6-T10 were used for validation, confirming the predictive capability of the kinetics model.

[FIGURE 8 OMITTED]

Phenol degradation in the presence of 2-cp and 4-cp was modelled by Equation (6), while the cometabolic transformation of 2-cp and 4-cp in the ternary substrate system was modelled by Equation (5). Figure 9 shows the comparison of the model predictions and the experimental data for two of the ternary substrate systems (Experiments T2 and T14). It can be seen that the model predicts phenol degradation very well. The corroboration between the simulations of 2-cp and 4-cp transformation profiles and the experimental results are also very good, ascertaining the validity of our previous simplification as described in the Model Development section.

[FIGURE 9 OMITTED]

The magnitude of the model parameters provides a quantitative indication of the extent of inhibition and toxicity of phenol and the chlorophenols investigated in this research. Firstly, the interaction between phenol and chlorophenols (2-cp and 4-cp) could be regarded as competitive inhibition as the models revealed. Biodegradation of phenol, 2-cp and 4-cp each occurred via the meta-cleavage pathway, as shown in Figure 5. Based on the degradative pathway depicted, it seems that the point of departure between 2-cp and 4-cp transformation lied in the transformation of the associated chlorocatechol, while the initial hydroxylation was less specific (Haggblom and Valo, 1995). On closer examination however, it can be seen that in both cases, the purpose of mono-oxygenase in the first step was to add a hydroxyl to the ortho position of the chlorophenol. It is speculated that since chlorine was occupying one of the ortho position in 2-cp, competitive inhibition between phenol and the chlorophenols favoured the initial hydroxylation step over 2-cp. The inhibition exerted by 4-cp to cell growth on phenol (the growth supporting substrate) can therefore be expected to be stronger than that by 2-cp. This bared out in the model parameters, [K.sub.2] and [K.sub.3] in the growth kinetics ([K.sub.2] = 5.62 mg/L > [K.sub.3] = 3.57 mg/L).

In the case of the degradation profile observed, interactions between the substrates could be a result of the toxicity of the intermediates of cometabolic transformation, as reported by Alvarez-Cohen and Speitel (2001). This has been generally reported as the toxicity effect. As shown in Table 2, the toxicity coefficient of 2-cp ([k.sub.d2]) is smaller than that of 4-cp ([k.sub.d3]), indicating that the toxicity of 2-cp to biodegradation rate was more intense than that of 4-cp. To this support, it has been reported that the presence of ortho substituents could increase the toxicity of phenol derivatives (Beltrame et al., 1988). On the contrary, Liu et al. (1982) reported a negative correlation of the chlorophenols' toxicity to a bacterial culture in the presence of ortho substituents. In fact, Beltrame et al. (1988) found that if a hydrophobic or electrophilic effect intervenes in the interaction of inhibitors with enzymes, factors affecting lipophilicity and electrophilicity might also affect the inhibiting action. In the case of chlorophenols, ortho chloro substituent could give a reduced contribution to lipophilicity and electrophilicity. It could also be possible that the intermediates of 2-cp were more inhibiting to biodegradation than that of 4-cp, a similar mechanism to that reported in the literature (Beltrame et al., 1988; Farrell and Quilty, 1999).

The overall effect of 2-cp and 4-cp on cell growth and phenol degradation should be determined by an overall evaluation of competitive inhibition and toxicity. As shown by the magnitude of [k.sub.d2] (17.8 mg/L) and [k.sub.d3] (51.5 mg/L), [k.sub.d3] is approximately three times larger than [k.sub.d2], implying a significant difference in toxicity level between 2-cp and 4-cp. Therefore, in our system it could be possible that toxicity effect on biodegradation outweighed the competitive inhibition effect on cell growth.

CONCLUSIONS

Cometabolism has been found to be an inexpensive and effective technology in the treatment of hazardous and recalcitrant industrial wastes, in which different substrates often coexist. In this work, Pseudomonas putida ATCC 49451 was used to transform 2-chlorophenol and 4-chlorophenol cometabolically in the presence of phenol. Batch experiments were performed to study the cell growth and substrates transformation kinetics and to understand the various substrate interactions in a ternary substrate system. As non-growth substrates, both 2-cp and 4-cp inhibited cell growth and phenol degradation severely due to the toxicity and substrate interactions. Among the three substrates, phenol was completely degraded within the shortest time, followed by 4-cp and then 2-cp. Very often, 2-cp and 4-cp could not be degraded completely due to the limited active cell mass and the intense toxicity pressure. During the biotransformation of 2-cp, a brown colouration resulted which remained in the medium. This is a manifest of the incomplete degradation of 2-cp via the meta-degradation pathway. A set of models that involved the substrate interactions and toxicity effect on cell growth and substrate transformation was developed. The parameter values gave some indications of the different degree of toxicity and substrate interactions of the monochlorophenols and the experimental observations were rationalized by means of the reaction mechanism and transformation pathways.

NOMENCLATURE

[k.sub.d] toxicity coefficient caused by interaction of 2-cp
 and 4-cp on cell growth (mg/L)
[k.sub.d0] endogenous decay coefficient ([h.sup.-1])
[k.sub.d2] toxicity coefficient of 2-cp on cell growth (mg/L)
[k.sub.d3] toxicity coefficient of 4-cp on cell growth (mg/L)
[K.sub.1] parameter in Andrews kinetics on phenol (mg/L)
[K.sub.11] self-inhibition constant of phenol (mg/L)
[K.sub.2] inhibition coefficient of 2-cp to cell growth on
 phenol (mg/L)
[K.sub.3] inhibition coefficient of 4-cp to cell growth on
 phenol (mg/L)
[K.sub.23.sup.*] substrate interaction coefficient of 2-cp and
 4-cp (mg/L)
[K.sub.c2] half-saturation constant for 2-cp transformation
 (mg/L)
[K.sub.c3] half-saturation constant for 4-cp transformation
 (mg/L)
[k.sub.I, 2-cp] inhibition coefficient of 2-cp to phenol degradation
 (mg/L)
[k.sub.I, 4-cp] inhibition coefficient of 4-cp to phenol degradation
 (mg/L)
[K.sub.I, 2-cp] inhibition constant of 2-cp (mg/L)
[K.sub.I, 4-cp] inhibition constant of 4-cp (mg/L)
[k.sub.2, ph] inhibition coefficient of phenol to 2-cp
 transformation (mg/L)
[k.sub.3, ph] inhibition coefficient of phenol to 4-cp
 transformation (mg/L)
[R.sub.m1] maximum specific degradation rate of phenol
 (mg/(mg.h))
[R.sub.m2] maximum specific consumption rate of 2-cp
 (mg/(mg.h))
[R.sub.m3] maximum specific consumption rate of 4-cp
 (mg/(mg.h))
S substrate concentration (mg/L)
[S.sub.i0] initial substrate concentration (mg/L)
t time (h)
X biomass concentration (mg/L)
[X.sub.0] initial biomass concentration (mg/L)

Greek Symbols

[mu] overall specific growth rate ([h.sup.-1])
[[mu].sub.d] cell decay rate ([h.sup.-1])
[[mu].sub.m] parameter in Andrews kinetics on phenol ([h.sup.-1])

REFERENCES

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Kai-Chee Loh * and Tingting Wu

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576

* Author to whom correspondence may be addressed.

E-mail address: chelohkc@nus.edu.sg

Table 1. Summary of batch biotransformation experiments

Experiment no. Initial nominal concentration (mg/L)

 Phenol 2-cp 4-cp

D1-D5 150 10, 20, 30, 40, 50 0
D6-D10 200 10, 20, 30, 40, 50 0
D11-D15 300 10, 20, 30, 40, 50 0
D16-D18 200 0 50, 70, 100
T1-T5 200 10, 20, 30, 40, 50 50
T6-T10 200 10, 20, 30, 40, 50 70
T11-T15 200 10, 20, 30, 40, 50 100

Table 2. Summary of model parameter values for mixtures of phenol and
chlorophenols

 Model
 parameters Value

Phenol [R.sub.m1] 0.819 mg/(mgxh)
degradation
 [K.sub.s] 2.19 mg/L
 [K.sub.p] 810 mg/L

Mixture of [k.sub.l, 2-cp] 4.41 mg/L ([+ or -]1.08)
phenol and 2-cp 0.0321 mg/(mgxh) ([+ or -]0.0076)
 [R.sub.m2] 2.83 mg/L ([+ or -]0.52)
 [K.sub.c2] 3.36 mg/L ([+ or -]1.06)
 [k.sub.2, ph] 117 mg/L ([+ or -]9)
 [K.sub.l, 2-cp] 0.857 mg/L ([+ or -]0.184)
 [k.sub.l, 4-cp] 0.101 mg/(mgxh) ([+ or -]0.004)

phenol and 4-cp [R.sub.m3] 3.10 mg/L ([+ or -]0.75)
 [K.sub.c3] 3.66 mg/L ([+ or -]0.45)
 [k.sub.3, ph] 141 mg/L ([+ or -]20)
 [K.sub.l, 4-cp]

Values in parentheses are one standard deviation from the mean.

Table 3. Summary of the model parameter values for ternary
substrate system (phenol, 2-cp and 4-cp)

 Model Value
 parameters

Cell growth in phenol alone [micro]m 0.900h-1
 [K.sub.1] 6.93 mg/L
 [K.sub.11] 284 mg/L

Inhibition of 2-cp [K.sub.2] 5.62 mg/L ([+ or -]1.64)

Inhibition of 4-cp [K.sub.3] 3.57 mg/L

Interaction of 2-cp and 4-cp [K.sup.*. 202 mg/L ([+ or -]39)
 sub.23]
 [k.sub.d2] 17.8 mg/L ([+ or -]4.9)

Toxicity coefficient [k.sub.d3] 51.5 mg/L
 [k.aub.d] 35.7 mg/L ([+ or -]4.3)

Endogeneous decay [k.sub.d0] 0.002[h.sup.-1]

Values in parentheses are one standard deviation from the mean.